TW202426060A - Engineered retrons and methods of use - Google Patents

Abstract

Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g., mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivoto the host (e.g., mammalian) cell.

Description

Engineered retrotransposons and methods of use

The present disclosure generally relates to systems, methods and compositions for precise genome editing, including nucleic acid insertion, substitution and deletion at targeted and precise genomic sites, wherein the systems, methods and compositions are based on novel and/or engineered retrotransposons.

Precise genome editing by programmable nucleases (e.g., RNA-guided nucleases (e.g., CRISPR nucleases), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENS)) generally relies on homology-directed repair (HDR) and the presence of a donor DNA template at the site of a double-strand break (DSB) induced by the programmable nuclease. It is generally believed that a limiting step of HDR-dependent precise genome editing is the delivery of the donor DNA template to the nuclease-induced DSB (e.g., see Ling et al., “Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates,” Science Advances, 2020, Vol. 6, No. 15, pp. 1-8). A variety of approaches have been reported to enhance the efficiency of HDR-dependent editing, many of which involve physically tethering the DNA donor to components of the precise editing system. Exemplary methods are discussed in: K. Lee et al., “Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering,” eLife 6, e25312 (2017); J. Carlson-Stevermer et al., “Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing,” Nat. Commun. 8, 171 1 (2017); N. Savic et al., “Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair,” eLife 7, e33761 (2018); and E. J. Aird et al., “Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.,” Commun. Biol. 1, 54 (2018), each of which is incorporated herein by reference. Despite these efforts, the efficiency of accurate editing of HDR dependencies remains unsatisfactory.

Retrotranscriptons are defined by their unique ability to generate unusual satellite DNA, known as msDNA (multi-copy single-stranded DNA). The DNA encoding the retrotranscripton includes a reverse transcriptase (RT) encoding gene ( ret ) and a nucleic acid sequence encoding a non-coding RNA (ncRNA) containing two consecutive and inverted non-coding sequences, known as msr and msd . The ret gene and the non-coding RNA (including msr and msd ) are transcribed as a single RNA transcript, which is folded into a specific secondary structure after post-transcriptional processing. Once transcribed, RT binds to an RNA template downstream of the msd locus, thereby initiating reverse transcription of the RNA towards its 5' end with the help of the 2'OH group present in the conserved branch-chain guanosine residue that acts as a primer. Reverse transcription stops before reaching the msr locus, and the resulting DNA (msDNA) remains covalently linked to the RNA template via 2'-5' phosphodiester bonds and base pairing between the msDNA and the 3' end of the RNA template. The external regions at the 5' and 3' ends of the msd/msr transcripts (a1 and a2, respectively) are complementary and can hybridize, leaving structures located in the msr and msd regions in internal positions (see Figure 1A). The msr locus does not undergo reverse transcription, forming one to three short stem-loops of variable size (ranging from 3 to 10 base pairs), while the msd locus folds into a single/double long hairpin with a highly variable stem of 10-50 bp in length, which is also present in the final msDNA form.

It has recently been reported that retrotranscripts can be used as a means to provide donor DNA templates for HDR-dependent genome editing (e.g., see Lopez et al., “Precise genome editing across kingdoms of life using retron-derived DNA,” Nature Chemical Biology , Dec. 12, 2021, 18 , pp. 199-206 (2022)), however, generating sufficient levels of donor DNA templates in cells to fully support efficient HDR-dependent editing remains a major challenge. Improved retrotranscript-based genome modification systems are highly desirable in this technology.

In one aspect, the present disclosure provides recombinant retrotransposons comprising one or more genetic modifications that improve the function and/or properties of the retrotransposons. Such genetic modifications may include mutations, insertions, deletions, inversions, substitutions, replacements or translocations of one or more consecutive or non-consecutive nucleotides in a nucleic acid molecule encoding a retrotransposon or a retrotransposon component (such as an ncRNA or a reverse transcriptase). In various aspects, a retrotranscript modified with one or more genetic modifications (i.e., a "pre-modified" or "unmodified" retrotranscript or retrotranscript component) is a naturally occurring retrotranscript or retrotranscript component (e.g., a naturally occurring ncRNA or RT of Table A) that is capable of promoting homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentration or amount of msDNA comprising a DNA donor template. In certain embodiments, the recombinant retrotranscript is based on and/or derived from a naturally occurring retrotranscript, such as any retrotranscript-related sequence provided by Table X (introducing one or more genetic modifications into a collection of 7257 previously unknown retrotranscripts discovered by the computational methods described herein (e.g., see Examples). In other embodiments, the recombinant retrotranscript is based on introducing one or more genetic modifications into a previously available retrotranscript sequence (e.g., "Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems , Nucleic Acids Research, Vol. 48, No. 22, Dec. 16, 2020, 12632-12647" (incorporated herein by reference) to obtain recombinant retrotranscripts having an enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.

In another aspect, the present disclosure further provides nucleic acid molecules encoding recombinant retrotranscripts and/or recombinant retrotranscript components (e.g., recombinant ncRNA and/or recombinant retrotranscript RT). In yet another aspect, the present disclosure provides a genome editing system comprising a recombinant retrotranscript component (e.g., recombinant ncRNA and/or recombinant RT), a programmable nuclease (e.g., RNA-guided nuclease, such as CRISPR-Cas proteins, ZFPs, and TALENS), and a guide RNA (in the case where RNA-guided nucleases are used in the genome editing systems). In another aspect, the present disclosure provides nucleic acid molecules encoding the genome editing systems and the components thereof, and polypeptides constituting the components of the genome editing systems. In another aspect, the present disclosure provides vectors for transferring and/or expressing such genome editing systems, e.g., in vitro , ex vivo , and in vivo conditions. In another aspect, the present disclosure provides cell delivery compositions and methods, including compositions for passive and/or active delivery to cells (e.g., plasmids), delivery by viral-based recombinant vectors (e.g., AAV and/or lentiviral vectors), delivery by non-viral-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retrotransposon-based genome editing systems described herein can be delivered in the form of DNA (e.g., plasmids or DNA-based viral vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), mixtures of DNA and RNA, proteins (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combination of methods for delivering the components of the retrotransposon-based genome editing systems disclosed herein can be employed. In one embodiment, each component of the retrotranscript-based genome editing system is delivered by a total RNA system, for example, one or more RNA molecules (e.g., mRNA and/or ncRNA) are delivered by one or more LNPs, wherein the one or more RNA molecules form ncRNA and guide RNA (as needed) and/or are translated into polypeptide components (e.g., RT and programmable nuclease). In another aspect, the present disclosure provides a method for genome editing by introducing the retrotranscript-based genome editing system described herein into a cell comprising a target editing site (e.g., in vitro, in vivo , or in vitro conditions), thereby causing editing to be performed at the target editing site. In other aspects, the disclosure provides formulations comprising any of the foregoing components for delivery to cells and/or tissues, including in vitro , in vivo and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retrotranscript-based genome modification systems and methods described herein, and methods of modifying cells by performing genome editing and related DNA donor-dependent methods (such as recombineering or cell transcription) using the retrotranscript-based genome modification systems disclosed herein. The present disclosure also provides methods for preparing the recombinant retrotransposons, retrotransposons-based genome modification systems, vectors, compositions and formulations described herein, as well as pharmaceutical compositions and kits for modifying cells in vitro , in vivo and ex vivo , which comprise the genome editing and/or modification systems disclosed herein.

In one embodiment, the present disclosure or the invention herein provides a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprises an RNA cargo; the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscript reverse transcriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the programmable nuclease; and each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c); whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b) and (c).

In one embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a single mRNA molecule encoding a nucleic acid programmable nuclease and a retrotransposase.

In one embodiment, in the gene editing system, (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.

In one embodiment, in a gene editing system, (a)(i) and (a)(ii) are encoded and expressed as a fusion protein, and the fusion protein comprises the C terminus of a nucleic acid programmable nuclease fused to the N terminus of a retrotranscriptase (nuclease:RT fusion); or the fusion protein comprises the N terminus of a nucleic acid programmable nuclease fused to the C terminus of a retrotranscriptase (RT:nuclease fusion).

In one embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a first mRNA molecule encoding a nucleic acid programmable nuclease and a second mRNA molecule encoding a retrotransposase.

In one embodiment, in the gene editing system, (c) is separated from (a)(i), (a)(ii) and (b) or provided in trans .

In one embodiment, in the gene editing system, (b) the engineered retrotran ncRNA and (c) the guide RNA are fused or provided in cis .

In one embodiment, in the gene editing system, (b) the engineered retrotranscript ncRNA and (c) the guide RNA are fused or provided in cis , and the guide RNA is fused to the 5' end of the retrotranscript ncRNA.

In one embodiment, in the gene editing system, (b) the engineered retrotranscript ncRNA and (c) the guide RNA are fused or provided in cis , and the guide RNA is fused to the 3' end of the retrotranscript ncRNA.

In one embodiment, in a gene editing system, (b) an engineered retrotranscript ncRNA and (c) a guide RNA are fused or provided in tandem , and the engineered ncRNA comprises a first guide RNA fused to the 5' end of the retrotranscript ncRNA and a second guide RNA fused to the 3' end of the retrotranscript ncRNA, and the first and second guide RNAs target different sequences. Therefore, in a broader scope, in one embodiment, in a gene editing system, (c) a guide RNA for a programmable nuclease may comprise one or more guides targeting the same or different target sequences. In one embodiment, such a guide RNA may be a single guide RNA or sgRNA; for example, when the nucleic acid programmable nuclease comprises Cas9.

In one embodiment, in a gene editing system, one or more delivery vehicles comprise liposomes or lipid nanoparticles (LNPs).

In one embodiment, in a gene editing system, (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscriptase and (b) an engineered retrotranscript ncRNA are in the same delivery vehicle.

In one embodiment, in a gene editing system, (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscriptase and (b) an engineered retrotranscript ncRNA are in separate delivery vehicles.

In one embodiment, in the gene editing system, the nucleic acid programmable nuclease and the retrotranscriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle.

In one embodiment, in the gene editing system, the nucleic acid programmable nuclease and the retrotransposase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.

In one embodiment, in a gene editing system, an engineered retrotranscript ncRNA includes a sequence encoding a donor polynucleotide comprising the desired edit at a target sequence to be integrated into a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at 5' of the target sequence and a 3' homology arm hybridized to a sequence at 3' of the target sequence. In one embodiment, the donor polynucleotide may be heterologous to the cell. In one embodiment, the donor polynucleotide may be endogenous to the cell; for example, the cell may contain a sequence that is unique to those in a population with a disease state, and the donor polynucleotide may be a sequence that is not unique to those in a population without a disease state (for example, the donor may be used for gene correction or repair of the cell to modify the cell from having a mutation or modification that causes a disease state to having a sequence that is not unique to the disease state). This may be done in an animal cell or a mammalian cell (e.g., a primate, a non-human primate, or a domesticated mammal, such as a cat or dog or horse) or a human cell; for example, to correct, resolve, treat, alleviate a genetic disease in an animal, mammal, domesticated mammal, cat, dog, horse, or human. This can be done in plant cells to introduce mutations that result in beneficial phenotypic traits such as disease resistance or other beneficial plant traits.

In one embodiment, in the gene editing system, the nucleic acid programmable nuclease comprises Cas9 nuclease, TnpB nuclease or Cas12a nuclease.

In one embodiment, in a gene editing system, an engineered retrotransposon ncRNA comprises: A) a leading msr sequence having a first complementary region of the retrotransposon ncRNA; B) an msr sequence including an msr stem-loop structure; C) an msd sequence including an msd stem-loop structure and related sequences, wherein the msd sequence templates a single-stranded DNA product (RT-DNA) in the presence of a retrotransposon reverse transcriptase; and D) an msd post-sequence having a second complementary region, wherein the first and second complementary regions form an a1/a2 duplex region of the retrotransposon ncRNA, wherein the msr stem-loop structure, The msd stem-loop structure or a1/a2 duplex comprises a modification that results in increased editing efficiency in the presence of a nucleic acid programmable nuclease associated with one or more guide RNAs, and wherein, as the case may be, one or more guide RNAs of (c) are coupled to the leading msr sequence, the msd post sequence, or both the leading msr sequence and the msd post sequence. In such embodiments where the engineered retrotranscript ncRNA comprises A), B), C) and D), wherein the sequence of interest may encode a donor polynucleotide comprising the intended editing at a target sequence to be integrated into a cell, wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at 5' of the target sequence and a 3' homology arm hybridized to a sequence at 3' of the target sequence. In such embodiments where the engineered retrotranscript ncRNA comprises A), B), C) and D) (and a related sequence encoding a donor polynucleotide or simply a related sequence), the ncRNA has a nucleotide sequence of Table B, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with a sequence of Table B. The donor polynucleotide may be heterologous to the cell. Alternatively, the donor polynucleotide may be endogenous to the cell. For example, the cell may contain sequences that are unique to those in a population with a disease state, and the donor polynucleotide may be a sequence that is unique to those in a population that does not have a non-disease state (e.g., the donor may be used for gene correction or repair of the cell to modify the cell from having a mutation or modification that causes a disease state to having a sequence that is not unique to the disease state).

In one embodiment of the gene editing system, the gene editing system may include any combination of the previously described embodiments of the gene editing system.

In one embodiment, the disclosure or invention herein provides a cell, such as an isolated cell comprising a gene editing system disclosed herein (such as in any of the preceding paragraphs). In one embodiment, the cell (e.g., an isolated cell) may be a eukaryotic cell. In one embodiment, the eukaryotic cell may be a plant cell or an animal cell or a mammalian cell, such as an isolated plant cell or an isolated animal cell or an isolated mammalian cell. In one embodiment, the mammalian cell (e.g., an isolated mammalian cell) may be a human cell. In one embodiment, the cell may be a prokaryotic cell, such as a bacterial cell. In such embodiments where the cell is a bacterial cell, the donor polynucleotide may encode antibiotic sensitivity; and thus, the invention may be directed to a means of addressing such bacteria by rendering antibiotic-resistant bacteria sensitive to antibiotics (and the individual to whom the gene editing system is administered may then also receive the antibiotic, the gene editing system rendering the bacteria sensitive to the antibiotic).

In one embodiment, the disclosure or invention herein provides a composition comprising: a) a gene editing system disclosed herein (such as in any of the preceding paragraphs); and b) a pharmaceutically or veterinarily acceptable carrier. In one embodiment, in the composition, the delivery vehicle may comprise a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids. In one embodiment, in the composition, the one or more ionizable lipids comprise the ionizable lipids set forth in Table 2.

In one embodiment, the present disclosure or the invention herein provides the use of the gene editing system embodiments and/or compositions disclosed herein (such as in any of the preceding paragraphs); for example, the use of in vivo , in vitro or ex vivo modified cells or genetically modified cells, such as eukaryotic or prokaryotic cells and/or animal cells and/or mammalian cells and/or human cells and/or bacterial cells and/or plant cells (such as any cells discussed herein, wherein the cells include isolated cells). In one embodiment, the present disclosure or the invention herein provides the use of the gene editing system embodiments and/or compositions disclosed herein (such as in any of the preceding paragraphs); for example, the use of treating or resolving a genetic disease in an individual,

In one embodiment, the disclosure or invention herein provides methods for genetically modifying cells, the methods comprising: contacting a gene editing system as discussed herein (such as in any of the preceding paragraphs), or a composition as discussed herein (such as in any of the preceding paragraphs) (which comprises a gene editing system as discussed herein (such as in any of the preceding paragraphs)), advantageously, a gene editing system comprising a sequence encoding a donor polynucleotide, the donor polynucleotide comprising the gene to be modified The method comprises contacting the composition or the gene editing system with a cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with a guide RNA, wherein the guide RNA guides the complex to the target sequence; the nucleic acid programmable nuclease generates a double-strand break in the target sequence; the retrotranscriptase and the engineered retrotranscript ncRNA generate RT DNA comprising a donor polynucleotide; and the donor polynucleotide is integrated into the target sequence; thereby editing the cell to be genetically modified. In one embodiment, the cell can be a eukaryotic cell or a prokaryotic cell or an animal cell or a mammalian cell or a human cell or a bacterial cell or a plant cell.

Exemplary and non-limiting aspects and embodiments of the present disclosure are summarized below in numbered paragraphs.

1. A gene editing system comprising one or more delivery vehicles, wherein: The delivery vehicle(s) comprises an RNA cargo, The RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the nucleic acid programmable nuclease, Each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), and (a)(i), (a)(ii), (b) and (c) are delivered by one or more delivery vehicles.

2.    The gene editing system of paragraph 1, wherein the engineered retrovirus ncRNA comprises an HDR nucleotide sequence substituted into the retrovirus ncRNA; wherein the retrovirus reverse transcriptase has an amino acid sequence having at least 90% sequence identity with the retrovirus reverse transcriptase of Table A; wherein the retrovirus ncRNA has about 85% to 98% sequence identity with the retrovirus ncRNA of Table B.

3. The gene editing system of paragraph 2, wherein the retrotranscript ncRNA and the retrotranscript reverse transcriptase are from the same evolutionary branch.

4. The gene editing system of paragraph 2, wherein the retrotranscript ncRNA nucleotide sequence has about 85% to 98% sequence identity with SEQ ID NO:15327, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type I-C retrotranscript reverse transcriptase.

5. The gene editing system of paragraph 4, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:1262.

6. The gene editing system of paragraph 4, wherein the retrotranscript ncRNA nucleotide sequence has about 85% to 98% sequence identity with SEQ ID NO:16411, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type III retrotranscript reverse transcriptase.

7. The gene editing system of paragraph 6, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:2781.

8. The gene editing system of paragraph 6, wherein the retrotranscript ncRNA nucleotide sequence has about 85% to 98% sequence identity with SEQ ID NO:18731, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type XIII retrotranscript reverse transcriptase.

9. The gene editing system of paragraph 8, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:6342.

10. The gene editing system of paragraph 1, wherein the retrovirus reverse transcriptase comprises at least one amino acid substitution that increases processibility and/or fidelity.

11. The gene editing system of paragraph 10, wherein the retrovirus reverse transcriptase comprises an amino acid substitution in an amino acid residue corresponding to the following amino acid residue in Eco1 RT: Q190, E302 or T306.

12. The gene editing system of paragraph 10, wherein the retrovirus reverse transcriptase comprises an amino acid substitution in an amino acid residue corresponding to the following amino acid substitutions in Eco1 RT: Q190F, E302R or T306K.

13. A gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprises an RNA cargo, the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered retrotranscript reverse transcriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), (a)(i), (a)(ii), (b) and (c) are delivered by one or more delivery vehicles, and The engineered retrotransposase comprises a processability-enhancing domain or a fidelity-enhancing domain.

14. The gene editing system of paragraph 13, wherein the processability enhancing domain comprises Sso7d or Sac7d.

15. The gene editing system of paragraph 13, wherein the fidelity enhancing domain comprises a 3’ to 5’ exonuclease domain.

16. The gene editing system of paragraph 15, wherein the exonuclease domain comprises POLE1, POLD1, POLG, Pfu or KOD.

17. The gene editing system of paragraph 13, wherein the engineered retroviral ncRNA comprises an HDR nucleotide sequence substituted into the retroviral ncRNA; wherein the retroviral reverse transcriptase has an amino acid sequence having at least 90% sequence identity with the retroviral reverse transcriptase of Table A; wherein the retroviral ncRNA has about 85% to 98% sequence identity with the retroviral ncRNA of Table B.

18. The gene editing system of paragraph 13, wherein the retrotranscript ncRNA and the retrotranscript reverse transcriptase are from the same evolutionary branch.

19. A gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprises an RNA cargo, the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered reverse transcriptase, (b) an engineered reverse transcriptase ncRNA, and (c) a guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), (a)(i), (a)(ii), (b) and (c) are delivered by one delivery vehicle or more than one delivery vehicle, and The engineered reverse transcriptase comprises a Y region domain from a reverse transcriptase RT corresponding to the engineered reverse transcriptase ncRNA.

20. The gene editing system of paragraph 19, wherein the engineered reverse transcriptase is a chimera comprising MMLV RT fused to the Y region of the retrotranscript RT.

21. The gene editing system of paragraph 1, wherein (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retrotransposase.

22. The gene editing system of paragraph 21, wherein (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.

23. The gene editing system of paragraph 22, wherein the fusion protein comprises the C-terminus of the nucleic acid programmable nuclease fused to the N-terminus of the retrotranscriptase (nuclease:RT fusion).

24. The gene editing system of paragraph 22, wherein the fusion protein comprises the N-terminus of the nucleic acid programmable nuclease fused to the C-terminus of the reverse transcriptase of the retrovirus (RT: nuclease fusion).

25. The gene editing system of paragraph 1, wherein (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retrotransposase.

26. The gene editing system of paragraph 1, wherein (c) is separated from (a)(i), (a)(ii) and (b) or provided in trans .

27. The gene editing system of paragraph 1, wherein (b) the engineered retrotran ncRNA and (c) the guide RNA are fused or provided in cis form .

28. The gene editing system of paragraph 27, wherein the guide RNA is fused to the 5' end of the retrotranscript ncRNA.

29. The gene editing system of paragraph 27, wherein the guide RNA is fused to the 3' end of the retrotranscript ncRNA.

30. The gene editing system of paragraph 27, wherein the engineered ncRNA comprises a first guide RNA fused to the 5' end of the retrotranscript ncRNA and a second guide RNA fused to the 3' end of the retrotranscript ncRNA, and the first and second guide RNAs target different sequences.

31. The gene editing system of paragraph 1, wherein the one or more delivery vehicles comprise liposomes or lipid nanoparticles (LNPs).

32. The gene editing system of paragraph 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in the same delivery medium.

33. The gene editing system of paragraph 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in a separate delivery medium.

34. The gene editing system of paragraph 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery medium.

35. The gene editing system of paragraph 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.

36. The gene editing system of paragraph 1, wherein the engineered retrotranscript ncRNA comprises a sequence encoding a donor polynucleotide comprising the desired edit at a target sequence to be integrated into a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at the 5' position of the target sequence and a 3' homology arm hybridized to a sequence at the 3' position of the target sequence.

37. The gene editing system of paragraph 1, wherein the nucleic acid programmable nuclease comprises Cas9 nuclease, TnpB nuclease or Cas12a nuclease.

38. The gene editing system of paragraph 1, wherein the nucleic acid programmable nuclease comprises a Cas9 nuclease.

39. The gene editing system of paragraph 1, wherein the nucleic acid programmable nuclease comprises a Cas9 nickase.

40. An isolated cell comprising the gene editing system of paragraph 1.

41. The isolated cell of paragraph 40, wherein the isolated cell is a mammalian cell.

42. The isolated cell of paragraph 41, wherein the mammalian cell is a human cell.

43. A composition comprising: a) the gene editing system of paragraph 1; and b) a pharmaceutically or veterinarily acceptable carrier.

44. The composition of paragraph 43, wherein the delivery vehicle is a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structured lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.

45. The composition of paragraph 44, wherein the one or more ionizable lipids comprise the ionizable lipids described in Table 2.

46.    A method for genetically modifying a cell, the method comprising: contacting the gene editing system of paragraph 1 with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein the guide RNA guides the complex to the target sequence, the nucleic acid programmable nuclease generates a double-strand break in the target sequence, the retrotranscriptase and the engineered retrotranscript ncRNA generate RT DNA comprising the donor polynucleotide, and the donor polynucleotide is integrated into the target sequence, thereby editing the cell to be genetically modified.

Other exemplary and non-limiting aspects and embodiments of the present disclosure are summarized below in numbered paragraphs.

1. An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus, wherein the ncRNA comprises: (I) ncRNAs listed in Table B, or ncRNAs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with the ncRNAs listed in Table B; and/or (II) ncRNAs having a conservative structure of any of the ncRNA structures of Figures 2-27; and wherein the ncRNAs exclude any ncRNAs associated with any of the retrotransposons in Table X in nature, as the case may be.

2. The engineered nucleic acid construct of paragraph 1, further comprising a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT or a portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

3.    The engineered nucleic acid construct of paragraph 2, wherein the second polynucleotide comprises: III)       a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polynucleotide listed in Table A; and/or IV)    Encoding a consensus amino acid sequence of Table C, or encoding an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with an amino acid sequence listed in Table C; and/or wherein the second polynucleotide encodes: V) A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table A; and/or VI)     A polypeptide comprising a consensus sequence of the polypeptides listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with an amino acid sequence listed in Table C; and/or wherein the second polynucleotide does not encode an amino acid sequence listed in Table X as the case may be.

4. An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of multiple copies of single-stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; b) one or more heterologous nucleic acids inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT or a portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the non-coding RNA (ncRNA) of the first polynucleotide optionally has a conserved structure of any one of the ncRNA structures of Figures 2-27; wherein the second polynucleotide comprises: 1) The polynucleotides listed in Table A, or polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the polynucleotides listed in Table A; and/or wherein the second polynucleotide encodes: II) A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or IV) A polypeptide comprising a consensus sequence of a polypeptide listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table C; and wherein the second polynucleotide optionally does not encode the amino acid sequence of Table X.

4a. An engineered nucleic acid construct comprising: 1) an msr locus (which encodes the msr RNA portion of the msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retrotranscriptase (RT), wherein the msd RNA can be reverse transcribed by the retrotranscriptase (RT) to form the msDNA; and 4) a heterologous nucleic acid inserted at the msd locus, upstream of the msr locus, upstream or downstream of the msd locus, or within the msd locus; wherein the engineered nucleic acid construct has, as the case may be, (a) the secondary structure of the wild-type ncRNA of any one of Figures 2-27, or b) a variant of a), which has: i) up to 1, 2 or 3 (e.g., up to 1) nucleotide changes for every 10 red letter nucleotides; ii) up to 4, 5 or 6 (e.g., up to 1 or 2) nucleotide changes for every 10 black lettered nucleotides; and/or iii) up to 7, 8 or 9 (e.g., up to 3 or 4) nucleotide changes for every 10 grey lettered nucleotides; and/or further comprising, as appropriate: i) 7, 8, 9 or 10 (e.g., 9 or 10) nucleotides for every 10 red circled nucleotides; ii) 6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides for every 10 black circled nucleotides; iii) 4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides for every 10 grey circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides for every 10 white circled nucleotides.

5. The engineered nucleic acid construct of any one of paragraphs 1 to 4a, comprising one or more sequence modifications (e.g., insertion, deletion and/or substitution of one or more nucleotides) in the msr locus and/or msd locus, which one or more sequence modifications: a) modulate (e.g., enhance) reverse transcription, processability, accuracy/fidelity and/or production of msDNA (e.g., in mammalian cells); b) modulate (e.g., reduce) the immunogenicity of ncRNA encoded by the engineered retrotranscript (e.g., msr locus and/or msd locus) in a host (e.g., a host comprising mammalian cells); c) modulate (e.g., permanently or transiently inhibit) the function of msDNA; and/or d) modulate (e.g., improve) the efficiency of targeted genome editing/engineering.

6.    An engineered nucleic acid construct of any of paragraphs 1 to 4, wherein the engineered nucleic acid construct has a secondary structure of a wild-type retrotranscript encoding a wild-type retrotranscript ncRNA covered below: a)  Any structure as depicted in Figure 2-27, or b)  A variant of a) having: i)   Up to 1, 2 or 3 (e.g., up to 1) nucleotide changes per 10 red-letter nucleotides; ii)  Up to 4, 5 or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black-letter nucleotides; and/or iii) Up to 7, 8 or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey-letter nucleotides; and/or further comprising, as the case may be: i)    7, 8, 9 or 10 (e.g., 9 or 10) nucleotides are present for every 10 red circled nucleotides; ii)    6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides are present for every 10 black circled nucleotides; iii)    4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides are present for every 10 grey circled nucleotides; and/or iv)    2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present for every 10 white circled nucleotides.

7. The engineered nucleic acid construct of any of paragraphs 1-6, wherein the nucleic acid construct is engineered by introducing the one or more sequence modifications into a wild-type retrotranscript of a wild-type ncRNA listed in Coding Table B.

8.    The engineered nucleic acid construct of any of paragraphs 1-7, wherein the one or more sequence modifications in the ncRNA include one or more of the following: (i) a modified (e.g., mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii) an extension or shortening of a1, a2, or both a1 and a2; (iii)      an extension or shortening of the spacer sequence between the hairpin loops (e.g., S1, S2, S3, and/or S4); (iv)      an additional or modified (e.g., mutated or eliminated) bulge in the hairpin loop (e.g., L2 and/or L3 (e.g., by removing unpaired bases in the bulge, or by replacing unpaired bases with an equal number of base pairs)); (v) Modified (e.g., extended or shortened) hairpin length (e.g., L1, L2, L3 and/or L4); (vi)      Alternative L1 and/or L2 with complement, reverse or reverse complement sequence; (vii)      Modified (e.g., increased) number of unpaired bases at the tip of the hairpin (e.g., L1, L2, L3 and/or L4); (viii)    Modified (e.g., increased or decreased) GC content in the hairpin (e.g., L1, L2, L3 and/or L4); (ix)     Insertion of the heterologous nucleic acid in the spacer sequence between the hairpin loops (e.g., S1, S2, S3 and/or S4) or at the tip of the hairpin loops (e.g., L1, L2, L3 and/or L4); (x) Deletion of one or more hairpin loops (e.g., L1, L2, L3 and/or L4); (xi)      Addition of new loops in the spacer sequence between the hairpin loops (e.g., S1, S2, S3 and/or S4); (xii)     Circularization of the ncRNA, wherein the 5' end and the 3' end of the ncRNA are directly or via the spacer sequence; (xiii)    Repositioned branched guanosine capable of initiating reverse transcription initiation; (xiv)   a staggered end sequence that reduces the immunogenicity of the retrotranscript ncRNA, generated by, for example, adding or removing 5' a1 nucleotides and/or 3' a2 nucleotides; and/or, (xv)     an antisense sequence that is complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes with and inhibits the gRNA in the encoded retrotranscript ncRNA, and wherein the antisense sequence is removed after reverse transcription of the msDNA.

9. The engineered nucleic acid construct of any one of paragraphs 1-8, wherein the one or more heterologous nucleic acid sequences comprise: a) a heterologous nucleic acid (such as a coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as in the S region (e.g., S1, S2, S3 and/or S4), or at the tip of the L region (e.g., L1, L2, L3 and/or L4), or upstream or downstream of the msr locus or the msd locus; or b) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a guide RNA.

10. An engineered nucleic acid construct of any one of paragraphs 1-9, wherein the heterologous nucleic acid encodes: (a) a protein or peptide of interest, or wherein the heterologous nucleic acid comprises; (b) a DNA donor template sequence; (c) a functional DNA element selected from promoters, enhancers, protein binding sequences, methylation sites, homologous regions for assisting gene editing, and similar elements; or (d) a coding sequence for a functional RNA element selected from guide RNA and ncRNA.

11. The engineered nucleic acid construct of paragraph 10, wherein the related protein or peptide comprises a therapeutic protein that can be used to treat a disease.

12. The engineered nucleic acid construct of paragraph 10, wherein the DNA donor template sequence corrects/repairs/removes a mutation at a target genomic site.

13. The engineered nucleic acid construct of any of paragraphs 1-12, further comprising or encoding a sequence-specific nuclease (such as a CRISPR/Cas effector enzyme, ZFN, TALEN, meganuclease, TnpB, IscB or restriction endonuclease (RE)) and/or a DNA repair regulatory biomolecule.

13b. The engineered nucleic acid construct of paragraphs 1-13, wherein the engineered nucleic acid is an all-RNA component system.

13c. The engineered nucleic acid construct of paragraphs 1-13, wherein the engineered nucleic acid is an all-DNA molecule system.

14. The engineered nucleic acid construct of paragraph 13, wherein the sequence-specific nuclease is fused to the RT via a flexible linker (e.g., a flexible linker comprising Gly and Ser-rich sequences such as G4S repeats or GS repeats) or by a generally disordered protein sequence (e.g., an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer), as appropriate.

15. The engineered nucleic acid construct of paragraph 13 or 14, wherein the nuclease is a CRISPR/Cas effector enzyme that forms a complex with a guide RNA (gRNA) that recognizes a target sequence, wherein the gRNA is linked to the ncRNA and/or the msDNA directly or via a linker/spacer polynucleotide.

16. The engineered nucleic acid construct of paragraph 13, wherein the DNA repair regulatory biomolecule is a regulatory protein that regulates (e.g., enhances) HDR, and the regulatory protein is fused to the RT or the sequence-specific nuclease via a flexible linker (e.g., a flexible linker comprising Gly and Ser-rich sequences (such as G4S repeats or GS repeats)) or by a generally disordered protein sequence (such as an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer), as the case may be.

17. A vector system comprising one or more vectors comprising an engineered nucleic acid construct of any one of paragraphs 1-16, wherein the vector system is optionally all-RNA.

18. The vector system of paragraph 17, wherein the msr locus, the msd locus and the polynucleotide encoding the RT are contained in the same vector.

19. The vector system of paragraph 17 or 18, wherein the same vector further comprises a promoter operably linked to the msr locus and/or the msd locus.

20. The vector system of paragraph 19, wherein the promoter is further operably linked to the polynucleotide encoding the RT.

21. A vector system comprising one or more vectors, comprising the engineered nucleic acid construct of paragraph 1 or 2, wherein the vector system further comprises a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the msr locus, the msd locus and the second polynucleotide encoding the RT are provided by at least two different vectors.

22. The vector system of paragraph 21, wherein: a) the second polynucleotide comprises: i)   a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polynucleotide listed in Table A; and/or b) the second polynucleotide encodes: i)  A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table A; and/or ii) A polypeptide listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table C; and wherein the second polynucleotide does not encode a polypeptide listed in Table X, as the case may be.

23. The vector system of paragraph 21 or 22, wherein the polynucleotide encoding the RT is provided in trans relative to the msr gene and/or the msd gene.

24. A vector system as described in any of paragraphs 17-23, wherein the one or more vectors comprise a viral vector.

25. The vector system of paragraph 24, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a poxvirus vector or a herpes simplex viral vector.

26. A vector system as described in any of paragraphs 17-23, wherein the one or more vectors comprise a non-viral vector.

27. The vector system of paragraph 26, wherein the non-viral vector comprises a plasmid.

28. The vector system of paragraph 26, wherein the non-viral vector comprises a liposome, a lipid nanoparticle (LNP), a cationic polymer, a vesicle or a gold nanoparticle.

29. A vector system as described in any of paragraphs 17-28, which comprises a vector encoding a sequence-specific nuclease.

30. The vector system of paragraph 29, wherein the sequence-specific nuclease comprises an RNA-guided sequence-specific nuclease (e.g., CRISPR/Cas effector enzyme, engineered RNA-guided FokI-nuclease (e.g., dCas-FokI), RNA-guided DNA endonuclease, TnpB, IscB or translocon-associated nuclease) or a non-RNA-guided sequence-specific nuclease (e.g., meganuclease, zinc finger nuclease (ZFN), TALE nuclease (TALEN) or restriction endonuclease (RE)).

31. The vector system of paragraph 30, wherein the Cas effector enzyme is class 1, type I, type II or type III Cas; class 2, type II Cas (e.g. Cas9); or class 2, type V Cas (e.g. Cpf1).

32. The vector system of paragraph 30, wherein: 1) the RNA-guided sequence-specific nuclease comprises the CRISPR/Cas effector enzyme, the engineered RNA-guided FokI nuclease (e.g., dCas-FokI), the RNA-guided DNA endonuclease, TnpB, IscB, IsrB, or a translocon-associated nuclease; or, 2) the non-RNA-guided sequence-specific nuclease comprises the meganuclease, the zinc finger nuclease (ZFN), the TALE nuclease (TALEN), or the restriction endonuclease (RE).

33. The vector system of any of paragraphs 17-32, further comprising a vector encoding a homologous recombination enhancer protein.

34. An RNA molecule encoded by an engineered nucleic acid construct of any of paragraphs 1-16.

35. An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a sequence encoding a reverse transcriptase (RT) or a domain thereof comprising: i) A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) The polypeptides listed in Table C, or polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the polypeptides listed in Table C; and wherein the RT optionally does not comprise a polypeptide listed in Table X.

36. An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding an msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding an msd RNA portion of the msDNA; wherein the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and/or ii) 2-27; and wherein the ncRNA excludes any ncRNA associated with any reverse transcriptase of Table X in nature as the case may be; b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus ; and c) reverse transcriptase (RT) or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

37. An engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding a msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding a msd RNA portion of the msDNA; wherein the ncRNA comprises: i) ncRNAs listed in Table B, or ncRNAs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the ncRNAs listed in Table B; and/or ii) ncRNAs having a conservative structure of any ncRNA structure of Figures 2-27; and wherein the ncRNA excludes any ncRNA associated with any retrotransposase of Table X in nature as the case may be; b) A heterologous nucleic acid inserted at or within a position selected from the group consisting of: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in Table A, or an RT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an RT listed in Table A; and/or ii) The consensus sequence listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence listed in Table C; and wherein the RT optionally does not include the RT listed in Table X.

38. An isolated host cell comprising an engineered nucleic acid construct of any of paragraphs 1-16, a vector system of any of paragraphs 17-33, an RNA molecule of paragraph 34, or an engineered nucleic acid-enzyme construct of any of paragraphs 35-37.

39. The isolated host cell of paragraph 38, wherein the host cell is a prokaryotic, archeon or eukaryotic host cell.

40. The isolated host cell of paragraph 38, wherein the eukaryotic host cell is a mammalian host cell.

41. The isolated host cell of paragraph 39, wherein the eukaryotic host cell is a non-human host cell.

42. The isolated host cell of paragraph 40, wherein the mammalian host cell is a human host cell.

43. An isolated host cell as described in any one of paragraphs 38-42, wherein the host cell is an artificial cell or a genetically modified cell.

44. A pharmaceutical composition comprising: a) an engineered nucleic acid construct of any one of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, an engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, and/or an isolated host cell of any one of paragraphs 38-43; and b) a pharmaceutically acceptable carrier.

45. A pharmaceutical composition comprising: a) lipid nanoparticles (LNPs); and b) an engineered nucleic acid construct of any one of paragraphs 1-16, a ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, and/or an engineered nucleic acid-enzyme construct of any one of paragraphs 35-37.

46. The pharmaceutical composition of paragraph 45, wherein the LNP encapsulates the engineered nucleic acid construct, the ncRNA, the vector system, the RNA molecule and/or the engineered nucleic acid-enzyme construct.

47. The pharmaceutical composition of paragraph 45 or 46, wherein the lipid nanoparticles comprise: a) one or more ionizable lipids; b) one or more structured lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.

48. The pharmaceutical composition of paragraph 47, wherein the one or more ionizable lipids are selected from the group consisting of those disclosed in Table 2.

49. The pharmaceutical composition of paragraph 47 or 48, wherein the one or more structured lipids are selected from the group consisting of: cholesterol, natriuretic acid, β-myristol, sterol, ergosterol, campesterol, stigmasterol, sterosterol, tomatine, tomatine, ursolic acid, α-tocopherol, prednisolone, dexamethasone, prednisolone and hydrocortisone.

50. The pharmaceutical composition of any of paragraphs 47-49, wherein the one or more PEGylated lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

51. The pharmaceutical composition of any one of paragraphs 47-50, wherein the one or more phospholipids are selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC ), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycero) sodium salt (DOPG) and sphingomyelin.

52. The pharmaceutical composition of any of paragraphs 47-51, wherein the lipid nanoparticles comprise about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 40 mol% structural lipids and about 1.5 mol% PEG lipids.

53. The pharmaceutical composition of any of paragraphs 47-52, wherein the lipid nanoparticles comprise about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 39 mol% structural lipids and about 2.5 mol% PEG lipids.

54. The pharmaceutical composition of any of paragraphs 47-53, wherein the LNP further comprises a targeting portion operably linked to the LNP.

55. The pharmaceutical composition of any of paragraphs 47-54, wherein the LNP further comprises one or more additional components selected from the group consisting of: DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP and C12-200.

56. The pharmaceutical composition of paragraph 45, wherein the lipid nanoparticles comprise at least one cationic lipid selected from the group consisting of lipids in Table 2, lipids having a structure of formula (I), lipids having a structure of formula (II), lipids having a structure of formula (III), lipids having a structure of formula (IV), lipids having a structure of formula (V), lipids having a structure of formula (VI), and combinations thereof.

57. A kit comprising an engineered nucleic acid construct of any of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any of paragraphs 1-16, a vector system of any of paragraphs 17-33, an RNA molecule of paragraph 34, an engineered nucleic acid-enzyme construct of any of paragraphs 35-37, a host cell of any of paragraphs 38-43, or a pharmaceutical composition of any of paragraphs 44-56, and instructions for genetically modifying a cell using the engineered nucleic acid construct, the ncRNA, the vector system, the host cell, or the pharmaceutical composition.

58. A method for modifying a target DNA sequence in a host (e.g., mammal) cell, the method comprising introducing an engineered nucleic acid construct of any of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any of paragraphs 1-16, a vector system of any of paragraphs 17-33, an RNA molecule of paragraph 34, an engineered nucleic acid-enzyme construct of any of paragraphs 35-37, or a pharmaceutical composition of any of paragraphs 44-56 into the mammalian cell to allow production of the msDNA in the host (e.g., mammal) cell, wherein the heterologous nucleic acid in the msDNA is integrated into the target DNA sequence in the genome of the host (e.g., mammal) cell by homology-dependent recombination.

59. The method of paragraph 58, wherein the modification comprises introducing insertions, deletions and/or substitutions into the target DNA sequence.

60. A method for treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an engineered nucleic acid construct of any one of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, an engineered nucleic acid-enzyme construct of any one of paragraphs 35-37, a host cell of any one of paragraphs 38-43, or a pharmaceutical composition of any one of paragraphs 44-56, thereby treating the disease or condition in the individual.

61. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a host cell of any one of paragraphs 38-43, thereby treating the disease or condition in the subject.

62. The method of paragraph 61, wherein the host cell is autologous to the individual.

63. The method of paragraph 61, wherein the host cell is allogeneic to the individual.

Related applications

This application is based on U.S. Provisional Application No. 63/373,545 filed on August 25, 2022 (RTX003-P1), U.S. Application No. 18/087,673 filed on December 22, 2022 (RTX004-T1), U.S. Provisional Application No. 63/476,900 filed on December 22, 2022 (RTX005-P1), International PCT Application No. PCT/US2023/6 filed on January 20, 2023, and U.S. Provisional Application No. 63/476,900 filed on December 22, 2022 (RTX005-P1). No. 1038 filed on March 3, 2023 (RTX006-PCT1), U.S. Provisional Application No. 63/488,317 filed on March 3, 2023 (RTX007-P1), U.S. Provisional Application No. 63/491,603 filed on March 22, 2023 (RTX008-P1), and U.S. Provisional Application No. 63/515,783 filed on July 26, 2023 (RTX009-P1), each of which is incorporated herein by reference in its entirety.

This application refers to the following applications: U.S. Provisional Application No. 63/301,936 filed on January 21, 2022 (RTX001-P1) and U.S. Provisional Application No. 63/370,880 filed on August 9, 2022 (RTX002-P1), each of which is incorporated herein by reference in its entirety.

The above-mentioned applications and all documents cited therein or during the prosecution thereof ("Documents cited in the Applications"), and all documents cited or referenced in documents cited in the Applications, and all documents cited or referenced herein ("Documents cited herein"), and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Sequence Listing

This application as originally filed includes/contains a sequence listing filed electronically in XML format, titled J0356-99004.xml, created on August 24, 2023 and 36,884,841 bytes in size. The contents of the sequence listing are incorporated herein in their entirety. Definitions

All technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and 62/1005 Marham, The Harper Collins Dictionary of Biology (1991).

General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift and Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the disclosure unless the context clearly dictates otherwise. The upper and lower limits of such smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of those limits, ranges excluding either or both of those included limits are also included in the disclosure.

It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "ncRNA" includes plural ncRNAs, and reference to "reverse transcriptase" includes reference to one or more RTs and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as an antecedent basis for the use of such exclusive terms as "only", "only", and the like, or the use of a "negative" limitation in the description of elements of the associated claims. For example, claims may be drafted to exclude certain RT sequences.

It will be appreciated that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments of the invention are specifically contemplated by the invention and disclosed herein, as if each combination were individually and expressly disclosed. Furthermore, all subcombinations of the multiple embodiments and elements thereof are also specifically contemplated by the invention and disclosed herein, as if each such subcombination were individually and expressly disclosed herein. Biological Activity

As used herein, the term "biological activity" refers to the characteristic of an agent (e.g., DNA, RNA , or protein) that is active in biological systems (including in vitro and in vivo biological systems), and particularly in living organisms (such as in mammals, including humans and non-human mammals). For example, an agent that has a biological effect on an organism when administered to that organism is considered biologically active.

As used herein, the term "bulge" refers to a small region of unpaired bases of the "stem" of the nucleotides that interrupt the base pairing. The bulge may comprise one or two single stranded or non-base paired nucleotides joined at both ends by base paired nucleotides of the stem. The bulge may be symmetrical ( i.e. , the two non-base paired single stranded regions have the same number of nucleotides), or asymmetrical ( i.e. , the non-base paired single stranded regions have different or unequal numbers of nucleotides), or there may be only one non-base paired nucleotide on one strand. The bulge may be described as A/B (e.g., "2/2 bulge" or "1/0 bulge"), where A represents the number of unpaired nucleotides on the upstream strand of the stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem. In the primary nucleotide sequence, the upstream strand of the protrusion is closer to the 5' of the downstream strand of the protrusion.

As used herein, the term "cDNA" refers to a DNA strand that has been copied from an RNA template, for example by reverse transcriptase .

The term " homologs " refers to two biological molecules that usually interact or coexist in nature.

As used herein, the term "complementary" or "substantially complementary" is intended to mean that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to non-covalently bind (i.e., form Watson-Crick base pairs and/or G/U base pairs), "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., nucleic acid specifically binds to a complementary nucleic acid), under appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C) [DNA, RNA]. Additionally, for hybridization between two RNA molecules (e.g., dsRNA), and hybridization between a DNA molecule and an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, in the context of base pairing of a tRNA anticodon with a codon in an mRNA, G/U base pairing is at least partially responsible for the parsimony (i.e., redundancy) of the genetic code. Thus, in the context of the present disclosure, guanine (G) is considered complementary to both uracil (U) and adenine (A). For example, when a G/U base pair can be generated at a given nucleotide position of the dsRNA duplex of a guide RNA molecule, that position is not considered non-complementary, but rather is considered complementary.

It is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. In addition, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments do not participate in the hybridization event (e.g., bulges, loop structures, or hairpin structures, etc.). A polynucleotide may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity with a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of the 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize would represent 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered with or interspersed with complementary nucleotides and need not be adjacent to each other or to complementary nucleotides. Any convenient method may be used to determine the percentage of complementarity between a particular stretch of nucleic acid sequences within a nucleic acid. Example methods include BLAST (Basic Local Alignment Search Tool) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656); Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), for example, using default settings, and similar programs. DNA- guided nuclease

As used herein, "DNA-guided nucleases" are a class of "programmable nucleases" and a specific class of "nucleic acid-guided nucleases." Examples of DNA-guided nucleases are reported in Varshney et al., DNA-guided genome editing using structure-guided endonucleases, Genome Biology, 2016, 17(1), 187, which may be used in the context of the present disclosure and is incorporated herein by reference. As used herein, the term "DNA-guided nuclease" or "DNA-guided endonuclease" refers to a nuclease that covalently or non-covalently binds to a guide RNA, thereby forming a complex between the guide RNA and the DNA-guided nuclease. The guide RNA comprises a spacer sequence comprising a nucleotide sequence that is complementary to a strand of a target DNA sequence. Thus, a DNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule by its association with a guide RNA which directly binds or anneals to one strand of the target DNA through its complementary region via Watson -Crick base pairing .

As used herein, the terms "DNA regulatory sequence,""controlelement," and "regulatory element" are used interchangeably herein to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, which provide for and/or regulate the transcription of non-coding sequences (e.g., guide RNA) or coding sequences and/or regulate the translation of mRNA into the encoded polypeptide. Donor Nucleic Acid

"Donor nucleic acid" or "donor polynucleotide" or "donor DNA" or "HDR donor DNA" means a single strand of DNA to be inserted at a site to be cleaved by a programmable nuclease (e.g., CRISPR/Cas effector protein; TALEN; ZFN; meganuclease) (e.g., after dsDNA cleavage, after nicking of the target DNA, after double nicking of the target DNA, and the like). The donor polynucleotide can contain sufficient homology to the genome sequence at the target site, such as 70%, 80%, 85%, 90%, 95% or 100% homology to the nucleotide sequence flanking the target site, such as within about 200 bases or less of the target site, such as within about 190 bases or less of the target site, such as within about 100 bases or less of the target site. The target site is within about 180 alkali groups or less, such as within about 170 alkali groups or less of the target site, such as within about 160 alkali groups or less of the target site, such as within about 150 alkali groups or less of the target site, such as within about 140 alkali groups or less of the target site, such as within about 130 alkali groups or less of the target site. The target site is within about 120 alkali groups or less, such as within about 110 alkali groups or less, such as within about 100 alkali groups or less, such as within about 90 alkali groups or less, such as within about 80 alkali groups or less, such as within about 100 alkali groups or less, such as within about 90 alkali groups or less, such as within about 80 alkali groups or less, such as within about 100 alkali groups or less. Within about 70 bases or less, such as within about 60 bases or less of the target site, such as within 50 bases or less of the target site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or directly flanking the target site to support homology-directed repair between it and a genomic sequence with homology thereto.

As used herein, a DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein (and thus both the DNA and the mRNA encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.). In the case of a retrotranscript, the retrotranscript DNA may encode the ncRNA locus (which includes the msr and msd regions) as well as the retrotranscript RT. Engineered Retrotranscripts

As used herein, the term "engineered retrotranscript" or equivalently "recombinant retrotranscript" refers to a retrotranscript that does not exist in nature. In one embodiment, the engineered retrotranscript can include a wild-type or naturally occurring retrotranscript that has been modified to contain at least one modification, including a single nucleotide substitution, insertion or deletion, or a substitution, insertion or deletion of more than one nucleotide, that is, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 ,93,94, 95, 96, 97, 98, 99 or up to 100 or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or up to 2000 nucleotides are substituted, inserted or deleted. When more than one nucleotide of the starting point retrotranscript (e.g., wild-type retrotranscript) is substituted, deleted or inserted, the nucleotides may be consecutive or non-consecutive. Although the engineered retrotranscript as a whole does not occur naturally, it may include components that do occur in nature, such as nucleotide sequences. For example, an engineered retrotranscript may have nucleotide sequences from different organisms ( e.g. , from different bacterial species) or from completely synthetic/artificial/recombinant nucleic acid sequences. Thus, an engineered retrotranscript may have bacterial nucleotide sequences, human nucleotide sequences, viral nucleotide sequences, and/or synthetic/artificial/recombinant nucleotide sequences and/or combinations of such sequences. Examples of modifications of the recombinant retrotranscript disclosed herein include inserting a heterologous nucleic acid sequence into the retrotranscript, for example, into a ncRNA locus, such as an msr or msd locus. Attaching a guide RNA molecule to the 5' and/or 3' end (i.e., attaching a molecule to the 5' end of the ncRNA and/or attaching a molecule to the 3' end of the ncRNA) also represents another modification contemplated for the recombinant retrotranscript disclosed herein. In such embodiments, the guide RNA molecule may also be classified or more generally referred to as a type of heterologous nucleic acid sequence used to modify the start site of a retrotransposon .

As used herein, the term "exosome" refers to a small membrane-bound vesicle of endocytic origin. Without wishing to be bound by theory, exosomes are typically released from host/progenitor cells into the extracellular environment following fusion of multicellular bodies with the cytoplasmic membrane. Thus, in addition to designed components (e.g., engineered retrotransposons), exosomes may also include components of the progenitor cell membrane. Exosome membranes are generally lamellar, composed of a lipid bilayer with aqueous inter-nanoparticle spaces. Expression vectors

As used herein, the term "expression vector" or "expression construct" refers to a vector that includes one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, bacilli, tobacco mosaic viruses, herpes viruses, cytomegaloviruses, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available, such as from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). The present invention encompasses recombinant vectors, which may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof. Guide RNA

The RNA molecule that binds to the programmable nuclease of the retrotranscript-based gene editing system and targets the nuclease to a specific position within the targeted polynucleotide sequence is referred to herein as a "guide RNA" or "guide RNA polynucleotide" (also referred to herein as "guide RNA" or "gRNA" or "crRNA"). In certain embodiments (depending on the specific nuclease with which it interacts), the guide RNA comprises two segments, a "DNA targeting segment" and a "protein binding segment." "Segment" means a segment/portion/region of a molecule, such as a stretch of contiguous nucleotides in an RNA. As an illustrative, non-limiting example, the protein binding segment of the guide RNA may comprise base pairs 5-20 of an RNA molecule of 40 base pairs in length; and the DNA targeting segment may comprise base pairs 21-40 of an RNA molecule of 40 base pairs in length. Unless otherwise specifically defined in a particular context, the definition of "segment" is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of individual molecules within a complex, and may include regions of RNA molecules of any overall length, and may or may not include regions that are complementary to other molecules.

The DNA targeting segment (or "DNA targeting sequence") comprises a nucleotide sequence that is complementary to a specific sequence within the targeting polynucleotide sequence (the complementary strand of the targeting polynucleotide sequence) designated herein as a "protospacer-like" sequence. The protein binding segment (or "protein binding sequence") interacts with the site-directed modifying polypeptide. When the site-directed modifying polypeptide is a CRISPR nuclease, site-specific cleavage of the targeting polynucleotide sequence can occur at a position determined by: (i) base pairing complementarity between the guide RNA and the targeting polynucleotide sequence; and (ii) a short motif in the targeting polynucleotide sequence (called a protospacer adjacent motif (PAM)). Heterologous Nucleic Acid Sequence

As used herein, the term "heterologous nucleic acid" refers to a genotypically different entity that is different from the rest of the entity with which it is compared or introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide ( e.g. , DNA or RNA) and, if expressed, can encode a heterologous polypeptide. Similarly, a cellular sequence ( e.g. , a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector. In some embodiments, a heterologous sequence inserted into a wild-type retrotranscript region is not naturally inserted into such a region ( e.g. , an engineered retrotranscript with an inserted heterologous sequence does not occur naturally). For example, the heterologous sequence can be from a bacterium of the same species in which the wild-type retrotranscript is normally found, as long as the heterologous sequence is not naturally inserted into the wild-type retrotranscript at the position where the heterologous sequence is inserted. In certain embodiments, the heterologous sequence is a mammalian sequence ( e.g. , a human sequence) or a reverse complement thereof. The heterologous nucleic acid sequence introduced into the retrotranscript may include, but is not limited to, a guide RNA sequence, an HDR donor template, a protein coding gene, or a non-coding functional RNA element (e.g., a stem-loop, a hairpin, and a bulge). Homology-directed repair

As used herein, "homologous directed repair (HDR)" refers to a specific form of DNA repair that occurs, for example, during the repair of a double-strand break in a cell. This process requires nucleotide sequence homology, uses a "donor" molecule to template the repair of a "target" molecule (i.e., the molecule that has undergone a double-strand break), and results in the transfer of genetic information from the donor to the target. If the donor polynucleotide is different from the target molecule and part or all of the sequence of the donor polynucleotide is incorporated into the sequence of the targeting polynucleotide, homology-directed repair can result in an alteration (e.g., insertion, deletion, mutation ) in the sequence of the target molecule.

As used herein, the term "identical" refers to two or more identical sequences or subsequences. In addition, as used herein, the term "substantially identical" refers to two or more sequences having a certain percentage of identical contiguous units when compared and aligned over a comparison window or specified region for maximum correspondence, as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages describe the "percentage of identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 contiguous units in length, over a region that is about 50 contiguous units in length, or, where not specified, over the entire sequence. This definition also refers to complements of test sequences.

Alternatively, substantial identity or similarity exists when a nucleic acid or fragment thereof is hybridized with another nucleic acid, a strand of another nucleic acid, or a complementary strand thereof under strict hybridization conditions. "Strict hybridization conditions" and "strict wash conditions" in the context of nucleic acid hybridization experiments depend on a variety of different physical parameters. Nucleic acid hybridization will be affected by conditions such as salt concentration, temperature, solvent, basic composition of the hybridization medium, length of the complementary region, and the number of nucleotide base mismatches between the hybridized nucleic acids, as should be readily understood by those skilled in the art. Those of ordinary skill know how to vary these parameters to achieve a particular hybridization stringency. Lipid Nanoparticles (LNP)

As used herein, the term "lipid nanoparticle" or LNP refers to a class of lipid particle delivery systems formed by small solid or semisolid particles having an outer lipid layer with a hydrophilic outer surface exposed to the non-LNP environment; an interior space that can be aqueous (vesicle-like) or non-aqueous (micell-like); and at least one hydrophobic intermembrane space. The LNP membrane can be lamellar or non-lamellar and can comprise 1, 2, 3, 4, 5 or more layers. In some embodiments, the LNP can comprise a nucleic acid ( e.g. , an engineered retrotransposon) that enters its interior space, enters the intermembrane space, reaches its outer surface, or any combination thereof. In some embodiments, the LNP of the present disclosure comprises ionizable lipids, structural lipids, PEGylated lipids (also referred to as PEG lipids) and phospholipids. In alternative embodiments, the LNP comprises ionizable lipids, structural lipids, PEGylated lipids (also referred to as PEG lipids) and zwitterionic amino acid lipids.

Further discussion of liposomes can be found in, for example, Tenchov et al., "Lipid Nanoparticles - From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement," ACS Nano , 2021, 15, pp. 16982-17015 (incorporated by reference).

As used herein, the term "linker" refers to a molecule that connects or joins two other molecules or parts. In the case where the linker joins two fusion proteins, the linker can be an amino acid sequence. For example, an RNA-guided nuclease (e.g., Cas12a) can be fused to a retrotranscriptase by an amino acid linker sequence. In the case of joining two nucleotide sequences together, the linker can also be a nucleotide sequence. For example, in the present case, ncRNA can be connected to one or more guide RNAs at its 5' and/or 3' end by a nucleotide sequence linker. In other embodiments, the linker is an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, the linker is 5-100 amino acids long, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids long. Longer or shorter linkers are also contemplated. Liposomes

As used herein, the term "liposome" refers to a class of lipid particle delivery systems comprising vesicles containing at least one lipid membrane surrounding an aqueous nanoparticle interior space that is generally not derived from progenitor cells/host cells. Liposomes are universal carrier platforms because they can transport hydrophobic or hydrophilic molecules (including small molecules, proteins, and nucleic acids) into cells. It is one of the earliest developed nanoscale drug delivery platforms. A variety of liposomal drug formulations have been approved for use in human drugs, such as Doxil, a lipid nanoparticle formulation of the anti-tumor agent doxorubicin. Further discussion of liposomes can be found in, for example, Tenchov et al., “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp. 16982-17015 (incorporated by reference ).

As used herein, the term "loop" in a polynucleotide refers to a single-stranded stretch of one or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, wherein the 5'-most nucleotide and the 3'-most nucleotide of the loop are each linked to a base-paired nucleotide in the stem.

As used herein, the term " micell " refers to small particles that do not have aqueous intraparticle space.

As used herein, the term "nanoparticle" refers to any nanoscale particle with a size generally ranging from about 1 nm to 1000 nm. Nuclear Localization Sequence (NLS)

As used herein, the term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates the import of a protein (e.g., an RNA-guided nuclease) into the cell nucleus, such as by nuclear transport. Nuclear localization sequences are known in the art. For example, NLS sequences are described in International PCT Application No. PCT/EP2000/011690 to Plank et al., filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. Nucleic Acids

As used herein, the term "nucleic acid" or "nucleic acid molecule" or "nucleic acid sequence" or "polynucleotide" generally refers to a deoxyribonucleic acid or ribonucleic acid oligonucleotide in single- or double-stranded form. The term also encompasses oligonucleotides containing known analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. The term encompasses both ribonucleic acid (RNA) and DNA, including cDNA (including RT DNA), genomic DNA, synthetic, synthetic (e.g., chemically synthesized) DNA, and/or DNA (or RNA) containing nucleic acid analogs. The nucleotides adenine (A), thymine (T), guanine (G) and cytosine (C) may or may not also include nucleotide modifications, such as methylated and/or hydroxylated nucleotides, for example, cytosine (C) includes 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic acid-guided nuclease

As used herein, the term "nucleic acid-guided nuclease" or "nucleic acid-guided endonuclease" refers to a nuclease that covalently or non-covalently binds to a guide nucleic acid (e.g., a guide RNA or a guide DNA), thereby forming a complex between the guide nucleic acid and the nucleic acid-guided nuclease. The guide nucleic acid comprises a spacer sequence comprising a nucleotide sequence that is complementary to a strand of a target DNA sequence. Thus, the nucleic acid-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule by its association with a guide nucleic acid that directly binds or anneals to a strand of the target DNA through its complementary region via Watson-Crick base pairing. In some embodiments, the nucleic acid-guided nuclease will include DNA binding activity (e.g., as in the case of CRISPR Cas9). Most commonly, nucleic acid-guided nucleases are programmed by binding to a guide RNA molecule and in such cases, the nuclease may be referred to as an "RNA-guided nuclease." When programmed by a guide DNA, the nuclease may be referred to as a "DNA-guided nuclease." Nucleic acid-guided, RNA-guided, or DNA-guided nucleases may also be referred to as "programmable nucleases," which also include other classes of programmable nucleases that recognize by amino acid/nucleotide sequence (e.g., zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)) rather than by binding to a specific DNA sequence via a guide RNA. In addition, any of the nucleases contemplated herein may also be engineered to remove, inactivate, or otherwise eliminate one or more nuclease activities (e.g., by introducing a nuclease-inactivating mutation in the active site of the nuclease). Nucleases that have been modified to remove, inactivate, or otherwise eliminate all nuclease activity may be referred to as "dead" nucleases. Dead nucleases are unable to cleave either strand of a double-stranded DNA molecule. Nucleases that have been modified to remove, inactivate, or otherwise eliminate at least one nuclease activity but still retain at least one nuclease activity may be referred to as "nickase" nucleases. Nickase nucleases cleave one strand of a double-stranded DNA molecule, not both strands. For example, CRISPR Cas9 naturally comprises two different nuclease activity domains, the HNH domain and the RuvC domain. The HNH domain cleaves the DNA strand bound to the guide RNA and the RuvC domain cleaves the protospacer strand. Nickase Cas9 can be obtained by inactivating the HNH domain or the RuvC domain. Dead Cas9 can be obtained by inactivating both the HNH domain and the RuvC domain. Likewise, other RNA-guided nucleases can be converted into nickases and /or dead nucleases by inactivating one or more of the existing nuclease domains.

As used herein, the term "operably linked" or "under transcriptional control" when used in conjunction with the description of a promoter refers to the correct position and orientation relative to a polynucleotide ( e.g. , a coding sequence) to control the initiation of transcription by RNA polymerase and the expression of the coding sequence (e.g., the coding sequence of the msr gene, msd gene, and/or ret gene). Other transcriptional control regulatory elements (e.g., enhancer sequences, transcription factor binding sites) are also operably linked to a gene if their position relative to the gene controls or regulates the expression of the gene. Programmable Nucleases

As used herein, the term "programmable nuclease" is intended to refer to a polypeptide having the property of selectively localizing to a specific desired nucleotide sequence target (e.g., a specific gene target) in a nucleic acid molecule due to one or more targeting functions. Such targeting functions may include one or more DNA binding domains, such as zinc finger domains unique to a variety of different types of DNA binding proteins or TALE domains unique to TALEN proteins. Such targeting functions may also include the ability to bind and/or form a complex with a guide RNA, which then localizes to a specific site on the DNA having a sequence that is complementary to a portion of the guide RNA (i.e., a spacer base of the guide RNA). In some embodiments, a programmable nuclease may be a single protein comprising a domain that binds directly (e.g., a ZF protein) or indirectly (e.g., an RNA-guided protein) to a target DNA site and a nuclease domain. In other embodiments, a programmable nuclease may be a complex of two or more separate proteins or domains (from different proteins) that together provide the necessary functions of selective DNA binding and nuclease activity. For example, a programmable nuclease may comprise (a) a nuclease-inactive RNA-guided nuclease (which is still able to bind guide RNA, localize to target DNA, and bind to target DNA, but is unable to cut or nick strands), fused to (b) a nuclease protein or domain, such as the FokI nuclease. Promoter

As used herein, the term "promoter" is art-recognized and refers to a nucleic acid molecule having a sequence recognized by the cellular transcription machinery and capable of initiating transcription of downstream genes. Promoters can be constitutively active, meaning that the promoter is always active in a given cellular background, or conditionally active, meaning that the promoter is only active under specific conditions. For example, a conditional promoter may be active only in the presence of a specific protein that connects the protein associated with the regulatory element in the promoter to the basic transcription machinery, or only in the absence of an inhibitory molecule. Within the promoter sequence will be found a transcription start site and a protein binding domain responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box. Various promoters, including inducible promoters, can be used to drive expression of the various vectors of the present disclosure. Recombinant Nucleic Acids

"Recombinant nucleic acid" or "recombinant nucleotide" refers to a molecule constructed by joining nucleic acid molecules that can, if appropriate, replicate themselves in living cells.

As used herein, the term "retrotranscriptome" refers to a specific type of naturally occurring and unique DNA sequence found in the genome of a variety of bacteria, which typically encodes three different components, namely, (a) noncoding RNA ("ncRNA") (including consecutive reverse sequences (msr and msd), (b) a reverse transcriptase (RT) encoding gene (ret), and (c) in many cases, a retrotranscriptome-related gene of unknown function. Retrotranscriptomes are defined, among other things, by their unique ability to generate satellite DNA, which is called msDNA (multi-copy single-stranded DNA). ncRNA (comprising msr and msd elements) and the ret gene are transcribed as a single polycistronic RNA transcript, which is processed into a ncRNA transcript and a transcript encoding the ret gene. The ncRNA is then folded into a specific secondary structure. Once translated, RT then binds to the folded ncRNA and reverse transcribes the msd region to form a single strand of cDNA (msDNA), which remains covalently linked to the RNA template via a 2'-5' phosphodiester bond and base pairing between the msDNA and the 3' end of the RNA template. See Figure 1A, which provides a schematic diagram of the production of msDNA from a naturally occurring retrotranscript. Retrotranscript components

As used herein, the term "retrotranscriptome components" refers to unique elements or features of a retrotranscriptome, namely, (a) noncoding RNA ("ncRNA") (comprising consecutive reverse sequences (msr and msd), (b) a reverse transcriptase (RT) encoding gene (ret), and (c) in many cases, a retrotranscriptome-associated gene of unknown function. RNA- guided nuclease

As used herein, "RNA-guided nuclease" is a type of "programmable nuclease" and a specific type of "nucleic acid-guided nuclease." As used herein, the term "RNA-guided nuclease" or "RNA-guided endonuclease" refers to a nuclease that covalently or non-covalently binds to a guide RNA, thereby forming a complex between the guide RNA and the RNA-guided nuclease. The guide RNA comprises a spacer sequence comprising a nucleotide sequence that is complementary to a strand of the target DNA sequence. Thus, the RNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule by its binding to the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementary region via Watson-Crick base pairing. Sequence identity

As used herein, the term "sequence identity" refers to the overall relatedness between polymer molecules, such as between polynucleotide molecules ( e.g. , DNA molecules and/or RNA molecules) and/or between polypeptide molecules. For example, the calculation of the percent identity of two sequences can be performed by aligning two polynucleotide sequences for optimal comparison purposes ( e.g. , gaps can be introduced in one or both of the first and second nucleic acid sequences to achieve optimal alignment and non-identical sequences can be ignored for comparison purposes). For example, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residual table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software suite using the NWSgapdna.CMP matrix. Methods commonly used to determine the percent identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are incorporated into publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG suite of programs (Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215, 403 (1990).

It should be noted that when the present disclosure refers to a polypeptide having a percent identity to another amino acid sequence (reference amino acid sequence) (including anywhere in the specification, including Table A and in the Examples), such as a polypeptide that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to another amino acid sequence (reference amino acid sequence), it is advantageous to refer to a polypeptide that is at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to the reference amino acid sequence. In a polypeptide having a percent identity to an amino acid sequence, a conserved region of a reference amino acid sequence is retained (e.g., when compared to other retrotranscripts RT, such as those identified herein), and/or the polypeptide has at least one activity selected from reverse transcriptase; endonuclease activity; endoribonuclease activity or RNA-guided DNase activity, and/or the polypeptide comprises: a. one or more α-helical recognition lobe (REC) and nuclease lobe (NUC); b. a wedge (WED), an α-helical recognition lobe (REC), a PAM interaction (PI), a RuvC nuclease, a bridging helix (BH) and a NUC domain; or c. one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains, and/or the polypeptide recognizes or binds to a guide RNA or ncRNA, as appropriate. Similarly, when the disclosure refers to a nucleic acid sequence or molecule having a percent identity relative to a nucleic acid sequence having a percent identity to another nucleic acid sequence or molecule (a reference nucleic acid sequence), such as a nucleic acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the other nucleic acid sequence, it is advantageous to retain conserved regions of the reference nucleic acid sequence in the nucleic acid sequence having a percent identity to the reference nucleic acid sequence (e.g., when compared to the reference nucleic acid sequence). other retrotranscripts, such as those identified herein), and/or in a polypeptide represented by a nucleic acid sequence having a percent identity with a reference nucleic acid sequence, the polypeptide contains a conserved region (e.g., conserved when compared to other retrotranscript sequences, such as those identified herein), and/or the polypeptide has at least one activity selected from reverse transcriptase; endonuclease activity; endoribonuclease activity or RNA-guided DNase activity, And/or its polypeptide comprises: a. one or more α-helical recognition lobe (REC) and nuclease lobe (NUC); b. wedge (WED), α-helical recognition lobe (REC), PAM interaction (PI), RuvC nuclease, bridge helix (BH) and NUC domains; or c. one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains, and/or the polypeptide recognizes or binds to a guide RNA. Individual

As used herein, the term "subject" refers to an individual organism, such as an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, such as a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. The terms "individual", "subject", "host" and "patient" are used interchangeably herein.

As used herein, the term "stem" refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs formed by the inverted repeat sequences connected at the "tip", wherein the more 5' or "upstream" strand of the stem bends to allow the more 3' or "downstream" strand to base pair with the upstream strand. The number of base pairs in the stem is the "length" of the stem. The tip of the stem is usually at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. Larger tips with more than 5 nucleotides are also called "loops." In other cases the continuous stem may be interrupted by one or more protrusions as defined herein. The number of unpaired nucleotides in the protrusions is not included in the length of the stem. The position of the protrusion closest to the tip can be described by the number of base pairs between the protrusion and the tip ( e.g. , the protrusion is 4 bp from the tip). The positions of other protrusions (if any) distal to the tip can be described by the number of base pairs in the stem between the protrusion in question and the tip, excluding any unpaired bases of other protrusions in between. Synthetic or artificial nucleic acids

"Synthetic or artificial nucleic acid" refers to a nucleic acid that is a sequence that does not occur naturally. Such sequences are not derived from any living organism or are known not to exist in any living organism ( e.g. , based on sequence searches in existing sequence databases). Recombinant nucleic acids and synthetic nucleic acids also include those molecules produced by replication of any of the foregoing. The engineered nucleic acid constructs of the present disclosure (such as the engineered retrotranscripts described herein) can be encoded by a single molecule ( e.g. , encoded by or present on the same plasmid or other suitable vector) or by multiple different molecules ( e.g. , multiple independently replicating vectors). Target site

As used herein, a "target site" as used herein is a polynucleotide (e.g., DNA, such as genomic DNA) that includes a site or specific locus ("target site" or "target sequence") that is targeted by the recombinant retrotranscript genome modification system disclosed herein. In the context of the retrotranscript genome modification system comprising an RNA-guided nuclease disclosed herein, the target sequence is a sequence that will hybridize with the guide sequence of a guide nucleic acid (e.g., a guide RNA). For example, the target site (or target sequence) 5'-GTCAATGGACC-3' (SEQ ID NO: 19933) within the target nucleic acid is targeted (or bound by, hybridized with, or complemented with) the sequence 5'-GGTCCATTGAC-3' (SEQ ID NO: 19934). Suitable hybridization conditions include physiological conditions that normally exist in cells. For a double-stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is called the "complementary strand" or "target strand", and the strand of the target nucleic acid that is complementary to the "target strand" (and therefore not complementary to the guide RNA) is called the "non-target strand" or "non-complementary strand ".

As used herein, the terms "treatment", "treat", or "treating" refer to clinical intervention intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after the development of one or more symptoms and/or after the disease is diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., based on a history of symptoms and/or based on genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. Upstream and Downstream

As used herein, the terms "upstream" and "downstream" are relative terms that define the linear position of at least two elements located in a nucleic acid molecule (single or double stranded) oriented in a 5'-3' direction. In a nucleic acid molecule in which a first element is located somewhere 5' to a second element, the first element is said to be upstream of the second element. Conversely, in a nucleic acid molecule in which a first element is located somewhere 3' to a second element, the first element is downstream of the second element. Variants

As used herein, the term "variant" is understood to mean a property that exhibits a pattern that deviates from that which occurs in nature, for example, a variant retrotranscript RT is a retrotranscript RT that comprises one or more amino acid residue changes compared to the wild-type retrotranscript RT amino acid sequence. The term "variant" encompasses homologous proteins that have a percent identity of at least 75% or at least 80% or at least 85% or at least 90% or at least 95% or at least 99% with a reference sequence and have one or more functional activities that are the same or substantially the same as the reference sequence. The term also encompasses mutants, truncations or domains of a reference sequence that exhibit one or more functional activities that are the same or substantially the same as the reference sequence. Vector

As used herein, the term "vector" allows or facilitates the transfer of a polynucleotide from one environment to another. It is a replicon, such as a plasmid, phage, or cohesin, into which another DNA segment can be inserted so as to cause replication of the inserted segment ( e.g. , an engineered retrotransposon of the present invention). Typically, a vector is capable of replication when combined with appropriate control elements. The term "vector" can include cloning and expression vectors, as well as viral vectors and integration vectors. Wild type

As used herein, the term "wild type" is a technical term understood by skilled artisans and means the typical form of an organism, strain, gene, protein or trait occurring in nature, as distinguished from mutant or variant forms.

The present disclosure provides systems, methods and compositions for precise genome editing, including installation of nucleic acid insertions, substitutions and deletions at targeted and precise genomic sites, wherein the systems, methods and compositions are based on novel and/or modified retrotranscripts or components thereof, such as modified forms of the retrotranscript RTs of Table X, modified forms of the ncRNAs of Table A, and modified forms of the RTs of Table B.

In one aspect, the present disclosure provides recombinant retrotransposons comprising one or more genetic modifications that improve the function and/or properties of the retrotransposons. Such genetic modifications may include mutations, insertions, deletions, inversions, substitutions, replacements or translocations of one or more consecutive or non-consecutive nucleotides in a nucleic acid molecule encoding a retrotransposon or a retrotransposon component (such as an ncRNA or a reverse transcriptase). In various aspects, a retrotranscript modified with one or more genetic modifications (i.e., a "pre-modified" or "unmodified" retrotranscript or retrotranscript component) is a naturally occurring retrotranscript or retrotranscript component (e.g., a naturally occurring ncRNA or RT of Table A) that is capable of promoting homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentration or amount of msDNA comprising a DNA donor template. In certain embodiments, the recombinant retrotranscript is based on and/or derived from a naturally occurring retrotranscript, such as any retrotranscript-related sequence provided by Table X (introducing one or more genetic modifications into a collection of 7257 previously unknown retrotranscripts discovered by the computational methods described herein (e.g., see Examples). In other embodiments, the recombinant retrotranscript is based on introducing one or more genetic modifications into a previously available retrotranscript sequence (e.g., "Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems , Nucleic Acids Research, Vol. 48, No. 22, Dec. 16, 2020, 12632-12647" (incorporated herein by reference) to obtain recombinant retrotranscripts having an enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.

In another aspect, the present disclosure further provides nucleic acid molecules encoding recombinant retrotranscripts and/or recombinant retrotranscript components (e.g., recombinant ncRNA and/or recombinant retrotranscript RT). In yet another aspect, the present disclosure provides a genome editing system comprising a recombinant retrotranscript component (e.g., recombinant ncRNA and/or recombinant RT), a programmable nuclease (e.g., RNA-guided nuclease, such as CRISPR-Cas proteins, ZFPs, and TALENS), and a guide RNA (in the case where RNA-guided nucleases are used in the genome editing systems). In another aspect, the present disclosure provides nucleic acid molecules encoding the genome editing systems and the components thereof, and polypeptides constituting the components of the genome editing systems. In another aspect, the present disclosure provides vectors for transferring and/or expressing such genome editing systems, e.g., in vitro , ex vivo , and in vivo conditions. In another aspect, the present disclosure provides cell delivery compositions and methods, including compositions for passive and/or active delivery to cells (e.g., plasmids), delivery by viral-based recombinant vectors (e.g., AAV and/or lentiviral vectors), delivery by non-viral-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retrotransposon-based genome editing systems described herein can be delivered in the form of DNA (e.g., plasmids or DNA-based viral vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), mixtures of DNA and RNA, proteins (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combination of methods for delivering the components of the retrotransposon-based genome editing systems disclosed herein can be employed. In one embodiment, each component of the retrotranscript-based genome editing system is delivered by a total RNA system, for example, one or more RNA molecules (e.g., mRNA and/or ncRNA) are delivered by one or more LNPs, wherein the one or more RNA molecules form ncRNA and guide RNA (as needed) and/or are translated into polypeptide components (e.g., RT and programmable nuclease). In another aspect, the present disclosure provides a method for genome editing by introducing the retrotranscript-based genome editing system described herein into a cell comprising a target editing site (e.g., in vitro, in vivo , or in vitro conditions), thereby causing editing to be performed at the target editing site. In other aspects, the disclosure provides formulations comprising any of the foregoing components for delivery to cells and/or tissues, including in vitro , in vivo and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retrotranscript-based genome modification systems and methods described herein, and methods of modifying cells by performing genome editing and related DNA donor-dependent methods (such as recombineering or cell transcription) using the retrotranscript-based genome modification systems disclosed herein. The present disclosure also provides methods for preparing the recombinant retrotransposons, retrotransposons-based genome modification systems, vectors, compositions and formulations described herein, as well as pharmaceutical compositions and kits for modifying cells in vitro , in vivo and ex vivo , which comprise the genome editing and/or modification systems disclosed herein.

Described herein are engineered retrotranscripts comprising one or more heterologous nucleic acids. The one or more heterologous nucleic acids can be inserted, for example, at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus. In some embodiments, the engineered retrotranscript has structural improvements over its naturally occurring counterpart or wild-type retrotranscript in at least the encoded ncRNA and/or reverse transcriptase (RT), such that the engineered retrotranscript or its encoded ncRNA exhibits multiple functional improvements over its naturally occurring/wild-type retrotranscript elements when delivered to a host cell (such as a mammalian host cell).

Exemplary (non-limiting) functional improvements may include any one or more of the features described herein. For example, in some embodiments, the engineered retrotranscript may comprise a sequence modification ( e.g. , insertion, deletion, and/or substitution of one or more nucleotides) in the msr locus and/or the msd locus that: i) modulates ( e.g. , enhances) the reverse transcription, processibility, accuracy/fidelity, and/or production of msDNA ( e.g. , in mammalian cells); ii) modulates ( e.g. , reduces) the immunogenicity of ncRNA encoded by the engineered retrotranscript ( e.g. , encoded by the msr locus and/or the msd locus) in a host ( e.g. , a host comprising mammalian cells); iii) comprises a nucleotide sequence that modulates ( e.g. , inhibits or antagonizes) msDNA function; and/or iv) modulates ( e.g. , improves) the efficiency of targeted genomic engineering.

Thus, in general, an engineered retrotranscript is an engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: i) an msr locus encoding the msr RNA portion of multiple copies of single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus.

The engineered nucleic acid construct (eg, engineered retrotranscript) can further comprise a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

In certain embodiments, the engineered retrotranscript of the present invention encodes a reverse transcriptase (RT) or a functional domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in any one of Table C. In some embodiments, RT does not comprise a polypeptide listed in Table X.

In certain embodiments, the engineered retrotranscript of the present invention encodes a reverse transcriptase (RT) or a functional domain thereof comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; and/or ii) a consensus polynucleotide sequence listed in Table C. In some embodiments, the polynucleotide encoding RT does not comprise a polynucleotide of Table X.

In certain embodiments, the engineered retrotranscripts of the present invention encode ncRNAs comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA in Table B.

In certain embodiments, the engineered retrotranscript of the present invention encodes ncRNA and reverse transcriptase (RT) or its functional domain, wherein the ncRNA and RT or its functional domain are as described above.

Specifically, in such embodiments, the ncRNA may include: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B.

In such embodiments, the reverse transcriptase (RT) or its functional domain comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, RT does not comprise a polypeptide listed in Table X; or (B) i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide in Table A; and/or, as the case may be, the polynucleotide encoding RT does not comprise a polynucleotide in Table X.

In certain embodiments, the engineered nucleic acid construct comprises: 1) an msr locus (which encodes the msr RNA portion of msDNA); 2) an msd locus, which encodes the msd RNA portion of msDNA; 3) a sequence encoding a retrotranscriptase (RT), wherein the msd RNA is capable of being reverse transcribed by the retrotranscriptase (RT) to form msDNA; and 4) a heterologous nucleic acid inserted at the msd locus, upstream of the msr locus, upstream or downstream of the msd locus, or within the msd locus; wherein the engineered nucleic acid construct is based on and/or is similar to the secondary structure of a wild-type or common retrotranscript encoding a wild-type or common retrotranscript ncRNA covered by: a) any one of SEQ ID NO: in Table B and/or any one of the sequences and/or structures depicted in Figures 2-27; or b) a) having: i) up to 1, 2 or 3 (e.g., up to 1) nucleotide changes for every 10 nucleotides in red letters; ii) up to 4, 5 or 6 (e.g., up to 1 or 2) nucleotide changes for every 10 nucleotides in black letters; and/or iii) up to 7, 8 or 9 (e.g., up to 3 or 4) nucleotide changes for every 10 nucleotides in grey letters; and/or further comprising, as appropriate: i) 7, 8, 9 or 10 (e.g., 9 or 10) nucleotides for every 10 nucleotides in red circles; i i) 6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides are present for every 10 black circled nucleotides; iii) 4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides are present for every 10 grey circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present for every 10 white circled nucleotides; wherein the ncRNA does not include an ncRNA related to a sequence of Table X.

An engineered nucleic acid construct (e.g., an engineered retrotranscript) can comprise one or more sequence modifications (e.g., insertion, deletion, and/or substitution of one or more nucleotides) in the msr locus and/or the msd locus that: a) modulates (e.g., enhances) the reverse transcription, processibility, accuracy/fidelity, and/or production of msDNA (e.g., in mammalian cells); b) modulates (e.g., reduces) the immunogenicity of ncRNA encoded by the engineered retrotranscript (e.g., msr locus and/or msd locus) in a host (e.g., a host comprising mammalian cells); c) modulates (e.g., permanently or transiently inhibits) the function of msDNA; and/or d) modulates (e.g., improves) the efficiency of targeted genome editing/engineering.

In some embodiments, the engineered nucleic acid construct (e.g., engineered retrotranscript) is based on and/or is engineered to resemble the secondary structure of a wild-type or consensus retrotranscript encoding a wild-type or consensus retrotranscript ncRNA encompassed below: a) any of the sequences in Table B ncRNA sequences and/or the structures depicted in any of Figures 2-27; or b) a variant of a) having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes for every 10 red lettered nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes for every 10 black lettered nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes for every 10 grey lettered nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., up to 1) nucleotide changes for every 10 red circled nucleotides. , 9 or 10) nucleotides; ii) there are 6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides for every 10 black circled nucleotides; iii) there are 4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides for every 10 grey circled nucleotides; and/or iv) there are 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides for every 10 white circled nucleotides.

Another aspect of the disclosure provides a vector system comprising a vector comprising an engineered retrotransposon as described herein.

Another aspect of the disclosure provides an isolated host cell comprising an engineered retrotransposon described herein or a vector system described herein.

Another aspect of the present disclosure provides a pharmaceutical composition comprising the engineered retrotransposons described herein or the vector systems described herein.

Another aspect of the present disclosure provides a delivery vehicle comprising an engineered retrotranscript described herein or an ncRNA encoded by an engineered retrotranscript described herein, a vector or vector system described herein, a host cell described herein, or a pharmaceutical composition described herein.

Another aspect of the disclosure provides a kit comprising an engineered retrotranscript described herein or an ncRNA encoded by an engineered retrotranscript described herein, and, as appropriate, instructions for genetically modifying a cell using the engineered retrotranscript described herein or the ncRNA encoded by an engineered retrotranscript described herein.

Another aspect of the present disclosure provides a method for modifying a target DNA sequence in a host cell ( e.g. , a mammalian cell), the method comprising introducing an engineered retrotranscript of the present invention, an ncRNA encoded by an engineered retrotranscript of the present invention, or a vector/vector system described herein into a host cell ( e.g. , a mammalian cell) to allow production of msDNA in the host cell ( e.g. , a mammalian cell), wherein at least a portion of the heterologous nucleic acid in the msDNA is integrated into the target DNA sequence in the host ( e.g. , mammalian) cell genome. Optionally, the target sequence is recognized by a suitable nuclease, such as CRISPR/Cas effector enzyme, ZFN, TALEN, meganuclease, TnpB, IscB or restriction endonuclease (RE), and a double strand break (DSB) is generated by the nuclease to promote/facilitate the insertion of the heterologous nucleic acid portion into the target sequence. Further, as the case may be, the target sequence modified/inserted by the heterologous nucleic acid portion can no longer be recognized by the nuclease to regenerate a DSB.

Another aspect of the disclosure provides for use of the engineered retrotranscriptors in the various methods described herein.

Another aspect of the present disclosure provides a genome editing system comprising: a) a nuclease capable of acting on a target site on a genome ( e.g. , a human genome), such as a CRISPR/Cas effector, ZFN, TALEN, meganuclease, TnpB, IscB, or a restriction endonuclease (RE); and b) an engineered retrotranscript described herein, or an ncRNA encoded thereby, or a vector or vector system comprising or encoding an engineered retrotranscript or ncRNA. Optionally, the nuclease may be linked to one or more elements of the engineered retrotranscript or the encoded ncRNA. For example, in one embodiment, the nuclease may be linked ( e.g. , fused or bound) to a reverse transcriptase of an engineered retrotranscript described herein. In another embodiment, the nuclease can be tethered/bound to form a complex with a nucleic acid guide sequence (such as a single guide RNA of a Cas enzyme), wherein the guide sequence is linked to the ncRNA and/or msDNA of the engineered retrotranscript described herein.

Another aspect of the present disclosure provides an enhanced genome editing system comprising the genome editing system of the present disclosure linked to a biomolecule that regulates host DNA repair so as to, for example, regulate ( e.g. , enhance) the incorporation of a heterologous nucleic acid sequence into a genome ( e.g. , a human genome).

Using the general aspects of the disclosure described herein, specific aspects and embodiments of the disclosure are further described in the following sections. It should be understood that any embodiment of the disclosure (including those described only in the examples or patent application or only in one section below) can be combined with any one or more additional embodiments of the invention, unless such combination is explicitly denied or inappropriate. A. Recombinant Retrotranscript

The present disclosure provides engineered retrotranscripts, as well as compositions, systems and methods comprising or utilizing engineered retrotranscripts for genome modifications such as genome editing, cellular transcription and recombineering.

Retrotranscripts were first discovered in 1984 in the bacterium Myxococcus xanthus , when short, multi-copy single-stranded DNA (msDNA) was identified as being present in large quantities in bacterial cells. Since then, a variety of naturally occurring retrotranscripts have been discovered in many prokaryotes, such as bacteria.

As depicted in Figure 1A, the retrotranscript is encoded and transcribed as a single RNA, which includes a non-coding RNA (ncRNA) portion and a portion encoding a specific reverse transcriptase (RT). The retrotranscript ncRNA ( msr and msd ) is the precursor of the final hybrid molecule, and it is initially folded into a typical RNA secondary structure, which is recognized by the accompanying RT. The translated RT usually recognizes certain secondary structures in the ncRNA and binds to the RNA template downstream of the msd region. RT starts from the 2' end of the conserved guanosine (G) residue found immediately after the double-stranded RNA structure (a1/a2 region) in the ncRNA and initiates reverse transcription of the RNA towards its 5' end. A portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the ncRNA segment that serves as a template, but does not degrade other parts of the ncRNA. As a result of reverse transcription, the msDNA remains covalently linked to the RNA template via a 2'-5' phosphodiester bond, and uses the 3' end of the msDNA for base pairing with the RNA template. See Figure 1A for the general or typical organization of the retrotranscript coding sequence (including the RT coding sequence and the msr and msd loci), and the synthesis of msDNA by reverse transcription of the initial ncRNA transcript.

Many retrotranscripts also contain accessory proteins (not depicted in Figure 1A) that may have variable functions that may not be fully understood. In certain embodiments, the engineered retrotranscripts described herein do not include accessory proteins that are naturally associated with the wild-type or template retrotranscript.

The applicant has discovered, analyzed, and phylogenetically classified 7,257 previously unknown retrotranscripts from nature based on multiple criteria, including sequence homology and conserved predicted secondary structure, and has grouped these retrotranscripts into different phylogenetic clades based on sequence homology and/or conserved predicted secondary structure. These clades include IA_IIA1 (Fig. 2), 1B1 (Fig. 3), IB2 (Fig. 4), 1C (Fig. 5), other IIA1 (Fig. 6), IIA2 (Fig. 7), IIA3 (Fig. 8), IIA4 (Fig. 9), IIA5 (Fig. 10), IIIA1 (Fig. 11), IIIA2 (Fig. 12), IIIA3 (Fig. 13), IIIA4 (Fig. 14), IIIA5 (Fig. 15), IIIunk (Fig. 16), IV (Fig. IX), V (Fig. 19), VI (Fig. 20), XI group 1 (Fig. 21), XI (group 2) (Fig. 22), XII (Fig. 23), XIII (Fig. 24), XIV (Fig. 25), Eco107-like (Fig. 26) and outgroup A (Fig. 27). This disclosure further describes the engineering and/or modification of these newly discovered retrotranscript sequences as a starting point for obtaining useful recombinant retrotranscripts such as those depicted in FIG. 1B .

FIG. 1B.1 depicts one embodiment of a recombinant retrotranscript construct contemplated by the present disclosure (e.g., a nucleotide sequence cloned into an expression vector). In the upper left schematic, a single thin black line represents a double-stranded nucleotide sequence (e.g., as cloned into an expression vector such as a plasmid). By modifying the encoding ncRNA The starting point retrotranscript DNA sequence of (msr/msd region) (such as any of the 7257 newly discovered retrotranscript sequences disclosed herein, and in particular any of the 7257 ncRNA sequences in Table B) is used to construct a recombinant retrotranscript. The starting point retrotranscript DNA sequence encoding the ncRNA can be modified in a variety of ways, and can include one modification or more than one modification. For example, the retrotranscript DNA can be modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion or deletion, or a substitution, insertion or deletion of more than one nucleotide, that is, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,5 3, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, 99 or up to 100 or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or up to 2000 nucleotides are substituted, inserted or deleted. When more than one nucleotide of a starting point retrotran (e.g., a wild-type retrotran) is substituted, deleted or inserted, the nucleotides may be consecutive or non-consecutive. Although an engineered retrotran is not naturally occurring as a whole, it may include components that do exist in nature, such as nucleotide sequences. For example, an engineered retrotran may have nucleotides from different organisms. ( e.g. , from different bacterial species) or from a nucleotide sequence of a completely synthetic/artificial/recombinant nucleic acid sequence. Therefore, the engineered retrotransposons may have bacterial nucleotide sequences, human nucleotide sequences, viral nucleotide sequences and/or synthetic/artificial/recombinant nucleotide sequences and/or combinations of such sequences. Examples of modifications of the recombinant retrotransposons disclosed herein include inserting a heterologous nucleic acid sequence into the retrotransposons, for example, into a ncRNA locus, such as an msr or msd locus. Connecting a guide RNA molecule to the 5' and/or 3' end (i.e., connecting a molecule to the 5' end of the ncRNA and/or connecting a molecule to the 3' end of the ncRNA) also represents another modification considered for the recombinant retrotransposons disclosed herein. In such embodiments, the guide RNA molecule may also be classified or more generally referred to as the type of heterologous nucleic acid sequence used to modify the starting point retrotransposons. These modifications are depicted in Figure 1B.

In addition to DNA encoding ncRNA, DNA encoding RT can also be modified to obtain recombinant RT. For example, the DNA encoding RT can be modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion or deletion, or a substitution, insertion or deletion of more than one nucleotide, that is, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or up to 2000 nucleotides are substituted, inserted or deleted.

Such modifications to the DNA encoding the ncRNA and/or RT can modulate the function of the ncRNA and/or RT in a variety of ways, including i) modulating ( e.g. , enhancing) the reverse transcription, processibility, accuracy/fidelity and/or production of msDNA ( e.g. , in mammalian cells); ii) modulating ( e.g. , reducing) the immunogenicity of the ncRNA ( msr locus and msd locus) encoded by the engineered retrotranscript in a host ( e.g. , a host comprising mammalian cells); iii) modulating ( e.g. , permanently or transiently inhibiting) the function of msDNA; and/or iv) modulating ( e.g. , improving) the efficiency of targeted genome editing/engineering.

In one embodiment, the present disclosure provides a recombinant retrotranscriptase having the following general structure: a) an msr locus; b) an msd locus encoding the msd RNA portion of the msDNA; c) a sequence encoding a retrotranscriptase (RT) (optionally in trans relative to the ncRNA), wherein the msd RNA is capable of being reverse transcribed ( e.g. , in a host cell, such as a mammalian cell) by the retrotranscriptase (RT) to form msDNA; d) a heterologous nucleic acid ( e.g. , heterologous DNA) capable of being transcribed through the msr locus and/or the msd locus, optionally inserted at the msd locus, upstream of the msr locus, upstream or downstream of the msd locus, or within the msd locus.

The engineered retrotranscripts of the present invention are optionally further modified in structure to include one or more heterologous nucleic acids. The engineered retrotranscripts can be further modified to provide various functional improvements, such as (but not limited to) enhancing msDNA production in cells ( e.g., mammalian cells, including human cells).

In certain embodiments, the present disclosure provides engineered retrotranscripts based on their conservative predicted secondary structures, such as those in Figures 2-27.

In other embodiments, the disclosure provides engineered retrotransposons based on their sequence identity. Table A provides exemplary RT amino acid sequences and ret gene nucleic acid sequences. Table C provides exemplary RT consensus amino acid sequences and/or ret gene nucleic acid sequences. Table B provides exemplary ncRNA sequences.

Table X provides retrotranscript sequences that may, in some embodiments, be provided outside the scope of the present invention.

In certain embodiments, exemplary engineered retrotranscripts of the invention (1) are engineered based on a secondary structure as depicted in any of Figures 2-27 or engineered similar to such secondary structures, and/or (2) are provided in Table B. Sequences having a significant percentage of sequence identity ( e.g. , at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity) are also within the scope of the invention.

In certain embodiments, the engineered nucleic acid construct comprises: 1) an msr locus (which encodes the msr RNA portion of msDNA); 2) an msd locus, which encodes the msd RNA portion of msDNA; 3) a sequence encoding a retrotranscriptase (RT), wherein the msd RNA is capable of being reverse transcribed by the retrotranscriptase (RT) to form msDNA; and 4) a heterologous nucleic acid inserted at the msd locus, upstream of the msr locus, upstream or downstream of the msd locus, or within the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or similar to the secondary structure of a wild-type or common retrotranscript encoding a wild-type or common retrotranscript ncRNA covered by: a) any sequence and/or structure depicted in any SEQ ID No of Table B and/or Figures 2-27; or b) a) having: i) up to 1, 2 or 3 (e.g., up to 1) nucleotide changes for every 10 nucleotides in red letters; ii) up to 4, 5 or 6 (e.g., up to 1 or 2) nucleotide changes for every 10 nucleotides in black letters; and/or iii) up to 7, 8 or 9 (e.g., up to 3 or 4) nucleotide changes for every 10 nucleotides in grey letters; and/or further comprising, as appropriate: i) 7, 8, 9 or 10 (e.g., 9 or 10) nucleotides for every 10 nucleotides in red circles; i i) 6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides are present for every 10 black circled nucleotides; iii) 4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides are present for every 10 grey circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present for every 10 white circled nucleotides; wherein the ncRNA does not comprise an ncRNA related to a sequence of Table X.

In certain embodiments, the insertion of heterologous nucleic acid includes the deletion of retrotransposons. In certain embodiments, the inserted heterologous nucleic acid replaces the deleted retrotransposons. It is not required that the inserted heterologous nucleic acid and the deleted retrotransposons have the same or similar size. In certain embodiments, the replacement comprises replacing a portion of the hairpin loop region of the retrotransposons. In certain embodiments, the replacement comprises replacing a portion of the stem-loop region of the retrotransposons. In certain embodiments, the replacement comprises replacing a region of the retrotransposons that does not include a stem or loop. In certain cases, the identification of the retrotransposons structure involves comparison with the model ncRNA structures provided herein, for example, comparison with one or more of Figures 2-27. In some cases, identification of retrotranscript structure involves modeling by nucleic acid folding algorithms, such as but not limited to RNAfold (Gruber AR et al., The Vienna RNA Websuite. Nucleic Acids Research , Vol. 36, No. suppl_2, July 1, 2008, pp. W70-W74), UNAFold (Markham NR et al., UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol. Vol. 453, 2008, pp. 3-31), and SPOT-RNA (Singh J et al., RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning. Nature communications , Vol. 10, 2019, pp. 1-13).

In certain embodiments, the engineered retrotranscript of the present invention encodes a reverse transcriptase (RT) or a functional domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A. In some embodiments, RT does not comprise a polypeptide identified in Table X.

In certain embodiments, the engineered retrotranscript of the present invention encodes a reverse transcriptase (RT) or a functional domain thereof comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide of Table A; and/or ii) a consensus polynucleotide sequence listed in Table A. In some embodiments, the polynucleotide encoding RT does not comprise a polynucleotide identified in Table X.

In certain embodiments, the engineered retrotranscripts of the present invention encode ncRNAs comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the ncRNA of Table B.

The engineered ncRNA of the present invention may be different in size from the ncRNA on which it is based, and the proportion of ncRNA retained in the engineered ncRNA may vary. In certain embodiments, the retention of the ncRNA is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 50% to 80%, or 60% to 85%, 70% to 90%, or 80% to 95%, or 85% to 98%, or 90% to 99%, or all of the ncRNA.

In certain embodiments, the engineered retrotranscript of the present invention encodes ncRNA and reverse transcriptase (RT) or its functional domain, wherein the ncRNA and the RT or its functional domain are as described above.

Specifically, in such embodiments, the ncRNA may include: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and wherein the ncRNA optionally excludes ncRNAs related to sequences identified in Table X.

In such embodiments, the reverse transcriptase (RT) or its functional domain comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, RT does not comprise a polypeptide identified in Table X; or (B) i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; optionally, the polynucleotide encoding RT does not include a polynucleotide related to a sequence identified in Table X.

In certain embodiments, the heterologous nucleic acid is between >20 nucleotides and about 10,000 nucleotides.

The engineered retrotranscript may further comprise a sequence modification ( e.g. , insertion, deletion and/or substitution of one or more nucleotides) in the msr locus and/or msd locus that: i) modulates ( e.g. , enhances) the reverse transcription, processibility, accuracy/fidelity and/or production of msDNA ( e.g. , in mammalian cells); ii) modulates ( e.g. , reduces) the immunogenicity of ncRNA ( msr locus and msd locus) encoded by the engineered retrotranscript in a host ( e.g. , a host comprising mammalian cells); iii) comprises a nucleotide sequence that modulates ( e.g. , permanently or transiently inhibits) msDNA function; and/or iv) modulates ( e.g. , improves) the efficiency of targeted genome editing/engineering.

Retrotranscript msr gene, msd gene and RT nucleic acid sequence ( e.g. , ret gene) and the encoded retrotranscript reverse transcriptase protein sequence that can serve as a template for the engineered retrotranscript described herein can be derived from any source, such as those in Table A, excluding those related to the sequences of Table X as appropriate.

In some embodiments, the template or wild-type (wt) sequence of the msr gene, msd gene, and RT coding sequence ( ie , ret gene) used in the engineered retrotranscript is derived from a bacterial retrotranscript.

In some embodiments, the representative template/wild-type retrotransposons are from Gram-negative bacteria. In some embodiments, the retrotransposons are from bacteria listed in Table X.

In some embodiments, the engineered retrotranscript is engineered based on an evolutionary branch defined for retrotranscripts/retrotranscript RTs, wherein the retrotranscript is associated with a tripartite system consisting of ncRNA, RT, and an additional protein or RT fusion domain with different enzymatic functions. See, e.g., "Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems , Nucleic Acids Research, Vol. 48, No. 22, Dec. 16, 2020, pp. 12632-12647" (incorporated herein by reference). Although these evolutionary branches are mainly based on naturally occurring ncRNAs and retrotransposons/retrotransposons RTs, as well as additional proteins or RT fusion domains, for the purpose of serving as templates for the engineered retrotransposons of the present invention, these evolutionary branches are not limited to naturally occurring sequences. Instead, these evolutionary branches can also cover non-naturally occurring ncRNAs and RTs, including but not limited to recombinant, modified or altered, chimeric, hybrid, synthetic, artificial, etc.

Therefore, according to the present disclosure, retrotranscripts can be considered to be germline related based on a neighbor-joining algorithm of at least 75% (at least 1000 replicates) and a Poisson-corrected distance measure of no more than 0.05 (based on alignment of retrotranscript RTs). Alternatively or additionally, retrotranscripts can be considered to be germline related when/if the same RT or closely related RTs can recognize the secondary structure of the ncRNA of the retrotranscript and transcription of the retrotranscript is preserved to generate msDNA.

In certain embodiments, sequence alignment or secondary structure generation between different retrotranscript sequences ( eg , ncRNA and/or RT (protein and/or nucleic acid) sequences) is based on software known to those of ordinary skill in the art.

Retrotranscript ncRNA sequences (including msr and msd sequences) within the same evolutionary branch may be highly conserved at certain positions but less conserved at other positions.

Exemplary consensus sequences based on clade members (see corresponding Figures 2-27, respectively) were generated to show these conserved sequences and/or secondary structures, including highly conserved nucleotides with at least 97% sequence conservation at red letter nucleotides, those with between 90-97% sequence conservation at black letter nucleotides, and those with 75-90% nucleotide sequence identity at gray letter nucleotides. Further structural constraints of the clade consensus sequences are provided in the form of colored circles, indicating the probability of having a base at that particular position, including red circles representing bases in 97% of the cases, black circles representing bases in 90-97% of the cases, and gray circles representing bases in 75-90% of the cases.

In some embodiments, the template ncRNA (including msr and msd region sequences) as the basis for modifying the engineered retrotranscript of the present invention is a consensus sequence of various retrotranscript ncRNA (including msr and msd nucleic acid sequences) evolutionary branches, as provided in Table B and any SEQ ID NO: of the corresponding Figures 2-27, respectively, including highly conserved bases depicted by letters or circles of specific colors, and optionally further including bases represented by circles of specific colors that may be present at specific positions.

In some embodiments, the engineered retrotranscript is based on and/or engineered to resemble the secondary structure of a wild-type or consensus retrotranscript encoding a wild-type or consensus retrotranscript ncRNA covered below: 1) a sequence and/or structure as depicted in any one of SEQ ID NOs: in Table B and corresponding Figures 2-27, respectively; or 2) a variant of 1) having: A) up to 1, 2, or 3 ( e.g. , up to 1) nucleotide changes per 10 red-letter nucleotides; B) up to 4, 5, or 6 ( e.g. , up to 1 or 2) nucleotide changes per 10 black-letter nucleotides; and/or C) up to 7, 8, or 9 ( e.g. , up to 3 or 4) nucleotide changes per 10 grey-letter nucleotides. Optionally, the variant of 1) further comprises: a) 7, 8, 9 or 10 ( e.g. , 9 or 10) nucleotides for every 10 red circled nucleotides; b) 6, 7, 8, 9 or 10 ( e.g. , 8, 9 or 10) nucleotides for every 10 black circled nucleotides; c) 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 6, 7, 8, 9 or 10) nucleotides for every 10 grey circled nucleotides; and/or d) 2, 3, 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 4, 5, 6, 7, 8, 9 or 10) nucleotides for every 10 white circled nucleotides.

An engineered retrotranscript can be engineered by introducing a sequence modification ( eg , a deletion, addition, or substitution) into a wild-type retrotranscript encoding a wild-type retrotranscript ncRNA or into a retrotranscript encoding a consensus retrotranscript ncRNA.

For example, a variant retrotransposons may not meet the sequence and/or structural requirements of any SEQ ID NO: in Table B and corresponding Figures 2-27, respectively, but may still be a suitable template for the engineered retrotransposons described herein, as long as one or more of the conditions set forth in A) - C) and/or a) - d) are met.

In certain embodiments, highly conserved sequences in the template retrotranscripts are retained/conserved or substantially retained/conserved in the engineered retrotranscripts described herein.

In certain embodiments, all or substantially all red-letter nucleotides ( i.e. , those conserved in about 97% or more retrotranscripts in the same evolutionary branch) are retained/conserved in the engineered retrotranscripts described herein. In certain embodiments, no more than 1, 2, or 3 ( e.g. , up to 1) nucleotide changes ( e.g. , deletions or substitutions) occur for every 10 red-letter nucleotides in the engineered retrotranscripts described herein. In certain embodiments, no more than about 0.3%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the red-letter nucleotides are changed ( e.g. , deleted or substituted) in the engineered retrotranscripts described herein.

In certain embodiments, all or substantially all black letter nucleotides ( i.e. , those conserved in about 90-97% of retrotransposons in the same evolutionary branch) are retained/conserved in the engineered retrotransposons described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ( e.g. , up to 1 or 2) nucleotide changes ( e.g. , deletions or substitutions) occur for every 10 black letter nucleotides in the engineered retrotransposons described herein. In certain embodiments, no more than about 3%, 4%, 5%, or 10% of the black letter nucleotides are changed ( e.g. , deleted or substituted) in the engineered retrotransposons described herein.

In certain embodiments, all or substantially all grey lettered nucleotides ( i.e. , those conserved in about 75-90% of retrotransposons in the same evolutionary branch) are retained/conserved in the engineered retrotransposons described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ( e.g. , up to 3 or 4, or up to 7, 8, or 9) nucleotide changes ( e.g. , deletions or substitutions) occur for every 10 grey lettered nucleotides in the engineered retrotransposons described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, or 25% of the grey lettered nucleotides are changed ( e.g. , deleted or substituted) in the engineered retrotransposons described herein.

In certain embodiments, all or substantially all red circle nucleotides ( i.e. , those having nucleotides in about 97% or more retrotranscripts in the same evolutionary branch) are present in the engineered retrotranscripts described herein. In certain embodiments, no more than 1, 2, or 3 ( e.g. , 0.3, 0.5, or up to 1) nucleotides are absent ( e.g. , deleted) for every 10 red circle nucleotides in the engineered retrotranscripts described herein. In certain embodiments, 7, 8, 9, or 10 ( e.g., 9 or 10) nucleotides are present for every 10 red circle nucleotides in the engineered retrotranscripts described herein. In certain embodiments, no more than about 0.3%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the red circle nucleotides are absent (e.g. , deleted) in the engineered retrotranscripts described herein.

In certain embodiments, all or substantially all black circled nucleotides ( i.e. , those having nucleotides in about 90-97% of retrotransposons in the same clade) are present in the engineered retrotransposons described herein. In certain embodiments, no more than 1, 2, 3, or 4 ( e.g. , up to 1 or 2) nucleotides are absent ( e.g. , deleted) for every 10 black circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, 6, 7, 8, 9, or 10 (e.g., 8, 9, or 10) nucleotides are present for every 10 black circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, no more than about 1%, 2%, 3%, 5%, or 10% of the black circled nucleotides are absent ( e.g. , deleted) in the engineered retrotransposons described herein .

In certain embodiments, all or substantially all of the grey circled nucleotides ( i.e. , those having nucleotides in about 75-90% of retrotransposons in the same clade) are present in the engineered retrotransposons described herein. In certain embodiments, no more than 1, 2, 3, 4, or 5 ( e.g. , up to 2, 3, or 4) nucleotides are absent ( e.g. , deleted) for every 10 grey circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, 4, 5, 6, 7, 8, 9, or 10 ( e.g., 6, 7, 8, 9, or 10) nucleotides are present for every 10 grey circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, or 25% of the grey circled nucleotides are absent (e.g. , deleted) in the engineered retrotransposons described herein.

In certain embodiments, all or substantially all white circled nucleotides ( i.e. , those having nucleotides in about 50-75% of retrotransposons in the same clade) are present in the engineered retrotransposons described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, or 6 ( e.g. , up to 2, 3, 4, 5, 6) nucleotides are absent ( e.g. , deleted) for every 10 white circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ( e.g. , 4, 5, 6, 7, 8, 9, or 10) nucleotides are present for every 10 grey circled nucleotides in the engineered retrotransposons described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the white circled nucleotides are absent ( eg , deleted) in an engineered retrotranscript described herein.

In some embodiments, the engineered retrotranscript is synthetically produced. In other embodiments, the synthetically produced engineered retrotranscript comprises a sequence and/or secondary structure as depicted in any one of SEQ ID NOs: in Table B and corresponding Figures 2-27, respectively, and conserved colored letter nucleotides ( e.g. , red, black, and gray letters) at least according to their respective sequence identity levels, and/or conserved colored circle nucleotides ( e.g. , red, black, and gray circles) at least according to their respective sequence occurrence probability levels.

In some embodiments, sequence modifications in the engineered retrotran result in desired improvements in the function of the encoded retrotran ncRNA.

In certain embodiments, in the ncRNA, the one or more sequence modifications include one or more of the following: (i) a modified (e.g., mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii) (iii) an extension or shortening of a1, a2, or both a1 and a2; (iv) an additional or modified (e.g., mutated or eliminated) protrusion in a hairpin (e.g., L2 and/or L3 in FIG. 2, or any L region in FIG. 2-27 (e.g., by removing unpaired bases in the protrusion, or by replacing unpaired bases with an equal number of base pairs)); (v) a modified (e.g., extended or shortened) length of a hairpin (e.g., L1, L2, L3 and/or L4 in FIG. 2, or any L region in FIG. 2-27) ; (vi) an alternative L1 and/or L2 (in FIG. 2 , or any L region of any of FIG. 2-27 ) with a complement, reverse, or reverse complement sequence; (vii) a modified (e.g., increased) number of unpaired bases at the tip of a hairpin loop (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any L region of any of FIG. 2-27 ); (viii) a modified (e.g., increased or decreased) GC content in a hairpin loop (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any L region of any of FIG. 2-27 ); (ix) in the spacer sequence between hairpin loops (e.g., S1, S2 in FIG. 2 ); 2-27) or inserting the heterologous nucleic acid at the tip of a hairpin loop (e.g., L1, L2, L3 and/or L4 in FIG. 2, or any L region in FIG. 2-27); (x) deleting one or more hairpin loops (e.g., L1, L2, L3 and/or L4 in FIG. 2, or any L region in FIG. 2-27); (xi) adding a new loop to the spacer sequence between hairpin loops (e.g., S1, S2, S3 and/or S4 in FIG. 2, or any S region in FIG. 2-27); (xii) circularization of the ncRNA, wherein the 5' and 3' ends of the ncRNA are linked directly or via a spacer sequence; (xiii) a relocated branched chain guanosine capable of initiating retrotranscriptional initiation; (xiv) a staggered end sequence that reduces the immunogenicity of the retrotranscript ncRNA, which is generated by, for example, adding or removing 5' a1 nucleotides and/or 3' a2 nucleotides; and/or (xv) an antisense sequence that is complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes with and inhibits the gRNA in the encoded retrotranscript ncRNA, and wherein the antisense sequence is removed after reverse transcription of the msDNA.

Unless otherwise specifically indicated, the a1 and a2 regions are both single-stranded and substantially anti-complement each other, forming a stem interrupted by symmetrical or asymmetrical bulges, as appropriate, with one or more 5' and/or 3' overhanging/unpaired nucleotides, as appropriate, wherein the a1 region generally ends before ( e.g. , immediately 5' to) a conserved branching guanosine (G) that provides a 2'-OH for reverse transcription initiation.

In some embodiments, the sequence variation comprises a mutation in the a1/a2 stem region, a reduction or elimination of an overhang, including sequence variation in one ( ie , a1 or a2) strand or both a1 and a2 strands.

For example, in some embodiments, the sequence change comprises deleting nucleotides from a1, a2, or both a1 and a2, such that the size of the bulge is reduced, or a symmetric bulge becomes asymmetric, or vice versa, or the bulge is eliminated.

In some embodiments, the sequence change comprises a replacement/substitution of a nucleotide in a1, a2, or both a1 and a2 such that a previously unpaired base in the bulge becomes base-paired.

In some embodiments, the sequence changes comprise replacing an unpaired purine base with one or more unpaired pyrimidine bases.

In some embodiments, the sequence changes comprise replacing an unpaired pyrimidine base with one or more unpaired purine bases.

In some embodiments, the sequence change comprises replacing one unpaired purine base ( e.g. , A or G) with another unpaired purine base ( e.g. , G or A, respectively).

In some embodiments, the sequence change comprises replacing one unpaired pyrimidine base ( e.g. , T/U or C) with another unpaired pyrimidine base ( e.g. , C or T/U, respectively).

In some embodiments, the sequence change comprises an extension or shortening of a1, a2, or both a1 and a2.

For example, the length of a1 can be shortened by deleting a 5' overhang, deleting any upstream protruding nucleotides, or deleting bases that participate in base pairing. Similarly, the length of a1 can be extended by adding a 5' overhang, adding any upstream protruding nucleotides, or adding bases that participate in base pairing.

In some embodiments, the length of a2 can be shortened by deleting a 5' overhang, deleting any downstream protruding nucleotides, or deleting bases that participate in base pairing. Similarly, the length of a2 can be extended by adding a 5' overhang, adding any downstream protruding nucleotides, or adding bases that participate in base pairing.

In some embodiments, the spacer sequence between the hairpin loops as depicted in any of Figures 2-27 ( e.g. , S1, S2, S3, and/or S4 in Figure 2) can be lengthened or shortened. In some embodiments, the modification can be performed by inserting a heterologous nucleic acid sequence into the spacer sequence between the hairpin loops ( e.g. , S1, S2, S3, and/or S4 in Figure 2). In certain embodiments, one or more heterologous nucleic acid sequences are inserted into the spacer sequence in the msd region. In some embodiments, the modification of the spacer region can be performed by interrupting the spacer base with an additional protrusion or hairpin loop.

In other embodiments, a protrusion in a hairpin is mutated or eliminated in the protrusion ( e.g. , by removing unpaired bases), such that, for example, a symmetric protrusion becomes an asymmetric protrusion, or an asymmetric protrusion becomes a symmetric protrusion or even more asymmetric protrusion. In certain embodiments, unpaired bases in a protrusion are replaced with an equal number of base pairs. The additional base pairs may be incorporated into the stem at one or both ends of the previous protrusion, or the previous protrusion may be bisected to create two protrusions.

In some embodiments, the length of one or more hairpin loops ( e.g. , L1, L2, L3, and/or L4 of FIG. 2 ) as depicted in any of FIGS. 2-27 can be extended or shortened. For example, the number of unpaired bases in the tip or loop can be increased or decreased. In addition, a related heterologous nucleic acid sequence can be inserted into the tip or hairpin loop. In certain embodiments, a related heterologous nucleic acid sequence is inserted into the tip or hairpin loop in the msd locus.

In other embodiments, the GC content in the tip or hair clip is increased or decreased.

In other embodiments, the hair clip ring may be absent.

In some embodiments, the ncRNA can be circularized by joining the 5' and 3' ends of the ncRNA directly or through a spacer sequence.

In some embodiments, one or more hairpin loops ( e.g. , L1, L2, L3, and/or L4 of FIG. 2) are modified to have a complement, reverse, or reverse complement sequence.

In certain embodiments, a branched guanosine (G) capable of initiating reverse transcription is relocated. For example, G can be further downstream of the end of the a1 sequence, such as 1, 2, 3, 4 or 5 additional nucleotides.

In certain embodiments, the immunogenicity of a retrotran ncRNA is reduced by, for example, adding or removing 5' a1 nucleotides and/or 3' a2 nucleotides.

In certain embodiments, the one or more heterologous nucleic acid sequences (inserted into the engineered retrotransposons of the present invention) comprise: a) a heterologous nucleic acid (such as an RNA aptamer or a ribozyme encoding sequence) inserted into the msr locus or the msd locus (such as an S region (e.g., S1, S2, S3 and/or S4 in FIG. 2 , or any S region of any one of FIGS. 2-27 ), or at the tip of the L region (e.g., L1, L2, L3 and/or L4 in FIG. 2 , or any L region of any one of FIGS. 2-27 ), or upstream or downstream of the msr locus or the msd locus; or b) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a CRISPR/Cas guide RNA (gRNA).

In certain embodiments, an antisense sequence may be included that is complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes with and inhibits the gRNA in the encoded retrotranscript ncRNA, and wherein the antisense sequence is removed after retrotranscription of the msDNA.

In certain embodiments, the heterologous nucleic acid encodes a protein or peptide of interest, or wherein the heterologous nucleic acid comprises or encodes a donor/template sequence (e.g., a donor that corrects/repairs/removes a mutation at a target genome site (e.g., a mutant exon in a disease gene); a functional DNA element (e.g., a promoter, an enhancer, a protein binding sequence, a methylation site, a homologous region for assisting gene editing, etc.); or a coding sequence for a functional RNA element (ncRNA, etc.)).

In certain embodiments, the protein or peptide of interest comprises a therapeutic protein that can be used to treat a disease (eg, a wild-type protein that is defective in a diseased cell, or a therapeutic antibody or antigen-binding fragment thereof).

Other heterologous nucleic acids of the present invention are described in other sections of this specification, which are incorporated herein by reference.

In some embodiments, the template/wild-type retrotranscript for an engineered retrotranscript encodes a wild-type or common retrotranscript ncRNA polynucleotide having a common secondary structure as shown in any of Figures 2-27, which are described individually as follows: Variants of this template, which may also be used in the engineered retrotranscripts of the present invention, include variants having: A) up to 1, 2, or 3 ( e.g. , up to 1) nucleotide changes for every 10 red-letter nucleotides; B) up to 4, 5, or 6 ( e.g. , up to 1 or 2) nucleotide changes for every 10 black-letter nucleotides; and/or C) up to 7, 8, or 9 ( e.g. , up to 3 or 4) nucleotide changes for every 10 gray-letter nucleotides; and/or further comprising, as appropriate: a) up to 10 red-circled nucleotides a) there are 7, 8, 9 or 10 ( e.g. , 9 or 10) nucleotides per 10 black circled nucleotides; c) there are 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 6, 7, 8, 9 or 10) nucleotides per 10 grey circled nucleotides; and/or d) there are 2, 3, 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 4, 5, 6, 7, 8, 9 or 10) nucleotides per 10 white circled nucleotides.

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in SEQ ID NO. XX and in Figures 2-27.

In some embodiments, the non-coding RNA (ncRNA) portion of the engineered retrotransposons comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to an ncRNA listed in Table B. In some embodiments, the ncRNA does not comprise an ncRNA related to a sequence of Table X.

For example, the amplification of the engineered retrotranscript described herein can be performed before transfecting cells or joining into a vector. Any method for amplifying engineered retrotranscripts can be used, including but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand substitution amplification (SDA), and ligase chain reaction (LCR). In one embodiment, the engineered retrotranscript comprises a common 5' and 3' activating site to allow amplification of retrotranscript sequences in parallel with a set of universal primers. In another embodiment, a set of selective primers is used to selectively amplify a subset of retrotranscript sequences from a pooled mixture.

In some embodiments, the template/wild-type retrotranscript for the engineered retrotranscript encodes a wild-type or consensus retrotranscript ncRNA polynucleotide having a consensus secondary structure as shown in Figures 2-27, and the Figures are described separately below: Type IA/IIA1 Retrotranscript (Figure 2)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-a2-S4-L4, wherein: a1/a2 is a stem of 8 bp in length; L1 is a stem of 5 bp with a 10-nt tip; L2 is a stem of 7 bp with a 5-nt tip and a 1/1 bulge 3 nt from the tip; L3 is a stem of 23 bp with a 22-nt tip and a 2/2 bulge 21 bp from the tip; L4 is a stem of 11 bp with a 5-nt tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1, wherein there is no spacer between L1 and L2; S2 is a single-stranded spacer between L2 and L3; S3 is a single-stranded spacer between L3 and a1/a2 stem; and S4 is a single-stranded spacer between a1/a2 stem and L4, and the conserved nucleotides are as shown in SEQ ID NO: 1 and Figure 2, and wherein the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 2. Type IB1 retrotranscript (Figure 3)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a 6 bp long stem with a 2/2 bulge 3 bp from the tip, wherein a1 has a 2-nt overhang and a2 has a 6-nt overhang; L1 is a 14 bp stem with a 3-nt tip, a 1/0 bulge 4 bp from the tip, and a 0/6 bulge 10 bp from the tip; L2 is a 23 bp stem with a 5-nt tip, a 1/1 bulge 4 bp from the tip, and a 0/1 bulge 18 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2. Conserved nucleotides are shown in FIG3 , and the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 3. Type IB2 retrotranscript (Figure 4)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-L4-S4-a2, wherein: the a1/a2 stem is 16 bp long with a 17-base 5' overhang and a 16-base 3' overhang; L1 is a 6 bp stem with a 4-nt tip; L2 is a 4 bp stem with a 4-nt tip and a 2/2 protrusion 2 nt from the tip; L3 is a 3 bp stem with a 5-nt tip; L4 is a 9 bp stem with a 5-nt tip and a 1/1 protrusion 4 nt from the tip; S1, S2, S3 and S4 are single-stranded spacer regions between a1/a2 stem and L1, L2 and L3, L3 and L4, and L4 and a1/a2 stem, respectively, wherein there is no spacer between L1 and L2; wherein the last 5 nt of S1 and the 5th to 9th nt of S2 form a 5-bp stem, and the conserved nucleotides are as shown in FIG4, and wherein the colored circled nucleotides are present at respective certainty levels (e.g., at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 4. Type 1C retrotranscript (Figure 5)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem of 13 bp in length; L1 is a stem of 9 bp with a 3-nt tip; L2 is a stem of 10 bp with a 5-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2, Conserved nucleotides are shown in FIG. 5 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 5. Type IIA1 retrotranscript (Figure 6)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a 10 bp long stem with a 1-nt overhang on a2; L1 is a 10 bp stem with a 3-nt tip; L2 is a 7 bp stem with a 5-nt tip; L3 is a 27 bp stem with an 8-nt tip and a 0/2 bulge 26 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and a1/a2. The conserved nucleotides are shown in FIG6 , and the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 6. Type IIA2 retrotranscript (Figure 7)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-L3-S3-a2, wherein: a1/a2 is a 7 bp long stem without an overhang; L1 is an 8 bp stem with a 3-nt tip; L2 is a 30 bp stem with an 8-nt tip, a 1/1 bulge 2 bp from the tip, and a 1/1 bulge 27 bp from the tip; L3 is an 8 bp stem with a 5-nt tip and a 0/1 bulge 3 nt from the tip; S1 is a single-stranded spacer between the a1/a2 stem and L1; S2 is a single-stranded spacer between L1 and L2; and S3 is a single-stranded spacer between L3 and a1/a2 stems; the conserved nucleotides are shown in FIG7 , and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 7. Type IIA3 retrotranscript (Figure 8)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a 6 bp long stem; L1 is an 8 bp stem with a 9-nt tip; L2 is an 8 bp stem with a 3-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L2 and L3; S3 is a single-stranded spacer between L3 and a1/a2, Conserved nucleotides are shown in FIG. 8 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotransposons are completely synthetically produced and have conserved nucleotides represented by colored letters as shown in Figure 8. Type IIA4 retrotransposons (Figure 9)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-a2-L1-S1-L2-L3-S2-L4-S3, wherein: a1/a2 is a 3 bp long stem without an overhang and with a 7-nt tip; L1 is a 7 bp stem with a 3-nt tip; L2 is a 6 bp stem with a 4-nt tip; L3 is a 40 bp stem with a 5-nt tip and a 2/2 bulge 3 bp from the tip, a 5/4 bulge 10 bp from the tip, and a 12/15 bulge 30 bp from the tip; L4 is a 4 bp stem with a 9-nt tip; S1 is a single-stranded spacer between L1 and L2/L3; S2 is a single-stranded spacer between L2/L3 and L4; S3 is a single-stranded spacer between L4 and the 3' end of the ncRNA; and the conserved nucleotides are as shown in FIG. 9 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 9. Novel retrotranscript of type IIA5 (Figure 10)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a 15 bp long stem with a 1-nt overhang on a1, a 13-nt overhang on a2, and a 7/5 protrusion 13-nt from the tip; L1 is a 10 bp stem with a 3-nt tip; L2 is a 35 bp stem with a 3-nt tip; L3 is a 6 bp stem with a 5-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and a1/a2. Conserved nucleotides are shown in FIG. 10 , and the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 10. Type IIIA1 retrotranscript (Figure 11)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a 2 bp long stem with a 1-nt overhang on a2; L1 is an 8 bp stem with a 4-nt tip; L2 is a 9 bp stem with a 3-nt tip and a 1/1 bulge 3 bp from the tip; L3 is a 20 bp stem with a 3-nt tip and a 1/2 bulge 3 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L2 and L3; S3 is a single-stranded spacer between L3 and a1/a2, Conserved nucleotides are shown in FIG. 11 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 11. Type IIIA2 retrotranscript (Figure 12)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-L4-S5-a2, wherein: a1/a2 is a stem of 15 bp in length; L1 is a stem of 6 bp with a 4-nt tip; L2 is a stem of 13 bp with a 5-nt tip; L3 is a stem of 4 bp with an 8-nt tip; L4 is a stem of 20 bp with a 4-nt tip and a 2/2 bulge 6 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and L4; S5 is a single-stranded spacer between L4 and a1/a2; Conserved nucleotides are shown in FIG. 12 , and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 12. Type IIIA3 retrotranscript (Figure 13)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-L4-L5-L6-S5-a2, wherein: a1/a2 is a stem of 24 bp in length and has a 1/0 bulge 15 bp from the tip and a 1/1 bulge 19 bp from the tip; L1 is a stem of 7 bp with a 4-nt tip; L2 is a stem of 9 bp with an 8-nt tip; L3 is a stem of 8 bp with a 4-nt tip; L4 is a stem of 4 bp with a 9-nt tip and a 2/2 bulge 3 bp from the tip; L5 is a stem of 19 bp with an 18-nt tip; L6 is a stem of 5 bp stem with a 3-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and L4; S5 is a single-stranded spacer between L6 and a1/a2; Conserved nucleotides are shown in Figure 13, and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 13. Type IIIA4 retrotranscript (Figure 14)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a 5 bp long stem with a 1/2 bulge 2 bp from the tip; L1 is an 8 bp stem with a 6-nt tip; L2 is an 8 bp stem with a 5-nt tip; L3 is a 13 bp stem with a 14-nt tip and a 1/0 bulge 2 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3: S4 is a single-stranded spacer between L3 and a1/a2, and the conserved nucleotides are shown in SEQ ID NO: 19 and Figure 20, and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 14. Type IIIA5 retrotranscript (Figure 15)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-L3-L4-S3-a2, wherein: a1/a2 stem is 11 bp long and has no overhang; L1 is a 9 bp stem with a 3-nt tip; L2 is a 14 bp stem with a 5-nt tip; L3 is a 9 bp stem with a 7-nt tip; L4 is a 15 bp stem with a 7-nt tip; S1, S2 and S3 are single-stranded spacer regions between a1/a2 stem and L1, L2 and L3, and L4 and a1/a2 stem, respectively, wherein there is no spacer between L1 and L2, and there is no spacer between L2 and L3; wherein the 5th to 2nd last nt of S2 and the 3rd to 6th nt of S3 form a 4-bp stem, and the conserved nucleotides are shown in FIG15, and wherein the colored circled nucleotides are present at respective certainty levels (e.g., at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 15. IIIunk type retrotranscript (Figure 16)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem of 11 bp in length; L1 is a stem of 12 bp with a 2-nt tip; L2 is a stem of 21 bp with a 1-nt tip; L3 is a stem of 20 bp with a 4-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and a1/a2, Conserved nucleotides are shown in FIG. 16 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 16. Type IV Retrotranscript (Figure 17)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-a2, wherein: a1/a2 is a 9 bp long stem without an overhang; L1 is a 5 bp stem with a 6-nt tip; L2 is a 9 bp stem with a 4-nt tip; L3 is a 26 bp stem with a 5-nt tip, a 0/1 bulge 7 bp from the tip, and a 0/1 bulge 9 bp from the tip; S1 is a single-stranded spacer between the a1/a2 stem and L1, wherein there is no spacer between L1 and L2; S2 is a single-stranded spacer between L2 and L3; and S3 is a single-stranded spacer between L3 and a1/a2 stems; the conserved nucleotides are shown in FIG. 17 , and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotransposons are completely synthetically produced and have conserved nucleotides represented by colored letters as shown in Figure 17. Type IX retrotransposons (Figure 18)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-L1-S1-L2-S2-a2, wherein: a1/a2 is a stem of 12 bp in length, wherein a1 has a 14-nt overhang and a2 has a 2-nt overhang; L1 is a stem of 11 bp with a 3-nt tip and a 1/3 bulge 7 bp from the tip; L2 is a stem of 25 bp with a 7-nt tip; S1 is a single-stranded spacer between L1 and L2; S2 is a single-stranded spacer between L2 and a1/a2; Conserved nucleotides are shown in FIG. 18 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotransposons are completely synthetically produced and have conserved nucleotides represented by colored letters as shown in Figure 18. V- type retrotransposons (Figure 19)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a stem of 13 bp in length; L1 is a stem of 20 bp with a 4-nt tip and a 6/4 protrusion 6 bp from the tip; L2 is a stem of 14 bp with a 4-nt tip and a 1/0 protrusion 5 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2, Conserved nucleotides are shown in FIG. 19 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 19. Type VI Retrotranscript (Figure 20)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-L4-S4-a2, wherein: a1/a2 is a 4 bp long stem with a 1 bp 5'overhang; L1 is a 7 bp stem with a 4-nt tip; L2 is an 8 bp stem with a 4-nt tip; L3 is a 16 bp stem with a 6-nt tip, a 3/4 bulge 3 bp from the tip, a 2/3 bulge 5 bp from the tip, and a 3/1 bulge 8 bp from the tip; S1 is a single-stranded spacer between the a1/a2 stem and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3, wherein there is no spacer between L3 and L4; S4 is a single-stranded spacer between L4 and a1/a2 stems; and the conserved nucleotides are as shown in FIG. 20 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 20. Type XI Group 1 Retrotranscript (Figure 21)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a 16 bp long stem with a 5-nt overhang on a1 and a 3-nt overhang on a2; L1 is a 9 bp stem with a 3-nt tip; L2 is a 7 bp stem with a 13-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2, Conserved nucleotides are shown in FIG. 21 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 21. Type XI Group 2 Retrotranscript (Figure 22)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a 13 bp long stem with a 1-nt overhang on a2; L1 is a 7 bp stem with a 3-nt tip and a 2/2 protrusion 1 bp from the tip; L2 is an 8 bp stem with a 20-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2, Conserved nucleotides are shown in FIG. 22 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 22. Type XII Retrotranscript (Figure 23)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-a2, wherein: a1/a2 is a 13 bp long stem with a 1-nt overhang on a2; L1 is a 7 bp stem with a 3-nt tip and a 2/2 protrusion 1 bp from the tip; L2 is an 8 bp stem with a 19-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and a1/a2, Conserved nucleotides are shown in FIG. 23 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 23. Type XIII Retrotranscript (Figure 24)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-L3-S3-a2, wherein: a1/a2 is a 7 bp long stem without an overhang; L1 is an 8 bp stem with a 3-nt tip; L2 is a 30 bp stem with an 8-nt tip, a 1/1 bulge 2 bp from the tip, and a 1/1 bulge 27 bp from the tip; L3 is an 8 bp stem with a 5-nt tip and a 0/1 bulge 3 nt from the tip; S1 is a single-stranded spacer between the a1/a2 stem and L1; S2 is a single-stranded spacer between L1 and L2; and S3 is a single-stranded spacer between L3 and a1/a2 stems; the conserved nucleotides are shown in FIG. 24 , and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 24. Type XIV Retrotranscript (Figure 25)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a common secondary structure that can be described as follows: a1-S1-L1-L2-S2-L3-S3-a2, wherein: the a1/a2 stem is 15 bp long, has no overhang, and has a 4/2 protrusion 7 bp from the 5' end of a1; L1 is an 8 bp stem with a 5-nt tip; L2 is a 7 bp stem with a 5-nt tip; L3 is a 13 bp stem with a 2-nt tip, a 5/9 protrusion 5 bp from the tip, and a 5/5 protrusion 8 bp from the tip; S1, S2 and S3 are single-stranded spacer regions between a1/a2 stem and L1, L2 and L3, and L3 and a1/a2 stem, respectively, wherein there is no spacer between L1 and L2; wherein the 5th to 3rd last nt of S1 and the 2nd to 5th 4 nt of S2 form a 3-bp stem, and the conserved nucleotides are shown in Figure 25, and wherein the colored circled nucleotides are present at their respective certainty levels (e.g., at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides are present).

In some embodiments, the engineered retrotranscript is entirely synthetically produced and has conserved nucleotides represented by colored letters as shown in FIG. 25 . Ec107 -like retrotranscript (FIG. 26) In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a stem of 12 bp in length; L1 is a stem of 4 bp with an 8-nt tip; L2 is a stem of 8 bp with a 3-nt tip; L3 is a stem of 22 bp with a 3-nt tip, a 4/6 bulge 6 bp from the tip, a 3/3 bulge 13 bp from the tip, and a 1/1 bulge 18 bp from the tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; and S4 is a single-stranded spacer between L3 and a1/a2. The conserved nucleotides are shown in FIG. 26 , and the colored circled nucleotides are present at their respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is completely synthetically produced and has conserved nucleotides represented by colored letters as shown in Figure 26. Outgroup A retrotranscript (Figure 27)

In some embodiments, the template/wt retrotranscript used in the engineered retrotranscript of the present invention encodes a wild-type retrotranscript ncRNA polynucleotide having a consensus secondary structure that can be described as follows: a1-S1-L1-S2-L2-S3-L3-S4-a2, wherein: a1/a2 is a 5 bp long stem with a 2-nt overhang on a2; L1 is an 11 bp stem with a 4-nt tip; L2 is an 8 bp stem with a 3-nt tip; L3 is an 18 bp stem with a 3-nt tip; S1 is a single-stranded spacer between a1/a2 and L1; S2 is a single-stranded spacer between L1 and L2; S3 is a single-stranded spacer between L2 and L3; S4 is a single-stranded spacer between L3 and a1/a2, Conserved nucleotides are shown in FIG. 27 , wherein the colored circled nucleotides are present at respective certainty levels ( e.g. , at least about 97% of the red circled nucleotides, at least about 90-97% of the black circled nucleotides, at least about 75-90% of the gray circled nucleotides, and at least about 50% of the white circled nucleotides).

In some embodiments, the engineered retrotranscript is produced entirely synthetically and has conserved nucleotides represented by colored letters as shown in FIG. 27 .

For example, amplification of the engineered retrotranscripts described herein (e.g., in Figures 2-27) can be performed prior to transfection of cells or ligation into a vector. Any method for amplifying engineered retrotranscripts can be used, including but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand-swap amplification (SDA), and ligase chain reaction (LCR). In one embodiment, the engineered retrotranscript comprises a common 5' and 3' priming site to allow amplification of retrotranscript sequences in parallel with a set of universal primers. In another embodiment, a set of selective primers is used to selectively amplify a subset of retrotranscript sequences from a pooled mixture.

Variants of these templates, which may also be used in the engineered retrotransposons of the present invention, include variants having: A) up to 1, 2 or 3 ( e.g. , up to 1) nucleotide changes for every 10 red lettered nucleotides; B) up to 4, 5 or 6 ( e.g. , up to 1 or 2) nucleotide changes for every 10 black lettered nucleotides; and/or C) up to 7, 8 or 9 ( e.g. , up to 3 or 4) nucleotide changes for every 10 grey lettered nucleotides; and/or optionally further comprising: a) up to 10 red circled nucleotides a) there are 7, 8, 9 or 10 ( e.g. , 9 or 10) nucleotides per 10 black circled nucleotides; c) there are 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 6, 7, 8, 9 or 10) nucleotides per 10 grey circled nucleotides; and/or d) there are 2, 3, 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 4, 5, 6, 7, 8, 9 or 10) nucleotides per 10 white circled nucleotides.

In some embodiments, the non-coding RNA (ncRNA) portion of the engineered retrotransposons comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to an ncRNA listed in Table B. In some embodiments, the ncRNA does not comprise an ncRNA related to an RT sequence of Table X. Forms of ncRNA and Guide RNA

In some embodiments, the ncRNA and guide RNA can be delivered as a single molecule, i.e., wherein the guide RNA is fused to the 5' and/or 3' end of the ncRNA. In some embodiments, the ncRNA can have guide RNAs at both ends.

In other embodiments, the guide RNA and ncRNA can be provided and/or delivered as separate components. As shown in Example 4, the separation of the guide RNA and ncRNA can result in increased editing efficiency.

In other embodiments, the ncRNA-gRNA fusion can be co-delivered with a separate guide RNA.

In other embodiments, the ncRNA disclosed herein may be modified by introducing additional RNA motifs into the ncRNA, such as at the 5' and 3' ends of the ncRNA, or even at positions therebetween (e.g., in the msr or msd regions), to improve transcriptional production and/or stability and/or function (e.g., RT-DNA production). Such structures may include, but are not limited to, RNA hairpins, RNA stem-loops, RNA quadruplexes, cap structures, and poly(A) tails or ribozyme functions and the like. In addition, the ncRNA may also be modified to include one or more nuclear localization sequences.

Additional RNA motifs may also improve the RT processibility of ncRNAs or enhance ncRNA activity by enhancing RT binding. Addition of dimerization motifs (such as kissing loops or GNRA tetraloop/tetraloop receptor pairs) to the 5' and 3' ends of ncRNAs may also lead to efficient circularization of ncRNAs, thereby improving stability. In addition, it is expected that the addition of these motifs may enable physical separation of ncRNA components, such as separation of msr and msd regions. Short 5' or 3' extensions of ncRNAs form small foothold hairpins at either or both ends of the ncRNA, which may also advantageously compete with annealing of internal complementary regions along the length of the ncRNA. Finally, kissing loops may also be used to recruit other RNAs or proteins to genome sites and enable exchange of RT activity from one RNA to another.

ncRNAs may be further improved through directed evolution in a manner similar to how protein function can be improved. Directed evolution may enhance ncRNA recognition by RT, and/or reduce off-site targeting and/or indels, and/or improve precision editing efficiency.

The present disclosure contemplates any such means to further improve the stability and/or functionality of the ncRNAs disclosed herein.

In some embodiments, the RNA (including guide RNA and ncRNA) used in the compositions of the present disclosure has been chemically or biologically modified to make it more stable. Exemplary modifications to RNA include depletion of bases (e.g., by deletion or by substitution of one nucleotide for another) or modification of bases (e.g., chemical modification of bases). As used herein, the phrase "chemical modification" includes modifications that introduce chemical properties that are different from those found in naturally occurring RNA, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or the inclusion of side groups that are not naturally found in such mRNA molecules).

Other suitable polynucleotide modifications that can be incorporated into the RNA used in the compositions of the present disclosure include, but are not limited to, 4'-thio modified bases: 4'-thio-adenosine, 4'-thio-guanosine, 4'-thio-cytidine, 4'-thio-uridine, 4'-thio-5-methyl-cytidine, 4'-thio-pseudouridine and 4'-thio-2-thiouridine, pyridin-4-one ribonucleosides, 5- Aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, 1 -taurine methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-methylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- Methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- Methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycolylaminomethyladenosine, N6-succinimidylaminomethyladenosine, 2-methylthio-N6-succinimidylaminomethyladenosine, N6,N6-dimethyladenosine, 7-methyl Adenine, 2-methylthio-adenine and 2-methoxy-adenine, inosine, 1-methyl-inosine, yadosine, yadosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6 The term modification also includes, for example, incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequence of the present invention (e.g., modification of one or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include adding bases to the mRNA sequence (e.g., including a poly A tail or a longer poly A tail), altering the 3'UTR or 5'UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and including elements that alter the structure of the RNA molecule (e.g., which form a secondary structure).

In some embodiments, RNA (e.g., ncRNA) includes a 5' cap structure. The 5' cap is generally added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphate groups; then, guanosine triphosphate (GTP) is added to the terminal phosphate group via a guanosyltransferase, thereby generating a 5'5'5 triphosphate bond; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. The naturally occurring cap structure comprises a 7-methylguanosine linked via a triphosphate bridge to the 5' end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added to the cell nucleus and is catalyzed by the enzyme guanosyltransferase. Immediately after transcription is initiated, the cap is added to the 5' end of the RNA. The terminal nucleoside is usually guanosine and is in the opposite orientation to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

Additional cap analogs include, but are not limited to, chemical structures selected from the group consisting of: m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9: 1108-1122 (2003)).

Typically, the presence of a "tail" is used to protect RNA (e.g., ncRNA) from exonuclease degradation. Poly A or poly U tails are believed to stabilize natural messengers and synthetic sense RNAs. Therefore, in certain embodiments, long poly A or poly U tails can be added to RNA molecules, thereby making the RNA more stable. Poly A or poly U tails can be added using a variety of technically recognized techniques. For example, poly A polymerase can be used to add a long poly A tail to synthetic or in vitro transcribed RNA (Yokoe et al. Nature Biotechnology. 1996; 14: 1252-1256). Transcription vectors can also encode long poly A tails. In addition, poly A tails can be added by directly transcribing from PCR products. Poly A can also be joined to the 3' end of the sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd edition, Sambrook, Fritsch and Maniatis, eds. (Cold Spring Harbor Laboratory Press: 1991 edition)).

Typically, the length of the poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 or at least 500 nucleotides. In some embodiments, the poly A tail at the 3' end of the mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA includes a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of the mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A or poly-U tail, or may replace the poly-A or poly-U tail.

RNA (e.g., ncRNA) according to the present disclosure may be synthesized according to any of a variety of known methods. For example, RNA according to the present invention may be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary depending on the specific application. An improved ncRNA IVT method is disclosed in Example 5 herein.

In a specific embodiment (as exemplified in Example 6 herein), the ncRNA may include an MS2 modification, as a specific RNA hairpin structure recognized by a certain MS2 binding protein in nature. This domain may help stabilize the ncRNA and improve editing efficiency. The present disclosure contemplates other similar modifications. Other such MS2-like domains are described in the art, for example, in Johansson et al., "RNA recognition by the MS2 phage coat protein," Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., "Organization of intracellular reactions with rationally designed RNA assemblies," Science, 2011, Vol. 333: 470-474; Mali et al., "Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nat. Biotechnol., 2013, Vol. 31: 833- 838; and Zalatan et al., "Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds," Cell, 2015, Vol. 160: 339-350, each of which is incorporated herein by reference in its entirety. Other systems include the PP7 hairpin, which specifically recruits PCP proteins; and the "com" hairpin, which specifically recruits Com proteins. See Zalatan et al. The nucleotide sequence of the MS2 hairpin (or equivalently, the "MS2 aptamer") is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 19935). B. Heterologous Nucleotide Sequence (HNS)

The engineered retrotransposons may comprise or encode heterologous nucleic acids (e.g., DNA or RNA) within the msr locus or msd locus ( e.g. , in the tip of the S region or L region in the consensus structure in any of Figures 2-27 and variants thereof) or upstream or downstream of the msr locus or msd locus. In some embodiments, the heterologous nucleic acid is inserted into the msd locus. In some embodiments, the heterologous nucleic acid is inserted upstream of the msr locus. In some embodiments, the heterologous nucleic acid is inserted upstream or downstream of the msd locus. In other embodiments, the heterologous nucleic acid is inserted into a spacer sequence ( e.g. , S1, S2, S3, and/or S4 as depicted in Figure 2) between and/or within the hairpin loops ( e.g. , L1, L2, L3, and/or L4 as depicted in Figure 2) ( e.g. , the tip or loop region), or within the bulge. In some embodiments, the heterologous nucleic acid sequence can be inserted into the tip of the L region or hairpin ( e.g. , L1, L2, L3 and/or L4 as depicted in Figure 2). In some embodiments, one or more heterologous nucleic acids are inserted into the msd locus. In some embodiments, the first heterologous nucleic acid is inserted into the msd locus, and the second heterologous nucleic acid is inserted upstream of the msr locus or upstream or downstream of the msd locus.

In some embodiments, the heterologous nucleic acid comprises flanking contiguous nucleotides (also referred to as homology arms) that are substantially complementary to the target site genome sequence of the cell to facilitate insertion of at least a portion of the heterologous nucleic acid into the target site in the cell genome via homology directed repair (HDR). In some embodiments, the heterologous nucleic acid is between >20 nucleotides and 10,000 nucleotides ( e.g. , including the flanking homology arms).

In some embodiments, one or both homology arms on the heterologous nucleic acid are 100% identical to the target genome sequence. In some embodiments, one or both homology arms on the heterologous nucleic acid are less than 100% complementary to the target genome sequence, such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identical to the target genome sequence.

The portion of the heterologous nucleic acid to be inserted into the target genome sequence is sometimes referred to as a donor sequence. The donor sequence may be partially identical to the entire length of the target genome sequence or a portion thereof, or may be unrelated to the target genome sequence. The donor sequence can be used, for example, to introduce modifications ( e.g. , substitutions, deletions, insertions, or combinations thereof), such as mutations or other genetic changes ( e.g. , genetic elements (such as stop codons) or open reading frame shifts on the target polynucleotide) into its target sequence.

In some embodiments, the heterologous nucleic acid sequence is or encodes a biologically active molecule, such as but not limited to a therapeutic protein.

Any therapeutic protein can be encoded by a heterologous nucleic acid.

In some embodiments, the heterologous nucleic acid sequence encodes one or more prophylactically or therapeutically active proteins, polypeptides, or other factors.

As a non-limiting example, the heterologous sequence may be or encode an agent that enhances tumoricidal activity in cancer, such as, but not limited to, TRAIL or tumor necrosis factor (TNF). As another non-limiting example, the heterologous sequence may be or encode an agent suitable for treating the following diseases: such as muscle atrophy ( e.g. , the heterologous sequence is or encodes dystrophin or a functional fragment or variant thereof, such as the various dystrophin minigenes or mini-dystrophin encoding sequences known in the art), cardiovascular disease ( e.g. , the heterologous sequence is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc. ), neurodegenerative disease ( e.g. , the heterologous sequence is or encodes NGF, BDNF, GDNF, NT-3 , etc. ).

As an additional non-limiting example, the heterologous nucleic acid sequence may be or encode an agent that enhances tumoricidal activity in cancer, such as, but not limited to, TRAIL or tumor necrosis factor (TNF). As another non-limiting example, the heterologous nucleic acid sequence may be or encode an agent suitable for treating the following diseases: such as muscle atrophy ( e.g. , the heterologous nucleic acid sequence is or encodes anti-dystrophy protein), cardiovascular disease ( e.g. , the heterologous nucleic acid sequence is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd , etc. ), neurodegenerative disease ( e.g. , the heterologous nucleic acid sequence is or encodes NGF, BDNF, GDNF, NT-3 , etc. ), chronic pain ( e.g. , the heterologous nucleic acid sequence is or encodes GlyRal), enkephalin or glutathione. amino acid decarboxylase ( e.g. , the heterologous nucleic acid sequence is or encodes GAD65, GAD67 or another isoform), lung disease ( e.g. , the heterologous nucleic acid sequence is or encodes CFTR), hemophilia ( e.g. , the heterologous nucleic acid sequence is or encodes factor VIII or factor IX), tumor formation ( e.g. , the heterologous nucleic acid sequence is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notch1, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HIF Fla, HIF3a, Met, HRG, Bcl2, PPARα, PPARγ, WT1 (Wilm's tumor), FGF receptor family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (androgen receptor), TSG101, IGF, IGF receptor, Igf1 (4 variants), Igf2 (3 variants), Igf1 receptor, Igf2 receptor, Bax, Bcl2, caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape), age-related macular degeneration ( e.g. , heterologous nucleic acid sequences are or encode Aber, Ccl2, Cc2, cp (plasma cuprocyanin), Timp3, cathepsin D, Vldlr), schizophrenia ( e.g. , neuromodulin (Nrgl), Erb4 (neuromodulin receptor), Complexin-1 (Cplxl), Tphl tryptophan hydroxylase, Tph2 tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Daol)), trinucleotide repeat disorders ( e.g. , HTT (Huntington's Dx), SBMA/SMAXI/AR (Kennedy's Dx), FXN/X25 (Friedrich's movement disorder), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar disorders), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP global instability), VLDLR (Alzheimer's), Atxn7, Atxn10), fragile X syndrome ( e.g. , the heterologous nucleic acid sequence is or encodes FMR2, FXRI, FXR2, mGLUR5), secretase-related disorders ( e.g. , the heterologous nucleic acid sequence is or encodes APH-1 (α and β), presenilin (Psen1), nicastrin (Ncstn), PEN-2), ALS ( e.g. , the heterologous nucleic acid sequence is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism ( e.g. , the heterologous nucleic acid sequence is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1), Alzheimer's disease ( e.g. , heterologous nucleic acid sequences are or encode El, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PS1, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl, aquaporin 1), Uchll, Uchl3, APP), inflammation ( e.g. , heterologous nucleic acid sequences are or encode IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll), Parkinson's disease ( e.g. , x-synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders ( e.g. , anemia, bare lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis, hemophilia A, hemophilia B, bleeding disorders, leukocytopenias and disorders, sickle cell anemia, and thalassemia) ( For example , the heterologous nucleic acid sequence is or encodes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A. FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, 3-4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, E IF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1), B cell non-Hodgkin lymphoma or leukemia ( e.g. , the heterologous nucleic acid sequence is or encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2 , BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2 X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCR A, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN), inflammation and immune-related diseases and disorders ( e.g. , heterologous nucleic acid sequences are or encode KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation ( e.g. , heterologous nucleic acid sequences are or encode IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4), metabolic, liver, kidney and protein diseases and disorders ( e.g. , heterologous nucleic acid sequences are or encode TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAM P2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCO1, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), musculoskeletal diseases and disorders ( e.g. , heterologous nucleic acid sequences are or encode DMD, BMD, MYF6, LMNA, L MN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DM DA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, C MD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1), neural and neuronal diseases and disorders ( e.g. , heterologous nucleic acid sequences are or encode SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A , Neurexin 1. GLOl, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA 17. SNCA, NACP, PARK1, PA RK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-synaptotagmin, DJ-1, neuromodulin-1 (Nrgl), Erb4 (neuromodulatory protein receptor), Complexin-1 (Cplxl), Tphl tryptophan hydroxylase, Tph2, tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A, DAOA, DTNBP1, Dao (Daol), APH-1 (α and β), Presenilin (Psenl), Nicastrin, (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA/SMAX1/AR, FXN/X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atnl, CBP, VLDLR, Atxn7 and AtxnlO) and ocular diseases and disorders ( e.g. , Aber, Ccl2, Cc2, cp (plasma copper cyanin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, C RYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLCIA, JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12 , LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD and VMD2).

In some embodiments, the heterologous nucleic acid sequence is or encodes a factor that can affect cell differentiation. As a non-limiting example, the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant negative p53, Nanog, Glis1, Lin28, TFIID, mir-302/367 or other miRNAs can make cells become induced enriched potential stem (iPS) cells.

In some embodiments, the heterologous nucleic acid sequence is or encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatocytes.

In certain embodiments, the heterologous nucleic acid encodes a therapeutic antibody or an antigen-binding fragment thereof.

In certain embodiments, the heterologous nucleic acid encodes a protein for replacement therapy. The protein may be defective in a diseased cell or diseased organism/individual, and when delivered to the diseased cell/tissue/organism/individual by the heterologous nucleic acid, the wild-type protein or a functional fragment or variant thereof at least partially or completely restores the function of the protein lost in the diseased cell/tissue/organism/individual. HNS = donor DNA template

In some embodiments, the heterologous nucleic acid sequence is a donor DNA template that can be integrated into the host genome via HDR.

In some embodiments, the heterologous nucleic acid sequence is a donor DNA that can serve as a template or primer for recombination engineering during replication.

In certain embodiments, the heterologous nucleic acid comprises or encodes a donor/template sequence, wherein the donor/template corrects/repairs/removes a mutation at a target genome site. For example, the mutation may be a mutated exon in a disease gene.

In certain embodiments, the donor/template may encode or contain functional DNA elements, such as promoters, enhancers, protein binding sequences, methylation sites, or homology regions for assisting gene editing.

"Donor DNA" or "donor DNA template" means a single strand of DNA to be inserted at a site to be cleaved by a gene editing nuclease (e.g., CRISPR/Cas effector protein; TALEN; ZFN) (e.g., after dsDNA cleavage, after nicking of the target DNA, after double nicking of the target DNA, and the like). The donor DNA template can contain sufficient homology to the genomic sequence at the target site, e.g., 70%, 80%, 85%, 90%, 95% or 100% homology to the nucleotide sequence flanking the target site, e.g., within about 50 bases or less of the target site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or directly flanking the target site, to support homology-directed repair between the donor DNA template and the genomic sequence to which it carries homology.

Sequence homology of about 25, 50, 100 or 200 nucleotides or more (or any integer value between 10 and 200 nucleotides or more) between the donor DNA template and the genome sequence can support homology-directed repair. The donor DNA template can have any length, such as 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc. Suitable donor DNA templates can be 50 nucleotides to 100 nucleotides, 100 nucleotides to 500 nucleotides, 500 nucleotides to 1000 nucleotides, 1000 nucleotides to 5000 nucleotides, or 5000 nucleotides to 10,000 nucleotides or more than 10,000 nucleotides in length.

As described above, the donor DNA template comprises a first homology arm and a second homology arm. The first homology arm is located at or near the 5' end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to the first nucleotide sequence in the target nucleic acid. The second homology arm is located at or near the 3' end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to the second nucleotide sequence in the target nucleic acid. The first and second homology arms can each independently have a length of about 10 nucleotides to 400 nucleotides; for example, 10 nucleotides (nt) to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, 45 nt to 50 nt, 50 nt to 75 nt, 75 nt to 100 nt, 100 nt to 125 nt, 125 nt to 150 nt, 150 nt to 175 nt, 175 nt to 200 nt, 200 nt to 225 nt, 225 nt to 250 nt, 250 nt to 275 nt, 275 nt to 300 nt, 325 nt to 350 nt, 350 nt to nt to 375 nt or 375 nt to 400 nt.

In certain embodiments, the donor DNA template is used to edit the target nucleotide sequence. In certain embodiments, the donor DNA template comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the mutation causes an open reading frame shift on the target polynucleotide. In certain embodiments, the donor polynucleotide changes the stop codon in the target polynucleotide. In certain embodiments, the donor polynucleotide corrects a premature stop codon. Correction can be achieved by deleting the stop codon or by introducing one or more sequence changes to change the stop codon to a codon. In certain embodiments, the donor polynucleotide addresses loss of function mutations, deletions or translocations that may occur, for example, in certain disease settings by inserting or restoring a functional copy of a gene or a functional fragment thereof, or a functional regulatory sequence or a functional fragment of a regulatory sequence. Functional fragments include fragments of nucleotide sequence that are less than a complete copy of a gene but that otherwise provide sufficient nucleotide sequence to restore the functionality of a wild-type gene or non-coding regulatory sequence ( e.g. , a sequence encoding a long non-coding RNA).

In some embodiments, the donor DNA template can be used to replace a single allele of a defective gene or a defective fragment thereof. In another embodiment, the donor DNA template is used to replace both alleles of a defective gene or a defective gene fragment. A "defective gene" or "defective gene fragment" is a gene or a portion of a gene that, when expressed, fails to generate a functional protein or non-coding RNA having the functionality of the corresponding wild-type gene.

In certain exemplary embodiments, these defective genes may be associated with one or more disease phenotypes. In certain exemplary embodiments, the defective gene or gene fragment is not replaced, but a donor polynucleotide encoding a gene or gene fragment that compensates or exceeds the expression of the defective gene is inserted using a heterologous nucleic acid, so that the cell phenotype associated with the expression of the defective gene is eliminated or changed to a different or desired cell phenotype. This can be achieved by including a coding sequence for a therapeutic protein (such as a therapeutic antibody or a functional fragment thereof, or a wild-type form of a defective protein associated with one or more disease phenotypes).

In certain embodiments, the donor may include, but is not limited to, a gene or gene fragment, an RNA transcript encoding a protein or to be expressed, a regulatory element, a repair template, and the like. According to the present invention, the donor polynucleotide may include left and right sequence elements that function with the transposition component mediating insertion.

In some embodiments, the donor DNA template manipulates the splice site on the target polynucleotide. In some embodiments, the donor DNA template disrupts the splice site. Disruption can be achieved by inserting a polynucleotide into the splice site and/or introducing one or more mutations into the splice site. In some embodiments, the donor polynucleotide can restore the splice site. For example, the polynucleotide can include a splice site sequence.

In certain embodiments, the donor DNA template to be inserted has a size of 10 bp to 50 kb in length, such as 50 bp to about 40 kb, 100 bp to about 30 kb, 100 bp to about 10 kb, 100 bp to 300 bp, 200 bp to 400 bp, 300 bp to 500 bp, 400 bp to 600 bp, 500 bp to 700 bp, 600 bp to 800 bp, 700 bp to 900 bp, 800 bp to 1000 bp, 900 bp to 1100 bp, 1000 bp to 1200 bp, 1100 bp to 1300 bp, 1200 bp to 1400 bp, 1300 bp to 1500 bp, 1400 bp to 1600 bp, or bp, 1500 bp to 1700 bp, 1600 bp to 1800 bp, 1700 bp to 1900 bp, 1800 bp to 2000 bp nucleotide length.

In certain embodiments, the homology arms at one or both ends of the sequence to be inserted are independently about 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp or 150 bp.

The first homology arm and the second homology arm of the donor DNA are flanked by nucleotide sequences to be introduced into the target nucleic acid ("related nucleotide sequences" or "intervening nucleotide sequences"). The related nucleotide sequences may include: i) nucleotide sequences encoding related polypeptides; ii) nucleotide sequences encoding gene exons; iii) promoter sequences; iv) enhancer sequences; v) nucleotide sequences encoding non-coding RNA; or vi) any combination of the foregoing.

Donor DNA can provide gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc. For example, donor DNA can be used to add (e.g., insert or replace) nucleic acid material to target DNA (e.g., to "knock in" nucleic acids encoding proteins, siRNAs, miRNAs, etc.), add tags (e.g., 6xHis, fluorescent proteins (e.g., green fluorescent proteins; yellow fluorescent proteins, etc.), hemagglutinin (HA), FLAG, etc.), add regulatory sequences to genes (e.g., promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, enhancers, etc.), modify nucleic acid sequences (e.g., introduce mutations), and the like. For example, the donor DNA can be used to modify DNA in a site-specific (i.e., "targeted") manner; for example, gene knockout, gene knock-in, gene editing, gene tagging, etc., such as for use in, for example, gene therapy, e.g., to treat disease; or as an antiviral, antipathogenic, or anticancer therapeutic, to generate genetically modified organisms in agriculture, to mass produce proteins by cells for therapeutic, diagnostic, or research purposes, to induce high-potential stem cells, for biological research, to target pathogenic genes for deletion or replacement, etc.

In some cases, the donor DNA comprises a nucleotide sequence encoding a polypeptide of interest. The polypeptide of interest includes, for example, a) a functional form of a polypeptide comprising one or more amino acid substitutions, insertions and/or deletions and exhibiting reduced function, for example, where the reduced function is associated with or causes a pathological disorder; b) a fluorescent polypeptide; c) a hormone; d) a receptor for a ligand; e) an ion channel; f) a neurotransmitter; g) and the like.

In some cases, the donor DNA comprises a nucleotide sequence encoding a wild-type protein lacking in the recipient cell. In some cases, the donor DNA encodes a wild-type factor involved in coagulation (e.g., factor VII, factor VIII, factor IX and the like). In some cases, the donor DNA comprises a nucleotide sequence encoding a therapeutic antibody. In some cases, the donor DNA comprises a nucleotide sequence encoding an engineered protein or receptor. In some cases, the engineered receptor is a T cell receptor (TCR), a natural killer (NK) receptor (NKR), or a B cell receptor (BCR). In some cases, the engineered TCR or NKR targets a cancer marker (e.g., a polypeptide expressed (e.g., overexpressed) on the surface of a cancer cell). In some cases, the donor DNA comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some cases, the CAR targets a cancer marker. Donor DNA encoding the CAR, TCR and/or NCR proteins can be folded into a DNA origami structure (DNA nanostructure) and delivered to T cells or NK cells in vitro or in vivo.

Non-limiting examples of polypeptides that may be encoded by the donor DNA include, for example, IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostaglandin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP binding cassette, subfamily G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostaglandin) receptor (IP)), KCNJ11 (potassium inward rectifier channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB (platelet-derived growth factor receptor, beta polypeptide), CCNA2 (cytokine A2), PDGFB (platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassium inward rectifier channel, subfamily J, member 5), KCNN3 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3), CAPN10 (calcification 10), PTGES (prostaglandin E synthetase), ADRA2B (adrenaline-stimulated alpha-2B-receptor), ABCG5 (ATP-binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calcification 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (Cryptidys elegans)), ACE angiotensin I convertase (peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon beta 2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (connexin, fibronectin activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, containing C1Q and collagen domains), APOB (apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E), LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriuretic peptide pro-actin B), NOS3 (nitric oxide synthase 3 (endothelial cells)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (fibroblast lysinogen activator, tissue), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (cholesterol ester transfer protein, plasma), AGTR1 (angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (insulin-like growth factor 1 (interleukin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (peroxisomal phosphatase 1), KNG1 (kininogen 1), CCL2 (chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), vWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, β1), NPPA (natriuretic peptide propromoter A), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA (lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (heme phenotype), F3 (coagulation factor III (thrombin, tissue factor)), CST3 (cystatin C), COG2 (oligomeric Golgi complex component 2), MMP9 (matrix metalloproteinase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa Type IV collagenase)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (ring opening) 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8), PROK1 (prokinesin 1), CBS (cystathionine-β-synthase), NOS2 (nitric oxide synthase 2, inducing), TLR4 (toll-like receptor 4), SELP (selectin P (granule membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP binding cassette, subfamily A (ABC1), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low-density lipoprotein receptor), GPT (glutamine-pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interferon-γ-inducing factor)), NOS1 (nitric oxide synthase 1 (neuron)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrillogen beta chain), HGF (hepatocyte growth factor (hepatocyte poi-toiin A; proliferating factor)), ILIA (interleukin 1, alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogene homolog 1), LIPC (lipase, liver), HSPD1 (heat shock protein 60 kDa 1 (chaperone)), MAPK14 (mitogen activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, β3 (thrombopoietin 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (coagulation factor X), CP (plasma cucocyanin (ferrooxidase)), TNFRSF11B (tumor necrosis factor receptor subfamily, member lib), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (erythroblastic leukemia virus (v-erb-b) oncogene homolog, avian)), MMP2 (matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)), PLG (fibrolysinogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytoma viral oncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymotrypsin 1, mast cell), PLAU (fibronectin activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), beta polypeptide 3), ADRB2 (adrenaline-stimulated beta-2-receptor, surface), APOA5 (apolipoprotein A-V), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (prothrombin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonic acid 5-lipoxygenase), HLA-DRB1 (major histocompatibility complex, class II, DRβ1), PARP1 (poly (ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (peroxisomal 2), AGER (advanced glycation end product specific receptor), IRS1 (insulin receptor receptor 1), PTGS1 (prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin convertase 1), F7 (coagulation factor VII (plasma prothrombin conversion accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor subfamily, member 6)), ABCB1 (ATP binding cassette, subfamily B (MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor subfamily member 5), LCAT (lecithin-cholesterol transferase), CCR 5 (chemokine (C-C motif) receptor 5), MMP1 (matrix metalloproteinase 1 (interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM (adrenomedulin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription 3 (acute phase response factor)), MMP3 (matrix metalloproteinase 3 (matrilysin 1, procollagen)), ELN (elastin), USF1 (upstream transcription factor 1), CFH (complement factor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrix metalloproteinase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenaline stimulating beta-1-receptor), CYBA (cytochrome b-245, alpha polypeptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamicinyltransferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia-inducing factor 1, alpha subunit (basal helix-loop-helix transcription factor)), CXCR4 (interleukin (C-X-C motif) receptor 4), PROC (protein C (inactivator of coagulation factors Va and Villa)), SCARB1 (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-related alpha), PLTP (phospholipid transfer protein), ADD1 (adductin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid Al), KCNH2 (potassium voltage-gated channel, subfamily H (eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A) (atrial natriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viral oncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isomerase G (cyclophilin G)), IL1R1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinol binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (Cell cycle protein-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basal)), EDNRB (endothelin receptor type B), ITGA2 (integrin, α2 (CD49B, α2 subunit of VLA-2 receptor)), CAB INI (calcineurin phosphatase binding protein 1), SHBG (sex hormone binding globulin), HMGB1 (high mobility group box 1), HSP90B2P (heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subgroup A, polypeptide 4), GJA1 (gap junction protein, α1, 43 kDa), CAV1 (cavolin 1, caveolin, 22 kDa), ESR2 (estrogen receptor 2 (ERβ)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (neural growth factor (β polypeptide)), SP1 (Sp 1 transcription factor), TGIF1 (TGFB inducing factor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), EGF (epidermal growth factor (β-urogastatin)), PIK3CG (phosphoinositide-3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like family, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (protofibrillin 1), CHKA (choleline kinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursor protein), CTNNB1 (trencin (calcified mucin-related protein), β1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, β1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member Al), CX3CR1 (interleukin (C-X3-C motif) receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IE17A (interleukin 17A), PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferase μ1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor XIII, Al polypeptide), TTR (transthyretin), FABP4 (fatty acid binding protein 4, adipocyte), PON3 (peroxisomal phosphatase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor subfamily, member IB), HTR2A (5-hydroxytryptamine (serotonin) receptor 2A), CSF3 (colony stimulating factor 3 (granulocyte)), CYP2C9 (cytochrome P450, family 2, subgroup C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, subgroup B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulating factor 2 (granulocyte-macrophage)), KDR (kinase insert domain receptor (type III body tyrosine kinase)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondria)), TCF7L2 (transcription factor 7-like 2 (T cell-specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor (erythroid 2)-like 2), NOTCH1 (Notch homolog 1, translocation-related (Drosophila)), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide Al), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin (silent mate information regulator 2 homolog) 1 (brew yeast)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing hormone-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, pappa lysine 1), ARR3 (arrestin 3, retinal (X-(arrestin)), NPPC (natriuretic peptide promotor C), AHSP (alpha heme stabilizer), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanistic target of rapamycin (serine/thiocyanate kinase)), ITGB2 (integrin, β2 (complement component 3 receptor 3 and 4 subunits)), GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signal transducer (gpl30, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solutolytic carrier family 8 (sodium/calcium exchanger), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldodeductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member Al), BGLAP (bone gamma-carboxyglutamine (gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferred, member 3), RAGE (kidney tumor antigen), C4B (complement component 4B (Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), RNLS (nephrin, FAD-dependent amine oxidase), CREB1 (cAMP responsive element binding protein 1), POMC (promorphin melanocortin), RAC1 (ras-related C3 botulinum toxin substrate 1 (rho family, small TP binding protein Racl)), LMNA (nuclear laminin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V alpha subunit), CYP1B1 (cytochrome P450, family 1, subgroup B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation inhibitor)), MMP13 (matrix metalloproteinase 13 (collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subgroup A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subgroup A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 2 (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin deficiency)), SELPLG (selectin P ligand), AOC3 (copper-containing amine oxidase 3 (vascular adhesion protein 1)), CTSL1 (histatinase LI), PCNA (proliferating cell nuclear antigen), IGF2 (insulin-like growth factor 2 (interleukin A)), ITGB1 (integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 including MDF2, MSK12)), CAST (calcified protein), CXCL12 (chemointerleukin (C-X-C motif) ligand 12 (stromal cell-derived factor 1)), IGHE (immunoglobulin heavy chain homeostasis epsilon), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen, type I, α1), COL1A2 (collagen, type I, α2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor type 1), SLC2A1 (solute carrier family 2 (glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (Cellulin (C-C motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALC A (calcitonin-related polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathione S-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subgroup A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (interleukin (C-C motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenaline-stimulated beta receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metalloproteinase 8 (neutrophil globulinogenase)), NPR2 (natriuretic peptide receptor B/guanylate cyclase B (atrial natriuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamicin-prolyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PEC AMI (platelet/endothelial cell adhesion molecule), CCL4 (chemointerferon (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (α-1 antiprotease, antitrypsin), member 3), CASR (calcium-sensitive receptor), GJA5 (gap junction protein, α 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcriptional termination factor, RNA polymerase II), PROS1 (protein S (α)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, β (43 kDa dystrophin-related glycoprotein)), YME1L1 (YME1-like 1 (brew yeast)), CAMP (antibacterial peptide antimicrobial peptide), ZC3H12A (zinc finger CCCH type 12A), AKR1B1 (aldose reductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrix metalloproteinase 7 (matrilysin, uterine)), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (conjunctive tissue growth factor), KCNMA1 (potassium large conductance calcium-activated channel, subfamily M, alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol-b-methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calcimodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL11 (chemointerleukin (C-C motif) ligand 11), PGF (placental growth factor), THPO (thrombopoietin), GP6 (glycoprotein VI (platelets)), TACR1 (tachykinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, Shal-related subfamily, member 1), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelets)), CYP2J2 (cytochrome P450, family 2, subgroup J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ALOX12 (arachidonic acid 12-lipoxygenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteine methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocation factor), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonic acid 5-lipoxygenase activating protein), NUMA1 (nuclear mitotron protein 1), CYP27B1 (cytochrome P450, family 27, subgroup B, polypeptide 1), CYSLTR2 (cysteamine leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obesitystatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (kelch repeat and BTB (POZ) domain 10), TNC (tenascin C), TYMS (thymidylate synthase), SHC1 (SHC (Src homology 2 domain) conversion protein 1), LRP1 (low-density lipoprotein receptor-related protein 1), SOCS3 (suppressor of interleukin signaling 3), ADH1B (alcohol dehydrogenase IB (class I), beta polypeptide), KLK3 (pancreatic vasodilator-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor, evolutionary clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, α M (complement component 3 receptor 3 subunit)), PITX2 (paired homology domain-like 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (IgG Fc fragment, low affinity 111a receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (glutamine-oxalyltransferase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histaminic receptor HI), NR112 (nuclear receptor subfamily 1, group I, member 2), CRH (epinephrine-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-dependent anion channel 1), HPSE (acetylheparinase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP-binding cassette, subfamily B (MDR/TAP)), RNF123 (RING finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5, group A, member 1), GJB2 (gap junction protein, β2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin/kexin type 9), FCGR2A (IgG Fc fragment, low affinity Ila receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, evolutionary clade F (alpha-2 antifibrotic enzyme, pigment epithelium-derived factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest-specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acid lysosome), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)), TFAP2A (transcription factor AP-2 α (activating enhancer binding protein 2 α)), C4BPA (complement component 4 binding protein, α), SERPINF2 (serpin peptidase inhibitor, evolutionary clade F (α-2 antifibrinolytic enzyme, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)), CXCR2 (chemoattractant (C-X-C motif) receptor 2), SLC39A3 (solute carrier family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, subfamily G (WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), Fll (coagulation factor XI), ATP7A (ATPase, Cu++ transporter, alpha polypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops blood type)), GFAP (fibroblast acidic protein), ROCK1 (Rho-associated coiled coil containing protein kinase 1), MECP2 (methyl CpG binding protein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCF1E (butyryl cholinesterase), LIPE (lipase, hormone sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (C-X-C motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CF1GA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloliquefacial polypeptide), RFIO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTF1LF1 (parathyroid hormone-like hormone), NRG1 (neuromodulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamicin (aminopeptidase A)), CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), NAGLU (N-acetylglucosidase, α), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (interleukin (C-X3-C motif) ligand 1), BDKRB1 (bradykinin receptor Bl), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil expressed), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISF1 (interleukin-induced SF12-containing protein), GAST (gastrin), MYOC (myosin, trabecular meshwork-induced glucocorticoid response), ATP1A2 (ATPase, Na+/K+ transporter, α2 polypeptide), NF1 (neurofibroma protein 1), GJB1 (gap junction protein, β1, 32 kDa), MEF2A (myocyte enhancing factor 2A), VCL (focal adhesion protein), BMPR2 (bone morphogenetic protein receptor, type II (serine/thiocyanine kinase)), TUBB (tubulin, beta), CDC42 (mitotic cycle 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), F1SF1 (heat shock transcription factor 1), MYB (v-myb myeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase, AMP-activated, alpha 2 catalytic subunit), ROCK2 (Rho-associated coiled-coil containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (calcificin (calcificin-related protein), delta 1), CTF1 (cystathionase (cystathionine gamma-lyase)), CTSS (histatenase S), VAV2 (vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1), PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOF1 (apolipoprotein FI (beta-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonic acid 15-lipoxygenase), FBLN1 (fibroin 1), NR1F13 (nuclear receptor subfamily 1, FI group, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide), CF1GB (chromogranin B (secretogranin 1)), PRKCB (protein kinase C, β), SRD5A1 (steroid-5-α-reductase, α polypeptide 1 (3-hydroxy-5α-steroid delta 4-dehydrogenase α1)), F1SD11B2 (hydroxysteroid (11-β) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4), PIK3C2A (phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DRβ5), BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium-binding protein A 12), PADI4 (peptidylarginine deaminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemolysin (C-X-C motif) receptor 1), H19 (H19, imprinted maternal transcript (non-protein coding)), KRTAP19-3 (keratin-associated protein 19-3), insulin, RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP-binding protein Rac2)), RYR1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homolog (mouse)), NGFR (neural growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (choleline stimulatory receptor, niacin, alpha 4), CACNA1C (calcium channel, voltage-dependent, L-type, alpha 1C subunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalytic subunit), CHAT (choleline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11 A (tumor necrosis factor receptor subfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (life span protein)), CYSLTR1 (cysteine leukotriene receptor 1), MAT1A (methionine adenosine transferase I, alpha), OPRL1 (opium receptor-like 1), IMPA1 (inositol (muscle)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoic acid amide dehydrogenase), PSMA6 (proteasome (precursor, megalin factor) subunit, alpha type, 6), PSMB8 (proteasome (precursor, megalin factor) subunit, beta type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (chondroitin-39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member Bl), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette, subfamily C (CFTR/MRP), member 6), RGS2 (regulator of G protein signaling 2, 24 kDa), EFNB2 (adrenaline-B2), cystic fibrosis transmembrane conductance regulator (CFTR), GJB6 (gap junction protein, β6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferlin, limb-girdle muscular atrophy 2B (somatic recessive)), FDFT1 (farnesyl-diphosphate farnesyltransferase 1), EDN2 (endothelin 2), CCR6 (interleukin (C-C motif) receptor 6), GJB3 (gap junction protein, β3, 31 kDa), IL1RL1 (interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (calcified mucin, EGF LAG seven-channel G-type receptor 2 (red rooster homolog, fruit fly)), F11R (Fll receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronosidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (activating transcription factor 6), KΉK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine Nl-acetyltransferase 1), GGFI (γ-glutamicin hydrolase (binding enzyme, pyrrolyl poly-γ-glutamicin hydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute carrier family 4, sodium bicarbonate co-transporter, member 4), PDE2A (phosphodiesterase 2 A, cGMP stimulated), PDE3B (phosphodiesterase 3B, cGMP inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and senescent cell antigen-like domain 1), RFIOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), F-side 1 (forkhead box 01), PNPLA2 (patatin-like phospholipase domain-containing 2), TRH (thyroid-stimulating hormone-releasing hormone), GJC1 (gap junction protein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17 (anion/sugar transporter), member 5), FTO (fat mass and obesity-related), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1 (proline/serine-rich coiled coil 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tricholoma homolog 1 (Drosophila)), PBX4 (pre-B cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa selenoprotein), CILP2 (chondrocyte intermediate layer protein 2), TERC (telomerase RNA component), GGT2 (gamma-glutamyl transferase 2), MT-CO1 (mitochondrial encoded cytochrome c oxidase I), UOX (urate oxidase, pseudogene), CRISPR/Cas effector polypeptide, enzymatically active CRISPR/Cas effector polypeptide (e.g., capable of cleaving a target nucleic acid), and enzymatically inactive CRISPR/Cas effector polypeptide (e.g., does not cleave a target nucleic acid, but retains binding to a target nucleic acid). In some cases, the donor DNA encodes a wild-type form of any of the foregoing polypeptides; that is, the donor DNA may encode a "normal" form that does not include mutations that result in reduced function, lack of function, or pathogenicity.

In some cases, the donor DNA comprises a nucleotide sequence encoding a fluorescent polypeptide. Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEGFP), destabilized EY ...EFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2 (12), mRFPl, pocilloporin, sea GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliprotein and phycobiliprotein conjugates (including B-phycoerythrin, R-phycoerythrin and isophycocyanin). Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909) and the like. Any of a variety of fluorescent and colored proteins from coral polyp species may be encoded, as described, e.g., in Matz et al. (1999) Nature Biotechnol. 17:969-973.

In some cases, the donor DNA encodes RNA, such as siRNA, microRNA, short hairpin RNA (shRNA), antisense RNA, riboswitch, ribozyme, aptamer, ribosomal RNA, transfer RNA, and the like.

In addition to nucleotide sequences encoding one or more gene products (e.g., RNA and/or polypeptides), the donor DNA may also include one or more transcriptional control elements, such as promoters, enhancers, and the like. In some cases, the transcriptional control element is an inducible type. In some cases, the promoter is reversible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in eukaryotic cells. In some cases, the promoter is a cell type-specific promoter. In some cases, the promoter is a tissue-specific promoter.

The nucleotide sequence of the donor DNA is generally inconsistent with the target nucleic acid (e.g., genome sequence) that it replaces. More specifically, the donor DNA may contain at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the target nucleic acid (e.g., genome sequence), as long as there is sufficient homology to support homology-directed repair (e.g., for gene correction, such as to convert pathogenic base pairs or non-pathogenic base pairs). In some cases, the donor DNA comprises a non-homologous sequence flanked by two homologous regions, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence into the target region. The donor DNA may also comprise a vector backbone containing a sequence that is not homologous to the relevant DNA region (target nucleic acid) and is not intended to be inserted into the relevant DNA region (target nucleic acid). Generally, the homologous region of the donor sequence will have at least 50% sequence identity with the target nucleic acid (e.g., genomic sequence) to be recombined. In some cases, there is 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% sequence identity. Any value between 1% and 100% sequence identity may exist, depending on the length of the donor polynucleotide.

As compared to the target nucleic acid (e.g., genomic sequence), the donor DNA may include certain nucleotide sequence differences, where such differences include, for example, restriction sites, nucleotide polymorphisms, selection markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), etc., which can be used to assess the successful insertion of the donor DNA at the cleavage site, or in some cases can be used for other purposes (e.g., to indicate expression at the targeted genomic locus). In some cases, if located in the coding region, such nucleotide sequence differences will not cause changes in the amino acid sequence, or will produce silent amino acid changes (i.e., changes that do not affect protein structure or function). Alternatively, such sequence differences may include flanking recombination sequences, such as FLP, loxP sequences, or the like, which can be activated at a later time to remove the marker sequence. In some cases, the donor DNA will include one or more nucleotide sequences to aid in localization of the donor to the nucleus of the recipient cell or to aid in integration of the donor DNA into the target nucleic acid. For example, in some cases, the donor DNA may include one or more nucleotide sequences encoding one or more nuclear localization signals (e.g., PKKKRKV (SEQ ID NO: 19399), VSRKRPRP (SEQ ID NO: 19400), QRKRKQ (SEQ ID NO: 19401), and the like (Frietas et al. (2009) Cun-Genomics 10:550-7). In some cases, the donor DNA will include nucleotide sequences to recruit DNA repair enzymes to increase insertion efficiency. Fiuman enzymes involved in homology-directed repair include MRN-CtIP, BLM-DNA2, Exol, ERCC1, Rad51, Rad52, Ligase 1, RoIQ, PARP1, Ligase 3, BRCA2, RecQ/BLM-ToroIIIa, RTEL, RoId, and RoIh (Verma and Greenburg (2016) Genes Dev. 30 (10): 1138-1154). In some cases, donor DNA is delivered as reconstituted chromatin (Cruz-Becerra and Kadonaga (2020) eLife 2020;9:e55780 DOI: 10.7554/eLife.55780).

In some cases, the ends of the donor DNA are protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those skilled in the art. For example, one or more bi-deoxynucleotide residues can be added to the 3' end of the linear molecule and/or a self-complementary oligonucleotide can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amine groups, and using modified internucleotide linkages, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the ends of the linear donor DNA, additional lengths of sequence can be included outside the homology region that can be degraded without affecting recombination. HNS = functional RNA element

In certain embodiments, the donor/template comprises a coding sequence for a functional RNA element (ncRNA, siRNA, shRNA, sgRNA, etc.).

In some embodiments, the heterologous nucleic acid sequence encodes a functional non-translated RNA. In some embodiments, the functional non-translated RNA is an RNA aptamer or a ribozyme.

In some embodiments, the heterologous nucleic acid of the engineered retrotransposons further comprises a unique barcode to facilitate multiplexing. The barcode may comprise one or more nucleotide sequences for identifying the nucleic acid or cell associated with the barcode. Such barcodes may be inserted, for example, into the tip/loop region of the msd- encoded DNA. The barcodes can be 3-1000 or more nucleotides long, 10-250 nucleotides long, or 10-30 nucleotides long, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides long.

In some embodiments, barcodes are also used to identify the location of the cell, colony, or sample from which the retrotransposons originated ( i.e. , positional barcodes), such as colony location in a cell array, well location in a multiwell plate, tube location in a rack, or sample location in a laboratory. Specifically, barcodes can be used to identify the location of genetically modified cells containing retrotransposons. The use of barcodes allows retrotransposons from different cells to be pooled in a single reaction mixture for sequencing while still being able to trace a specific retrotransposon back to its colony of origin.

In addition, linker sequences can be added to engineered retrotranscripts to facilitate high-throughput amplification or sequencing. For example, a pair of linker sequences can be added to the 5' and 3' ends of the retrotranscript construct to allow multiple engineered retrotranscripts to be amplified or sequenced simultaneously by the same set of primers. HNS = guide RNA

In some embodiments, the functional non-translated RNA is a CRISPR/Cas guide RNA (gRNA) specific for a target sequence in a mammalian cell. FIG. 1G depicts various configurations of a recombinant retrotranscript ncRNA modified by inserting a guide RNA at the 5' end or 3' of the ncRNA. The guide RNA can also be provided in trans as a separate construct. In addition, the guide RNA can be placed at both ends of the recombinant retrotranscript ncRNA.

The skilled artisan will appreciate that the selection of the appropriate guide RNA is informed by which RNA-guide nuclease is utilized.

The guide RNA provides target specificity to the complex (RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to the target nucleic acid sequence. As used herein, the term "guide RNA" refers to an RNA that includes: i) an "activator" nucleotide sequence that binds to a CRISPR/Cas effector polypeptide (e.g., a type 2 CRISPR/Cas effector polypeptide, such as a type II, type V, or type VI CRISPR/Cas endonuclease) and activates the CRISPR/Cas effector polypeptide; and ii) a "targeter" nucleotide sequence that includes a nucleotide sequence that hybridizes with the target nucleic acid. The "activator" nucleotide sequence and the "targeter" nucleotide sequence can be on separate RNA molecules (e.g., "dual guide RNAs"); or can be on the same RNA molecule ("single guide RNA"). In some cases, the guide nucleic acid includes only ribonucleotides. In some cases, the guide nucleic acid includes ribonucleotides and deoxyribonucleotides.

In some cases, the CRISPR/Cas guide RNA comprises one or more modifications, such as base modifications, backbone modifications, sugar modifications, etc., to provide new or enhanced characteristics to the nucleic acid (e.g., improved stability, such as improved in vivo stability). Suitable nucleic acid modifications include, but are not limited to: 2'O-methyl modified nucleotides, 2' fluorine modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and 5' caps (e.g., 7-methylguanylate caps (m7G)). Suitable modified nucleic acid backbones containing phosphorus atoms include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoamidates (including 3'-aminophosphoamidates and aminoalkylphosphoamidates with normal 3'-5' linkages, phosphodiamidates, phosphothioamidates, phosphothioalkylphosphonates, phosphothioalkylphosphotriesters, selenophosphates and boranophosphates), 2'-5' linked analogs of these, as well as those with reverse polarity wherein one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages. Suitable oligonucleotides with reverse polarity comprise a single 3' to 3' linkage proximal to the 3' internucleotide linkage, i.e., a single reverse nucleoside residue that may be basic (nucleobases are absent or have hydroxyl groups in their place). Various salts (e.g., potassium or sodium), mixed salts, and free acid forms are also included. The CRISPR-Cas guide RNA may also include one or more substituted sugar moieties. Suitable polynucleotides comprise sugar substituents selected from the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. _Particularly suitable _are 0(( _CH2 ) _n0 ) _mCH3 , ₀ ( _{CH2 ) n0CH3} , ₀ ( _CH2 ) _nNH2 , 0( _CH2 ) _nCH3 , 0( _CH2 ) _nONH2 and 0( _CH2 ) _nON (( _CH2 ) _nCH3 ) ₂ , wherein n and m are 1 to about 10 _. Other suitable polynucleotides include sugar substituents selected from the following: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , OCF3, _SOCH3 , S02CH3, _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl _, aminoalkylamine, polyalkylamine, substituted _{silyl ,} RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-0-CH ₂ CH ₂ OCH ₃ , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-0-CH ₂ -0-CH ₂ -N(CH ₃ ) ₂ .

Examples of various CRISPR/Cas effector proteins and CRISPR/Cas guide RNAs (as well as information on requirements related to the presence of a protospacer adjacent motif (PAM) sequence in the target nucleic acid) can be found in the art, e.g., see Jinek et al., Science. 2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol. 2013 May;10(5):726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al. Cell. 2013 Feb 28;152(5):1173-83; Wang et al. Cell. 2013 May 9;153(4):910-8; Auer et al. Genome Res. 2013 Oct 31; Chen et al. Nucleic Acids Res. 2013 Nov 1;41(20):el9; Cheng et al. Cell Res. 2013 Oct;23(10):1163-71; Cho et al. Genetics. 2013 Nov;195(3):1177-80; DiCarlo et al. Nucleic Acids Res. 2013 Apr;41(7):4336-43；Dickinson et al., Nat Methods. 2013 Oct;10(10):1028-34；Ebina et al., Sci Rep. 2013;3:2510；Fujii et al., Nucleic Acids Res. 2013 Nov 1;41(20):el87；Hu et al., Cell Res. 2013 Nov;23(ll):1322-5；Jiang et al., Nucleic Acids Res. 2013 Nov 1;41(20):el88；Larson et al., Nat Protoc. 2013 Nov;8(ll):2180-96；Mali et al., Nat Methods. 2013 Oct;10(10):957-63；Nakayama et al., Genesis. 2013 Dec;51(12):835-43；Ran et al., Nat Protoc. 2013 Nov;8(ll):2281-308；Ran et al., Cell. 2013 Sep 12;154(6):1380-9；Upadhyay et al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8；Walsh et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15514-5；Xie et al., Mol Plant. 2013 Oct 9；Yang et al., Cell. 2013 Sep 12;154(6):1370-9; Briner et al., Mol Cell. 2014 Oct 23;56(2):333-9; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 2014 20140242699; 20140242700; 20140242702; 20140 248702;20140256046;20140273037;20140273226;20140273230;2014 20140273231 309487;20140310828;20140310830;20140315985;20140335063;2014 0335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; which are hereby incorporated by reference in their entirety.

Examples and instructions for Type V or Type VI CRISPR/Cas endonucleases and guide RNAs (as well as information regarding requirements for protospacer adjacent motif (PAM) sequences present in the target nucleic acid) can be found in the art, e.g., Zetsche et al., Cell. 2015 Oct 22;163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 Nov;13(ll):722-36; and Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97. C. Reverse transcriptase (RT)

Reverse transcriptase (RT, also known as RNA-directed DNA polymerase) is an enzyme present in all three domains of life, which is a DNA polymerase that uses RNA as a template. The reverse transcriptase of the present disclosure is used to reverse transcribe the template msd RNA into single-stranded msDNA.

Reverse transcriptases or functional domains thereof useful in the present invention include prokaryotic and eukaryotic RTs, provided that the RT functions within a host to generate a donor polynucleotide sequence from an RNA template ( eg , an RNA template from a retrotranscriptome ncRNA).

In certain embodiments, suitable RT sequences (including amino acid sequences and encoding polynucleotide sequences) are provided in Table A.

In certain embodiments, for example, the nucleotide sequence of a native or wild-type RT is modified using known codon optimization techniques to optimize expression in a desired host.

In certain embodiments, the RT domain of a reverse transcriptase is used in the present invention as long as it is compatible with the engineered retrotranscript of the present invention. The domain may include only RNA-dependent DNA polymerase activity. In certain embodiments, the RT domain is non-mutation-induced, that is , it will not cause mutations in the donor polynucleotide ( e.g. , during the reverse transcriptase process). In certain embodiments, the RT domain may originate from a non-retrotranscript RT, such as a viral RT or a human endogenous RT. In certain embodiments, the RT domain is a retrotranscript RT or a DGR RT. In certain embodiments, RT may have lower mutation inducibility than the partner wild-type RT. In certain embodiments, RT is not mutation-induced.

In some embodiments, the reverse transcriptase is encoded by the retrotranscriptase ret gene, which can be associated with cognate msr and msd loci and specifically recognize the secondary structure of cognate ncRNA transcripts.

In some embodiments, RT can be obtained from prokaryotic or eukaryotic cells. Most reverse transcriptases (80%) can be divided into three major lineages in terms of germline occurrence: group II introns, diversity-generating retrotransposons (DGRs), and retrotransposons. Other clades of RT include abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, group II-like (G2L), unknown group (UG), and rvt elements.

In some embodiments, the RT gene is a homologous RT, a retrotranscript RT from a species in the same species or clade as the homologous RT, or a retrotranscript RT that is not in the same clade as the homologous RT (such as an unrelated RT or an engineered RT). In some embodiments, non-retrotranscript related RTs are RTs from group II introns, diversity-generating retrotranscript elements (DGRs), abortive infection (Abi) RTs, CRISPR-Cas related RTs, group II-like (G2L), unknown group (UG), and rvt elements. See Mestre et al. , Nucleic Acids Research, Vol. 48, No. 22, December 16, 2020, pp. 12632-12647; and Mestre et al. , UG/Abi: “A Highly Diverse Family of Prokaryotic Reverse Transcriptases Associated With Defense Functions,” doi.org/10.1101/2021.12.02.470933 (incorporated by reference) .

In some embodiments, the RT is from a clade associated with a retrotransposons/retrotransposons-like sequence. In some embodiments, the RT is selected from the RTs provided in Table A. In some embodiments, the RT is not an RT associated with a sequence identified in Table X.

In prokaryotic retrotransposons, the RT gene is usually located downstream of the ncRNA ( msr and msd) loci. In engineered retrotransposons, the RT position may be different from that of natural or wild-type retrotransposons. In some embodiments, the RT gene can be provided in cis , such as upstream or downstream of the msr locus or the msd locus. In certain embodiments, the RT gene is provided in trans , such as provided separately in a vector of a vector system described herein, wherein the ncRNA encoding the msr and msd sequences is provided in different vectors of the vector system described herein.

In some embodiments, RT is modified ( e.g. , insertion, deletion and/or substitution of one or more nucleotides) or codon optimized to enhance activity or processability.

In certain embodiments, the cryptic stop signal is removed from the RT, thereby allowing the generation of longer ssDNA.

In certain embodiments, the RT is derived from a retrotranscript encoding msDNA as described in US 6,017,737; US 5,849,563; US 5,780,269; US 5,436,141; US 5,405,775; US 5,320,958; CA 2,075,515; all of which are incorporated herein by reference in their entirety.

In some embodiments, the engineered retrotranscript further comprises a polynucleotide (e.g., a DNA molecule) encoding a reverse transcriptase (RT) or a portion thereof. In some embodiments, the encoded RT or a portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding msDNA. In some embodiments, the polynucleotide (e.g., a DNA molecule) encoding RT comprises a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a polynucleotide listed in Table A.

In some embodiments, the polynucleotide encoding RT encodes a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or a polypeptide of Table C.

In some embodiments, the polynucleotides encoding RT do not include the polynucleotides listed in Table X.

Once translated, RT binds to the ncRNA template downstream of the msd locus, thereby forming an RT-RNA complex and initiating reverse transcription of the RNA toward its 5' end. Thus, in certain aspects, the present disclosure relates to an engineered nucleic acid-enzyme construct comprising: a. a non-coding RNA (ncRNA) comprising: i) an msr locus encoding a portion of an msr RNA of multiple copies of a single-stranded DNA (msDNA); and ii) an msd RNA encoding the msDNA. a. a heterologous nucleic acid inserted at or within a position selected from the group consisting of the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus ; and c. a reverse transcriptase (RT) or a domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C. In some embodiments, RT does not comprise a polypeptide listed in Table X.

In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a noncoding RNA (ncRNA) comprising: i) an msr locus encoding a msr RNA portion of multiple copies of a single-stranded DNA (msDNA); and ii) an msd locus encoding a msd RNA portion of the msDNA, b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT) or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the ncRNA and/or the RT is any one of the inventions described herein.

In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a noncoding RNA (ncRNA) comprising: i) an msr locus encoding an msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding an msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in Table A, or has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 0%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity; and/or ii) the consensus sequence listed in Table C; and wherein the RT does not comprise a sequence from Table X.

In some embodiments of the nuclease constructs described herein, the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and/or optionally, wherein the ncRNA is not an ncRNA from a retrotranscript of Table X.

In some embodiments, RT is linked to components such as RNA-guided and non-RNA-guided nucleases. Linking can be via a peptide bond or a short linker peptide in a fusion protein. Suitable linker peptides include flexible linkers, such as those containing G or S repeats, such as _G4S repeat units or GS repeat units, with 1-20 repeats, such as 1, 2, 3, 4, 5, 6, 7 or 8 repeats.

In certain embodiments, RT is chemically linked or conjugated to RNA-guided and non-RNA-guided nucleases via non-peptide bonds. Such protein conjugates can be delivered directly to host cells together with or alone the nucleic acid component of the engineered retrotranscript described herein.

In some embodiments, the RT is linked to a DNA repair regulatory biomolecule ( e.g. , an NHEJ peptide inhibitor. D. Programmable nucleases (RNA -guided nucleases )

In certain embodiments, an engineered retrotranscript (e.g., an engineered nucleic acid construct or an engineered nucleic acid-enzyme construct as described herein) may comprise or encode, as a heterologous nucleic acid, a guide RNA (gRNA) suitable for directing a nuclease to target a specific genome sequence to be modified. The gRNA includes a sequence that is complementary to the genome sequence and can thus mediate the binding of the nuclease-gRNA complex to the genome target site by hybridization between the guide sequence and the target site sequence.

In certain embodiments, the gRNA may be linked to the ncRNA and/or msDNA at the 5' end of the ncRNA and/or msDNA encoded by the engineered retrotranscript described herein. In certain embodiments, the gRNA may be linked to the ncRNA and/or msDNA at the 3' end of the RNA and/or msDNA of the ncRNA encoded by the engineered retrotranscript described herein after reverse transcription.

In some embodiments, the nuclease that can form a complex with the gRNA can be any of the Cas effectors of the technically recognized clustered regularly interspaced short palindromic repeat (CRISPR) system, which can be used , for example, for genome editing, including genome editing in mammalian cells or human cells. For example, the gRNA that can be loaded into Cas9 or its variants can be encoded by an engineered retrotranscript so that the gRNA is transcribed as part of the msDNA. In some embodiments, the gRNA can be linked to the 5' end of the a1 region of the msr coding sequence in the retrotranscript ncRNA, and the msDNA after reverse transcription. In some embodiments, the gRNA can be part of the modified msr region present in the ncRNA and msDNA produced after reverse transcription ( that is , the encoded gRNA has not been degraded due to the synthesis of msDNA by reverse transcription of the ncRNA).

Any technically recognized CRISPR/Cas effector enzyme or variant thereof ("Cas enzyme") known to be useful for CRISPR-based genome editing may be used with an engineered retrotranscript, although such Cas enzymes may not necessarily be part of an engineered retrotranscript, and may (but are not required to) be provided separately. For example, the Cas enzyme may be provided as part of the vector system described herein. The coding sequence of a suitable Cas enzyme may be present on the same vector that provides the engineered retrotranscript, or on a different vector. When the engineered retrotranscript and the Cas enzyme are present on the same vector, they may be under the transcriptional control of the same promoter, enhancer, or other transcriptional regulatory element, or may be regulated separately by different promoters, enhancers, and/or other transcriptional regulatory sequences in the vector system.

In some embodiments, the Cas enzyme is a Class 2, Type II CRISPR/Cas effector enzyme, such as Cas9. In some embodiments, Cas9 is from Streptococcus pyogenes (SpCas9), and the gRNA encoded by the engineered retrotranscript comprises both a crisprRNA (crRNA) and a tracrRNA linked together as a single guide RNA (gRNA).

In some embodiments, Cas9 is from Staphylococcus aureus Cas9 (SaCas9) or an engineered variant thereof, such as SaCas9-HF (a high-fidelity variant with genome-wide activity), KKHSaCas9 (which recognizes 5'-NNGRRT-3' PAM and has a 2-4-fold wider range of target sites in the human genome than wild-type SaCas9), and microABE1744 (an engineered SaCas9 variant tuned for adenine base editing (ABE), with significantly improved targeted editing compared to other nucleases and reduced RNA off-target footprints).

In some embodiments, Cas9 is from Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), or Curculigo jejuni (CjCas9).

In some embodiments, Cas9 is from Streptococcus canis (ScCas9), which has a less stringent PAM sequence requirement of 5'-NNG-3' (rather than the more stringent 5'-NGG-3' of SpCas9).

In some embodiments, Cas9 is from Staphylococcus auris (SauriCas9) (which recognizes a 5'-NNGG-3' PAM sequence and has high editing activity).

In some embodiments, the Cas enzyme is a Cas9 variant with a mutant catalytic domain that retains cleavage specificity but only nicks a single DNA strand at a desired target sequence rather than generating a double-strand break (DSB). Two such Cas9 nickase variants targeting different strands of the same target sequence can be used together to increase the fidelity of generating DSBs, each variant using a different gRNA that can be provided by an engineered retrotranscript.

In some embodiments, the Cas enzyme is a high-fidelity Cas9 variant (such as SpCas9-HF1) with weakened DNA phosphate backbone interactions, which exhibits genome-wide specificity and undetectable off-target effects.

In some embodiments, the Cas enzyme is a Cas9 variant, referred to as eSpCas9, which weakens the interaction between eSpCas9 and its gRNA, and is not precisely complementary to the target DNA sequence, thereby providing improved specificity and lower off-target editing rates.

In some embodiments, the Cas enzyme is a hyperprecise Cas9 variant (HypaCas9), which improves proofreading before cleavage and thus greatly reduces off-target cleavage.

In some embodiments, the Cas enzyme is a Cas9 variant (FokI-fused dCas9), which combines the DNA recognition ability of dCas9 with the specificity of the active nuclease FokI. The resulting nuclease cleaves the target sequence only after dimerization, which is more difficult to occur at off-target sites, thereby enhancing specificity.

In some embodiments, the Cas enzyme is a Cas9 variant xCas9, which recognizes multiple PAM sequences, thereby increasing the number of target sites in the genome by one quarter. In addition, the xCas9 variant also exhibits a lower off-target rate than the commonly used SpCas9.

In some embodiments, the Cas enzyme is a Cas9 variant with altered PAM sequence specificity, including SpG variants with expanded target range of PAM sequences and SpRY variants that can target nearly all PAM sequences.

In some embodiments, the Cas enzyme is dCas9, which has an inactive catalytic nuclease domain while maintaining a recognition domain that allows guide RNA-mediated targeting of specific DNA sequences. dCas9 can be further linked to functional domains with unique biological functions, such as transcriptional activation/inhibition, DNA methylation, demethylation, nucleases (such as FokI) or fluorescent dyes. Representative (non-limiting) dCas9-linked functional domains include dCas9-SAM, dCas9-SunTag, dCas9-VPR, and dCas9-KREB.

In some embodiments, dCas9 is fused to a catalytic enzyme with cytidine deaminase activity, which converts GC base pairs to AT base pairs.

In some embodiments, dCas9 is fused to an engineered RNA adenosine deaminase, which converts AT base pairs to GC base pairs.

In some embodiments, the Cas enzyme is a Class 2, V-type CRISPR/Cas effector enzyme, such as Cas12a (Cpf1) (V-A type), Cas12b (C2c1) (V-B type), Cas12c (C2c3) (V-C type), Cas12d (CasY) (V-D type), Cas12e (CasX) (V-E type), Cas12f (Cas14, C2c10) (V-F type), Cas12g (V-G type), Cas12h (V-H type), Cas12i (V-I type), Cas12k (C2c5) (V-K type) or C2c4/C2c8/C2c9 (V-U type).

In some embodiments, the Cas enzyme is Cas12a or Cpf1 (CRISPR from Prevotella and Francisella 1). Unlike Cas9, Cas12a is well suited for targeting AT-rich DNA sequences due to its AT-rich PAM sequence. In some embodiments, Cas12a is FnCas12a (which recognizes the PAM sequence 5'-TTN-3'), or AsCas12a or LbCas12a (which recognizes the 5'-TTTV-3' PAM sequence), where V is an A, G, or C nucleotide. In addition, Cas12a produces staggered double-strand breaks in the target DNA, rather than the blunt ends generated by SpCas9, making it more suitable for HDR repair. In this embodiment, the engineered retrotranscript of the present invention encodes a gRNA suitable for use with Cas12a because the gRNA does not require a tracer RNA and only requires a crRNA.

In some embodiments, the Cas enzyme is a Cas12a variant (enAsCas12a) from Aminococcus , which has an expanded target range of PAM sequences and significantly higher editing activity compared to wild-type Cas12a.

In some embodiments, the Cas enzyme is a high-fidelity Cas12a variant with reduced off-target editing (enAsCas12a-HF1).

In some embodiments, the Cas enzyme is Cas12b or C2c1. In some embodiments, Cas12b is from Acidococcus terrestris (AacCas12b), or from Acidococcus acidophilus Cas12b (AapCas12b).

In some embodiments, the Cas enzyme is a Cas12b variant that functions at 37°C, such as a form of Bacillus hisashii (BhCas12b).

In some embodiments, the Cas enzyme is a BhCas12b variant with higher specificity than SpCas9.

In some embodiments, the Cas enzyme is CasX or Cas12d.

In some embodiments, the Cas enzyme is CasY or Cas12e, and the engineered retrotranscript of the present invention encodes a short complementary non-translated RNA (scoutRNA) and crRNA (rather than tracrRNA used in other CRISPR-Cas systems).

In some embodiments, the Cas enzyme is Cas14 from Archaea. Cas14 targets single-stranded (ss) DNA target sequences, does not require a PAM sequence for activation, and has incidental activity ( i.e. , non-specifically cleaves other non-target ssDNA strands after binding to the target sequence). Unlike Cas12a, Cas14a requires high-fidelity complementarity with the target ssDNA and is extremely sensitive to mismatches in the internal seed region of the ssDNA target substrate.

In some embodiments, the gRNA nuclease is an engineered RNA-guided FokI nuclease. The RNA-guided FokI nuclease comprises a fusion of an inactive Cas9 (dCas9) and a FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting to FokI. For a description of engineered RNA-guided Fold nucleases, see , e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; incorporated herein by reference.

In other embodiments, the RNA-guided nuclease may be a non-CRISPER/Cas related nuclease, such as a transposon-encoded nuclease, IscB, IscR, or TnpB.

In some embodiments, the Cas enzyme is Cas9

In some embodiments, the retrotransposons-based editing systems described herein may include any Cas9 equivalents. Cas9 equivalents are Cas9-like proteins that provide the same or substantially the same function as Cas9. For example, if Cas9 refers to a type II enzyme of a CRISPR-Cas system, a Cas9 equivalent may refer to a type V or type VI enzyme of a CRISPR-Cas system.

For example, Cas12e (CasX) is a Cas9 equivalent that is reported to have the same function as Cas9, but evolved by convergent evolution. Therefore, it is expected that the Cas12e (CasX) protein is used with the retrotransposons-based editing system described herein. In addition, any variants or modifications of Cas12e (CasX) are conceivable and within the scope of the present disclosure.

In some embodiments, a Cas9 equivalent may refer to Cas12e (CasX) or Cas12d (CasY), which have been described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. Feb. 21, 2017. doi: 10.1038/cr.2017.21, the entire contents of which are hereby incorporated by reference. Using genome-resolved metagenomics, a variety of CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This exotic Cas9 protein was found in a little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems have been discovered: CRISPR-Cas12e and CRISPR-Cas12d, which are among the most compact systems discovered to date. In some embodiments, Cas9 refers to Cas12e or a variant of Cas12e. In some embodiments, Cas9 refers to Cas12d or a variant of Cas12d. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% consistent with a naturally occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% consistent with a wild-type Cas protein or any Cas portion provided herein.

In various embodiments, nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. One example of a nucleic acid programmable DNA binding protein with a different PAM specificity from Cas9 is the clustered regularly interspaced short palindromic repeats from Prevotella and Francisella 1 (i.e., Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of the Type V enzyme subgroup, not a member of the Type II subgroup. Cas12a (Cpf1) has been shown to mediate potent DNA interference with features distinct from those of Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease that lacks a tracrRNA and utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Among the 16 Cpf1-family proteins, two enzymes from Aminococcus and Lachnospiraceae were shown to have potent genome editing activity in human cells. The Cpf1 protein is known in the art and has been described previously, for example, in Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, pp. 949-962; the entire contents of which are hereby incorporated by reference.

In other embodiments, the Cas protein may include any CRISPR-related protein, including but not limited to Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof.

In various other embodiments, the RNA-guided nuclease can be any of the following proteins: Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), GeoCas9, CjCas9, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, Csn2, xCas9, SpCas9-NG or an Argonaute (Ago) domain or a variant thereof.

The amino acid sequences of RNA-guided nucleases are readily available and known in the art. Exemplary RNA-guided nucleases and their amino acid sequences can be found, for example, in WO 2017/070633, US 2020/0010835, US 2022/0204975, US 11071790, WO 2020/191233, US 11447770, US 10858639, and US 10947530, each of which is incorporated herein by reference in its entirety. E. Programmable Nucleases ( Others )

In addition to the CRISPR/Cas system, the engineered retrotranscripts of the present invention can also be used in combination with sequence-specific nucleases that do not use guide RNA to recognize the target sequence, such as non-CRISPR/Cas sequence-specific nucleases, including TALEN, ZFN, meganuclease and restriction enzymes, and other sequence-specific nucleases guided by other RNAs, such as transposon-encoded IscB, IscR or TnpB.

For example, the engineered retrotranscript of the present invention can encode or provide msDNA, which can serve as a donor or template sequence for HDR-mediated genome editing. Optionally, the RT of the engineered retrotranscript is fused to such sequence-specific nucleases, so that the msDNA generated by the RT close to the HDR-mediated genome editing site can more effectively participate in HDR-mediated genome editing.

In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE nuclease, a TALE nickase, a zinc finger (ZF) nuclease, a ZF nickase, a meganuclease, or a combination thereof. In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or comprises two, three, four or more of independently selected TALE nucleases, TALE nickases, zinc finger (ZF) nucleases, ZF nickases, meganucleases, restriction enzymes, or a combination thereof. In some embodiments, the combination is or comprises a TALE nuclease/ZF nuclease; a TALE nickase/ZF nickase.

In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE nuclease (transcription activator-like effector nuclease (TALEN)). TALEN is a restriction enzyme engineered to cleave a specific target DNA sequence. TALEN comprises a TAL effector (TALE) DNA binding domain (which binds at or near the target DNA), fused to a DNA cleavage domain that cleaves the target DNA. TALE is engineered to bind to almost any desired DNA sequence. Therefore, in some embodiments, TALEN comprises an N-terminal capping region, a DNA binding domain, and a C-terminal capping region, and the DNA binding domain may comprise at least one or more TALE monomers or half-monomers specifically ordered to target a relevant genomic locus, wherein these three parts are arranged in a predetermined N-terminal to C-terminal orientation. Optionally, TALEN includes at least one or more regulatory or functional protein domains.

In some embodiments, the TALE monomer or half-monomer may be a variant TALE monomer derived from a natural or wild-type TALE monomer but having altered amino acids at positions that are usually highly conserved in nature, and in particular having a combination of amino acids as RVDs that do not occur in nature and that can recognize nucleotides with higher activity, specificity and/or affinity than naturally occurring RVDs. Such variants may include deletions, insertions and substitutions at the amino acid level, as well as transpositions, conversions and inversions at one or more positions at the nucleic acid level. Such variants may also include truncations.

In some embodiments, TALE monomer/half-monomer variants include homologous and functional derivatives of the parent molecule. In some embodiments, the variants are encoded by polynucleotides capable of hybridizing with the wild-type nucleotide sequence encoding the parent molecule under highly stringent conditions.

In some embodiments, the DNA binding domain of a TALE has at least 5 or more TALE monomers and at least one or more half-monomers that are specifically ordered or arranged to target a relevant genomic locus. The construction and production of the TALE or polypeptide of the present invention may involve any method known in the art.

Naturally occurring TALE or "wild-type TALE" is a nucleic acid binding protein secreted by multiple mutant species. TALE contains a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides, which are mainly 33, 34 or 35 amino acids long and differ from each other mainly at amino acid positions 12 and 13. The TALE monomer contained in the DNA binding domain is generally represented as Xl-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid. X12X13 indicates an RVD. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such monomers, the RVD consists of a single amino acid. In such cases, the RVD can be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent. The DNA binding domain may comprise several repeats of the TALE monomer and this may be represented as (Xl-11-(X12X13)-X14-33 or 34 or 35)z, wherein z is optionally at least 5-40, such as 10-26.

TALE monomers have nucleotide binding affinity determined by the amino acid identity in their RVDs. Polypeptide monomers with NI RVDs preferentially bind to adenine (A), monomers with NG RVDs preferentially bind to thymine (T), monomers with HD RVDs preferentially bind to cytosine (C), monomers with NN RVDs preferentially bind to both adenine (A) and guanine (G), monomers with IG RVDs preferentially bind to T, and monomers with NS RVDs recognize all four base pairs and can bind to A, T, G, or C. Therefore, the number and order of polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity. The structure and function of TALEs are further described in, e.g., Moscou et al. , Science 326: 1501 (2009); Boch et al. , Science 326: 1509-1512 (2009); and Zhang et al. , Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety.

In some embodiments, the TALE is a dTALE (or designer TALE), see Zhang et al. , Nature Biotechnology 29:149-153 (2011), incorporated herein by reference.

In some embodiments, the TALE monomer comprises an HN or NH RVD that preferentially binds to guanine, and the TALE has high binding specificity to a target nucleic acid sequence containing guanine. In the following embodiments, polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH, and SS preferentially bind to guanine. In some embodiments, polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS, and SN preferentially bind to guanine. In some embodiments, polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN, and SS preferentially bind to guanine. In some embodiments, the RVDs with high binding specificity to guanine are RN, NH RH, and KH. In some embodiments, polypeptide monomers with NV RVD preferentially bind to adenine and guanine, as do monomers with RVD HN. Monomers with NC RVD preferentially bind to adenine, guanine, and cytosine, and monomers with S (or S*) RVD bind to adenine, guanine, cytosine, and thymine with comparable affinity. In further embodiments, monomers with H*, HA, KA, N*, NA, NC, NS, RA, and S* RVDs bind to adenine, guanine, cytosine, and thymine with comparable affinity. Such polypeptide monomers allow for the generation of degenerate TALEs capable of binding to a repertoire of related but non-identical target nucleic acid sequences.

In certain embodiments, the TALE polypeptide has a nucleic acid binding domain comprising polypeptide monomers arranged in a predetermined N-terminal to C-terminal order, such that each polypeptide monomer binds to a nucleotide of a predetermined target nucleic acid sequence, and wherein at least one polypeptide monomer has an HN or NH RVD and preferentially binds to guanine, has an NV RVD and preferentially binds to adenine and guanine, has an NC RVD and preferentially binds to adenine, guanine and cytosine, or has an S RVD and binds to adenine, guanine, cytosine and thymine.

In some embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to adenine has a NI, NN, NV, NC, or S RVD.

In certain embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to guanine has an HN, NH, NN, NV, NC, or S RVD.

In certain embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to cytosine has a HD, NC, or S RVD.

In some embodiments, each polypeptide monomer that binds to thymine has a NG or S RVD.

In some embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to adenine has a NI RVD.

In certain embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to guanine has an HN or NH RVD.

In certain embodiments, each polypeptide monomer in the nucleic acid binding domain that binds to cytosine has an HD RVD.

In some embodiments, each polypeptide monomer that binds to thymine has a NG RVD.

In certain embodiments, the RVDs specific for adenine are NI, RI, KI, HI, and SI.

In certain embodiments, the RVDs specific for adenine are HN, SI, and RI, and the most preferred RVD for adenine specificity is SI.

In certain embodiments, the RVDs specific for thymine are NG, HG, RG, and KG.

In certain embodiments, the RVDs specific for thymine are KG, HG, and RG, and the most preferred RVD for thymine specificity is KG or RG.

In certain embodiments, the RVDs specific for cytosine are HD, ND, KD, RD, HH, YG, and SD.

In certain embodiments, the RVDs specific for cytosine are SD and RD. Representative RVDs and their targeted nucleotides are provided in Figure 4B of WO 2012/067428, the entire contents of which are hereby incorporated herein by reference.

In certain embodiments, the variant TALE monomer may comprise any RVD that exhibits specificity for a nucleotide as depicted in Figure 4A of WO2012/067428. All such TALE monomers allow for the generation of degenerate TALEs that are capable of binding to a repertoire of related but non-identical target nucleic acid sequences.

In certain embodiments, RVD SH may be specific for G, RVD IS may be specific for A, and RVD IG may be specific for T.

In certain embodiments, RVD NT can bind to G and A. In certain embodiments, RVD NP can bind to A, T, and C. In certain embodiments, at least one selected RVD can be NI, HD, NG, NN, KN, RN, NH, NQ, SS, SN, NK, KH, RH, HH, KI, HI, RI, SI, KG, HG, RG, SD, ND, KD, RD, YG, HN, NV, NS, HA, S*, N*, KA, H*, RA, NA, or NC.

The predetermined N-terminal to C-terminal sequence of one or more polypeptide monomers of a nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the TALE or polypeptide of the present invention can bind.

As used herein, monomers and at least one or more half-monomers are "specifically ordered to target" a genomic locus or gene of interest. In plant genomes, the natural TALE binding site always begins with thymine (T), which can be specified by a cryptic signal within the non-repetitive N-terminus of the TALE polypeptide; in some cases, this region may be referred to as repeat 0. In animal genomes, the TALE binding site does not necessarily have to begin with thymine (T), and the polypeptides of the present invention can target DNA sequences that begin with T, A, G, or C. The tandem repeats of a TALE monomer always end with a half-length repeat or sequence stretch that may only share identity with the first 20 amino acids of the repeating full-length TALE monomer, and this half-repeat may be referred to as a half-monomer (Figure 8 of WO 2012/067428). Therefore, it can be seen that the length of the targeted nucleic acid or DNA is equal to the number of complete monomers plus 2 (see Figure 44 of WO 2012/067428).

In certain embodiments, the nucleic acid binding domain is engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more polypeptide monomers arranged in an N-terminal to C-terminal direction to bind to a nucleic acid sequence of a predetermined length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.

In certain embodiments, the nucleic acid binding domain is engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more full-length polypeptide monomers specifically ordered or arranged to target nucleic acid sequences of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 nucleotides in length, respectively. In certain embodiments, the polypeptide monomers are contiguous. In some embodiments, half-monomers can be used to replace one or more monomers, especially if they are present at the C-terminus of the TALE.

The polypeptide monomer is usually 33, 34 or 35 amino acids long. Except for the RVD, the amino acid sequence of the polypeptide monomer is highly conserved or as described herein, the amino acids in the polypeptide monomer (except for the RVD) show a pattern that affects TALE activity, and its identification can be used in preferred embodiments of the present invention.

In certain embodiments, when the DNA binding domain may comprise (X1-11-X12X13-X14-33 or 34 or 35) z, wherein X1-11 is a chain of 11 consecutive amino acids, wherein X12X13 is a repeating variable diresidue (RVD), wherein X14-33 or 34 or 35 is a chain of 21, 22 or 23 consecutive amino acids, wherein z is at least 5 to 26, then the preferred amino acid combination is LTLD (SEQ ID NO: 19936) or LTLA (SEQ ID NO: 19937) or LTQV (SEQ ID NO: 19938) at X1-4, or EQHG (SEQ ID NO: 19939) or RDHG (SEQ ID NO: 19940) at positions X30-33 or X31-34 or X32-35. NO:19940). In addition, when the monomer is 34 amino acids long, the other relevant amino acid combinations in the monomer are LTPD (SEQ ID NO:19941) at X1-4 and NQALE (SEQ ID NO:19942) at X16-20 and DHG at X32-34. When the monomer is 33 or 35 amino acids long, the corresponding shift occurs in the positions of the consecutive amino acids NQALE (SEQ ID NO:19942) and DHG. In certain embodiments, NQALE (SEQ ID NO:19942) is at X15-19 or X17-21 and DHG is at X31-33 or X33-35.

In certain embodiments, when the monomer is 34 amino acids long, the relevant amino acid combination in the monomer is LTPD at X1-4 and KRALE (SEQ ID NO: 19943) at X16-20 and AHG at X32-34, or LTPE (SEQ ID NO: 19944) at X1-4 and KRALE (SEQ ID NO: 19943) at X16-20 and DHG at X32-34. When the monomer is 33 or 35 amino acids long, the corresponding shift occurs in the position of the consecutive amino acids KRALE (SEQ ID NO: 19943), AHG and DHG. In certain embodiments, the positions of the consecutive amino acids may be (LTPD at X1-4 and KRALE (SEQ ID NO: 19943) at X15-19 and AHG at X31-33) or (LTPE at X1-4 and KRALE (SEQ ID NO: 19943) at X15-19 and DHG at X31-33) or (LTPD at X1-4 and KRALE at X17-21 and AHG at X33-35) or (LTPE at X1-4 and KRALE (SEQ ID NO: 19943) at X17-21 and DHG at X33-35).

In certain embodiments, the consecutive amino acids [NGKQALE] (SEQ ID NO: 19945) are present at positions X14-20 or X13-19 or X15-21. These representative positions suggest various embodiments of the present invention and provide guidance for identifying additional relevant amino acids or relevant amino acid combinations in all TALE monomers (see Figures 24A-24F and Figure 25 of WO 2012/067428).

Exemplary amino acid sequences of the conserved portion of the polypeptide monomer are provided below. The RVD position in each sequence is represented by XX or X* (where (*) indicates that the RVD is a single amino acid and residue 13 (X13) is absent). LTPAQVVAIASXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 19402) LTPAQVVAIASX*GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 19403) LTPDQVVAIANXXGGKQALATVQRLLPVLCQDHG (SEQ ID NO: 19404) LTPDQVVAIANXXGGKQALETLQRLLPVLCQDH G (SEQ ID NO: 19405) LTPDQVVAIANXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 19406) LTPDQVVAIASXXGGKQALATVQRLLPVLCQDHG (SEQ ID NO: 19407) LTPDQVVAIASXXGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 19408) LTPDQVVAIASXXGGKQALETVQRVLPVLCQDHG (SEQ ID NO: 19409) LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 19410) LTPYQVVAIASXXGSKQALETVQRLLPVLCQDHG (SEQ ID NO: 19411) LTREQVVAIASXXGG KQALETVQRLLPVLCQDHG (SEQ ID NO: 19412) LSTAQVVAIASXXGGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 19413) LSTAQVVAVASXXGGKPALEAVRAQLLALRAAPYG (SEQ ID NO: 19414)

Figures 24A-F of WO 2012/067428 (which is incorporated herein by reference) provide a further list of TALE monomers that exclude RVDs that can be represented by the sequence (X1-11-X14-34 or X1-11-X14-35), where X is any amino acid and the subscript is the amino acid position.

In certain embodiments, TALE polypeptide binding efficiency is increased by including an amino acid sequence from a "capping region" directly N-terminal or C-terminal to the DNA binding region of a naturally occurring TALE at the N-terminus or N-terminal position of the engineered TALE DNA binding region of the engineered TALE. Thus, in certain embodiments, the TALE polypeptides described herein further comprise an N-terminal capping region and/or a C-terminal capping region.

An exemplary amino acid sequence for the N-terminal capping region is: MDPIRSRTPSPARELLSGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIV ALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN (SEQ ID NO: 19415)

An exemplary amino acid sequence of the C-terminal capping region is: RPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTRAS (SEQ ID NO: 19416)

As used herein, the predetermined "N-terminal" to "C-terminal" orientation of the N-terminal capping region, the DNA binding domain comprising the repeat TALE monomer, and the C-terminal capping region provide the structural basis for the organization of the different domains in the d-TALE or polypeptide of the present invention.

The complete N-terminal and/or C-terminal capping region is not necessary to enhance the binding activity of the DNA binding region. Therefore, in certain embodiments, fragments of the N-terminal and/or C-terminal capping region are included in the TALE polypeptides described herein.

In certain embodiments, the TALE (including a plurality of TALE) polypeptides described herein contain an N-terminal capping region fragment that includes at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 amino acids of the N-terminal capping region. In certain embodiments, the N-terminal capping region fragment amino acids are at the C-terminus (DNA binding region proximal) of the N-terminal capping region. The N-terminal capping region fragment including the C-terminal 240 amino acids enhanced binding activity equivalent to the full-length capping region, while the fragment including the C-terminal 147 amino acids retained greater than 80% of the efficacy of the full-length capping region, and the fragment including the C-terminal 117 amino acids retained greater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain a C-terminal capping region fragment comprising at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, 180 amino acids of the C-terminal capping region. In certain embodiments, the C-terminal capping region fragment amino acids belong to the N-terminus (DNA binding region proximal) of the C-terminal capping region. In certain embodiments, the C-terminal capping region fragment comprising the C-terminal 68 amino acids enhances binding activity equivalent to the full-length capping region, while the fragment comprising the C-terminal 20 amino acids retains greater than 50% of the efficacy of the full-length capping region.

In certain embodiments, the capping region of the TALE polypeptides described herein need not have a sequence that is identical to the capping region sequence provided herein. Thus, in some embodiments, the capping region of the TALE polypeptides described herein has a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or shares identity with the capping region amino acid sequence provided herein. Sequence identity is related to sequence homology. Homology comparisons can be made by eye, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent (%) of homology between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. In some preferred embodiments, the capping region of the TALE polypeptides described herein has a sequence that is at least 95% identical or shares identity with the capping region amino acid sequences provided herein.

Sequence homology can be generated by any of a variety of computer programs known in the art, including but not limited to BLAST or FASTA. Suitable computer programs for alignment, such as the GCG Wisconsin Bestfit suite, can also be used. Once the software produces the best alignment, the homology %, preferably the sequence identity %, can be calculated. The software usually does this as part of a sequence comparison and generates a numerical result. Homology % can be calculated on a continuous sequence, that is , one sequence is aligned with another sequence, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed only on a relatively small number of residues.

The conserved portions of the polypeptide monomers and additional sequences of the N-terminal and C-terminal capping regions are included in the sequences with the following gene accession numbers: AAW59491.1, AAQ79773.2, YP_450163.1, YP_001912778.1, ZP_02242672.1, AAW59493.1, AAY54170.1, ZP_02245314.1, ZP_02243372.1, AAT46123.1, AAW59492.1, YP_451030.1, YP_001915105.1, ZP_02242534.1, AAW77510.1, ACD11364.1, ZP_02245056.1, ZP_ 02245055.1, ZP_02242539.1, ZP_02241531.1, ZP_02243779.1, AAN01357.1, ZP_02245177.1, ZP_02243366.1, ZP_02241530.1, AAS58130.3, ZP_0224 2537.1 , YP_200918.1, YP_200770.1, YP_451187.1, YP_451156.1, AAS58127.2, YP_451027.1, UR_451025.1, AAA92974.1, UR_001913755.1, ABB70183.1, UR_4518 93 .1, UR_450167.1, ABY60855.1, UR_200767.1, ZR_02245186.1, ZR_02242931.1, ZR_02242535.1, AAU54169.1, UR_450165.1, UR_001913452.1, AAS58129.3 , ACM44927.1, ZR_02244836.1, AAT46125.1, UR_450161.1, ZR_02242546.1, AAT46122.1, UR_451897.1, AAF98343.1, UR_001913484.1, AAY54166.1, UR_0019 15 093.1, UR_001913457.1, ZR_02242538.1, UR_200766.1, UR_453043.1, UR_001915089.1, UR_001912981.1, ZR_02242929.1, UR_001911730.1, UR_201654. 1. UR_199877.1, ABB70129.1, UR_451696.1, UR_199876.1, AAS75145.1, AAT46124.1, UR_200914.1, UR001915101.1, ZR_02242540.1, AAG02079.2, UR_451895. 1、YP451189.1、UR_200915.1、AAS46027.1、UR_001913759.1、UR_001912987.1、AAS58128.2、AAS46026.1、UR_201653.1、UR_202894.1、UR_001913480.1、ZR_02242666.1、R_001912775.1、ZR_02242662.1、AAS46025.1、AAC43587.1、BAA37119.1、NPJ544725.1、AB077779.1、BAA37120.1、ACZ62652.1、BAF46271.1、AC Z62653.1, NPJ544793.1, ABO77780.1, ZR_02243740.1, ZR_02242930.1, AAB69865.1, AAY54168.1, ZR_02245191.1, UR_001915097.1, ZR_02241539.1, UR_ 45 1158.1, BAA37121.1, UR_001913182.1, UR_200903.1, ZR_02242528.1, ZR_06705357.1, ZR_06706392.1, ADI48328.1, ZR_06731493.1, ADI48327.1, AB07 778 2.1、ZR06731656.1、NR_942641.1、AAY43360.1、ZR_06730254.1、ACN39605.1、UR_451894.1、UR_201652.1、UR_001965982.1、BAF46269.1、NPJ544708.1、ACN82432.1、AB077781.1、P14727.2、BAF46272.1、AAY43359.1、BAF46270.1、NR_644743.1、ABG37631.1、AAB00675.1、YP199878.1、ZR_02242536.1、CAA48680.1 , ADM80412.1, AAA27592.1, ABG37632.1, ABP97430.1, ZR_06733167.1, AAY43358.1, 2KQ5_A, BAD42396.1, ABO27075.1, UR_002253357.1, UR_002252977.1, ABO27074.1, ABO27067.1, ABO27072.1, ABO27068.1, UR_003750492.1, ABO27073.1, NR_519936.1, ABO27071.1, AB027070.1, and ABO27069.1, each of which is hereby incorporated by reference.

In some embodiments, the TALEs described herein also include a nuclear localization signal and/or a cytosolic uptake signal. Such signals are known in the art and can target the TALE to the nucleus and/or intracellular compartment of a cell. Such cytosolic uptake signals include, but are not limited to, the minimal Tat protein transduction domain spanning residues 47-57 of the human immunodeficiency virus Tat protein: YGRKKRRQRRR (SEQ ID NO: 19952).

In some embodiments, the TALEs described herein include nucleic acids or DNA binding domains that are non-TALE nucleic acids or non-TALE DNA binding domains. As used herein, the term "non-TALE DNA binding domain" refers to a DNA binding domain having a nucleic acid sequence that corresponds to a nucleic acid sequence that is substantially non-originating from a nucleic acid encoding a TALE protein or a fragment thereof, for example , a nucleic acid sequence that is different from a nucleic acid encoding a TALE protein and that originates from the same or a different organism.

In certain embodiments, a TALE described herein comprises a nucleic acid or DNA binding domain linked to a non-TALE polypeptide.

"Non-TALE polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially derived from a TALE protein or a fragment thereof, for example , a protein that is different from the TALE protein and originates from the same or a different organism. As used herein, the term "linked" is intended to include any means by which the nucleic acid binding domain and the non-TALE polypeptide may be linked to each other, including, for example, by peptide bonds that are part of the same polypeptide chain or by other covalent interactions, such as chemical linkers. The non-TALE polypeptide may be linked, for example, to the N-terminus and/or C-terminus of the nucleic acid binding domain, may be linked to a C-terminal or N-terminal cap region, or may be indirectly linked to the nucleic acid binding domain.

In certain embodiments, the TALE or polypeptide of the present invention comprises a chimeric DNA binding domain. A chimeric DNA binding domain can be generated by fusing a complete TALE (including the N-terminal and C-terminal capping regions) to another TALE or a non-TALE DNA binding domain such as a zinc finger (ZF), a helix-loop-helix, or a catalytically inactive DNA endonuclease ( e.g. , EcoRI, meganuclease, etc.), or a portion of a TALE can be fused to other DNA binding domains. A chimeric domain can have a novel DNA binding specificity that combines the specificities of both domains.

In certain embodiments, the TALE polypeptides of the invention include a nucleic acid binding domain linked to one or more effector domains. In certain embodiments, the effector domain is a nickase or a nuclease.

In certain embodiments, the sequence-specific nuclease is a zinc finger nuclease (ZFN), such as an artificial zinc finger nuclease having an array of zinc finger (ZF) modules to target a new DNA binding site in a target sequence ( e.g. , a target sequence or target site in a genome). Each zinc finger module in the ZF array targets three DNA bases. Customized arrays of individual zinc finger domains are assembled into ZF proteins (ZFPs). The resulting ZFPs can be linked to functional domains, such as nucleases.

ZF nucleases (ZFNs) can be used as alternative programmable nucleases to RNA-guided nucleases for retrotranscript-based editing. ZFN proteins have been extensively described in this technology, for example, Carroll et al., "Genome Engineering with Zinc-Finger Nucleases," Genetics, August 2011, Vol. 188: 773-782; Durai et al., "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells," Nucleic Acids Res, 2005, Vol. 33: 5978-90; and Gaj et al., "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering," Trends Biotechnol. 2013, Vol. 31: 397-405, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the ZF-linked nuclease is the catalytic domain of the type IIS restriction enzyme FokI (see Kim et al. , PNAS USA 91:883-887, 1994; Kim et al. , PNAS USA 93:1156-1160, 1996, each of which is incorporated herein by reference).

In certain embodiments, the ZFNs comprise paired ZFN heterodimers, resulting in increased cleavage specificity and/or reduced off-target activity. In this embodiment, each ZFN in the heterodimer targets a different nucleotide sequence separated by a short spacer (see Doyon et al. , Nat. Methods 8:74-79, 2011, incorporated herein by reference).

In certain embodiments, a ZFN comprises a polynucleotide binding domain (comprising multiple sequence-specific ZF modules) and a polynucleotide cleavage nickase domain.

In certain embodiments, a library of two finger templates is used to engineer ZFs.

In certain embodiments, strings of two-finger units are used in ZFNs to improve the DNA binding specificity of multi-zinc finger peptides (see PNAS USA 98: 1437-1441, incorporated herein by reference).

In some embodiments, the ZFN has more than 3 fingers. In some embodiments, the ZFN has 4, 5, or 6 fingers. In some embodiments, the ZF modules in the ZFN are separated by one or more linkers to improve specificity.

In certain embodiments, the ZFs of the ZFNs include substitutions in the dimer interface of the cleavage domain that prevent homodimerization between the ZFs but allow heterodimer formation.

In certain embodiments, the ZFs of the ZFNs have a design that retains activity while inhibiting homodimerization.

In certain embodiments, the ZFN is any one of the ZF nucleases in Table 1 of Carroll et al ., Genetics 188(4):773-782, 2011 (incorporated herein by reference).

General principles and guidance for generating ZFs, ZF arrays, and ZFNs can be found in this technology, such as modular design of ZFs or ZF arrays in ZFNs (where different modules can be rearranged and assembled into new combinations for new targets), as taught in Carroll et al. , Nat. Protoc. 1: 1329-1341, 2006 (incorporated herein by reference); new three-finger sets of engineered ZFs generated using partially random libraries; using a rapid cell-based method to profile the DNA binding specificity of engineered Cys2His2 zinc finger domains (see Nucleic Acids Res. 35: e81, incorporated by reference). Certain DNA triplet ZFs that perform well in neighbor assembly are described in Sander et al ., 2011. Selection-free zinc finger nuclease engineering by neighbor assembly (CoDA) is taught in Nat. Methods 8: 67-69). ToolGen describes individual fingers in its collection that perform best in modular assembly (Kim et al., 2011). Preassembled zinc finger arrays for rapid construction of ZFNs are taught in Nat. Methods 8:7.

Additional non-limiting ZFs and AFNs that may be adapted for use in the present invention include those described in WO2010/065123, WO2000/041566, WO2003/080809, WO2015/143046, WO2016/183298, WO2013/044008, WO2015/031619, WO2017/136049, WO2016/014794, WO2017 /091512, WO1995/009233, WO2000/023464, WO2000/042219, WO2002/026960, WO2001/083793; US9428756, US9145565, US8846578, US8524874, US6777185, US659 9692, US7235354, US6503717, US749153 1. US7943553, US7262054, US8680021, US7705139, US7273923, US6780590, US6785613, US7788044, US7177766, US6453242, US6794136, US7358085, US8383766, US7 030215、US7013219、US7361635、US79 39327, US8772453, US9163245, US7045304, US8313925, US9260726, US6689558, US8466267, US7253273, US7947873, US9388426, US8153399, US8569253, US852422 1. US7951925, US9115409, US8772008, US9121072, US9624498, US6979539, US9491934, US6933113, US9567609, US7070934, US9624509, US8735153, US9567573, US6919204, US2002-0081614, US2004-020 3064、US2006-0166263、US2006-029 2621, US2003-0134318, US2006-0294617, US2007-0287189, US2007-0065931, US2003-0105593, US2003-0108880, US2009-0305402, US2008-0209587, US201 3-0123484, US2004-0091991, US2009-0305 977, US2008-0233641, US2014-0287500, US2011-0287512, US2009-0258363, US2013-0244332, US2007-0134796, US2010-0256221, US2005-0267061, US2012 -0204282, US2012-0252122, US2010-03111 24. US2016-0215298, US2008-0031109, US2014-0017214, US2015-0267205, US2004-0235002, US2004-0204345, US2015-0064789, US2006-0063231, US2011-0265198, and US2017-0218349 are all incorporated herein by reference.

Also provided herein are polynucleotides and vectors capable of expressing one or more ZFNs, which can be part of the vector systems of the invention. Such polynucleotides and vectors can be expressed in cells, such as eukaryotic cells, mammalian cells, or human cells. Suitable vectors, cells, and expression systems are described in more detail elsewhere herein and may be suitable for use with TALEs, meganucleases, and CRISPR-Cas nucleases.

In certain embodiments, the sequence-specific nuclease is a meganuclease.

Meganucleases are a class of sequence-specific endonucleases that recognize large DNA target sites (>12 bp). These proteins can cleave unique chromosomal sequences without affecting overall genomic integrity. Meganucleases generate site-specific DNA DSBs and, in the presence of donor DNA (such as that present in a heterologous nucleic acid encompassed or encoded by an engineered retrotranscript of the invention), promote integration of the donor DNA at the cleavage site by homologous recombination (HR).

In certain embodiments, the meganuclease is a homing endonuclease, which is a class of ubiquitous proteins found in eukaryotes, bacteria, and archaea. In certain embodiments, the meganuclease belongs to the LAGLIDADG (SEQ ID NO: 19953) family of homing endonucleases.

In certain embodiments, the meganuclease is I-Scel, I-Cre-I, I-Dmol, or an engineered or naturally occurring variant thereof. The hallmark of these proteins is a well-conserved LAGLIDADG (SEQ ID NO: 19953) peptide motif found in one or two copies, referred to as a (do) decapeptide. Nesting endonucleases that have only one such motif (such as I-Crel or I-Ceu1) function as homodimers. In contrast, larger proteins that carry two (do) decapeptide motifs (such as I-Scel, Pl-Scel, and I-Dmol) are single-chain proteins.

Additional homing nucleases can be found at the homingendonuclease.net website, which provides a database listing the basic properties of the known LAGLIDADG (SEQ ID NO: 19953) homing endonuclease. See also Taylor et al. , Nucleic Acids Research 40 (W1): W110-W116, 2012 (both incorporated herein by reference).

In certain embodiments, the specificity (or polynucleotide recognition) of a meganuclease is modified by changing amino acids within the meganuclease and/or by fusing additional effector domains to the meganuclease.

In certain embodiments, the meganuclease is a megaTAL, which includes a DNA binding domain from a TALE.

In certain embodiments, the meganuclease is engineered to have nickase activity.

Additional suitable natural and engineered meganucleases and megaTALs are described in WO2006/097853, WO2004/067736, WO2012/030747, WO2007/123636, WO2010/001189, WO2018/071565, WO2007/049095, WO2009/068937, WO2010/001189 ... O2005/105989、WO2008/102198、WO2007/057781、WO2019/126558、WO2010/046786、US2010-0151556、US2014-0121115、US2011-0207199、US2012-0301456、US 2013-0189759、 US2011-0158974, US2010-0144012, US2014-0112904, US2013-0196320, US2010-0203031, US2010-0167357, US2012-0272348, US2012-0258537, US2011-0072 527、US2013-01 83282, US2014-0178942, US2012-0260356, US2013-0236946, US2010-0325745, US2011-0041194, US2014-0004608, US2011-0263028, US2011-0225664, US201 3-0145487、US2 013-0045539, US2012-0171191, US2015-0315557, US2014-0017731, US2011-0091441, US2014-0038239, US2010-0229252, US2009-0222937, US2010-014665 1. US2013-00593 87. US2011-0179507, US2013-0326644, US2006-0078552, US2004-0002092, US2012-0052582, US2009-0162937, US2010-0086533, US2009-0220476, US88024 37. US7842489, U S8715992, US8426177, US8476072, US9365864, US9540623, US9273296, US9290748, US8163514, US8148098, US8143016, US8143015, US8133697, US8129134, US8124 369、US811 9361, US7897372, US9683257, US10287626, US10273524, US10000746, US10006052, US7919605, US9018364, US10407672, US8211685, US9365864, US7476500, all of which are incorporated herein by reference.

In certain embodiments, the sequence-specific nuclease is TnpB, which is a programmable RNA-guided DNA endonuclease. It is believed that TnpB is a functional progenitor cell of CRISPR-Cas nuclease.

Transposons are mobile genetic elements that contain only the genes required for their transposition and its regulation. These elements encode the tnpA transposase, which is essential for mobilization, and usually carry an auxiliary tnpB gene, which is dispensable for transposition. TnpB has been shown to be a nuclease that is guided by RNA derived from the right-hand element of the transposon, cleaves DNA next to the 5'-TTGAT transposon-associated motif, and can be reprogrammed to cleave DNA target sites in human cells.

In certain embodiments, TnpB is from the IS200/IS605 family of D. radiodurans ISDra2.

In certain embodiments, TnpB is from transposon PsiTn554.

In certain embodiments, the sequence-specific nuclease is a TnpB-like protein, such as Fanzor1 or Fanzor2. These proteins are widely present in a variety of eukaryotic transposable elements (TEs) and large double-stranded DNA (dsDNA) viruses that infect eukaryotes. Fanzor and TnpB proteins share the same conserved amino acid motif in their C-terminal half: D-X(125, 275)-[TS]-[TS]-X-X-[C4 zinc finger]-X(5,50)-RD, but their N-terminal regions are highly variable. Fanzor1 proteins are frequently captured by DNA transposons from different superfamilies, including Helitron, Mariner, IS4-like, Sola, and MuDr. In contrast, Fanzor2 proteins are only found in some IS607-type elements.

In certain embodiments, the sequence-specific nuclease is IscB.

ISC (Insertion Sequence Cas9-like) is a group of emerging bacterial and archaeal DNA transposons that encode Cas9 homologs. The protein containing two nuclease domains encoded by the ISC transposon may be the ancestor of the CRISPR-related Cas9. The homology region includes an arginine-rich helix and an HNH nuclease domain inserted into the RuvC-like nuclease domain. However, the ISC gene is not related to the Cas gene or CRISPR. It represents a unique group of non-autonomous transposons with many different full-length ISC transposon families. Its terminal sequence (in detail, the 3' end) is similar to those of the IS605 superfamily transposons, which are activated by the Y1 tyrosine translocase encoded by the TnpA gene and usually also encode TnpB proteins containing a RuvC-like nuclease domain. The terminal regions of ISC and IS605 transposons contain palindromic structures that are likely recognized by Y1 transposase. These two sets of transposons precisely insert into the middle or upstream of the specific 4-bp target site without duplication of the target site.

In certain embodiments, the sequence-specific nuclease is a restriction endonuclease (RE), such as an RE having a strict/long recognition sequence of at least 8 nt.

In certain embodiments, the RE is a rare-cutting RE with seven and eight base pair recognition sequences. Exemplary rare-cutting RE enzymes include NotI, which cuts after the first GC of the 5'-GCGGCCGC-3' sequence (SEQ ID NO: 19417).

In certain embodiments, the components of the system ( e.g. , ncRNA or msDNA encoded by a retrotranscript in complex with RT, sequence-specific nucleases, and DNA repair regulatory biomolecules) can form multiple complexes in a so-called split-complex configuration. Multiple complexes can be combined together to form a functional complex.

For example, in some embodiments, the first component of the system can be a split protein or domain. A fragment of the split protein or domain can be combined with a second component of the system, and another fragment of the split protein or domain can be combined with a third component of the system. The two fragments of the split protein or domain can be combined together ( e.g. , together with other components of the system) to form a functional complex.

In certain embodiments, the split protein or domain is a sequence-specific nuclease, such as a CRISPR/Cas effector enzyme ( e.g., a Cas protein such as Cas9 or Cas12), a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE).

In certain embodiments, the split protein or domain is a reverse transcriptase domain.

In certain embodiments, the split protein or domain is a DNA repair regulatory biomolecule. For example, a first fragment of a sequence-specific nuclease can be conjugated to a reverse transcriptase domain, and a second fragment of a sequence-specific nuclease can be conjugated to a DNA repair regulatory biomolecule. The two fragments of the split protein or domain can be combined together ( e.g. , together with the reverse transcriptase domain and the DNA repair regulatory biomolecule) to form a functional complex. The combination between the parts of the split protein or domain can be carried out by an adaptor protein or linker described herein ( e.g. , those used to combine the Cas protein with the functional domain).

In certain embodiments, the split protein or domain is split in the sense that the two parts of the split protein or domain substantially comprise a functional split protein or domain. Ideally, the split should always leave the catalytic domain unaffected. The split protein or domain may act as a sequence-specific nuclease or it may be a dead Cas, which is essentially an RNA binding protein with little or no catalytic activity due to typical mutations in its catalytic domain.

Each fragment of the split protein or domain can be fused to a dimerization partner. For example, a rapamycin-sensitive dimerization domain enables a chemically induced split protein or domain to temporally control the activity of the split protein or domain. Thus, the split protein or domain can become chemically induced by splitting into two fragments, and the rapamycin-sensitive dimerization domain can be used for controlled reassembly of the split protein or domain. The two parts of the split protein or domain can be considered as the N' terminal portion and the C' terminal portion of the split protein or domain. The fusion is usually at the split point of the split protein or domain. In other words, the C' terminus of the N' terminal portion of the split protein or domain is fused to one half of the dimer, and the N' terminus of the C' terminal portion is fused to the other half of the dimer.

The split protein or domain is not necessarily split in the sense of newly creating a break. The split point is usually designed in silico and cloned into a construct. The two parts of the split protein or domain (the N' terminal and C' terminal parts) together form a complete split protein or domain, which preferably comprises at least 70% or more wild-type amino acids (or nucleotides encoding the amino acids), at least 80% or more, at least 90% or more, at least 95% or more, and at least 99% or more wild-type amino acids (or nucleotides encoding the amino acids). When the two parts are combined together, the desired split protein or domain function is restored or reconstructed. The dimer can be a homodimer or a heterodimer.

In certain embodiments, the protein components of the system ( e.g. , RT, sequence-specific nuclease (CRISPR/Cas effector enzyme, ZFN, TALEN, meganuclease, TnpB, IscB or restriction endonuclease (RE)), and DNA repair regulatory biomolecule) may further comprise one or more additional functional domains.

In some embodiments, the functional domain comprises a nuclear localization signal (NLS). In some embodiments, one or more C-terminal or N-terminal NLSs are linked. In some embodiments, a C-terminal NLS is linked for expression and nuclear targeting in eukaryotic cells ( e.g. , human cells). In some embodiments, the NLS may be at a position that is not at the C-terminus or N-terminus, for example, the NLS may be between two polypeptides.

Non-limiting examples of NLSs include NLS sequences derived from: NLS of SV40 virus large T antigen; NLS from nucleoplasmin ( e.g. , nucleoplasmin bipartite NLS); c-myc NLS; hRNPAl M9 NLS; NLS from the IBB domain of importin-α; NLS of myoma T protein; NLS of human p53; NLS of mouse c-abl IV; NLS of influenza virus NS1; NLS of hepatitis virus delta antigen; NLS of mouse Mxl protein; NLS of human poly (ADP-ribose) polymerase; and NLS of steroid hormone receptor (human) glucocorticoid. Exemplary NLS sequences include those described in paragraph [00106] of Feng Zhang et al . (WO2016/106236), all of which are incorporated herein by reference.

In certain embodiments, the functional domain comprises at least two NLS domains. One or more NLS domains may be located at or near or near the end of the polypeptide, and if there are two or more NLS, each of the two may be located at or near or near the end of the polypeptide.

In any fusion protein, the fusion between two domains (such as RT and Cas enzymes, or Cas and DNA repair regulatory biomolecules) can be connected by a linker. As used herein, "linkers" include peptides that join two proteins or domains to form a fusion protein. Typically, such molecules do not have specific biological activity other than joining or preserving some minimum distance or other spatial relationship between proteins/domains. However, in some embodiments, the linker can be selected to affect some properties of the linker and/or fusion protein, such as the folding, net charge or hydrophobicity of the linker. Suitable linkers for use in the present disclosure are well known to those skilled in the art and include, but are not limited to, linear or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. However, as used herein, the linker may also be a covalent bond (carbon-carbon bond or carbon-heteroatom bond).

In certain embodiments, the linker is used to separate a sequence-specific nuclease (CRISPR/Cas effector, ZFN, TALEN, meganuclease, TnpB, IscB, or restriction endonuclease (RE)) and a RT and/or DNA repair regulatory biomolecule at a distance sufficient to ensure that each protein domain retains its desired functional properties.

Preferred peptide linker sequences adopt a flexible extended conformation and do not show a tendency to develop an ordered secondary structure. In certain embodiments, the linker can be a chemical moiety, which can be a monomer, dimer, multimer or polymer. In certain embodiments, the linker comprises amino acids. Typical amino acids in flexible linkers include Gly, Asn and Ser. Therefore, in specific embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near-neutral amino acids (such as Thr and Ala) can also be used in linker sequences.

In certain embodiments, the linker comprises a GlySer-rich sequence, such as a _GnS linker (n = 1, 2, 3, 4 or 5, such as GS or _G4S ) (SEQ ID NO: 19946) or repeats thereof (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 repeats, optionally with a total length of about 4-30 residues, 4-20 residues or 4-10 residues).

In some embodiments, the linker comprises a G ₄ S linker having 3, 6, 9, or 12 repeats.

In certain embodiments, the linker is a linker disclosed in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., PNAS USA 83: 8258-62, 1986; US Pat. No. 4,935,233; or US Pat. No. 4,751,180, all of which are incorporated by reference.

In certain embodiments, the linker comprises a GlySer linker, such as GGS, GGGS (SEQ ID NO: 19947), GSG, GGGGS (SEQ ID NO: 19948), optionally with 3 (such as (GGS) ₃ , (SEQ ID NO: 19949) (GGGGS) ₃ ) (SEQ ID NO: 19950), 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more repeats to provide an appropriate length.

In certain embodiments, the linker comprises (GGGGS) _3-15 (SEQ ID NO: 19951), such as (GGGGS) _3-11 (SEQ ID NO: 19951), for example , having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 repeats of GGGGS (SEQ ID NO: 19951).

In certain embodiments, the linker comprises LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19418).

In another embodiment, the linker is an XTEN linker.

In certain embodiments, the N-terminal and/or C-terminal NLS also serves as a linker ( e.g. , PKKKRKVEASSPKKRKVEAS (SEQ ID NO: 19420)).

gRNA and various nucleases and fusions thereof can be provided in the form of proteins, where the nuclease is complexed with the gRNA, or can be provided by nucleic acids encoding RNA-guided nucleases, such as RNA ( e.g. , messenger RNA) or DNA (expression vector). In some embodiments, RNA-guided nucleases and gRNA are both provided by vectors. Both can be expressed by a single vector, or separately on different vectors. Vectors encoding RNA-guided nucleases and gRNAs can be included in a vector system comprising engineered retrotransposons msr gene, msd gene, and ret gene sequences.

Codon usage can be optimized to improve the production of engineered retrotranscripts, such as retrotranscriptases, ncRNAs, and/or RNA-guided nucleases, in a particular cell or organism. For example, a nucleic acid encoding an ncRNA, an RNA-guided nuclease, or a reverse transcriptase can be modified to replace codons with a higher usage frequency in a particular cell, such as a eukaryotic cell (e.g., a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other relevant host cell), as compared to a naturally occurring polynucleotide sequence. When a nucleic acid encoding a reverse transcriptase or ncRNA is introduced into a cell, the protein can be expressed transiently, conditionally, or constitutively in the cell. F. RT-PN fusion protein

The editing system based on recombinant retrotransposons described herein contemplates a fusion protein comprising a programmable nuclease (PN) and RT optionally joined by a linker. The present application contemplates combining any suitable programmable nuclease and RT (e.g., the retrotransposons RT of Table A) in a single fusion protein. In one embodiment, RT is joined to the N-terminus of PN. In another embodiment, RT is joined to the C-terminus of PN. Examples of PN and RT are each defined herein.

In various embodiments, the fusion proteins may comprise any suitable structural configuration. For example, the fusion protein may comprise PN fused to RT from the N-terminus to the C-terminus. In other embodiments, the fusion protein may comprise RT fused to NP from the N-terminus to the C-terminus. The fusion domains may be joined by a linker (e.g., an amino acid sequence) as appropriate. G. Retrotranscript-based gene editing system

The present disclosure relates to novel genome editing systems including retrotranscripts (including retrotranscript RT and ncRNA). In exemplary embodiments, the editing systems include: (a) one or more retrotranscript RT polypeptide sequences, which contain at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% sequence identity with any one of the amino acid sequences or polynucleotide sequences encoding the amino acid sequences in Table A (also provided in Table A); (b) (a) one or more retrotranscript ncRNA polynucleotide sequences comprising at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% sequence identity to any one of the amino acid sequences of Table B, wherein in one embodiment, the retrotranscript RT and the ncRNA are a homologous pair; (c) one or more programmable nucleases (e.g., TnpB, Cas12a or Cas9 as disclosed herein); and (d) one or more polynucleotide sequences comprising a guide RNA, wherein the guide RNA comprises a complementary sequence to the targeting polynucleotide sequence and is bound to the programmable nuclease.

In other aspects, the gene editing system based on the retrotranscript may include one or more additional auxiliary proteins with genome modification functions, including recombinases, invertases, nucleases, polymerases, ligases, deaminases, reverse transcriptases or epigenetic modification functions. In various embodiments, the auxiliary protein can be provided separately. In other embodiments, the auxiliary protein can be fused to the retrotranscript component (e.g., fused to the retrotranscript RT) with a linker as appropriate.

In various embodiments, the genome editing system may include a guide RNA that hybridizes to one or more targeting polynucleotide sequences. In a preferred embodiment, the guide RNA of the genome editing system comprises 12-40 nucleotides.

In various embodiments, the genome editing system comprises a targeting polynucleotide sequence comprising one or more protospacer adjacent motif (PAM) recognition domains selected from 5'-TTTN-3', 5'-TTN-3', 5'-TNN-3', 5'-TTV-3' or 5'-TTTV-3', wherein N = A, T, C or G and V = A, C or G. In additional embodiments, the targeting polynucleotide sequence comprises one or more relaxed PAM recognition domains. Jacobsen, Thomas et al. "Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs." Nucleic acids research Vol. 48, 10 (2020): 5624-5638. doi:10.1093/nar/gkaa272. Previous work has demonstrated that the requirement for an extended TTTV protospacer base-adjacent motif (PAM) was overcome by expanding the targeting range of non-canonical PAMs such as ATTA, CTTA, GTTA, and TCTA. Kleinstiver, Benjamin P, et al. “Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.” Nature biotechnology, vol. 37, 3 (2019): 276-282. doi:10.1038/s41587-018-0011-0. Most Cpf1 nucleases require a thymine-rich PAM. Different studies have demonstrated an increase in the Cpf1 targeting range using in vitro and in vivo ( E. coli) PAM characterization assays. Zhang, Xiaochun et al. "Multiplex gene regulation by CRISPR-ddCpf1." Cell discovery 3.1 (2017): 1-9. Two Cpf1 endonucleases, AsCpf1 and LbCpf1, require TTTV as the PAM sequence, where V can be an A, C, or G nucleotide. Mutations at positions S542R/K607R and S542R/K548V/N552R generate AsCpf1 variants that recognize TYCV and TATV PAMs, respectively, where Y can be C or T. Gao, Linyi et al. "Engineered Cpf1 variants with altered PAM specificities." Nature biotechnology 35.8 (2017): 789-792. AsCpf1 shows increased activity toward TTTV PAM and decreased activity toward TTTT PAM Kim, Hui K. et al. “In vivo high-throughput profiling of CRISPR–Cpf1 activity.” Nature methods 14.2 (2017): 153-159.

Therefore, it is within the scope of the present disclosure to design the editing system to recognize altered PAM recognition domains for genome editing. In preferred embodiments, the programmable polypeptide assembly recognizes one or more non-canonical PAM sequences in a targeted polynucleotide sequence, the PAM being upstream of a crRNA complementary DNA sequence on a non-target strand. In a related embodiment, the gRNA has an eight nucleotide seed sequence located 5' to the spacer and adjacent to a PAM sequence on a targeted polynucleotide sequence.

In further embodiments, one or more polypeptide sequences and one or more polynucleotide sequences comprising a cognate guide RNA of the genome editing system form a ribonucleoprotein complex.

In various embodiments in which the programmable nuclease is a V-type enzyme (e.g., Cas12a), one or more polypeptide sequences of the genome editing system include: ●   One or more α-helical recognition lobe (REC) and nuclease lobe (NUC); ●   Wedge (WED), α-helical recognition lobe (REC), PAM interaction (PI), ●   RuvC nuclease, bridge helix (BH), and NUC domains; or ●   One or more domains selected from RuvC, REC, WED, BH, PI, and NUC domains.

In various embodiments in which the programmable nuclease is a type II enzyme (e.g., Cas9), one or more polypeptide sequences of the genome editing system comprises: ●     an α-helical lobe, and ●     a nuclease lobe comprising two nuclease domains (a RuvC domain that cleaves non-target DNA strands and a HNH domain that cleaves target strands).

In various embodiments, the molecular weight of the programmable nuclease component is characterized by a molecular weight of about 50 kDa - 100 kDa, 100 kDa - 200 kDa, 200 kDa - 500 kDa.

In additional embodiments, the polypeptide sequence comprises at least one activity selected from the following: endonuclease activity; endoribonuclease activity, or RNA-guided DNase activity. In such embodiments, the homologous guide RNA and Cas12a protein modify the target polynucleotide sequence of the host cell genome. In some cases, the target polynucleotide sequence is modified by inserting, deleting or changing one or more base pairs at the target polynucleotide sequence in the host cell genome.

In related embodiments, the genome editing system is characterized by enhanced efficiency and accuracy of site-directed integration. Preferably, the efficiency and accuracy of site-directed integration achieved by the genome editing system is enhanced by staggered overhangs on the donor nucleic acid sequence. In certain embodiments, the targeting polynucleotide sequence is double-stranded and contains a 5' overhang, wherein the overhang preferably comprises five nucleotides.

In various other embodiments, the polypeptide of the genome editing system comprises one or more mutations. More preferably, the mutation encodes a nuclease-deficient polypeptide. In various embodiments, the genome editing system comprises a fusion of one or more deaminases and a nuclease-deficient polypeptide. Preferably, one or more deaminases of the genome editing system are selected from adenine deaminases or cytosine deaminases. The use of cytidine deaminases and adenosine deaminases alkali editing is disclosed in U.S. Patent No. 9,840,699. One method is to produce a Cas12a fusion protein (preferably, an inactive or nickase variant) and an alkali editor or an active domain of an alkali editor. Cytidine deaminase and adenosine deaminase base editing are disclosed in U.S. Patent No. 9,840,699. In various embodiments, the compositions include contacting a targeting polynucleotide sequence with a fusion protein comprising Cas12a and one or more base editing polypeptides (such as deaminase); and a gRNA that targets the fusion protein to a targeting polynucleotide sequence of a DNA strand. Therefore, the fusion of one or more deaminases with the nuclease-deficient polypeptide of the Cas12a genome editing system enables base editing of DNA and/or RNA. In selected embodiments, the system modifies one or more nucleobases on DNA and RNA. In related embodiments, the system enables multiple gene editing. Preferably, the genome editing system comprises a single crRNA. Preferably, the system enables the simultaneous targeting of multiple genes.

In other embodiments, the programmable polypeptide is operably linked to a nuclear localization signal (NLS). Preferably, the programmable polypeptide comprises an NLS at the N-terminus or C-terminus or both or multiple NLSs on the Cas12a polypeptide. In some embodiments, the polypeptide connected to the NLS further comprises crRNA to form a ribonucleoprotein complex. In some embodiments, the polypeptide comprises one or more NLS repeats at the N-terminus or C-terminus of the polypeptide.

In selected embodiments, one or more polypeptide sequences of the genome editing system comprises a modification, wherein the modification comprises a nuclease-deficient polypeptide (dCas). In related embodiments, the guide RNA of the genome editing system comprises a priming editing guide RNA (pegRNA). Preferably, the pegRNA of the genome editing system hybridizes with the target polynucleotide sequence and acts as a primer for one or more reverse transcriptases. More preferably, the pegRNA of the genome editing system binds to the nicked strand to initiate repair by a reverse transcriptase using a repair template.

In various additional embodiments, the nuclease-deficient polypeptide of the genome editing system comprises nickase activity. Preferably, the genome editing system comprises a fusion of one or more reverse transcriptases and a nuclease-deficient Cas (dCas). In certain embodiments, the fusion of one or more reverse transcriptases is selected from a component/modification donor template selected as appropriate .

In one embodiment, the compositions and systems herein may further include one or more donor templates for editing. In some cases, the donor template may include one or more polynucleotides. In some cases, the donor template may include the coding sequence of one or more polynucleotides. The donor template may be a DNA template. It may be single-stranded or double-stranded. It may also be circular single-stranded or double-stranded. It may also be linear single-stranded or double-stranded. Without being bound by theory, the donor template may be integrated into the genome after targeted cleavage by the programmable nuclease cutter described herein through the cell repair machinery including HDR and NHEJ. In various embodiments, the HDR donor is formed by reverse transcription of ncRNA and forms its RT-DNA portion.

Donor templates that can be integrated into ncRNA and expressed as RT-DNA products can be used to edit target polynucleotides. In some cases, the donor polynucleotides include one or more mutations to be introduced into the target polynucleotides. Examples of such mutations include substitutions, deletions, insertions, or combinations thereof. Mutations can cause open reading frame shifts on target polynucleotides. In some cases, the donor templates change the stop codons in the target polynucleotides. For example, the donor template can correct premature stop codons. Correction can be achieved by deleting the stop codon or introducing one or more mutations into the stop codon. In other exemplary embodiments, the donor template solves the loss of functional mutations, deletions, or translocations that may occur, for example, in certain disease backgrounds by inserting or restoring a functional copy of a gene or a functional fragment thereof, or a functional regulatory sequence or a functional fragment of a regulatory sequence. A functional fragment refers to a less than complete copy of a gene achieved by providing a nucleotide sequence sufficient to restore the functionality of a wild-type gene or a non-coding regulatory sequence (e.g., a sequence encoding a long non-coding RNA). In certain exemplary embodiments, the system disclosed herein can be used to replace a single allele of a defective gene or a defective fragment thereof. In another exemplary embodiment, the system disclosed herein can be used to replace two alleles of a defective gene or a defective gene fragment. A "defective gene" or "defective gene fragment" is a gene or a portion of a gene that, when expressed, is unable to generate a functional protein or non-coding RNA having the functionality of the corresponding wild-type gene. In certain exemplary embodiments, these defective genes may be associated with one or more disease phenotypes. In certain exemplary embodiments, the defective gene or gene fragment is not replaced, but the system described herein is used to insert a donor template encoding a gene or gene fragment that compensates or exceeds the expression of the defective gene, so that the cell phenotype associated with the defective gene expression is eliminated or changed to a different or desired cell phenotype.

In one embodiment of the present invention, the donor template may include but is not limited to a gene or gene fragment, an RNA transcript encoding a protein or to be expressed, a regulatory element, a repair template and the like. According to the present invention, the donor template may include left and right sequence elements that function together with the transposition component mediating insertion.

In some cases, the donor template manipulates a splice site on the target polynucleotide. In some instances, the donor template disrupts the splice site. Disruption can be achieved by inserting a polynucleotide into the splice site and/or introducing one or more mutations into the splice site. In some instances, the donor template can restore the splice site. For example, the polynucleotide can include a splice site sequence.

The donor template to be inserted can have a size of 10 base pairs or nucleotides to 50 kb in length, for example, 50 to 40 k, 100 and 30 k, 100 to 10000, 100 to 300, 200 to 400, 300 to 500, 400 to 600, 500 to 700, 600 to 800, 700 to 900, 800 to 1000, 900 to 1100, 1000 to 1200, 1100 to 1300, 1200 to 1400, 1300 to 1500, 1400 to 1600, 1500 to 1700, 600 to 1800, 1700 to 1900, 1800 to 2000 base pairs (bp) or nucleotides in length.

In some embodiments, the heterologous nucleic acid sequence is a donor DNA template that can be integrated into the host genome via HDR. In other embodiments, the heterologous nucleic acid sequence is a donor DNA template that can be integrated into the host genome via NHEJ.

In certain embodiments, the heterologous nucleic acid comprises or encodes a donor/template sequence, wherein the donor/template corrects/repairs/removes a mutation at a target genome site. For example, the mutation may be a mutant exon in a disease gene.

In certain embodiments, the donor/template may encode or contain functional DNA elements, such as promoters, enhancers, protein binding sequences, methylation sites, or homology regions for assisting gene editing.

"Donor DNA" or "donor DNA template" means a DNA segment (which can be single-stranded or double-stranded DNA) to be inserted at a site to be cleaved by a gene-editing nuclease (e.g., Cas9 or Cas12a nuclease) (e.g., after dsDNA cleavage, after nicking of the target DNA, after double nicking of the target DNA, and the like). The donor DNA template may contain sufficient homology to the genome sequence at the target site, such as 70%, 80%, 85%, 90%, 95% or 100% homology to the nucleotide sequence flanking the target site, such as within about 50 bases or less of the target site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases or directly flanking the target site to support homology-directed repair between the donor DNA template and the genome sequence carrying homology thereto. In the case of repair by NHEJ, the donor DNA template does not require homology to the site of its targeted editing. The donor template may be integrated into the ncRNA of the retrotranscript and appear as an RT product in the form of RT-DNA.

Sequence homology of about 25, 50, 100 or 200 nucleotides or more (or any integer value between 10 and 200 nucleotides or more) between the donor DNA template and the genome sequence can support homology-directed repair. The donor DNA template can have any length, such as 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc. Suitable donor DNA templates can be 50 nucleotides to 100 nucleotides, 100 nucleotides to 500 nucleotides, 500 nucleotides to 1000 nucleotides, 1000 nucleotides to 5000 nucleotides, or 5000 nucleotides to 10,000 nucleotides or more than 10,000 nucleotides in length.

As described above, in some embodiments, the donor DNA template comprises a first homology arm and a second homology arm. The first homology arm is located at or near the 5' end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to the first nucleotide sequence in the target nucleic acid. The second homology arm is located at or near the 3' end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to the second nucleotide sequence in the target nucleic acid. The first and second homology arms can each independently have a length of about 10 nucleotides to 400 nucleotides; for example, 10 nucleotides (nt) to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, 45 nt to 50 nt, 50 nt to 75 nt, 75 nt to 100 nt, 100 nt to 125 nt, 125 nt to 150 nt, 150 nt to 175 nt, 175 nt to 200 nt, 200 nt to 225 nt, 225 nt to 250 nt, 250 nt to 275 nt, 275 nt to 300 nt, 325 nt to 350 nt, 350 nt to nt to 375 nt or 375 nt to 400 nt.

In certain embodiments, the donor DNA template is used to edit the target nucleotide sequence. In certain embodiments, the donor DNA template comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the mutation causes an open reading frame shift on the target polynucleotide. In certain embodiments, the donor polynucleotide changes the stop codon in the target polynucleotide. In certain embodiments, the donor polynucleotide corrects a premature stop codon. Correction can be achieved by deleting the stop codon or by introducing one or more sequence changes to change the stop codon to a codon. In certain embodiments, the donor polynucleotide addresses loss of function mutations, deletions or translocations that may occur, for example, in certain disease settings by inserting or restoring a functional copy of a gene or a functional fragment thereof, or a functional regulatory sequence or a functional fragment of a regulatory sequence. Functional fragments include fragments of nucleotide sequence that are less than a complete copy of a gene but that otherwise provide sufficient nucleotide sequence to restore the functionality of a wild-type gene or non-coding regulatory sequence ( e.g. , a sequence encoding a long non-coding RNA).

In some embodiments, the donor DNA template can be used to replace a single allele of a defective gene or a defective fragment thereof. In another embodiment, the donor DNA template is used to replace both alleles of a defective gene or a defective gene fragment. A "defective gene" or "defective gene fragment" is a gene or a portion of a gene that, when expressed, fails to generate a functional protein or non-coding RNA having the functionality of the corresponding wild-type gene.

In certain exemplary embodiments, these defective genes may be associated with one or more disease phenotypes. In certain exemplary embodiments, the defective gene or gene fragment is not replaced, but a donor polynucleotide encoding a gene or gene fragment that compensates or exceeds the expression of the defective gene is inserted using a heterologous nucleic acid, so that the cell phenotype associated with the expression of the defective gene is eliminated or changed to a different or desired cell phenotype. This can be achieved by including a coding sequence for a therapeutic protein (such as a therapeutic antibody or a functional fragment thereof, or a wild-type form of a defective protein associated with one or more disease phenotypes).

In certain embodiments, the donor may include, but is not limited to, a gene or gene fragment, an RNA transcript encoding a protein or to be expressed, a regulatory element, a repair template, and the like. According to the present invention, the donor polynucleotide may include left and right sequence elements that function with the transposition component mediating insertion.

In some embodiments, the donor DNA template manipulates the splice site on the target polynucleotide. In some embodiments, the donor DNA template disrupts the splice site. Disruption can be achieved by inserting a polynucleotide into the splice site and/or introducing one or more mutations into the splice site. In some embodiments, the donor polynucleotide can restore the splice site. For example, the polynucleotide can include a splice site sequence.

In certain embodiments, the donor DNA template to be inserted has a size of 10 bp to 50 kb in length, such as 50 bp to about 40 kb, 100 bp to about 30 kb, 100 bp to about 10 kb, 100 bp to 300 bp, 200 bp to 400 bp, 300 bp to 500 bp, 400 bp to 600 bp, 500 bp to 700 bp, 600 bp to 800 bp, 700 bp to 900 bp, 800 bp to 1000 bp, 900 bp to 1100 bp, 1000 bp to 1200 bp, 1100 bp to 1300 bp, 1200 bp to 1400 bp, 1300 bp to 1500 bp, 1400 bp to 1600 bp, or bp, 1500 bp to 1700 bp, 1600 bp to 1800 bp, 1700 bp to 1900 bp, 1800 bp to 2000 bp nucleotide length.

In certain embodiments, the homology arms at one or both ends of the sequence to be inserted are independently about 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp or 150 bp.

The first homology arm and the second homology arm of the donor DNA are flanked by nucleotide sequences to be introduced into the target nucleic acid ("related nucleotide sequences" or "intervening nucleotide sequences"). The related nucleotide sequences may include: i) nucleotide sequences encoding related polypeptides; ii) nucleotide sequences encoding gene exons; iii) promoter sequences; iv) enhancer sequences; v) nucleotide sequences encoding non-coding RNA; or vi) any combination of the foregoing.

Donor DNA can provide gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc. For example, donor DNA can be used to add (e.g., insert or replace) nucleic acid material to target DNA (e.g., to "knock in" nucleic acids encoding proteins, siRNAs, miRNAs, etc.), add tags (e.g., 6xHis, fluorescent proteins (e.g., green fluorescent proteins; yellow fluorescent proteins, etc.), hemagglutinin (HA), FLAG, etc.), add regulatory sequences to genes (e.g., promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, enhancers, etc.), modify nucleic acid sequences (e.g., introduce mutations), and the like. For example, the donor DNA can be used to modify DNA in a site-specific (i.e., "targeted") manner; for example, gene knockout, gene knock-in, gene editing, gene tagging, etc., such as for use in, for example, gene therapy, e.g., to treat disease; or as an antiviral, antipathogenic, or anticancer therapeutic, to generate genetically modified organisms in agriculture, to mass produce proteins by cells for therapeutic, diagnostic, or research purposes, to induce high-potential stem cells, for biological research, to target pathogenic genes for deletion or replacement, etc.

In some cases, the donor DNA comprises a nucleotide sequence encoding a polypeptide of interest. The polypeptide of interest includes, for example, a) a functional form of a polypeptide comprising one or more amino acid substitutions, insertions and/or deletions and exhibiting reduced function, for example, where the reduced function is associated with or causes a pathological disorder; b) a fluorescent polypeptide; c) a hormone; d) a receptor for a ligand; e) an ion channel; f) a neurotransmitter; g) and the like.

In some cases, the donor DNA comprises a nucleotide sequence encoding a wild-type protein lacking in the recipient cell. In some cases, the donor DNA encodes a wild-type factor involved in coagulation (e.g., factor VII, factor VIII, factor IX and the like). In some cases, the donor DNA comprises a nucleotide sequence encoding a therapeutic antibody. In some cases, the donor DNA comprises a nucleotide sequence encoding an engineered protein or receptor. In some cases, the engineered receptor is a T cell receptor (TCR), a natural killer (NK) receptor (NKR), or a B cell receptor (BCR). In some cases, the engineered TCR or NKR targets a cancer marker (e.g., a polypeptide expressed (e.g., overexpressed) on the surface of a cancer cell). In some cases, the donor DNA comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some cases, the CAR targets a cancer marker. Donor DNA encoding the CAR, TCR and/or NCR proteins can be folded into a DNA origami structure (DNA nanostructure) and delivered to T cells or NK cells in vitro or in vivo.

Non-limiting examples of polypeptides that may be encoded by the donor DNA include, for example, IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostaglandin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP binding cassette, subfamily G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostaglandin) receptor (IP)), KCNJ11 (potassium inward rectifier channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB (platelet-derived growth factor receptor, beta polypeptide), CCNA2 (cytokine A2), PDGFB (platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassium inward rectifier channel, subfamily J, member 5), KCNN3 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3), CAPN10 (calcification 10), PTGES (prostaglandin E synthetase), ADRA2B (adrenaline-stimulated alpha-2B-receptor), ABCG5 (ATP-binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calcification 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (Cryptidys elegans)), ACE angiotensin I convertase (peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon beta 2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (connexin, fibronectin activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, containing C1Q and collagen domains), APOB (apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E), LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriuretic peptide pro-actin B), NOS3 (nitric oxide synthase 3 (endothelial cells)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (fibroblast lysinogen activator, tissue), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (cholesterol ester transfer protein, plasma), AGTR1 (angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (insulin-like growth factor 1 (interleukin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (peroxisomal phosphatase 1), KNG1 (kininogen 1), CCL2 (chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), vWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, β1), NPPA (natriuretic peptide propromoter A), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA (lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (heme phenotype), F3 (coagulation factor III (thrombin, tissue factor)), CST3 (cystatin C), COG2 (oligomeric Golgi complex component 2), MMP9 (matrix metalloproteinase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa Type IV collagenase)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (ring opening) 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8), PROK1 (prokinesin 1), CBS (cystathionine-β-synthase), NOS2 (nitric oxide synthase 2, inducing), TLR4 (toll-like receptor 4), SELP (selectin P (granule membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP binding cassette, subfamily A (ABC1), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low-density lipoprotein receptor), GPT (glutamine-pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interferon-γ-inducing factor)), NOS1 (nitric oxide synthase 1 (neuron)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrillogen beta chain), HGF (hepatocyte growth factor (hepatocyte poi-toiin A; proliferating factor)), ILIA (interleukin 1, alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogene homolog 1), LIPC (lipase, liver), HSPD1 (heat shock protein 60 kDa 1 (chaperone)), MAPK14 (mitogen activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, β3 (thrombopoietin 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (coagulation factor X), CP (plasma cucocyanin (ferrooxidase)), TNFRSF11B (tumor necrosis factor receptor subfamily, member lib), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (erythroblastic leukemia virus (v-erb-b) oncogene homolog, avian)), MMP2 (matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)), PLG (fibrolysinogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytoma viral oncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymotrypsin 1, mast cell), PLAU (fibronectin activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), beta polypeptide 3), ADRB2 (adrenaline-stimulated beta-2-receptor, surface), APOA5 (apolipoprotein A-V), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (prothrombin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonic acid 5-lipoxygenase), HLA-DRB1 (major histocompatibility complex, class II, DRβ1), PARP1 (poly (ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (peroxisomal 2), AGER (advanced glycation end product specific receptor), IRS1 (insulin receptor receptor 1), PTGS1 (prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin convertase 1), F7 (coagulation factor VII (plasma prothrombin conversion accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor subfamily, member 6)), ABCB1 (ATP binding cassette, subfamily B (MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor subfamily member 5), LCAT (lecithin-cholesterol transferase), CCR 5 (chemokine (C-C motif) receptor 5), MMP1 (matrix metalloproteinase 1 (interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM (adrenomedulin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription 3 (acute phase response factor)), MMP3 (matrix metalloproteinase 3 (matrilysin 1, procollagen)), ELN (elastin), USF1 (upstream transcription factor 1), CFH (complement factor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrix metalloproteinase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenaline stimulating beta-1-receptor), CYBA (cytochrome b-245, alpha polypeptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamicinyltransferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia-inducing factor 1, alpha subunit (basal helix-loop-helix transcription factor)), CXCR4 (interleukin (C-X-C motif) receptor 4), PROC (protein C (inactivator of coagulation factors Va and Villa)), SCARB1 (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-related alpha), PLTP (phospholipid transfer protein), ADD1 (adductin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid Al), KCNH2 (potassium voltage-gated channel, subfamily H (eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A) (atrial natriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viral oncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isomerase G (cyclophilin G)), IL1R1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinol binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (Cell cycle protein-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basal)), EDNRB (endothelin receptor type B), ITGA2 (integrin, α2 (CD49B, α2 subunit of VLA-2 receptor)), CAB INI (calcineurin phosphatase binding protein 1), SHBG (sex hormone binding globulin), HMGB1 (high mobility group box 1), HSP90B2P (heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subgroup A, polypeptide 4), GJA1 (gap junction protein, α1, 43 kDa), CAV1 (cavolin 1, caveolin, 22 kDa), ESR2 (estrogen receptor 2 (ERβ)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (neural growth factor (β polypeptide)), SP1 (Sp 1 transcription factor), TGIF1 (TGFB inducing factor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), EGF (epidermal growth factor (β-urogastatin)), PIK3CG (phosphoinositide-3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like family, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (protofibrillin 1), CHKA (choleline kinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursor protein), CTNNB1 (trencin (calcified mucin-related protein), β1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, β1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member Al), CX3CR1 (interleukin (C-X3-C motif) receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IE17A (interleukin 17A), PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferase μ1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor XIII, Al polypeptide), TTR (transthyretin), FABP4 (fatty acid binding protein 4, adipocyte), PON3 (peroxisomal phosphatase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor subfamily, member IB), HTR2A (5-hydroxytryptamine (serotonin) receptor 2A), CSF3 (colony stimulating factor 3 (granulocyte)), CYP2C9 (cytochrome P450, family 2, subgroup C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, subgroup B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulating factor 2 (granulocyte-macrophage)), KDR (kinase insert domain receptor (type III body tyrosine kinase)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondria)), TCF7L2 (transcription factor 7-like 2 (T cell-specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor (erythroid 2)-like 2), NOTCH1 (Notch homolog 1, translocation-related (Drosophila)), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide Al), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin (silent mate information regulator 2 homolog) 1 (brew yeast)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing hormone-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, pappa lysine 1), ARR3 (arrestin 3, retinal (X-(arrestin)), NPPC (natriuretic peptide promotor C), AHSP (alpha heme stabilizer), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanistic target of rapamycin (serine/thiocyanate kinase)), ITGB2 (integrin, β2 (complement component 3 receptor 3 and 4 subunits)), GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signal transducer (gpl30, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solutolytic carrier family 8 (sodium/calcium exchanger), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldodeductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member Al), BGLAP (bone gamma-carboxyglutamine (gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferred, member 3), RAGE (kidney tumor antigen), C4B (complement component 4B (Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), RNLS (nephrin, FAD-dependent amine oxidase), CREB1 (cAMP responsive element binding protein 1), POMC (promorphin melanocortin), RAC1 (ras-related C3 botulinum toxin substrate 1 (rho family, small TP binding protein Racl)), LMNA (nuclear laminin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V alpha subunit), CYP1B1 (cytochrome P450, family 1, subgroup B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation inhibitor)), MMP13 (matrix metalloproteinase 13 (collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subgroup A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subgroup A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 2 (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin deficiency)), SELPLG (selectin P ligand), AOC3 (copper-containing amine oxidase 3 (vascular adhesion protein 1)), CTSL1 (histatinase LI), PCNA (proliferating cell nuclear antigen), IGF2 (insulin-like growth factor 2 (interleukin A)), ITGB1 (integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 including MDF2, MSK12)), CAST (calcified protein), CXCL12 (chemointerleukin (C-X-C motif) ligand 12 (stromal cell-derived factor 1)), IGHE (immunoglobulin heavy chain homeostasis epsilon), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen, type I, α1), COL1A2 (collagen, type I, α2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor type 1), SLC2A1 (solute carrier family 2 (glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (Cellulin (C-C motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALC A (calcitonin-related polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathione S-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subgroup A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (interleukin (C-C motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenaline-stimulated beta receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metalloproteinase 8 (neutrophil globulinogenase)), NPR2 (natriuretic peptide receptor B/guanylate cyclase B (atrial natriuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamicin-prolyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PEC AMI (platelet/endothelial cell adhesion molecule), CCL4 (chemointerferon (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (α-1 antiprotease, antitrypsin), member 3), CASR (calcium-sensitive receptor), GJA5 (gap junction protein, α 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcriptional termination factor, RNA polymerase II), PROS1 (protein S (α)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, β (43 kDa dystrophin-related glycoprotein)), YME1L1 (YME1-like 1 (brew yeast)), CAMP (antibacterial peptide antimicrobial peptide), ZC3H12A (zinc finger CCCH type 12A), AKR1B1 (aldose reductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrix metalloproteinase 7 (matrilysin, uterine)), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (conjunctive tissue growth factor), KCNMA1 (potassium large conductance calcium-activated channel, subfamily M, alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol-b-methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calcimodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL11 (chemointerleukin (C-C motif) ligand 11), PGF (placental growth factor), THPO (thrombopoietin), GP6 (glycoprotein VI (platelets)), TACR1 (tachykinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, Shal-related subfamily, member 1), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelets)), CYP2J2 (cytochrome P450, family 2, subgroup J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ALOX12 (arachidonic acid 12-lipoxygenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteine methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocation factor), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonic acid 5-lipoxygenase activating protein), NUMA1 (nuclear mitotron protein 1), CYP27B1 (cytochrome P450, family 27, subgroup B, polypeptide 1), CYSLTR2 (cysteamine leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obesitystatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (kelch repeat and BTB (POZ) domain 10), TNC (tenascin C), TYMS (thymidylate synthase), SHC1 (SHC (Src homology 2 domain) conversion protein 1), LRP1 (low-density lipoprotein receptor-related protein 1), SOCS3 (suppressor of interleukin signaling 3), ADH1B (alcohol dehydrogenase IB (class I), beta polypeptide), KLK3 (pancreatic vasodilator-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor, evolutionary clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, α M (complement component 3 receptor 3 subunit)), PITX2 (paired homology domain-like 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (IgG Fc fragment, low affinity 111a receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (glutamine-oxalyltransferase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histaminic receptor HI), NR112 (nuclear receptor subfamily 1, group I, member 2), CRH (epinephrine-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-dependent anion channel 1), HPSE (acetylheparinase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP-binding cassette, subfamily B (MDR/TAP)), RNF123 (RING finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5, group A, member 1), GJB2 (gap junction protein, β2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin/kexin type 9), FCGR2A (IgG Fc fragment, low affinity Ila receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, evolutionary clade F (alpha-2 antifibrotic enzyme, pigment epithelium-derived factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acid lysosome), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)), TFAP2A (transcription factor AP-2 α (activating enhancer binding protein 2 α)), C4BPA (complement component 4 binding protein, α), SERPINF2 (serpin peptidase inhibitor, evolutionary clade F (α-2 antifibrotic enzyme, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)), CXCR2 (chemoattractant (C-X-C motif) receptor 2), SLC39A3 (solute carrier family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, subfamily G (WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), Fll (coagulation factor XI), ATP7A (ATPase, Cu++ transporter, alpha polypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops blood type)), GFAP (fibroblast acidic protein), ROCK1 (Rho-associated coiled coil containing protein kinase 1), MECP2 (methyl CpG binding protein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCF1E (butyryl cholinesterase), LIPE (lipase, hormone sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (C-X-C motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CF1GA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloliquefacial polypeptide), RFIO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTF1LF1 (parathyroid hormone-like hormone), NRG1 (neuromodulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamicin (aminopeptidase A)), CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), NAGLU (N-acetylglucosidase, α), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (interleukin (C-X3-C motif) ligand 1), BDKRB1 (bradykinin receptor Bl), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil expressed), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISF1 (interleukin-induced SF12-containing protein), GAST (gastrin), MYOC (myosin, trabecular meshwork-induced glucocorticoid response), ATP1A2 (ATPase, Na+/K+ transporter, α2 polypeptide), NF1 (neurofibroma protein 1), GJB1 (gap junction protein, β1, 32 kDa), MEF2A (myocyte enhancing factor 2A), VCL (focal adhesion protein), BMPR2 (bone morphogenetic protein receptor, type II (serine/thiocyanine kinase)), TUBB (tubulin, beta), CDC42 (mitotic cycle 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), F1SF1 (heat shock transcription factor 1), MYB (v-myb myeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase, AMP-activated, alpha 2 catalytic subunit), ROCK2 (Rho-associated coiled-coil containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (calcificin (calcificin-related protein), delta 1), CTF1 (cystathionase (cystathionine gamma-lyase)), CTSS (histatenase S), VAV2 (vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1), PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOF1 (apolipoprotein FI (beta-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonic acid 15-lipoxygenase), FBLN1 (fibroin 1), NR1F13 (nuclear receptor subfamily 1, FI group, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide), CF1GB (chromogranin B (secretogranin 1)), PRKCB (protein kinase C, β), SRD5A1 (steroid-5-α-reductase, α polypeptide 1 (3-hydroxy-5α-steroid delta 4-dehydrogenase α1)), F1SD11B2 (hydroxysteroid (11-β) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4), PIK3C2A (phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DRβ5), BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium-binding protein A 12), PADI4 (peptidylarginine deaminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemolysin (C-X-C motif) receptor 1), H19 (H19, imprinted maternal transcript (non-protein coding)), KRTAP19-3 (keratin-associated protein 19-3), insulin, RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP-binding protein Rac2)), RYR1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homolog (mouse)), NGFR (neural growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (choleline stimulatory receptor, niacin, alpha 4), CACNA1C (calcium channel, voltage-dependent, L-type, alpha 1C subunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalytic subunit), CHAT (choleline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11 A (tumor necrosis factor receptor subfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (life span protein)), CYSLTR1 (cysteine leukotriene receptor 1), MAT1A (methionine adenosine transferase I, alpha), OPRL1 (opium receptor-like 1), IMPA1 (inositol (muscle)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoic acid amide dehydrogenase), PSMA6 (proteasome (precursor, megalin factor) subunit, alpha type, 6), PSMB8 (proteasome (precursor, megalin factor) subunit, beta type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (chondroitin-39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member Bl), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette, subfamily C (CFTR/MRP), member 6), RGS2 (regulator of G protein signaling 2, 24 kDa), EFNB2 (adrenaline-B2), cystic fibrosis transmembrane conductance regulator (CFTR), GJB6 (gap junction protein, β6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferlin, limb-girdle muscular atrophy 2B (somatic recessive)), FDFT1 (farnesyl-diphosphate farnesyltransferase 1), EDN2 (endothelin 2), CCR6 (interleukin (C-C motif) receptor 6), GJB3 (gap junction protein, β3, 31 kDa), IL1RL1 (interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (calcified mucin, EGF LAG seven-channel G-type receptor 2 (red rooster homolog, fruit fly)), F11R (Fll receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronosidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (activating transcription factor 6), KΉK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine Nl-acetyltransferase 1), GGFI (γ-glutamicin hydrolase (binding enzyme, pyrrolyl poly-γ-glutamicin hydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute carrier family 4, sodium bicarbonate co-transporter, member 4), PDE2A (phosphodiesterase 2 A, cGMP stimulated), PDE3B (phosphodiesterase 3B, cGMP inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and senescent cell antigen-like domain 1), RFIOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), F-side 1 (forkhead box 01), PNPLA2 (patatin-like phospholipase domain-containing 2), TRH (thyroid-stimulating hormone-releasing hormone), GJC1 (gap junction protein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17 (anion/sugar transporter), member 5), FTO (fat mass and obesity-related), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1 (proline/serine-rich coiled coil 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tricholoma homolog 1 (Drosophila)), PBX4 (pre-B cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa selenoprotein), CILP2 (chondrocyte intermediate layer protein 2), TERC (telomerase RNA component), GGT2 (gamma-glutamyl transferase 2), MT-CO1 (mitochondrial encoded cytochrome c oxidase I), UOX (urate oxidase, pseudogene), CRISPR/Cas effector polypeptide, enzymatically active CRISPR/Cas effector polypeptide (e.g., capable of cleaving a target nucleic acid), and enzymatically inactive CRISPR/Cas effector polypeptide (e.g., does not cleave a target nucleic acid, but retains binding to a target nucleic acid). In some cases, the donor DNA encodes a wild-type form of any of the foregoing polypeptides; that is, the donor DNA may encode a "normal" form that does not include mutations that result in reduced function, lack of function, or pathogenicity.

In some cases, the donor DNA comprises a nucleotide sequence encoding a fluorescent polypeptide. Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEGFP), destabilized EY ...EFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized EYFP (dEFP), destabilized (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2 (12), mRFPl, pocilloporin, sea GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliprotein and phycobiliprotein conjugates (including B-phycoerythrin, R-phycoerythrin and isophycocyanin). Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909) and the like. Any of a variety of fluorescent and colored proteins from coral polyp species may be encoded, as described, e.g., in Matz et al. (1999) Nature Biotechnol. 17:969-973.

In some cases, the donor DNA encodes RNA, such as siRNA, microRNA, short hairpin RNA (shRNA), antisense RNA, riboswitch, ribozyme, aptamer, ribosomal RNA, transfer RNA, and the like.

In addition to nucleotide sequences encoding one or more gene products (e.g., RNA and/or polypeptides), the donor DNA may also include one or more transcriptional control elements, such as promoters, enhancers, and the like. In some cases, the transcriptional control element is an inducible type. In some cases, the promoter is reversible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in eukaryotic cells. In some cases, the promoter is a cell type-specific promoter. In some cases, the promoter is a tissue-specific promoter.

The nucleotide sequence of the donor DNA is generally inconsistent with the target nucleic acid (e.g., genome sequence) that it replaces. More specifically, the donor DNA may contain at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the target nucleic acid (e.g., genome sequence), as long as there is sufficient homology to support homology-directed repair (e.g., for gene correction, such as to convert pathogenic base pairs or non-pathogenic base pairs). In some cases, the donor DNA comprises a non-homologous sequence flanked by two homologous regions, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence into the target region. The donor DNA may also comprise a vector backbone containing a sequence that is not homologous to the relevant DNA region (target nucleic acid) and is not intended to be inserted into the relevant DNA region (target nucleic acid). Generally, the homologous region of the donor sequence and the target nucleic acid (e.g., genomic sequence) to be recombined will have at least 50% sequence identity. In some cases, there is 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% sequence identity. Any value between 1% and 100% sequence identity may exist, depending on the length of the donor polynucleotide.

As compared to the target nucleic acid (e.g., genomic sequence), the donor DNA may include certain nucleotide sequence differences, where such differences include, for example, restriction sites, nucleotide polymorphisms, selection markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), etc., which can be used to assess the successful insertion of the donor DNA at the cleavage site, or in some cases can be used for other purposes (e.g., to indicate expression at the targeted genomic locus). In some cases, if located in the coding region, such nucleotide sequence differences will not cause changes in the amino acid sequence, or will produce silent amino acid changes (i.e., changes that do not affect protein structure or function). Alternatively, such sequence differences may include flanking recombination sequences, such as FLP, loxP sequences, or the like, which can be activated at a later time to remove the marker sequence. In some cases, the donor DNA will include one or more nucleotide sequences to help the donor localize to the nucleus of the recipient cell or to help the donor DNA integrate into the target nucleic acid. For example, in some cases, the donor DNA may include one or more nucleotide sequences encoding one or more nuclear localization signals and their analogs (Frietas et al. (2009) Cun-Genomics 10:550-7). In some cases, the donor DNA will include nucleotide sequences to recruit DNA repair enzymes to increase insertion efficiency. Fiuman enzymes involved in homology-directed repair include MRN-CtIP, BLM-DNA2, Exol, ERCC1, Rad51, Rad52, Ligase 1, RoIQ, PARP1, Ligase 3, BRCA2, RecQ/BLM-ToroIIIa, RTEL, Roΐd and Roΐh (Verma and Greenburg (2016) Genes Dev. 30 (10): 1138-1154). In some cases, donor DNA is delivered as reconstructed chromatin (Cruz-Becerra and Kadonaga (2020) eLife 2020;9:e55780 DOI: 10.7554/eLife.55780).

In some cases, the ends of the donor DNA are protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those skilled in the art. For example, one or more bi-deoxynucleotide residues can be added to the 3' end of the linear molecule and/or a self-complementary oligonucleotide can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amine groups, and using modified internucleotide linkages, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the ends of the linear donor DNA, additional lengths of sequence can be included outside the homology region that can be degraded without affecting recombination .

In some embodiments, the polypeptides of the retrotranscript-based gene editing system are coupled to one or more auxiliary functions via linkers. Such auxiliary functions may include deaminases, nucleases, reverse transcriptases, and recombinases. For example, the retrotranscript RT can be linked to a programmable nuclease, such as a type II or type V CRISPR nuclease. The term linker as used in reference to fusion proteins refers to a molecule that joins proteins to form a fusion protein. Typically, such molecules do not have specific biological activity other than joining or preserving some minimum distance or other spatial relationship between proteins. However, in one embodiment, the linker can be selected to affect some properties of the linker and/or fusion protein, such as the folding, net charge, or hydrophobicity of the linker.

Suitable linkers for use in the methods of the present invention are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a tendency to develop an ordered secondary structure. In one embodiment, the linker may be a chemical moiety that may be a monomer, dimer, multimer, or polymer. Preferably, the linker comprises an amino acid. Typical amino acids in flexible linkers include Gly, Asn, and Ser.

Thus, in certain embodiments, the linker comprises a combination of one or more of the amino acids Gly, Asn, and Ser. Other near-neutral amino acids such as Thr and Ala may also be used in the linker sequence. Exemplary linkers are disclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l. Acad. Sci. USA 83: 8258-62; U.S. Patent No. 4,935,233; and U.S. Patent No. 4,751,180. For example, a GlySer linker may be based on a repeat unit of GGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or even 12 or more repeat units, including but not limited to: SEQ ID describe sequence GlySer linker based on GGS repeat unit GGS 19469 GlySer linker based on GGS repeat unit GGS GGS 19470 GlySer linker based on GGS repeat unit GGS GGS GGS 19471 GlySer linker based on GGS repeat unit GGS GGS GGS GGS 19472 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS 19473 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS 19474 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS 19475 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS 19476 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS 19477 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS 19478 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS 19479 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS 19480 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS 19481 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS 19482 GlySer linker based on GGS repeat unit GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS GGS

In another example, the GlySer linker can be based on repeat units of GSG, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeat units, including but not limited to: SEQ ID describe sequence GlySer linker based on GSG repeat unit GSG 19483 GlySer linker based on GSG repeat unit GSG GSG 19484 GlySer linker based on GSG repeat unit GSG GSG GSG 19485 GlySer linker based on GSG repeat unit GSG GSG GSG GSG 19486 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG 19487 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG 19488 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG 19489 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG 19490 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG 19491 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG 19492 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG 19493 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG 19494 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG 19495 GlySer linker based on GSG repeat unit GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG GSG

In another example, the GlySer linker can be based on repeat units of GGGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeat units, including but not limited to: SEQ ID describe sequence 19496 GlySer linker based on GGGS repeat unit GGGS 19497 GlySer linker based on GGGS repeat unit GGGS GGGS 19498 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS 19499 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS 19500 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS 19501 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS 19502 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19503 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19504 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19505 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19506 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19507 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19508 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19509 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS 19510 GlySer linker based on GGGS repeat unit GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS

In another example, the GlySer linker can be based on repeat units of GGGGS, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 or more repeat units, including but not limited to: SEQ ID describe sequence 19511 GlySer linker based on GGGGS repeat unit GGGGS 19512 GlySer linker based on GGGGS repeat unit GGGGS GGGGS 19513 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS 19514 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS 19515 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS 19516 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19517 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19518 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19519 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19520 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19521 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19522 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19523 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19524 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS 19525 GlySer linker based on GGGGS repeat unit GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS

In another embodiment, LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19526) is used as a linker.

In another additional embodiment, the linker is an XTEN linker, which is TCGGGATCTGAGACGCCTGGGACCTCGGAATCGGCTACGCCCGAAAGT (SEQ ID NO: 19527). In other specific embodiments, the Cas12a polypeptide is linked to the N-terminus of the deaminase protein or its catalytic domain at the C-terminus by means of a LEPGEKPYKCPECGKSFSQSGALTRHQRTHTRLEPGEKPYKCPECGKSFSQSGALTRHQRTHTRLEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 19528) linker. In addition, N-terminal and C-terminal NLSs can also serve as linkers (e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NO: 19420)).

The above description of linkers is intended to be non-limiting and includes any combination of the above linkers or heterologous combinations of repeat GlySer linkers.

The linker can be as simple as a covalent bond, or it can be a polymeric linker of multiple atoms in length. In some embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is non-peptide-like. In some embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In some embodiments, the linker is an amide-linked carbon-nitrogen bond. In some embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In some embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In some embodiments, the linker comprises a monomer, dimer or polymer of aminoalkanoic acid. In some embodiments, the linker comprises aminoalkanoic acid (e.g., glycine, acetic acid, alanine, β-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In some embodiments, the linker comprises a monomer, dimer or polymer of aminohexanoic acid (Ahx). In some embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a benzene ring. The linker can include a functionalized portion to facilitate attachment of a nucleophilic agent (e.g., thiol, amine group) from the peptide to the linker. Any electrophilic agent can be used as part of the linker. Exemplary electrophilic agents include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

The linker can be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of a F2A linker, a P2A linker, a T2A linker, an E2A linker, and combinations thereof. This family of self-cleaving peptide linkers is called 2A peptides and has been described in the art (see, for example, Kim, J. H. et al. (2011) PLoS ONE 6: e18556). In some embodiments, the linker is a F2A linker. In some embodiments, the linker is a GGGS linker. In some embodiments, the fusion protein contains three domains with an intervening linker, having the structure: domain-linker-domain-linker-domain.

Cleavable linkers known in the art may be used in conjunction with the present disclosure. Exemplary such linkers include: F2A linker, T2A linker, P2A linker, E2A linker (see, e.g., WO2017127750). A skilled artisan will appreciate that other art-recognized linkers may be suitable for use with the constructs of the present disclosure (e.g., encoded by the nucleic acids of the present disclosure). A skilled artisan will also appreciate that other polycistronic constructs (mRNAs encoding more than one nucleobase editing system component/polypeptide in the same molecule) may be suitable for use as provided herein. Nuclear Localization Domain

In various embodiments, the gene editing systems or any components thereof can be fused to one or more nuclear localization sequences (NLS), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In one embodiment, the gene editor component (e.g., a nucleic acid programmable DNA binding protein or an editing accessory protein) comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the carboxyl terminus, or a combination thereof (e.g., zero or at least one or more NLSs at the amino terminus and zero or one or more NLSs at the carboxyl terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In one embodiment of the invention, the editor component polypeptide comprises up to 6 NLSs. In one embodiment, an NLS is considered to be near the N-terminus or C-terminus when the most recent amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain starting from the N-terminus or C-terminus. Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 19399); an NLS from a nucleoplasmic protein (e.g., a nucleoplasmic protein bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 19529); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 19530) or RQRRNELKRSP (SEQ ID NO: 19531); an hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSS GPYGGGGQYFAKPRNQGGY (SEQ ID NO: 19532); a sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 19533) from the IBB domain of importin-alpha. NO: 19533); the sequence of myoma T protein VSRKRPRP (SEQ ID NO: 19400) and PPKKARED (SEQ ID NO: 19534); the sequence of human p53 PQPKKKPL (SEQ ID NO: 19535); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO: 19536); the sequence of influenza virus NS 1 DRLRR (SEQ ID NO: 19537) and PKQKKRK (SEQ ID NO: 19538); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO: 19539); the sequence of mouse Mxl protein REKKKFLKRR (SEQ ID NO: 19540); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 19541); and the sequence of the steroid hormone receptor (human) glucocorticoid RI＜CLQAGMNLEARI＜TI＜I＜ (SEQ ID NO: 19542).

Generally, one or more NLSs are strong enough to drive the accumulation of the relevant polypeptide (e.g., retrotranscript RT) in detectable amounts in the nucleus of eukaryotic cells. Generally, the strength of nuclear localization activity can be derived from the number of NLSs in the relevant polypeptide (e.g., retrotranscript RT), the specific NLS used, or a combination of these factors. Detection of accumulation in the cell nucleus can be performed by any suitable technique.

For example, a detectable marker can be fused to a polypeptide of interest (e.g., a retrotranscript RT) so that the location within the cell can be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus, such as DAPI). The nucleus can also be isolated from the cell, and the contents of the nucleus can then be analyzed by any suitable method for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity analysis. Accumulation in the nucleus can also be determined indirectly, such as by an assay for determining the effect of complex formation (e.g., an assay for determining DNA cleavage or mutation at a target sequence, or an assay for determining altered gene expression activity affected by complex formation and/or polypeptide activity) as compared to a control not exposed to the polypeptide or complex or exposed to a polypeptide lacking one or more NLSs. In one embodiment of the polypeptide protein complexes and systems described herein, the codon-optimized polypeptide protein comprises an NLS linked to the C-terminus of the protein. In one embodiment, other localization tags can be fused to the polypeptide, such as but not limited to localizing the polypeptide to a specific site in the cell, such as an organelle, such as mitochondria, plastids, chloroplasts, vesicles, Golgi apparatus, (nuclear or cell) membrane, ribosomes, nucleoli, ER, cytoskeleton, vacuoles, centrosomes, nucleosomes, granules, centrioles, etc.

In one embodiment of the invention, at least one nuclear localization signal (NLS) is linked to a nucleic acid sequence encoding a polypeptide of interest (e.g., a retrotranscript RT). In a preferred embodiment, at least one or more C-terminal or N-terminal NLSs are linked (and thus, a nucleic acid molecule encoding a polypeptide may include an encoding NLS such that the expressed product is linked to the NLS). In a preferred embodiment, the C-terminal NLS is linked for optimal expression and nuclear targeting in eukaryotic cells (preferably, human cells). The invention also encompasses methods for delivering multiple nucleic acid components, each of which is specific for a different target locus of interest, thereby modifying multiple target loci of interest. The nucleic acid component of the complex may include one or more protein-binding RNA aptamers. The one or more aptamers are capable of binding to bacteriophage coat proteins.

In other examples, a fusion protein comprising a polypeptide of interest (eg, a retrotranscript RT) and another accessory protein (eg, a nuclease) contains one or more nuclear localization signals selected from or derived from SV40, c-Myc or NLP-1.

The above NLS examples are non-limiting. The proteins and/or fusions contemplated herein may comprise any known NLS sequence, including any of those described in Cokol et al., "Finding nuclear localization signals," EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., "Mechanisms and Signals for the Nuclear Import of Proteins," Current Genomics, 2009, 10(8): 550-7, each of which is incorporated herein by reference.

In various embodiments, any polypeptide component of the retrotransposons-based editing system can be engineered with one or more nuclear localization signals, which helps promote protein translocation into the nucleus. The polypeptides of the retrotransposons-based editing system can comprise any known NLS sequence, including any of those described in Cokol et al., "Finding nuclear localization signals," EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., "Mechanisms and Signals for the Nuclear Import of Proteins," Current Genomics, 2009, 10(8): 550-7, each of which is incorporated herein by reference.

In various embodiments, the polypeptide disclosed herein may include one or more, preferably at least two, nuclear localization signals. The NLS may be any known NLS sequence in the art. The NLS may also be any NLS discovered in the future for nuclear localization. The NLS may also be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations). The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that promotes protein import into the cell nucleus, such as by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to a skilled artisan. For example, NLS sequences are described in Plank et al., International PCT Application PCT/EP2000/011690, filed on November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 19399).

Another representative nuclear localization signal is a peptide sequence that directs a protein to the nucleus of a cell in which the sequence is expressed. Nuclear localization signals are primarily alkaline and can be located at almost any position in the amino acid sequence of a protein, generally comprising a short sequence of four amino acids (Autieri and Agrawal, (1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference) to eight amino acids, and are typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274: 11-16, incorporated herein by reference). Nuclear localization signals typically comprise proline residues. A number of nuclear localization signals have been identified and have been used to affect the transport of biomolecules from the cytoplasm of a cell to the nucleus. See, e.g., Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34, which are incorporated by reference. It is currently believed that translocation involves nucleoporins.

Most NLSs can be classified into three general groups: (i) monopartite NLSs, exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 19399); (ii) bipartite motifs, consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus laevis nucleoplasmic protein NLS (KRXXXXXXXXXXKKKL) (SEQ ID NO: 19419); and (iii) non-canonical sequences, such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991). Nuclear localization signals occur at different points in the protein amino acid sequence. NLSs have been identified at the N-terminal, C-terminal, and central regions of proteins. Thus, the present disclosure provides polypeptides that can be modified with one or more NLSs at the C-terminus, N-terminus, and internal regions of the polypeptide (including fusion proteins).

The present disclosure encompasses any suitable means for modifying a polypeptide to include one or more NLSs. In one aspect, a polypeptide (e.g., a programmable nuclease) can be engineered to express a translationally fused NLS at its N-terminus or its C-terminus (or both), i.e., to form a polypeptide-NLS fusion construct. Additionally, the NLS can include various amino acid linkers or spacers encoded between the polypeptide and, for example, the N-terminus, C-terminus, or internally linked NLS amino acid sequences, as well as in the central region of the protein.

Therefore, the present disclosure also provides nucleotide constructs, vectors and host cells for expressing fusion proteins comprising a polypeptide and one or more NLSs.

In some embodiments, the retrotransposon-based editing system or its components may comprise a polypeptide tag, such as an affinity tag (chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), SBP tag, Strep tag, AviTag, calcimodulin tag); a solubilization tag; a chromatography tag (polyanionic amino acid tag, such as FLAG tag); an antigenic determinant tag (a short peptide sequence that binds to a high-affinity antibody, such as V5 tag, Myc tag, VSV tag, Xpress tag, E tag, S tag and HA tag); a fluorescent tag (e.g., GFP). In some embodiments, retrotranscript-based editing system peptides may include an amino acid tag, such as one or more lysine, histidine, or glutamine, which can be added to the polypeptide sequence (e.g., at the N-terminus or C-terminus). Lysine can be used to increase peptide solubility or allow biotinylation. Protein and amino acid tags are peptide sequences that are genetically transplanted onto recombinant proteins. Sequence tags are attached to proteins for various purposes, such as peptide purification, identification, or localization, for use in various applications, including, for example, affinity purification, protein arrays, Western blotting, immunofluorescence, and immunoprecipitation. Such tags can then be removed by chemicals or by enzymatic means, such as by specific proteolysis or intein splicing.

Alternatively, amino acid residues at the carboxyl and amino terminal regions of the amino acid sequence of a peptide or protein may be deleted, as appropriate, to provide a truncated sequence. Certain amino acids (e.g., the C-terminal or N-terminal residue) may alternatively be deleted, depending on the purpose of the sequence, e.g., to express the sequence as part of a soluble larger sequence, or to attach to a solid support.

In certain embodiments, the nucleic acid component of the retrotransposons-based editing system (e.g., guide RNA or retrotransposons ncRNA) may further comprise a functional structure designed to improve the molecular structure, architecture, stability, genetic expression, or any combination thereof of the nucleic acid component. Such structures may include aptamers.

Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science 1990, 249: 505-510). For example, nucleic acid aptamers can be selected from a pool of random sequence oligonucleotides that have high binding affinity and specificity for a wide range of biomedically relevant targets, indicating that aptamers have broad therapeutic uses (Keefe, Anthony D., Supriya Pai and Andrew Ellington. "Aptamers as therapeutics." Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also indicate the wide application of aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar et al. "Nanotechnology and aptamers: applications in drug delivery." Trends in biotechnology 26.8 (2008): 442-449; and Hicke BJ, Stephens AW. "Escort aptamers: a delivery service for diagnosis and therapy." J Clin Invest 2000, 106: 923-928.). Aptamers can also be constructed to act as molecular switches, changing properties in response to que, such as RNA aptamers that bind fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu and Sarnie R. Jaffrey. "RNA mimics of green fluorescent protein." Science 333.6042 (2011): 642-646). It has also been suggested that aptamers can be used as components of targeted siRNA therapeutic delivery systems, such as targeting cell surface proteins (Zhou, Jiehua and John J. Rossi. "Aptamer-targeted cell-specific RNA interference." Silence 1.1 (2010): 4).

Thus, in certain embodiments, retrotranscript-based gene editing nucleic acid components are modified, for example, by one or more aptamers designed to improve delivery of RNA or DNA component molecules, including delivery across cell membranes to intracellular compartments, or into the nucleus. Such structures may include (a variety of) moieties in addition to or in the absence of one or more aptamers, so as to render the nucleic acid component molecule deliverable, inducible, or responsive to a selected effector. Thus, the present invention encompasses reRNA component molecules that respond to normal or pathological physiological conditions, including but not limited to pH , hypoxia, oxygen concentration, temperature, protein concentration, enzyme concentration, lipid structure, light exposure, mechanical damage (e.g., ultrasound), magnetic field, electric field or electromagnetic radiation.

In certain embodiments, the retrotransposon-based gene editing system described herein further comprises or encodes a DNA repair regulatory biomolecule, which can further enhance the integration efficiency of the repair outcome.

In certain embodiments, the DNA repair modulating biomolecule comprises a non-homologous end joining (NHEJ) inhibitor.

In certain embodiments, the DNA repair regulatory biomolecule comprises a homology-directed repair (HDR) promoter.

In certain embodiments, the DNA repair modulating biomolecule comprises an NHEJ inhibitor and an HDR promoter.

In certain embodiments, a DNA repair modulating biomolecule enhances or improves more precise genome editing and/or homologous recombination efficiency compared to an otherwise consistent embodiment without the DNA repair modulating biomolecule.

In some embodiments, the HDR promoter and/or NHEJ inhibitor may comprise one or more small molecules. Systems carrying recombinant enhancers (such as small molecules that locally activate HDR and inhibit NHEJ at genomic sites of DNA damage) can be adapted to further enhance their efficiency when placed on an engineered system. In general, small molecule recombinant enhancers can be synthesized to carry linkers and functional groups (such as maleimide for reacting with thiol groups on Cys residues of proteins) to chemically bind to engineered systems. Commercially available functionalized PEG linkers (alkynes, azides, cyclooctynes, etc.) can also be used for conjugation, and orthogonal conjugation chemistry can be used for multivalent presentation.

In cases where the modification does not affect the potency of the selected recombinant enhancer, the binding site can be readily identified.

In certain embodiments, multivalent presentation of one or more DNA repair modulating biomolecules can be achieved, including multiple portions of NHEJ inhibitors, HDR promoters, or combinations thereof. See, e.g., "Genomic targeting of epigenetic probes using a chemically tailored Cas9 system" Liszczak et al. , Proc Natl Acad Sci USA 114: 681-686, 2017 (incorporated herein by reference). In certain embodiments, multivalent presentation of small molecule compounds can be achieved by using a sortase cycloprotein as a scaffold for presentation thereof.

In some embodiments, the DNA repair regulatory biomolecule may comprise an HDR promoter. An HDR promoter may comprise a small molecule, such as RSI or its analog. In certain embodiments, an HDR promoter stimulates RAD51 activity or RAD52 motif protein 1 (RDM1) activity. In certain embodiments, an HDR promoter comprises nocodazole, which may result in higher HDR selection.

In some embodiments, an HDR enabler may be administered prior to delivering the reverse transcription based editing system described herein.

In some embodiments, the HDR promoter locally enhances HDR without inhibiting NHEJ. For example, RAD51 is a protein involved in homology region search during strand exchange and HDR repair. In some embodiments, the HDR promoter is phenylbenzamide RSI, identified as a small molecule RAD51 stimulator (see [0200]-[0204] of WO2019/135816, specifically incorporated herein by reference).

In certain embodiments, the DNA repair regulatory biomolecule comprises a C-terminal binding protein interacting protein (CtIP) or a functional fragment or homolog thereof. CtIP is a key protein in the early steps of homologous recombination. According to this embodiment, CtIP or a functional fragment or homolog thereof can be linked ( e.g. , fused) to RT or sequence-specific nucleases ( e.g. , CRISPR/Cas effectors, ZFNs, TALENs, meganucleases, TnpB, IscB, or restriction endonucleases (RE)), and transgene integration is stimulated by HDR.

In certain embodiments, the CtIP fragment is a minimal N-terminal fragment of wild-type CtIP, such as an N-terminal fragment comprising residues 1-296 of full-length CtIP (HE of the HDR enhancer), as described in Charpentier et al . (Nature Comm., DOI: 10.1038/s41467-018-03475-7, incorporated herein by reference), which was shown to be sufficient to stimulate HDR. The activity of this fragment depends on the CDK phosphorylation sites ( e.g. , S233, T245 and S276) and the multimerization domain required for CtIP activity in homologous recombination. Therefore, alternative fragments comprising CDK phosphorylation sites and the multimerization domain required for CtIP activity are also within the scope of the present invention.

In certain embodiments, the DNA repair modulating biomolecule comprises dominant negative 53BP1.

In certain embodiments, the DNA repair regulatory biomolecule comprises a cell cycle specific degradation tag, such as the degradation domain of (human) Geminin and (murine) CyclinB2.

In some embodiments, the DNA repair regulatory biomolecule comprises CyclinB2, which is a member of the B-type cell cycle proteins that associate with p34cdc2 and is an essential component of the cell cycle regulatory machinery. The efficiency of CRISPR-mediated knock-in can be increased by promoting a relative increase in Cas9 activity in the G2 phase of the cell cycle (when HDR is more active). In some embodiments, the degradation domains of (human) Geminin and (murine) CyclinB2 can be used as N-terminal or C-terminal fusions to serve as DNA repair regulatory biomolecules. These domains are known to determine the cell cycle-specific profile of the chimeric proteins, i.e., their relative concentrations are increased in S and G2 compared to G1, thereby high-jacking the known CyclinB2 and Geminin degradation pathways. This results in active Geminin-Cas9 and CyclinB2-Cas9 chimeric proteins that are degraded in a cell cycle-dependent manner. Compared to the commonly used Cas9, these chimeras shift the repair of DSBs to the HDR repair pathway.

While not wishing to be bound by a particular theory, it is believed that the application of such cell cycle-specific degradation tags allows/facilitates more efficient/safe gene editing.

In certain embodiments, the DNA repair regulatory biomolecule comprises a Rad family member protein, such as Rad50, Rad51, Rad52, etc., which is used to promote the integration of exogenous DNA into host chromosomes. Specifically, Rad52 is an important homologous recombination protein, and its complex with Rad51 plays a key role in HDR, mainly participating in the regulation of exogenous DNA in eukaryotes. The key steps in the HR process include Rad51-mediated repair and strand exchange. The co-expression of Rad52 as a DNA repair regulatory biomolecule significantly enhances the possibility of HDR, for example , by three times.

In certain embodiments, the DNA repair modulating biomolecule comprises a RAD52 protein, for example, in the form of an N-terminal or C-terminal fusion.

In certain embodiments, the DNA repair regulatory biomolecule comprises a RAD52 motif protein 1 (RDM1) that functions similarly to RAD52. RDM1 has been shown to be able to repair DSBs caused by DNA replication, prevent G2 or M cell cycle arrest, and improve HDR selection.

In certain embodiments, the DNA repair regulatory biomolecule comprises a dominant negative form of the tumor suppressor p53 binding protein 1 (53BP1). The wild-type protein 53BP1 is a key regulator in the choice between NHEJ and HDR - it is a pro-NHEJ factor that limits HDR by blocking DNA end resection and also by inhibiting BRCA1 recruitment to DSB sites. It has been shown that global inhibition of 53BP1 by ubiquitin variants significantly improves the frequency of Cas9-mediated HDR in non-hematopoietic and hematopoietic cells using single-stranded oligonucleotide delivery or double-stranded donors in AAV.

In certain embodiments, the dominant negative (DN) form of 53BP1 comprises a minimal focus-forming region, but lacks domains outside of this region ( e.g. , toward the N-terminus and tandem C-terminal BRCT repeats) that recruit key effectors involved in NHEJ, such as RIF1-PTIP and EXPAND, respectively. 53BP1 binds proteins through this minimal focus-forming region, which is recruited to specific histone markers at DSB sites, and comprises several conserved domains, including an oligomerization domain (OD), a glycine-arginine-rich (GAR) motif, a Tudor domain, and an adjacent ubiquitin-dependent recruitment (UDR) motif. The Tudor domain mediates interaction with histone H4 dimethylated at K2023.

In certain embodiments, the dominant negative form (DN1S) of 53BP1 inhibits the accumulation of endogenous 53BP1 and downstream NHEJ proteins at DNA damage sites, while upregulating the recruitment of BRCA1 HDR proteins. Such DN forms of 53BP1 can be used as DNA repair regulatory biomolecules in the form of N-terminal or C-terminal fusions (such as Cas9 fusions to locally inhibit NHEJ at the Cas9 target site defined by its gRNA, while promoting the increase of HDR, and will not affect NHEJ globally, thereby improving cell viability).

In certain embodiments, the DNA repair modulating biomolecule comprises an NHEJ inhibitor, such as a DNA ligase IV inhibitor, a KU inhibitor ( e.g. , KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor.

In certain embodiments, the NHEJ inhibitor inhibits the NHEJ pathway, enhances HDR, or modulates both. In certain embodiments, the NHEJ inhibitor is a small molecule inhibitor.

In certain embodiments, the small molecule inhibitor of the NHEJ pathway comprises an SCR7 analog, such as PK66, PK76, PK409.

In certain embodiments, the NHEJ inhibitor comprises a KU inhibitor, such as KU5788 and KU0060648.

In certain embodiments, small molecule NHEJ inhibitors are linked to polyglycine tripeptides via PEG for sortase-mediated conjugation, as described in WO2019/135816, Guimaraes et al. , Nat Protoc 8:1787-99, 2013; Theile et al. , Nat Protoc 8:1800-7, 2013; and Schmohl et al. , Curr Opin Chem Biol 22:122-8, 2014 (all incorporated herein by reference). The same approach can also be used to link small molecule HDR enhancers to proteins.

An exemplary method for conjugating small molecule DNA repair modulating biomolecules without loss of activity is described in WO2019135816, wherein conjugation of a poly-glycine peptide to SCR-7 of the carboxyl portion at loop 4 retains inhibitor activity, wherein loops 1, 2, and 3 of the molecule are involved in target binding, thereby providing a simple and effective strategy for conjugating small molecule NHEJ inhibitors to the systems described herein ( e.g. , to sequence-specific nucleases including Cas enzymes, or to RT) to precisely enhance the HDR pathway near the nucleic acid target site.

In certain embodiments, nucleic acid targeting moiety conjugates based on small molecule inhibitors of DNA-dependent protein kinase (DNA-PK) or heterodimeric Ku (KU70/KU80) can be utilized. KU-0060648 is a potent KU inhibitor that can also be functionalized with polyglycine and used to enhance recombination.

In certain embodiments, the DNA repair regulatory biomolecule comprises the tumor suppressor p53. p53 plays a direct role in DNA repair, including HR regulation, where it affects the extension of new DNA, thereby affecting HDR selection. In vivo, p53 is bound to the nuclear matrix and is the rate-limiting factor for repairing DNA structure. p53 regulates DNA repair processes in almost all eukaryotic organisms via transactivation-dependent and non-transactivation-dependent pathways, but only the non-transactivation-dependent function of p53 is involved in HR regulation. Wild-type p53 protein can connect double-strand breaks to form intact DNA, and also plays a role in inhibiting NHEJ. p53 interacts with HR-related proteins, including Rad51, where it controls HR by directly interacting with Rad51. Accessory domains

In other aspects, the gene editing system based on retrotransposons may include one or more additional auxiliary proteins with genome modification functions, including recombinases, invertases, nucleases, polymerases, ligases, deaminases, reverse transcriptases or epigenetic modification functions. In various embodiments, auxiliary proteins can be provided separately. In other embodiments, auxiliary proteins can be fused to the polypeptide components of the editing system based on retrotransposons using a linker as appropriate.

Retroviral gene editing systems can further include additional polypeptides, proteins and/or peptides known in the art. Non-limiting classes of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides and chimeric polypeptides, receptors, enzymes, hormones, transcription factors, ligands, membrane transporters, structural proteins, nucleases, or components, variants or fragments (e.g., biologically active fragments) thereof.

As used herein, the term "peptide" generally refers to a shorter polypeptide of about 50 amino acids or less. A peptide having only two amino acids may be referred to as a "dipeptide". A peptide having only three amino acids may be referred to as a "tripeptide". A polypeptide generally refers to a polypeptide having about 4 to about 50 amino acids. Peptides may be obtained by any method known to those skilled in the art. In some embodiments, peptides may be expressed in culture. In some embodiments, peptides may be obtained by chemical synthesis (e.g., solid phase peptide synthesis).

In some embodiments, RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more cognate coding products, or non-coding RNAs such as guide RNAs) may encode user-programmable DNA binding proteins or gene editor accessory proteins such as, but not limited to, deaminases, nucleases, transposases, polymerases, and reverse transcriptases, among others.

In some embodiments, RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more cognate coding products, such as the starting constructs and baseline constructs described herein) may encode simple proteins associated with non-proteins. Non-limiting examples of associated proteins include glycoproteins, heme, phosphatidylcholine, nucleoproteins, and phosphoproteins.

In some embodiments, RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more cognate coding products, such as the starting constructs and baseline constructs described herein) may encode proteins derived from simple or conjugated proteins by chemical or physical means. Non-limiting examples of derived proteins include denatured proteins and peptides.

In some embodiments, the polypeptide, protein or peptide may be unmodified.

In some embodiments, the polypeptide, protein or peptide may be modified. Types of modification include, but are not limited to, phosphorylation, glycosylation, acetylation, ubiquitination/ubiquitination, methylation, palmitylation, quinone, acylation, myristoylation, pyrrolidonecarboxylic acid, hydroxylation, phosphopantetheinylethylamine, prenylation, GPI anchoring, oxidation, ADP-ribosylation, sulfation, S-nitrosylation, citrullination, nitration, γ-carboxyglutamate, formylation, putrescine lysine (hypusine), tropaquinone (TPQ), bromination, lysine tropaquinone (LTQ), tryptophanyl quinone (TTQ), iodination and cysteine tropaquinone (CTQ). In some aspects, a polypeptide, protein or peptide may be modified by post-transcriptional modifications that affect its structure, subcellular localization and/or function.

In some embodiments, phosphorylation can be used to modify a polypeptide, protein, or peptide. Phosphorylation, or the addition of phosphate groups to serine, threonine, or tyrosine residues, is one of the most common forms of protein modification. Protein phosphorylation plays an important role in the fine-tuning of signals in intracellular signaling cascades.

In some embodiments, polypeptides, proteins or peptides may be modified using ubiquitination, which is the covalent attachment of ubiquitin to a target protein. Ubiquitination-mediated protein turnover has been shown to play a role in driving cell cycle as well as protein degradation-independent intracellular signaling pathways.

In some embodiments, acetylation and methylation can be used to modify polypeptides, proteins or peptides, and acetylation and methylation can play a role in the regulation of gene expression. As a non-limiting example, acetylation and methylation may mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin), which may have an impact on mediating gene silencing.

In some embodiments, glycosylation can be used to modify a polypeptide, protein, or peptide. Glycosylation is the attachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins and plays a role in biological processes including, but not limited to, embryonic development, cell division, and regulation of protein structure. The two main types of protein glycosylation are N-glycosylation and O-glycosylation. For N-glycosylation, the glycans are attached to asparagine; and for O-glycosylation, the glycans are attached to serine or thixoamine.

In some embodiments, paraubiquitination can be used to modify polypeptides, proteins, or peptides. Paraubiquitination is the addition of SUMO (small ubiquitin-like modifier) to proteins and is a post-translational modification similar to ubiquitination.

In other embodiments, RNA payloads (e.g., linear and/or circular mRNA payloads encoding one or more cognate coding products, such as the starting constructs and baseline constructs described herein) can encode therapeutic proteins, such as those exemplified below.

In other embodiments, the RNA payload (e.g., linear and/or circular mRNA payloads encoding one or more relevant products, such as the starting constructs and baseline constructs described herein) can encode a gene editing system, such as those exemplified below. As used herein, a "nucleobase editing system" is a protein, DNA or RNA composition that is capable of editing, modifying or altering one or more relevant targeted genes. According to the present invention, one or more nucleobase editing systems currently commercially available or under development can be encoded by the RNA payloads described herein of the present invention (e.g., linear and/or circular mRNA payloads encoding one or more relevant coding products). Induced Modification

In one embodiment, the editing system based on retrotranscripts can be configured as an inducible gene editing system. The inducing properties of the system will allow the use of energy forms to control gene editing or gene expression in time and space. Energy forms may include but are not limited to electromagnetic radiation, sound energy, chemical energy, and thermal energy. Examples of inducing systems include tetracycline-induced promoters (Tet-On or Tet-Off), small molecule double hybrid transcription activation systems (FKBP, ABA, etc.) or light-induced systems (phytochrome, LOV domain or cryptochrome). In one embodiment, the editing system based on retrotranscripts or its components may include light-induced transcription effectors (LITEs) to guide changes in transcriptional activity in a sequence-specific manner. The light component may include a retrotranscript editing system component, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation/repression domain. Further examples of inducible DNA binding proteins and methods of use thereof are provided in U.S. Provisional Application Nos. 61/736,465 and 61/721,283 and International Patent Publication No. WO 2014/018423 A2, which are hereby incorporated by reference in their entirety.

Once all copies of a gene in the genome of a cell have been edited, the system no longer needs to continue to express it in that cell. In fact, if off-target effects occur at unexpected genomic sites, etc., continued expression will be undesirable. Therefore, time-limited expression will be useful. Induced expression provides one approach, but in addition, the applicants have engineered a self-inactivating system that relies on the use of non-coding nucleic acid component molecular target sequences within the vector itself. Therefore, after expression begins, the system will cause its own destruction, but before the destruction is completed, it will have time to edit the genomic copies of the target gene (in the case of normal point mutations in diploid cells, it requires at most two edits). Briefly, the self-inactivation system includes an additional RNA (e.g., a nucleic acid component molecule) that targets the coding sequence of the Cas12a polypeptide itself or targets one or more non-coding nucleic acid component molecule target sequences that are complementary to a unique sequence present in one or more of the following: (a) within a promoter that drives expression of a non-coding RNA element, (b) within a promoter that drives expression of a Cas12a polypeptide gene, (c) within 100 bp of the ATG translation start codon in the Cas12a polypeptide coding sequence, (d) within the inverted terminal repeats (iTR) of a viral delivery vector, such as in the AAV genome.

In some aspects, a single nucleic acid component molecule is provided that can hybridize with a sequence downstream of the Cas12a polypeptide start codon, whereby after a period of time, loss of Cas12a polypeptide expression occurs. In some aspects, one or more nucleic acid component molecules are provided that can hybridize with one or more coding regions or non-coding regions of a polynucleotide encoding the system, whereby after a period of time, one or more or all of the system is inactivated. In some aspects of the system, and without intending to be limited by theory, a cell may comprise a plurality of complexes, wherein a first subset of the complexes comprises a first nucleic acid component molecule capable of targeting one or more genomic loci to be edited, and a second subset of the complexes comprises at least one second nucleic acid component molecule capable of targeting a polynucleotide encoding the system, wherein the first subset of the complexes mediates editing of the one or more targeted genomic loci and the second subset of the complexes ultimately inactivates the system, thereby inactivating further expression in the cell.

The various coding sequences (Cas12a polypeptides and nucleic acid component molecules) can be included on a single vector or on multiple vectors. For example, it is possible to encode the enzyme on one vector and various RNA sequences on another vector, or to encode the enzyme and one nucleic acid component molecule on one vector and the remaining nucleic acid component molecules on another vector, or any other arrangement. In general, systems using a total of one or two different vectors are preferred. H. Expression vectors of engineered retrotransposons

Delivery of engineered retrotranscripts to cells can generally be accomplished with or without the use of vectors. Delivery of ncRNA encoded by an engineered retrotranscript generally does not require a vector for producing the ncRNA from the engineered retrotranscript. For example, the ncRNA can be directly encapsulated into a delivery vehicle (such as lipid nanoparticles) and delivered to a host cell, as described in other sections.

Engineered retrotransposons (or vectors containing such retrotransposons) can be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeal organism, including bacteria, archaea, fungi, protists, plants ( e.g. , monocots and dicots); and animals ( e.g. , vertebrates and invertebrates). Examples of animals that can be transfected with engineered retrotransposons include, but are not limited to, vertebrates (e.g., fish, birds, mammals ( e.g. , humans and non-human primates, farm animals, pets, and experimental animals)), reptiles, and amphibians.

Engineered retrotransposons can be introduced into single cells or cell populations. Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro , and artificial cells ( e.g. , nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) can be transfected with engineered retrotransposons.

The engineered retrotransposons can be introduced into cellular fragments, cellular components, or cellular organelles ( e.g. , mitochondria in animal and plant cells, plastids ( e.g. , chloroplasts) in plant cells and algae).

Following transfection with the engineered retrotransposons, cells can be cultured or expanded.

Methods for introducing nucleic acids into host cells are well known in the art. Common methods include chemically induced transformation, usually using divalent cations ( e.g. , _CaCl ), dextran-mediated transfection, polybrene-mediated transfection, lipofectamine and LT-1-mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of nucleic acids containing engineered retrotransposons into cell nuclei. See , e.g., Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197; incorporated herein by reference in their entirety.

Methods for genetic transformation of plant cells are known in the art and include those described in US2022/0145296, and U.S. Patent Nos. 8,575,425; 7,692,068; 8,802,934; and 7,541,517; each of which is incorporated herein by reference in its entirety. See also Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, G. W. (1999) Method s in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; and Jones et al. (2005) Plant Methods 1:5.

Transformed plant cells can be grown into transgenic organisms, such as plants, according to known methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84.

Plant materials that can be transformed with the engineered retrotransposons described herein include plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant wound tissue, plant clumps, and intact plant cells in plants or plant parts (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stems, roots, root tips, anthers, and the like). Progeny, variants, and mutants of the regenerated plants are also included within the scope of the present disclosure, as long as such parts contain the genetic modification introduced by the engineered retrotransposons. Further provided are processed plant products or by-products that retain the genetic modification introduced by the engineered retrotransposons.

The engineered retrotransposons described herein can be used to produce transgenic plants with desired phenotypes including, but not limited to, increased disease resistance (e.g., increased viral, bacterial or fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil content, and color.

In some embodiments, the retrotransposons msr gene, msd gene and/or ret gene are expressed in an in vitro self-vector, such as in an in vivo transcription system. The resulting ncRNA or msDNA can be isolated and then packaged and/or formulated for direct delivery to a host cell. For example, the isolated ncRNA or msDNA can be packaged/formulated in a delivery medium such as lipid nanoparticles, as described in other sections.

In some embodiments, the retrotransposons msr gene, msd gene and/or ret gene are expressed in vivo from a vector in a cell. Retrotransposons msr gene, msd gene and/or ret gene can be introduced into cells using a single vector or multiple separate vectors to produce msDNA in a host individual.

In other embodiments, the retrotranscript msr gene, msd gene and/or ret gene and any other components (e.g., trans- guide RNA, programmable nuclease (e.g., trans- )) of the retrotranscript-based genome editing system described herein can be expressed in vivo by RNA delivered to cells. Retrotranscript msr gene, msd gene and/or ret gene can be introduced into cells using a single vector or multiple separate vectors to produce msDNA in a host individual.

Vectors and/or nucleic acid molecules encoding a recombinant retrotranscript-based genome editing system or its components may include control elements operably linked to the retrotranscript sequence that allow the production of msDNA in vitro or in vivo in an individual species. For example, a retrotranscript msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retrotranscript reverse transcriptase and/or msDNA product. In some embodiments, a heterologous sequence encoding a desired related product ( e.g. , a polynucleotide encoding a polypeptide or regulatory RNA, a donor polynucleotide for gene editing, or a protospacer DNA for molecular recording) can be inserted into the msr gene and/or msd gene.

Any eukaryotic, archaeal, or prokaryotic cell that can be transfected with a vector or retrotransposon delivery system containing an engineered retrotranscript sequence can be used to produce msDNA in vivo . The ability of a construct to produce msDNA and other retrotranscript-encoded products can be determined empirically. For example, transfected cells can be analyzed by phenotypic changes due to the introduced sequence or by direct DNA sequencing.

In some embodiments, the engineered retrotransposons are produced by a vector system comprising one or more vectors. In the vector system, the msr gene, the msd gene and/or the ret gene can be provided by the same vector ( that is , the cis-arrangement of all such retrotransposons), wherein the vector comprises a promoter operably linked to the msr gene and/or the msd gene. In some embodiments, the promoter is further operably linked to the ret gene. In other embodiments, the vector further comprises a second promoter operably linked to the ret gene. Alternatively, the ret gene can be provided by a second vector that does not include the msr gene and/or the msd gene ( that is , the trans-arrangement of msr-msd and ret). In other embodiments, the msr gene, the msd gene and the ret gene are each provided by different vectors ( that is , the trans- arrangement of all retrotransposons).

A variety of vectors can be used for the vector or vector system, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses.

Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. The expression construct can be replicated in living cells, or it can be produced synthetically.

In some embodiments, the nucleic acid comprising an engineered retrotranscript sequence is under the transcriptional control of a promoter. In some embodiments, the promoter is competent to initiate transcription of an operably linked coding sequence by RNA polymerase I, II or III.

Exemplary promoters for mammalian cell expression include the SV40 early promoter, the CMV promoter (such as the CMV immediate early promoter) (see U.S. Pat. Nos. 5,168,062 and 5,385,839, which are incorporated herein by reference in their entirety), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, etc. Other non-viral promoters (such as the promoter derived from the murine metallothionein gene) will also be used for mammalian expression.

Exemplary promoters for plant cell expression include the CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171); the ubiquitin promoter (Christensen et al., 1989, Plant Mol. Biol. 12:619-632; and Christensen et al., 1992, Plant Mol. Biol. 18:675-689); the pEMU promoter (Last et al., 1991, Theor. Appl. Genet. 81:581-588); and the MAS promoter (Velten et al., 1984, EMBO J. 3:2723-2730).

In additional embodiments, retroviral-based vectors may also contain tissue-specific promoters so that the protein is expressed only after it has been delivered to a specific tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

These and other promoters can be obtained from or incorporated into commercially available plasmids using techniques well known in the art. See , e.g., Sambrook et al. , supra.

In some embodiments, one or more enhancer elements are used in conjunction with a promoter to increase the expression level of the construct. Examples include the SV40 early gene enhancer, as described in Dijkema et al. , EMBOJ (1985) 4:761; enhancer/promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus, as described in Gorman et al ., Proc. Natl. Acad. Sci. USA (1982b) 79:6777; and elements derived from human CMV, as described in Boshart et al. , Cell (1985) 41:521, such as elements included in the CMV intron A sequence. All such sequences are incorporated herein by reference.

In one embodiment, an expression vector for expressing an engineered retrotranscript (including msr gene, msd gene and/or ret gene) comprises a promoter operably linked to a polynucleotide encoding msr gene, msd gene and/or ret gene.

In some embodiments, the vector or vector system also comprises a transcriptional terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al. , supra, and the bovine growth hormone terminator sequence (see , e.g., U.S. Patent No. 5,122,458).

Additionally, a 5'-UTR sequence may be placed adjacent to the coding sequence to further enhance expression. Such sequences may include a UTR comprising an internal ribosome entry site (IRES). The inclusion of an IRES allows translation of one or more open reading frames from the vector. The IRES element attracts the eukaryotic ribosome translation initiation complex and promotes translation initiation. See , e.g., Kaufman et al ., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al. , Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al. , BioTechniques (1996) 20:102-110; Kobayashi et al. , BioTechniques (1996) 21:399-402; and Mosser et al. , BioTechniques (199722 ISO-161)c. A variety of IRES sequences are known and include sequences derived from a variety of viruses, such as the leader sequences of picorna viruses, such as the encephalomyocarditis virus (EMCV) UTR (Jang et al . Virol. (1989) 63:1651-1660), the polio leader, the hepatitis A virus leader, the hepatitis C virus IRES, the human rhinovirus type 2 IRES (Dobrikova et al. , Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301)), the IRES element from foot-and-mouth disease virus (Ramesh et al. , Nucl. Acid Res. (1996) 24:2697-2700), the pyriformis virus IRES (Garlapati et al. , J Biol. Chem. (2004) 279(51):3389-33971) and similar sequences. A variety of non-viral IRES sequences will also be used herein, including but not limited to IRES sequences from yeast and human angiotensin II type 1 receptor IRES (Martin et al. , Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRES (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al . (2008) Proc. Natl. Acad Sci. USA 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083) and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1):37-44).

Such elements can be purchased in the form of plasmids sold , for example, by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA), and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). IRES sequences can be included in vectors, for example, to express a variety of phage recombinant proteins for recombineering or RNA-guided nucleases ( e.g. , Cas9) for HDR in combination with a retrotranscriptase from an expression cassette.

In some embodiments, polynucleotides encoding viral self-cleaving 2A peptides (such as T2A peptides) can be used to allow the production of multiple protein products ( e.g. , Cas9, phage recombinant proteins, retrotranscriptase) from a single vector or a single transcription unit under one promoter. One or more 2A linker peptides can be inserted between the coding sequences in the polycistronic construct. The 2A peptide is self-cleaving, allowing the production of proteins co-expressed in the polycistronic construct at equimolar levels. 2A peptides from various viruses can be used, including but not limited to 2A peptides from foot-and-mouth disease virus, equine rhinitis virus A, Jhosea asigna virus, and porcine tegus virus-1. See , e.g., Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10): 625-629, Furler et al. (2001) Gene Ther. 8(11):864-873; each of which is incorporated herein by reference in its entirety.

In some embodiments, the expression construct comprises a plasmid suitable for transformation of a bacterial host. A variety of bacterial expression vectors are known to those skilled in the art, and the selection of an appropriate vector is a matter of choice. Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31. Bacterial plasmids may contain antibiotic selection markers ( e.g. , ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), the lacZ gene (b-galactosidase produces a blue pigment from the x-gal substrate), fluorescent markers ( e.g., GFP. mCherry), or other markers for selection of transformed bacteria. See , e.g., Sambrook et al. , supra.

In other embodiments, the expression construct comprises a plasmid suitable for transforming yeast cells. Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and a nutritional selection marker ( e.g. , HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), an antibiotic selection marker ( e.g. , kanamycin resistance), a fluorescent marker ( e.g. , mCherry), or other markers for selecting transformed yeast cells. Yeast plasmids may further contain components that allow shuttling between bacterial hosts ( e.g. , E. coli) and yeast cells. Many different types of yeast plasmids are available, including yeast integrating plasmids (Yip), which lack an ORI and integrate into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromeric plasmids (YCp), which are low-copy vectors containing an ARS portion and a centromeric sequence (CEN) portion; and yeast episomal plasmids (YEp), which are high-copy number plasmids that contain a fragment from the 2-micron circle (a native yeast plasmid) that allows for the stable propagation of 50 or more copies per cell.

In other embodiments, the expression construct does not comprise a plasmid suitable for transformation of yeast cells.

In other embodiments, the expression construct comprises a virus or an engineered construct derived from a viral genome. A variety of virus-based systems have been developed to transfer genes into mammalian cells. These include adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, bacilli, and herpes simplex viruses (see , e.g., Wamock et al. (2011) Methods Mol. Biol. 737: 1-25; Walther et al. (2000) Drugs 60(2): 249-271; and Lundstrom (2003) Trends Biotechnol. 21(3): 117-122; incorporated herein by reference in their entirety). Certain viruses are able to enter cells via receptor-mediated endocytosis, integrate into the host cell genome and stably and efficiently express viral genes, making them attractive candidates for transferring foreign genes into mammalian cells.

For example, retroviruses provide a convenient platform for gene delivery systems. The selected sequence can be inserted into a vector and encapsulated in a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of an individual in vivo or in vitro. A variety of retroviral systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr. Pharm. Des. 17(24): 2516-2527). Lentiviruses are a type of retrovirus that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and non-dividing cells (see , e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; incorporated herein by reference).

A variety of adenoviral vectors have also been described. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thus minimizing the risks associated with the induction of insertional mutagenesis.

In addition, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be easily constructed using techniques well known in the art. See , e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. , Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al. , Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, BJ Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol and Immunol. (1992) 158:97-129; Kotin, RM Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al. , J. Exp. Med. (1994) 179:1867-1875.

Another vector system that can be used to deliver nucleic acids encoding engineered retrotransposons is the enterally administered recombinant poxvirus vaccine described by Small, Jr., PA et al. (U.S. Patent No. 5,676,950, issued October 14, 1997, incorporated herein by reference).

Other viral vectors include those derived from the pox family of viruses, including vaccinia virus and fowlpox virus. For example, a vaccinia virus recombinant expressing a nucleic acid molecule of interest ( e.g. , an engineered retrotransposons) can be constructed as follows. First, DNA encoding a specific nucleic acid sequence is inserted into an appropriate vector so that it is adjacent to the vaccinia promoter and flanked by vaccinia DNA sequences, such as sequences encoding thymidine kinase (TK). This vector is then used to transfect cells simultaneously infected with vaccinia. Homologous recombination is used to insert the vaccinia promoter plus a gene encoding a sequence of interest into the viral genome. The resulting TK recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and selecting viral plaques that are resistant to it.

In some embodiments, avipox viruses (such as chickenpox virus and canarypox virus) can also be used to deliver relevant nucleic acid molecules. The use of avipox vectors is particularly desirable in humans and other mammalian species because members of the genus Avipox can only replicate productively in susceptible avian species and are therefore not infectious in mammalian cells. Methods for generating recombinant avipox viruses are known in the art and employ genetic recombination, as described above for the generation of vaccinia viruses. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

Molecularly conjugated vectors can also be used for gene delivery, such as the adenovirus chimeric vectors described by Michael et al ., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. , Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103.

Members of the alphavirus genus may also be used as viral vectors to deliver the polynucleotides of the invention, such as, but not limited to, vectors derived from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE). For a description of Sindbis virus-derived vectors that can be used to practice the methods of the present invention, see Dubensky et al . (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; and Dubensky, Jr., TW et al. , U.S. Patent No. 5,843,723, issued December 1, 1998, and Dubensky, Jr., TW, U.S. Patent No. 5,789,245, issued August 4, 1998, both of which are incorporated herein by reference. Particularly preferred are chimeric alphavirus vectors that contain sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See , e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; incorporated herein by reference in their entirety.

The vaccinia-based infection/transfection system can be conveniently used to provide induced transient expression of relevant nucleic acids ( e.g. , engineered retrotransposons) in host cells. In this system, cells are first infected in vitro with vaccinia virus recombinants that encode bacteriophage T7 RNA polymerase. This polymerase exhibits strong specificity in that it only transcribes templates carrying the T7 promoter. After infection, cells are transfected with relevant nucleic acids driven by the T7 promoter. The polymerase expressed in the cytoplasm by the vaccinia virus recombinants transcribes the transfected DNA into RNA. This method provides high-level, transient, cytoplasmic production of large amounts of RNA. See , e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. , Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

In other methods of delivering nucleic acids using recombinant infection of cowpox or fowlpox viruses or using other viral vectors, an amplification system can be used that will result in high levels of expression after introduction into host cells. Specifically, the T7 RNA polymerase promoter preceding the T7 RNA polymerase coding region can be engineered. Translation of RNA derived from this template will produce T7 RNA polymerase, which in turn will transcribe more templates. At the same time, there will be cDNA, whose expression is under the control of the T7 promoter. Therefore, some T7 RNA polymerase generated by the amplified template RNA will result in transcription of the desired gene. Because some T7 RNA polymerase is needed to initiate amplification, the T7 RNA polymerase can be introduced into the cell together with the template to initiate the transcription reaction. The polymerase can be introduced as a protein, or introduced into the plasmid encoding the RNA polymerase. For further discussion of the T7 system and its use for transforming cells, see , e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al. , Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al. , Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Patent No. 5,135,855.

Insect cell expression systems, such as the baculovirus system, may also be used and are known to those skilled in the art and are described, for example, in Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, DW Murhammer, ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for the baculovirus/insect cell expression system are available in kit form, inter alia, from Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA).

Plant expression systems can also be used to transform plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For descriptions of such systems, see, for example, Porta et al. , Mol. Biotech. (1996) 5:209-221; and Hackland et al. , Arch. Virol. (1994) 139:1-22.

In order to obtain expression of an engineered retrotranscript or the ncRNA encoded thereby, the expression construct or ncRNA must be delivered to the cell. This delivery can be accomplished in vitro , such as in laboratory procedures used to transform cell lines, or in vivo or ex vivo , such as in the treatment of certain disease states. One delivery mechanism is through viral infection, in which the expression construct is encapsulated in infectious viral particles.

Several non-viral methods for transferring expression constructs into cultured cells are also contemplated. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high-speed microprojectiles, and receptor-mediated transfection (see , e.g., Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol. 7:2745-2752; Rippe et al. (1990) Mol. Cell Biol. 10:689-695; Gopal (1985) Mol. Cell Biol. 5:1188-1190; Tur-Kaspa et al. (1986) Mol. Cell. Biol. 6:716-718; Potter et al. (1984) Proc. Natl. Acad. USA 81:7161-7165); Harland and Weintraub (1985) J. Cell Biol. 101:1094-1099); Nicolau and Sene (1982) Biochim. Biophys. Acta 721:185-190; Fraley et al. (1979) Proc. Natl. Acad. Sci. USA 76:3348-3352; Fechheimer et al. (1987) Proc. Natl. Acad. Sci. USA 84:8463-8467; Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572; Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; Wu and Wu (1988) Biochemistry 27:887-892; incorporated herein by reference). Some of these techniques can be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered to the cell, the nucleic acid comprising the engineered retrotranscript sequence can be localized at different sites and expressed. In some embodiments, the nucleic acid comprising the engineered retrotranscript sequence can be stably integrated into the genome of the cell. This integration can be in a homologous position and orientation via homologous recombination (gene replacement), or it can be integrated in a random, non-specific position (gene enhancement). In other embodiments, the nucleic acid can be stably maintained in the cell as an independent, additional DNA segment. Such nucleic acid segments or episomes encode sequences sufficient to allow maintenance and replication independent of or in sync with the host cell cycle. How the expression construct is delivered to the cell and where in the cell the nucleic acid is retained depends on the type of expression construct used.

In some embodiments, the expression construct may simply consist of naked recombinant DNA or plasmids containing an engineered retrotransposon. Transfer of the construct may be performed by any of the methods mentioned above that physically or chemically permeate the cell membrane. This is particularly useful for in vitro transfer, but it may also be applied for in vivo use. Dubensky et al. (Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533) successfully injected polyomavirus DNA in the form of a calcium phosphate precipitate into the liver and spleen of adult and newborn mice, demonstrating active viral replication and acute infection. Benvenisty and Neshif (Proc. Natl. Acad. Sci. USA (1986) 83:9551-9555) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids resulted in expression of transfected genes. It is expected that DNA encoding related engineered retrotranscripts can also be transferred in vivo in a similar manner and express retrotranscript products.

In another embodiment, naked DNA expression constructs can be transferred into cells by particle bombardment. This method relies on the ability to accelerate DNA-coated microprojectiles to high speeds, allowing them to pierce the cell membrane and enter the cell without killing the cell (Klein et al. (1987) Nature 327:70-73). Several devices have been developed for accelerating small particles. One such device relies on a high-voltage discharge to generate an electric current, which in turn provides the motive force (Yang et al . (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572). The microprojectiles can be composed of biologically inert materials such as tungsten or gold beads.

In another embodiment, liposomes can be used to deliver the expression construct. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat (1991) Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al . (eds.), Marcel Dekker, NY, 87-104). The use of lipofectamine-DNA complexes is also contemplated.

In some embodiments, the liposomes may be complexed with hemagglutinating virus (HVJ). This has been shown to promote fusion with cell membranes and facilitate entry of liposome-encapsulated DNA into cells (Kaneda et al. (1989) Science 243:375-378). In other embodiments, the liposomes may be complexed or used in combination with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem. 266(6):3361-3364).

In other embodiments, liposomes may be complexed or used in combination with both HVJ and HMG-I. When a bacterial promoter is used in the DNA construct, it will also be necessary to include an appropriate bacterial polymerase in the liposomes.

Other expression constructs that can be used to deliver nucleic acids into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in nearly all eukaryotic cells. Due to the cell type-specific distribution of the various receptors, delivery can be highly specific (Wu and Wu (1993) Adv. Drug Delivery Rev. 12:159-167). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most widely characterized ligands are asialo-seromucoid (ASOR) and transferrin (see , e.g., Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):3410-3414). Synthetic neoglycoproteins that recognize the same receptor as ASOR have been used as gene delivery vehicles (Ferkol et al. (1993) FASEB J. 7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci. USA 91(9):4086-4090), and epidermal growth factor (EGF) has also been used to deliver genes to squamous cell carcinomas (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may include a ligand and a liposome. For example, Nicolau et al. (Methods Enzymol. (1987) 149: 157-176) used lactose 1-ceramide (a galactose-terminal desialoganglioside) incorporated into liposomes and observed an increase in the uptake of insulin genes by hepatocytes. Therefore, it is feasible that nucleic acids encoding specific genes can also be specifically delivered to cells by any number of receptor-ligand systems (with or without liposomes). In addition, antibodies to cell surface antigens can also be used as targeting moieties.

In some embodiments, promoters useful in the retrotransposons delivery systems described herein can be constitutive, inducible, or tissue-specific. In some embodiments, the promoter can be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or combinations of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics or alcohol. In some embodiments, the inducible promoter may be a promoter with a low basal (non-induced) expression level, such as the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter. I. Overview of Delivery Systems and Delivery Methods

In another aspect, the present disclosure provides vectors for transferring and/or expressing the retrotransposons-based gene editing systems, e.g., in vitro , ex vivo , and in vivo conditions. In another aspect, the present disclosure provides cell delivery compositions and methods, including compositions for passively and/or actively delivering the retrotransposons-based gene editing systems described herein to cells (e.g., plasmids), delivery by viral-based recombinant vectors (e.g., AAV and/or lentiviral vectors), delivery by non-viral-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retrotransposons-based gene editing systems described herein can be delivered in the form of DNA (e.g., plasmids or DNA-based viral vectors), RNA (e.g., guide RNA and mRNA delivered by LNPs), mixtures of DNA and RNA, proteins (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combination of methods for delivering the components of the retrotransposons-based gene editing systems disclosed herein can be employed.

Retroviral gene editing systems and/or their components can be delivered by any known delivery system, such as those described above, including (a) vector-free (e.g., electroporation), (b) viral delivery systems, and (c) non-viral delivery systems. Viral delivery systems include expression vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. Expression constructs can be replicated in living cells, or they can be made synthetically. Non-viral delivery systems include, but are not limited to, lipid particles ( e.g., lipid nanoparticles (LNPs)), non-lipid nanoparticles, exosomes, liposomes, micelles, virosomes, stable nucleic acid-lipid particles (SNALPs), lipid complexes/polyplexes, DNA nanowires, gold nanoparticles, iTOP, streptolysin O (SLO), multifunctional coated nanodevices (MENDs), lipid-coated mesoporous silica particles, inorganic nanoparticles, and polymer delivery technologies ( e.g. , polymer-based particles).

Delivery of nucleic acid forms, including RNA therapeutics, is further described in Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev Genet. 2022 May;23(5):265-280. doi: 10.1038/s41576-021-00439-4. Epub 2022 Jan 4. PMID: 34983972; PMCID: PMC8724758；Hong CA, Nam YS. Functional nanostructures for effective delivery of small interfering RNA therapeutics. Theranostics. 2014 Sep 19;4(12):1211-32. doi: 10.7150/thno.8491. PMID: 25285170; PMCID: PMC4183999; Liu F, Wang C, Gao Y, Li X, Tian F, Zhang Y, Fu M, Li P, Wang Y, Wang F. Current Transport Systems and Clinical Applications for Small Interfering RNA (siRNA) Drugs. Mol Diagn Ther. 2018 Oct;22(5):551-569. doi: 10.1007/s40291-018-0338-8. ID: 29926308; Zhang Y, Almazi JG, Ong HX, Johansen MD, Ledger S, Traini D, Hansbro PM, Kelleher AD, Ahlenstiel CL. Nanoparticle Delivery Platforms for RNAi Therapeutics Targeting COVID-19 Disease in the Respiratory Tract. Int J Mol Sci. 2022 Feb 22; 23(5):2408. doi: 10.3390/ijms23052408. PMID: 35269550; PMCID: PMC8909959；Zhang M, Hu S, Liu L, Dang P, Liu Y, Sun Z, Qiao B, Wang C. Engineered exosomes from different sources for cancer-targeted therapy. Signal Transduct Target Ther. 2023年3月15日; 8(1):124. doi: 10.1038/s41392-023-01382-y. PMID: 36922504; PMCID: PMC10017761；Hastings ML, Krainer AR. RNA therapeutics. RNA. 2023年4月; 29(4):393-395. doi: 10.1261/rna.079626.123. PMID: 36928165; PMCID: PMC10019368; Miele E, Spinelli GP, Miele E, Di Fabrizio E, Ferretti E, Tomao S, Gulino A. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy. Int J Nanomedicine. 2012;7:3637-57. doi: 10.2147/IJN.S23696. Epub 2012 Jul 20. PMID: 22915840; PMCID: PMC3418108, each of which is incorporated by reference in its entirety.

Engineered retrotransposon-based gene editing systems (or vectors containing such retrotransposon) can be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeal organism, including bacteria, archaea, fungi, protists, plants ( e.g. , monocots and dicots); and animals ( e.g. , vertebrates and invertebrates). Examples of animals that can be transfected with engineered retrotransposon-based gene editing systems include, but are not limited to, vertebrates (such as fish, birds, mammals ( e.g. , humans and non-human primates, farm animals, pets, and experimental animals)), reptiles, and amphibians.

The engineered retrotransposon-based gene editing system (or its components) can be introduced into a single cell or a population of cells. Cells from tissues, organs and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro , and artificial cells ( e.g. , nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) can be transfected with the engineered retrotransposon-based gene editing system.

The engineered retrotransposon-based gene editing system (or components thereof) can be introduced into cellular fragments, cellular components, or cellular organelles ( e.g. , mitochondria in animal and plant cells, plastids ( e.g. , chloroplasts) in plant cells and algae).

Following transfection with the engineered retroviral-based gene editing system, cells can be cultured or expanded.

Methods for introducing nucleic acids into host cells are well known in the art. Common methods include chemically induced transformation, dextran-mediated transfection, polybrene-mediated transfection, lipofectamine and LT- ₁ -mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of nucleic acids containing the Cas12a editing system into the nucleus. See, for example, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al . (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197; incorporated herein by reference in their entirety.

The retroviral-based gene editing system (or its components) disclosed herein can also be targeted to plant cells. Methods for genetic transformation of plant cells are known in the art and include those described in US2022/0145296, and U.S. Patent Nos. 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is incorporated herein by reference in its entirety. See also Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, G. W. (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; and Jones et al. (2005) Plant Methods 1:5.

Transformed plant cells can be grown into transgenic organisms, such as plants, according to known methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84.

Plant materials that can be transformed with the retrotransposon-based gene editing system described herein (or its components) include plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant wound tissue, plant clusters, and intact plant cells in plants or plant parts (such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, shells, stems, roots, root tips, anthers, and the like). Progeny, variants, and mutants of regenerated plants are also included in the scope of the present disclosure, as long as these parts contain the genetic modifications introduced by the retrotransposon-based gene editing system. Further provided are processed plant products or by-products that retain the genetic modifications introduced by the retrotransposon-based gene editing system.

The retroviral-based gene editing system described herein can be used to produce transgenic plants with desired phenotypes, including but not limited to increased disease resistance (e.g., increased viral, bacterial or fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil content, and color.

In some embodiments involving retrotranscript-based gene editing systems, retrotranscript msr genes, msd genes, and/or ret genes can be expressed in ex vivo self-vectors, such as in ex vivo transcription systems. The resulting ncRNA or msDNA can be isolated and then packaged and/or formulated for direct delivery to host cells. For example, the isolated ncRNA or msDNA can be packaged/formulated in a delivery medium such as lipid nanoparticles, as described in other sections.

In some embodiments involving retrotransposons-based gene editing systems, retrotransposons msr genes, msd genes, and/or ret genes are expressed in vivo from vectors in cells. Retrotransposons msr genes, msd genes, and/or ret genes can be introduced into cells using a single vector or multiple separate vectors to produce msDNA in a host individual.

In other embodiments, the retrotranscript msr gene, msd gene and/or ret gene and any other components (e.g., trans- guide RNA, programmable nuclease (e.g., trans- )) of the retrotranscript-based genome editing system described herein can be expressed in vivo by RNA delivered to cells. Retrotranscript msr gene, msd gene and/or ret gene can be introduced into cells using a single vector or multiple separate vectors to produce msDNA in a host individual.

Vectors and/or nucleic acid molecules encoding a recombinant retrotranscript-based genome editing system or its components may include control elements operably linked to the retrotranscript sequence that allow the production of msDNA in vitro or in vivo in an individual species. For example, in embodiments involving retrotranscript-based gene editing systems, the retrotranscript msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retrotranscript reverse transcriptase and/or msDNA product. In some embodiments, a heterologous sequence encoding a desired related product ( e.g. , a polynucleotide encoding a polypeptide or regulatory RNA, a donor polynucleotide for gene editing, or a protospacer DNA for molecular recording) can be inserted into the msr gene and/or msd gene.

In some embodiments, a retroviral-based gene editing system is generated by a vector system comprising one or more vectors.

A variety of vectors can be used for the vector or vector system, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids , and viruses.

In various embodiments, the retrotranscript-based gene editing system disclosed herein (or its components) can be delivered in "all RNA" form. As used herein, the term "all RNA" form refers to the fact that each component of the retrotranscript editing system (e.g., retrotranscript RT, programmable nuclease, sgRNA and ncRNA) is delivered and/or administered in the form of RNA (e.g., coding RNA or non-coding RNA). In some embodiments, the RNA components can be delivered to cells and/or tissues by direct means (such as electroporation or transfection). In other embodiments, the RNA components can be delivered to cells and/or tissues with the aid of delivery vehicles (such as LNPs or liposomes).

In various embodiments, the retrotranscript editing system described herein may include a coding RNA (e.g., a linear or circular mRNA) encoding a retrotranscript reverse transcriptase (e.g., any RT from Table X or Table A), a coding RNA (e.g., a linear or circular mRNA) encoding a programmable nuclease (e.g., Cas9, Cas12a or TnpB nuclease), a retrotranscript ncRNA (e.g., an ncRNA from Table B), and a guide RNA for targeting the programmable nuclease to a specific desired target sequence.

In some embodiments, the RT and nuclease components can be encoded on the same coding RNA molecule. The protein can also be expressed by a separate coding RNA molecule. In other embodiments, the RT and nuclease components can be fused together as a single fusion polypeptide having an RT domain and a nuclease domain optionally joined by a linker.

In addition, in some embodiments, the ncRNA and the guide RNA can be fused together as a single RNA molecule. For example, the guide RNA can be located at the 5' end of the ncRNA. In other embodiments, the guide RNA can be located at the 3' end of the ncRNA. In some embodiments, the ncRNA can include guide RNAs at both the 3' and 5' ends of the ncRNA.

In other embodiments, the ncRNA and guide RNA can be separate molecules, i.e., delivered separately.

In other embodiments, the retrotranscript editing system may include a ncRNA-guide RNA fusion and an additional guide RNA provided as a separate molecule.

In various embodiments, the different RNA components of the total RNA retrotranscript editing system can be combined and administered (e.g., directly or in a delivery vehicle) in different ratios. In some embodiments, the ratios of such RNA components or substances can be expressed as molar ratios.

For example, the molar ratio of RT encoding RNA to nuclease encoding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of nuclease encoding RNA to RT encoding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of ncRNA or ncRNA-guide RNA fusion to guide RNA alone can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of guide RNA alone to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of ncRNA to guide RNA alone can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the guide RNA to the ncRNA alone can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the coding RNA (e.g., encoding RT and/or nuclease) to the ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20, or any other ratio. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the coding RNA encoding the retrotranscript RT to the ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20, or any other ratio. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the coding RNA encoding the programmable nuclease to the ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20, or any other ratio. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the coding RNA encoding the reverse transcriptase RT or nuclease to the guide RNA alone can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In another example, the molar ratio of the guide RNA alone to the RNA encoding the reverse transcriptase RT or the nuclease can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Available ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40.

In some embodiments, the amount of ncRNA-sgRNA is increased relative to the amount of RT mRNA. In some embodiments, the RT mRNA:ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40. In certain embodiments, the RT-Cas9 (or Cas9-RT) fusion is encoded by mRNA. In certain embodiments, the RT-Cas9 mRNA:ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include 1:1 to 1:2, 1:1.5 to 1:4, 1:2 to 1:4, 1:2 to 1:8, 1:2 to 1:10, 1:3 to 1:9, 1:3 to 1:12, 1:3 to 1:15, 1:4 to 1:8, 1:4 to 1:12, 1:4 to 1:20, 1:5 to 1:10, 1:5 to 1:15, 1:5 to 1:20, 1:10 to 1:20, or 1:10 to 1:40. In certain embodiments, multiple genetic loci are targeted, and thus the ncRNA-sgRNA includes a mixture of ncRNA-sgRNA species and the same ratios and ranges are applicable. Viral Vector Delivery

In various embodiments, the retroviral-based gene editing systems described herein can be delivered in viral vectors.

Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. The expression construct can be replicated in living cells, or it can be produced synthetically.

In some embodiments, the nucleic acid comprising the retrotransposon-based gene editing system (or its components) is under the transcriptional control of a promoter. In some embodiments, the promoter is competent to initiate transcription of an operably linked coding sequence by RNA polymerase I, II or III.

Exemplary promoters for mammalian cell expression include the SV40 early promoter, the CMV promoter (such as the CMV immediate early promoter) (see U.S. Pat. Nos. 5,168,062 and 5,385,839, which are incorporated herein by reference in their entirety), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, etc. Other non-viral promoters (such as the promoter derived from the murine metallothionein gene) will also be used for mammalian expression.

Exemplary promoters for plant cell expression include the CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171); the ubiquitin promoter (Christensen et al., 1989, Plant Mol. Biol. 12:619-632; and Christensen et al., 1992, Plant Mol. Biol. 18:675-689); the pEMU promoter (Last et al., 1991, Theor. Appl. Genet. 81:581-588); and the MAS promoter (Velten et al., 1984, EMBO J. 3:2723-2730).

In additional embodiments, retroviral-based vectors may also contain tissue-specific promoters so that the protein is expressed only after it has been delivered to a specific tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

These and other promoters can be obtained from or incorporated into commercially available plasmids using techniques well known in the art. See , e.g., Sambrook et al. , supra.

In some embodiments, one or more enhancer elements are used in conjunction with a promoter to increase the expression level of the construct. Examples include the SV40 early gene enhancer, as described in Dijkema et al. , EMBOJ (1985) 4:761; enhancer/promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus, as described in Gorman et al ., Proc. Natl. Acad. Sci. USA (1982b) 79:6777; and elements derived from human CMV, as described in Boshart et al. , Cell (1985) 41:521, such as elements included in the CMV intron A sequence. All such sequences are incorporated herein by reference.

In one embodiment, an expression vector for expressing a retrotranscript-based gene editing system (or its components) comprises a promoter operably linked to a polynucleotide encoding the components. The components of the retrotranscript-based gene editing system can be configured as individual gene transcripts or fusion constructs. For example, a nuclease component can be fused to a reverse transcriptase component. In another example, a ncRNA component can be fused to a guide RNA component. In another example, a nuclease component can be fused to a reverse transcriptase component, but the guide RNA and ncRNA are separate. In other embodiments, the guide RNA and ncRNA components can be fused, but the reverse transcriptase and nuclease components are provided separately. Any functional combination of fusion components and non-fusion components is contemplated.

In some embodiments, the vector or vector system also comprises a transcriptional terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al. , supra, and the bovine growth hormone terminator sequence (see , e.g., U.S. Patent No. 5,122,458).

Additionally, a 5'-UTR sequence may be placed adjacent to the coding sequence to further enhance expression. Such sequences may include a UTR comprising an internal ribosome entry site (IRES). The inclusion of an IRES allows translation of one or more open reading frames from the vector. The IRES element attracts the eukaryotic ribosome translation initiation complex and promotes translation initiation. See , e.g., Kaufman et al ., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al. , Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al. , BioTechniques (1996) 20:102-110; Kobayashi et al. , BioTechniques (1996) 21:399-402; and Mosser et al. , BioTechniques (199722 ISO-161)c. A variety of IRES sequences are known and include sequences derived from a variety of viruses, such as the leader sequences of picorna viruses, such as the encephalomyocarditis virus (EMCV) UTR (Jang et al . Virol. (1989) 63:1651-1660), the polio leader, the hepatitis A virus leader, the hepatitis C virus IRES, the human rhinovirus type 2 IRES (Dobrikova et al. , Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301)), the IRES element from foot-and-mouth disease virus (Ramesh et al. , Nucl. Acid Res. (1996) 24:2697-2700), the pyriformis virus IRES (Garlapati et al. , J Biol. Chem. (2004) 279(51):3389-33971) and similar sequences. A variety of non-viral IRES sequences will also be used herein, including but not limited to IRES sequences from yeast and human angiotensin II type 1 receptor IRES (Martin et al. , Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRES (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al . (2008) Proc. Natl. Acad Sci. USA 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083) and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1):37-44).

Such elements can be purchased in the form of plasmids sold , for example, by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA), and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). IRES sequences can be included in vectors, for example, to express a variety of phage recombinant proteins for recombineering or RNA-guided nucleases ( e.g. , Cas9) for HDR in combination with a retrotranscriptase from an expression cassette.

In some embodiments, polynucleotides encoding viral self-cleaving 2A peptides (such as T2A peptides) can be used to allow the production of multiple protein products ( e.g. , Cas9, phage recombinant proteins, retrotranscriptase) from a single vector or a single transcription unit under one promoter. One or more 2A linker peptides can be inserted between the coding sequences in the polycistronic construct. The 2A peptide is self-cleaving, allowing the production of proteins co-expressed in the polycistronic construct at equimolar levels. 2A peptides from various viruses can be used, including but not limited to 2A peptides from foot-and-mouth disease virus, equine rhinitis virus A, Jhosea asigna virus, and porcine tegus virus-1. See , e.g., Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10): 625-629, Furler et al. (2001) Gene Ther. 8(11):864-873; each of which is incorporated herein by reference in its entirety.

In some embodiments, the expression construct comprises a plasmid suitable for transformation of a bacterial host. A variety of bacterial expression vectors are known to those skilled in the art, and the selection of an appropriate vector is a matter of choice. Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31. Bacterial plasmids may contain antibiotic selection markers ( e.g. , ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), the lacZ gene (b-galactosidase produces a blue pigment from the x-gal substrate), fluorescent markers ( e.g., GFP. mCherry), or other markers for selection of transformed bacteria. See , e.g., Sambrook et al. , supra.

In other embodiments, the expression construct comprises a plasmid suitable for transforming yeast cells. Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and a nutritional selection marker ( e.g. , HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), an antibiotic selection marker ( e.g. , kanamycin resistance), a fluorescent marker ( e.g. , mCherry), or other markers for selecting transformed yeast cells. Yeast plasmids may further contain components that allow shuttling between bacterial hosts ( e.g. , E. coli) and yeast cells. Many different types of yeast plasmids are available, including yeast integrating plasmids (Yip), which lack an ORI and integrate into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromeric plasmids (YCp), which are low-copy vectors containing an ARS portion and a centromeric sequence (CEN) portion; and yeast episomal plasmids (YEp), which are high-copy number plasmids that contain a fragment from the 2-micron circle (a native yeast plasmid) that allows for the stable propagation of 50 or more copies per cell.

In other embodiments, the expression construct does not comprise a plasmid suitable for transformation of yeast cells.

In other embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. A variety of virus-based systems have been developed to transfer genes into mammalian cells. These include adenovirus, retrovirus (g-retrovirus and lentivirus), poxvirus, adeno-associated virus, bacilli, and herpes simplex virus (see , e.g., Wamock et al. (2011) Methods Mol. Biol. 737: 1-25; Walther et al. (2000) Drugs 60(2): 249-271; and Lundstrom (2003) Trends Biotechnol. 21(3): 117-122; incorporated herein by reference in their entirety). Certain viruses are able to enter cells via receptor-mediated endocytosis, integrate into the host cell genome and stably and efficiently express viral genes, making them attractive candidates for transferring foreign genes into mammalian cells.

For example, retroviruses provide a convenient platform for gene delivery systems. The selected sequence can be inserted into a vector and encapsulated in a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of an individual in vivo or in vitro. A variety of retroviral systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al . (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr. Pharm. Des. 17(24): 2516-2527). Lentiviruses are a type of retrovirus that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and non-dividing cells (see , e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; incorporated herein by reference).

A variety of adenoviral vectors have also been described. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thus minimizing the risks associated with the induction of insertional mutagenesis.

In addition, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be easily constructed using techniques well known in the art. See , e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. , Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al. , Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, BJ Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol and Immunol. (1992) 158:97-129; Kotin, RM Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al. , J. Exp. Med. (1994) 179:1867-1875.

Another vector system that can be used to deliver nucleic acids encoding components of the Cas12a editing system is the enterally administered recombinant poxvirus vaccine described by Small, Jr., PA et al. (U.S. Patent No. 5,676,950, issued October 14, 1997, incorporated herein by reference).

Other viral vectors include those derived from the pox family of viruses including vaccinia virus and fowlpox virus. For example, a vaccinia virus recombinant expressing a related nucleic acid molecule ( e.g. , Cas12a editing system) can be constructed as follows. First, the DNA encoding a specific nucleic acid sequence is inserted into an appropriate vector so that it is adjacent to the vaccinia promoter and flanking vaccinia DNA sequences, such as sequences encoding thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination is used to insert the vaccinia promoter plus a gene encoding a related sequence into the viral genome. The resulting TK recombinant can be selected by culturing cells in the presence of 5-bromodeoxyuridine and selecting viral plaques that are resistant to it.

In some embodiments, avipox viruses (such as chickenpox virus and canarypox virus) can also be used to deliver relevant nucleic acid molecules. The use of avipox vectors is particularly desirable in humans and other mammalian species because members of the genus Avipox can only replicate productively in susceptible avian species and are therefore not infectious in mammalian cells. Methods for generating recombinant avipox viruses are known in the art and employ genetic recombination, as described above for the generation of vaccinia viruses. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

Molecularly conjugated vectors can also be used for gene delivery, such as the adenovirus chimeric vectors described by Michael et al ., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. , Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103.

Members of the alphavirus genus may also be used as viral vectors to deliver the polynucleotides of the invention, such as, but not limited to, vectors derived from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE). For a description of Sindbis virus-derived vectors that can be used to practice the methods of the present invention, see Dubensky et al . (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; and Dubensky, Jr., TW et al. , U.S. Patent No. 5,843,723, issued December 1, 1998, and Dubensky, Jr., TW, U.S. Patent No. 5,789,245, issued August 4, 1998, both of which are incorporated herein by reference. Particularly preferred are chimeric alphavirus vectors that contain sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See , e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; incorporated herein by reference in their entirety.

The infection/transfection system based on cowpox can be conveniently used to provide induced transient expression of relevant nucleic acids ( e.g. , Cas12a editing system) in host cells. In this system, cells are first infected in vitro with cowpox virus recombinants that encode bacteriophage T7 RNA polymerase. This polymerase exhibits strong specificity because it only transcribes templates carrying the T7 promoter. After infection, the cells are transfected with relevant nucleic acids driven by the T7 promoter. The polymerase expressed by the cowpox virus recombinant in the cytoplasm transcribes the transfected DNA into RNA. This method provides high-level, transient, cytoplasmic production of large amounts of RNA. See , e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. , Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

In other methods of delivering nucleic acids using recombinant infection of cowpox or fowlpox viruses or using other viral vectors, an amplification system can be used that will result in high levels of expression after introduction into host cells. Specifically, the T7 RNA polymerase promoter preceding the T7 RNA polymerase coding region can be engineered. Translation of RNA derived from this template will produce T7 RNA polymerase, which in turn will transcribe more templates. At the same time, there will be cDNA, whose expression is under the control of the T7 promoter. Therefore, some T7 RNA polymerase generated by the amplified template RNA will result in transcription of the desired gene. Because some T7 RNA polymerase is needed to initiate amplification, the T7 RNA polymerase can be introduced into the cell together with the template to initiate the transcription reaction. The polymerase can be introduced as a protein, or introduced into the plasmid encoding the RNA polymerase. For further discussion of the T7 system and its use for transforming cells, see , e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al. , Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al. , Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Patent No. 5,135,855.

Insect cell expression systems, such as the baculovirus system, may also be used and are known to those skilled in the art and are described, for example, in Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, DW Murhammer, ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for the baculovirus/insect cell expression system are available in kit form, inter alia, from Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA).

Plant expression systems can also be used to transform plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For descriptions of such systems, see, for example, Porta et al. , Mol. Biotech. (1996) 5:209-221; and Hackland et al. , Arch. Virol. (1994) 139:1-22.

In order to obtain expression of a retroviral-based editing system (or its components), the expression construct and/or RNA components must be delivered to the cell. This delivery can be accomplished in vitro , as in laboratory procedures used to transform cell lines, or in vivo or ex vivo , as in the treatment of certain disease states. One mechanism of delivery is via viral infection, where the expression construct is encapsulated in infectious viral particles. Non-viral delivery methods

Several non-viral methods for transferring expression constructs are also contemplated that can be used to deliver retroviral-based editing systems or components thereof into cells. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high-speed microprojectiles, and receptor-mediated transfection (see , e.g., Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol. 7:2745-2752; Rippe et al. (1990) Mol. Cell Biol. 10:689-695; Gopal (1985) Mol. Cell Biol. 5:1188-1190; Tur-Kaspa et al. (1986) Mol. Cell. Biol. 6:716-718; Potter et al . (1984) USA 81:7161-7165); Harland and Weintraub (1985) J. Cell Biol. 101:1094-1099); Nicolau and Sene (1982) Biochim. Biophys. Acta 721:185-190; Fraley et al . (1979) Proc. Natl. Acad. Sci. USA 76:3348-3352; Fechheimer et al. (1987) Proc. Natl. Acad. Sci. USA 84:8463-8467; Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572; Wu and Wu (1987) J. Biol. Chem. 262: 4429-4432; Wu and Wu (1988) Biochemistry 27:887-892; incorporated herein by reference). Some of these techniques can be successfully adapted for in vivo or ex vivo use.

In some embodiments, nucleic acid molecules encoding a retrotranscript-based editing system or its components can be stably integrated into the genome of a cell. This integration can be in a homologous position and orientation via homologous recombination (gene replacement), or it can be integrated in a random, non-specific position (gene enhancement). In other embodiments, the nucleic acid can be stably maintained in the cell as an independent, additional DNA segment. Such nucleic acid segments or episomes encode sequences sufficient to allow maintenance and replication independent of or in sync with the host cell cycle. How the expression construct is delivered to the cell and where the nucleic acid is retained in the cell depends on the type of expression construct used.

In some embodiments, the expression construct encoding the retrotransposon-based editing system or its components may simply consist of naked recombinant DNA or plasmids comprising nucleotide sequences encoding the retrotransposon-based editing system or its components. Transfer of the construct may be performed by any of the methods mentioned above, which physically or chemically permeate the cell membrane. This is particularly applicable to in vitro transfer, but it can also be applied for in vivo use. Dubensky et al. (Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into the liver and spleen of adult and newborn mice, demonstrating active viral replication and acute infection. Benvenisty and Neshif (Proc. Natl. Acad. Sci. USA (1986) 83:9551-9555) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids resulted in expression of the transfected gene. It is expected that DNA encoding a related retrotranscript-based editing system or its components can also be transferred in vivo in a similar manner and express retrotranscript products.

In another embodiment, DNA expression constructs encoding retrotranscript-based editing systems or components thereof can be transferred into cells by particle bombardment. This method relies on the ability to accelerate DNA-coated microprojectiles to high speeds, allowing them to pierce cell membranes and enter cells without killing them (Klein et al. (1987) Nature 327:70-73). Several devices have been developed for accelerating small particles. One such device relies on a high-voltage discharge to generate an electric current, which in turn provides the motive force (Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572). Microprojectiles can be composed of biologically inert materials such as tungsten or gold beads. Liposomes

In another embodiment, liposomes can be used to deliver constructs encoding retrotranscript-based editing systems or components thereof. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat (1991) Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (eds.), Marcel Dekker, NY, 87-104). The use of lipofectamine-DNA complexes is also contemplated.

In some embodiments, the liposomes may be complexed with hemagglutinating virus (HVJ). This has been shown to promote fusion with cell membranes and facilitate entry of liposome-encapsulated DNA into cells (Kaneda et al. (1989) Science 243:375-378). In other embodiments, the liposomes may be complexed or used in combination with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem. 266(6):3361-3364).

In other embodiments, liposomes may be complexed or used in combination with both HVJ and HMG-I. When a bacterial promoter is used in the DNA construct, it will also be necessary to include an appropriate bacterial polymerase in the liposomes.

Liposomes can be made from several different types of lipids (e.g., phospholipids). Liposomes can include natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylinositol choline (DSPC), sphingomyelin, phosphatidylcholine, monosialoganglioside, or any combination thereof.

A variety of other additives can be added to liposomes in order to modify their structure and properties. For example, liposomes can further comprise cholesterol, sphingomyelin and/or 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), for example to increase stability and/or prevent leakage of cargo inside the liposomes.

In one embodiment, the liposome comprises a transport polymer, which may be branched, comprising at least 10 amino acids and a ratio of histidine to non-histidine amino acids greater than 1.5 and less than 10. The branched transport polymer may comprise one or more backbones, one or more terminal branches, and optionally one or more non-terminal branches. See U.S. Patent No. 7,070,807, which is incorporated herein by reference in its entirety. In one embodiment, the transport polymer is a histidine-lysine copolymer (HKP), which is used to encapsulate and deliver mRNA and other cargoes. See U.S. Patent Nos. 7,163,695 and 7,772,201, which are incorporated herein by reference in their entirety.

In one embodiment, the lipid particles may be stable nucleic acid lipid particles (SNALP). SNALP may include ionizable lipids (DLinDMA) (e.g., cationic at low pH), neutral auxiliary lipids, cholesterol, diffusible polyethylene glycol (PEG)-lipids, or any combination thereof. In some examples, SNALP may include synthetic cholesterol, dimalmitoylphosphatidylcholine, 3-N-[(w-methoxypolyethylene glycol)2000)aminomethyl]-1,2-dimyristyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane. In some examples, SNALP can include synthetic cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-eDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA). Polymer-based media

In one embodiment, a retroviral-based editing system or components thereof can be encapsulated by a delivery vehicle comprising polymer-based particles (e.g., nanoparticles). In one embodiment, the polymer-based particles can mimic the viral membrane fusion mechanism. The polymer-based particles can be synthetic copies of the influenza virus machinery and form transfection complexes with various types of nucleic acids (siRNA, miRNA, plasmid DNA or nucleic acid components, mRNA) that are taken up by the cell via the endocytic pathway, a process that involves the formation of an acidic compartment. The low pH in the late endosome acts as a chemical switch, making the particle surface hydrophobic and facilitating membrane permeation. Once in the cytosol, the particles release their payload for cellular action. This active endosomal escape technology is safe and maximizes transfection efficiency because it uses the natural uptake pathway. In one embodiment, the polymer-based particles can comprise alkylated and carboxyalkylated branched-chain polyethyleneimine. In some examples, the polymer-based particles are VIROMER, such as VIROMER RNAi, VIROMER RED, VIROMER mRNA. Exemplary methods for delivering the systems and compositions herein include those described in: Bawage SS et al., Synthetic mRNA expressed Cast 3a mitigates RNA virus infections, biorxiv.org/content/10.1101/370460vl. Full doi: doi.org/10.1101/370460, Viromer® RED, A powerful tool for transfecting keratinocytes . doi: 10.13140/RG.2.2.16993.61281, Viromer® Transfection - Factbook 2018: Technology, Product Overview, User Information, doi: 10.13140/RG.2.2.23912.16642.

The delivery vehicle may comprise an exosome. Exosomes include membrane-bound extracellular vesicles that can be used to contain and deliver various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids and complexes thereof (e.g., RNPs). Examples of exosomes include those described below: Schroeder A et al., J Intern Med. 2010 Jan;267(l):9-21; El-Andaloussi S et al., Nat Protoc. 2012 Dec;7(12):2112-26; Uno Y et al., Hum

Gene Ther. 2011 Jun;22(6):711-9; Zou W et al., Hum Gene Then 2011 Apr;22(4):465-75. Exemplary exosomes can be generated by 293F cells, where in some cases, mRNA-loaded exosomes drive higher mRNA expression than mRNA-loaded LNPs. See, e.g., J. Biol. Chem. (2021) 297(5) 101266

In some examples, the exosome can form a complex with one or more components of the cargo (e.g., by binding directly or indirectly). In certain examples, the exosome molecule can be fused to a first anchor protein and the cargo component can be fused to a second anchor protein. The first and second anchor proteins can specifically bind to each other, thereby associating the cargo with the exosome. Examples of such exosomes include those described below: Ye Y et al., Biomater Sci. 2020 Apr 28. doi: 10.1039/d0bm00427h. Receptor-mediated delivery

Other expression constructs encoding retrotranscript-based editing systems or their components are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in nearly all eukaryotic cells. Due to the cell type-specific distribution of the various receptors, delivery can be highly specific (Wu and Wu (1993) Adv. Drug Delivery Rev. 12:159-167). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most widely characterized ligands are asialo-seromucoid (ASOR) and transferrin (see , e.g., Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):3410-3414). Synthetic neoglycoproteins that recognize the same receptor as ASOR have been used as gene delivery vehicles (Ferkol et al. (1993) FASEB J. 7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci. USA 91(9):4086-4090), and epidermal growth factor (EGF) has also been used to deliver genes to squamous cell carcinomas (Myers, EPO 0273085).

In other embodiments, the delivery vehicle comprising one or more expression constructs encoding retrotranscript-based editing systems or components thereof may comprise a ligand and a liposome. For example, Nicolau et al. (Methods Enzymol. (1987) 149: 157-176) used lactose 1-ceramide (a galactose-terminal desialoganglioside) incorporated into liposomes and observed an increase in the uptake of insulin genes by hepatocytes. Thus, it is feasible that nucleic acids encoding specific genes can also be specifically delivered to cells by any number of receptor-ligand systems (with or without liposomes). In addition, antibodies to cell surface antigens can also be used as targeting moieties.

In some embodiments, promoters useful in the retrotransposons-based editing systems described herein or components thereof can be constitutive, induced, or tissue-specific. In some embodiments, the promoter can be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or combinations of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be a promoter with a low basal (non-induced) expression level, such as the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is expressed exclusively or primarily in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter and WASP promoter. Lipid Nanoparticles (LNP)

The retrotranscript-based editing systems or components thereof described herein (e.g., linear and circular mRNAs; nucleobase editing systems and/or components thereof) can be encapsulated and delivered by lipid nanoparticles (LNPs) and compositions and/or formulations comprising RNA-encapsulated LNPs.

Described below are LNPs that can be used as payload delivery vehicles contemplated herein, as well as various ionizable lipids, structured lipids, PEGylated lipids, and phospholipids that can be used to prepare LNPs for delivery of payloads to cells herein. In addition, contemplated additional LNP components, such as targeting moieties and other lipid components, are described below.

In one aspect, the present disclosure further provides a delivery system for delivering a therapeutic payload disclosed herein (e.g., an RNA payload described herein, which may encode a polypeptide of interest, such as a nucleobase editing system or a therapeutic protein). In some embodiments, a delivery system suitable for delivering a therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.

In some embodiments, the LNP of the present disclosure comprises ionizable lipids, structural lipids, PEGylated lipids (also referred to as PEG lipids) and phospholipids. In alternative embodiments, the LNP comprises ionizable lipids, structural lipids, PEGylated lipids (also referred to as PEG lipids) and zwitterionic amino acid lipids. In some embodiments, in addition to any of the aforementioned lipid components, the LNP further comprises a fifth lipid. In some embodiments, the LNP encapsulates one or more elements of the active agent of the present disclosure. In some embodiments, the LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is a targeting moiety that is bound to the cells of a specific organ system or otherwise promotes the uptake of the cells.

In some embodiments, the LNP has a diameter of at least about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm. In some embodiments, the LNP has a diameter of less than about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or 160 nm. In some embodiments, the LNP has a diameter of less than about 100 nm. In some embodiments, the LNP has a diameter of less than about 90 nm. In some embodiments, the LNP has a diameter of less than about 80 nm. In some embodiments, the LNP has a diameter of about 60-100 nm. In some embodiments, the LNP has a diameter of about 75-80 nm.

In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the molar ratio of the component lipids in the formulation. As a non-limiting example, the mol% of the ionizable lipids can be about 10 mol% to about 80 mol%. As a non-limiting example, the mol% of the ionizable lipids can be about 20 mol% to about 70 mol%. As a non-limiting example, the mol% of the ionizable lipids can be about 30 mol% to about 60 mol%. As a non-limiting example, the mol% of the ionizable lipids can be about 35 mol% to about 55 mol%. As a non-limiting example, the mol% of the ionizable lipids can be about 40 mol% to about 50 mol%.

In some embodiments, the mol% of phospholipids may be about 1 mol% to about 50 mol%. In some embodiments, the mol% of phospholipids may be about 2 mol% to about 45 mol%. In some embodiments, the mol% of phospholipids may be about 3 mol% to about 40 mol%. In some embodiments, the mol% of phospholipids may be about 4 mol% to about 35 mol%. In some embodiments, the mol% of phospholipids may be about 5 mol% to about 30 mol%. In some embodiments, the mol% of phospholipids may be about 10 mol% to about 20 mol%. In some embodiments, the mol% of phospholipids may be about 5 mol% to about 20 mol%.

In some embodiments, the mol% of structural lipids may be about 10 mol% to about 80 mol%. In some embodiments, the mol% of structural lipids may be about 20 mol% to about 70 mol%. In some embodiments, the mol% of structural lipids may be about 30 mol% to about 60 mol%. In some embodiments, the mol% of structural lipids may be about 35 mol% to about 55 mol%. In some embodiments, the mol% of structural lipids may be about 40 mol% to about 50 mol%.

In some embodiments, the mol% of PEG lipids may be about 0.1 mol% to about 10 mol%. In some embodiments, the mol% of PEG lipids may be about 0.2 mol% to about 5 mol%. In some embodiments, the mol% of PEG lipids may be about 0.5 mol% to about 3 mol%. In some embodiments, the mol% of PEG lipids may be about 1 mol% to about 2 mol%. In some embodiments, the mol% of PEG lipids may be about 1.5 mol%. In some embodiments, the mol% of PEG lipids may be about 2.5 mol%. i. Ionizable lipids

In some embodiments, the LNP disclosed herein comprises an ionizable lipid. In some embodiments, the LNP comprises two or more ionizable lipids.

Various exemplary ionizable lipids of the present disclosure are described below.

In some embodiments, the LNP of the present disclosure comprises an ionizable lipid disclosed in one of the following: US 2023/0053437; US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated herein by reference in its entirety.

In some embodiments, the LNPs described herein comprise lipids, such as ionizable lipids, disclosed in U.S. Application Publication No. US2017/0119904, which is incorporated herein by reference in its entirety.

In some embodiments, the LNPs described herein comprise lipids, such as ionizable lipids, disclosed in PCT application publication WO2021/204179, which is incorporated herein by reference in its entirety.

In some embodiments, the LNPs described herein comprise lipids disclosed in PCT application WO2022/251665A1, such as ionizable lipids, which are incorporated herein by reference in their entirety. In some embodiments, the LNPs described herein comprise ionizable lipids of Table Z: Table Z - Exemplary ionizable lipids Compound# Structure L-1 L-2 L-3 L-4 L-5 L-6 L-7 L-8 L-9 L-10

In some embodiments, the ionizable lipid is MC3.

In some embodiments, the LNP of the present disclosure comprises an ionizable lipid disclosed in PCT application publication WO2023044343A1, which is incorporated herein by reference in its entirety. Formula (VII-A)

In some embodiments, the lipids of the present disclosure have the structure of formula (VII-A): (VII-A), or a pharmaceutically acceptable salt thereof, wherein: A is -N(-X ¹ R ¹ )-, -C(R ^' )(-L ¹ -N(R")R ⁶ )-, -C(R')(-OR ^7a )-, -C(R')(-N(R")R ^8a )-, -C(R')(-C(=O)OR ^9a )-, -C(R')(-C(=O)N(R")R ^10a )-, or -C(=NR ^11a )-; T is -X ^2a -Y ^1a -Q ^1a or -X ³ -C(=O)OR ⁴ ; X ¹ is an optionally substituted C ₂ -C ₆ alkylene group; R ¹ is -OH, -R ^1a , or , ^Z1 is an optionally substituted _C1 - _C6 alkyl group; ^Z1a is hydrogen or an optionally substituted _C1 - _C6 alkyl group; ^X2 and ^X2a are independently an optionally substituted _C2 - _C14 alkylene group or an optionally substituted _C2 - _C14 alkenylene group; ^X3 is an optionally substituted _C2 - _C14 alkylene group or an optionally substituted _C2 - _C14 alkenylene group; (i) ^Y1 is , , , , , or ; The key marked with "*" is connected to X ² ; Y ^1a is , , , , , or ; wherein the bond marked with "*" is connected to X ^2a ; each Z ² is independently H or an optionally substituted C ₁ -C ₈ alkyl group; each Z ³ is independently an optionally substituted C ₁ -C ₆ alkylene group; Q ¹ is -NR ² R ³ , -CH(OR ² )(OR ³ ), -CR ² ═C(R ³ )(R ¹² ) or -C(R ² )(R ³ )(R ¹² ); Q ^1a is -NR ^2' R ^3' , -CH(OR ^2' )(OR ^3' ), -CR ² ═C(R ³ )(R ¹² ) or -C(R ^2' )(R ^3' )(R ^12' ); or (ii) Y ¹ is , , or , where the key marked with "*" is connected to X ² ; Y ^1a is , , or , wherein the bond marked with "*" is connected to X ^2a ; each Z ² is independently H or an optionally substituted C ₁ -C ₈ alkyl group; each Z ³ is independently an optionally substituted C ₁ -C ₆ alkylene group; Q ¹ is -NR ² R ³ ; Q ^1a is -NR ^2' R ^3' ; R ² , R ³ and R ¹² are independently hydrogen, an optionally substituted C ₁ -C ₁₄ alkyl group, an optionally substituted C ₂ -C ₁₄ alkenyl group or -(CH ₂ ) _m -G-(CH ₂ ) _n H; R ^2' , R ^3' and R ^12' are independently hydrogen, an optionally substituted C ₁ -C ₁₄ alkyl group, an optionally substituted C ₂ -C each n is independently 0, 1, ₂ , ₃ , 4, 5, 6, 7 _, 8, 9, _{10, 11 or 12; X3 is an optionally substituted C2-C14} ^alkylene _{group ; R4} ^is an optionally substituted _C4 - _C14 alkylene group; ^L1 is a _C1 - _C8 alkylene group; ^R6 is a _{C1 - C6} alkyl group, a (hydroxy) _C1 - _C6 alkyl group or a (amino) _{C1 - C6} alkyl group; ^R7a is -C(=O)N(R'") ^R7b , -C(=S)N(R'")R ^7b , -N=C(R ^7b )(R ^7c ) or ; R ^7b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^7c is hydrogen or C ₁ -C ₆ alkyl; R ^8a is -C(=O)N(R'")R ^8b , -C(=S)N(R'")R ^8b , -N=C(R ^8b )(R ^8c ) or , R ^8b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^8c is hydrogen or C ₁ -C ₆ alkyl; R ^9a is -N=C(R ^9b )(R ^9c ); R ^9b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^9c is hydrogen or C ₁ -C ₆ alkyl; R ^10a is -N=C(R ^10b )(R ^10c ); R ^10b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^10c is hydrogen or C ₁ -C ₆ alkyl; R ^11a is -OR ^11b , -N(R")R ^11b , -OC(=O)R ^11b or -N(R")C(=O)R ^11b ; R ^11b is C ₁ -C ₆ alkyl, (hydroxy)C ₁ -C ₆ alkyl or (amino)C ₁ -C ₆ alkyl; R' is hydrogen or C ₁ -C ₆ alkyl; R" is hydrogen or C ₁ -C ₆ alkyl; and R'" is hydrogen or C ₁ -C ₆ alkyl. Formula (VIII-A)

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-A), wherein the lipid of the present disclosure has a structure of formula (VIII-A): (VIII-A), or a pharmaceutically acceptable salt thereof. Formula (VII-B)

In some embodiments, the lipids of the present disclosure have the structure of formula (VII-B): (VII-B), or a pharmaceutically acceptable salt thereof, wherein: A is -C(R ^' )(- ^L1 -N(R") ^R6 )-, -C(R')(- ^OR7a )-, -C(R')(-N(R") ^R8a )-, -C(R')(-C(=O) ^OR9a )-, -C(R')(-C(=O)N(R") ^R10a )-, or -C(= ^NR11a )-; T is ^-X2a - ^Y1a - ^Q1a or ^-X3- C(=O) ^OR4 ; ^X2 and ^X2a are independently _C2 - _C14 alkylene or C2- _C14 alkenylene; ^X3 is _C1 -C14 alkylene or _C1 -C14 alkenylene; ₁₄ -alkylene or optionally substituted C ₂ -C ₁₄ -alkenylene; Y ¹ is , , or , where the key marked with "*" is connected to X ² ; Y ^1a is , , or , wherein the bond marked with "*" is connected to ^X2a ; each ^Z3 is independently an optionally substituted _C1 - _C6 alkylene group or an optionally substituted _C2 - _C14 alkenylene group; ^Q1 is ^-NR2R3 , -CH( ^OR2 )(OR3), ^-CR2 =C( ^R3 )( ^R12 ), or -C( ^R2 )( ^R3 )( ^R12 ); ^Q1a is ^{-NR2'R3 '} , -CH( ^OR2' )(OR3 ^' ), ^-CR2 =C( ^R3 )( ^R12 ), or -C(R2 ^' )( ^{R3' )} ( ^{R12 '} ); ^R2 , ^R3 , and ^R12 are independently hydrogen, an optionally substituted C1- _C6 alkylene group, or an optionally substituted C2-C14 alkenylene group; R ^2' , R ^3' and R ^12' are independently hydrogen, optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl or -(CH ₂ ) _m -G-(CH ₂ ) _n H; G is C ₃ -C _{8 cycloalkylene} ; each m is independently 0, 1, 2 _, 3, 4, 5, 6, 7, ₈ , 9, 10, 11 or 12; each n is independently 0, 1, _2, 3, 4, 5, 6, ₇ , 8, 9, ₁₀ , 11 or 12; X ³ is optionally substituted C _{2 -C 14} alkylene; R ^R4 is an optionally substituted _C4 - _C14 alkyl group; ^L1 is a _C1 - _C8 alkylene group; ^R6 is a (hydroxy) _C1 - _C6 alkyl group or an (amino) _C1 - _C6 alkyl group. ^R7a is -C(=O)N(R'") ^R7b , -C(=S)N(R'") ^R7b , -N=C( ^R7b )( ^R7c ), , or ; Z ¹ is an optionally substituted C ₁ -C ₆ alkyl group; R ¹⁰ is a C ₁ -C ₆ alkylene group; R ^7b is a C ₁ -C ₆ alkyl group, a (hydroxy) C ₁ -C ₆ alkyl group or a (amino) C ₁ -C ₆ alkyl group; R ^7c is hydrogen or a C ₁ -C ₆ alkyl group; R ^8a is -C(=O)N(R'")R ^8b , -C(=S)N(R'")R ^8b , -N=C(R ^8b )(R ^8c ), or ; R ^8b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^8c is hydrogen or C ₁ -C ₆ alkyl; R ^9a is -N=C(R ^9b )(R ^9c ); R ^9b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^9c is hydrogen or C ₁ -C ₆ alkyl; R ^10a is -N=C(R ^10b )(R ^10c ); R ^10b is C ₁ -C ₆ alkyl, (hydroxy) C ₁ -C ₆ alkyl or (amino) C ₁ -C ₆ alkyl; R ^10c is hydrogen or C ₁ -C ₆ alkyl; R ^11a is -OR ^11b , -N(R")R ^11b , -OC(=O)R ^11b or -N(R")C(=O)R ^11b ; R ^11b is C ₁ -C ₆ alkyl, (hydroxy)C ₁ -C ₆ alkyl or (amino)C ₁ -C ₆ alkyl; R' is hydrogen or C ₁ -C ₆ alkyl; R" is hydrogen or C ₁ -C ₆ alkyl; and R'" is hydrogen or C ₁ -C ₆ alkyl.

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein A is -C(R ^' )(- ^L1 -N(R") ^R6 )-.

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein A is -C(R')(-OR ^7a )-.

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein A is -C(R')(-N(R")R ^8a ).

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein A is -C(R')(-C(=O)OR ^9a ).

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein A is -C(R')(-C(=O)N(R")R ^10a )-.

In some embodiments, the lipids of the present disclosure have a structure of Formula (VII-B), wherein A is -C(=NR ^11a )-.

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein T is -X ^2a -Y ^1a -Q ^1a .

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein T is -X ³ -C(=O)OR ⁴ .

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein X ² and/or X ^2a is an optionally substituted C ₂ -C ₁₄ alkylene (e.g., C ₂ -C ₁₀ alkylene, C ₂ -C ₈ alkylene, C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ or C ₈ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein X ² is a C ₂ -C ₁₄ alkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein X ^2a is a C ₂ -C ₁₄ alkylene.

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ and/or Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ and/or Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ and/or Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ and/or Y ^1a is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ¹ is .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein Y ^1a is .

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein Q ¹ and/or Q ^1a is -C(R ^2' )(R ^3' )(R ^12' ). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein Q ¹ is -C(R ^2' )(R ^3' )(R ^12' ). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein Q ^1a is -C(R ^2' )(R ^3' )(R ^12' ).

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein X ³ is an optionally substituted C ₁ -C ₁₄ alkylene group (e.g., C ₁ -C ₆ , C ₁ -C ₄ alkylene group). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein X ³ is a C ₁ -C ₁₄ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ² , R ³ , R ¹² , R ^2' , R ^3' and/or R ^12' are hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ² is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ³ is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ¹² is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^2' is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^3' is hydrogen. In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein R ^12′ is hydrogen.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ² , R ³ , R ¹² , R ^2' , R ^3' and/or R ^12' are optionally substituted C ₁ -C ₁₄ alkyl groups (e.g., C ₄ -C ₁₀ alkyl groups, C ₅ , C ₆ , C ₇ , C ₈ , C ₉ alkyl groups). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ² is a C ₄ -C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ³ is a C ₄ -C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ¹² is a C ₄ -C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^2' is a C ₄ -C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^3' is a C ₄ -C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^12' is a C ₄ -C ₁₀ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ⁴ is an optionally substituted C ₄ -C ₁₄ alkyl group (e.g., C ₈ -C ₁₄ alkyl group, linear C ₈ -C ₁₄ alkyl group, C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ or C ₁₄ alkyl group). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ⁴ is a linear C ₈ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ⁴ is a linear C ₁₁ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein L ¹ is a C ₁ -C ₃ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ⁶ is (hydroxy) C ₁ -C ₆ alkyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^7a is or In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein R ^7a is In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein R ^7a is .

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^7a is selected from the group consisting of -C(=O)N(R'")R ^7b , -C(=S)N(R'")R ^7b , and -N=C(R ^7b )(R ^7c ). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^7a is -C(=O)N(R'")R ^7b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^7a is -C(=S)N(R'")R ^7b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^7a is -N=C(R ^7b )(R ^7c ).

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^8a is selected from the group consisting of -C(=O)N(R'")R ^8b , -C(=S)N(R'")R ^8b , and -N=C(R ^8b )(R ^8c ). In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^8a is -C(=O)N(R'")R ^8b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^8a is -C(=S)N(R'")R ^8b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^8a is -C(=S)N(R ^{' "} )R 8b .

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^8a is .

In some embodiments, the lipids of the present disclosure have the structure of Formula (VII-B), wherein R ^9b is (hydroxy)C ₁ -C ₆ alkyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^10b is (amino) C ₁ -C ₆ alkyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -OR ^11b or -OC(=O)R ^11b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -OR ^11b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -OC(=O)R ^11b .

In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -N(R")R ^11b or -N(R")C(=O)R ^11b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -N(R")R ^11b . In some embodiments, the lipids of the present disclosure have a structure of formula (VII-B), wherein R ^11a is -N(R")C(=O)R ^11b .

In some embodiments, the lipid of the present disclosure has a structure of formula (VII-B), wherein R ^11b is (amino) C ₁ -C ₆ alkyl. Formula (III-C)

In some embodiments, the lipids of the present disclosure have the structure of formula (III-C): (III-C), or a pharmaceutically acceptable salt thereof, wherein R ²⁰ is C ₁ -C ₆ alkylene-NR ^20' C(O)OR ^20'' ; R ^20' is hydrogen or an optionally substituted C ₁ -C ₆ alkylene; R ^20'' is an optionally substituted C ₁ -C ₆ alkylene, phenyl or benzyl; Z ¹ is an optionally substituted C ₁ -C ₆ alkylene; X ² and X ^2a are independently an optionally substituted C ₂ -C ₁₄ alkylene; Y ¹ and Y ^1a are independently or ; wherein the bond marked with "*" is connected to X ² or X ^2a ; Z ³ is independently an optionally substituted C ₂ -C ₆ alkylene group; R ² and R ³ are independently an optionally substituted C ₄ -C ₁₄ alkyl group; and R ^2' and R ^3' are independently an optionally substituted C ₄ -C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R ²⁰ is -CH ₂ CH ₂ CH ₂ NHC(O)O-tert-butyl or -CH ₂ CH ₂ CH ₂ NHC(O)O-benzyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R ²⁰ is -CH ₂ CH ₂ CH ₂ NHC(O)O-tert-butyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R ²⁰ is -CH ₂ CH ₂ CH ₂ NHC(O)O-tert-butyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein X ² and X ^2a are independently C ₄ -C ₈ alkylene (e.g., C ₅ , C ₆ , C ₇ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein X ² is a C ₆ alkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein X ^2a is a C ₆ alkylene.

In some embodiments, the lipid of the present disclosure has a structure of formula (III-C), wherein Y ¹ and Y ^1a are , wherein Z ³ is C ₂ -C ₄ alkylene (e.g., C ₂ alkylene). In some embodiments, the lipid of the present disclosure has a structure of formula (III-C), wherein Y ¹ is , wherein Z ³ is C ₂ -C ₄ alkylene (e.g., C ₂ alkylene). In some embodiments, the lipid of the present disclosure has a structure of formula (III-C), wherein Y ^1a is , wherein Z ³ is C ₂ -C ₄ alkylene (eg, C ₂ alkylene).

In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R2, R3, R2' and R3' are independently optionally substituted C4-C10 alkyl (e.g., C6-C9 alkyl, C6, C7, C8, C9 alkyl). In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R2 is a C6-C9 alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R3 is a C6-C9 alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R ^2' is a C ₆ -C ₉ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-C), wherein R ^3' is a C ₆ -C ₉ alkyl. Formula (III-D)

In some embodiments, the lipids of the present disclosure have the structure of formula (III-D): (III-D), or a pharmaceutically acceptable salt thereof, wherein R ¹ is -OH; X ¹ is an optionally substituted C ₄ alkylene group; X ² and X ^2a are independently an optionally substituted C ₂ -C ₁₄ alkylene group; Y ¹ and Y ^1a are independently or ; Z ³ is independently an optionally substituted C ₂ -C ₆ alkylene group; R ² and R ³ are independently an optionally substituted C ₄ -C ₁₄ alkyl group or an optionally substituted C ₁ -C ₂ alkyl group substituted with a cyclopropyl group; or R ² and R ³ are independently an optionally substituted C ₄ -C ₁₄ alkyl group or an optionally substituted C ₁ -C ₂ alkyl group substituted with a cyclopropyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein X ¹ is a C ₄ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein X ² and X ^2a are independently optionally substituted C ₄ -C ₁₀ alkylene (e.g., C ₅ , C ₆ , C ₇ , C ₈ , C ₉ or C ₁₀ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein X ² is C ₄ -C ₁₀ alkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein X ^2a is C ₄ -C ₁₀ alkylene.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein Y ¹ and Y ^1a are independently , wherein Z ³ is independently C ₂ -C ₄ alkylene (eg, C ₂ , C ₄ alkylene).

In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² , R ³ , R ^2′ and R ^3′ are independently C ₆ -C ₁₄ alkyl (e.g., C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ or C ₁₄ alkyl) or C ₁ -C ₂ alkyl substituted with an optionally substituted cyclopropyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² , R ³ , R ^2′ and R ^3′ are independently C ₆ -C ₁₄ alkyl (e.g., C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ or C ₁₄ alkyl). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² is a C ₆ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ³ is a C ₆ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^2' is a C ₆ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^3' is a C ₆ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² is a C ₁ -C ₂ alkyl group substituted with a substituted cyclopropyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ³ is a C ₁ -C ₂ alkyl substituted by a substituted cyclopropyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^2' is a C ₁ -C ₂ alkyl substituted by a substituted cyclopropyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^3' is a C ₁ -C ₂ alkyl substituted by a substituted cyclopropyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² , R ³ , R ^2' and R ^3' are independently C ₁ -C ₂ alkyl substituted with cyclopropylene-(C ₁ -C ⁶ alkylene, optionally substituted with cyclopropylene substituted with C ₁ -C ₆ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ² is C ₁ -C ₂ alkyl substituted with cyclopropylene-(C ₁ -C ₆ alkylene, optionally substituted with cyclopropylene substituted with C ₁ -C ₆ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ³ is a C ₁ -C ₂ alkyl substituted with a cyclopropylene-(C ₁ -C ⁶ alkylene, optionally substituted with a cyclopropylene substituted with a C ₁ -C ₆ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^2' is a C ₁ -C ₂ alkyl substituted with a cyclopropylene-(C ₁ -C ⁶ alkylene, optionally substituted with a cyclopropylene substituted with a C ₁ -C ₆ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-D), wherein R ^3′ is a C ₁ -C ₂ alkyl substituted with a cyclopropyl-(C ₁ -C ⁶ alkylene, optionally substituted with a cyclopropyl substituted with a C ₁ -C ₆ alkylene). Formula (III-E)

In some embodiments, the lipids of the present disclosure have the structure of formula (III-E): (III-E), or a pharmaceutically acceptable salt thereof, wherein R ¹ is -OH; X ¹ is a branched C ₂ -C ₈ alkylene group; X ² and X ^2a are independently optionally substituted C ₂ -C ₁₄ alkylene groups; Y ¹ and Y ^1a are independently or ; Z ³ is independently an optionally substituted C ₂ -C ₆ alkylene group; R ² and R ³ are independently an optionally substituted C ₄ -C ₁₄ alkyl group; R ^2' and R ^{3' are} independently an optionally substituted C ₄ -C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ¹ is a branched C ₆ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ² and X ^2a are independently C ₄ -C ₁₀ alkylene (e.g., C ₆ , C ₇ , C ₈ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ² is C ₄ -C ₁₀ alkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ² is C ₄ -C ₁₀ alkylene.

In some embodiments, the lipid of the present disclosure has a structure of formula (III-E), wherein Y ¹ and Y ^1a are , wherein Z ³ is independently an optionally substituted C ₂ alkylene group. In some embodiments, the lipid of the present disclosure has a structure of formula (III-E), wherein Y ¹ is , wherein Z ³ is independently an optionally substituted C ₂ alkylene group. In some embodiments, the lipid of the present disclosure has a structure of formula (III-E), wherein Y ^1a is , wherein Z ³ is independently an optionally substituted C ₂ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ² , R ³ , R ^2' and R ^3' are independently C ₆ -C ₁₂ alkyl (e.g., C ₉ alkyl) or C ₄ -C ₁₀ alkyl (e.g., C ₄ , C ₆ alkyl) optionally substituted with C ₂ -C ₈ alkenyl (e.g., C _{4 ,} C ₆ alkenyl). In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ² is C ₆ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ³ is C ₆ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^2' is C ₆ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^3' is a C ₆ -C ₁₂ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ² is a C ₄ -C ₁₀ alkyl group optionally substituted with a C ₂ -C ₈ alkenyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ³ is a C ₄ -C ₁₀ alkyl group optionally substituted with a C ₂ -C ₈ alkenyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^2' is a C ₄ -C ₁₀ alkyl group optionally substituted with a C ₂ -C ₈ alkenyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^3′ is a C ₄ -C ₁₀ alkyl group optionally substituted with a C ₂ -C ₈ alkenyl group. Formula (III-F)

In some embodiments, the lipids of the present disclosure have the structure of formula (III-F): (III-F), or a pharmaceutically acceptable salt thereof, wherein R ¹ is -OH; X ¹ is an optionally substituted C ₂ -C ₆ alkylene group; X ² and X ^2a are independently an optionally substituted C ₂ -C ₁₄ alkylene group; Y ¹ and Y ^1a are each a bond; R ² and R ³ are independently an optionally substituted C ₄ -C ₁₄ alkylene group; and R ^2' and R ^3' are independently an optionally substituted C ₄ -C ₁₄ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ¹ is a C ₄ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ² and X ^2a are independently C ₄ -C ₁₀ alkylene (e.g., C ₆ -C ₈ alkylene, C ₆ , C ₇ , C ₈ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ² is C ₄ -C ₁₀ alkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein X ^2a is C ₄ -C ₁₀ alkylene.

In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ² , R ³ , R ^2' and R ^3' are independently C ₆ -C ₁₀ alkyl (e.g., C _{7 ,} C ₈ alkyl). In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ² is C ₆ -C ₁₀ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ³ is C ₆ -C ₁₀ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^2' is C ₆ -C ₁₀ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (III-E), wherein R ^3' is C ₆ -C ₁₀ alkyl. Formula (VIII-B)

In some embodiments, the lipids of the present disclosure have the structure of formula (VIII-B): (VIII-B), or a pharmaceutically acceptable salt thereof, wherein: ^X1 is a bond, ^R1 is a _C1 - _C6 alkyl group, ^X2 is a _C2 - _C6 alkylene group, ^X2a is a _C2 - _C14 alkylene group, wherein ^X2 or ^X2a is substituted with OH or _C1-4 alkylene-OH, and ^Y1 is , , or , where the key marked with "*" is connected to X ² ; Y ^1a is , , or , wherein the bond marked with "*" is connected to ^X2a ; each ^Z3 is independently a _C1 - _C6 alkylene group which is optionally substituted or a _C2 - _C14 alkenylene group which is optionally substituted; ^Q1 is -C( ^R2 )( ^R3 )( ^R12 ); ^Q1a is -C( ^R2' )(R3 ^' )(R12 ^' ); ^R2 , ^R3 and ^R12 are independently hydrogen, a _C1 - _C14 alkyl group which is optionally substituted or a _C2 - _C14 alkenylene group which is optionally substituted, and R2 ^' , ^R3' and R12 ^' are independently hydrogen, a _C1 - _C14 alkyl group which is optionally substituted or a _C2 - _C14 alkenylene group which is optionally substituted.

In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ¹ is methyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein X ² is C ₄ , C ₅ or C ₆ alkylene.

In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein X ^2a is C ₄ -C ₈ alkylene (eg, C ₅ , C ₆ or C ₇ alkylene).

In some embodiments, the lipid of the present disclosure has a structure of formula (VIII-B), wherein Y ¹ is or , and Y ^1a is or In some embodiments, the lipid of the present disclosure has a structure of formula (VIII-B), wherein Y ¹ is In some embodiments, the lipid of the present disclosure has a structure of formula (VIII-B), wherein Y ¹ is In some embodiments, the lipid of the present disclosure has a structure of formula (VIII-B), wherein Y ^1a is In some embodiments, the lipid of the present disclosure has a structure of formula (VIII-B), wherein Y ^1a is .

In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ² , R ³ , R ¹² , R ^2' , R ^3' and R ^12' are independently hydrogen or C ₅ -C ₁₂ alkyl (e.g., C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ alkyl). In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ² is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ³ is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ^2' is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ^3' is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ² is C ₅ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ³ is C ₅ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ^2' is C ₅ -C ₁₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (VIII-B), wherein R ^3' is C ₅ -C ₁₂ alkyl. Formula (X)

In some embodiments, the lipids of the present disclosure have a structure of formula (X): (X), or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 9; R ^xx is selected from hydrogen and an optionally substituted C ₁ -C ₆ alkyl group; and (i) ee is 1, each dd is independently selected from 1 to 4; and each R ^ww is independently selected from the group consisting of a C ₄ -C ₁₄ alkyl group, a branched chain C ₄ -C ₁₂ alkenyl group, a C ₄ -C ₁₂ alkenyl group containing at least two double bonds, and a C ₉ -C ₁₂ alkenyl group, wherein any -(CH ₂ ) ₂ - of the C ₄ -C ₁₄ alkyl group may be optionally replaced by a C ₂ -C ₆ cycloalkylene group; (ii) ee is 0, each dd is 1; and each R ^ww is a straight chain C ₄ -C ₁₂ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is H. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is an optionally substituted C ₁ -C ₆ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₁ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₃ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₄ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₅ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein R ^xx is a C ₆ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently selected from the group consisting of C ₄ -C ₁₄ alkyl, branched C ₄ -C ₁₂ alkenyl, C _{4 -C 12 alkenyl containing at least two double bonds, and C 9 -C 12 alkenyl} , wherein any -(CH ₂ ) ₂ - of the C ₄ -C ₁₄ alkyl may be optionally replaced by a C ₂ -C ₆ cycloalkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₄ -C ₁₄ alkyl, wherein any -(CH ₂ ) ₂ - of the C ₄ -C ₁₄ alkyl may be optionally replaced by a C ₂ -C ₆ cycloalkylene. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₄ -C ₁₄ alkyl group, wherein any -(CH ₂ ) ₂ - of the C ₄ -C ₁₄ alkyl group may be optionally replaced by a cyclopropene. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₄ -C ₁₂ alkenyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₄ -C 12 alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is _a C ₉ -C ₁₂ alkenyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a linear C ₄ -C ₁₂ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently selected from the group consisting of a C ₆ -C ₁₄ alkyl group, a branched C ₈ -C ₁₂ alkenyl group, a C ₈ -C ₁₂ alkenyl group containing at least two double bonds, and a C ₉ -C ₁₂ alkenyl group, wherein any -(CH ₂ ) ₂ - of the C ₆ -C ₁₄ alkyl group may be optionally replaced by a cyclopropane group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₆ -C ₁₄ alkyl group, wherein any -(CH ₂ ) ₂ - of the C ₆ -C ₁₄ alkyl group may be optionally replaced by a cyclopropyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched C ₈ -C ₁₂ alkenyl group, such as (straight or branched C ₃ -C ₅ alkylene)-(branched C ₅ -C ₇ alkenyl), such as (branched C ₅ alkylene)-(branched C ₅ alkenyl), such as .

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₈ -C ₁₂ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ -C ₁₂ alkenyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently selected from the group consisting of C ₆ -C ₁₄ alkyl (e.g., C ₆ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₃ alkyl), wherein any -(CH ₂ ) ₂ - of the C ₆ -C ₁₄ alkyl may be optionally substituted with a cyclopropyl group.

In some embodiments, the lipids of the present disclosure have a structure of Formula (X), wherein each R ^ww is independently a branched-chain C ₈ -C ₁₂ alkenyl (eg, a branched-chain C ₁₀ alkenyl).

In some embodiments, the lipids of the present disclosure have a structure of Formula (X), wherein each R ^ww is independently a C ₈ -C ₁₂ alkenyl group containing at least two double bonds (eg, a C ₉ or C ₁₀ alkenyl group containing two double bonds).

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently (C ₁ alkylene)-(cyclopropylene-C ₆ alkylene) or (C ₂ alkylene)-(cyclopropylene-C ₂ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently (C ₁ alkylene)-(cyclopropylene-C ₆ alkylene). In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is independently (C ₂ alkylene)-(cyclopropylene-C ₂ alkylene).

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₅ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₆ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₇ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₈ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₁ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₂ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₃ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₀ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₁ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₂ alkenyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₈ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₀ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₁ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₂ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₃ alkenyl group containing at least two double bonds. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₁₄ alkenyl group containing at least two double bonds.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkyl group, wherein one -(CH ₂ ) ₂ - of the C ₉ alkyl group is replaced by a C ₂ -C ₆ cycloalkylene group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkyl group, wherein one -(CH ₂ ) ₂ - of the C ₉ alkyl group is replaced by a cyclopropylene group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a C ₉ alkyl group, wherein two -(CH ₂ ) ₂ - of the C ₉ alkyl group are replaced by C ₂ -C ₆ cycloalkylene groups. In some embodiments, the lipids of the present disclosure have the structure of Formula (X), wherein each R ^ww is a C ₉ alkyl group, wherein two —(CH ₂ ) ₂ -cyclopropyl groups of the C ₉ alkyl group are replaced.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₅ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₆ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₇ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₈ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₉ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₁₁ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₁₂ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₁₃ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a straight chain C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₈ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₉ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₁₀ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₁₁ alkenyl. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each R ^ww is a branched chain C ₁₂ alkenyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is independently selected from 3 to 7. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 3. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 4. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 5. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 6. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 7. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 8. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each cc is 9.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each dd is independently selected from 1 to 4. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each dd is 1. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each dd is 2. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each dd is 3. In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein each dd is 4.

In some embodiments, the lipids of the present disclosure have a structure of formula (X), wherein ee is 1.

In some embodiments, the lipid of the present disclosure has a structure of formula (X), wherein ee is 0. Formula (X-A)

In some embodiments, the lipid of the present disclosure has a structure of formula (X), wherein the lipid of the present disclosure has a structure of formula (XA): (XA), or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 7; each dd is independently selected from 1 to 4; R ^xx is selected from hydrogen and optionally substituted C ₁ -C ₆ alkyl; and each R ^ww is independently selected from the group consisting of C ₄ -C ₁₄ alkyl or (straight or branched C ₃ -C ₅ alkylene)-(branched C ₅ -C ₇ alkenyl).

In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is hydrogen. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₁ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₂ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₃ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₄ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₅ alkyl. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein R ^xx is C ₆ alkyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 4, 5, 6 or 7. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 3. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 4. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 5. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 6. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each cc is 7.

In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each dd is 1 or 3. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each dd is 1. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each dd is 2. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each dd is 3. In some embodiments, the lipids of the present disclosure have a structure of formula (X-A), wherein each dd is 4.

In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₄ -C ₁₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₄ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₅ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₆ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₇ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₈ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₉ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₁₀ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₁₁ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₁₂ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₁₃ alkyl group. In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is a C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (XA), wherein each R ^ww is (straight or branched C ₃ -C ₅ alkylene)-(branched C ₅ -C ₇ alkenyl), for example (branched C ₅ alkylene)-(branched C ₅ alkenyl), for example .

In some embodiments, the lipids of the present disclosure comprise an acyclic core. In some embodiments, the lipids of the present disclosure are selected from any lipid in the following Table (I) or a pharmaceutically acceptable salt thereof: Table (I). Non-limiting examples of ionizable lipids having an acyclic core Structure Compound No. 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

In some embodiments, the LNP disclosed herein comprises an ionizable lipid disclosed in PCT application publication WO2023044333A1, which is incorporated herein by reference in its entirety. Formula (CY) In some embodiments, the LNP disclosed herein comprises an ionizable lipid of formula (CY) (CY), or a pharmaceutically acceptable salt thereof, wherein: R ¹ is selected from the group consisting of: -OH, -OAc, R ^1a , and ; Z ¹ is an optionally substituted C ₁ -C ₆ alkyl group; X ¹ is an optionally substituted C ₂ -C ₆ alkylene group; X ² is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -; X ^2' is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -; X ³ is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -; X ^3' is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -; X ⁴ and X ⁵ are independently an optionally substituted C ₂ -C ₁₄ alkylene group or an optionally substituted C ₂ -C ₁₄ alkenylene group; Y ¹ and Y ² are independently selected from the group consisting of: , , , , , , , , , , or ; wherein the bond marked with "*" is connected to X ⁴ or X ⁵ ; each Z ² is independently H or an optionally substituted C ₁ -C ₈ alkyl group; each Z ³ is independently an optionally substituted C ₁ -C ₆ alkylene group; R ² is selected from the group consisting of an optionally substituted C ₄ -C ₂₀ alkyl group, an optionally substituted C ₂ -C ₁₄ alkenyl group, and -(CH ₂ ) _p CH(OR ⁶ )(OR ⁷ ); R ³ is selected from the group consisting of an optionally substituted C ₄ -C ₂₀ alkyl group, an optionally substituted C ₂ -C ₁₄ alkenyl group, or -(CH ₂ ) _q CH(OR ⁸ )(OR ⁹ ); R ^1a is: , , or ; R ^2a , R ^2b and R ^2c are independently hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently hydrogen and C ₁ -C ₆ alkyl; R ⁶ , R ⁷ , R ⁸ and R ⁹ are independently optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl or -(CH ₂ ) _m -A-(CH ₂ ) _n H; each A is independently C ₃ -C ₈ cycloalkylene; Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; p is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6 and 7; and q is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6 and 7. Formula (CY-I), (CY-II), (CY-III), (CY-IV) and (CY-V)

In some embodiments, the present disclosure includes compounds of formula (CY-I), (CY-II), (CY-III), (CY-IV), or (CY-V): (CY-I), (CY-II), (CY-III), (CY-IV) and (CY-V) or a pharmaceutically acceptable salt thereof, wherein X ¹ , X ² , X ^2' , X ³ , X ^3' , X ⁴ , X ⁵ , Y ¹ , Y ² , R ¹ , R ² and R ³ are defined herein. Formula (CY-VI) and (CY-VII)

In some embodiments, the present disclosure includes compounds of formula (CY-VI) or (CY-VII): (CY-VI) (CY-VII) or a pharmaceutically acceptable salt thereof, wherein X ¹ , X ⁴ , X ⁵ , R ¹ , R ² and R ³ are as defined herein. Formula (CY-VIII) and (CY-IX)

In some embodiments, the present disclosure includes compounds of formula (CY-VIII) or (CY-IX): (CY-VIII) (CY-IX), or a pharmaceutically acceptable salt thereof. wherein ^X1 , ^X4 , ^X5 , ^R1 , ^R2 and ^R3 are as defined herein. Formula (CY-IV-a), (CY-IV-b) and (CY-IV-c)

In some embodiments, the present disclosure includes compounds of formula (CY-IV-a), (CY-IV-b) or (CY-IV-c) (CY-IV-a) (CY-IV-b) (CY-IV-c), or a pharmaceutically acceptable salt thereof. wherein X ¹ , X ⁴ , X ⁵ , R ² and R ³ are as defined herein. Formula (CY-IV-d), (CY-IV-e) and (CY-IV-f)

In some embodiments, the present disclosure includes compounds of formula (CY-IV-d), (CY-IV-e) or (CY-IV-f) (CY-IV-d) (CY-IV-e) (CY-IV-f), or a pharmaceutically acceptable salt thereof. wherein X ¹ , X ⁴ , X ⁵ , R ² and R ³ are as defined herein. Formula (CY-IV)

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-IV'): (CY-IV'), or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , R ³ , X ¹ , X ² , X ³ , X ⁴ , X ⁵ , Y ¹ and Y ² are as defined in the combination formula (CY-I').

In some embodiments, the lipid of the present disclosure has a structure of formula (CY-IV'), wherein: R ¹ is -OH, R ^1a , or , wherein Z ¹ is an optionally substituted C ₁ -C ₆ alkyl group; X ¹ is an optionally substituted C ₂ -C ₆ alkylene group; X ² and X ³ are independently a bond, -CH ₂ - or -CH ₂ CH ₂ -; X ⁴ and X ⁵ are independently an optionally substituted C ₂ -C ₁₄ alkylene group; Y ¹ and Y ² are independently , , , or ; R ² and R ³ are independently optionally substituted C ₄ -C ₂₀ alkyl; R ^1a is: , , or ; R ^2a , R ^2b and R ^2c are independently hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently hydrogen and C ₁ -C ₆ alkyl; and R ^5a , R ^5b and R ^5c are independently hydrogen and C ₁ -C ₆ alkyl

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-IV'), wherein R ¹ is -OH, or .

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-IV'), wherein ^Y1 and ^Y2 are independently: .

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-IV'), wherein R ² is -CH(OR ⁶ )(OR ⁷ ).

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-IV'), wherein R ³ is -CH(OR ⁸ )(OR ⁹ ).

Non-limiting examples of lipids having a structure of formula (CY-IV') include compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY60. Formula (CY-VI')

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'): (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , X ¹ , X ² , X ³ , X ⁴ , X ⁵ , Y ¹ and Y ² are as defined in the combination formula (CY-I').

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ¹ is -OH.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^X1 is a _C2 - _C6 alkylene group.

In some embodiments, the lipids of the present disclosure have _a structure of Formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^X2 is _-CH2CH2- .

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein X ⁴ is a C ₂ -C ₆ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^X5 is a _C2 - _C6 alkylene group.

In some embodiments, the lipid of the present disclosure has a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein Y ¹ is: .

In some embodiments, the lipid of the present disclosure has a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^Y2 is: .

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein each Z ³ is independently an optionally substituted C ₁ -C ₆ alkylene group.

In some embodiments, the lipids of the present disclosure have a structure of Formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein each Z ³ is -CH ₂ CH ₂ -.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁶ is a C ₅ -C ₁₄ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^R7 is a _C5 - _C14 alkyl group.

In some embodiments, the lipid of the present disclosure has a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁶ is C ₆ -C ₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein ^R7 is _C6 - _C14 alkenyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁸ is a C ₅ -C ₁₆ alkyl group.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁹ is a C ₅ -C ₁₄ alkyl group.

In some embodiments, the lipid of the present disclosure has a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁸ is C ₆ -C ₁₄ alkenyl.

In some embodiments, the lipids of the present disclosure have a structure of formula (CY-VI'), or a pharmaceutically acceptable salt thereof, wherein R ⁹ is C ₆ -C ₁₄ alkenyl.

In some embodiments, the lipid of the present disclosure comprises a heterocyclic core, wherein the hetero atom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. In some embodiments, the lipid of the present disclosure is selected from any lipid in Table (II) below or a pharmaceutically acceptable salt thereof: R ¹

In some embodiments, R ¹ is selected from the group consisting of: -OH, -OAc, R ^1a , and .

In some embodiments, R ¹ is -OH or -OAc. In some embodiments, R ¹ is OH. In some embodiments, R ¹ is -OAc. In some embodiments, R ¹ is R ^1a . In some embodiments, R ¹ is imidazolyl.

In some embodiments, ^R1 is . R ²

In some embodiments, R ² is selected from the group consisting of optionally substituted C ₄ -C ₂₀ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, and —(CH ₂ ) _p CH(OR ⁶ )(OR ⁷ ).

In some embodiments, R ² is an optionally substituted C ₄ -C ₂₀ alkyl. In some embodiments, R ² is an optionally substituted C ₈ -C ₁₇ alkyl. In some embodiments, R 2 is an optionally substituted C ₉ -C ₁₆ alkyl. In some embodiments, R ² is an optionally substituted C ₈ -C ₁₀ alkyl. In some embodiments, R ² is an optionally substituted C ₁₁ -C ₁₃ alkyl. In some embodiments, R ² is an optionally substituted C ₁₄ -C ₁₆ alkyl. In some embodiments, R ² is an optionally substituted C ₉ alkyl. In some embodiments, R ² is ^an optionally substituted C ₁₀ alkyl. In some embodiments, R ² is an optionally substituted C ₁₁ alkyl. In some embodiments, R ² is an optionally substituted C ₁₂ alkyl. In some embodiments, R ² is an optionally substituted C ₁₃ alkyl. In some embodiments, R ² is an optionally substituted C ₁₄ alkyl. In some embodiments, R ² is an optionally substituted C ₁₅ alkyl. In some embodiments, R ² is an optionally substituted C ₁₆ alkyl.

In some embodiments, R ² is an optionally substituted C ₂ -C ₁₄ alkenyl. In some embodiments, R ² is an optionally substituted C ₅ -C ₁₄ alkenyl. In some embodiments, R 2 is an optionally substituted C ₇ -C 14 alkenyl. In some embodiments, R ² is an optionally substituted C ₉ -C ₁₄ alkenyl. In some embodiments, R ² is ^an optionally substituted C ₁₀ -C ₁₄ alkenyl. In some embodiments, R ² is an optionally substituted C ₁₂ -C _{14 alkenyl} .

In some embodiments, R ² is –(CH ₂ ) _p CH(OR ⁶ )(OR ⁷ ). In some embodiments, R ² is –CH(OR ⁶ )(OR ⁷ ). In some embodiments, R ² is –CH ₂ CH(OR ⁶ )(OR ⁷ ). In some embodiments, R ² is –(CH ₂ ) ₂ CH(OR ⁶ )(OR ⁷ ). In some embodiments, R ² is –(CH ₂ ) ₃ CH(OR ⁶ )(OR ⁷ ). In some embodiments, R ² is –(CH ₂ ) ₄ CH(OR ⁶ )(OR ⁷ ).

In some embodiments, ^R is selected from the group consisting of: and . R ³

In some embodiments, R ³ is selected from the group consisting of optionally substituted C ₄ -C ₂₀ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, and —(CH ₂ ) _q CH(OR ⁶ )(OR ⁷ ).

In some embodiments, R ³ is an optionally substituted C ₄ -C ₂₀ alkyl. In some embodiments, R ³ is an optionally substituted C ₈ -C ₁₇ alkyl. In some embodiments, R 3 is an optionally substituted C ₉ -C ₁₆ alkyl. In some embodiments, R ³ is an optionally substituted C ₈ -C ₁₀ alkyl. In some embodiments, R ³ is an optionally substituted C ₁₁ -C ₁₃ alkyl. In some embodiments, R ³ is an optionally substituted C ₁₄ -C ₁₆ alkyl. In some embodiments, R ³ is an optionally substituted C ₉ alkyl. In some embodiments, R ³ is ^an optionally substituted C ₁₀ alkyl. In some embodiments, R ³ is optionally substituted C ₁₁ alkyl. In some embodiments, R ^{3 is optionally substituted C 12 alkyl. In some embodiments, R 3} _is ^optionally substituted C ₁₃ alkyl. In some embodiments, R ³ is optionally substituted C ₁₄ alkyl. In some embodiments, R ³ is optionally substituted C ₁₅ alkyl. In some embodiments, R ³ is optionally substituted C ₁₆ alkyl.

In some embodiments, R ³ is an optionally substituted C ₂ -C ₁₄ alkenyl. In some embodiments, R ³ is an optionally substituted C ₅ -C ₁₄ alkenyl. In some embodiments, R 3 is an optionally substituted C ₇ -C 14 alkenyl. In some embodiments, R ³ is an optionally substituted C ₉ -C ₁₄ alkenyl. In some embodiments, R ³ is ^an optionally substituted C ₁₀ -C ₁₄ alkenyl. In some embodiments, R ³ is an optionally substituted C ₁₂ -C _{14 alkenyl} .

In some embodiments, R ³ is -(CH ₂ ) _q CH(OR ⁸ )(OR ⁹ ). In some embodiments, R ³ is -CH(OR ⁸ )(OR ⁹ ). In some embodiments, R ³ is -CH ₂ CH(OR ⁸ )(OR ⁹ ). In some embodiments, R ³ is -(CH ₂ ) ₂ CH(OR ⁸ )(OR ⁹ ). In some embodiments, R ³ is -(CH ₂ ) ₃ CH(OR ⁸ )(OR ⁹ ). In some embodiments, R ³ is -(CH ₂ ) ₄ CH(OR ⁸ )(OR ⁹ ).

In some embodiments, ^R3 is selected from the group consisting of: and ^R6 , ^R7 , ^R8 , ^R9

In some embodiments, R ⁶ , R ⁷ , R ⁸ and R ⁹ are independently optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, or -(CH ₂ ) _m -A-(CH ₂ ) _n H. In some embodiments, R ⁶ , R ⁷ , R ⁸ and R ⁹ are independently optionally substituted C ₁ -C ₁₄ alkyl. In some embodiments, R ⁶ , R ⁷ , R ⁸ and R ⁹ are independently optionally substituted C ₂ -C ₁₄ alkenyl. In some embodiments, R ⁶ , R ⁷ , R ⁸ and R ⁹ are independently -(CH ₂ ) _m -A-(CH ₂ ) _n H.

In some embodiments, R ⁶ is optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, or -(CH ₂ ) _m -A-(CH ₂ ) _n H. In some embodiments, R 6 is optionally substituted C _{3 -C 10 alkyl. In some embodiments, R 6 is optionally substituted C 4 -C 10} ^alkyl _. In some embodiments, R ⁶ is independently optionally substituted C ₅ -C ₁₀ alkyl. In some embodiments, R ⁶ is optionally substituted C ₉ -C ₁₀ alkyl ^{. In some embodiments, R 6 is optionally substituted C 1 -C 14 alkyl. In some embodiments, R 6 is} _{optionally substituted} ^C _{2 -C 14} alkenyl. In some embodiments, R ⁶ is —(CH ₂ ) _m —A-(CH ₂ ) _n H.

In some embodiments, R ⁷ is optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, or -(CH ₂ ) _m -A-(CH ₂ ) _n H. In some embodiments, R 7 is optionally substituted C _{3 -C 10 alkyl. In some embodiments, R 7 is optionally substituted C 4 -C} ¹⁰ _{alkyl .} In some embodiments, R ⁷ is optionally substituted C ₅ -C ₁₀ alkyl ^{. In some embodiments, R 7 is optionally substituted C 9 -C 10 alkyl. In some embodiments, R 7 is} _{optionally substituted} ^C ₁ -C ₁₄ alkyl. In some embodiments, ^R7 is optionally substituted or optionally substituted _C2 - _C14 alkenyl. In some embodiments, ^R7 is -( _CH2 ) _m -A-( _CH2 ) _nH .

In some embodiments, R ⁸ is optionally substituted C ₁ -C ₁₄ alkyl, optionally substituted C ₂ -C ₁₄ alkenyl, or -(CH ₂ ) _m -A-(CH ₂ ) _n H. In some embodiments, R 8 is optionally substituted C _{3 -C 10 alkyl. In some embodiments, R 8 is optionally substituted C 4 -C} ¹⁰ _{alkyl .} In some embodiments, R ⁸ is optionally substituted C ₅ -C ₁₀ alkyl. In some embodiments, R ⁸ is optionally substituted C ₉ -C ₁₀ alkyl. In some embodiments, R ⁸ is optionally substituted C ₁ -C ₁₄ alkyl. In some embodiments, R ^{8 is} optionally substituted C ₂ -C ₁₄ alkenyl. In some embodiments, R ⁸ is —(CH ₂ ) _m —A-(CH ₂ ) _n H.

In some embodiments, R ⁹ is an optionally substituted C ₁ -C ₁₄ alkyl, an optionally substituted C ₂ -C ₁₄ alkenyl, or -(CH ₂ ) _m -A-(CH ₂ ) _n H. In some embodiments, R ⁹ is an optionally substituted C ₃ -C ₁₀ alkyl. In some embodiments, R 9 is an optionally substituted C ₄ -C ₁₀ alkyl. In some embodiments, R ⁹ is an optionally substituted C ₅ -C ₁₀ alkyl. In some embodiments, R ⁹ is an optionally substituted C ₉ -C ₁₀ alkyl. In some embodiments, R ⁹ is ^an optionally substituted C ₁ -C ₁₄ alkyl. In some embodiments, R ⁹ is an optionally substituted C ₂ -C ₁₄ alkenyl. In some embodiments, R ⁹ is —(CH ₂ ) _m —A—(CH ₂ ) _n H.

In some embodiments, each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, each m is 0. In some embodiments, each m is 1. In some embodiments, each m is 2. In some embodiments, each m is 3. In some embodiments, each m is 4. In some embodiments, each m is 5. In some embodiments, each m is 6. In some embodiments, each m is 7. In some embodiments, each m is 8. In some embodiments, each m is 9. In some embodiments, each m is 10. In some embodiments, each m is 11. In some embodiments, each m is 12.

In some embodiments, each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, each n is 0. In some embodiments, each n is 1. In some embodiments, each n is 2. In some embodiments, each n is 3. In some embodiments, each n is 4. In some embodiments, each n is 5. In some embodiments, each n is 6. In some embodiments, each n is 7. In some embodiments, each n is 8. In some embodiments, each n is 9. In some embodiments, each n is 10. In some embodiments, each n is 11. In some embodiments, each n is 12.

In some embodiments, each A ^is independently C ₃ -C ₈ cycloalkylene. In some embodiments, each A is cyclopropylene.

In some embodiments, X1 is an optionally substituted C2-C6 alkylene. In some embodiments, X1 is an optionally substituted C2-C5 alkylene. In some embodiments, X1 is an optionally substituted C2-C4 alkylene. In some embodiments, X1 is an optionally substituted C2-C3 alkylene. In some embodiments, X1 is an optionally substituted C2 alkylene. In some embodiments, X1 is an optionally substituted C3 alkylene. In some embodiments, X1 is an optionally substituted C4 alkylene. In some embodiments, X1 is an optionally substituted C5 alkylene. In some embodiments, X1 is an optionally substituted C6 alkylene. In some embodiments, X1 is an optionally substituted –(CH2)2-. In some embodiments, X1 is optionally substituted -(CH2)3-. In some embodiments, X1 is optionally substituted -(CH2)4-. In some embodiments, X1 is optionally substituted -(CH2)5-. In some embodiments, X1 is optionally substituted -( _CH2 ) _6- . ^X2

In some embodiments, X2 is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In some embodiments, X2 is a bond. In some embodiments, X2 is -CH2-. In some embodiments, X2 is -CH2CH2-. X ^2'

In some embodiments, X2' is selected from the group consisting of a bond, -CH2- and _-CH2CH2- . In some embodiments, X2 ^{' is a bond. In some embodiments, X2'} is _-CH2- . In some embodiments, ^{X2' is} _-CH2CH2- . ^X3

In some embodiments, X ³ is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -. In some embodiments, X ³ is a bond. In some embodiments, X ³ is -CH ₂ -. In some embodiments, X ³ is -CH ₂ CH ₂ -. X ^3'

In some embodiments, X ^3' is selected from the group consisting of a bond, -CH ₂ - and -CH ₂ CH ₂ -. In some embodiments, X ^3' is a bond. In some embodiments, X 3' is -CH ₂ -. In some embodiments, X ^{3' is} -CH ₂ CH ₂ -. X ⁴

In some embodiments, X ⁴ is selected from the group consisting of an optionally substituted C ₂ -C ₁₄ alkylene and an optionally substituted C ₂ -C ₁₄ alkenylene. In some embodiments, X ⁴ is an optionally substituted C ₂ -C ₁₄ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₂ -C ₁₀ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₂ -C ₈ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₂ -C ₆ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₃ -C ₆ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₃ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₄ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₅ alkylene. In some embodiments, X ⁴ is an optionally substituted C ₆ alkylene. In some embodiments, X ⁴ is an optionally substituted –(CH ₂ ) ₂ -. In some embodiments, X ⁴ is an optionally substituted –(CH ₂ ) ₃ -. In some embodiments, X ⁴ is an optionally substituted –(CH ₂ ) ₄ -. In some embodiments, X ⁴ is an optionally substituted –(CH ₂ ) ₅ -. In some embodiments, X ⁴ is an optionally substituted –(CH ₂ ) ₆ -. X ⁵

In some embodiments, X ⁵ is selected from the group consisting of an optionally substituted C ₂ -C ₁₄ alkylene and an optionally substituted C ₂ -C ₁₄ alkenylene. In some embodiments, X ⁵ is an optionally substituted C ₂ -C ₁₄ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₂ -C ₁₀ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₂ -C ₈ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₂ -C ₆ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₃ -C ₆ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₃ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₄ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₅ alkylene. In some embodiments, X ⁵ is an optionally substituted C ₆ alkylene. In some embodiments, X ⁵ is an optionally substituted –(CH ₂ ) ₂ -. In some embodiments, X ⁵ is an optionally substituted –(CH ₂ ) ₃ -. In some embodiments, X ⁵ is an optionally substituted –(CH ₂ ) ₄ -. In some embodiments, X ⁵ is an optionally substituted –(CH ₂ ) ₅ -. In some embodiments, X ⁵ is an optionally substituted –(CH ₂ ) ₆ -. Y ¹

In some embodiments, ^Y1 is selected from the group consisting of: , , , , , , , , , , and .

In some embodiments, ^Y1 is or . Y ²

In some embodiments, ^Y2 is selected from the group consisting of: , , , , , , , , , , and .

In some embodiments, ^Y2 is or Table (II). Non-limiting examples of ionizable lipids having a cyclic core Structure Compound No. CY1 CY2 CY3 CY4 CY5 CY6 CY7 CY8 CY9 CY10 CY11 CY12 CY13 CY14 CY15 CY16 CY17 CY18 CY19 CY20 CY21 CY22 CY23 CY24 CY25 CY26 CY27 CY28 CY29 CY30 CY31 CY32 CY33 CY34 CY35 CY36 CY37 CY38 CY39 CY40 CY41 CY42 CY43 CY44 CY45 CY46 CY47 CY48 CY49 CY50 CY51 CY52 CY53 CY54 CY55 CY56 CY57 CY58 CY59 CY60 CY61 CY62 CY63 CY64 CY65 CY66 CY67 CY68 CY69 CY70 CY71

In some embodiments, the LNPs of the present disclosure comprise ionizable lipids disclosed in PCT application PCT/US2022/082276 (WO2023122752), which is incorporated herein by reference in its entirety.

In one embodiment, the present disclosure provides a compound of formula IA : IA , or a pharmaceutically acceptable salt or solvent thereof, wherein: A is selected from the group consisting of: -N(R ^1a )- and -C(R')-OC(=O)(R ^8a )-; R ^1a is -L ¹ -R ¹ ; L ¹ is C ₂ -C ₆ alkylene or -(CH ₂ ) _2-6 -OC(=O)-; R ¹ is selected from the group consisting of: -OH, , , , , , , and ; R ^2a , R ^2b and R ^2c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^6a , R ^6b and R ^6c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^6a and R ^6b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^6c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^7a , R ^7b and R ^7c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^7a and R ^7b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^7c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R' is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^8a is -L ² -R ⁸ ; L ² is C ₂ -C ₆ alkylene; R ⁸ is selected from the group consisting of: -NR ^9a R ^9b , , , , , , , and R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^9a and R ^9b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; Q ¹ is C ₁ -C ₂₀ alkylene; W ¹ is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O- and -OC(=O)O-; R ^12a is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ¹ is an optionally substituted C ₁ -C ₁₅ alkylene; or X ¹ is a bond; Y ¹ is selected from the group consisting of: -(CH ₂ ) _m -, -O-, -S- and -SS-; m is 0, 1, 2, 3, 4, 5 or 6; Z ¹ is selected from the group consisting of: optionally substituted C ₄ -C ₁₂ cycloalkylene, and R ¹⁰ is selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl; Q ² is C ₁ -C ₂₀ alkylene; W ² is selected from the group consisting of -C(=O)O-, -C(=O)N(R ^12b )-, -OC(=O)N(R ^12b )- and -OC(=O)O-; R ^12b is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ² is an optionally substituted C ₁ -C ₁₅ alkylene; or X ² is a bond; Y ² is selected from the group consisting of -(CH ₂ ) _n -, -O-, -S- and -SS-; n is 0, 1, 2, 3, 4, 5 or 6; Z ² is selected from the group consisting of -(CH ₂ ) _p -, optionally substituted C ₄ -C ₁₂ cycloalkylene group, and ; p is 0 or 1; and R ¹¹ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; wherein one or more methylene bonds of X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ and R ¹¹ are, as the case may be, independently replaced by a group selected from -O-, -CH=CH-, -S- and C ₃ -C ₆ cycloalkylene.

In one embodiment, the present disclosure provides a compound of formula IB : IB , or a pharmaceutically acceptable salt or solvent thereof, wherein: A is selected from the group consisting of: -N(R ^1a )- and -C(R')-OC(=O)(R ^8a )-; R ^1a is -L ¹ -R ¹ ; L ¹ is C ₂ -C ₆ alkylene or -(CH ₂ ) _2-6 -OC(=O)-; R ¹ is selected from the group consisting of: -OH, , , , , , , and ; R ^2a , R ^2b and R ^2c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^6a , R ^6b and R ^6c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^6a and R ^6b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^6c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^7a , R ^7b and R ^7c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^7a and R ^7b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^7c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R' is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^8a is -L ² -R ⁸ ; L ² is C ₂ -C ₆ alkylene; R ⁸ is selected from the group consisting of: -NR ^9a R ^9b , , , , , , , and R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^9a and R ^9b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; Q ¹ is C ₁ -C ₂₀ alkylene; W ¹ is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O- and -OC(=O)O-; R ^12a is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ¹ is an optionally substituted C ₁ -C ₁₅ alkylene; or X ¹ is a bond; Y ¹ is selected from the group consisting of: -(CH ₂ ) _m -, -O-, -S- and -SS-; m is 0, 1, 2, 3, 4, 5 or 6; Z ¹ is selected from the group consisting of: optionally substituted C ₅ -C ₁₂ bridged cycloalkyl, and R ¹⁰ is selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl; Q ² is C ₁ -C ₂₀ alkylene; W ² is selected from the group consisting of -C(=O)O-, -C(=O)N(R ^12b )-, -OC(=O)N(R ^12b )- and -OC(=O)O-; R ^12b is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ² is an optionally substituted C ₁ -C ₁₅ alkylene; or X ² is a bond; Y ² is selected from the group consisting of -(CH ₂ ) _n -, -O-, -S- and -SS-; n is 0, 1, 2, 3, 4, 5 or 6; Z ² is selected from the group consisting of -(CH ₂ ) _p -, optionally substituted C ₄ -C ₁₂ cycloalkylene group, and ; p is 0 or 1; and R ¹¹ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; wherein one or more methylene bonds of X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ and R ¹¹ are, as the case may be, independently replaced by a group selected from -O-, -CH=CH-, -S- and C ₃ -C ₆ cycloalkylene.

In one embodiment, the present disclosure provides compounds of formula IC : IC , or a pharmaceutically acceptable salt or solvent thereof, wherein: A is selected from the group consisting of: -N(R ^1a )- and -C(R')-OC(=O)(R ^8a )-; R ^1a is -L ¹ -R ¹ ; L ¹ is C ₂ -C ₆ alkylene or -(CH ₂ ) _2-6 -OC(=O)-; R ¹ is selected from the group consisting of: -OH, , , , , , , and ; R ^2a , R ^2b and R ^2c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^6a , R ^6b and R ^6c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^6a and R ^6b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^6c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^7a , R ^7b and R ^7c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^7a and R ^7b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^7c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R' is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^8a is -L ² -R ⁸ ; L ² is C ₂ -C ₆ alkylene; R ⁸ is selected from the group consisting of: -NR ^9a R ^9b , , , , , , , and R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^9a and R ^9b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; Q ¹ is C ₁ -C ₂₀ alkylene; W ¹ is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O- and -OC(=O)O-; R ^12a is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ¹ is an optionally substituted branched chain C ₁ -C ₁₅ alkylene; or X ¹ is a bond; Y ¹ is selected from the group consisting of: -(CH ₂ ) _m -, -O-, -S- and -SS-; m is 0, 1, 2, 3, 4, 5 or 6; Z ¹ is selected from the group consisting of: optionally substituted C ₄ -C ₁₂ cycloalkylene, and R ¹⁰ is selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl; Q ² is C ₁ -C ₂₀ alkylene; W ² is selected from the group consisting of -C(=O)O-, -C(=O)N(R ^12b )-, -OC(=O)N(R ^12b )- and -OC(=O)O-; R ^12b is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ² is optionally substituted C ₁ -C ₁₅ alkylene; or Y ² is selected from the group consisting of -(CH ₂ ) _n -, -O-, -S- and -SS-; n is 0, 1, 2, 3, 4, 5 or 6; Z ² is -(CH ₂ ) _p -; p is 0 or 1; and R ¹¹ is C ₁ -C ₂₀ branched alkyl groups; wherein one or more methylene bonds of X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ and R ¹¹ are optionally and independently replaced by a group selected from -O-, -CH=CH-, -S- and C ₃ -C ₆ cycloalkylene groups.

In some embodiments, the present disclosure provides compounds of any one of Formulae IA , IB , IC, or a pharmaceutically acceptable salt or solvent thereof, wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In some embodiments, the present disclosure provides a compound of any one of Formulas IA , IB , IC, or a pharmaceutically acceptable salt or solvent thereof, wherein Z ¹ is not an adamantyl group.

In one embodiment, the present disclosure provides a compound of formula ID : ID , or a pharmaceutically acceptable salt or solvent thereof, wherein: A is selected from the group consisting of: -N(R ^1a )- and -C(R')-OC(=O)(R ^8a )-; R ^1a is -L ¹ -R ¹ ; L ¹ is C ₂ -C ₆ alkylene or -(CH ₂ ) _2-6 -OC(=O)-; R ¹ is selected from the group consisting of: -OH, , , , , , , and ; R ^2a , R ^2b and R ^2c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^6a , R ^6b and R ^6c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^6a and R ^6b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^6c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^7a , R ^7b and R ^7c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^7a and R ^7b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^7c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R' is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^8a is -L ² -R ⁸ ; L ² is C ₂ -C ₆ alkylene; R ⁸ is selected from the group consisting of: -NR ^9a R ^9b , , , , , , , and R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^9a and R ^9b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; Q ¹ is C ₁ -C ₂₀ alkylene; W ¹ is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O- and -OC(=O)O-; R ^12a is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ¹ is an optionally substituted branched chain C ₁ -C ₁₅ alkylene; or X ¹ is a bond; Y ¹ is selected from the group consisting of: -(CH ₂ ) _m -, -O-, -S- and -SS-; m is 0, 1, 2, 3, 4, 5 or 6; Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkyl group; R ¹⁰ is selected from the group consisting of: hydrogen, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl; Q ² is a C ₁ -C ₂₀ alkylene group; W ² is selected from the group consisting of: -C(=O)O-, -C(=O)N(R ^12b )-, -OC(=O)N(R ^12b )- and -OC(=O)O-; R ^12b is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ² is an optionally substituted C ₁ -C or Y ² is -(CH ₂ ) _n -; n is 0, 1, 2, 3, 4, 5 or 6; Z ² is -(CH ₂ ) _p -; p is 0 or 1; and R ¹¹ is _a C ₁ -C ₂₀ branched chain alkyl.

In some embodiments, the present disclosure provides a compound of formula ID or a pharmaceutically acceptable salt or solvent thereof, wherein Z ¹ is not an adamantyl group.

In one embodiment, the present disclosure provides a compound of formula I : I, or a pharmaceutically acceptable salt or solvent thereof, wherein: A is selected from the group consisting of: -N(R ^1a )- and -C(R')-OC(=O)(R ^8a )-; R ^1a is -L ¹ -R ¹ ; L ¹ is C ₂ -C ₆ alkylene; R ¹ is selected from the group consisting of: -OH, , , , , and ; R ^2a , R ^2b and R ^2c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^3a , R ^3b and R ^3c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^4a , R ^4b and R ^4c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^5a , R ^5b and R ^5c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^6a , R ^6b and R ^6c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^6a and R ^6b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^6c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^7a , R ^7b and R ^7c are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^7a and R ^7b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; and R ^7c is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R' is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; R ^8a is -L ² -R ⁸ ; L ² is C ₂ -C ₆ alkylene; R ⁸ is -NR ^9a R ^9b ; R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; or R ^9a and R ^9b together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; Q ¹ is C ₁ -C ₂₀ alkylene; W wherein ^{R 1} is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O-, and -OC(=O)O-; R ^12a is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ¹ is C ₁ -C ₁₅ alkylene; or X ¹ is a bond; Y ¹ is selected from the group consisting of -(CH ₂ ) _m -, -O-, -S-, and -SS-; m is 0, 1, 2, 3, 4, 5, or 6; Z ¹ is selected from the group consisting of C ₄ -C ₁₂ cycloalkylene, and R ¹⁰ is selected from the group consisting of hydrogen, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl; Q ² is C ₁ -C ₂₀ alkylene; W ² is selected from the group consisting of -C(=O)O-, -C(=O)N(R ^12b )-, -OC(=O)N(R ^12b )- and -OC(=O)O-; R ^12b is selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl; X ² is C ₁ -C ₁₅ alkylene; or X ² is a bond; Y ² is selected from the group consisting of -(CH ₂ ) _n -, -O-, -S- and -SS-; n is 0, 1, 2, 3, 4, 5 or 6; Z ² is selected from the group consisting of -(CH ₂ ) _p -, C ₄ -C _12- cycloalkylene, and ; p is 0 or 1; and R ¹¹ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl.

In another embodiment, the present disclosure provides a compound of formula II : II , or a pharmaceutically acceptable salt or solvent thereof, wherein ^R1 , ^R10 , ^R11 , ^Q1 , ^Q2 , ^W1 , ^W2 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 and ^Z2 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula III : III , or a pharmaceutically acceptable salt or solvent thereof, wherein R', ^R9a , ^R9b , ^R10 , ^R11 , ^L2 , ^Q1 , ^Q2 , ^W1 , ^W2 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 and ^Z2 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula IV : VI or a pharmaceutically acceptable salt or solvent thereof, wherein R ^9a , R ^9b , L ² , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula VI' : VI' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^9a , R ^9b , L ² , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula VI'' : VI'' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^9a , R ^9b , L ² , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of Formula VI''' : VI''' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^9a , R ^9b , L ² , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula VII : VII or a pharmaceutically acceptable salt or solvent thereof, wherein R ¹ , L ¹ , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below. In another embodiment, the present disclosure provides a compound of Formula VII' : VII' or a pharmaceutically acceptable salt or solvent thereof, wherein ^R1 , ^L1 , ^Q1 , ^Q2 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula VII'' : VII'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^R1 , ^L1 , ^Q1 , ^Q2 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of Formula VII''' : VII''' or a pharmaceutically acceptable salt or solvent thereof, wherein R ¹ , L ¹ , Q ¹ , Q ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , Z ² , R ¹⁰ , R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below. Formula IA, Formula IB, Formula IC, Formula I, in another embodiment, the present disclosure provides a compound of Formula VIII : VIII or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula VIII , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula VIII' : VIII' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula VIII' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula VIII'' : VIII'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula VIII'' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula VIII''' : VIII''' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula VIII''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula IX : IX or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of Formula IX , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula IX' : IX' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula IX' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula IX'' : IX'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula IX'' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula IX''' : IX''' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula IX''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula X : X or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R9a , ^R9b , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula X , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula X' : X' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R9a , ^R9b , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula X' , wherein ^Z1 is an optionally substituted _C5 - _C12 bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula X'' : X'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R9a , ^R9b , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula X″ , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula X''' : X''' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R9a , ^R9b , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In certain embodiments, the compound is of formula X''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XI : XI or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and A, ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula XI , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XI' : XI' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and A, ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XI' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XI'' : XI'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and A, ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XI″ , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XI''' : XI''' or a pharmaceutically acceptable salt or solvent thereof, wherein q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and A, X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XI''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XII : XII or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of Formula XII , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XII' : XII' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula XII' , wherein ^Z1 is an optionally substituted _C5 - _C12 bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XII'' : XII'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is of formula XII'' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XII''' : XII''' or a pharmaceutically acceptable salt or solvent thereof, wherein q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of Formula XII''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XIII : XIII or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R9a , ^R9b , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula I or below.

In certain embodiments, the compound is of formula XIII , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XIII' : XIII' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R9a , ^R9b , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XIII' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XIII'' : XIII'' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^r2 is 0, 1 or 2; ^s2 is 0, 1, 2, 3, 4, 5, 6; and ^L1 , ^X1 , ^Y1 , ^Z1 , ^R9a , ^R9b , ^R10 and ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XIII'' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of Formula XIII''' : XIII''' or a pharmaceutically acceptable salt or solvent thereof, wherein q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ^9a , R ^9b , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is of formula XIII''' , wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group.

In another embodiment, the present disclosure provides a compound of formula XIV : XIV or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11′ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and A, X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is a compound of formula XIV, wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group. In certain embodiments, Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of Formula XIV' : XIV' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and A, X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In some embodiments, the compound is a compound of formula XIV', wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group. In some embodiments, Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of Formula XIV'' : XIV'' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and A, X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is a compound of formula XIV'', wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group. In certain embodiments, Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of Formula XIV''' : XIV''' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and A, X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In certain embodiments, the compound is a compound of formula XIV''', wherein Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene group. In certain embodiments, Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of formula XV : XV or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11′ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula I or below; wherein Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of formula XV' : XV' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined in Formula IA, Formula IB, Formula IC, Formula I or below; wherein Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of formula XV'' : XV'' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined in Formula IA, Formula IB, Formula IC, Formula I or as described below; wherein Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of formula XV''' : XV''' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ¹⁰ and R ¹¹ are as defined in Formula IA, Formula IB, Formula IC, Formula I or as described below; wherein Z ¹ is not an adamantyl group.

In another embodiment, the present disclosure provides a compound of Formula XVI : XVI or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11′ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ^9a , R ^9b , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In another embodiment, the present disclosure provides a compound of Formula XVI' : XVI' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ^9a , R ^9b , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In another embodiment, the present disclosure provides a compound of Formula XVI'' : XVI'' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ^9a , R ^9b , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In another embodiment, the present disclosure provides a compound of Formula XVI''' : XVI''' or a pharmaceutically acceptable salt or solvent thereof, wherein R ^11' is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl and C ₂ -C ₁₀ alkenyl; q ¹ is 0, 1, 2 or 3; q ² is 0, 1, 2 or 3; r ² is 0, 1 or 2; s ² is 0, 1, 2, 3, 4, 5, 6; and L ¹ , X ¹ , Y ¹ , Z ¹ , R ^9a , R ^9b , R ¹⁰ and R ¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In another embodiment, the present disclosure provides a compound of Formula XVII : XVII or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is a compound of formula XVII , wherein one or more methylene bonds of X ² , Y ² , Z ² and R ¹¹ are not replaced by a group selected from —O—, —CH═CH—, —S— and C ₃ -C ₆ cycloalkylene.

In another embodiment, the present disclosure provides a compound of formula XVIII : XVIII or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z2 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In certain embodiments, the compound is a compound of formula XVIII , wherein one or more methylene bonds of X ² , Y ² , Z ² and R ¹¹ are not replaced by a group selected from —O—, —CH═CH—, —S— and C ₃ -C ₆ cycloalkylene.

In another embodiment, the present disclosure provides a compound of Formula XVIII ': XVIII' or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below.

In another embodiment, the present disclosure provides a compound of formula XIX : XIX or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as described below.

In another embodiment, the present disclosure provides a compound of formula XX : XX or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; ^L1 , ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z2 , ^R11 are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

In another embodiment, the present disclosure provides a compound of formula XXI : XXI or a pharmaceutically acceptable salt or solvent thereof, wherein ^q1 is 0, 1, 2 or 3; ^q2 is 0, 1, 2 or 3; A, ^X1 , ^X2 , ^Y1 , ^Y2 , ^Z1 , ^Z2 , ^R10 , ^R11 are as defined in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or as defined below. ^L1

In another embodiment, L1 is selected from the group consisting of -CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-. In another embodiment, L1 is -CH2CH2-. In another embodiment, L1 is -CH2CH2CH2-. In another embodiment, ^L1 is -CH ₂ CH ₂ CH ₂ CH ₂ -. In certain embodiments, ^L1 is -(CH ₂ ) _2-6 -OC(=O)-. In certain embodiments, ^L1 is -(CH ₂ ) ₂ -OC(=O)-. R ¹

In some embodiments, ^R1 is In another embodiment, R ¹ is -OH. In some embodiments, R ¹ is -N(R ^9a )(R ^9b ). In some embodiments, R ¹ is -NMe _{2 .} In some embodiments, R ¹ is -NEt _{2 .} In another embodiment, R ¹ is In another embodiment, R ¹ is . L ²

In another embodiment, ^L2 is selected _from the group consisting of _{-CH2CH2- , -CH2CH2CH2- , and -CH2CH2CH2CH2CH2-} . In another embodiment _, ^L2 _{is -CH2CH2-} . In _another embodiment _, ^L2 is _{-CH2CH2CH2- .} In _another embodiment _, ^L2 _{is -CH2CH2CH2CH2-} . ^R8

In some embodiments, ^R8 is In another embodiment, R ⁸ is -NR ^9a R ^9b . In some embodiments, R ⁸ is -NMe _{2 .} In some embodiments, R ⁸ is -NEt _{2 .} In another embodiment, R ⁸ is -OH. R ^9a , R ^9b

In another embodiment, R ^9a and R ^9b are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl. In another embodiment, R ^9a and R ^9b are each methyl. In another embodiment, R ^9a and R ^9b are each ethyl. R'

In another embodiment, R' is hydrogen. In some embodiments, R' is ^C ₁ -C ₆ alkyl.

In another embodiment, Q ¹ is a linear C ₁ -C ₂₀ alkylene group. In another embodiment, Q ¹ is a linear C ₁ -C ₁₀ alkylene group. In another embodiment, Q ¹ is a C ₁ -C _{10 alkylene group. In another embodiment, Q 1 is a C 2 -C 5} ^alkylene _{group .} Q ¹ is a C ₆ -C ₉ alkylene group. In another embodiment, Q ¹ is selected from the group consisting of —CH ₂ CH ₂ —, —CH 2 CH ₂ CH ₂ —, —CH ₂ (CH ₂ ) ₂ CH ₂ —, —CH ₂ (CH ₂ ) _{3 CH 2 —} , —CH ₂ (CH ₂ ) ₄ CH ₂ —, —CH ₂ (CH ₂ ) ₅ CH ₂ —, —CH ₂ (CH ₂ ) ₆ CH ₂ —, —CH ₂ (CH ₂ ) ₇ CH ₂ —, and —CH ₂ (CH ₂ ) ₈ CH ₂ —. In another embodiment, Q ¹ is —CH ₂ CH ₂ —. In another embodiment, Q ¹ is —CH ₂ CH ₂ CH ₂ —. In another embodiment, Q ¹ is —CH ₂ (CH ₂ ) ₂ CH ₂ —. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) ₃ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) ₄ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) ₅ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) ₆ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) ₇ CH ₂ -. In another embodiment, Q ¹ is -CH ₂ (CH ₂ ) _8. CH ₂ -. W ¹

In another embodiment, W ^is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R ^12a )-, -N(R ^12a )C(=O)-, -OC(=O)N(R ^12a )-, -N(R ^12a )C(=O)O-, and -OC(=O)O-. In another embodiment, ^W is -C(=O)O-. In another embodiment, ^W is -OC(=O)-. In another embodiment, W ^is -C(=O)N(R ^12a )-. In another embodiment, W ^is -N(R ^12a )C(=O)-. In another embodiment, ^W is -OC(=O)N(R ^12a )-. In another embodiment, W ¹ is -N(R ^12a )C(=O)O-. In another embodiment, W ¹ is -OC(=O)O-. X ¹

In another embodiment, ^X2 is optionally substituted _C1 - _C15 alkylene. In another embodiment, ^X2 is branched C1- _C15 alkylene. In another embodiment, ^X1 is a bond or _C1 -C15 alkylene. In another embodiment, ^X1 is a bond. In another embodiment, ^X1 is _C2 - _C5 alkylene. In another embodiment, ^X1 is _{C6 - C9} alkylene. In another embodiment, ^X1 is _-CH2- . In another embodiment, X2 is _{-CH2CH2- .} In another embodiment, ^X2 is _-CH2CH2CH2- . In _another embodiment _, ^X2 is _-CH2CH2CH2- . In _another embodiment _, ^X2 is _{-CH2CH2CH2CH2- .} In another embodiment, X ² is -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -. Y ¹

In another embodiment, ^Y1 is selected from the group consisting of: -( _CH2 ) _m- , -O-, -S-, and -SS-. In another embodiment, ^Y1 is -( _CH2 ) _m- . In some embodiments, ^Y1 is -O-. In some embodiments, ^Y1 is _-S- . In another embodiment, ^Y1 is _-CH2- . In another embodiment, ^Y2 is _-CH2CH2- . m

In another embodiment, m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4. In another embodiment, m is 5. In another embodiment, m is 6 .

In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6 .

In another embodiment, p is 0. In another embodiment, p is 1. Z ¹

In another embodiment, ^Z1 is selected from the group consisting of _C4 - _C12 cycloalkylene, and In certain embodiments, Z ¹ is optionally substituted.

In another embodiment, ^Z1 is or .

In another embodiment, Z ¹ is a C ₄ -C ₁₂ cycloalkylene. In another embodiment, Z ¹ is a monocyclic C ₄ -C ₈ cycloalkylene. In another embodiment, Z ¹ is a monocyclic C ₄ -C ₆ cycloalkylene. In another embodiment, Z ¹ is a monocyclic C ₄ cycloalkylene. In another embodiment, Z ¹ is a monocyclic C ₅ cycloalkylene. In another embodiment, Z ¹ is a monocyclic C ₆ cycloalkylene.

In another embodiment, Z ¹ is an optionally substituted bridged bicyclic or polycyclic cycloalkylene. In some embodiments, Z ¹ is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene. In some embodiments, Z ¹ is an optionally substituted C ₆ -C ₁₀ bridged cycloalkylene. In some embodiments, ^Z1 is an optionally substituted _C5 - _C10 bridged cycloalkylene selected from the group consisting of adamantyl, cubanyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[1.1.1]pentyl, bicyclo[3.2.1]octyl, and bicyclo[3.1.1]heptyl.

In another embodiment, ^Z1 is selected from the group consisting of: , , , , , , , or .

In another embodiment, ^Z1 is selected from the group consisting of: and . R ¹⁰

In another embodiment, R ¹⁰ is hydrogen.

In another embodiment, R ¹⁰ is C ₁ -C ₁₀ alkyl. In another embodiment, R ¹⁰ is C ₃ -C ₇ alkyl. In another embodiment, R ¹⁰ is C ₄ -C ₆ alkyl. In another embodiment, R 10 is C _4. In another embodiment, R ¹⁰ is C _5. In another embodiment ^{, R 10 is} C ₆ .

In another embodiment, R ¹⁰ is C ₂ -C ₁₂ alkenyl. In another embodiment, R ¹⁰ is C ₆ -C ₁₂ alkenyl. In another embodiment, R ¹⁰ is C ₂ -C ₈ alkenyl. R ¹¹

In another embodiment, R ¹¹ is C ₁ -C ₁₀ alkyl. In another embodiment, R ¹¹ is C ₁ -C ₂₀ alkyl, which may be substituted. In another embodiment, R ¹¹ is C ₁ -C ₂₀ alkyl, which may be substituted. In another embodiment, R ¹¹ is C ₁ -C ₁₅ alkyl, which may be substituted. In another embodiment, R ¹¹ is C ₁ -C ₁₅ branched alkyl, which may be substituted. In another embodiment, R ¹¹ is C ₁₀ -C ₁₅ alkyl, which may be substituted. In another embodiment, R ¹¹ is C ₁₀ -C ₁₅ branched alkyl, which may be substituted. In another embodiment, R ¹¹ is selected from the group consisting of -CH ₃ , -CH ₂ CH ₃ , and -CH ₂ CH ₂ CH ₃ . In another embodiment, R ¹¹ is selected from the group consisting of -CH ₂ (CH ₂ ) ₂ CH ₃ , -CH ₂ (CH ₂ ) ₃ CH ₃ , -CH ₂ (CH ₂ ) ₄ CH ₃ , -CH ₂ (CH ₂ ) ₅ CH ₃ , -CH ₂ (CH ₂ ) ₆ CH ₃ , -CH ₂ (CH ₂ ) ₇ CH ₃ , and -CH ₂ (CH ₂ ) ₈ CH ₃ . In another embodiment, R ¹¹ is -CH ₃ . In another embodiment, R ¹¹ is -CH ₂ CH ₃ . In another embodiment, R ¹¹ is -CH ₂ CH _{3 .} In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₂ CH _3. In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₃ CH _3. In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₄ CH _3. In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₅ CH _3. In another embodiment, R ¹¹ is CH ₂ (CH ₂ ) ₆ CH _3. In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₇ CH _3. In another embodiment, R ¹¹ is -CH ₂ (CH ₂ ) ₈ CH ₃ .

In another embodiment, R ¹¹ is C ₂ -C ₁₀ alkenyl. In another embodiment, R ¹¹ is C ₂ -C ₁₂ alkenyl. In another embodiment, R ¹¹ is C ₆ -C ₁₂ alkenyl. In another embodiment, R ¹¹ is C ₂ -C ₈ alkenyl.

In another embodiment, the present disclosure provides a compound of any one of Formulas IA, IB, IC or I - XXI , or a pharmaceutically acceptable salt or solvent thereof, wherein ^{R 11} is hydrogen.

In another embodiment, Q ² is a straight chain C ₁ -C ₂₀ alkylene group. In another embodiment, Q ² is a straight chain C ₁ -C ₁₀ alkylene group. In another embodiment, Q ² is a C ₂ -C ₁₀ alkylene group. In another embodiment, Q ² is selected from the group consisting of -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH ₂ (CH ₂ ) ₂ CH ₂ -, -CH ₂ (CH ₂ ) ₃ CH ₂ -, -CH ₂ (CH ₂ ) ₄ CH ₂ -, -CH ₂ (CH ₂ ) ₅ CH ₂ -, -CH ₂ (CH ₂ ) ₆ CH ₂ -, -CH ₂ (CH ₂ ) ₇ CH ₂ -, and -CH ₂ (CH ₂ ) _8. CH ₂ -. In another embodiment, Q ² is -CH ₂ CH ₂ -. In another embodiment, Q ² is -CH ₂ CH ₂ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) ₃ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) ₄ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) ₅ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) ₆ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) ₇ CH ₂ -. In another embodiment, Q ² is -CH ₂ (CH ₂ ) _8. CH ₂ -. W ²

In another embodiment, W ² is selected from the group consisting of -C(=O)O- and -OC(=O)-. In another embodiment, W ² is -C(=O)O-. In another embodiment, W ² is -OC(=O)-. X ²

In another embodiment, ^X2 is an optionally substituted _C1 - _C15 alkylene. In another embodiment, ^X2 is a _C1 - _C15 branched chain alkylene. In another embodiment, ^X2 is a _C1 - _C6 alkylene or a bond. In another embodiment, ^X2 is a _C2 - _C4 alkylene. In another embodiment, ^X2 is a _C3 - _{C5 alkylene} . In another embodiment, ^X2 is selected from the _group consisting of: _{-CH2CH2- , -CH2CH2CH2-} , _{-CH2 ( CH2} ) _{2CH2- , -CH2} ( _CH2 ) _{3CH2- ,} and _{-CH2(CH2)4CH2- . In} another embodiment, _X2 is ^-CH2- . In another embodiment, X ² is a bond. In another embodiment, X ² is a branched C ₁ -C ₁₅ alkylene group, wherein one or more methylene bonds of X ² are optionally and independently replaced by a group selected from -O-, -CH=CH-, -S- and C ₃ -C ₆ cycloalkylene groups. Y ²

In another embodiment, Y ² is selected from the group consisting of -(CH ₂ ) _m - and -S-. In another embodiment, Y ² is -(CH ₂ ) _m -. In another embodiment, Y ² is -S-. Z ²

In another embodiment, Z ² is -(CH ₂ ) _p -. In another embodiment, Z ² is -CH ₂ -. In another embodiment, Z ² is -CH ₂ CH ₂ -. In another embodiment, Z ² is C ₄ -C ₁₂ cycloalkylene. In another embodiment, Z ² is monocyclic C ₄ -C ₈ cycloalkylene. In certain embodiments, Z ² is optionally substituted.

In another embodiment, Z ² is an optionally substituted bridged bicyclic or polycyclic cycloalkylene. In some embodiments, Z ² is an optionally substituted C ₅ -C ₁₂ bridged cycloalkylene. In some embodiments, Z ² is an optionally substituted C ₆ -C ₁₀ bridged cycloalkylene. In some embodiments, ^Z2 is an optionally substituted _C5 - _C10 bridged cycloalkylene selected from the group consisting of adamantyl, cubanyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[1.1.1]pentyl, bicyclo[3.2.1]octyl, and bicyclo[3.1.1]heptyl.

In another embodiment, ^Z2 is selected from the group consisting of: , , , , , , , or .

In another embodiment, ^Z2 is selected from the group consisting of: and .

In another embodiment, the present disclosure provides a compound selected from any one or more compounds of Table (III) or a pharmaceutically acceptable salt or solvent thereof. Table (III). Non-limiting examples of ionizable lipids with constrained arms Compound No. Structure C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73 C74 C75 C76 C77 C78 C79 C80 C81

In some embodiments, the LNPs of the present disclosure comprise ionizable lipids disclosed in PCT application PCT/US2023/065477, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I): (CX-I) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ or -(CH ₂ ) _1-6 OH; R ² is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1 to 6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-; R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1 to 6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-; each R ^{a is} independently an optionally substituted C ₁ -C ₆ alkyl group; or two ^Ras are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-i): (CX-i) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a key, , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ ; R ² is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted C ₁ -C ₆ alkyl group; or two Ra ^s are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I'), (CX-I''), (CX-I'''), (CX-I') (CX-I'') (CX-I''') or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present disclosure are represented by Formula (CX-Ia), (CX-Ib), (CX-Ic) or (CX-Id): (CX-Ia) (CX-Ib) (CX-Ic) (CX-Id) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-a'), (CX-I-b'), (CX-I-c') or (CX-I-d'): (CX-I-a') (CX-I-b') (CX-I-c') (CX-I-d') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-a''), (CX-I-b''), (CX-I-c'') or (CX-I-d''): (CX-I-a'') (CX-I-b'') (CX-I-c'') (CX-I-d'') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-a'''), (CX-I-b'''), (CX-I-c'''), or (CX-I-d'''): (CX-I-a''') (CX-I-b''') (CX-I-c''') (CX-I-d''') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-Ie) or (CX-If): (CX-Ie) (CX-If) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-e') or (CX-I-f'): (CX-I-e') (CX-I-f') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-e'') or (CX-I-f''): (CX-I-e'') (CX-I-f'') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-I-e''') or (CX-I-f'''): (CX-I-e''') (CX-I-f''') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II): (CX-II) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N( ^Ra ) ₂ or -(CH ₂ ) _1-6 OH; R ² is an optionally substituted C ₅ -C ₃₆ alkyl group or an optionally substituted C ₅ -C ₃₆ alkenyl group, wherein 2 methylene units of R ² are replaced by -O- to form a carboxaldehyde in R ² , and wherein 1-6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₅ -C ₃₆ alkenyl group, wherein R 1-6 methylene units of ^2' are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted _C1 - _C6 alkyl; or two ^Ra are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-ii): (CX-ii) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N( ^Ra ) ₂ ; R ² is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1 to 6 methylene units of R ² are optionally substituted by a group selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-; R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group, wherein 1 to 6 methylene units of R ^2' are optionally substituted by a group selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted C ₁ -C ₆ alkyl group; or two R ^a and the nitrogen to which it is attached form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II'), (CX-II''), (CX-II'''), (CX-II') (CX-II'') (CX-II''') or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-a): (CX-II-a) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-a'), (CX-II-a'') or (CX-II-a'''), (CX-II-a') (CX-II-a'') (CX-II-a''') or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-b), (CX-II-c) or (CX-II-d): (CX-II-b) (CX-II-c) (CX-II-d) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-b'), (CX-II-c') or (CX-II-d'): (CX-II-b') (CX-II-c') (CX-II-d') or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-b''), (CX-II-c'') or (CX-II-d''): (CX-II-b'') (CX-II-c'') (CX-II-d'') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-b'''), (CX-II-c''') or (CX-II-d'''): (CX-II-b''') (CX-II-c''') (CX-II-d''') or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-II-e): (CX-II-e) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-III): (CX-III) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N( ^Ra ) ₂ or -(CH ₂ ) _1-6 OH; R ² is an optionally substituted C ₅ -C ₃₆ alkyl group or an optionally substituted C ₅ -C ₃₆ alkenyl group, wherein 2 methylene units of R ² are replaced by -O- to form a carboxaldehyde in R ² , and wherein 1-6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein R ² is optionally substituted by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted _C1 - _C6 alkyl; or two ^Ra are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; and n is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-iii): (CX-iii) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ ; each R ² is independently an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted C ₁ -C ₆ alkyl group; or two Ra ^s are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; and n is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-III-a), (CX-III-b) or (CX-III-c): (CX-III-a) (CX-III-b) (CX-III-c) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-III-d) or (CX-III-e): (CX-III-d) (CX-III-e) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-IV): (CX-IV) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , , , , , and ; R ¹ is -(CH ₂ ) _1-6 N( ^Ra ) ₂ or -(CH ₂ ) _1-6 OH; R ² is a C ₃ -C ₃₆ branched alkyl group or an optionally substituted C ₃ -C ₃₆ branched alkenyl group, wherein 1-6 methylene units of R ² are optionally substituted by a cyclopropane group and -O-; R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1-6 methylene units of R ² are optionally substituted by a cyclopropane group, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted C ₁ -C or two ^Ra _are taken together with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; and n is 1 or 2.

In some embodiments, the compound is represented by formula (CX-IV-a), (CX-IV-b) or (CX-IV-c): (CX-IV-a) (CX-IV-b) (CX-IV-c) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CX-IV-d) or (CX-IV-e): (CX-IV-d) (CX-IV-e) or their pharmaceutically acceptable salts.

In some embodiments, Z is selected from the group consisting of: a key, , , , , and .

In some embodiments, Z is selected from the group consisting of: , and In some embodiments, Z is .

In some embodiments, Z is selected from the group consisting of: a key, , , , , and , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is selected from the group consisting of: , , and , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *. In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *. In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *.

In some embodiments, each Y is independently selected from the group consisting of: , , , , , , and .

In some embodiments, Y is selected from the group consisting of: , , and .

In some embodiments, Y is selected from the group consisting of: , , and , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is .

In some embodiments, Y is or .

In some embodiments, Y is .

In some embodiments, Y is . R ¹

In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ or -(CH ₂ ) _1-6 OH. In some embodiments, R ¹ is -(CH ₂ ) _1-6 OH. In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(R ^a ) _2. In some embodiments, R ¹ is -(CH ₂ ) ₂ N(R ^a ) _2. In some embodiments, R ¹ is -(CH ₂ ) ₃ N(R ^a ) _2. In some embodiments, R ¹ is -(CH ₂ ) ₄ N(R ^a ) _2. In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(n-Pr) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(i-Pr) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₂ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₃ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₄ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₂ N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₃ N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₄ N(Et) ₂ .

In some embodiments, R is ^selected from the group consisting of: , , and

In some embodiments, R is ^selected from the group consisting of: , , and .

In some embodiments, R is ^selected from the group consisting of: , , and . R ² and R ^2'

In some embodiments, R ² is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ² is an optionally substituted C ₁ -C ₃₂ alkyl group or an optionally substituted C ₂ -C ₃₂ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C30 alkyl group or an optionally substituted C2-C30 _alkenyl group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _{C24 alkyl} group or an optionally substituted C2- _{C24 alkenyl} group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C24 alkyl group or an optionally substituted C2- _C24 alkenyl group, wherein 1-6 methylene units of ^R2 are replaced by groups independently selected from cyclopropene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C24 alkyl group or an optionally substituted C2- _C24 alkenyl group. In some embodiments, ^R2 is an optionally substituted _C10 - _{C24 alkyl} group or an optionally substituted _C10 - _{C24 alkenyl} group, wherein 1-6 methylene units of ^R2 are replaced by -O-.

In some embodiments, ^R2 is an optionally substituted _C5 - _C36 alkyl group or an optionally substituted _C5 - _C36 alkenyl group, wherein 2 methylene units of ^R2 are replaced by -O- to form an acetal in ^R2 , and wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; and R2 ^' is an optionally substituted _C1 - _C36 alkyl group or an optionally substituted _C2 - _C36 alkenyl group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-.

In some embodiments, ^R2 is an optionally substituted _C10 - _C24 alkyl group or an optionally substituted _C10 -C24 _alkenyl group, wherein 2 methylene units of ^R2 are replaced by —O— to form an acetal in ^R2 , and wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, —O—, —OC(O)—, and —C(O)O—; and R2 ^′ is an optionally substituted _C10 - _C36 branched chain alkyl group or an optionally substituted _C10 -C36 _alkenyl group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, —O—, —OC(O)—, and —C(O)O—.

In some embodiments, ^R2 is a _C3 - _C36 branched alkyl group or an optionally substituted _C3 - _C36 branched alkenyl group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene and -O-; and R2 ^' is an optionally substituted _C1 - _C36 alkyl group or an optionally substituted _C2 - _{C36 alkenyl} group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C10 - _C24 branched chain alkyl or an optionally substituted _C10 - _C24 branched chain alkenyl, wherein 1-3 methylene units of ^R2 are optionally replaced by -O-; and R2 ^' is an optionally substituted _C10 - _C36 alkyl or an optionally substituted _C10 - _C36 alkenyl, wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-.

In some embodiments, R ² and/or R ^2' is wherein each q is independently selected from 0-12 and each R ^° is independently selected and as described and defined herein.

In some embodiments, R ² and/or R ^2' is where each q is independently selected from 0-12.

In some embodiments, ^R2 is an optionally substituted _C5 - _C36 alkyl group or an optionally substituted _C5 - _C36 alkenyl group, wherein 2 methylene units of ^R2 are replaced by -O- to form an acetal in ^R2 , and wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-; R2 ^' is an optionally substituted _C1 - _C36 alkyl group or an optionally substituted _C5 - _C36 alkenyl group, wherein 1-6 methylene units of R2 ^' are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)- and -C(O)O-.

In some embodiments, ^R2 is an optionally substituted _C10 - _C24 alkyl group wherein 2 methylene units of ^R2 are replaced by —O— to form an acetal within ^R2 , and R2 ^′ is an optionally substituted _C10 - _C24 alkyl group wherein 2 methylene units of R2 ^′ are replaced by —O— to form an acetal within R2 ^′ .

In some embodiments, each q is independently selected from 0-6. In some embodiments, each q is independently selected from 0-8. In some embodiments, each q is independently selected from 0-10. In some embodiments, each q is independently selected from 0-12.

In some embodiments, ^R2 is an optionally substituted _C10 - _C24 alkyl or an optionally substituted _C10 - _C24 alkenyl, wherein 2 methylene units of ^R2 are replaced by -O- to form an acetal in ^R2 , and wherein 1-6 methylene units of ^R2 are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; and R2 ^' is an optionally substituted _C10 - _C24 alkenyl, wherein 1-3 methylene units of R2 ^' are optionally replaced by -O-.

In some embodiments, ^R is selected from the group consisting of: , , , , , , , , , , , and .

In some embodiments, ^R2 is or .

In some embodiments, ^R2 is .

In some embodiments, ^R2 is .

In some embodiments, ^R is selected from the group consisting of: , and .

In some embodiments, R ^2' is an optionally substituted C ₁ -C ₃₆ alkyl group, wherein 1-6 methylene units of R ^2' are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ^2' is an optionally substituted C ₁ -C ₃₂ alkyl group, wherein 1-6 methylene units of R ^2' are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ^2' is an optionally substituted C ₁ -C ₃₀ alkyl group, wherein 1-6 methylene units of R ^2' are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ^2' is an optionally substituted C ₁ -C ₂₄ alkyl group, wherein 1-6 methylene units of R ^2' are optionally replaced by a group independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ^2' is an optionally substituted C ₁ -C ₂₄ alkyl group, wherein 1-6 methylene units of R ^2' are replaced by a group independently selected from -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ^2' is an optionally substituted C ₁ -C ₂₄ alkyl group. In some embodiments, R ^2' is an optionally substituted C ₁₀ -C ₂₄ alkyl group, wherein 1-6 methylene units of R ^2' are replaced by -O-.

In some embodiments, R ^2' is selected from the group consisting of: , , , , , , , and .

In some embodiments, R ² and R ^2' are each independently selected from the group consisting of: , , , and .

In some embodiments, R ^2' is selected from the group consisting of: , , , , and .

In some embodiments, R ^2' is .

In some embodiments, R ^2' is .

In some embodiments, R ^2' is selected from the group consisting of: , and .

In some embodiments, ^R is selected from the group consisting of: , , , and .

In some embodiments, R ^2' is selected from the group consisting of: , , and .

In some embodiments, the present disclosure includes a compound selected from any lipid or a pharmaceutically acceptable salt thereof in Table (IV) below: Table (IV). Non-limiting Examples of Ionizable Lipids Structure Compound No. CX-1 CX-2 CX-3 CX-4 CX-5 CX-6 CX-7 CX-8 CX-8a CX-8b CX-8c CX-9 CX-10 CX-11 CX-12 CX-13 CX-14 CX-15 CX-16 CX-17 CX-18 CX-19 CX-20 CX-21 CX-22 CX-23 CX-24 CX-25 CX-26 CX-27 CX-28 CX-29 CX-30 CX-30a CX-30b CX-30c

In some embodiments, the lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-I): (CZ-I) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , and ; R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ ; each R ² is independently an optionally substituted C ₁ -C ₃₆ alkyl or an optionally substituted C ₂ -C ₃₆ alkenyl, wherein 1-6 methylene units of R ² are optionally replaced by a group independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O-; each Ra ^is independently an optionally substituted C ₁ -C ₆ alkyl; or two Ra ^s are combined with the nitrogen to which they are attached to form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-Ia), (CZ-Ib), (CZ-Ic) or (CZ-Id): (CZ-Ia) (CZ-Ib) (CZ-Ic) (CZ-Id) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-Ie) or (CZ-If) (CZ-Ie) (CZ-If) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-Ig): (CZ-Ig) or its pharmaceutically acceptable salt.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-II): (CZ-II) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of: a , , , , and ; Each Y is independently selected from the group consisting of: , , and ; R ¹ is -(CH ₂ ) _1-6 N( ^Ra ) ₂ ; each R ² is independently an optionally substituted C ₁ -C ₃₆ alkyl or an optionally substituted C ₂ -C ₃₆ alkenyl, wherein 1-6 methylene units of R ² are independently selected from cyclopropene, -O-, -OC(O)- and -C(O)O- group replacement; each Ra ^is independently an optionally substituted C ₁ -C ₆ alkyl; or two ^Ras and the nitrogen to which they are attached form an optionally substituted 4-7 membered heterocyclic ring; m is 0, 1 or 2; n is 1 or 2; and p is 1 or 2. Or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-II-a), (CZ-II-b), (CZ-II-c) or (CZ-II-d): (CZ-II-a) (CZ-II-b) (CZ-II-c) (CZ-II-d) or their pharmaceutically acceptable salts.

In some embodiments, the compounds of the present disclosure are represented by formula (CZ-II-e): (CZ-II-e) or its pharmaceutically acceptable salt.

In some embodiments, Z is selected from the group consisting of: a key, , , , , and .

In some embodiments, Z is selected from the group consisting of: , and In some embodiments, Z is .

In some embodiments, Z is selected from the group consisting of: a key, , , , , and , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is selected from the group consisting of: , , and , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is .

In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Z is , wherein R ¹ is connected at the position indicated by *.

In some embodiments, Y is selected from the group consisting of: , , and In some embodiments, Y is or .

In some embodiments, Y is selected from the group consisting of: , , and , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *. In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is , wherein R ² is connected at the position indicated by *.

In some embodiments, Y is .

In some embodiments, Y is or .

In some embodiments, Y is .

In some embodiments, Y is . R ¹

In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(R ^a ) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₂ N(R ^a ) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₃ N(R ^a ) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₄ N(R ^a ) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(n-Pr) ₂ . In some embodiments, R ¹ is -(CH ₂ ) _1-6 N(CZ-I-Pr) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₂ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₃ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₄ N(Me) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₂ N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₃ N(Et) ₂ . In some embodiments, R ¹ is -(CH ₂ ) ₄ N(Et) ₂ .

In some embodiments, ^R1 is selected from the group consisting of: , , and

In some embodiments, R is ^selected from the group consisting of: , , and .

In some embodiments, ^R1 is selected from the group consisting of: , , and . R ²

In some embodiments, R ² is an optionally substituted C ₁ -C ₃₆ alkyl group or an optionally substituted C ₂ -C ₃₆ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R ² is an optionally substituted C ₁ -C ₃₂ alkyl group or an optionally substituted C ₂ -C ₃₂ alkenyl group, wherein 1-6 methylene units of R ² are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C30 alkyl group or an optionally substituted C2-C30 _alkenyl group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _{C24 alkyl} group or an optionally substituted C2- _{C24 alkenyl} group, wherein 1-6 methylene units of ^R2 are optionally replaced by a group selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C24 alkyl group or an optionally substituted C2- _C24 alkenyl group, wherein 1-6 methylene units of ^R2 are replaced by a group independently selected from -O-, -OC(O)-, and -C(O)O-. In some embodiments, ^R2 is an optionally substituted _C1 - _C24 alkyl group or an optionally substituted _C2 - _C24 alkenyl group. In some embodiments, ^R2 is an optionally substituted _C10 - _C24 alkyl group or an optionally substituted C10- _{C24 alkenyl} group, wherein _1-6 methylene units of ^R2 are replaced by -O-.

In some embodiments, ^R2 is wherein each q is independently selected from 0-12 and each R ^° is independently selected and defined herein.

In some embodiments, ^R2 is where each q is independently selected from 0-12.

In some embodiments, each q is independently selected from 0-6. In some embodiments, each q is independently selected from 0-8. In some embodiments, each q is independently selected from 0-10. In some embodiments, each q is independently selected from 0-12.

In some embodiments, ^R is selected from the group consisting of: , , , , , , , , , , , and .

In some embodiments, ^R is selected from the group consisting of: , , and .

In some embodiments, the present disclosure includes a compound selected from any lipid or a pharmaceutically acceptable salt thereof in Table (V) below: Table (V). Non-limiting Examples of Ionizable Lipids Structure Compound No. CZ-1 CZ-2 CZ-3 CZ-4 CZ-5 CZ-6 CZ-7 CZ-8 CZ-9 CZ-10 CZ-11 CZ-12 CZ-13 CZ-14 CZ-15 CZ-16 CZ-17 CZ-18 ii. Structural lipids

In some embodiments, the LNP comprises a structural lipid. The structural lipid may be selected from the group consisting of, but not limited to, cholesterol, natriol, fucosterol, β-mysterol, mysterol, ergosterol, campesterol, stigmasterol, stigmasterol, tomatosterol, lycoside, cholic acid, glutanostanol, litocholic acid, tomatin, ursolic acid, α-tocopherol, vitamin D3, vitamin D2, calcipotriol, botulinum toxin, lupeol, dianthin, β-mysterol-acetate, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structured lipid is a cholesterol analog disclosed in Patel et al., Nat Commun., 11, 983 (2020), which is incorporated herein by reference in its entirety. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone and hydrocortisone) or any combination thereof. In some embodiments, the structured lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

In some embodiments, the structural lipid is a cholesterol analog. The use of cholesterol analogs can enhance endosomal escape as described in Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications (2020), which is incorporated herein by reference.

In some embodiments, the structured lipid is a plant sterol. Use of plant sterols can enhance endosomal escape as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference.

In some embodiments, the structured lipids contain plant sterol mimetics for enhanced endosomal release. iii. PEGylated lipids

PEGylated lipids are lipids modified with polyethylene glycol.

In some embodiments, the LNP comprises one, two or more PEGylated lipids or PEGylated lipids. The PEGylated lipids can be selected from a non-limiting group consisting of PEGylated phosphatidylethanolamine, PEGylated phosphatidic acid, PEGylated ceramide, PEGylated dialkylamine, PEGylated diacylglycerol, PEGylated dialkylglycerol and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

In some embodiments, the PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propyl carbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000 -DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N (carbonyl-methoxy polyethylene glycol-2000)-l, 2-stearyl-sn-glycero-3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l, 2-stearyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, folic acid PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000 maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG -PEGMA, DOPE-PE G2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, Distearyl-glycerol-polyethylene glycol, CI8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propyl carbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

In some embodiments, the LNP comprises a PEGylated lipid as disclosed in one of the following: US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828A1, each of which is incorporated herein by reference in its entirety.

In some embodiments, the LNP comprises a PEGylated lipid substitute to replace the PEGylated lipid. All embodiments disclosed herein that consider PEGylated lipids should be understood to also apply to PEGylated lipid substitutes. In some embodiments, the LNP comprises a poly(lactic acid)-lipid conjugate, such as those disclosed in US 2022/0001025 A1, which is incorporated herein by reference in its entirety. iv. Phospholipids

In some embodiments, the LNPs of the present disclosure comprise phospholipids. The phospholipids useful in the compositions and methods can be selected from a non-limiting group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- 2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di(undecanyl)-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycero) sodium salt (DOPG), (S)-2-ammonium-3-((((R)-2-(oleoyl)-3-phosphoethanolamine, =Sodium (( ... (DOPS), cell-free fusion phospholipids (DPhPE), dimalmitoylphosphatidylethanolamine (DPPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (DEPE), dimalmitoylphosphatidylglycerol (DPPG), dimalmitoylphosphatidylserine (DPPS), distearylphosphatidylcholine (DSPC), distearyl-phosphatidyl-ethanolamine (DSPE), distearylphosphoethanolamine imidazole (DSPEI), 1,2-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), bis((S)-2-hydroxy-3-(oleyloxy)propyl)ammonium phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-inositol) (DOPI; 18:1 PI), 1,2-distearyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 In some embodiments, the LNP comprises DSPC. In some embodiments, the LNP comprises DOPE. In some embodiments, the LNP comprises both DSPC and DOPE.

In some embodiments, the LNP comprises a phospholipid selected from the group consisting of 1-pentadecanyl-2-oleyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, Choline phosphate, 1-palmitoyl-2-oleyl-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1-stearyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-stearyl-2-palmitoyl sn-glycero-3-phosphocholine, 1-stearyl-2-oleyl-sn-glycero-3-phosphocholine, 1-stearyl-2-linoleyl-sn-glycero-3-phosphocholine, 1-stearyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-stearyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1-oleyl-2-myristoyl-sn-glycero-3-phosphocholine 、1-oleyl-2-palmitoyl-sn-glycero-3-phosphocholine、1-oleyl-2-stearoyl-sn-glycero-3-phosphocholine、1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine、1,2-dioleoyl-sn-glycero-3-phosphocholine-(1'-inositol-3',4'-diphosphate)、1,2-dioleoyl-sn-glycero-3-phosphocholine-(1'-inositol-3',5'-diphosphate)、 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-4',5'-diphosphate), 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-3',4',5'-triphosphate), 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-3'-phosphate), 1,2-Dioleoyl-sn-glycerol-3-phosphate-( 1'-inositol-4'-phosphate), 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-inositol-5'-phosphate), 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-inositol), 1,2-dioleoyl-sn-glycero-3-phosphate-L-serine and 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine.

In some embodiments, the phospholipid tail can be modified to promote endosomal escape, as described in U.S. Application Publication No. 2021/0121411, which is incorporated herein by reference.

In some embodiments, the LNP comprises a phospholipid disclosed in one of the following: US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated herein by reference in its entirety.

In some embodiments, the phospholipid disclosed in US 2020/0121809 has the following structure: Wherein R1 and R2 are each independently a branched or straight chain, a saturated or unsaturated carbon chain (e.g., an alkyl, alkenyl, alkynyl). vi. Targeting moiety

In some embodiments, the lipid nanoparticles further comprise a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen.

In some embodiments, the pharmaceutical composition comprises a targeting moiety operably linked to a lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed more in the target organ than in the liver.

In some embodiments, the targeting moiety is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeting particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows the siRNA encapsulated in the particles to be specifically delivered to B cell lymphocytic malignancies (such as MCL) at a greater percentage than to other white blood cell subtypes.

In some embodiments, lipid nanoparticles can be targeted when bound/linked/conjugated to a targeting moiety such as an antibody. vii. Zwitterionic amino lipids

In some embodiments, the LNP comprises a zwitterionic lipid. In some embodiments, the LNP comprising a zwitterionic lipid does not comprise a phospholipid.

Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs in the absence of phospholipids for mRNA loading, stabilization, and release within cells, as described in U.S. Patent Application No. 20210121411, which is incorporated herein by reference in its entirety. Zwitterionic, ionizable cation-, and permanent cation-assisted lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in the spleen, liver, and lung, as described in Liu et al., Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety.

Zwitterionic lipids can have head groups containing cationic amines and anionic carboxylate groups, as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long-chain dialkylamine via an amide linkage at the α-amine of the lysine can reduce immunogenicity, as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013). viii. Additional Lipid Components

In some embodiments, the LNP composition of the present disclosure further comprises one or more additional lipid components capable of affecting the tropism of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP and C12-200 (see Cheng et al. Nat Nanotechnol. 2020 April; 15(4): 313–320.; Dillard et al. PNAS 2021 Vol. 118 No. 52.).

In some embodiments, the LNP composition of the present disclosure comprises or further comprises one or more lipids selected from the following: 1,2-di-O-octadecene-sn-glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleyl-sphingomyelin (SPM) (C18:1), N-xylyl SPM (C18:2), N-hydroxysphingomyelin (SPM) (C18:3), N-hydroxysphingomyelin (SPM) (C18:1), N-hydroxysphingomyelin (SPM) ... (C24:0), N-neuroyl sphingomyelin (C24:1), cardiolipin (CL), l,2-bis(tricosyl-10,12-diynylyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain di-n-heptadecanylphospholipid acylcholine (DHPC), dihexadecanoyl-phosphoethanolamine (DHPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauryl-sn-glycero-3-PE (DLPE), dimyristylglycerol hemisuccinate (DMGS), dimyristylphosphatidylcholine (DMPC), dimyristylphosphoethanolamine (DMPE), dimyristylphosphatidylglycerol (DMPG), dioleyloxybenzyl alcohol (DOBA), 1,2-dioleylglycerol-3 -hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-1-tetrahydro-pyran-2-ylthio)-propionamide (DOGP4αMan), dioleylphosphatidylcholine (DOPC), dioleylphosphatidylethanolamine (DOPE), dioleyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleylphosphatidylglycerol (DOPG), 1,2-dioleyl-sn-glycero-3-(phospho-L-serine) (DOPS), cell-free phospholipids (DPhPE), dimalmitoylphosphatidylethanolamine (DPPE), dimalmitoylphosphatidylglycerol (DPPG), dimalmitoylphosphatidylserine (DPPS), distearylphosphatidylcholine (DSPC), distearyl-phosphatidyl-ethanolamine (DSPE), distearylphosphoethanolamine imidazole (DSPEI), 1,2-di(undecanoyl)-sn-glycero-phosphate Choline (DUPC), phosphatidylcholine (EPC), histamine distearyl glycerol (HDSG), 1,2-dipalmitoylglycerol-hemisuccinate-Nα-histidyl-hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-pentacosadiamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyrodemagnesium chlorophyllide-a (HPPH), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dipalmitoylglycerol-O-α-histidyl-Nα-hemisuccinate (IsohistsuccDG), mannosylated dimalmitoylphosphatidylethanolamine (ManDOG), l,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-formamide] (MCC-PE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), thiol-reactive maleimide head group lipids (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide (MPB-PE)), neuraminic acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N-dodecyl (NC12-DOPE), 1-Oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipids (PE), PE lipids conjugated with polyethylene glycol (PEG) (e.g., polyethylene glycol-distearylphosphatidylethanolamine lipids (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soybean phosphatidylcholine (PHSPC), phosphatidylinositol lipids (PI), phosphatidylinositol-4-phosphate (PIP), palmitoyloleylphospholipids Phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleylphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamine B-phosphatidylethanolamine lipid (Rh-PE), purified soybean derived phospholipid mixture (SIOO), phosphatidylcholine (SM), 18-1-trans-PE, 1-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelin (SPM), α, α-Trehalose-6,6'-dibehenate (TDB), l,2-di-antioleyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dihydroxy-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl methyl phosphate, 1,2-diamidoyl-sn-glycero-3-phosphocholine, 1,2-diamidoyl- sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE and dioleyl phosphatidylethanolamine. viii. LNP pharmaceutical composition

In some embodiments, the nanoparticles include ionizable lipids, phospholipids, PEG lipids and structural lipids. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol% to about 60 mol% ionizable lipids, about 0 mol% to about 30 mol% phospholipids, about 18.5 mol% to about 48.5 mol% structural lipids and about 0 mol% to about 10 mol% PEG lipids, with the limitation that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol% to about 55 mol% ionizable lipids, about 5 mol% to about 25 mol% phospholipids, about 30 mol% to about 40 mol% structural lipids and about 0 mol% to about 10 mol% PEG lipids. In a specific embodiment, the lipid component includes about 50 mol% ionizable lipids, about 10 mol% phospholipids, about 38.5 mol% structural lipids, and about 1.5 mol% PEG lipids. In another specific embodiment, the lipid component includes about 40 mol% ionizable lipids, about 20 mol% phospholipids, about 38.5 mol% structural lipids, and about 1.5 mol% PEG lipids. In another specific embodiment, the lipid component includes about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 40 mol% structural lipids, and about 1.5 mol% PEG lipids. In another specific embodiment, the lipid component includes about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 39 mol% structural lipids and about 2.5 mol% PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol. The amount of the active agent in the nanoparticle composition may depend on the size, composition, desired target and/or application or other characteristics of the nanoparticle composition, as well as the characteristics of the active agent. For example, the amount of the active agent available in the nanoparticle composition may depend on the size, sequence and other characteristics of the active agent. The relative amounts of the active agent and other elements (e.g., lipids) in the nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to the enzyme in the nanoparticle composition can be about 5: 1 to about 60: 1, such as 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1 and 60: 1. The amount of enzyme in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-Vis spectroscopy).

In some embodiments, the nanoparticle composition comprising the active agent of the present disclosure is formulated to provide a specific E:P ratio. The E:P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA active agent. In general, lower E:P ratios are preferred. One or more enzymes, lipids, and amounts thereof may be selected to provide an E:P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the E:P ratio may be about 2:1 to about 8:1. In other embodiments, the E:P ratio is about 5: 1 to about 8: 1. For example, the E:P ratio can be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1.

The characteristics of a nanoparticle composition may depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid may have different characteristics from a nanoparticle composition comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher molar fraction of phospholipids may have different characteristics from a nanoparticle composition comprising a lower molar fraction of phospholipids. Characteristics may also vary depending on the preparation method and conditions of the nanoparticle composition. Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure various characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

The average size of the nanoparticle composition can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). For example, the average size can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the nanoparticle composition may have an average size of about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the nanoparticle composition may have an average size of about 70 nm to about 100 nm. In a particular embodiment, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.

The zeta potential of a nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with relatively low charge (positive or negative) are generally desirable because higher charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be from about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV, to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV, to about +15 mV, or about +5 mV. mV to approximately +10 mV.

The encapsulation efficiency of a payload describes the amount of payload that is encapsulated or otherwise associated with a nanoparticle composition after preparation relative to the initial amount provided. A high encapsulation efficiency is desirable (e.g., close to 100%). For example, encapsulation efficiency can be measured by comparing the amount of payload in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free payload in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and/or prophylactic agent may be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

Lipids and methods for preparing the same are disclosed in, for example, U.S. Patent Nos. 8,569,256 and 5,965,542 and U.S. Patent Publication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257 , No. 2013/086373, No. 2013/0338210, No. 2013/0323269, No. 2013/0245107, No. 2013/0195920, No. 2013/0123338, No. 2013/0022649, No. 2013/0017223, No. 2012/0295832, No. 2012/0183581, No. 2012/0172411, No. 012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 200 7/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Publication No. WO 99/39741, WO 2017/117528, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO 2011/141705 and WO 2001/07548 and Semple et al., Nature Biotechnology, 2010, 28, 172-176, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

Nanoparticle compositions may include any substance that can be used in pharmaceutical compositions. For example, nanoparticle compositions may include one or more pharmaceutically acceptable excipients or adjunct ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives and other substances. Excipients such as waxes, creams, colorants, coatings, flavorings and fragrances may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy , 21st ed., AR Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006). Other various lipid or liposomal formulations and administration methods including nanoparticles include, but are not limited to, U.S. Patent Publications 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent that they disclose formulations and other relevant aspects of nucleic acid administration and delivery. Methods for forming particles are also disclosed in U.S. Patent Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for their aspects.

In some embodiments, the LNP encapsulates an engineered retrotranscript, e.g., an engineered nucleic acid construct, ncRNA, vector system, RNA molecule, and/or engineered nucleic acid-enzyme construct as described herein.

In some embodiments, lipid nanoparticles include: one or more ionizable lipids; one or more structural lipids; one or more PEGylated lipids; and one or more phospholipids. In some embodiments, the one or more ionizable lipids are selected from the group consisting of those disclosed in Table X.

In some embodiments, the one or more structural lipids are selected from the group consisting of: cholesterol, natriol, β-mysterol, mysterol, ergosterol, campesterol, stigmasterol, sterosterol, tomatine, tomatin, ursolic acid, α-tocopherol, prednisolone, dexamethasone, prednisolone and hydrocortisone. In some embodiments, the one or more PEGylated lipids are selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

In some embodiments, the one or more phospholipids are selected from the group consisting of 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- Dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di(undecanyl)-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycero) sodium salt (DOPG) and sphingomyelin.

In some embodiments, the lipid nanoparticles comprise about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 40 mol% structural lipids, and about 1.5 mol% PEG lipids.

In some embodiments, the lipid nanoparticles comprise about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 39 mol% structural lipids, and about 2.5 mol% PEG lipids. In some embodiments, the LNP further comprises a targeting moiety operably linked to the LNP. In some embodiments, the LNP further comprises one or more additional components selected from the group consisting of: DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200.

In some embodiments, engineered retrotransposons can be used for gene transfer, which can be performed in vitro or in vivo. Ex vivo gene therapy refers to isolating cells from an individual, delivering nucleic acids to the cells ex vivo , and returning the modified cells to the individual. This may involve collecting a biological sample containing cells from the individual. For example, blood can be obtained by venous puncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.

Typically, but not always, the individual receiving the cells ( e.g. , the recipient) is also the individual from whom the cells were harvested or obtained, which provides the advantage that the donated cells are autologous. However, the cells can be obtained from another individual ( e.g. , a donor), a cell culture from a donor, or an established cell culture. Thus, in some embodiments, the cells are allogeneic to the recipient. The cells can be obtained from the same or a different species as the individual to be treated, but are preferably of the same species as the individual, and more preferably have the same immunotype as the individual. Such cells can be obtained, for example, from a biological sample comprising cells from a close relative or matched donor, then transfected with a nucleic acid ( e.g. , comprising an engineered retrotransposon), and administered to an individual in need of genetic modification, e.g., for treatment of a disease or disorder.

In other embodiments, an engineered retrotransposon can be introduced in vivo (e.g., for gene therapy) by physically delivering the engineered retrotransposon to an individual. Examples of physically introducing an engineered retrotransposon include via injection, electroporation, and transfection (e.g., calcium-mediated or liposome transfection or the like). J. Payload

Retrotran-based gene editing systems and/or components thereof can be delivered with the aid of LNPs as described herein. In various embodiments, retrotran-based gene editing systems can be delivered to cells, tissues, organs or organisms via LNPs. Depending on the selected form, the retrotran-based gene editing system and/or its individual or combined components can be delivered as a DNA molecule (e.g., encoded on one or more plastids), an RNA molecule (e.g., a guide RNA for targeting a programmable nuclease encoding a retrotran RT or a linear or circular mRNA or a programmable nuclease component of a retrotran-based gene editing system), a protein (e.g., a retrotran polypeptide, an accessory protein with other functions (e.g., a recombinase, a nuclease, a polymerase, a ligase, a deaminase or a reverse transcriptase) or The LNP cargo or payload is a nucleic acid payload that is delivered by a protein-nucleic acid complex (e.g., a complex between a guide RNA and a programmable nuclease protein or a fusion protein comprising a retrotranscript RT). Such DNA, RNA, protein or nucleoprotein corresponding to and/or encoding a retrotranscript-based gene editing system or its components comprises an LNP cargo or payload. In various embodiments, the LNP cargo or payload may comprise a nucleic acid payload, including encoding payloads such as linear and circular mRNAs for encoding various components of a retrotranscript-based editing system. 1. Nucleic acid payload

In various embodiments, the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, such as a linear or circular mRNA.

In various embodiments, the retrotranscript-based editing compositions described herein may include a nucleic acid or polynucleotide payload, such as a linear or circular mRNA. For example, a retrotranscript gene editing system may include one or more mRNAs (circular or linear) encoding a retrotranscript RT and other accessory proteins (e.g., a programmable nuclease), and these RNA components may be delivered by LNP.

In some embodiments, LNPs are capable of delivering polynucleotides to target cells, tissues or organs. In the broadest sense of the term, polynucleotides include any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (including messenger mRNA (mRNA)), hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc. RNA that can be used in the compositions and methods described herein can be selected from the group consisting of, but not limited to, shortimer, antagomir, antisense, ribozyme, short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is a circular RNA. In some embodiments, the polynucleotide encodes a protein, such as a nucleobase editor. The polynucleotide can encode any relevant polypeptide, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell. In other embodiments, the polynucleotide is siRNA. siRNA may be able to selectively knock down or downregulate the expression of related genes. For example, after administering a nanoparticle composition comprising siRNA to an individual in need, siRNA may be selected to silence a gene associated with a specific disease, condition, or disorder. siRNA may include a sequence that is complementary to an mRNA sequence encoding a related gene or protein. In some embodiments, siRNA may be an immunomodulatory siRNA.

In some embodiments, the polynucleotide is shRNA or a vector or plasmid encoding shRNA. After the appropriate construct is delivered to the cell nucleus, shRNA can be produced inside the target cell. The constructs and mechanisms associated with shRNA are well known in the relevant field.

A polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region at the 5' end of the first region (e.g., a 5'-UTR), a second flanking region at the 3' end of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the polynucleotide further includes a poly A region or a Kozak sequence (e.g., in a 5'-UTR). In some cases, the polynucleotide may contain one or more intronic nucleotide sequences that can be excised from the polynucleotide. In some embodiments, a polynucleotide (e.g., an mRNA) may include a 5' cap structure, a chain termination nucleotide, a stem loop, a poly A sequence, and/or a polyadenylation signal. Any region of a nucleic acid may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilizing region may contain alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and/or the coding region, 5'-UTR, 3'-UTR, or cap region may include alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methylpseudouridine or 1-ethyl-pseudouridine), and/or 5-substituted cytidines (e.g., 5-methyl-cytidine). In some embodiments, the polynucleotide contains only naturally occurring nucleosides.

In some cases, the polynucleotide is greater than 30 nucleotides long. In another embodiment, the polynucleotide molecule is greater than 35 nucleotides long. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.

In some embodiments, the polynucleotide molecules, formulas, compositions or methods related thereto comprise one or more polynucleotides comprising the features of WO2002/098443, WO2003/051401, WO2008/052770, WO2009/127230, WO2006/122828, WO2008/083949, WO2010/088927, WO2010/037539, WO2004/004743, WO2005/006743, WO2006 ... 05/016376、WO2006/024518、WO2007/095976、WO2008/014979、WO2008/077592、WO2009/030481、WO2009/095226、WO2011/069586、WO2011/026641、WO2011/1 44358、WO2012/019780、WO2012/013326、WO2012/089338、WO2012/ 113513、WO2012/116811、WO2012/116810、WO2013/113502、WO2013/113501、WO2013/113736、WO2013/143698、WO2013/143699、WO2013/143700、WO2013/1206 26. WO2013/120627, WO2013/120628, WO2013/120629, WO2013/174 409, WO2014/127917, WO2015/024669, WO2015/024668, WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664, WO2015/101415, WO2015/101414, WO2015/024667, WO2015/062738, WO2015/101416, all of which are incorporated herein by reference.

In some embodiments, the polynucleotide comprises one or more microRNA binding sites. In some embodiments, the microRNA binding sites are identified by microRNAs in non-target organs. In some embodiments, the microRNA binding sites are identified by microRNAs in the liver. In some embodiments, the microRNA binding sites are identified by microRNAs in hepatocytes.

In certain embodiments, the RNA of the present disclosure comprises one or more phosphonate modifications selected from the group consisting of phosphorothioate linkage (PS), phosphorodithioate linkage (PS2), methylphosphonate linkage (MP), methoxypropylphosphonate linkage (MOP), 5'-(E)-vinylphosphonate linkage (5'-(E)-VP), 5'-methylphosphonate linkage (5'-MP), (S)-5'-C-methyl with phosphate linkage, 5'-phosphorothioate linkage (5'-PS), and peptide nucleic acid linkage (PNA). In certain embodiments, the RNA of the present disclosure comprises one or more ribose modifications selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy-2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), locked nucleic acid (LNA), (S)-cET-BNA, tricyclic-DNA (tcDNA), PMO, unblocked nucleic acid (UNA), and glycol nucleic acid (GNA). In certain embodiments, the RNA comprises a locked nucleic acid (LNA) comprising a methyl bridge, an ethyl bridge, a propyl bridge, a butyl bridge, or a substituted variant of any of the foregoing. In certain embodiments, the RNA of the present disclosure comprises one or more modified bases selected from the group consisting of pseudouridine (ψ), 2'thiouridine (s2U), N6'-methyladenosine ( ^m6A ), 5'methylcytidine ( ^m5C ), 5'fluoro-2'-deoxyuridine, adenine modified with N-ethylpiperidinium 7'-EAA triazole, adenine modified with N-ethylpiperidinium 6' triazole, 6'phenylpyrrolo-cytosine (PhpC), 2',4'-difluorotoluoyl ribonucleoside (rF), and 5'-nitroindole. 2. Single-stranded DNA payload

In various embodiments, the LNP of the present disclosure may include a payload having at least one single strand of DNA. In certain embodiments, the single strand of DNA is a linear single strand of DNA. In certain embodiments, the single strand of DNA is a circular single strand of DNA. In certain embodiments, the payload further includes a nucleobase editing system, such as an enzyme or a polynucleotide encoding an enzyme that can independently or co-dependently edit, modify or alter a target polynucleotide sequence or a target transcript comprising a nucleic acid sequence.

In some embodiments, the circular single-stranded DNA (CiSSD) payload is a payload described in PCT Publication WO2020142730A1, which is incorporated herein by reference in its entirety. In some embodiments, CiSSD is used as a donor template as part of a nucleobase editing system for targeted genome modification. In some embodiments, CiSSD comprises a DNA insert, a 5' homology arm, and a 3' homology arm. In some embodiments, the DNA insert is located between the 5' homology arm and the 3' homology arm. As used herein, a homology arm refers to a series of nucleotides that complement a series of nucleotides in an endogenous DNA sequence in a target region. The homology arms flanking the DNA insert allow the DNA insert to be specifically inserted into the target region. The target region is a nucleic acid sequence where the desired insertion or modification occurs.

In some embodiments, the DNA insert is at least 1 nucleotide. In some embodiments, the DNA insert is at least about 0.5 kb, 2 kb, 2.5 kb, 5 kb, 10 kb, 20 kb, 40 kb, 80 kb, 100 kb, 150 kb, or 200 kb. In some embodiments, the DNA insert is about 0.5 kb to 5 kb, about 1 kb to 5 kb, about 1 kb to 10 kb, about 1.6 kb to 5 kb, about 1.6 kb to 10 kb, about 2 kb to 5 kb, about 2 kb to 20 kb, about 2.5 kb to 5 kb, about 2.5 kb to 10 kb, about 2.5 kb to 20 kb, and about 5 kb to 100 kb in length. In some embodiments, the DNA insert size can range from about 1 kb to about 3 kb, about 3 kb to about 6 kb, about 6 kb to about 9 kb, about 9 kb to about 12 kb, about 12 kb to about 15 kb, about 15 kb to about 18 kb, or about 18 kb to about 21 kb.

In some embodiments, the DNA insert may include a nucleotide sequence encoding a marker or reporter (e.g., a fluorescent marker, an antibiotic marker, or any suitable marker). As used herein, a "marker" or "reporter" means a characteristic that allows identification and selection of desired cells, such as by fluorescence or antibiotic resistance. For example, the insert may include a nucleotide sequence encoding a reporter (e.g., GFP, RFP, or any suitable reporter) or a recombinase. For example, the reporter is an N-terminal GFP fusion reporter.

In some embodiments, the DNA insert may include a nucleotide sequence encoding a transcription unit, wherein each transcription unit can produce a cellular product (e.g., a protein or RNA). In some embodiments, the DNA insert may include a nucleotide sequence encoding a protein, such as an immunomodulatory protein (e.g., a cytokine), an antibody, a chimeric antigen receptor (CAR), a growth factor, a T cell receptor, or another protein.

In some embodiments, the CiSSD comprises a DNA insert that can be inserted into a nucleotide break in a target region of genomic DNA. In some embodiments, the break is a double-strand break (DSB). In some embodiments, the break is a single-strand DNA break or incision. Precision gene editing technology (such as CRISPR) produces breaks near the desired sequence change (target sequence). CRISPR can be used to produce deletions, disruptions, insertions, substitutions, and repairs. The components of the template donors used for these different modifications are generally the same, consisting of three basic elements: 5' homology arms, DNA inserts, and 3' homology arms. CRISPR-based gene editing can generate gene knockouts by disrupting gene sequences, however, the efficiency of inserting exogenous DNA (knock-in) or replacing genomic sequences using current methods is very poor. In certain embodiments, CiSSD can be used with CRISPR by generating knock-in modifications. Double-strand breaks can be introduced by any suitable mechanism, including, for example, by using a gene editing system of CRISPR, zinc finger nucleases, TALEN nucleases (transcription activator-like effector nucleases), or meganucleases as previously described. In short, the CRISPR genome editing system uses CRISPR programmable DNA endonucleases to generate targeted DSBs, which can be targeted to specific DNA sequences (target sequences) by small "guide" RNAs (crRNAs). Guide RNAs (e.g., (z.e.), crRNAs and tracrRNAs) for CRISPR-based modifications can be generated by any suitable method. In certain embodiments, crRNAs and tracrRNAs can be chemically synthesized. In other embodiments, a single guide RNA (sgRNA) can be constructed and synthesized by in vitro transcription.

In certain embodiments, the LNP of the present disclosure comprises a CiSSD disclosed herein and further comprises a component of a precise gene editing system, such as CRISPR, zinc finger nuclease, TALEN nuclease (transcription activator-like effector nuclease) or meganuclease, or any other nucleobase editing system known in the art.

In certain embodiments, the single-stranded DNA (SSD) payload is a payload described in PCT Publication WO2020232286A1, which is incorporated herein by reference in its entirety.

In some embodiments, the SSD comprises an engineered start sequence and an engineered stop sequence from a filamentous phage, and a related DNA sequence, wherein the related DNA sequence is located 3' to the engineered start sequence and 5' to the engineered stop sequence. In some embodiments, the SSD comprises a selectable marker.

In some embodiments, the single-stranded DNA (SSD) payload is prepared by the method described in PCT Publication WO2020232286A1. In some embodiments, the SSD is prepared by a method comprising the following steps: (a) culturing the host cell of technical solution 11 under conditions suitable for producing ssDNA from the relevant DNA sequence in the engineered nucleic acid and producing a plurality of phage proteins from the nucleic acid-assisted plasmid; (b) assembling the ssDNA and the plurality of phage proteins into an engineered phage; and (c) collecting the engineered phage. In some embodiments, the method further comprises extracting the SSD from the engineered phage.

In some embodiments, at least 90% of the SSD is the same length as the associated DNA sequence. In some embodiments, at least 95% of the ssDNA is the same length as the associated DNA sequence. In some embodiments, the length of the SSD is between 100 and 20,000 nucleotides. In some embodiments, the ssDNA is circular.

In some embodiments, the single-stranded DNA (SSD) payload is a payload described in PCT Publication WO2022011082A1, which is incorporated herein by reference in its entirety. In some embodiments, the SSD comprises a first sequence from a filamentous phage, the first sequence having initiator and terminator functions; a second sequence identical to the first sequence; and a related single-stranded DNA sequence between the first sequence and the second sequence. In some embodiments, the SSD further comprises a selectable marker. In some embodiments, the SSD is circular. In some embodiments, the SSD is linear.

In some embodiments, the single-stranded DNA (SSD) payload is made by the method described in PCT Publication WO2022011082A1. In some embodiments, the method includes culturing host cells under conditions suitable for producing single-stranded DNA from the relevant single-stranded DNA sequence in the isolated nucleic acid and producing phage proteins from the nucleic acid-assisted plasmid; assembling the single-stranded DNA and phage proteins into engineered phages; and collecting the engineered phages. In some embodiments, the host cell comprises an isolated nucleic acid, the nucleic acid comprising: a first sequence from a filamentous phage, the first sequence having initiator and terminator functions; a second sequence identical to the first sequence; and a related single-stranded DNA sequence between the first sequence and the second sequence; and a nucleic acid helper plasmid for expressing a phage protein capable of assembling single-stranded DNA into phage. In some embodiments, the method further comprises extracting SSD from the engineered phage.

In some embodiments, at least 90% of the SSD is the same length as the associated DNA sequence. In some embodiments, at least 95% of the ssDNA is the same length as the associated DNA sequence. In some embodiments, the length of the SSD is between 100 and 20,000 nucleotides. In some embodiments, the SSD is circular.

In certain embodiments, the single-stranded DNA (SSD) payload is a payload described in PCT Publication WO2021055616A1, which is incorporated herein by reference in its entirety. 3. Linear mRNA payload

In various embodiments, the LNP-based pharmaceutical compositions described herein (e.g., LNP-based gene editing systems) may include one or more linear mRNA molecules or linear mRNA payloads. In various embodiments, the mRNA payload may encode one or more components of the gene editing system described herein. For example, the mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., a retrotranscript RT or a programmable nuclease) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).

Depending on the nature of the gene editing system, the mRNA payload may also encode one or more effector domains that provide various functions that promote changes in nucleotide sequence and/or gene expression, such as but not limited to single-stranded DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., as well as fusion proteins comprising one or more functional domains linked together.

Ribonucleic acid (RNA) is a molecule composed of nucleotides, which are linked to a ribose sugar containing a nitrogenous base and a phosphate group. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally speaking, RNA mostly exists in a single-stranded form, but in some cases it can also exist in a double-stranded form. The length, form, and structure of RNA vary depending on the purpose of the RNA. For example, the length of RNA can vary from short sequences (e.g., siRNA) to long sequences (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can be a coding (e.g., mRNA) or non-coding (e.g., lncRNA) sequence.

In various embodiments, the LNP-based gene editing systems, RNA therapeutics, and pharmaceutical compositions thereof described herein can be used to deliver mRNA payloads as linear mRNA molecules. In embodiments, the mRNA payload can include one or more nucleotide sequences encoding related products, such as, but not limited to, components of a gene editing system (e.g., endonucleases, primer editors, etc.) and/or therapeutic proteins.

In some embodiments, the RNA payload may be a linear mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein of interest and can be translated in vitro , in vivo , in situ or ex vivo to produce the encoded protein of interest.

In general, an mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly A tail. In some aspects, one or more structural and/or chemical modifications or alterations may be included in the RNA, which may reduce the innate immune response of the cell into which the mRNA is introduced. As used herein, a "structural" feature or modification is a feature or modification in which two or more linked nucleotides are inserted, deleted, repeated, inverted, or randomized in a nucleic acid without causing significant chemical modification to the nucleotides themselves. Because chemical bonds are necessarily destroyed and reformed to affect structural modifications, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications will result in different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G".

In general, the relevant coding region in the mRNA used herein can encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide or a decapeptide. In another embodiment, the mRNA can encode a peptide of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25 or 10-20 amino acids. The mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide of no longer than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

Generally, the length of the mRNA region encoding the product of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 2 70,000, 80,000, 90,000, or up to and including 100,000 nucleotides).

In some embodiments, the mRNA has a total length spanning from about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50 ,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,5 00 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

In some embodiments, the length of one or more regions flanking the region encoding the product of interest may independently range from 15-1,000 nucleotides (e.g., greater than or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides).

In some embodiments, the mRNA comprises a tailing sequence, which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). When the tailing region is a poly A tail, the length can be determined in units of poly A binding protein binding or as a function of poly A binding protein binding. In this embodiment, the poly A tail is long enough to bind at least 4 monomers of the poly A binding protein. The poly A binding protein monomer binds to an extension of about 38 nucleotides. Thus, poly A tails of about 80 nucleotides and 160 nucleotides have been observed to be functional.

In some embodiments, the mRNA comprises a capping sequence comprising a single cap or a series of nucleotides that form a cap. The capping sequence can be 1 to 10 (e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2 or 10 or less) nucleotides long. In some embodiments, the capping sequence is absent.

In some embodiments, the mRNA includes a region comprising a start codon. The length of the region comprising the start codon may be between 3 and 40 (e.g., 5-30, 10-20, 15 or at least 4 or 30 or less) nucleotides.

In some embodiments, the mRNA includes a region comprising a stop codon. The length of the region comprising the stop codon may be between 3 and 40 (e.g., 5-30, 10-20, 15 or at least 4 or 30 or less) nucleotides.

In some embodiments, the mRNA includes a region comprising a restriction sequence. The length of the region comprising the restriction sequence can range from 3 to 40 (e.g., 5-30, 10-20, 15 or at least 4 or 30 or less) nucleotides. Untranslated region (UTR)

In various embodiments, the mRNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may include at least one untranslated region (UTR) flanking the region encoding the product of interest and/or incorporated into the mRNA molecule. UTRs are transcribed but not translated. The mRNA payload may include 5'UTR sequences and 3'UTR sequences as well as internal UTRs.

The RNA payload of the present disclosure may include one or more regions or portions that serve or function as non-translated regions. When a nucleic acid is designed to encode at least one polypeptide of interest, the nucleic acid may include one or more of these non-translated regions (UTRs). The wild-type non-translated regions of a nucleic acid are transcribed but not translated. In an mRNA, the 5' UTR begins at the transcription start site and continues to, but not including, the start codon; while the 3' UTR begins immediately following the stop codon and continues until the transcription stop signal. There is increasing evidence for the regulatory role that UTRs play in the stability and translation of nucleic acid molecules. UTR regulatory features can be incorporated into the RNA payload molecules (e.g., linear and circular mRNA molecules) of the present disclosure to, among other things, enhance molecular stability. Specific features can also be incorporated to ensure controlled downregulation of transcripts to prevent them from being misdirected to undesired organ sites. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

In various embodiments, the mRNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may comprise at least one UTR, which may be selected from any of the UTR sequences listed in Table 19 or 20 of U.S. Patent No. 10,709,779, which is incorporated herein by reference. 5' UTR region

In various embodiments, the mRNA payload of the LNP-based gene editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may comprise at least one 5'UTR.

The 5'UTR is the region of the mRNA that is immediately upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein (is non-coding). The natural 5'UTR has the characteristic of playing a role in translation initiation. It carries imprints, such as the Kozak sequence, which are generally known to be involved in the process of ribosome initiation of translation of various genes. The Kozak sequence has the consensus CCR(A/G)CCAUGG (SEQ ID NO: 19421), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. The 5'UTR is also known to form a secondary structure that is involved in the binding of elongation factors. It is also known that the 5'UTR sequence is important for ribosome recruitment to mRNA and is reported to play a role in translation (Hinnebusch A et al., (2016) Science, 352:6292: 1413-6). In addition, the 5'UTR sequence can confer increased half-life, increased expression and/or increased activity to the polypeptide encoded by the RNA payload described herein.

In various embodiments, the RNA payload constructs contemplated herein may include 5'UTRs found in nature and those 5'UTRs not found in nature. For example, the 5'UTR may be synthetic and/or may have sequence alterations relative to the naturally occurring 5'UTR. Such altered 5'UTRs may include one or more modifications relative to the naturally occurring 5'UTR, such as insertions, deletions, or altered sequences, or substitutions of naturally occurring nucleotides with one or more nucleotide analogs.

The 5'UTR begins at the transcription start site and continues to the start codon, but not including the start codon, while the 3'UTR begins immediately after the stop codon and continues until the transcription stop signal. Although not wishing to be bound by theory, UTRs may have regulatory roles in nucleic acid translation and stability.

The natural 5'UTR generally includes features that have a role in translation initiation, as these features tend to include the Kozak sequence, which is generally known to be involved in the process by which ribosomes initiate translation of a variety of genes. The Kozak sequence has a consensus of CCR(A/G)CCAUGG (SEQ ID NO: 19421), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. The 5'UTR is also known to form a secondary structure that is involved in the binding of elongation factors.

In one embodiment, the 5'UTR comprises a sequence provided in Table X or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5'UTR sequence provided in Table Y, or a variant or fragment thereof (e.g., a fragment lacking the first one, two, three, four, five or six nucleotides of a 5'UTR sequence provided in Table Y). In one embodiment, the 5'UTR comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the sequences in Table Y. Table Y - Exemplary Nucleotide Sequences of 5'UTR 5'UTR nucleotide sequence Sequence Identifier ggaaaucgca aaauuugcuc uucgcguuag auuucuuuua guuuucucgc aacuagcaag cuuuuuguuc ucgccgccgc c SEQ ID NO: 19422 ggaaaucgca aaauuugcuc uucgcguuag auuucuuuua guuuucucgc aacuagcaag cuuuuuguuc ucgccgccgc c SEQ ID NO: 19423 ggaaaucgca aaauuuucuu uucgcguuag auuucuuuua guuuucuuuc aacuagcaag cuuuuuguuc ucgccgccgc c SEQ ID NO: 19424 ggaaaucgca aaauuuuugc ucuuuuucgc guuagauuuc uuuuaguuuu cuykcaacua gcaagcuuuu uguucucgcc rcc SEQ ID NO: 19425 ggaaaucccc acaaccgccu cauauccagg cucaagaaua gagcucagug uuuuguuguu uaaucauucc gacguguuuu gcgauauucg cgcaaagcag ccagucgcgc gcuugcuuuu aaguagaguu guuuuuccac ccguuugcca ggcaucuuua auuuaacaua uuuuuauuuu ucagg cuaac cuacgccgcc acc SEQ ID NO: 19426 ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga ucucccugag cuucagggag ccccggcgcc gccacc SEQ ID NO: 19427 ggaaaccccc cacccccgua agagagaaaa gaagaguaag aagaaauaua agaucucccu gagcuucagg gagccccggc gccgccacc SEQ ID NO: 19428 ggagaacuuc cgcuuccguu ggcgcaagcg cuuucauuuu uucugcuacc gugacuaag SEQ ID NO: 19429 ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga gccacc SEQ ID NO: 19430 ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga ccccggcgcc gccacc SEQ ID NO: 19431 ggaaacuuua uuuaguguua cuuuauuuuc uguuuauuug uguuucuuca guggguuugu ucuaauuucc uuggccgcc SEQ ID NO: 19432 ggaaaaucug uauuagguug gcguguucuu uggucgguug uuaguauugu uguugauucg uuuguggucg guugccgcc SEQ ID NO: 19433 ggaaaauuau uaacaucuug guauucucga uaaccauucg uuggauuuua uuguauucgu aguuuggguu ccugccgcc SEQ ID NO: 19434 ggaaauuauu auuauuucua gcuacaauuu aucauuguau uauuuuagcu auucaucauu auuuacuugg ugaucaaca SEQ ID NO: 19435 ggaaauaggu uguuaaccaa guucaagccu aauaagcuug gauucuggug acuugcuuca ccguuggcgg gcaccgauc SEQ ID NO: 19436 ggaaaucgua gagagucgua cuuaguacau aucgacuauc gguggacacc aucaagauua uaaaccaggc caga SEQ ID NO: 19437 ggaaacccgc ccaagcgacc ccaacauauc agcaguugcc caaucccaac ucccaacaca auccccaagc aacgccgcc SEQ ID NO: 19438 ggaaagcgau ugaaggcguc uuuucaacua cucgauuaag guuggguauc gucgugggac uuggaaauuu guuguuucc SEQ ID NO: 19439 ggaaacuaau cgaaauaaaa gagccccgua cucuuuuauu ucuauuaggu uaggagccuu agcauuugua ucuuaggua SEQ ID NO: 19440 ggaaauguga uuuccagcaa cuucuuuuga auauauugaa uuccuaauuc aaagcgaaca aaucuacaag ccauauacc SEQ ID NO: 19441 ggaaaucgua gagagucgua cuuacguggu cgccauugca uagcgcgcga aagcaacagg aacaagaacg cgcc SEQ ID NO: 19442 ggaaaucgua gagagucgua cuuagaauaa acagagucgg gucgacuugu cucugauacu acgacgucac aauc SEQ ID NO: 19443 ggaaaauuug ccuucggagu ugcguauccu gaacugccca gccuccugau auacaacugu uccgcuuauu cgggccgcc SEQ ID NO: 19444 ggaaaucuga gcaggaaucc uuugugcauu gaagacuuua gauuccucuc ugcgguagac gugcacuuau aaguauuug SEQ ID NO: 19445 ggaaagcgau ugaaggcguc uuuucaacua cucgauuaag guuggguauc gucgugggac uuggaaauuu guugccacc SEQ ID NO: 19446 ggaaaauuuu agccuggaac guuagauaac uguccuguug ucuuuauaua cuuggucccc aaguaguuug ucuuccaaa SEQ ID NO: 19447 ggaaauuuuu uuuugauauu auaagaguuu uuuuuugaua uuaagaaaau uuuuuuuuga uauuagaaga guaagaagaa auauaagacc ccggcgccgc cacc SEQ ID NO: 19448 ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga gccaaaaaaa aaaaacc SEQ ID NO: 19449 ggaaaucucc cugagcuuca gggaguaaga gagaaaagaa gaguaagaag aaauauaaga ccccggcgcc gccacc SEQ ID NO: 19450

In some embodiments of the present disclosure, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature and associated with a different mRNA. In another embodiment, the 5'UTR is a synthetic UTR, i.e., does not exist in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., such UTRs increase gene expression; and those that are completely synthetic. Exemplary 5'UTRs include Xenopus or human-derived α-globin or β-globin (e.g., US8,278,063 and US9,012,219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and tobacco etch virus. The CMV immediate early 1 (IE1) gene (see US20140206753 and WO2013/185069), sequence GGGAUCCUACC (SEQ ID NO: 19451) (WO2014144196) can also be used. In another embodiment, the 5'UTR of the TOP gene is a 5'UTR of a TOP gene lacking a 5'TOP motif (oligopyrimidine tract) (e.g., WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667, WO2015024667; a 5'UTR element derived from the ribosomal protein large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738)), a 5'UTR element derived from the 5'UTR of the hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667) or a 5'UTR derived from the 5'UTR of ATP5A1 can be used. UTR element (WO2015024667). In one embodiment, an internal ribosome entry site (IRES) is used as a substitute for the 5' UTR.

In some embodiments, the 5'UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 19452 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC) and SEQ ID NO: 19453 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC). 3'UTR Region

In various embodiments, the mRNA payload of the LNP-based base editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may include at least one 3'UTR. The 3'UTR may be heterologous or synthetic.

The 3'UTR is a region of mRNA that is immediately downstream (3') of the stop codon (the codon of the mRNA transcript that signals the end of translation). The 3'UTR does not encode protein (is non-coding). The native or wild-type 3'UTR is known to have stretches of adenosine and uridine embedded therein. These AU-rich imprints are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, these AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III ARES are not well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize the message, but members of the ELAV family, especially HuR, have been documented to increase mRNA stability. HuR binds to AREs of all three classes. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and, therefore, message stabilization in vivo.

The 3'UTR is known to have stretches of adenosine and uridine embedded in it. These AU-rich imprints are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, these AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III ARES are not well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize the message, while members of the ELAV family, especially HuR, have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and, therefore, message stabilization in vivo.

The introduction, removal or modification of the 3'UTR AU-rich element (ARE) can be used to modulate the stability of the mRNA payload described herein. For example, one or more copies of the ARE can be introduced to make the mRNA less stable and thereby reduce translation and reduce production of the resulting protein. Alternatively, the ARE can be identified and removed or mutated to increase intracellular stability and thereby increase translation and production of the resulting protein.

In some embodiments, the introduction of a feature that is normally expressed in a target organ gene in a specific organ and/or tissue can enhance the stability of mRNA and protein production. As a non-limiting example, the feature can be a UTR. As another example, the feature can be an intron or part of an intron sequence.

One of ordinary skill in the art will appreciate that a heterologous or synthetic 5'UTR can be used with any desired 3'UTR sequence. For example, a heterologous 5'UTR can be used with a synthetic 3'UTR having a heterologous 3'UTR.

Non-UTR sequences can also be used as regions or subregions within RNA payload constructs. For example, introns or portions of intronic sequences can be incorporated into regions of the nucleic acids of the present disclosure. Incorporation of intronic sequences can increase protein production and nucleic acid levels.

Combinations of features may be included in the flanking regions and may be contained within other features. For example, the relevant polypeptide coding region in the mRNA payload may be flanked by a 5'UTR, which may contain a strong Kozak translation initiation signal; and/or a 3'UTR, which may include an oligo(dT) sequence for templated addition of a poly A tail. The 5'UTR may comprise a first polynucleotide segment and a second polynucleotide segment from the same and/or different genes, such as the 5'UTR described in U.S. Patent Application Publication No. 20100293625 and PCT/US2014/069155, each of which is incorporated herein by reference in its entirety.

It should be understood that any UTR from any gene can be incorporated into a region of an RNA payload molecule (e.g., a linear mRNA). In addition, multiple wild-type UTRs of any known gene can be utilized. It is also within the scope of the present disclosure to provide artificial UTRs that are variants of non-wild-type regions. These UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected, or the orientation or position can be changed. Thus, a 5' or 3' UTR can be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the term "altered" in relation to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. For example, a 3'UTR or 5'UTR may be altered by a change in orientation or position as taught above, or may be altered by the inclusion of additional nucleotides, nucleotide deletions, nucleotide exchanges, or transpositions relative to a wild-type or native UTR. Any of these changes that result in an "altered" UTR (whether 3' or 5') includes a variant UTR.

In some embodiments, a double, triple, or quadruple UTR may be used, such as a 5'UTR or a 3'UTR. As used herein, a "double" UTR is a UTR in which two copies of the same UTR are encoded in tandem or substantially in tandem. For example, a double β-globin 3'UTR may be used as described in U.S. Patent Publication No. 20100129877, the contents of which are incorporated herein by reference in their entirety.

It is also within the scope of the present disclosure to have patterned UTRs. As used herein, "patterned UTRs" are those UTRs that reflect a repeating or alternating pattern, such as ABABAB or AABBAABBABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter A, B, or C represents a different UTR at the nucleotide level.

In some embodiments, the flanking regions are selected from a family of transcripts whose proteins share a common function, structure, characteristic, or property. For example, related polypeptides may belong to a family of proteins that are expressed in a particular cell, tissue, or at a certain time during development. A UTR from any of these genes may be exchanged with any other UTR of the same or different protein family to generate a new polynucleotide. As used herein, "protein family" is used in the broadest sense to refer to a group of two or more related polypeptides that share at least one function, structure, characteristic, location, origin, or expression pattern.

The non-translated region may also include a translation enhancing element (TEE). As a non-limiting example, TEEs may include those described in U.S. Application No. 20090226470 (incorporated herein by reference in its entirety), as well as those known in the art. 5' capping

In various embodiments, the mRNA payload of the LNP-based base editing system, RNA therapeutics, and pharmaceutical compositions thereof described herein may comprise a 5' cap structure.

The 5' cap structure of mRNA is involved in nuclear export, increases polynucleotide stability and binds mRNA cap binding protein (CBP), which is responsible for mRNA stability and translation competence in the cell through the formation of mature circular mRNA species by CBP and poly(A) binding protein. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5'-capped, resulting from a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or pre-terminal transcribed nucleotides at the 5' end of the mRNA can also be 2'-0-methylated as appropriate. 5'-decapping achieved by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

Modification of mRNA can generate a non-hydrolyzable cap structure, thereby preventing decapping and thus increasing the half-life of the mRNA. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to generate phosphorothioate linkages in the 5'-ppp-5' cap.

Additional modified guanosine nucleotides may be used, such as α-methyl-phosphonic acid and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminal and/or 5'-pre-terminal nucleotide of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. A variety of different 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule (such as an mRNA molecule).

Cap analogs are also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, whose chemical structure is different from the natural (i.e., endogenous, wild-type or physiological) 5'-cap while retaining the cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or attached to a nucleic acid molecule.

For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which contains an N7-methyl group and a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine ( ^m7G -3'mppp-G; which can be equivalently referred to as 3'O-Me-m7G(5') ppp(5')G). The 3'-0 atom of the other unmodified guanine is linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA). The N7- and 3'-0-methylated guanines provide the terminal portion of the capped nucleic acid molecule (e.g., mRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (ie, N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, ^m7Gm -ppp-G).

Although cap analogs allow simultaneous capping of nucleic acid molecules during in vitro transcription reactions, up to 20% of transcripts may remain uncapped. This, along with structural differences between cap analogs and the endogenous 5'-cap structure of nucleic acids produced by the endogenous cellular transcription machinery, can lead to reduced translational competence and reduced cellular stability.

mRNA can also be capped after transcription using enzymes to generate more realistic 5'-cap structures. As used herein, the term "more realistic" refers to features that closely reflect or mimic endogenous or wild-type features in structure or function. That is, "more realistic" features are more representative of endogenous, wild-type, natural or physiological cellular functions and/or structures, such as compared to synthetic features or analogs of the prior art, or they outperform the corresponding endogenous, wild-type, natural or physiological features in one or more aspects. Non-limiting examples of more realistic 5' cap structures are those structures that, among other things, have enhanced binding of cap-binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping, such as compared to synthetic 5' cap structures (or wild-type, natural or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-0-methyltransferases can generate a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of an mRNA and a guanine cap nucleotide, wherein the cap guanine contains an N7 methylation and the 5' terminal nucleotide of the mRNA contains a 2'-0-methyl group. Such structures are referred to as Capl structures. This cap results in higher translational competence and cell stability and reduced activation of cellular pro-inflammatory cytokines, as compared to other 5' cap analog structures known in the art, for example. Cap structures include, but are not limited to, 7mG(5 ^* )ppp(5 ^* )N,pN2p (cap 0), 7mG(5 ^* )ppp(5 ^* )NlmpNp (cap 1), and 7mG(5 ^* )-ppp(5')NlmpN2mp (cap 2).

In some embodiments, the 5' terminal cap may include an endogenous cap or a cap analog.

In some embodiments, the 5' end cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. IRES sequence

In various embodiments, the mRNA payload of the LNP-based gene editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may comprise one or more IRES sequences.

In some embodiments, the mRNA may contain an internal ribosome entry site (IRES). IRES was originally identified as a feature of picornavirus RNAs and plays an important role in initiating protein synthesis in the absence of a 5' cap structure. IRES can serve as the only ribosome binding site, or can serve as one of multiple ribosome binding sites for an mRNA. An mRNA containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by the ribosome. Non-limiting examples of IRES sequences that can be used include, but are not limited to, those from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

In some embodiments, the IRES is from Taura syndrome virus, cone bug virus, Theilerian encephalomyelitis virus, simian virus 40, red fire ant virus 1, rice aphid virus, reticuloendotheliosis virus, human poliovirus 1, Persian enterovirus, cashmere honey bee virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid microRNA virus-like virus, encephalomyocarditis virus, fruit fly C virus, human coxsackievirus B3, cruciferous tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, fruit fly tentacles, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c -IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kip1, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, canine Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, brewing yeast TFIID, brewing yeast YAP1, tobacco etch virus, cyanocobalamin virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirus, HCV QC64, Human Cossavirus E/D, Human Cossavirus F, Human Cossavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sabiavirus A SH1, Sabiavirus FHB, Sabiavirus NG-J1, Human coleus virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human coleus virus 5, Aichi virus, Hepatitis A virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picornaviral Virus, CRPV, Salivirus A BNS, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12, EV24 or eIF4G aptamer. Poly A tail and 3' stabilizing region

In various embodiments, the mRNA payload of the LNP-based gene editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may include a poly A tail.

During RNA processing, long chains of adenine nucleotides (poly A tails) can be added to polynucleotides (such as mRNA molecules) to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to release the 3' hydroxyl group. Poly A polymerase then adds chains of adenine nucleotides to the free 3' hydroxyl end. This process is called polyadenylation, and a certain length of poly A tail is added.

In some embodiments, the length of the poly A tail is greater than 30 nucleotides. In another embodiment, the poly A tail is greater than 35 nucleotides long (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 3,400, 4,500, 5 700, 800, 900, 1000, 2000, or 3000 nucleotides in length. In some embodiments, the mRNA includes about 30 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 1 from 1,000 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500, and 2,500 to 3,000).

In some embodiments, the length of the poly A tail relative to the total mRNA is designed. This design can be based on the length of the region encoding the target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the final product expressed from the mRNA.

In this case, the length of the poly A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater than the mRNA or its feature. The poly A tail can also be designed as part of the mRNA to which it belongs. In this case, the poly A tail can be 10, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total length of the construct or the total length of the construct minus the poly A tail. In addition, engineered binding sites and mRNA binding for poly A binding proteins can enhance expression.

Alternatively, multiple different mRNAs can be linked together to PABP (poly A binding protein) via the 3' end using modified nucleotides at the 3' end of the poly A tail. Transfection experiments can be performed in relevant cell lines and protein production can be analyzed by ELISA at 12 h, 24 h, 48 h, 72 h and 7 days after transfection.

In some embodiments, the mRNA is designed to include a poly-AG tetrad. A G-tetrad is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G-tetrad is incorporated into the end of the poly-A tail. Stop codon

In various embodiments, the mRNA payload of the LNP-based gene editing system, RNA therapeutic agent, and pharmaceutical composition thereof described herein may include one or more translational termination codons. The translational termination codons UAA, UAG, and UGA are important components of the genetic code and signal the termination of mRNA translation. During protein synthesis, the termination codon interacts with the protein release factor and this interaction can regulate ribosome activity, thereby affecting translation (Tate WP et al., (2018) Biochem Soc Trans, 46(6):1615-162).

As used herein, a termination element refers to a nucleic acid sequence comprising a stop codon. In the case of DNA, the stop codon may be selected from TGA, TAA and TAG, or in the case of RNA, may be selected from UGA, UAA and UAG. In one embodiment, the termination element comprises two consecutive stop codons. In one embodiment, the termination element comprises three consecutive stop codons. In one embodiment, the termination element comprises four consecutive stop codons. In one embodiment, the termination element comprises five consecutive stop codons.

In some embodiments, the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As a non-limiting example, the stop codon may be selected from TGA, TAA and TAG.

In other embodiments, the stop codon can be selected from one or more of the following termination elements in Table Z: Table Z: Additional termination elements for linear mRNA Nucleotide sequence (5' to 3') Sequence Identifier UGAUAAUAG SEQ ID NO: 19454 UAAUAGUAA SEQ ID NO: 19455 UAAGUCUAA SEQ ID NO: 19456 UAAAGCUAA SEQ ID NO: 19457 UAAGUCUCC SEQ ID NO: 19458 UAAGGCUAA SEQ ID NO: 19459 UAAGCCCCUCCGGGG SEQ ID NO: 19460 UAAAGCUCCCCGGGG SEQ ID NO: 19461 UAAGCCCCU SEQ ID NO: 19462 UAAAGCUCC SEQ ID NO: 19463 UAAAGCUCC SEQ ID NO: 19464 UAGGGUUAA SEQ ID NO: 19465 UAAGCACCC SEQ ID NO: 19466 UGAUAGUAA SEQ ID NO: 19467 UAAAGCGCU SEQ ID NO: 19468

In some embodiments, the mRNA includes the stop codon TGA and an additional stop codon. In another embodiment, the additional stop codon may be TAA. MicroRNA Binding Sites and Other Regulatory Elements

In various embodiments, the mRNA payload of the LNP-based gene editing systems, RNA therapeutics, and pharmaceutical compositions thereof described herein may comprise one or more regulatory elements, including but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and/or motifs, artificial binding sites that bind to endogenous nucleic acid binding molecules, and combinations thereof .

In some embodiments, the mRNA payload of the LNP-based gene editing systems, RNA therapeutics, and pharmaceutical compositions thereof described herein is not chemically modified and comprises standard ribonucleotides composed of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT). Chemically Modified Nucleotides

In some embodiments, the mRNA payload of the LNP-based gene editing systems, RNA therapeutics, and pharmaceutical compositions thereof described herein comprises (in some embodiments comprises) at least one chemical modification.

The terms "chemical modification" and "chemically modified" refer to modifications in at least one aspect of the position, pattern, percentage or group of ribonucleosides or deoxyribonucleosides relative to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C). Generally, these terms do not refer to naturally occurring ribonucleotide modifications in the 5' terminal mRNA cap portion. With respect to polypeptides, the term "modification" refers to modifications relative to the canonical set of 20 amino acids. A polypeptide as provided herein is also considered "modified" if it contains amino acid substitutions, insertions, or a combination of substitutions and insertions.

In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a variety (more than one) different modifications. In some embodiments, a specific region of a polynucleotide contains one, two, or more (as the case may be) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or an organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or an organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

Modifications of polynucleotides include, but are not limited to, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may include naturally occurring modifications, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include any useful modification, such as modifications of sugars, nucleobases, or internucleoside linkages (e.g., attachment to phosphates, phosphodiester linkages, or phosphodiester backbones).

In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises non-naturally modified nucleotides introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. The modification may be present at the internucleotide linkage, the purine or pyrimidine base, or the sugar. The modification may be introduced by chemical synthesis or by a polymerase at the end of the chain or elsewhere in the chain. Any region of a polynucleotide may be chemically modified.

The present disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or its derivatives combined with an organic base (e.g., purine or pyrimidine) or its derivatives (also referred to herein as "nucleobase"). "Nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any available method, such as chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides. A polynucleotide may include one or more regions linking nucleosides. Such regions may have variable backbone linkages. Such linkages may be standard phosphodiester linkages, in which case the polynucleotide will include nucleotide regions.

Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bond formation between a non-standard base and a standard base or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotides inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker can be incorporated into the polynucleotides of the present disclosure.

In some embodiments, a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) includes a combination of at least two (eg, 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the modified nucleobase in a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) is selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (m ¹ ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyluridine. In some embodiments, a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) includes a combination of at least two (eg, 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the modified nucleobase in a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is selected from the group consisting of 1-methyl-pseudouridine (m ¹ ψ), 5-methoxy-uridine (mo ⁵ U), 5-methyl-cytidine (m ⁵ C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the polynucleotide comprises a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (m ⁵ C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m ¹ ψ). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m ¹ ψ) and 5-methyl-cytidine (m ⁵ C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine (s ² U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m ⁵ C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy-uridine (mo ⁵ U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (mo ⁵ U) and 5-methyl-cytidine (m ⁵ C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyluridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyluridine and 5-methyl-cytidine (m ⁵ C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m ⁶ A). In some embodiments, a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) comprises N6-methyl-adenosine ( ^m6A ) and 5-methyl-cytidine (mC).

In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., completely modified, modified throughout the sequence) to carry out a specific modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine ( ^m5C ), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine ( ^m5C ). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacing it with a modified residue (such as those described above).

Exemplary nucleobases and nucleosides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halogen-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and in some embodiments, the modified nucleobase is a modified cytosine. Nucleosides having modified uridine include 5-cyanouridine and 4′-thiouridine.

The polynucleotides of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, in a polynucleotide of the present invention, or in a given predetermined sequence region thereof (e.g., in an mRNA including or excluding a poly A tail), one or more or all or a given type of nucleotide (e.g., a purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified. In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may be any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

The polynucleotide may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentages (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 1 % to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It is understood that any remaining percentages are explained by the presence of unmodified A, G, U, or C.

The polynucleotide may contain at least 1% and at most 100% modified nucleotides, or any intermediate percentages, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotide may contain a modified pyrimidine, such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the polynucleotide is replaced by a modified uracil (e.g., a 5-substituted uracil). The modified uracil may be replaced by a compound having a single unique structure, or may be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced by modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). 4. Circular mRNA Payload

In various embodiments, the LNP-based pharmaceutical compositions described herein (e.g., LNP-based gene editing systems) may include one or more circular mRNA molecules or "oRNAs". In various embodiments, the circular mRNA payload may encode one or more components of the gene editing system described herein or other related therapeutic proteins. For example, the circular mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., retrotranscript RT, CRISPR nuclease, TALEN, and zinc finger binding domain) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).

Depending on the nature of the gene editing system, the circular mRNA payload may also encode one or more effector domains that provide various functions that promote changes in nucleotide sequence and/or gene expression, such as but not limited to single-stranded DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminase or adenosine deaminase), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., as well as fusion proteins comprising one or more functional domains linked together.

The circular RNA described herein is a polyribonucleotide that forms a continuous structure by covalent or non-covalent bonds. Due to the circular structure, oRNA has improved stability, increased half-life, reduced immunogenicity and/or improved functionality (e.g., the functions described herein) compared to the corresponding linear RNA.

In some embodiments, the oRNA binds to a target. In some embodiments, the oRNA binds to a substrate. In some embodiments, the oRNA binds to a target and binds to a substrate of the target. In some embodiments, the oRNA binds to a target and mediates regulation of the substrate of the target. In some embodiments, the oRNA binds to a target and its substrate to mediate modification of the substrate, such as post-translational modification. In some embodiments, the oRNA binds to a target and its substrate to mediate a cellular process involving the substrate (e.g., altering protein degradation or signal transduction). In some embodiments, the target is a target protein and the substrate is a substrate protein.

In some embodiments, the oRNA comprises a binding portion for binding to a compound. The binding portion may be a modified polyribonucleotide. The compound may be bound to the oRNA by a binding portion. In some embodiments, the compound binds to a target and mediates the regulation of the target's substrate. In some embodiments, the oRNA binds to the target's substrate, and the compound bound to the oRNA by a binding portion binds to the target to bind the target and its substrate together, thereby mediating the modification of the substrate, such as post-translational modification. In some embodiments, the oRNA binds to the target's substrate, and the compound bound to the oRNA by a binding portion binds to the target to bind the target and its substrate together, thereby mediating the modification of the substrate to mediate a cellular process involving the substrate (e.g., altering protein degradation or signal transduction). In some embodiments, the target is a target protein and the substrate is a substrate protein.

In some embodiments, the oRNA can be non-immunogenic in mammals (e.g., humans, non-human primates, rabbits, rats, and mice).

In some embodiments, in cells from aquaculture animals (e.g., fish, crab, shrimp, oysters, etc.), mammalian cells, cells from pet or zoo animals (e.g., cats, dogs, lizards, birds, lions, tigers, and bears, etc.), cells from farm or working animals (e.g., horses, cows, pigs, chickens, etc.), human cells, cultured cells, The oRNA may be capable of replication, or undergo replication, in primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.

In one aspect, provided herein is a pharmaceutical composition comprising: a circular RNA comprising, in the following order, a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or a component thereof), and a 5' group I intron fragment; and a transfer vehicle comprising at least one of the following: (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a cell (e.g., a human cell, such as an immune cell present in a human individual), so that the polypeptide is translated in the cell.

In some embodiments, the pharmaceutical composition is formulated for intravenous administration to a human subject in need thereof. In some embodiments, the 3' group I intron fragment and the 5' group I intron fragment are Anabaena group I intron fragments.

In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L9a-5 arrangement site in the complete intron. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 arrangement site in the complete intron.

In some embodiments, the IRES is from Taura syndrome virus, cone bug virus, Theilerian encephalomyelitis virus, simian virus 40, red fire ant virus 1, rice aphid virus, reticuloendotheliosis virus, human poliovirus 1, Persian enterovirus, cashmere honey bee virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid microRNA virus-like virus, encephalomyocarditis virus, fruit fly C virus, human coxsackievirus B3, cruciferous tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, fruit fly tentacles, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c -IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFlα, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, canine Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, brewer's yeast TFIID, brewer's yeast YAP1, tobacco etch virus, cyanocobalamin virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirus, HCV QC64, Human Cossavirus E/D, Human Cossavirus F, Human Cossavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sabiavirus A SHI, Sabiavirus FHB, Sabiavirus NG-J1, Human coleus virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human coleus virus 5, Aichi virus, Hepatitis A virus HA 16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picornavirus-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G aptamer.

In some embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof. In some embodiments, the pharmaceutical composition comprises a first internal spacer between the 3' group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5' group I intron fragment. In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

In some embodiments, the circular mRNA comprises a nucleotide sequence encoding a polypeptide of interest, such as a nucleobase editing system or a therapeutic protein (e.g., a CAR or TCR complex protein).

In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein further comprise a targeting moiety. In certain embodiments, the targeting moiety mediates receptor-mediated endocytosis or directly fuses the delivery vehicle (LNP) to a selected cell population or tissue in the absence of cell separation or purification. In certain embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CDS, CD7, PD-1, 4-1BB, CD28, Clq, and CD2. In certain embodiments, the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen. In certain embodiments, the targeting moiety comprises a scFv, a nanobody, a peptide, a minibody, a polynucleotide aptamer, a heavy chain variable region, a light chain variable region, or a fragment thereof.

In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein are administered in an amount effective to treat a disease in a human subject (e.g., where the disease may be cancer, a muscle disorder, or a CNS disorder, etc.). In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions have an enhanced safety profile when compared to pharmaceutical compositions comprising T cells or vectors comprising exogenous DNA encoding the same polypeptide.

In some embodiments, LNP-based nucleotide editing systems and pharmaceutical compositions thereof are administered in an amount effective to induce the desired precise editing in the genome. In some embodiments, LNP-based nucleotide editing systems and pharmaceutical compositions have an enhanced safety profile when compared to prior art gene editing delivery compositions.

In another aspect, the present disclosure provides a circular RNA comprising, in the following order, a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or a component thereof), and a 5' group I intron fragment.

In some embodiments, the 3' group I intron fragment and the 5' group I intron fragment are Anabaena group I intron fragments. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L9a-5 arrangement site in the complete intron. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 arrangement site in the complete intron. In certain embodiments, the IRES comprises CVB3 IRES or a fragment or variant thereof.

In some embodiments, the circular RNA comprises a first internal spacer between the 3' group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5' group I intron fragment.

In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein is composed of natural nucleotides. In some embodiments, the circular RNA further comprises a second expression sequence encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a checkpoint inhibitor. In certain embodiments, the therapeutic protein comprises an interleukin.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein is composed of natural nucleotides.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein comprises a partially or completely codon-optimized nucleotide sequence. In some embodiments, the circular RNA is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one endonuclease sensitive site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one RNA editing sensitive site present in an equivalent pre-optimized polynucleotide.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein has a functional half-life in vivo in the human body that is greater than the functional half-life in vivo of an equivalent linear RNA with the same expression sequence. In some embodiments, the circular RNA has a length of about 100 nucleotides to about 10 kilobases. In some embodiments, the circular RNA has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA has a therapeutic effect duration of at least about 20 hours in human cells. In some embodiments, the duration of the therapeutic effect of the circular RNA in human cells is greater than or equal to the duration of the therapeutic effect of an equivalent linear RNA comprising the same expression sequence. In some embodiments, the functional half-life of the circular RNA in human cells is greater than or equal to the functional half-life of an equivalent linear RNA comprising the same expression sequence.

In some embodiments, the half-life of the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein is at least the half-life of the linear partner. In some embodiments, the half-life of the oRNA is increased compared to the half-life of the linear partner. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In some embodiments, the half-life or persistence of the oRNA in the cell is at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer, or any time in between. In some embodiments, the half-life or persistence of the oRNA in the cell is no more than about 10 min to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein has a half-life or persistence in the cell when the cell is dividing. In some embodiments, the oRNA has a half-life or persistence after cell division.

In certain embodiments, the half-life or persistence of the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein in dividing cells is greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or more or any period in between.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein modulates cellular function, for example transiently or chronically. In certain embodiments, the alteration in cellular function is stable, such as lasting at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer of modulation. In certain embodiments, the cellular function is altered transiently, such as lasting no more than about 30 minutes to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days of modulation.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5, In some embodiments, the oRNA may be of sufficient size to accommodate the binding site of a ribosome.

In some embodiments, the maximum size of the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein may be limited by the ability to package and deliver the RNA to the target. In some embodiments, the size of the oRNA is a length sufficient to encode a polypeptide, and thus, a length of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from each other by a spacer sequence or a linker. In some embodiments, the elements can be separated from each other by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, and at least about 1000 nucleotides.

In some embodiments, one or more elements are adjacent to each other, e.g., lacking a spacing element.

In some embodiments, one or more elements are conformationally flexible. In some embodiments, conformational flexibility is due to the sequence being substantially free of secondary structure.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises a secondary or tertiary structure that accommodates a binding site for ribosomes, translation, or circular translation.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein comprises a specific sequence feature. For example, the oRNA may comprise a specific nucleotide composition. In some such embodiments, the oRNA may comprise one or more purine-rich regions (adenine or guanosine). In some such embodiments, the oRNA may comprise one or more purine-rich regions (adenine or guanosine). In some embodiments, the oRNA may comprise one or more AU-rich regions or elements (ARE). In some embodiments, the oRNA may comprise one or more adenine-rich regions.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more modifications described elsewhere herein.

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein comprises one or more expression sequences and is configured to be continuously expressed in vivo in an individual cell. In some embodiments, the oRNA is configured so that the expression of one or more expression sequences in the cell at a later time point is equal to or higher than that at an earlier time point. In such embodiments, the expression of one or more expression sequences can be maintained at a relatively stable level or can increase over time. The expression of the expression sequence can be relatively stable over an extended period of time. For example, in some cases, expression of one or more expression sequences in a cell does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more. In some cases, in some cases, expression of one or more expression sequences in a cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or more. Regulatory Elements

In some embodiments, the circular RNA payload of the LNP-based nucleobase editing system, RNA therapeutic agent, and pharmaceutical composition described herein comprises one or more regulatory elements. As used herein, a "regulatory element" is a sequence that modifies the expression of an expressed sequence (e.g., a nucleotide sequence encoding a nucleobase editing system or a therapeutic protein, i.e., a cognate coding region (CROI)). The regulatory element may include a sequence located adjacent to the cognate coding region encoded on the circular RNA payload. The regulatory element can be operably linked to a nucleotide sequence of a circular RNA encoding a cognate coding region (e.g., a nucleobase editing system or a therapeutic polypeptide).

In some embodiments, a regulatory element can increase the amount of expression of the relevant coding region encoded in the circular RNA payload, as compared to the amount of expression in the absence of the regulatory element.

In some embodiments, the regulatory element may comprise a sequence that selectively initiates or activates the translation of a cognate coding sequence encoded on the circular RNA payload.

In some embodiments, the regulatory element may comprise a sequence that initiates degradation of an oRNA or an effective load or cargo. Non-limiting examples of sequences that initiate degradation include, but are not limited to, riboswitch aptamers and miRNA binding sites.

In some embodiments, the regulatory element can regulate the translation of the relevant coding region encoded on the oRNA. Regulation can produce an increase (enhancer) or decrease (repressor) in the expression of the relevant coding region. The regulatory element can be located adjacent to the CROI (e.g., on one or both sides of the CROI).

In some embodiments, the translation initiation sequence serves as a regulatory element. In some embodiments, the translation initiation sequence comprises AUG/ATG codons. In some embodiments, the translation initiation sequence comprises any eukaryotic initiation codon, such as but not limited to AUG/ATG, CUG/CTG, GUG/GTG, UUG/TTG, ACG, AUC/ATC, AUU, AAG, AUA/ATA or AGG. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, translation starts at an alternative translation initiation sequence (e.g., a translation initiation sequence other than AUG/ATG codons) under selective conditions (e.g., stress-induced conditions). As a non-limiting example, the translation of a cyclic polyribonucleotide can start at an alternative translation initiation sequence (e.g., ACG). As another non-limiting example, circular polyribonucleotide translation can start at alternative translation initiation sequence CUG/CTG. As another non-limiting example, translation can start at alternative translation initiation sequence GUG/GTG. As another non-limiting example, translation can start at alternative translation initiation sequence of repeating related non-AUG (RAN) sequence, such as comprising short extension of repeat RNA (e.g. CGG, GGGGCC, CAG or CTG).

In some embodiments, the oRNA encodes a polypeptide or peptide and may include a translation initiation sequence. The translation initiation sequence may include, but is not limited to, a start codon, a non-coding start codon, a Kozak sequence, or a Shine-Dalgarno sequence. The translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one or both sides of the relevant coding region).

In some embodiments, the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is in a substantially single-stranded region of the oRNA.

The oRNA may include more than one start codon, such as but not limited to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation may start at the first start codon or may start downstream of the first start codon.

In some embodiments, oRNA can start at a codon (e.g., AUG) that is not the first start codon. The translation of the cyclic polyribonucleotide can start at an alternative translation start sequence, such as, but not limited to, ACG, AGG, AAG, CUG/CTG, GUG/GTG, AUA/ATA, AUU/ATT, UUG/TTG. In some embodiments, translation starts at an alternative translation start sequence under selective conditions (e.g., stress-induced conditions). As a non-limiting example, the translation of oRNA can start at an alternative translation start sequence (e.g., ACG). As another non-limiting example, oRNA translation can start at an alternative translation start sequence CUG/CTG. As another non-limiting example, oRNA translation can start at an alternative translation start sequence GTG/GUG. As another non-limiting example, oRNAs can initiate translation at an alternative translation initiation sequence that includes a short stretch of repetitive RNA (e.g., CGG, GGGGCC, CAG, CTG). IRES sequence

In some embodiments, the oRNA described herein comprises an internal ribosome entry site (IRES) element capable of attaching to a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides. In one embodiment, the IRES element is derived from the DNA of an organism, including but not limited to viruses, mammals, and fruit flies. Such viral DNA can be derived from, but not limited to, microRNA virus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. In one embodiment, the fruit fly DNA from which the IRES element is derived includes, but is not limited to, the Antlerpedia gene from Drosophila melanogaster.

In some embodiments, the IRES element is at least partially derived from a virus, for example, it can be derived from a viral IRES element, such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_1-748, FeLV-Notch2, FMDV_C_type, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_1a_type, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, KBV _IGRpred,LINE-1_ORF1_-101_to_-1,L INE-1_ORF1-302_to_-202, LINE-1_ORF2-138_to_-86, LINE-1_ORF1_-44_to_-1, PSIV_IGR, PV_type 1_Mahoney, PV_type 3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR, or TSV_IGR. In some embodiments, the IRES element is derived at least in part from a cellular IRES, such as AML1/RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, AT1R_var2, AT1R_var3, AT1R_var4, BAG1_p36delta236 nt, BAG1_p36, BCL2, BiP_-222_-3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMR 1. Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, L-myc, LamB1_- 335_-1, LEF1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC, NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSF1, ODC1, p27kip1, 03_128-269, PDGF 2/c-sis, Pim-1, PITSLRE_p58, Rbm3, reaper, Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_373-961, UNR, Ure2, UtrA, VEGF-A-133-1, XIAP_5-464, XIAP_305-466, or YAP1.

In another embodiment, the IRES is an IRES sequence from Coxsackievirus B3 (CVB3), the protein coding region encodes Guassia luciferase (Gluc) and the spacer sequence is poly A-C.

In some embodiments, the IRES, if present, is at least about 50 nucleotides long. In one embodiment, the vector comprises an IRES comprising a native sequence. In one embodiment, the vector comprises an IRES comprising a synthetic sequence.

IRES can serve as the only ribosome binding site, or can serve as one of multiple ribosome binding sites for an mRNA. A polynucleotide molecule containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by the ribosome (e.g., a polycistronic mRNA). When the polynucleotide has an IRES, a second translatable region is further provided as appropriate. Examples of IRES sequences that can be used according to the present disclosure include, but are not limited to, those from microRNA viruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV). Termination element

In some embodiments, oRNA includes one or more relevant coding regions (i.e., also referred to as product expression sequences), which encode relevant polypeptides, including but not limited to ribobase editing systems and therapeutic proteins. In various embodiments, the product expression sequence may or may not have a termination element.

In some embodiments, the oRNA includes one or more product expression sequences that lack termination elements, such that the oRNA is continuously translated.

Excluding the termination element can result in circular translation or continuous expression of the encoded peptide or polypeptide because the ribosomes will not stall or fall off. In such embodiments, circular translation is continuously expressed by the product expression sequence.

In some embodiments, one or more product expression sequences in the oRNA include a termination element. In some embodiments, not all product expression sequences in the oRNA include a termination element. In such cases, when the ribosome encounters the termination element and terminates translation, the product expression sequence can fall off the ribosome .

In some embodiments, once the translation of the oRNA is initiated, the ribosome bound to the oRNA will not disengage from the oRNA before completing at least one round of translation of the oRNA. In some embodiments, the oRNA as described herein is competent for circular translation. In some embodiments, during circular translation, once translation of an oRNA is initiated, a ribosome bound to the oRNA does not dissociate from the oRNA before at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 1500, at least 2000, at least 5000, at least 10000, at least 10 ⁵ , or at least 10 ⁶ rounds of translation of the oRNA are completed.

In some embodiments, circular translation of an oRNA results in the production of a polypeptide resulting from more than one round of translation of the oRNA. In some embodiments, the oRNA comprises a staggered element, and circular translation of the oRNA results in the production of a polypeptide product resulting from a single round of translation of the oRNA or less than a single round of translation.

In one embodiment, the linear RNA can be circularized or concatemerized. In some embodiments, the linear RNA can be circularized in vitro prior to formulation and/or delivery. In some embodiments, the linear RNA can be circularized in cells.

In some embodiments, the cyclization or concatemerization mechanism can occur by at least 3 different pathways: 1) chemical, 2) enzymatic, and 3) ribozyme catalysis. The newly formed 5'-/3'-linkage can be intramolecular or intermolecular.

In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when brought together, form a new covalent bond between the 5' and 3' ends of the molecule. The 5' end may contain an NHS-ester reactive group and the 3' end may contain a 3'-amine terminated nucleotide, such that in an organic solvent, the 3'-amine terminated nucleotide at the 3' end of the synthetic mRNA molecule will undergo nucleophilic attack on the 5'-NHS-ester moiety, thereby forming a new 5'-/3'-amide bond.

In the second pathway, T4 RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule to a 3'-hydroxyl group of a nucleic acid, thereby forming a new phosphodiester bond. In an exemplary reaction, 2 g of nucleic acid molecule is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) at 37°C for 1 hour according to the manufacturer's protocol. The ligation reaction can occur in the presence of a split oligonucleotide capable of base pairing with the juxtaposed 5'- and 3'-regions to assist the enzymatic ligation reaction.

In the third approach, the 5' or 3' end of the cDNA template encodes a ligase ribozyme sequence, so that during in vitro transcription, the resulting nucleic acid molecule may contain an active ribozyme sequence capable of joining the 5' end of a nucleic acid molecule to the 3' end of a nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, a group I intron, hepatitis D virus, a hairpin ribozyme, or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take from 1 to 24 hours at a temperature between 0 and 37°C.

In some embodiments, oRNA is produced by circularization of linear RNA.

In some embodiments, the following elements are operably linked to each other and, in some embodiments, are arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) protein coding or non-coding region, d.) 5' group I intron fragment containing 5' splice site dinucleotide, and e.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells. In some embodiments, the biologically active RNA is, for example, a miRNA sponge or a long non-coding RNA.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) optionally, 5' spacer sequence, d.) optionally, internal ribosome entry site (IRES), e.) protein coding or non-coding region, f.) optionally, 3' spacer sequence, g.) 5' group I intron fragment containing 5' splice site dinucleotide, and h.) 3' homology arm. In certain embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) 5' spacer sequence, d.) internal ribosome entry site (IRES), e.) protein coding or non-coding region, f.) 5' group I intron fragment containing 5' splice site dinucleotide, and g.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) 5' spacer sequence, d.) protein coding or non-coding region, e.) 3' spacer sequence, f.) 5' group I intron fragment containing 5' splice site dinucleotide, and g.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) internal ribosome entry site (IRES), d.) protein coding or non-coding region, e.) 3' spacer sequence, f) 5' group I intron fragment containing 5' splice site dinucleotide, and g.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) protein coding or non-coding region, d.) 3' spacer sequence, e.) 5' group I intron fragment containing 5' splice site dinucleotide, and f.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) 5' spacer sequence, d.) protein coding or non-coding region, e.) 5' group I intron fragment containing 5' splice site dinucleotide, and f.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) internal ribosome entry site (IRES), d.) protein coding or non-coding region, e.) 5' group I intron fragment containing 5' splice site dinucleotide, and f) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In some embodiments, the following elements are operably linked to each other and arranged in the following order: a.) 5' homology arm, b.) 3' group I intron fragment containing 3' splice site dinucleotide, c.) 5' spacer sequence, d.) internal ribosome entry site (IRES), e.) protein coding or non-coding region, f) 3' spacer sequence, g.) 5' group I intron fragment containing 5' splice site dinucleotide, and h.) 3' homology arm. In some embodiments, the vector allows the production of circular RNA that is translatable and/or biologically active inside eukaryotic cells.

In one embodiment, the 3' group I intron fragment and/or the 5' group I intron fragment is from the cyanobacterium Anabaena pre-tRNA-Leu gene or the T4 phage Td gene.

In one embodiment, the 3' group I intron fragment and/or the 5' group I intron fragment is from the cyanobacterium Anabaena pre-tRNA-Leu gene.

In one embodiment, the protein coding region encodes a protein of eukaryotic or prokaryotic origin. In another embodiment, the protein coding region encodes a human protein or a non-human protein. In some embodiments, the protein coding region encodes one or more antibodies. For example, in some embodiments, the protein coding region encodes a human antibody. In one embodiment, the protein coding region encodes a protein selected from the following: hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase (hMUT), CFTR, cancer autoantigens, and additional gene editing enzymes, such as Cpf1, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). In another embodiment, the protein coding region encodes a protein for therapeutic use. In one embodiment, the human antibody encoded by the protein coding region is an anti-HIV antibody. In one embodiment, the antibody encoded by the protein coding region is a bispecific antibody. In one embodiment, the bispecific antibody is specific for CD19 and CD22. In another embodiment, the bispecific antibody is specific for CD3 and CLDN6. In one embodiment, the protein coding region encodes a protein for diagnostic purposes. In one embodiment, the protein coding region encodes Gaussia luciferase (Gluc), firefly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO) or Cas9 endonuclease.

In one embodiment, the 5' homology arm is about 5-50 nucleotides long. In another embodiment, the 5' homology arm is about 9-19 nucleotides long. In some embodiments, the 5' homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides long. In some embodiments, the 5' homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides long. In some embodiments, the 5' homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides long.

In one embodiment, the 3' homology arm is about 5-50 nucleotides long. In another embodiment, the 3' homology arm is about 9-19 nucleotides long. In some embodiments, the 3' homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides long. In some embodiments, the 3' homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides long. In some embodiments, the 3' homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides long.

In one embodiment, the 5' spacer sequence is at least 10 nucleotides long. In another embodiment, the 5' spacer sequence is at least 15 nucleotides long. In another embodiment, the 5' spacer sequence is at least 30 nucleotides long. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the length of the 5' spacer sequence is between 20 and 50 nucleotides. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly A-C sequence.

In one embodiment, the 3' spacer sequence is at least 10 nucleotides long. In another embodiment, the 3' spacer sequence is at least 15 nucleotides long. In another embodiment, the 3' spacer sequence is at least 30 nucleotides long. In some embodiments, the 3' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the 3' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the 3' spacer sequence is between 20 and 50 nucleotides long. In certain embodiments, the 3' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 3' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly AC sequence.

In some embodiments, linear RNAs are circularized or concatemerized using chemical methods to form oRNAs. In some chemical methods, the 5' and 3' ends of a nucleic acid (e.g., a linear RNA) include chemically reactive groups that, when brought together, can form new covalent bonds between the 5' and 3' ends of the molecule. The 5' end can contain an NHS-ester reactive group and the 3' end can contain a 3'-amine terminated nucleotide, such that in an organic solvent, the 3'-amine terminated nucleotide at the 3' end of the linear RNA will undergo nucleophilic attack on the 5'-NHS-ester moiety, thereby forming a new 5'-/3'-amide bond.

In one embodiment, a DNA or RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., a linear RNA) to a 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid), thereby forming a new phosphodiester bond. In an exemplary reaction, the linear RNA is incubated with 1-10 units of T4 RNA ligase at 37C for 1 hour according to the manufacturer's protocol. The ligation reaction can occur in the presence of a linear nucleic acid capable of base pairing with the juxtaposed 5'- and 3'-regions to assist the enzymatic ligation reaction. In one embodiment, the ligation is a splint ligation, in which a single-stranded polynucleotide (the splint, such as a single-stranded RNA) can be designed to hybridize with both ends of the linear RNA so that the two ends can be juxtaposed after hybridization with the single-stranded splint. Thus, splint ligase catalyzes the joining of two juxtaposed ends of a linear RNA to generate oRNA.

In one embodiment, DNA or RNA ligase can be used for the synthesis of oRNA. As a non-limiting example, the ligase can be a circ ligase (circ ligase/circular ligase).

In one embodiment, the 5' or 3' end of the linear RNA may encode a ligase ribozyme sequence, such that during in vitro transcription, the resulting linear RNA includes an active ribozyme sequence capable of ligating the 5' end of the linear RNA to the 3' end of the linear RNA. The ligase ribozyme may be derived from a group I intron, hepatitis D virus, a hairpin ribozyme, or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).

In one embodiment, a linear RNA can be circularized or concatemerized by using at least one non-nucleic acid portion. In one aspect, the at least one non-nucleic acid portion can react with a region or feature near the 5' end and/or near the 3' end of the linear RNA to circularize or concatemerize the linear RNA. In another aspect, the at least one non-nucleic acid portion can be located at the 5' end and/or 3' end of the linear RNA, or connected to the 5' end and/or 3' end of the linear RNA, or located near the 5' end and/or 3' end of the linear RNA. The non-nucleic acid portion considered can be homologous or heterologous. As a non-limiting example, the non-nucleic acid portion can be a linkage, such as a hydrophobic linkage, an ionic linkage, a biodegradable linkage, and/or a cleavable linkage. As another non-limiting example, the non-nucleic acid portion is a binding portion. As another non-limiting example, the non-nucleic acid portion can be an oligonucleotide or peptide portion, such as an aptamer or non-nucleic acid linker described herein.

In one embodiment, a linear RNA may be cyclized or concatemerized due to a non-nucleic acid portion that causes attractive forces between the 5' and 3' ends of the linear RNA, nearby or attached atoms, or molecular surfaces. As non-limiting examples, one or more linear RNAs may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipole bonds, conjugation, hyperconjugation, and antibonding.

In one embodiment, the linear RNA may include a ribozyme RNA sequence near the 5' end and near the 3' end. When the ribozyme RNA sequence is exposed to the remaining portion of the ribozyme, the sequence can be covalently linked to a peptide. In one aspect, the peptides covalently linked to the ribozyme RNA sequence near the 5' end and the 3' end can be attached to each other, resulting in circularization or multimerization of the linear RNA. In another aspect, the peptides covalently linked to the ribozyme RNA near the 5' end and the 3' end can cause the linear RNA to be circularized or multimerized after being attached using various methods known in the art (such as but not limited to protein attachment).

In some embodiments, the linear RNA may include a 5' triphosphate of the nucleic acid, which is converted to a 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (adenosine triphosphate diphosphatase). Alternatively, the 5' triphosphate of the linear RNA may be converted to a 5' monophosphate by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., a thermosensitive phosphatase, a phosphatase, or a calf intestinal phosphatase) to remove all three phosphates; (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., a polynucleotide kinase) that adds a single phosphate.

In some embodiments, RNA can be circularized using the methods described in WO2017222911 and WO2016197121, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, RNA can be circularized, for example, by back splicing of non-mammalian exogenous introns or splint ligation of the 5' and 3' ends of linear RNA. In one embodiment, circular RNA is produced by a recombinant nucleic acid encoding a target RNA to be made circular. As a non-limiting example, the method includes: a) generating a recombinant nucleic acid encoding a target RNA to be made circular, wherein the recombinant nucleic acid comprises, in 5' to 3' order: i) a 3' portion of an exogenous intron comprising a 3' splice site, ii) a nucleic acid sequence encoding the target RNA, and iii) a 5' portion of an exogenous intron comprising a 5' splice site; b) performing transcription, thereby generating RNA from the recombinant nucleic acid; and c) performing splicing of the RNA, thereby circularizing the RNA to generate oRNA.

Although not wishing to be bound by theory, circular RNA produced with foreign introns is recognized by the immune system as "non-self" and triggers an innate immune response. On the other hand, circular RNA produced with endogenous introns is recognized by the immune system as "self" and generally does not elicit an innate immune response, even if it carries exons containing foreign RNA.

Thus, circular RNAs can be generated using endogenous or exogenous introns to control immunological self/non-self distinction as desired. A variety of intron sequences from a variety of organisms and viruses are known, and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA).

Circular RNA can be produced from linear RNA in a variety of ways. In some embodiments, circular RNA is produced from linear RNA by back splicing the downstream 5' splice site (splicing donor) to the upstream 3' splice site (splicing acceptor). Circular RNA can be generated in this way by any non-mammalian splicing method. For example, linear RNA containing various types of introns (including self-splicing group I introns, self-splicing group II introns, spliceosomal introns and tRNA introns) can be circularized. In detail, group I and group II introns have the following advantages: they can be easily used to produce circular RNA in vitro and in vivo because they can perform self-splicing due to their self-catalytic ribozyme activity.

In some embodiments, circular RNA can be generated in vitro from linear RNA by chemical or enzymatic ligation of the 5' and 3' ends of the RNA. Chemical ligation can be performed, for example, using cyanogen bromide (BrCN) or ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) to activate the nucleotide phosphate monoester groups, thereby allowing phosphodiester bond formation. See, for example, Sokolova (1988) FEBS Lett 232: 153-155; Dolinnaya et al. (1991) Nucleic Acids Res., 19:3067-3072; Fedorova (1996) Nucleosides Nucleotides Nucleic Acids 15: 1 137-1 147; incorporated herein by reference. Alternatively, enzymatic ligation can be used to circularize the RNA. Exemplary ligases that can be used include T4 DNA ligase (T4 Dn1), T4 RNA ligase 1 (T4 Rn1 1), and T4 RNA ligase 2 (T4 Rn1 2).

In some embodiments, splint ligation using an oligonucleotide splint that hybridizes to both ends of a linear RNA can be used to bring the ends of a linear RNA together for ligation. The splint can be DNA or RNA, with hybridization orienting the 5'-phosphate and 3'-OH at the RNA termini for ligation. Subsequent ligation can be performed using chemical or enzymatic techniques as described above. For example, enzymatic ligation can be performed using T4 DNA ligase (requires a DNA splint), T4 RNA ligase 1 (requires an RNA splint), or T4 RNA ligase 2 (DNA or RNA splint). If the structure of the hybridized splint-RNA complex interferes with enzyme activity, in some cases chemical ligation (such as with BrCN or EDC) is more effective than enzymatic ligation.

In some embodiments, the oRNA may further comprise an internal ribosome entry site (IRES) operably linked to the RNA sequence encoding the polypeptide. The inclusion of an IRES allows one or more open reading frames to be translated from the circular RNA. The IRES element attracts the eukaryotic ribosome translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161).

In some embodiments, the cyclization efficiency of the cyclization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the cyclization efficiency of the cyclization methods provided herein is at least about 40%. Splicing elements

In some embodiments, the oRNA includes at least one splicing element. The splicing element may be a complete splicing element that can mediate oRNA splicing, or the splicing element may be a residual splicing element from a completed splicing event. For example, in some cases, the splicing element of a linear RNA can mediate a splicing event that causes the linear RNA to be circularized, and the resulting oRNA includes residual splicing elements from such splicing-mediated circularization events. In some cases, the residual splicing element cannot mediate any splicing. In other cases, the residual splicing element can still mediate splicing in certain cases. In some embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the oRNA includes splicing elements adjacent to each expression sequence. In some embodiments, splicing elements flank one or both sides of each expression sequence, thereby resulting in separation of the expression products (e.g., peptides and or polypeptides).

In some embodiments, the oRNA includes an internal splicing element, and when replicated, the splice ends are joined together. Some examples may include mini-introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt), such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in cis sequence elements close to the reverse splicing event (suptable4 enriched motifs), such as 200 bp sequences before (upstream) or after (downstream) the reverse splicing site with flanking exons. In some embodiments, the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include a repetitive sequence from the intronic Alu family. See, e.g., U.S. Patent No. 11,058,706.

In some embodiments, an oRNA can include canonical splice sites flanking the head-to-tail junction of the oRNA.

In some embodiments, the oRNA may include a bulge-helix-bulge motif comprising a 4-basic stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating a characteristic fragment with a terminal 5'-hydroxyl group and a 2', 3'-cyclic phosphate group. Cyclization occurs by nucleophilic attack of the 5'-OH group on the 2', 3'-cyclic phosphate group of the same molecule, forming a 3', 5'-phosphodiester bridge.

In some embodiments, the oRNA may include a sequence that mediates self-joining. Non-limiting examples of sequences that may mediate self-joining include self-circularizing introns, such as 5' and 3' splice junctions; or self-circularizing catalytic introns, such as group I, group II, or group III introns. Non-limiting examples of group I intron self-splicing sequences may include self-splicing arranged intron-exon sequences derived from the T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena. Other Circularization Methods

In some embodiments, linear RNA may include complementary sequences, including repetitive or non-repetitive nucleic acid sequences within or across a single intron. In some embodiments, oRNA includes repetitive nucleic acid sequences. In some embodiments, the repetitive nucleotide sequence includes a poly-CA or poly-UG sequence. In some embodiments, oRNA includes at least one repetitive nucleic acid sequence, which is hybridized with a complementary repetitive nucleic acid sequence in another segment of the oRNA, wherein the hybridized segment forms an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate oRNAs are hybridized to generate a single oRNA, wherein the hybridized segment forms an internal double strand. In some embodiments, complementary sequences are found at the 5' and 3' ends of the linear RNA. In some embodiments, the complementary sequence includes about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired nucleotides.

In some embodiments, chemical methods of cyclization can be used to generate oRNA. Such methods may include, but are not limited to, click chemistry (e.g., alkyne-based and azide-based methods, or bases can be clicked), olefin metathesis, aminophosphoester ligation, hemiamine acetal-imine cross-linking, base modification, and any combination thereof. In some embodiments, enzymatic methods of cyclization can be used to generate oRNA. In some embodiments, a ligase (e.g., DNA or RNA ligase) can be used to generate an oRNA or a complement, an oRNA complement, or a template for an oRNA.

Any circular polynucleotides as taught in, for example, U.S. Provisional Application No. 61/873,010 filed on September 3, 2013 or U.S. Patent No. 10,709,779 can be used herein. The contents of these references are incorporated herein by reference in their entirety. In addition, any of the circular RNAs, methods for preparing circular RNAs, and circular RNA compositions described in the following publications are contemplated herein and are incorporated by reference in their entirety as part of this specification: U.S. Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; U.S. Application Publications US 2022/0177540, US 2021/0371494, US 2022/0090137, US 2019/0345503, and US 2015/0299702; and PCT Application Publication WO 2021/226597, WO 2019/236673, WO 2017/222911, WO 2016/187583, WO 2014/082644 and WO 1997/007825. K. Kit

Also provided are kits comprising an engineered retrotransposons (eg, engineered nucleic acid constructs or engineered nucleic acid-enzyme constructs) as described herein.

In some embodiments, the kit provides engineered retrotranscript constructs or vector systems comprising such retrotranscript constructs. In some embodiments, the engineered retrotranscript constructs included in the kit include heterologous sequences capable of providing nucleic acids encoding proteins of interest or regulatory RNAs to cells, cell barcodes, donor polynucleotides suitable for gene editing ( e.g. , by homology-directed repair (HDR) or recombination-mediated genetic engineering (recombination engineering)), or CRISPR protospacer DNA sequences for molecular recording. Other agents may also be included in the kit, such as transfection agents, host cells, suitable media for culturing cells, buffers, and the like.

In the context of a kit, the agent may be provided in liquid or solid form in any convenient package ( e.g. , a strip pack, a dose pack , etc. ). The agents of the kit may be present in the same or separate containers. The kit may contain any one or more of the components described herein in one or more containers. The components may be prepared aseptically, packaged in a syringe and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have other components prepared aseptically. Alternatively, the kit may include active agents premixed and shipped in a vial, tube, or other container.

The kit may have a variety of forms, such as a blister bag, shrink wrap bag, vacuum sealed bag, sealable thermoformed tray or similar bag or tray form, wherein the accessories are loosely packaged in a bag, one or more tubes, containers, boxes or bags. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be unpacked in an otherwise manner. The kit may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization or other sterilization methods known in the art. Depending on the particular application, the kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (such as gauze) for applying or removing disinfectants, disposable gloves, supports for the agent prior to administration, etc. Some aspects of the disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding various components of the retrotransposon-based editing system described herein.

In addition to the above components, the kit of the present invention may further include (in some embodiments) instructions for practicing the method of the present invention. Such instructions may be present in the kit of the present invention in a variety of forms, one or more of which may be present in the kit. One form in which such instructions may be present is as printed information on a suitable medium or substrate ( e.g. , one or more sheets of paper with printed information on it), in a kit package, a package insert, and the like. Another form of such instructions is a computer-readable medium, such as a disk, a compact disc (CD), a flash drive, and the like, on which information is recorded. Another form in which such instructions may be present is a website address, which can be used to access information from a remote data site via the Internet. In some embodiments, the information provided on the website is regularly updated to provide, for example, the latest information. Written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and may also reflect approval by that agency for manufacture, use or sale for animal administration. As used herein, "promotion" includes all methods of conducting business, including educational methods related to this disclosure, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activities including the sale of drugs, and any advertising or other promotional activities, including any form of written, oral and electronic communications.

Other aspects of the present disclosure provide kits comprising one or more nucleic acid constructs (e.g., one or more mRNA or circular RNA molecules encoding components of a retrotranscript-based genome editing system). In various embodiments, all nucleic acid constructs can be based on RNA molecules, i.e., an "all-RNA system." For example, each component of the editing system may be expressed by an mRNA molecule, which is delivered to a target cell by one or more delivery methods (e.g., LNP delivery). L. Cells

One aspect of the present disclosure provides an isolated host cell comprising one or more compositions described herein, including but not limited to engineered retrotranscripts and/or retrotranscript components, engineered ncRNAs, engineered msDNAs, engineered RTs, nucleic acid molecules encoding engineered retrotranscripts and/or retrotranscript components, and vectors or vector systems encoding engineered retrotranscripts and/or retrotranscript components, and any combination thereof. In some embodiments, the host cell is a prokaryotic cell, an archaeal cell, or a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a mammalian cell, such as a human cell, a non-human cell, or a non-human mammalian cell. In some embodiments, the host cell is an artificial cell or a genetically modified cell. In some embodiments, the host cell is in vitro , such as a tissue culture cell. In some embodiments, the host cell is in a living host organism.

The cell may contain any of the compositions described herein. The recombinant retrotransposons or components thereof are delivered to eukaryotic cells (e.g., mammalian cells, such as human cells) using the methods described herein. In some embodiments, the cell is in vitro (e.g., cultured cells). In some embodiments, the cell is in vivo (e.g., in an individual such as a human individual). In some embodiments, the cell is ex vivo (e.g., isolated from an individual and can be administered back to the same or a different individual).

The present disclosure contemplates the use of any suitable host cell. For example, the cell host can be a mammalian cell. The mammalian cells of the present disclosure include human cells, primate cells (e.g., Vero cells), rat cells (e.g., GH3 cells, OC23 cells), or mouse cells (e.g., MC3T3 cells). There are many human cell lines, including but not limited to human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (neuroglioblastoma) cells, SHSY5Y human neuroblastoma cells (selected from myeloma), and Saos-2 (bone cancer) cells. In some embodiments, the cell may be a human embryonic kidney (HEK) cell (e.g., HEK 293 or HEK 293T cell). In some embodiments, the cell may be a stem cell (e.g., a human stem cell), such as a high-potential stem cell (e.g., a human high-potential stem cell, including human induced high-potential stem cells (hiPSC)). Stem cells refer to cells that can divide indefinitely in culture and produce specialized cells. High-potential stem cells refer to a type of stem cell that can differentiate into all tissues of an organism but cannot sustain the development of the entire organism alone. Human induced high-potential stem cells refer to somatic cells (e.g., mature or adult) that have been reprogrammed into an embryonic stem cell-like state by being forced to express genes and factors that are important for maintaining the defining properties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663–76, 2006, incorporated herein by reference). Human induced high-potential stem cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).

Some aspects of the disclosure provide cells comprising any of the compositions disclosed herein, including but not limited to engineered retrotransposons and/or retrotransposons components, engineered ncRNAs, engineered msDNAs, engineered RTs, nucleic acid molecules encoding engineered retrotransposons and/or retrotransposons components, and vectors or vector systems encoding engineered retrotransposons and/or retrotransposons components, and any combination thereof. In some embodiments, host cells are transiently or non-transiently transfected with one or more of the delivery systems described herein, including viral-based systems, virus-like particle systems, and non-viral-based delivery, including LNPs and liposomes. In some embodiments, cells are transfected as they naturally exist in an individual. In some embodiments, the transfected cells are taken from an individual, i.e., ex vivo transfection. In some embodiments, the cells are derived from cells taken from an individual, such as a cell line. A variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB/3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells , Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI- H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR and their transgenic varieties.

Cell lines are available from a variety of sources known to those skilled in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, cells transfected with one or more of the retrotransposon delivery systems described herein are used to establish new cell lines comprising one or more nucleic acid molecules encoding a recombinant retrotransposon-based gene editing system described herein, or at least a component of such a system (e.g., a recombinant ncRNA or a recombinant retrotransposon RT). M. Pharmaceutical Compositions

The engineered retrotran-based genome editing system described herein or one or more components thereof (e.g., engineered ncRNA, engineered msDNA, engineered RT, nucleic acid molecules encoding engineered retrotran and/or retrotran components, guide RNA, programmable nuclease) can be provided as a pharmaceutical composition. For example, one or more LNPs or other non-viral-based delivery systems comprising one or more circular or linear RNA molecules encoding each component of the retrotran-based genome editing system can be formulated as a pharmaceutical composition for administration to an individual in need (e.g., a human in need of gene editing).

Formulations may include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors ( e.g. , for transfer or transplantation into a subject), and combinations thereof.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or later developed in the field of pharmacology. As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active ingredient and, if appropriate, one or more pharmaceutically acceptable excipients.

Generally, such preparation methods include the step of combining the active ingredient with a substituent and/or one or more other auxiliary ingredients. As used herein, the term "active ingredient" generally refers to an engineered retrotransposon as described herein.

The pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk as a single unit dose and/or as a plurality of single unit doses. As used herein, a "unit dose" refers to an individual amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to an individual, and/or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

Other aspects of the present disclosure are related to pharmaceutical compositions, which include any of the various components of the genome editing system based on recombinant retrotranscripts described herein, including but not limited to engineered retrotranscripts and/or retrotranscript components, engineered ncRNAs, engineered msDNAs, engineered RTs, nucleic acid molecules encoding engineered retrotranscripts and/or retrotranscript components, programmable nucleases (e.g., RNA-guided nucleases), guide RNAs, and vectors or vector systems encoding engineered retrotranscripts and/or retrotranscript components, and any combination thereof. As used herein, the term "pharmaceutical composition" refers to a composition formulated for medical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an additional agent (e.g., for specific delivery, increasing half-life or other therapeutic compounds).

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium stearate, calcium stearate or zinc stearate or stearic acid) or solvent encapsulating material, which material, composition or vehicle participates in carrying or transporting the compound from one part of the body (e.g., a delivery site) to another part (e.g., an organ, tissue or part of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.).

Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils. , such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffering solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. Terms such as "excipients", "carriers", "pharmaceutically acceptable carriers" or similar terms are used interchangeably herein.

In some embodiments, the pharmaceutical composition is formulated for delivery to an individual, for example, for gene editing. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, intragingival, intradental, intraotonic, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

In some embodiments, the pharmaceutical compositions described herein are administered locally to a diseased site (e.g., a tumor site). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, which is a porous, non-porous, or gel-like material, including a membrane (e.g., a sialic acid membrane) or a fiber.

In other embodiments, the pharmaceutical compositions described herein are delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymer material can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed, e.g., in Langer, supra.

In some embodiments, the pharmaceutical composition is formulated as a composition adapted for intravenous or subcutaneous administration to an individual (e.g., human) according to conventional procedures. In some embodiments, the pharmaceutical composition administered by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the medicament may also include a solubilizer and a local anesthetic (such as lignocaine) to reduce pain at the injection site. Generally speaking, the components are supplied separately or mixed together in a unit dosage form, such as a dry lyophilized powder or anhydrous concentrate in an airtight container (such as an ampoule or a sachet) indicating the amount of the active agent. In the case where the medicament is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or physiological saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or physiological saline may be provided so that the ingredients can be mixed prior to administration.

Pharmaceutical compositions for systemic administration may be liquids, such as sterile saline, lactated Ringer's solution or Hanks' solution. In addition, the pharmaceutical compositions may be in solid form and reconstituted or suspended immediately before use. Lyophilized forms are also contemplated.

The pharmaceutical composition may be contained in lipid particles or vesicles (such as liposomes or microcrystals or LNPs), which are also suitable for parenteral administration. The particles may have any suitable structure, such as monolayer or multilayer, as long as the composition is contained therein. The compound may be entrapped in "stabilized plasmid-lipid particles" (SPLPs) containing the fusogenic lipid dioleylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and stabilized by polyethylene glycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or "DOTAP" are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

In addition, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a recombinant retrotransposon-based genome editing system or one or more components thereof in lyophilized form, and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used to reconstitute or dilute the lyophilized system of the present invention. Optionally, associated with such a container may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which reflects the agency's approval of manufacture, use, or sale for human administration.

In another aspect, an article of manufacture containing materials useful for treating the above-mentioned diseases is included. In some embodiments, the article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Such containers can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container contains a composition effective for treating the diseases described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat a selected disease. The article of manufacture may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials that may be desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. N. Uses and Methods of Use

Engineered retrotransposons comprising heterologous nucleic acid sequences can be used in a variety of applications, several non-limiting examples of which are described herein. In general, engineered retrotransposons can be used in any suitable organism. In some embodiments, the organism is a eukaryotic organism.

In some embodiments, the organism is an animal. In some embodiments, the animal is a fish, an amphibian, a reptile, a mammal, or a bird. In some embodiments, the animal is a farm animal or an agricultural animal. Non-limiting examples of farm and agricultural animals include horses, goats, sheep, pigs, cattle, camels, alpacas, and birds, such as chickens, turkeys, ducks, and geese. In some embodiments, the animal is a non-human primate, such as a baboon, a capuchin monkey, a chimpanzee, a lemur, a macaque, a marmoset, a tamarin, a spider monkey, a squirrel monkey, and a vervet monkey. In some embodiments, the animal is a pet. Non-limiting examples of pets include dogs, cats, horses, wolves, rabbits, ferrets, gerbils, hamsters, chinchillas, chinchillas, guinea pigs, canaries, parrots, and parrots.

In some embodiments, the organism is a plant. Plants that can be transfected with engineered retrotransposons include monocots and dicots. Specific examples include, but are not limited to, corn/maize, sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley and rapeseed, sedge, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants and conifers. Vegetables include, but are not limited to crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas and members of the genus Cucurbita (such as cucumbers, cantaloupes and melons). Ornamental plants include but are not limited to azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, dwarf yews, carnations, poinsettias and chrysanthemums.

In some embodiments, heterologous nucleic acid sequences can be added to the engineered retrotranscripts of the present invention to provide cells with heterologous nucleic acids encoding proteins of interest or regulatory RNAs, cellular barcodes, donor polynucleotides suitable for gene editing ( e.g. , by homology-directed repair (HDR) or recombination-mediated genetic engineering (recombineering)), or CRISPR protospacer DNA sequences for molecular recording, as further discussed below. Such heterologous sequences can be inserted, for example, into the msr locus or msd locus, such that the heterologous sequences are transcribed by the retrotranscript reverse transcriptase as part of the msDNA product.

In some embodiments, the engineered retrotransposons described herein can be used in research tools (such as kits), functional genomic analysis, and the generation of engineered cell lines and animal models for research and drug screening. In addition to the engineered retrotransposons, the kits can also include one or more reagents, such as buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, and linkers for sequencing. The buffer can be, for example, a stabilization buffer, a reconstitution buffer, a dilution buffer, a wash buffer, or a buffer for introducing the polypeptides and/or polynucleotides of the kit into cells. In some cases, the kit may include one or more additional reagents specific to plants. The one or more additional reagents for plants may include, for example, soil, nutrients, plants, seeds, spores, Agrobacterium , T-DNA vectors, and pBINAR vectors .

In some embodiments, a retrotranscript is used to perform genome editing at a desired site. A retrotranscript is engineered with a heterologous nucleic acid sequence that encodes a donor polynucleotide suitable for use with a nuclease genome editing system. The nuclease is designed to specifically target a position close to the desired edit (the nuclease should be designed so that once the edit is correctly installed, the nuclease will not cut the target). The nuclease ( e.g. , CAS or non-CAS) is linked to the retrotranscript by direct fusion with RT or by fusion of msDNA with gRNA (only for RNA-guided nucleases). Insert the heterologous nucleic acid sequence into the retrotranscript msd. See, for example, FIG. 3, which shows markers representing edits.

In some embodiments, the heterologous nucleic acid sequence has 10-100 or more bp of nucleic acid sequence homologous to the genome on both sides of the desired edit. The desired edit (insertion, deletion or mutation) is between the homologous sequences.

In some embodiments, the donor polynucleotide includes a sequence comprising an intended genomic edit flanked by a pair of homology arms that are responsible for targeting the donor polynucleotide to a target locus to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm hybridized to the 5' genomic target sequence and a 3' homology arm hybridized to the 3' genomic target sequence. Further homology arms are referred to herein as 5' and 3' ( i.e. , upstream and downstream) homology arms, which refer to the relative positions of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which regions are referred to herein as "5' target sequence" and "3' target sequence," respectively.

The homology arms must be sufficiently complementary to hybridize with the target sequence, thereby mediating homologous recombination between the donor polynucleotide and the genomic DNA at the target locus. For example, the homology arms can comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence (including any percentage of identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto), wherein the nucleotide sequence comprising the desired edit can be integrated into the genomic DNA at the genomic target locus identified by the 5' and 3' homology arms ( i.e. , having sufficient complementarity to hybridize).

In some embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence ( i.e. , "5' target sequence" and "3' target sequence") flank a specific site for cleavage and/or a specific site for introduction of the desired edit. The distance between a specific cleavage site and the homologous nucleotide sequence ( e.g. , each homology arm) can be hundreds of nucleotides. In some embodiments, the distance between the homology arm and the cleavage site is 200 nucleotides or less ( e.g. , 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, smaller distances can produce higher gene targeting rates. In some embodiments, the donor polynucleotide is substantially identical to the target genome sequence throughout its entire length, except for the sequence changes to be introduced into a portion of the genome that includes the specific cleavage site and the portion of the genome target sequence to be altered.

The homology arms can be of any length, e.g., 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some cases, the lengths of the 5' and 3' homology arms are substantially equal to each other. However, in some cases, the lengths of the 5' and 3' homology arms are not necessarily equal to each other. For example, one homology arm may be 30% or less shorter than the other homology arm, 20% or less shorter than the other homology arm, 10% or less shorter than the other homology arm, 5% or less shorter than the other homology arm, 2% or less shorter than the other homology arm, or only a few nucleotides shorter than the other homology arm. In other cases, the lengths of the 5' and 3' homology arms are substantially different from each other, for example , one may be 40% or more shorter, 50% or more shorter, sometimes 60% or more shorter, 70% or more shorter, 80% or more shorter, 90% or more shorter, or 95% or more shorter than the other homology arm.

The donor polynucleotide can be used in combination with an RNA-guided nuclease that targets a specific genome sequence ( i.e. , the genome target sequence to be modified) via a guide RNA. The target-specific guide RNA comprises a nucleotide sequence that is complementary to the genome target sequence, and thereby mediates the binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed to have a sequence that is complementary to the sequence of the minor allele to target the nuclease-gRNA complex to the mutation site. The mutation may comprise an insertion, deletion, or substitution. For example, the mutation may comprise a single nucleotide variation, a gene fusion, a translocation, an inversion, a duplication, a frameshift, a missense, a nonsense, or other mutations associated with a relevant phenotype or disease. The targeted minor allele may be a common genetic variant or a rare genetic variant. In some embodiments, the gRNA is designed to selectively bind to a minor allele with a single base pair distinction, for example, to allow the nuclease-gRNA complex to bind to a single nucleotide polymorphism (SNP). In detail, the gRNA can be designed to target a relevant disease-related mutation for the purpose of performing genome editing to remove the mutation from the gene. Alternatively, the gRNA can be designed with a sequence that is complementary to the sequence of the major or wild-type allele to target the nuclease-gRNA complex to the allele for the purpose of performing genome editing to introduce a mutation (such as an insertion, deletion, or substitution) into a gene in the genomic DNA of the cell. Such genetically modified cells can be used, for example, to change the phenotype, confer new characteristics, or generate disease models for drug screening.

In some embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspaced short palindromic repeat (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease that can catalyze site-specific cleavage of DNA to allow integration of a donor polynucleotide by the HDR mechanism can be used for genome editing, including CRISPR system class 1, type I, type II, or type III Cas nucleases; class 2, type II nucleases (such as Cas9); class 2, type V nucleases (such as Cpf1), or class 2, type VI nucleases (such as C2c2). Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasSH, Csy1, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 and Cul966, and homologs or modified forms thereof.

In some embodiments, a Class 1, Type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity ( i.e. , catalyzing site-directed cleavage of DNA to generate double-strand breaks) can be used to perform genome modification as described herein. Cas9 need not be physically derived from an organism, but can be produced synthetically or recombinantly. Cas9 sequences from a variety of bacterial species are well known in the art and are listed in the National Center for Biotechnology Information (NCBI) database. See, e.g., the NCBI entries for Cas9 from: Streptococcus pyocyaneus (WP 002989955, WP_038434062, WP_011528583); Curvularia jejuni (WP_022552435, YP 002344900), Curvularia flexus (WP 060786116); Curvularia fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheriae (NC_016782, NC_016786); Enterococcus fecalis (WP 033919308); Spiroplasma aphidina (NC 021284); Prevotella intermedia (NC 017861); Taiwan spirochete (NC 021846); Streptococcus dolphinus (NC 021314); Balticella balticola (NC 018010); Psychrobacterium contortus (NC O 18721); Thermophilic Streptococcus ( YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as imported by the filing date of this application) are incorporated herein by reference in their entirety. Any of these sequences or variants thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percentage identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing as described herein. See also Fonfara et al . (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacterid. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105) for sequence comparisons and discussions of Cas9 genetic diversity and phylogeny analysis.

The genomic target site will typically include a nucleotide sequence that is complementary to the gRNA, and may further include a protospacer adjacent motif (PAM). In some embodiments, in addition to 3 or more base pair PAMs, the target site also includes 20-30 base pairs. Typically, the first nucleotide of the PAM can be any nucleotide, and the two or more other nucleotides will depend on the specific Cas9 protein selected. Exemplary PAM sequences are known to those skilled in the art, and include, but are not limited to, NNG, NGN, NAG, and NGG, where N represents any nucleotide. In some embodiments, the allele targeted by the gRNA includes a mutation that produces a PAM within the allele, wherein the PAM promotes the binding of the Cas9-gRNA complex to the allele.

In some embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides long, or any length between the ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. The guide RNA can be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA can comprise two RNA molecules, wherein the crRNA and tracrRNA sequences are present in separate RNA molecules.

In another embodiment, CRISPR nucleases (Cpf1 or Cas12a) from Prevotella and Francisella 1 are used. Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease that has similarity to Cas9 and can be used similarly. Unlike Cas9, Cpf1 does not require tracrRNA and relies only on crRNA in its guide RNA, which provides the following advantages: guide RNAs shorter than Cas9 can be used with Cpf1 for targeting. Cpf1 is capable of cleaving DNA or RNA. The PAM site recognized by Cpf1 has the sequence 5'-YTN-3' (wherein "Y" is pyrimidine and "N" is any nucleobase) or 5'-TTN-3', which is in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double-strand breaks with 4 or 5 nucleotide overhangs at the sticky ends. For a discussion of Cpf1, see , e.g., Ledford et al. (2015) Nature. 526(7571):17-17, Zetsche et al . (2015) Cell. 163(3):759-771, Murovec et al . (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; incorporated herein by reference.

C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that can be used. Similar to Cas9, C2c1 relies on crRNA and tracrRNA to guide to the target site. See , e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; incorporated herein by reference.

In another embodiment, an engineered RNA-guided FokI nuclease can be used. The RNA-guided FokI nuclease comprises a fusion of an inactive Cas9 (dCas9) and a FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting to FokI. For a description of engineered RNA-guided Fold nucleases, see , e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; incorporated herein by reference.

In other embodiments, any other Cas enzymes and variants described in other sections of this application (all incorporated herein) may be similarly used.

In some embodiments, RNA-guided nucleases are provided in the form of proteins, where the nucleases are complexed with gRNA to form ribonucleoprotein (RNP) complexes. In some embodiments, RNA-guided nucleases are provided by nucleic acids encoding RNA-guided nucleases, such as RNA ( e.g. , messenger RNA) or DNA (expression vectors). In some embodiments, RNA-guided nucleases and gRNA are provided by vectors, such as the vectors and vector systems described in other parts of this application (all incorporated herein by reference). Both can be expressed by a single vector, or expressed separately on different vectors. Vectors encoding RNA-guided nucleases and gRNAs can be included in a vector system, which includes engineered retrotransposons msr genes, msd genes, and ret gene sequences. In some embodiments, RNA-guided nucleases are fused with RT and/or msDNA.

The RNP complex can be administered to an individual or delivered to a cell by methods known in the art, such as those described in U.S. Pat. No. 11,390,884 (which is incorporated herein by reference in its entirety). In some embodiments, the endonuclease/gRNA ribonucleoprotein (RNP) complex is delivered to a cell by electroporation. Direct delivery of the RNP complex to an individual or cell eliminates the need for expression from nucleic acid (e.g., transfection of plasmids encoding Cas9 and gRNA). It also eliminates the unwanted integration of DNA segments derived from nucleic acid delivery (e.g., transfection of plasmids encoding Cas9 and gRNA). The endonuclease/gRNA ribonucleoprotein (RNP) complex is typically formed prior to administration.

Codon usage can be optimized to further improve the production of RNA-guided nucleases and/or reverse transcriptases (RTs) in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to replace codons with a higher frequency of usage in yeast cells, bacterial cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other relevant host cell, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase is introduced into a cell, the protein can be expressed transiently, conditionally, or constitutively in the cell.

In some embodiments, the engineered retrotranscript for genome editing using a nuclease genome editing system may further include an auxiliary protein or enhancer protein for recombination. Examples of recombination enhancers may include non-homologous end joining (NHEJ) inhibitors (e.g., DNA ligase IV inhibitors, KU inhibitors ( e.g. , KU70 or KU80), DNA-PKc inhibitors or artemis inhibitors) and homology-directed repair (HDR) promoters or both, which can enhance or improve more precise genome editing and/or homologous recombination efficiency. In some embodiments, the recombination auxiliary or enhancer may include C-terminal binding protein interacting protein (CtIP), cyclinB2, Rad family members ( e.g. , Rad50, Rad51, Rad52, etc.).

CtIP is a transcription factor containing a C2H2 zinc finger that is involved in the early steps of homologous recombination. Mammalian CtIP and its heterologous homologs in other eukaryotes promote the resection of double-stranded DNA breaks and are essential for meiotic recombination. HDR can be enhanced by using a Cas9 nuclease that is conjugated ( e.g. , fused) to the N-terminal domain of CtIP, which forces CtIP to reach the cleavage site and increases transgene integration by HDR. In some embodiments, the N-terminal fragment of CtIP (referred to as HE, an HDR enhancer) may be sufficient for HDR stimulation and requires the CtIP multimerization domain and CDK phosphorylation sites to be active. HDR stimulation caused by Cas9-HE fusion depends on the guide RNA used, and therefore the guide RNA will be designed accordingly.

Using the gene editing system described herein, any target gene or sequence in a host cell can be edited or modified to obtain desired traits, including but not limited to: myostatin ( e.g. , GDF8) to increase muscle growth; Pc POLLED to induce hair removal; KISS1R to induce hole contamination; dead-end protein (dnd) to induce sterility; Nano2 and DDX to induce sterility; CD163 to induce PRRSV resistance; RELA to induce ASFV recovery; CD18 to induce hemolytic Mannheimia (Pasteurella) recovery; NRAMPl to induce tuberculosis recovery; negative regulators of muscle mass ( e.g. , myostatin) to increase muscle mass. Diseases and Conditions

Provided herein are methods for treating a disease or condition, comprising administering to an individual in need thereof a pharmaceutical composition of the disclosure comprising a retrotransposon-based gene editing system and/or components thereof. In various embodiments of the invention, the target genome or epigenetic modification comprises a cell suffering from a single gene disease or condition. Various monogenic diseases include, but are not limited to: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's chorea; maple tree diabetes; Marfan syndrome; neurofibromatosis type 1; pachyonychia congenita; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; Tay-Sachs disease; hereditary tyrosinemia I; influenza; SARS-CoV-2; Alzheimer's disease; Parkinson's disease.

In some cases, target sequences associated with certain diseases and conditions are known. Target sequences or target editing sites include disease-associated mutations or pathogenic mutations for one or more of the 10,000 single-gene disorders. A list of target sequences can be generated based on single-gene disorders. Common genetic disorders that can be corrected by the gene editing system described herein include, but are not limited to: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's chorea; maple diabetes; Marfan syndrome; neurofibromatosis type 1; pachyonychia congenita; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. In other embodiments, the disease-associated gene may be associated with a polygenic disorder selected from the group consisting of: heart disease; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

The gene editing system disclosed herein can also be used to treat the following genetic diseases by editing defects in disease-related genes, as follows: Genetic diseases Disease genes Adrenoleukodystrophy (ALD) ABCD1 Non-Brukton's agammaglobulinemia IGHM Alport syndrome COL4A5 Amyloid neuropathy – Andrade disease TTR Vascular edema C1NH Alpha-1 antitrypsin deficiency SERPINEA 1 Bartter syndrome type 4 BSND Palpebral fissure - ptosis - inverted epicanthus epicanthus (BEPS) FOXL2 Brugada Syndrome- Long QT Syndrome-3 SCN5A Brunton's agammaglobulinemia tyrosine kinase BTK Neuronal waxy lipofuscinosis type 2 CLN2 Charcot Marie Tooth Type 1A (CMT1A) PMP22 Charcot Marie Tooth Type X(CMTX) CMTX Chronic granulomatous disease (CGD) CYBB Cystic Fibrosis (CF) CFTR Congenital adrenal hyperplasia (CAH) CYP21A2 Congenital disorder of glycosylation type Ia (CDG Ia) PMM2 Congenital fibrosis of extraocular muscles 1 (CFEOM1) KIF21A Crigler-Najjar syndrome UGT1A1 Deafness, somatic recessive CX26 Diamond-Blackfan Anemia (DBA) RPS19 Duchenne-Becker Muscle Dystrophy (DMD/DMB) DMD Duncan Disease - X-linked Lymphoproliferative Syndrome (XLPD) SH2D1A Eccentric dysplasia and cleft lip/palate syndrome (EEC) p63 Malnutrition/itchy epidermolysis bullosa COL7A1 Multiple exostoses type 1 (EXT1) EXT1 Multiple exostoses type II (EXT2) EXT2 Facioscapulohumeral atrophy FRG1 Factor VII deficiency F7 Familial Mediterranean fever (FMF) MEFV Fanconi Anemia A FANCA Fanconi Anemia G FANCG Fragility X FRAXA Gangliosidosis (GM1) GLB1 Gaucher disease (GD) GBA Thrombocytopenia ITGA2B Glucose-6-phosphate dehydrogenase deficiency G6PD Glutaric acidemia I GCDH Hemophilia A F8 Hemophilia B F9 Hand-Foot-Uterus Syndrome HOXD13 Familial hemophagocytic lymphohistiocytosis, type 2 (FHL2) PRF1 Primary hypomagnesemia CLDN16 Hypophosphatasia ALPL Holt-Oram syndrome (HOS) TBX5 Homocystinuria MTHFR Incontinence pigmenti NEMO Lesch-Nyhan syndrome HPRT Limb-girdle muscular dystrophy type 2C (LGMD2C) SGCG Long QT Syndrome-1 KCNQ1 Alpha-mannose syndrome MAN2B1 Marfan syndrome FBN1 Methacryluria, β-hydroxyisobutyryl-CoA deacylase deficiency HIBCH Mevalonic aciduria MVK Myotonic muscular dystrophy (DM) DMPK Myotonic dystrophy type 2 (DM2) ZNF9 Mucopolysaccharidosis Type I - Hurler Syndrome IDUA Mucopolysaccharidosis type IIIA-Sanfilippo syndrome A (MPS3A) SGSH Mucopolysaccharidosis type IIIB-Sanfilippo syndrome B (MPS3B) NAGLU Mucopolysaccharidosis Type VI (MPS VI) - Maroteaux-Lamy Syndrome ARSB Neurofibromatosis 1 - Batten's disease (CLN1) PPT1 Niemann-Pick disease SMPD1 Noonan syndrome PTPN11 Hereditary pancreatitis (PCTT) PRSS1 Paramyotonia congenita (PMC) SCN4A Phenylketonuria PAH Polycystic kidney disease type 1 (PKD1) PKD1 Polycystic kidney disease type 2 (PKD2) PKD2 Polycystic kidney and liver disease-1 (ARPKD) PKHD1 Schwartz-Jampel/Stuve-Wiedemann syndrome LIFR Sickle cell anemia HBB Polydactyly (SPD1) HOXA13 Smith-Lemli-Opitz syndrome DHCR7 Spastic paraplegia type 3 SPG3A Spinal Muscular Atrophy (SMA) SMN Spinocerebellar Ataxia 3 (SCA3) ATXN3 Spinocerebellar Ataxia 7 (SCA7) ATXN7 Stargardt disease ABCA4 Tay Sachs (TSD) HEXA Mediterranean anemia-alpha mental retardation syndrome ATRX Mediterranean anemia-β HBB Torsion dystonia, early onset type (EOTD) DYT1 Tyrosinemia type 1 FAH Tuberous sclerosis complex 1 TSC1 Tuberous sclerosis complex 2 TSC2 Wiskott-Aldrich syndrome (WAS) WAS

In addition, the gene editing system disclosed herein can also be used to treat the following genetic diseases by editing defects in a disease-associated gene or more than one gene associated with a particular disease, as follows: A B C D E F Genetic diseases Disease-related genes The most common (B) (C) Coded Product (C) Deposit Number (C) Product Type Adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD CAH) CYP21A2 CYP21A2 (196) Cytochrome P450 family 21 subgroup A member 2 P08686 Enzymes Aicardi-Goutières syndrome encephalopathy ADAR; IFIH1; RNASEH2A; RNASEH2B; RNASEH2C; SAMHD1; TREX1 RNASEH2B (28) RNase H2 subunit B Q5TBB1 Enzymes Alpha-1-antitrypsin (A1AT) deficiency (AATD) SERPINA1 SERPINA1 (83) Serpin family A member 1 P01009 Enzyme inhibitors Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC, ARVD) To date, 13 different genes have been linked to the condition. PKP2 (138) Plaquephilin 2 Q99959 Adhesion proteins in junctions and intermediate filaments. Autosomal dominant polycystic kidney disease (ADPKD) BICC1; GANAB; PKD1; PKD2 PKD1 (1154) Polycystin 1 P98161 Subunit of the ion channel complex Brugada syndrome ventricular fibrillation So far, 22 different genes have been linked to the condition. SCN5A (725) Sodium voltage gate channel α subunit 5 Q14524 Ion channel Catecholamine-induced polymorphic ventricular tachycardia (CPVT) CALM1; CALM2; CALM3; CASQ2; RYR2; TECRL; TRDN RYR2 (288) Ryanodine receptor 2 Q92736 Ion channel Charcot–Marie- ^Tooth disease/hereditary motor and sensory neuropathy To date, 75 different genes have been linked to the condition. PMP22 (63) Myelin protein 22 ^d Q01453 Ill-Myelin and Schwann cells have unclear roles Congenital adrenal hyperplasia (CAH) CYP11B1; CYP17A1; CYP21A2; HSD3B2; POR; STAR CYP21A2 (214) Cytochrome P450 family 21 subgroup A member 2 P08686 Enzymes Congenital sucrase-isomaltase deficiency (CSID) SI SI (23) Sucrase-Isomaltase P14410 Enzymes Congenital bilateral absence of the vas deferens CFTR;ADGRG2 CFTR (120) Cystic fibrosis transmembrane conductance regulator Q20BH0 Ion channel Cystic fibrosis CFTR; CLCA4; DCTN4; STX1A; TGFB1 CFTR (1053) Cystic fibrosis transmembrane conductance regulator Q20BH0 Ion channel Cystinuria-Lysinuria Syndrome/Cystinuria SLC3A1; SLC7A9 SLC7A9 (83) Solute carrier family 7 member 9 P82251 Membrane transporter Congenital adrenal hyperplasia giant cell type (AHC) (a subtype of congenital adrenal hyperplasia) NR0B1 NR0B1 (112) Nuclear receptor subfamily 0 group B member 1 P51843 Nuclear receptor Dentinogenesis Imperfecta (DGI) (all types) DSPP DSPP (11) Dentin sialophosphoprotein Q9NZW4 Seed biomineralization, dentin formation Duchenne Muscular Dystrophy (DMD) DMD;LTBP4 DMD (830) Dystrophin P11532 Structural proteins Abnormal betalipoproteinemia/hyperlipoproteinemia type 3 APOE APOE (42) Apolipoprotein E P02649 Lipoprotein Ehlers-Danlos syndrome COL1A1;COL5A1;COL5A2 COL5A1 (106) Type V collagen α1 chain, type V collagen α2 chain P20908, P05997 Structural proteins Familial Adenomatous Polyposis (FAP) APC; MUTYH APC (539) adenomatous polyposis coli protein P25054 Tumor suppressor factors, regulatory proteins Gardner syndrome (a subtype of familial adenomatous polyposis) APC APC (539) adenomatous polyposis coli protein P25054 Tumor suppressor factor, microtubule-associated Familial cavernous hemangioma CCM2;KRIT1;PDCD10 KRIT1 (80) Krev interacting trap protein 1 O00522 Regulatory proteins Familial Hypocalcemic Hypercalcemia Type 1 (FHH) CASR CASR (373) Calcium sensitive receptor P41180 G protein-coupled receptor Familial hypercholesterolemia APOB; LDLR; LDLRAP1; PCSK LDLR (1254) Low-density lipoprotein receptor P01130 Lipoprotein receptor Familial isolated dilated cardiomyopathy To date, 45 different genes have been linked to the condition. TTN (672) Titin Q8WZ42 Muscle Protein Familial long QT syndrome (LQTS), including Romano-Ward syndrome To date, 19 different genes have been linked to the condition. KCNQ1 (448) Potassium voltage gated channel subfamily Q member 1 P51787 Ion channel Fragile X Syndrome/Martin-bell Syndrome FMR1 FMR1 (7) Fragile X mental retardation 1 Q06787 mRNA biological regulators Glucose-6-phosphate dehydrogenase deficiency G6PD G6PD (218) Glucose-6-phosphate dehydrogenase P11413 Enzymes Glycogen storage disease To date, 27 different genes have been linked to the condition. AGL (117) Glycogen debranching enzyme P35573 Enzymes GM2 gangliosidosis GM2A; HEXA; HEXB HEXA (124) Hexosaminidase subunit alpha P06865 Enzymes Bloody BMP6; HAMP; HFE; HJV; SLC40A1; TFR2 HFE (43) Hereditary hemochromatin Q30201 Binds to transferrin receptor Hemolytic anemia due to erythrocyte pyruvate kinase deficiency PKLR PKLR (237) Pyruvate kinase P30613 Enzymes Hemophilia A and B F8; F9 F8 (1898) Coagulation Factor VIII P00451 Factor IXa cofactor Hemophilia A F8 F8 (3364) Coagulation Factor VIII P00451 Factor IXa cofactor Hemorrhagic telangiectasia/Osler Weder Rendu disease ACVRL1;ENG;GDF2;SMAD4 ENG (187) Endoglin P17813 Regulation of angiogenesis Hereditary angioedema (HAE)/angioneurotic edema ANGPT1; F12; PLG; SERPING1 SERPING1 (252) Serpin family G member 1 P05155 Enzyme inhibitors Hereditary breast and ovarian cancer syndrome To date, 14 different genes have been linked to the condition. BRCA1 (1262) Breast cancer susceptibility protein type 1 P38398 E3 ubiquitin protein ligase Hereditary fructose intolerance/fructoseemia ALDOB ALDOB (32) Aldolase, fructose-bisphosphate B P05062 Enzymes Hereditary xanthinuria/xanthine stone disease MOCOS;XDH MOCOS (8); XDH (17) Molybdenum cofactor sulfatase, xanthine dehydrogenase Q9C5X8, P47989 Enzymes Hypohidrotic ectodermal dysplasia (HED) To date, 10 different genes have been linked to the condition. EDA (199) Exogenous protein A Q92838 Interleukin Iminoglycinuria SLC36A2; SLC6A18; SLC6A19; SLC6A20 SLC36A2 (1) Solute carrier family 36 member 2 Q495M3 Membrane transporter Li-Fraumeni syndrome Sarcoma, Breast Cancer, Leukemia and Adrenal (SBLA) syndrome CDKN2A;CHEK2;MDM2;TP53 TP53 (417) Tumor protein p53 P04637 Tumor suppressors, gene regulators Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) HADHA HADHA (35) Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha P40939 Enzymes Lynch syndrome To date, 11 different genes have been linked to the condition. MSH2 (34) DNA mismatch repair protein Msh2 P43246 DNA repair, DNA binding, ATPase Marfan syndrome FBN1;TGFBR2 FBN1 (1893) Tropofibromin 1 P35555 Structural proteins, extracellular matrix Maternal phenylketonuria/phenylketonuria embryopathy PAH PAH (690) Phenylalanine hydroxylase P00439 Enzymes Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) ACADM ACADM (136) Acyl-CoA dehydrogenase middle chain P11310 Enzymes Mucolipidosis type III (ML3) α/β GNPTAB GNPTAB (68) N-acetylglucosamine 1-phosphotransferase, alpha and beta subunits Q3T906 Enzymes Mucopolysaccharidosis type 4A (MPS4A)/Morquio disease type A GALNS GALNS (269) Galactosamine (N-acetyl)-6-sulfatase P34059 Enzymes Multiple endocrine neoplasia type 2 RET RET (130) Ret proto-oncogene receptor tyrosine kinase P07949 Receptor tyrosine kinase Multiple Epiphyseal Dysplasia (MED) COL2A1; COL9A1; COL9A2; COL9A3/collagen type IX α3 chain; COMP; KIF7; MATN3; SLC26A2 COMP (155) Cartilage oligomeric matrix protein P49747 Structural proteins Neurofibromatosis type 1 (NF1)/Von Recklinghausen disease NF1 NF1 (1208) Neurofibromatosis protein 1 P21359 Regulators of Ras GTPase activity Oculocutaneous albinism (OCA) LRMDA; MC1R; OCA2; SLC24A5; SLC45A2; TYR; TYRP1 TYR (352) Tyrosinase P14679 Enzymes Osteogenesis imperfecta/brittle bone disease So far, 15 different genes have been linked to the condition. COL1A1 (547); COL1A2 (466) Type I collagen alpha 1 chain, type I collagen alpha 2 chain P02452、P08123 Structural proteins Pendred syndrome (PDS)/thyroid deafness FOXI1;KCNJ10;SLC26A4 SLC26A4 (404) Solute carrier family 26 member 4 O43511 Membrane transporter Phenylketonuria (PKU)/phenylalanine hydroxylase deficiency (PAH deficiency) PAH PAH (690) Phenylalanine hydroxylase P00439 Enzymes Proximal Spinal Muscular Atrophy (SMA) NAIP; SMN1; SMN2 SMN1 (47) Motor neuron survival protein Q16637 RNA splicing Retinitis Pigmentosa (RP) To date, 82 different genes have been linked to the condition. RHO (204) Rhodopsin P08100 G protein-coupled receptor XLI (Latent X-linked Scale Infection) STS STS (28) Steroid sulfatase P08842 Enzymes Retinoblastoma (RB bilateral (40% of cases) and unilateral (60% of cases—new mutation) NMYC;RB1 RB1 (292) RB transcriptional co-repressor 1 P06400 Tumor suppressor factor, cell cycle regulator Rett syndrome MECP2 MECP2 (246) Methyl-CpG binding protein 2 P51608 Binds to methylated DNA, gene regulation Sickle cell anemia HBB HBB (433) Heme β subunit P68871 Oxygen carrier Sotos syndrome/gigantism APC2; NSD1; SETD2 NSD1 (228) Nuclear receptor binding SET domain protein 1 Q96L73 Enzymes Stargardt disease/macular fundus ABCA4; CNGB3; ELOVL4; PROM1; PRPH2 ABCA4 (789) ATP binding cassette family A member 4 P78363 Membrane transporter Stickler syndrome/hereditary progressive arthropathy COL11A1; COL2A1; COL11A2; COL9A1; COL9A2; COL9A3; LOXL3 COL2A1 (335) Type II collagen alpha 1 chain P02458 Structural proteins Supravalvular aortic stenosis (SVAS) ELN ELN (25) Elastin P15502 Structural proteins β-Thalassemia HBB HBB (434) Heme B chain P68871 Oxygen carrier Tibial muscle atrophy/ascending myopathy TTN TTN (53) Titin Q8WZ42 Muscle Protein Tuberous sclerosis/Bourneville syndrome TSC1;TSC2 TSC2 (518) Potato protein P49815 Tumor suppressor, regulation of mTORC1 signaling Von-Hippel Lindau disease VHL VHL (218) Von Hippel–Lindau tumor suppressor factor P40337 Tumor suppressor factor, role in the E3 ubiquitin ligase complex Von Willebrand disease VWF VWF (636) Von Willebrand Factor P04275 Collagen binding, partner protein of factor VIII X-linked adrenoleukodystrophy (ALD) ABCD1 ABCD1 (425) ATP-binding cassette family D member 1 P33897 Membrane transporter

Therefore, in order to treat one or more such diseases or disorders, in various aspects of the present invention, one or more targeted polynucleotide sequences associated with certain diseases and disorders (e.g., gene mutations) are contacted with the retrotranscript-based gene editing system disclosed herein; and a guide RNA, wherein the guide RNA comprises a complementary sequence to the targeted polynucleotide sequence.

In some embodiments, the guide RNA guides the programmable nuclease of the retroviral editing system to the target site or the targeted polynucleotide sequence; and optionally forms a ribonucleoprotein complex with the polypeptide and the guide RNA.

Additional therapeutic applications of the genome editing systems disclosed herein include base editing, prime editing, gene insertion and/or deletion.

Diagnostic applications of genome editing systems include probes, diagnostics, and therapeutic diagnostics.

The editing system comprising heterologous nucleic acid sequences can be used in a variety of applications, several non-limiting examples of which are described herein. In general, the editing system can be used in any suitable organism. In some embodiments, the organism is a eukaryotic organism.

In some embodiments, the organism is an animal. In some embodiments, the animal is a fish, an amphibian, a reptile, a mammal, or a bird. In some embodiments, the animal is a farm animal or an agricultural animal. Non-limiting examples of farm and agricultural animals include horses, goats, sheep, pigs, cattle, camels, alpacas, and birds, such as chickens, turkeys, ducks, and geese. In some embodiments, the animal is a non-human primate, such as a baboon, a capuchin monkey, a chimpanzee, a lemur, a macaque, a marmoset, a tamarin, a spider monkey, a squirrel monkey, and a vervet monkey. In some embodiments, the animal is a pet. Non-limiting examples of pets include dogs, cats, horses, rabbits, ferrets, gerbils, hamsters, chinchillas, chinchillas, guinea pigs, canaries, parrots, and parrots.

In some embodiments, the organism is a plant. Plants that can be transfected by the Cas12a editing system include monocots and dicots. Specific examples include, but are not limited to, corn (corn/maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley and rapeseed, sedge, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants and conifers. Vegetables include, but are not limited to, cruciferous vegetables, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the cucurbit family (such as cucumbers, cantaloupes, and melons). Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, dwarf yews, carnations, poinsettias, and chrysanthemums.

In some embodiments, heterologous nucleic acid sequences can be added to the editing systems of the present invention to provide cells with heterologous nucleic acids encoding proteins of interest or regulatory RNAs, cell barcodes, donor polynucleotides suitable for gene editing ( e.g. , by homology-directed repair (HDR) or recombination-mediated genetic engineering (recombineering)), or CRISPR protospacer DNA sequences for molecular recording, as further discussed below. In embodiments involving retrotranscript-based gene editing systems, such heterologous sequences can be inserted, for example, into the msr locus or msd locus, such that the heterologous sequence is transcribed by the retrotranscript reverse transcriptase as part of the msDNA product.

In some embodiments, the editing systems described herein can be used for research tools (such as kits), functional genomic analysis, and the generation of engineered cell lines and animal models for research and drug screening. In addition to the editing system, the kit can also include one or more reagents, such as buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, and linkers for sequencing. The buffer can be, for example, a stabilization buffer, a reconstitution buffer, a dilution buffer, a wash buffer, or a buffer for introducing the polypeptides and/or polynucleotides of the kit into cells. In some cases, the kit may include one or more additional reagents specific to plants. The one or more additional reagents for plants may include, for example, soil, nutrients, plants, seeds, spores, Agrobacterium , T-DNA vectors, and pBINAR vectors. Production of protein or RNA

In some embodiments, single-stranded msDNA generated by the engineered retrotranscripts of the present invention can be used to produce desired products of interest in cells.

In some embodiments, a retrotranscript is engineered with a heterologous sequence encoding a polypeptide of interest to allow production of a polypeptide from the retrotranscript msDNA generated in a cell. The polypeptide of interest may be any type of protein/peptide, including but not limited to an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel or other membrane protein, a hormone, a neuropeptide, an antibody or a cytoskeletal protein, a functional fragment thereof or a related biologically active domain. In some embodiments, the protein is a therapeutic protein, a therapeutic antibody for treating a disease, or a template for fixing a mutation or mutant exon in a genome.

Non-limiting examples of related polypeptides include: growth hormone, insulin-like growth factor (IGF-1), Fat-1, phytase, xylanase, β-glucanase, lysozyme or lysostaphin, histone deacetylase, such as HDAC6, CD163 , etc.

In other embodiments, the retrotranscript is engineered with a heterologous sequence encoding a related RNA to allow RNA to be produced by the retrotranscript in the cell. The related RNA can be any type of RNA, including but not limited to RNA interference (RNAi) nucleic acids or regulatory RNAs, such as but not limited to microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), long noncoding RNA (IncRNA), antisense nucleic acids, and the like. Recombinant Engineering

Recombination (recombination-mediated genetic engineering) can be used to modify chromosomes and episomes in cells, for example, to produce gene replacement, gene knockout, deletion, insertion, inversion or point mutation. Recombination can also be used to modify plasmids or bacterial artificial chromosomes (BACs), for example, to clone genes or insert markers or tags.

The engineered retrotransposons described herein can be used in recombineering applications to provide linear single-stranded or double-stranded DNA for recombination. Homologous recombination can be mediated by phage proteins such as RecE/RecT from the Rac prophage or Redobd from the lambda phage. The linear DNA should have sufficient homology at the 5' and 3' ends to the target DNA molecule present in the cell ( e.g. , plasmid, BAC or chromosome) to allow recombination.

A linear single-stranded or double-stranded DNA molecule ( ie, donor polynucleotide) for recombineering comprises a sequence with the desired edit to be inserted flanked by two homology arms that target the linear DNA molecule to a target site for homologous recombination. Homology arms used for recombination engineering typically range from 13-300 nucleotides in length, or from 20 to 200 nucleotides in length, including any length within this range, such as 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 nucleotides in length. In some embodiments, the homology arms are at least 15, at least 20, at least 30, at least 40, or at least 50 or more nucleotides long. Homology arms in the range of 40-50 nucleotides in length generally have sufficient targeting efficiency for recombination; however, longer homology arms in the range of 150 to 200 bases or more can further improve targeting efficiency. In some embodiments, the 5' homology arm and the 3' homology arm are different in length. For example, a linear DNA may have about 50 bases at the 5' end and about 20 bases at the 3' end that have homology to the region to be targeted.

The phage homologous recombinant protein can be provided to the cell as a protein or by one or more vectors (such as vectors or vector systems) encoding the recombinant protein. In some embodiments, one or more vectors encoding the phage recombinant protein are included in a vector system, which comprises an engineered retrotransposon msr gene, msd gene and/or ret gene sequence. In addition, a variety of bacterial strains containing prophage recombination systems can be used for recombinant engineering, including but not limited to DY380, which contains a defective prophage with the recombination proteins exo, bet, and gam; EL250, which is derived from DY380 and contains, in addition to the recombination genes found in DY380, a tightly controlled arabinose-induced flpe gene (flpe mediates recombination between two consensus frt sites); EL350, which is also derived from DY380 and contains, in addition to the recombination genes found in DY380, a tightly controlled arabinose-induced ere gene ( ere mediates recombination between two consensus loxP sites; SW102, derived from DY380, is designed for BAC recombination engineering using galK positive/negative selection; SW105, derived from EL250, can also be used for galK positive/negative selection, but like EL250, contains an ara-inducible Flpe gene; and SW106, derived from EL350, can be used for galK positive/negative selection, but like EL350, contains an ara-inducible Cre gene. Recombination engineering can be performed by transfecting bacterial cells of such strains with an engineered retrotransposons containing heterologous sequences encoding linear DNA suitable for recombination engineering. For a discussion of recombination engineering systems and protocols, see, for example, Sharan et al. (2009) Nat Protoc. 4(2): 206-223, Zhang et al. (1998) Nature Genetics 20: 123-128, Muyrers et al . (1999) Nucleic Acids Res. 27: 1555-1557, Yu et al. (2000) Proc. Natl. Acad. Sci USA 97 (11): 5978-5983; incorporated herein by reference. Molecular Records

In some embodiments, the heterologous sequence in the engineered retrotranscript construct comprises a synthetic CRISPR protospacer DNA sequence to allow molecular recording. Bacteria and archaea often utilize endogenous CRISPR Cas1-Cas2 systems to track exogenous DNA sequences derived from viral infection by storing short sequences ( i.e., protospacers) that confer sequence-specific resistance to invasive viral nucleic acids in genome-based arrays. These arrays not only preserve the spacer sequence, but also record the order in which the sequence was captured, thereby generating a temporal record of the capture event.

This system can be adapted to record arbitrary DNA sequences into genomic CRISPR arrays in the form of "synthetic protospacers" that are introduced into cells using engineered retrotranscriptors. Engineered retrotranscriptors carrying protospacer sequences can be used to integrate synthetic CRISPR protospacer sequences into specific genomic loci using the CRISPR system Cas1-Cas2 complex. Molecular recording can be used to track certain biological events by generating stable genetic memory tracking codes. See , e.g., Shipman et al. (2016) Science 353(6298): aafl 175 and International Patent Application Publication No. WO/2018/191525; incorporated herein by reference in their entirety.

In some embodiments, a CRISPR-Cas system is used to record specific and arbitrary DNA sequences into the bacterial genome. These DNA sequences can be generated by engineered retrotransposons in cells. For example, engineered retrotransposons can be used to generate protospacers in cells, which are inserted into the CRISPR array in the cell. Cells can be modified to include one or more engineered returners (or vector systems encoding them) that can generate one or more synthetic protospacers in the cell, where these synthetic protospacers are added to the CRISPR array. Records of specified sequences that can be recorded over several days and in a variety of forms can be generated.

In some embodiments, the engineered retrotranscriptome comprises an msd protospacer nucleic acid region or an msr protospacer nucleic acid region. In the case of an msr protospacer nucleic acid region, the protospacer sequence is first incorporated into an msr RNA, which is reverse transcribed into a protospacer DNA. Double-stranded protospacer DNA is produced when two complementary protospacer DNA sequences with complementary sequences are hybridized, or when a double-stranded structure (such as a hairpin) is formed in a single-stranded protospacer DNA ( for example , a single msDNA can form an appropriate hairpin structure to provide a double-stranded DNA protospacer).

In some embodiments, single-stranded DNA generated in vivo by a first engineered retrotranscriptor can hybridize or form a hairpin structure with complementary single-stranded DNA generated in vivo by the same retrotranscriptor or a second engineered retrotranscriptor, and then used as a protospacer sequence for insertion into a CRISPR array as a spacer sequence. The engineered retrotranscriptor should provide sufficient levels of protospacer sequences in cells for incorporation into a CRISPR array. The use of protospacers generated in cells will expand the in vivo molecular recording system from capturing only information known to the user to capturing biological or environmental information that may not have been previously known to the user. For example, an msDNA protospacer sequence in an engineered retrotranscriptor construct may be driven by a promoter downstream of a sensor pathway for a biological phenomenon or environmental toxin. Capture and storage of the protospacer sequence in the CRISPR array records that event. If multiple msDNA protospacers are driven by different promoters, the activity of those promoters (and anything that may be upstream of the promoter) and the relative order of promoter activity (based on the relative position of the spacer sequences in the CRISPR array) are recorded. At any time after the recording occurs, the CRISPR array can be sequenced to determine whether a given biological or environmental event occurred and the order of multiple events given by the presence and relative position of msDNA-derived spacers in the CRISPR array.

In some embodiments, the synthetic protospacer further comprises an AAG PAM sequence at its 5' end. Protospacers comprising a 5' AAG PAM are captured by the CRISPR array with higher efficiency than those protospacers that do not include a PAM sequence.

In some embodiments, Cas1 and Cas2 are provided by a vector that expresses Cas1 and Cas2 at levels sufficient to allow the CRISPR array in the cell to capture the synthetic protospacer sequence produced by the engineered retrotranscript. Such vector systems can be used to perform molecular recording in cells that lack endogenous Cas proteins. Therapeutic Applications

Also provided herein are methods of using the engineered retrotransposons of the invention to diagnose, prognose, treat and/or prevent a disease, condition or disorder in or within an individual.

In general, such methods of diagnosing, prognosing, treating and/or preventing a disease, condition or disorder in or in an individual may comprise using an engineered retrotransposons composition, system or components thereof as described herein to modify polynucleotides in an individual or a cell thereof, and/or may comprise using an engineered retrotransposons composition, system or components thereof as described herein to detect diseased or healthy polynucleotides in an individual or a cell thereof.

In some embodiments, the treatment or prevention method may include using a composition, system or composition of an engineered retrotransposons to modify a polynucleotide of an infectious organism ( e.g. , a bacterium or virus) within an individual or a cell thereof.

In some embodiments, the treatment or prevention method may include using a composition, system or composition of an engineered retrotransposons to modify a polynucleotide of an infectious or commensal organism in an individual.

In some embodiments, engineered retrotran compositions, systems, and compositions can be used to develop models of a disease, condition, or disorder.

In some embodiments, engineered retrotranscript compositions, systems, and compositions may be used to detect disease states or correction thereof, such as by the treatment or prevention methods described herein.

In some embodiments, engineered retrotransposons compositions, systems, and compositions can be used to screen and select cells for use as treatments or preventions, for example, as described herein.

In some embodiments, the compositions, systems, and components thereof can be used to develop bioactive agents that can be used to modify one or more biological functions or activities in an individual or its cells.

In general, the method may include delivering a composition, system and/or component of an engineered retrotransposons to an individual or its cells, or to an infectious or symbiotic organism, by a suitable delivery technology and/or composition. Once administered, the components may be manipulated as described elsewhere herein to induce nucleic acid modification events. In some embodiments, nucleic acid modification events may occur at the genomic, epigenomic and/or transcriptomic level. DNA and/or RNA cleavage, gene activation and/or gene inactivation may occur.

The engineered retrotransposons compositions, systems and compositions as described elsewhere herein can be used to treat and/or prevent disease in an individual, such as a genetic and/or epigenetic disease; to treat and/or prevent a genetic infectious disease in an individual, such as a bacterial infection, a viral infection, a fungal infection, a parasitic infection, and combinations thereof; to alter the composition or pattern of the microbiome within an individual, which in turn can alter the health of the individual; to modify cells ex vivo, which can then be administered to an individual, whereby the modified cells can treat or prevent a disease or a symptom thereof; or to treat a mitochondrial disease, wherein the etiology of the mitochondrial disease involves mutations in mitochondrial DNA.

Also provided is a method of treating an individual ( e.g. , an individual in need thereof), comprising inducing gene editing by transforming the individual with any polynucleotide or vector described herein encoding a polynucleotide or engineered retrotransposon encoding one or more components of the composition, system, or complex, and administering it to the individual.

Also provided is a method of treating an individual ( e.g. , an individual in need thereof), the method comprising transcriptional activation or inhibition of multiple target gene loci by transforming the individual with a polynucleotide or vector described herein, wherein the polynucleotide or vector encodes or comprises one or more components of a composition, system, complex or component of an engineered retrotransposons and comprises multiple Cas effectors.

Also provided is a method of treating an individual ( e.g. , an individual in need thereof), comprising gene editing by transforming the individual with a Cas effector, and a composition, system ( e.g. , RNA, guide), complex or remainder of a component encoding and expressing the engineered retrotranscript in vivo. Suitable repair templates may also be provided by engineered retrotranscripts described elsewhere herein.

Also provided is a method of treating a subject ( eg , a subject in need thereof) comprising activating or inhibiting transcription by transforming the subject with a system or composition herein.

Also provided is a method of inducing modification of one or more polynucleotides in a eukaryotic or prokaryotic cell or component thereof ( e.g. , mitochondria) of an individual, infectious organism, and/or organism of the microbiome of an individual. The modification may include the introduction, deletion, or substitution of one or more nucleotides at a target sequence of a polynucleotide of the one or more cells. The modification may occur in vitro, ex vivo, in situ, or in vivo.

In some embodiments, the method of treating or inhibiting a disorder or disease caused by one or more mutations in a genomic locus in a eukaryotic or non-human organism may include manipulating the coding, non-coding or regulatory elements of the genomic locus in a target sequence in an individual or non-human individual in need thereof, including modifying the individual or non-human individual by manipulating the target sequence and wherein the disorder or disease is susceptible to treatment or inhibition by manipulating the target sequence, including providing a treatment comprising a delivery composition comprising a particle delivery system of any of the above embodiments or a delivery system or a viral particle or a cell of any of the above embodiments.

Also provided herein is a particle delivery system or a delivery system or a viral vector (in a viral particle) of any of the above embodiments or a cell of any of the above embodiments for in vitro or in vivo gene or genome editing; or for use in in vitro, in vitro or in vivo gene therapy.

Also provided herein are particle delivery systems, non-viral delivery systems and/or viral particles of any of the above embodiments or cells of any of the above embodiments for use in the manufacture of a medicament for use in vitro, ex vivo or in vivo gene or genome editing, or in vitro, ex vivo or in vivo gene therapy, or in a method of modifying an organism or a non-human organism by manipulating a target sequence in a genomic locus associated with a disease, or in a method of treating or inhibiting a disorder or disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non-human organism.

In some embodiments, modification of a target polynucleotide using the engineered retrotransposons and related compositions, vectors, systems and methods of the invention comprises adding, deleting or substituting 1 to about 10 k nucleotides at each target sequence of the polynucleotide in the cell(s). Modifications may include adding, deleting, or substituting at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 200, 250, 300, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more nucleotides per target sequence.

In some embodiments, formation of the system or complex results in cleavage, nicking, and/or another modification of one or both strands in or near ( e.g. , within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence.

In some embodiments, a method of modifying a target polynucleotide in a cell to treat or prevent a disease may include binding a composition, system or component of an engineered retrotransposons of the invention to a target polynucleotide, such as to effect cleavage, nicking or another modification of the target polynucleotide by the composition, system or component thereof, thereby modifying the target polynucleotide, wherein the composition, system or component thereof is complexed with a guide sequence, and the guide sequence is hybridized with a target sequence within the target polynucleotide, wherein the guide sequence is optionally linked to a tracr mate sequence, which in turn can hybridize with a tracr sequence. In some embodiments, modification may include cleavage or nicking of one or both strands at the location of the target sequence by one or more of the composition, system or component thereof.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses, and methods of use can be used to treat circulatory diseases. In some embodiments, treatment can be performed by using AAV or lentiviral vectors to deliver engineered retrotransposons, compositions, systems, and/or vectors described herein to modify hematopoietic stem cells (HSCs) or iPSCs in vivo or ex vivo . In some embodiments, treatment can be performed by using the compositions, systems, or components thereof herein to correct disease HSCs or iPSCs, wherein the compositions, systems, as appropriate, include a suitable HDR repair template ( e.g. , a template in the msDNA of an engineered retrotransposons).

In some embodiments, treatment or prevention for treating circulatory or blood diseases may include modifying human cord blood cells. In some embodiments, treatment or prevention for treating circulatory or blood diseases may include modifying granulocyte colony stimulating factor mobilized peripheral blood cells (mPB) with any modification described herein. In some embodiments, human cord blood cells or mPB may be CD34 ⁺ . In some embodiments, the modified cord blood cells or mPB cells are autologous. In some embodiments, the cord blood cells or mPB cells are allogeneic. In addition to modification of disease genes, the compositions and systems described herein can also be used to further modify allogeneic cells to reduce the immunogenicity of such cells when delivered to a recipient. Modified cord blood cells or mPB cells can be expanded in vitro as appropriate. Modified cord blood cells or mPB cells can be introduced into an individual in need using any appropriate delivery technology.

The compositions and systems can be engineered to target one or more genetic loci in HSCs. In some embodiments, the components of the systems can be codon optimized for eukaryotic cells and particularly mammalian cells ( e.g., human cells, such as HSCs or iPSCs) and sgRNAs targeting one or more loci in HSCs (e.g., circulatory diseases) can be prepared. These can be delivered via particles, such as the lipid nanoparticle delivery systems described herein. The particles can be formed by mixing the components of the systems herein.

In some embodiments, after ex vivo modification, HSCs or iPCS can be expanded before administration to an individual. HSCs can be expanded by any suitable method, such as the method described in Lee, "Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4." Blood. 2013 May 16;121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar 21.

In some embodiments, the modified HSC or iPCS is autologous. In some embodiments, the HSC or iPCS is allogeneic. In addition to the modification of disease genes, the compositions and systems described herein can also be used to further modify allogeneic cells to reduce the immunogenicity of the cells when delivered to a recipient.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat neurological diseases. In some embodiments, neurological diseases include brain and CNS diseases.

Delivery options for brain diseases include encapsulating the system in liposomes in the form of DNA or RNA and combining it with a molecular Trojan horse for delivery across the blood-brain barrier (BBB). Molecular Trojan horses have been shown to effectively deliver B-gal expression vectors into the brain of non-human primates. The same method can be used to deliver the vectors or vector systems of the present invention. In other embodiments, artificial viruses can be generated for CNS and/or brain delivery.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat hearing disease or hearing loss in one or both ears. Hearing loss is usually caused by the loss or damage of hair cells, which are unable to transmit signals to auditory neurons. In some embodiments, the composition, system or modified cells may be delivered to one or both ears to treat or prevent hearing disease or loss by any suitable method or technique known in the art, such as US20120328580 ( e.g. , otic administration), by intratympanic injection ( e.g. , into the middle ear) and/or injection into the outer ear, middle ear and/or inner ear; in situ administration via a catheter or pump (US 2006/0030837) and Jacobsen (U.S. Patent No. 7,206,639). See also US20120328580. The cells produced by such methods may then be transplanted or implanted into a patient in need of such treatment.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat diseases of non-dividing cells. Exemplary non-dividing cells include muscle cells or neurons. In such cells, homologous recombination (HR) is generally inhibited during the G1 cell cycle, but can be reversed using art-recognized methods, such as Orthwein et al. (Nature. 2015 Dec 17; 528(7582): 422–426).

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use may be used to treat eye diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat muscle diseases and cardiovascular diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat liver and kidney diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat epithelial and pulmonary diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat skin diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use may be used to treat cancer.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used in transfer cell therapy.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat infectious diseases.

In some embodiments, engineered retrotransposons and related compositions, systems, vectors, uses and methods of use can be used to treat mitochondrial diseases.

All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference. O. Sequence Summary Listing

The following sequence forms part of this specification. SEQ ID NO: describe 1-7257 Table A Sequence (AA) - Retroviral reverse transcriptase amino acid sequence calculated according to Example 3 and tested/evaluated throughout the Examples (sequence included in the sequence listing filed with this specification, identified as RTX009P1_Sequence_Listing_26Jul23.xml) 7258-14498 Table A Sequence (NT) - Nucleotide sequences of reverse transcriptases found computationally according to Example 3 and tested/evaluated throughout the Examples (sequences included in the sequence listing filed with this specification and identified as RTX009P1_Sequence_Listing_26Jul23.xml) 14499-19108 Table B Sequences (NT) - Retrotranscript non-coding RNA (ncRNA) nucleotide sequences found according to Example 3 and tested/evaluated throughout the Examples (sequences included in the sequence listing, which is filed with this specification and identified as RTX009P1_Sequence_Listing_26Jul23.xml) 19109-19125 Example: Selection of RT and ncRNA sequences (sequences included in this manual) 19126-19211 Example: RNA sequence (sequence included in this manual) 19212-19217 Example: Primer sequence (sequence included in this specification) 19218-19221 Example: qPCR primer sequence (sequence included in this manual) 19222-12261 Example: RT variant (coding sequence) (sequence included in this specification) 12262-19364 Example: Plasmid sequence (sequence included in this specification) 19365-19398 Table C - Consensus amino acid sequences of reverse transcriptases for most of the retrotransposons identified in Table A (sequences included in this specification) Sequences disclosed in references Table X - Sequences disclosed in: Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems , Nucleic Acids Research, Vol. 48, No. 22, December 16, 2020, pp. 12632-12647, which is incorporated herein by reference in its entirety. 19399-19401, 19420, 19529-19542 NLS sequence 19402-19415 TALE-related sequences 19416 Large range nuclease 19417 RE cleavage site 19418-19419, 19469-19528 Connector 19421 Kozak sequence 19422-19453 Table Y and related disclosure - Exemplary nucleotide sequences of 5'UTR 19454-19468 Table Z – Additional termination elements for linear mRNA 19543-19926 Table 31A - Optimized ncRNA library 19927-19930 RTX_6342 ncRNA Retrotranscript sequence

This specification discloses and advocates recombinant retrotranscripts and their components (e.g., recombinant ncRNA and recombinant retrotranscript RT), and genome modification systems comprising such recombinant retrotranscripts and/or their recombinant components. Such systems include but are not limited to genome editing systems, recombinant engineering systems, and cell recording systems based on recombinant retrotranscripts. This specification also discloses and advocates various components and aspects constituting such systems, as well as their uses and applications, including but not limited to: (a) recombinant nucleic acid molecules encoding recombinant retrotransposons and retrotransposons-based genome modification systems, (b) vector systems (including viral and non-viral) comprising one or more components of such retrotransposons-based genome modification systems, including all-DNA vector systems, all-RNA vector systems, and DNA/RNA vector systems, (c) delivery systems for delivering such vector systems and/or components of retrotransposons-based genome modification systems (e.g., lipid particles, lipid nanoparticles, and other forms of delivery vehicles). ), (d) formulations comprising any of the foregoing components for delivery to cells and/or tissues, including in vitro, in vivo and ex vivo delivery, (e) cells modified by the recombinant retrotransposons-based genome modification systems and methods described herein, and (f) methods of modifying cells by genome editing, recombinant engineering or cellular transcription using the retrotransposons-based genome modification systems disclosed herein, (g) methods of preparing the recombinant retrotransposons, retrotransposons-based genome modification systems, vectors and formulations described herein, and (h) pharmaceutical compositions and kits for modifying cells in vitro, in vivo and ex vivo.

In various embodiments, the recombinant retrotranscripts and retrotranscript-based genome modification systems described herein can be prepared by introducing one or more modifications into a known retrotranscript, e.g., a retrotranscript that is publicly known or previously published (e.g., Table X) or a novel retrotranscript sequence (Tables A and B, as identified in the Examples). Table X: Previously known retrotranscript reverse transcriptases

Table X provides non-limiting examples of 1928 retrotranscript reverse transcriptases that can be modified according to the methods herein to obtain recombinant retrotranscript reverse transcriptases for use in the compositions, systems, and methods described herein. These retrotranscript sequences are reported as follows: Mestre et al., "Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems," Nucleic Acids Research, Vol. 48, No. 22, December 16, 2020, pp. 12632-12647, the contents of which are incorporated herein by reference. In some embodiments, the retrotranscripts of Table X are intended to be excluded from the scope of the claimed subject matter. Table A with rows A1-A45: Novel Retrotranscript Reverse Transcriptase Sequences

Table A provides non-limiting examples of retrovirus reverse transcriptases that can be modified according to the methods herein to obtain recombinant retrovirus reverse transcriptases for use in the compositions, systems and methods described herein.

In detail, Table A provides sequence identifiers corresponding to novel retrotransposons RT identified as a result of the computational discovery work described in the Examples. The table provides sequence identifiers corresponding to the contents of the sequence listing included as part of this specification. The table includes RT amino acid sequences and RT nucleic acid sequences. The table is organized into forty-five rows, each row representing the sequences that form a single phylogenetic clade of the relevant retrotransposons RT, as determined by the computational work described in Example 3. surface Seq ID No ( Sequence number listed in the sequence list) Phylogenetic clades RT amino acid sequence RT nucleic acid sequence A1 3980-4178 11231-11429 I A A2 4671-4825 11922-12075 I-B1 A3 4980-5143 12229-12392 I-B2 A4 367-368, 427-441, 494-521, 526-527, 536, 626, 649, 660-668, 675, 679, 687-692, 695, 697, 703, 716, 721-722, 751-763, 767, 770-1411, 1456-1462 7624-7625, 7684-7698, 7751-7778, 7783-7784, 7793, 7883, 7906, 7917-7925, 7932, 7936, 7944-7949, 7952, 7954, 7960, 7973, 7978-7979, 8008-8 020, 8024, 8027-8667, 8712-8718 IC A5 1529-1569 8784-8823 ID A6 6697-6701 13943-13947 II A7 4179-4670 11430-11921 II-A1 A8 4884-4909 12134-12159 II-A1 Others A9 6919-6972 14163-14215 II-A2 A10 2786-2866, 2887-2938 10039-10119, 10140-10191 II-A3 A11 4826-4863 12076-12113 II-A4 A12 4864-4875 12114-12125 II-A4 Fusion A13 6974-7002 14217-14244 II-A5 A14 2598-2600, 2759-2785 9851-9853, 10012-10038 III A15 2445-2582 9699-9836 III-A1 A16 1983-2158 9237-9412 III-A2 A17 1612-1982 8866-9236 III-A3 A18 2601-2678 9854-9931 III-A4 A19 2679-2758 9932-10011 III-A5 A20 3442-3603 10694-10855 IV A21 3604-3708 10856-10959 V A22 2939-3441, 3709-3979, 5177-5192 10192-10693, 10960-11230, 12426-12441 VI A23 7003-7033 14245-14275 VII-A1 A24 7054-7133 14296-14374 VII-A2 A25 7034-7049 14276-14291 VIII A26 6835-6918 14079-14162 IX A27 6823-6834 14068-14078 X A28 298-366, 369-373, 442-493, 522-525, 528-535, 537, 551-554, 557, 560-625, 672-674, 680-681, 684-686, 696, 698, 702, 723-742, 764-766, 1412- 1450, 1452-1453, 1463-1466, 1571-1577 7555-7623, 2626-7630, 7699-7750, 7785-7792, 7794, 7808-7811, 7814, 7817-7882, 7929-7931, 7937-7938, 7941-7943, 7953, 7955, 7959, 7980-7 999, 8021-8023, 8668-8706, 8708-8709, 8719-8722, 8825-8831 XI A29 374-426, 539-550, 555-556, 558-559, 671, 682-683, 743, 745-750 7631-7683, 7796-7807, 7812-7813, 7815-7816, 7928, 7939-7940, 800, 8002-8007 XII A30 5942-6665 13189-13911 XIII A31 1-297, 715, 1580-1603 7258-7554 , 7972, 8834-8857 XIV A32 705-714 7962-7971 XV A33 6681-6694 13927-13940 XVI A34 6788-6803 14033-14048 XVII A35 1469-1526, 5147-5151 8725-8781, 12396-12400 CRISPR-related A36 2159-2428 9413-9682 Ec107-like A37 646-648 7903-7905 RT-ATPase A38 2592-2595 9846-9849 RT-DUF4116 A39 676-678, 717-720 7933-7935, 7974-7977 RT-HTH A40 538, 669, 704, 1454 7795, 7926, 7961, 8710 RT-pddex A41 670, 699-701 7927, 7956-7958 RT-unk A42 4917-4979 12167-12228 Bacteriophage A43 4910-4916 12160-12166 Giant phage A44 5195-5941 12444-13188 Outgroup A45 627-645, 650-659, 693-694, 744, 768-769, 1451, 1455, 1467-1468, 1527-1528, 1570, 1578, 1579, 1604-1611, 2429-2444, 2583-2591, 2596-2597, 2 867-2886, 4876-4883, 5144-5146, 5152-5176, 5193-5194, 6666-6680, 6695-6696, 6702-6787, 6804-6822, 6973, 7050-7053, 7134-7257 9 850, 10120- 10139, 12126-12133, 12393-12395, 12401-12425, 12442-12443, 13912-13926, 13941-13942, 13948-14032, 14049-14067, 14216, 14292-14295, 1 4375-14498 Uncategorized Table B with rows B1-B45: Exemplary ncRNA sequences

Table B provides non-limiting examples of retrotransposons ncRNA sequences that can be modified according to the methods herein to obtain recombinant retrotransposons ncRNA sequences for use in the compositions, systems and methods described herein. These sequences were discovered by the methods of Example 3.

In detail, Table B provides sequence identifiers corresponding to novel retrotranscript RTs identified as a result of the computational discovery work described in the Examples. The table provides sequence identifiers corresponding to the contents of the sequence listing included as part of this specification. The table is organized into forty-five rows, each row representing sequences that form a single phylogenetic clade of related retrotranscript ncRNAs, as determined by the computational work described in Example 3.

In various embodiments and claims, the present disclosure provides a genome editing system based on recombinant retrotransposons, which includes combining retrotransposons RT and ncDNA together in cells by various delivery strategies. In various aspects, the retrotransposons RT and ncDNA that constitute the genome editing system based on recombinant retrotransposons can be based on pairing such components together, which are naturally found in nature in association with each other, i.e., derived from the same bacterial species. These are referred to as "homologous" pairing of retrotransposons RT and retrotransposons ncRNA. By identifying that the ncRNA sequences in Table B and the RT sequences in Table A have the same genome accession number indicated for each sequence in the sequence listing , the homologous pairing of ncRNA and RT (amino acid or nucleotide sequence) can be easily identified from the information presented/included in the sequence listing. In various other aspects, the retrotranscript RT component and the ncRNA component can be from different bacterial species, that is, not found together as a homologous pair in nature. In other embodiments, the retrotranscript RT component and the ncRNA component can be from the same lineage at the same time to generate functional types (e.g., type IA, type I-B1, type I-B2, type IC, etc.). For example, a recombinant retrotranscript-based genome editing system may comprise a retrotranscript RT from type IA (i.e., SEQ ID Nos: 3980-4178 for AA and SEQ ID Nos: 11231-11429 for NT - see Table A) and a retrotranscript ncRNA also from type IA (i.e., SEQ ID Nos: 16886-17078 - see Table B). surface Seq ID No ( Sequence number listed in the sequence list) Phylogenetic clades B1 16886-17078 I A B2 17478-17622 I-B1 B3 17677-17756 I-B2 B4 14831-14833, 14838, 14847, 14850-15460 IC B5 N/A 1D B6 N/A II B7 17079-17477 II-A1 B8 17660-17676 II-A1 Others B9 19031-19080 II-A2 B10 16414-16516 II-A3 B11 17623-17659 II-A4 B12 N/A II-A4 Fusion B13 19081-19108 II-A5 B14 16397-16413 III B15 16195-16320 III-A1 B16 15779-15925 III-A2 B17 15476-15778 III-A3 B18 16321-16366 III-A4 B19 16367-16396 III-A5 B20 16705-16814 IV B21 16815-16885 V B22 16517-16704 VI B23 N/A VII-A1 B24 N/A VII-A2 B25 N/A VIII B26 18949-19030 IX B27 N/A X B28 14657-14716, 14778-14824, 14834, 14835-14836, 14839, 15461-15475 XI B29 14717-14777, 14841-14846 XII B30 18413-18936 XIII B31 14499 -14656 XIV B32 N/A XV B33 18939 XVI B34 N/A XVII B35 N/A CRISPR-related B36 15926-16178 Ec107-like B37 N/A RT-ATPase B38 N/A RT-DUF4116 B39 N/A RT-pddex B40 N/A RT-HTH B41 14837 RT-unk B42 N/A Bacteriophage B43 N/A Giant phage B44 17757-18412 Outgroup B45 14825-14830, 14840, 14848-14849, 16179-16194, 18937-18938, 18940-18948 Uncategorized Table C: Consensus RT amino acid sequences

Table C provides the consensus amino acid sequences for most of the RT germline occurrence types identified in Table A. RT type (based on phylogenetic clades) Consensus amino acid sequence SEQ ID NO: Type IA YKVYXIPKRXXGXRXIAXPXXXLKXXQXXXXXXXXXXXXXHXXXXAYXXXXXXIKXNAXXHXXXXYXLKXDXXXFFNSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXFWXXXXXXXXXXLXLSXGAPSSPXXSNXXMXXFDXXXXXXCXXXXXXYXRYADDXTFSTXXXXXLXXXPXXXXXXLXXXXXXXXXXNXXKTXFSSKAHNRH XTGXTXXNXXXXSXGRXXKRXIXXLXXXXXXX 19365 Type IB1 XXXXXXXXXXXXXXXXXXLKXXXXFXXXXXXXXXXXXXXXVXSYRKGXXXXXAVXXHXXXXXFXXXDXXXFFXSIXXXXXXXXXXXXXXXXP XXXXXXKXXXEXXXXXXXXX 19366 Type IB2 XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXRXDIXXFFXSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXP XXXXXXXXXXXXXX 19367 IC Type YXXFXIXKXXGXXRXIXAPXXXLKXXQXXLXXXLXXXXXXXXXXXXXXXXHXFXXXXXIXXNAXXHXXXXXVXNXDLXXFFXXXXFGRVXGXFXXXXXFXXXXXXAXXXAQXXCXXXXLPQGXPXSPXIXNXIXXXLDXXXXXXAXXXXXXYXRYADDXTFSTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXKTRXXXXXX RQXVTGLXVNXXXNXXXXYXXXXRXXXXXXXXX 19368 ID Type YXXXXXXKKXGGXRXIXXPXXXLXXXQXXLXXXLXXXYXXXXXXXXXGFXXXXXXXXXXXXIXXNAXXHXXKXXXLNXDXXXFFXSIXXXXXXXXXXXXFXXXXXXAXXXXLLXTXXXXLPXGAPXSPXXSNXXCXXXDXXLXXXXXXXXXXYXRYADLTFSXXXXXXXXXXXXXXXXIXXXXFXXNXKKXRXXXXXXXQX VTGXXVNXKXNXXRXXXXXXRAXXHXXXXX 19369 Type IIA1 YKXXXIXKXXGXXRXIXXPXXXXKXXQXXXXXXXXXXXXXHXXAXAYXXXXXIXXNAXXHXXXXXXXXXXDFXXFFXSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXLXIGXPXSPXXSNXXXXXXDXXXXXXXXXXXXXYXRYADDXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXNXXKTXXXXXXXXXXXTGXXXXXXXXXXX GRXXKRXXXXXXXXXXXX 19370 ID Type YXXXXXXKKXGGXRXIXXPXXXLXXXQXXLXXXLXXXYXXXXXXXXXGFXXXXXXXXXXXXIXXNAXXHXXKXXXLNXDXXXFFXSIXXXXXXXXXXXXFXXXXXXAXXXXLLXTXXXXLPXGAPXSPXXSNXXCXXXDXXLXXXXXXXXXXYXRYADLTFSXXXXXXXXXXXXXXXXIXXXXFXXNXKKXRXXXXXXXQX VTGXXVNXKXNXXRXXXXXXRAXXHXXXXX 19371 Type IIA1 YXXXXXXXXXXXXRXXXXPXXXLKXXQXWXXXXXXXXXXXXXXXXAYXXXXSXXXXAXXHXXXXXXXXXDIXXFFXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXYXXXXX 19372 Type IIA2 YRXFXXXKXXGXXRXIXXPRXFXKXXQXXXXDXXLXXLXXHXXXXXXXXXXSXXXNAXXHXXXXXXXXXXDIXXXFXXIXXXXXXXXXXXXXXXXXXXXXXXXTXXXXLPQGAPTSPXXSNXXLXXFDXXXXXXXXXXXXXYXRYXDDXTXSXXXXXXXXXXXXXXXXXLXXXXXXXNXXKXRXXXXXXXQXVTGXXXNXXXXPX RXXRXXXRAXXXXAXX 19373 Type IIA3 YXXXXXXKXXXXXRXIXXPXXXLKXXQXWILXXILXXXXXSXXXXXFXXXXXXXXNAXXHXXXXXXLXXDXXXFFXXXXXXXXVXXXFXXXGYXXXXXXXLXXXCXXXXXLPQGXXXSPXXXNLXXXXLDXRXXXXXXXXXXXYTRYADDXXXSXXXXXXXXXXXXXXXXIXXXEXXXXNXXKXXXXXXXXXXXXTGXXXXXXXXXXXXX XXXXRXXXXXXXX 19374 Type IIA4 XXXXXIXXXXXXRKIXTXXXXXXXXXXHXXXXXXXXXXXXXXXXFXKAYXXXXSIXXNAXXHMYNDXFXXXDIXXFFXXIXHXXLXXXLXXXXXXXXXXXXXXXXXXXXXXXXXXXXGLXXGXXXSPXLXNXYXKXFDXIXYGXLKXXXXXXXIYTRYADDXXISFKXXXXXXXXXXXIXXXXXXXLXXXXLXXNXXKXXXXXX XXSNHVXITGXXIXXXXXXXRXXXVGXXXXXXLXXXAXXXXXX 19375 Type IIA4 xxxxxxxxxxxxK XXXXXXXXXVGXXXXXXXXXXXXXXXXXX 19376 Type IIA5 YRXFXXXKXXGXXRXIXXPXTYLKVXQWWIIXDXIXXXXXXXXXXXGFXXGXXXXXNAXXHXXXXXXLNXDXXXFFXSXXXXXXXXXFXXXGXXXXXXXXLXXLXXXXXXXPXGAPTSPXXXNXXXXXXDXXLXXXXXXXXXXYXRYADDXTFSXXXXXXXXXXXXXXXXXXXXXXGFXXXXXKTXXXGXXXRXXVTGXXXXXXXXXXXXX RXXXRXXXHXXXXX 19377 Type IIIA1 YRXXXIXKXXGXXRXIXEPLPXLKXIQXWILXXILXXXXXSXXAKAXXXXXXXXXNXXXHXXXXXXXXXXDXXXFFXXIXXXXXXXXFXXXGYXXXXXXXLXXLCXXXXXLPQGAPTSPXLSNXXXXXXDXXXXXXXXXXXXXYTRYADDXXFSGXXXXXXXXXXXXXXXXXXXXXXNXXKXXXXXXXXXQXVTGXVVNXKXQX XXXXRXXXRXXXXXIXK 19378 Type IIIA2 YRXFXIXKXXG XXXXXXXXXXXX 19379 Type IIIA3 YXXXXXXKXXXXXRXIXXPXXXLXXXQXXIXXXXLXXXXXHXXXXAXXXXXXXXXXAXXHXXXXWXXKXDXXXFFXXXXEXXXXXXFXXXGYXXLXXXEXARXCTXXXXXXXXXXXXXXXXXXXXXXXXXXXGXLPQGAPTSXXLXNLXXXXXDXXXXXXAXXXXXXYTRYXDDXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXG XXXXXXKXXXXXPGXXXXVLGLXVXXXXXXLXXXXXXXXXXHXXXXXXX 19380 Type IIIA4 YXXXXXXKXXGGXRXIXXPXXXLXXXQXWIXXNILXXXXXXXXXXGFXXXXSIXXNAXXHXXXXXXLXXDLXXFXXXIXXXXXXXXFXXXGYXXXXXXXXAXXXTXXXXXXXXXXXXXXXXXXXXLPQGAPXSPXXXNXXXXXXDXRXXXXXXXXXXXYXRYADDXTFSXXXXXXXXXXXXXXIXXXEXXXXNXXKXXXXXXXXXXXXXVTGLXXXXX XXXXXXXXXXXXXXXXXXCXK 19381 Type IIIA5 YXXXXIXKXXGXXRXXXXPXXXLKXXQXWILXXILXXXXXXXXXXGFXXXXSIXXNAXXHXXXXXXXXXDXXXFFPXIXXXXXXXXFXXXGYXXXXXXXXXXXCTXXXXLPQGXPXSPXXXNXXXXXXDXRXXXXXXXXXXXYXRYADDXTXSGXXXXXXXXXXXXXIXXXXXXXXNXXKXXXXXXXXXQXVTGXXVNXXXXXXXXXX XXXXXXIYXXXKX 19382 IIIunk YXXXXXXXXXXXXRXXXXPXXXLKXXQXWIXXNILXXXXXXXXXXXXXXXXSIXXNAXXHXXXXXXXXXDIXXFFXSIXXXXXXXXFXXXXXXXXXXXXXXXXXXXXXXXLXQGXPXSPXXXNXXXXXXXXXXXXXXXXXXXXYXRYADDXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXNXXKTXXXXXXXXXXXTGXXXXXXXXXXXXXXXXXXXX CXX 19383 Type IV YXXXXXXKXXGXXRXXXXPXXXXRXXQXRINXRIFXXXXXWPXXXXGSXPXXXXXXXXXXXDYXXCAXXHCXXKXXLKXDIXXFFXNXXXXXVXXXFXXXXXXXXXXXXXLXXXCXXXXXXXQGXXTSSYXAXLXLXXXEXXXXXXXXXKXLXYTRXVDDITXSSXXXXXXFXXXXXXXXXMLXXXXLP XVHGLRXXXXXPRXPXXEXXXIRXXVXXXXXX 19384 V-type YXXXXXXXXXXXXKXRXXXXPXXXLKXXQKRINXXIFXXXXXPXYLXGGXXXXXXXRDYXXNXXXHXXXXXXIXLDXXXFYXXIXXXXVXXXXXXXXXFXXXVXXXLXXLXTXXXXXPQGXCTSSYXANLXXXXXEYXXXXXXXXXXXXYXRLLDDXTXSXXXXXXXXXXXXXIXXXXXXXXXXXLXXXXXKXXXXXXXXXXXX XXVTGLWXXXXXPXXXXXXRXXIRXXVXXCXXX 19385 Type VI XXXXXXXXXXXXXXRXXXXXXXXLXXXXXXXXXXXXXXXXPXXXXXXXXXXXXNAXXHXXXXXXXXXXDXXXFXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXKXXXXXXXXXXXXTGXXXXXXXXXXXXXXXXXXXXXXXXXXXX 19386 Type VIIA1 XXXXXXXXXXXXXXRXVWEXXXXXXXXXKXXXRXXXXFXXXXXXXXPHXXXXGYXXGRXXRXNAXXHXGXXXXXXXXDXXXFFPSIXXXRXXXXXXXXGXXXXXXXXLXXFXTIXXXLPLGLXXSPXXXNXXXXXXDXXLXXLAXXXXXXYXRYXDDXXXSXXXXXPXXXXXXXXXXXXXFXXXXXKXXXSKXGQXHXVTGLSXX XXXXPHXPRXXKXXLRQELXXXXXX 19387 Type VIIA2 XXXXXXXXXXXXRXXXXXXXXXXXXXKXXXXXXXXXXXXXXXXGFXXXXXXXXNAXXHXXXXXXXXXXDXXXFFXXIXXXXXXXXXXXXXXXXXXXXXXXXXXTXXXXLXXGXXTSPXXXNXXXXXXDXXXXXXXXXXXXXXRYXDDXXFSXXXXXXXXXXXXXXXXXXXXXXXXNXXKXXXXXXGXXQXVTGLXXXXXXXPRXXXXXXXKXXXRXXXXXXXX 19388 Type VIII YXXXXXXKRXXXXXGEXRXVXXAXXXXXXXXHRXXXXXXXXXXXFGXHVQGFXXXRSXXXNAXXHXXXXXXXHADIXXFFXXITXXQVXXXXXXXXXXXXXXAXXXAXXCTIDGXLXQGTRCSPXXXNXVCXXXDXXXLXLAXXXXXXXXRYADXXTFSGXXXXXXXXXXXXXXXXXXGFXLRXXXCYXQXXGXXQXVTGLXVXXXXXPRLPK XXKXXLRLXXXXXXKX 19389 Type IX YRXFXIXXXXXXXRXIXAPXVXLKXXQXWXXXXXXXXXXXXXXV GYRNXXRAXXHXXXXX 19390 X-Type YXXXPXXXXXXXXRWIEAPXXXLKXXQRXXLXXXXYXXXXXXXAHGFXXGRSIXXNAXXHXGXXXVVXXDXXXFFPXXXXXXXXXXXXXXXXXXXXXXXXLXXXXXXLPQGAPTSPXLXNLVXXXXDXXLXXXAXXXXXXYTRYADDLXFSXXXXXXXXXXXXXXXXXXIXXXXGXXXXXXKXXXXXXXQRQXVTGXVVNXX XXLPXXXRRXLRAXXXXXXXX 19391 Type XI YXXFXIXKXXGXXRXIXXPXXXLKXXQXXXXXXLXXXXXXXXXXXGFXXXXSXXXNAXXHXXXXXXXNXDLXXFFXXIXXXRXXXXXXXXXXXXXXXXAXXXXXXXXXLPQGAPXSPXXSNXXXXXXDXXLXXXAXXXXXXYTRYADDXTXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXFXXNXXKXXXXXXXXXXXVTGXXXNX XXNXXRXXXXXXXXXXXXXXXX 19392 Type XII YXXFXXXKXXGGXRXIXAPXXXLXXXQXXXXXXXXXXXXXXXXAHGFXXXXSXXXNAXXHXXXXXXXXDXXXFFPXXXXXRVXGXFXXXGYXXXXAXXXAXXXTXXXXXXXXXXXXXXXXXXXXRXLPQGAXXSPXXXNXXXXXLDXRLXXXAXXXXXXYTRYADDXTFSXXXXXXXXXXXXXXXXXXXXXXEGFXXXXXKXXXXXXXXXQ XVTGXXVNXXXXXXRXXXXXXRAXXXXXXXX 19393 Type XIII YXXFXXPKXXGGXRXIXAPXXXLXXXQXXXLXXXXXXXXXXXXAHGFXXXXSXXXNAXXHXXXXXXXXXXDXXXFFPXXXXXRVXGXFXXXGYXXXXAXXXXLXXTXXXXXXXXXXXXXXXXXXXXRXLPQGAXXSPXXXNXXXXXLDXRLXXXAXXXGXXYTRYADDLTFSXXXXXXXXXXXXXXXXXXXXXXEGFXXXXXKXXXXRXX XXQXVTGXVVNXXXXXXRXXXXXXRAXXXXXXXX 19394 Type XIV YXXFXIXKKXGXXRXIXXPXXXLXXXXXXXXXXXXXXXXXXXXGFXXXXSXXXNAXXHXXXXXVXNXDLXDFFXSXXXXRXXXXXXXXPXXXXXXXXAXXXXXLCXXXXXXXXXXXXXLPQGXPXSPXXXNXXCXXLDXXLXXXAXXXXXXYXRYADDXTFSXXXXXXXXXXXFXXXXXXIXXXXXXXXNXXKTRXXXXXXRQ EVTGXXVXXXXNVXXXYXXXXRXXLXXWXXX 19395 Type XV YXXFXXXKKSGGXRXIXXPXKSLXIXQXKLSQXLYXXYXPXXXVHGXXXXXSIXTNAXXHXXKXFXLNXDIXDFFXSINXGRVRGXFIAXPYXLXXXVATXXAXICCXXNKLPQGAPXSPIXSNLICXXXDXELQXFAXXXXXXYTRYADDITXSXXXXXLPXXLXXXXXXXXXXXXLGXELXXIIXXNGFXINXXKXRL XYXXQXQXVTGLXVNXXVNVXRKYIRNXXXXLHAWEKX 19396 Type XVI YXXFXXXKXXGXXRXIXAPXXXLKXXQXXILXXXLXXVXLXXXAXGFRXXRSIXTNAXXHXXXXXXXKXDXKXFFPSXXXXRVXGXXXXLGYPXXXXXXLTXLXTXXXXLPXGAPTSPXXXNXXXXRXDXRXXXLXXKXXFXYSRYADDXXXSSXXXXXXXXIPFFXXIXXXEGFXXNEXKXXIXRXGXRQXXTGXVVNX KXNXXXXEXXXLRAVXXNCXXX 19397 Type XVII YRXFXXXKXDGXXRXXXXPXXXLKXXQXXXXXXXLXXXXXHPXAXXFXXXXSXXXXAXXHAXXXXXXTXDXXDFFXXTXXXRVXXXXXXXXXXXXXXXXLXXLXXXXXXLPQGAPTSPXLSNXVNXXXDXXXXXXXXXXXXXYTRYXDDXXFSWXXXXXPXXFXXXXXXXLXXXGYXXXPXKXXXXXXXXXXPXXTGXXLXXX GXXXXPXXXXXXXXXXXX 19398 Table X - Previously known retrotransposons RT (accession number; organism)

The following sequence reveals the following: Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classi fi cation of The Encoded Tripartite Systems, Nucleic Acids Research, Vol. 48, No. 22, December 16, 2020, pp. 12632-12647, which is incorporated herein by reference in its entirety. The sequences are described by sequence reference number and source organism. Serial number deposit number; source organismfig|670897. 3. peg. 2382; E. coli 2362-75 WP_000111473. 1; Escherichia coli (retrotransformant-Eco7) fig|286156. 4. peg. 5031; Australian Light Rod Bacteria fig|171439. 3. peg. 1995; Photorhabditis luminescens subsp. luminescens fig|1004151. 3. peg. 110; Pseudomonas aeruginosa NC19 fig|1736225. 3. peg. 2969; Evansella Leaf53 fig|1897730. 3. peg. 2912; Citrobacter CFSAN044567 fig|286156. 4. peg. 5031; Aeromonas australis fig|1460083. 3. peg. 4429; Serratia liquefaciens FK01 fig|585. 10. peg. 2369; Proteobacterium vulgaris WP_140315795. 1; Vibrio parahaemolyticus (retrotransferase-Vpa1) fig|670. 147. peg. 3463; Vibrio parahaemolyticus fig|1516159. 4. peg. 4737; Vibrio corallii fig|190893. 12. peg. 246; Vibrio corallii fig|643674. 5. peg. 820; Human alkali-producing bacteria fig|1122619. 3. peg. 2381; Oligouricobiformis DSM 18253 fig|29489. 5. peg. 3423; Aeromonas enterica serovar Enterica fig|1899355. 18. peg. 3566; Marine Spirillaceae bacteria fig|49186. 3. peg. 4362; Haemophilus stany fig|672. 375. peg. 4377; Vibrio vulnificus fig|584. 202. peg. 1668; Proteobacterium mirabilis fig|394935. 10. peg. 4407; Chromobacterium haemolyticum fig|1196083. 117. peg. 637; Alveus nodigella fig|1196083. 120. peg. 2046; Alveus nodigella fig|1196083. 114. peg. 825; Alveus nodgrassii fig|550. 250. peg. 2975; Enterobacter vulvae fig|680. 27. peg. 793; Vibrio campbeni fig|1348393. 3. peg. 352; Pseudoalteromonas H105 fig|1234128. 4. peg. 4777; Vibrio parahaemolyticus SNUVpS-1 fig|69219. 6. peg. 2213; Enterococcus lysate subsp. vulvar fig|208224. 13. peg. 2962; Kobe Escherichia coli fig|672. 332. peg. 2758; Vibrio vulnificus fig|1777131. 3. peg. 2267; Chromobacterium F49 fig|945550. 3. peg. 1167; Vibrio sinarius DSM 21326 fig|648. 75. peg. 922; Aeromonas caviae fig|1238221. 3. peg. 2053; Vibrio parahaemolyticus VPTS-2009 fig|56192. 3. peg. 3860; Photorhabditis iliacis fig|1806667. 7. peg. 3169; Gallicarpospora fig|272773. 3. peg. 1019; Halobiobrio halophilus subsp. costico WP_073265166. 1; Pseudomonas pugnorum fig|1946584. 3. peg. 2789; Halomonas UBA3074 fig|2030880. 3. peg. 665; SAR86 cluster bacteria fig|80854. 14. peg. 530; Myxotrichum fig|1902503. 3. peg. 1072; Marinomonas QM202 fig|1122212. 3. peg. 1985; Minutus villosa DSM 6287 fig|40576. 4. peg. 4387; Pathogenic bacteria of cattle fig|287094. 3. peg. 78; Alteromonas fig|1805633. 3. peg. 1469; Acinetobacter SFA fig|1945927. 3. peg. 1017; Acinetobacter spp. UBA1497 fig|202956. 9. peg. 1680; Acinetobacter thompsonii fig|1811612. 3. peg. 155; Moraxellaceae Bacteria REDSEA-S32_B1 fig|573. 14330. peg. 438; Klebsiella pneumoniae fig|470. 1294. peg. 971; Acinetobacter baumanni fig|762966. 3. peg. 2452; Escherichia coli YIT 11859 fig|470. 3514. peg. 1550; Acinetobacter baumanni fig|470. 2538. peg. 3022; Acinetobacter baumanni fig|48296. 130. peg. 276; Acinetobacter pitei fig|663. 91. peg. 4688; Vibrio alginolyticus fig|296199. 3. peg. 4813; Giant Vibrio fig|1367490. 3. peg. 3583; Vibrio fischeri ETJB5C fig|326537. 3. peg. 3698;Colvera arctica fig|1175631. 4. peg. 4191; Pectinobacterium wasabiense CFBP 3304 WP_001403504. 1; Escherichia coli (retrotransformant-Eco4 / Ec83) fig|549. twenty one. peg. 1734; Pantoea agglomerata fig|140100. 3. peg. 2972; Vibrio cholerae fig|693153. 4. peg. 1176;Atlantic Vibrio fig|1238430. 3. peg. 1911; Vibrio nigromaculata AM115 fig|1123036. 3. peg. 144; Thermomonas arcticus DSM 14288 fig|173990. 3. peg. 3319; Rheinheimia pacifica fig|1869214. 4. peg. 3809; Rheinheimia fig|1898113. 7. peg. 1514; Bacteria of the family Osmundae fig|29484. 39. peg. 1876; Yersinia freundii fig|1761793. 3. peg. 274; Haemophilus sp. DSM 26671 fig|587. 48. peg. 2666; Providencia rettgeri fig|573. 4147. peg. 1684; Klebsiella pneumoniae fig|1263833. 3. peg. 2872; Serratia marcescens VGH107 fig|1690502. 3. peg. 467; Pantoea CFSAN033090 fig|1029989. 3. peg. 5037; Enteric Salmonella enterica subsp. Agona serotype 0292 fig|211759. 3. peg. 770; Serratia marcescens fig|29483. 5. peg. 2283; Yersinia fig|1268238. 3. peg. 3466; Escherichia coli O5:K4(L):H4 strain ATCC 23502 fig|548. 121. peg. 2368; Klebsiella aerogenes fig|196024. 6. peg. 1825; Aeromonas hydrophila fig|386429. 3. peg. 3784; Pseudoalteromonas BSi20495 fig|666. 2089. peg. 3167; Vibrio cholerae WP_159353404. 1; Vibrio cholerae (retrotransposon-Vch1 / Vc95) fig|670. 362. peg. 2186; Vibrio parahaemolyticus fig|615. 398. peg. 1671; Serratia marcescens fig|571. 188. peg. 5401; Klebsiella oxytoca fig|1389422. 3. peg. 2794; Klebsiella pneumoniae LAU-KP1 fig|1082704. 3. peg. 1242;Lonsdalea britannica fig|1686379. 3. peg. 3365; Bacillus citrobacter MGH104 fig|83655. 55. peg. 221; non-decarboxylating Luxenbergia fig|550. 532. peg. 617; Enterobacter vulvae fig|349965. 6. peg. 153; Yarrowia intermedia ATCC 29909 fig|1947028. 3. peg. 31; Pantoea UBA2708 fig|29484. 34. peg. 3725; Yersinia freundii fig|314608. 4. peg. 222; Shewanella abyssinica KT99 fig|585. 16. peg. 3620; Proteobacterium vulgaris fig|1117313. 3. peg. 4128; Pseudoalteromonas polaris A 37-1-2 fig|1236543. 3. peg. 1328; Shewanella putrefaciens JCM 20190 = NBRC 3908 fig|550. 520. peg. 1818; Enterobacter vulvae fig|592316. 4. peg. 43; Pantoea At-9b fig|1903177. 3. peg. 4556; Vibrio 10N. 261. 45. E1 fig|1435069. 3. peg. 925; Vibrio tritonius fig|666. 3258. peg. 1211; Vibrio cholerae fig|1579504. 3. peg. 1822; Shewanella spp.ECSMB14102 fig|727. 548. peg. 1576; Haemophilus influenzae EIJ70524. 1; Haemophilus parahaemolyticus HK385 fig|1121935. 3. peg. 14;Hahella ganghwensis DSM 17046 fig|400668. 8. peg. 2509; Marinomonas MWYL1 fig|1777491. 3. peg. 1212; Alteromonas Mac1 fig|2013797. 3. peg. 1728; γ-Proteobacteria HGW-γ-Proteobacteria-15 fig|1008297. 7. peg. 4158; Enteric Salmonella enterica subspecies serovar Typhimurium strain 798 EDM6246721. 1; Enteric Salmonella enterica subspecies Typhimurium (retrotranscript-Sen2 (St85)) fig|421. 19. peg. 3278; Methylomonas fig|758. 17. peg. 102; Pneumocystis jirovecii fig|726. 60. peg. 864; Haemophilus influenzae fig|1035188. 3. peg. 348; Haemophilus pitmanii HK 85 fig|670. 79. peg. 3738; Vibrio parahaemolyticus fig|1481663. 12. peg. 913; Meteorological Vibrio fig|1123402. 3. peg. 611; Anopheles trosella DSM 18579 fig|668. 83. peg. 3088; Vibrio fischeri fig|290110. 6. peg. 2319; Budapest pathogens fig|568766. 10. peg. 822; Dickie's bacterium NCPPB 3274 fig|470. 4268. peg. 2217; Acinetobacter baumanni fig|1977881. 3. peg. 1569; Acinetobacter ANC 4470 fig|548. 171. peg. 2395; Klebsiella aerogenes fig|584. 105. peg. 1823; Proteobacterium mirabilis fig|1275975. 3. peg. 1756; Enteric Salmonella enterica subspecies Newport serotype strain Henan_3 fig|615. 474. peg. 3994; Serratia marcescens fig|61647. 13. peg. 3699; Polybacterium gergoviniensis fig|549. twenty two. peg. 222; Pantoea agglomerata fig|991944. 3. peg. 3216; Vibrio cholerae HE-25 WP_001022871. 1; Vibrio cholerae (retrotransposon-Vch2 (Vc81)) fig|1638949. 3. peg. 1051; Vibrio spp.ECSMB14106 fig|73010. 3. peg. 2815; Aeromonas eel fig|1444141. 3. peg. 3893; Escherichia coli 3-373-03_S3_C1 fig|232. 5. peg. 1080; Alteromonas fig|1175295. 3. peg. 21; Pseudoalteromonas PAMC 22718 fig|265726. 7. peg. 3430; Halotoxinia halophila WP_009585554. 1; Acinetobacter fig|2004649. 3. peg. 1632; Acinetobacter WCHA29 fig|1324350. 3. peg. 2817; Acinetobacter equi fig|2048003. 3. peg. 1682; Alteromonas flavus fig|571. 171. peg. 5963; Klebsiella oxytoca fig|573. 4060. peg. 3574; Klebsiella pneumoniae fig|1173850. 3. peg. 2995; Salmonella enterica subspecies Indiana strain ATCC 51959 fig|1123516. 3. peg. 1267; Vibrio halophilus DSM 15072 fig|1981674. 3. peg. 1814; Pseudomonas R9 (2017) fig|1947311. 3. peg. 2053; Pseudomonas sp.UBA2684 fig|1198309. 3. peg. 4291; Pseudomonas fluorescens ICMP 11288 fig|715451. 3. peg. 1743;Alteromonas naphthalenifera fig|316. 285. peg. 730; Pseudomonas stutzeri fig|1190606. 3. peg. 313; Enterovibrio calvei 1F-211 WP_009176189. 1; WP_097050713. 1; Xiamen Conch fig|1208323. 3. peg. 893; Bacillus thuringiensis B30 KZK95863. 1; Pseudomycin Ad46 fig|101571. 310. peg. 3956; Uboburkholderia fig|1882791. 3. peg. 1790; Burkholderia CF099 fig|1736536. 3. peg. 4809; Root434 of the genus PIG30812. 1; Janssenella 35 fig|1798244. 3. peg. 1046; Trichoderma book bacteria GWA2_55_18 fig|1131551. 3. peg. 1124; Methylophilus 1P/1 fig|1843082. 3. peg. 1574; Macromonas BK-30 fig|279058. 16. peg. 4721; Monospora arenae fig|1548123. 6. peg. 1144; Brachymycetes T2 fig|380394. 4. peg. 276; Sulfobacillus ferrooxidans ATCC 53993 WP_080292858. 1; fig|101571. 162. peg. 3605; Uboburkholderia fig|1382803. 3. peg. 22; Chromobacterium amazonica fig|930. 4. peg. 3851; Thiobacillus thiosulfate-oxidizing fig|1261658. 3. peg. 1787; Algae B. berberis Y31 fig|1679001. 3. peg. 631; Pasteurellaceae NI1060 fig|1334187. 3. peg. 653; Haemophilus influenzae KR494 fig|1581107. 3. peg. 1286; Neisseria HMSC15G01 fig|486. twenty four. peg. 152; Neisseria lactis fig|1953412. 3. peg. 1956; Bacteria UBP10_UBA1160 WP_090322045. 1; Oligotrophic Nitrosomonas fig|2013740. 3. peg. 1400; δ-Proteobacteria HGW-δ-Proteobacteria-13 fig|1907413. 3. peg. 3170; Rhizobium RU33A fig|1817963. 3. peg. 856; Adenophora deserticola fig|2035448. 3. peg. 1752; Rhizobium C5 WP_014077019. 1; fig|1648404. 4. peg. 2797; Rhodopsinus atlanticus fig|359. 11. peg. 6331; Agrobacterium radiatum fig|887144. 4. peg. 573; Taibaishan rhizobium fig|1116389. 3. peg. 333; Isolation of Devosia DS-56 fig|121719. 10. peg. 3421; Panronia alkaline lake bacillus fig|34002. 6. peg. 3570; Paracoccus alkaliphilus fig|1940281. 4. peg. 1560; Hefflera fig|1040981. 5. peg. 1561; Mesorrhiza siceri WSM4083 fig|410764. 3. peg. 807; Multi-hospital rhizobium fig|1825934. 3. peg. 3111; Anhui Rhizobium fig|1952824. 3. peg. 3061; UBA3976 of the family A. cerevisiae fig|1871086. 3. peg. 2153; Brevundimonas fig|588932. 9. peg. 647; Brevundimonas neizangshanensis fig|1951751. 3. peg. 1538; Rhodopsinaceae bacteria UBA1460 fig|1843368. 3. peg. 904; Sphingolipids RAC03 fig|155892. 10. peg. 3219; Pseudomonas aeruginosa fig|43057. 4. peg. 4537; Rhodopsinus nitrogen-fixing fig|1514904. 3. peg. 974; Arendella marineensis fig|1338034. 3. peg. 722; Vibrio parahaemolyticus O1:Kuk strain FDA_R31 fig|150340. 18. peg. 1837;Archaevibrio fig|196024. 5. peg. 3821; Aeromonas hydrophila fig|244366. 32. peg. 1886; Klebsiella varicella fig|180957. 35. peg. 1654; Brazil fruit gum bacillus fig|55601. 149. peg. 665; Vibrio anguillarum fig|121723. 5. peg. 2901; Photorhabdus ska34 fig|584. 170. peg. 837; Proteobacterium mirabilis fig|40324. 136. peg. 3276; Oligotrophomonas maltophilia fig|1122188. 5. peg. 411; Spongiolyticus spongiolyticus DSM 21749 fig|2032566. 3. peg. 2826; Xanthomonadaceae bacteria NML93-0792 fig|287. 1731. peg. 2578; Green Pseudomonas aeruginosa fig|251702. 3. peg. 1529; Pseudomonas syringae pathogenic variant snapdragon fig|1960829. 3. peg. 5912; Pseudomonas MF6394 fig|76759. 17. peg. 5093; Pseudomonas monteri fig|1981678. 3. peg. 5241; Pseudomonas R45 (2017) fig|1699620. 3. peg. 3028; Pseudomonas RIT-PI-r fig|191391. 4. peg. 2140; Pseudomonas salmoides fig|1844093. 4. peg. 7190; Pseudomonas 22E 5 fig|287. 1744. peg. 1414; Green Pseudomonas aeruginosa fig|287. 1987. peg. 910; Green Pseudomonas aeruginosa fig|287. 4372. peg. 4481; Green Pseudomonas aeruginosa fig|1856685. 4. peg. 2159; Pseudomonas TCU-HL1 fig|1718920. 3. peg. 3357; Pseudomonas ICMP 8385 fig|1781066. 3. peg. 2816; Durum HH101 fig|95485. 5. peg. 60; Stable Burkholderia fig|1572871. 6. peg. 588; Janssenella BJB304 WP_034208069. 1; Burkholderia cepacia WP_074283015. 1; Burkholderia GAS332 fig|1168169. 3. peg. 2570; Methylomonas 11b fig|1899355. 16. peg. 1328; Marine Spirillaceae bacteria WP_093197597. 1; Pseudomonas aeruginosa YR750 fig|1660091. 3. peg. 1650; Bordetella SCN 67-23 fig|134375. 17. peg. 4387; Achromobacterium fig|426114. 10. peg. 1990; Arsenic-oxidizing Thiomonas fig|1947551. 3. peg. 1903; Oligotrophomonas spp. UBA2302 fig|1914330. 4. peg. 2242; Halococcus fig|1947037. 3. peg. 890; Pantoea UBA5707 WP_094422719. 1; Coxsackie WP_079496884. 1; WP_088126255. 1; Enterobacter cobaciformis WP_049614309. 1; Yersinia WP_048263135. 1; Peruvian gum mushroom WP_040197602. 1; Klebsiella pneumoniae fig|669. 34. peg. 1586; Vibrio harveyi fig|672. 219. peg. 1032; Vibrio vulnificus fig|670. 1028. peg. 1775; Vibrio parahaemolyticus WP_065207673. 1; Bright Light Rod Fungus fig|1869214. 3. peg. 2231; Rheinheimia WP_029795910. 1; Vibrio parahaemolyticus fig|1191302. 3. peg. 1081; Vibrio crassicae 9ZC77 fig|668. 70. peg. 1192; Vibrio fischeri fig|28229. 4. peg. 4229; Cold red Corveria fig|1855726. 3. peg. 270; Burkholderia KK1 fig|1674888. 3. peg. 829; Burkholderia Beta_02 fig|687412. 4. peg. 1108; Pseudomonas aquaticus WP_092465129. 1; P. ivory fig|1120653. 3. peg. 5479; Scutellaria LC384 fig|121719. 5. peg. 401; Panronia alkaline lake bacillus fig|1798804. 3. peg. 1597; Rhizobium 58 fig|1946675. 3. peg. 3089; Codymonas spp.UBA4487 fig|36861. 5. peg. 1400; Denitrificans fig|1115835. 3. peg. 1003; Universal Methylophilus 79 fig|1797188. 3. peg. 1508; Acidobacteria RIFCSPLOWO2_12_FULL_60_22 fig|57320. 3. peg. 123; Pseudodesulfovibrio deep fig|1267534. 3. peg. 1238; Acidobacteriaceae KBS 89 fig|1951344. 3. peg. 527; Acidobacteriaceae UBA1307 WP_006226461. 1; Achromobacterium mapulatum fig|1503054. 4. peg. 5764; Burkholderia stagnans WP_006159686. 1; Basel copper bacteria WP_090191767. 1; Unclassified Duchenne fig|539. 8. peg. 1698; Eikenella corrosiva fig|1946925. 3. peg. 2129; Mikavibrio spp. UBA5701 WP_047031309. 1; Hefflera IMCC20628 fig|1946134. 3. peg. 1092; Brevundimonas spp.UBA6547 WP_093914930. 1; Sulfatium marine bacillus fig|1862950. 3. peg. 1234; Rhizobium bacteria NRL2 fig|1166078. 4. peg. 1483; Goldenomonas phylloides fig|709015. 3. peg. 734; Anemone marinus DSM 19842 WP_092160028. 1; Desulfovibrio ferrosulfuricans fig|2026749. 3. peg. 3364; Ignavibacteriae bacteria WP_033771991. 1; Pantoea agglomerata WP_097097099. 1; Unclassified Enterobacteriaceae (mixed) fig|1444151. 3. peg. 2733; Escherichia coli 2-177-06_S3_C2 WP_137545672. 1; Escherichia coli retrotransposons-Eco3 (Ec73) fig|573. 14856. peg. 3852; Klebsiella pneumoniae WP_072021595. 1; Serratia marcescens fig|29571. 3. peg. 478; Halomonas subglacialis WP_004534676. 1; WP_095622523. 1; Halomonas WRN001 fig|376427. 4. peg. 3223; Halomonas gudaoensis fig|862908. 3. peg. 745; Marine halophilic bacteriophage Vibrio SJ SCJ40239. 1; Uncultured Clostridium spp. fig|717962. 3. peg. 287; Feline fecal cocci GD/7 WP_014642259. 1; Halophilic Bacillus halophilus fig|2009042. 3. peg. 2106; Pseudomonas Irchel 3H7 fig|1981718. 3. peg. 4346; Pseudomonas B39 (2017) fig|665135. 13. peg. 1401; Pseudomonas In5 fig|1949067. 3. peg. 5629; Pseudomonas PICF141 WP_007948552. 1; Pseudomonas GM21 SFB61662. 1; Tsuruhaeida delftia WP_011615687. 1; WP_014778098. 1; fig|1429083. 4. peg. 2612; Pseudomonas husseinensis WP_095024014. 1; Pseudomonas WP_090203690. 1; Pseudomonas aeruginosa fig|564423. 8. peg. 1646; Pseudomonas tora NCPPB 2192 WP_078802277. 1; Pseudomonas fluorescens WP_090453229. 1; Pseudomonas fig|1306420. 5. peg. 1032; Burkholderia pseudomallei MSHR5848 fig|1357270. 3. peg. 1923; Pseudomonas syringae UB246 fig|2018067. 3. peg. 2950; Pseudomonas FDAARGOS_380 fig|317. 311. peg. 3241; Pseudomonas syringae fig|287. 2309. peg. 126; Pseudomonas aeruginosa WP_039522442. 1; Brazil fruit gum bacillus WP_080861357. 1; Klebsiella pneumoniae WP_014542745. 1; Evansella sp. Ejp617 OSL25696. 1; Escherichia coli TA255 fig|1125693. 3. peg. 761; Proteobacterium mirabilis WGLW4 KMK80587. 1; Black rot pectin bacteria ICMP 1526 fig|550. 717. peg. 2037; Enterobacter vaginalis ACS86154. 1; Banana Dickie's bacteria Ech703 WP_050122514. 1; Yersinia freundii WP_081334048. 1; Alteromonas macledy WP_055016254. 1; Pseudoalteromonas P1-13-1a fig|56799. 5. peg. 478; Corveria spp. fig|666. 3375. peg. 2486; Vibrio cholerae PIW62005. 1; Shewanella CG12_big_fil_rev_8_21_14_0_65_47_15 OCA54994. 1; Pseudomonas australis CNK75559. 1; Yersinia freundii WP_024248662. 1; Escherichia coli WP_088618141. 1; Cold-tolerant Methyloomycete WP_051669880. 1; PCJ98666. 1; Alteromonadaceae bacteria WP_081919471. 1; Acidithiobacillus ferroferrophilus WP_055769167. 1; Oligotrophomonas WP_039422954. 1; Xanthomonas vesicatoria WP_078568253. 1; Xanthomonas campestris WP_093486747. 1; Unclassified Pseudocanthomonas WP_077445058. 1; Rhodobacterium C05 WP_092576562. 1; Achromobacterium NFACC18-2 fig|1330528. 3. peg. 2198; Escherichia coli NCCP 15656 fig|83655. 67. peg. 2965; non-decarboxylating Luxenbergia fig|573. 10044. peg. 2850; Klebsiella pneumoniae WP_071888955. 1; Enterobacteriaceae fig|1799789. 3. peg. 4357; Hydrolytic water-dwelling bacteria fig|2024839. 8. peg. 1563; Oomycetes fig|1381081. 7. peg. 1167; Vibrio pannuri fig|670. 908. peg. 3444; Vibrio parahaemolyticus fig|626887. 3. peg. 2431; South China Sea Bacillus D15-8W fig|1913989. 101. peg. 1616; Gammaproteobacteria fig|262489. 9. peg. 2938; δ-proteobacterium MLMS-1 fig|2035207. 3. peg. 545; Janssenella 67 fig|28095. 13. peg. 1040; Burkholderia gladiolus fig|941449. 3. peg. 1262; Desulfovibrio X2 fig|1768806. 3. peg. 778; Rhodospirillaceae bacteria CCH5-H10 WP_083634830. 1; Desulfovibrio DV fig|1231. 4. peg. 574; Nitrosospira polymorpha fig|604089. 3. peg. 1142; Chinese psychrotrophic yellow bacterium fig|357523. 3. peg. 1851; Flavobacterium 11 fig|1423323. 5. peg. 321; Flavobacterium AED fig|178356. 3. peg. 502;Xinjiang yellow bacterium fig|1946545. 3. peg. 3457; Flavobacterium UBA4120 fig|150146. 3. peg. 2822; Psoralea corylifolia fig|229203. 4. peg. 1981; Flavobacterium delbrueckii fig|280093. 5. peg. 432; Granular yellow bacterium fig|728056. 4. peg. 1154; Flavobacterium argentatum fig|143224. 8. peg. 2343; Xanthomonas zephyrans fig|1225176. 3. peg. 4300; Cecembia lonarensis LW9 fig|1434700. 3. peg. 581; Sediment of Bacillus mohei fig|996. 47. peg. 468; Flavobacterium columnaris fig|172045. 56. peg. 2231; Elizabethia milleri fig|2024823. 3. peg. 2086;Altibacter genus fig|2026728. 18. peg. 4090; Saffron yellow fungus family bacteria fig|980584. 3. peg. 2930; Agarophageal seawater bacteria fig|1946744. 3. peg. 1682;Lewenhoekia UBA1003 fig|1046627. 3. peg. 2526; Bizionia argentinensis JUB59 fig|906888. 15. peg. 37;Nonlabens ulvanivorans fig|407022. 4. peg. 2865; Domestic olive fungus fig|1500282. 3. peg. 3713; Aureobacterium CF365 WP_084550290. 1; Goldenrod fig|190304. 8. peg. 741; Fusobacterium nucleatum subsp. nucleatum ATCC 25586 fig|1352. 1731. peg. 603; Escherichia coli fig|1428. 658. peg. 666; Bacillus thuringiensis fig|1497681. 3. peg. 3095; Listeria monocytogenes fig|1396. 1440. peg. 4237; Bacillus cereus fig|1917876. 3. peg. 2997; Blautia Marseille-P3087 fig|1952168. 3. peg. 215; Lachnospiraceae bacteria UBA7480 fig|1907659. 3. peg. 1085; Blautia Marseille-P3201T fig|1265309. 16. peg. 461; Bacillus fumigatus F1926 fig|853. 163. peg. 215; Pseudomonas aeruginosa fig|1264. 5. peg. 4; White Ruminococcus fig|1500289. 3. peg. 4469; Aureobacterium OV705 fig|1197728. 3. peg. 2386; Concept Prevotella 9403948 fig|1947486. 3. peg. 2515; Sphingobacterium UBA1897 fig|529. 12. peg. 1303; Candida albicans fig|1523429. 3. peg. 2936; Rhizobium AAP116 fig|1761878. 3. peg. 469; Paenibacillus cl6col fig|1462996. 4. peg. 2634; Yongji Bacillus fig|582475. 4. peg. 4724; Xylose lysine Bacillus fig|1773. 7915. peg. 7638; Mycobacterium tuberculosis fig|360310. 3. peg. 4853; Bacillus CDB3 fig|1396. 515. peg. 2936; Bacillus cereus fig|662367. 4. peg. 242; Endophytic spiral bacteria fig|1895719. 3. peg. 2950; Pseudomonas 45-6 fig|906888. 9. peg. 926;Nonlabens ulvanivorans fig|694433. 3. peg. 2346; Giant saprophytic spirochetes DSM 2844 fig|1167006. 5. peg. 2941; Desulfocapsa sulfexigens DSM 10523 fig|649724. 3. peg. 304; Clostridium ATCC BAA-442 fig|1505. 32. peg. 2959; Clostridium sordellii fig|1953142. 3. peg. 1858; Pseudomonas aeruginosa UBA1947 fig|2029590. 3. peg. 2754; Myxobacterium MD40 fig|29581. 33. peg. 2300; Blue-black-purple fungi fig|40324. 292. peg. 236; Oligotrophomonas maltophilia fig|1403329. 3. peg. 287; Listeria monocytogenes Lm25180 fig|1121865. 3. peg. 1262; Enterococcus Columbiansis DSM 7374 = ATCC 51263 fig|1120746. 3. peg. 3113; Bacteria MS4 fig|1952299. 3. peg. 221; Ruminococcaceae bacteria UBA2656 fig|1965604. 3. peg. 686; Anaerobic Massimobacillum An250 fig|1673717. 3. peg. 805; Anaerobic Massimobaciella senegal WP_116884683. 1; Food Valley Fungus fig|1948697. 3. peg. 196; Lenticoccobacillus UBA4640 fig|1232460. 3. peg. 46; Clostridiales VE202-28 WP_007864340. 1; Clostridiales WP_055649738. 1;Hungatella hathewayi fig|1226325. 3. peg. 2005; Clostridium KLE 1755 fig|1432052. 10. peg. 3166; Eisenbergia tayii fig|208479. 8. peg. 4376; Clostridium portlandii fig|1298920. 3. peg. 1959; [Desulfocholine] DSM 4024 fig|1776047. 3. peg. 4241; Clostridium C105KSO15 fig|1946596. 3. peg. 2399; Hungatella genus UBA4396 fig|1946603. 3. peg. 924; Hungatella UBA7603 fig|1410651. 3. peg. 407; [Clostridium] oxygen-tolerant DSM 5434 fig|1697784. 3. peg. 9617; Clostridium UC5. 1-1D4 fig|1745713. 3. peg. 3865; Massilia fig|180332. 3. peg. 1515;Robinsoniella peoriensis WP_072851604. 1;Lactonifactor longoviformis WP_003507561. 1; Clostridiales fig|1111728. 3. peg. 587; Aquatic Bacteria DSM 5075 = ATCC 35567 fig|1122977. 4. peg. 2473; Springwater Prague bacteria DSM 5563 = ATCC 49100 fig|1950915. 3. peg. 189; Clostridiales UBA644 fig|1950927. 3. peg. 912; Clostridiales bacterium UBA7187 ERK60856. 1; Rhizobacterium KLE 1728 WP_009260579. 1; Clostridium prausnitzii fig|1235797. 3. peg. 2409; Rhizobacterium 1-3 fig|1520815. 3. peg. 1262; Ruminococcaceae bacteria D5 fig|1855302. 3. peg. 1138; Pseudomonas butyricivibrio JW11 fig|43305. 5. peg. 3631;Proteolytic Butyrivibrio fig|411463. 15. peg. 1791; Eubacterium caeruleum ATCC 27560 fig|1235792. 3. peg. 3837; Lachnospiraceae M18-1 fig|97139. 3. peg. 669;Sabdariffa arabica fig|1291051. 3. peg. 1165; Licorice-degrading Mediterranean bacillus JCM 13369 fig|1532. 6. peg. 4793; Blautrachomatis fig|1121114. 4. peg. 5478; Produces Blautia ATCC 27340 = DSM 2950 fig|1262776. 3. peg. 1908; Clostridium CAG:149 fig|1262792. 3. peg. 1164; Clostridium CAG:299 fig|1262995. 3. peg. 2852; Firmicutes CAG:646 fig|537007. 17. peg. 3146; Hansen's blotch DSM 20583 fig|1965569. 3. peg. 1928; Intestinal core bacteria genus An169 fig|1952411. 3. peg. 2018; Ruminococcaceae bacteria UBA6353 fig|1965578. 3. peg. 1947; Pseudoclofusobacterium An187 WP_001775049. 1; Escherichia coli retrotransposons-Eco5 (Ec107) WP_012602583. 1; WP_015962464. 1; Enterobacteriaceae strain FGI 57 fig|1005999. 3. peg. 3342; Leminoa grisea ATCC 33999 = DSM 5078 fig|1378073. 3. peg. 795; Enterobacter CC120223-11 fig|911023. 3. peg. 138; Yorkella reginsburg ATCC 49455 fig|1834193. 3. peg. 4113;Enterococcus 9E7_DIV0242 Fig | 1649188. 10. peg. 406; Listeria monocytogenes fig|1430899. 3. peg. 278; Listeria monocytogenes 1991 fig|1211844. 4. peg. 748; Candidate Stoquefichus Marseille AP9 fig|1658109. 3. peg. 34; Candidate Stoquefichus genus SB1 fig|1262793. 3. peg. 950; Clostridium CAG:302 fig|1262908. 3. peg. 1120; Mycoplasma CAG:956 fig|1674844. 3. peg. 242; Clostridiales Firm_06 fig|1410672. 3. peg. 2823; Ruminococcus flavus ND2009 fig|1947424. 3. peg. 1718; Ruminococcus UBA4310 fig|1265. 9. peg. 2602; yellow Ruminococcus fig|1336236. 3. peg. 1817; Ruminococcus flavus ATCC 19208 CDC65895. 1; Ruminococcus CAG:57 WP_092946213. 1; Ruminococcaceae bacteria YRB3002 fig|1307. 1644. peg. 1532; Streptococcus suis WP_050516365. 1; Escherichia coli WP_097505494. 1; Escherichia coli retrotransposon-Eco5 (Ec107) fig|573. 15585. peg. 2343; Klebsiella pneumoniae WP_023581669. 1; Proteobacterium houerneri WP_079656969. 1; Serratia marcescens WP_090085157. 1; Plant bacillus SCO41 fig|573. 15584. peg. 1543; Klebsiella pneumoniae WP_023330997. 1; Enterobacterium vulgare complex fig|72407. 673. peg. 2552; Klebsiella pneumoniae subsp. pneumoniae CNM01182. 1; Yersinia pseudotuberculosis CNG88012. 1; Yersinia coli fig|1925763. 3. peg. 649; Halobacterium salinarum PKW24121. 1; Haemophilus sp.LV10R510-8 WP_045597342. 1; Vibrio vulnificus WP_098972386. 1; Aeromonas CU5 WP_005172873. 1; Yersinia coli WP_052979504. 1; Enterobacteriaceae WP_083069261. 1; Pantoea agglomerata WP_053911905. 1; Pseudoalteromonas SW0106-04 fig|1916082. 18. peg. 39; Alteromonadaceae bacteria WP_046555216. 1; Arsukibacterium genus MJ3 KPW01986. 1; Pseudoalteromonas P1-8 WP_094277737. 1; Marine Mononas baumani fig|1414654. 3. peg. 2005; Psychrotolerant marine cocci WP_008133621. 1; Unclassified Pseudoalteromonas KQA22543. 1; Vibrio aeruginosa WP_000284440. 1; Vibrio cholerae WP_011261677. 1; Vibrio fischeri KEE40622. 1; WP_012982829. 1; ALL66139. 1; Paraburkholderia caribe MBA4 WP_093223969. 1; Pseudomonas vancomycin fig|2015553. 3. peg. 2940; Pseudomonas PGPPP1 WP_096082869. 1; Pseudomonas aeruginosa ONM67687. 1; Green Pseudomonas aeruginosa fig|316. 213. peg. 2906; Pseudomonas stutzeri WP_078734267. 1; Pseudomonas fluorescens WP_079384669. 1; Pseudomonas aeruginosa WP_095948157. 1; Polyborophage WP_011625020. 1; Shewanella MR-7 WP_100292553. 1; Aeromonas cavernosa WP_055021484. 1; Pseudoalteromonas P1-26 PHS01491. 1; Marine Bacillaceae fig|2024618. 3. peg. 1141; Acinetobacter BS1 WP_114139108. 1; Klebsiella pneumoniae WP_077749737. 1; Pseudomonas FSL W5-0299 WP_078451378. 1; Pseudomonas aeruginosa WP_007245785. 1; Pseudomonas syringae group fig|316. 280. peg. 1454; Pseudomonas stutzeri WP_086822222. 1; Pseudomonas aeruginosa WP_073268605. 1; Pseudomonas pugnorum WP_095280108. 1; Salted seafood Leucoderma WP_095715328. 1; TSA-1 of Citrobacter spp. WP_050111525. 1; Yersinia WP_013724211. 1; Aeromonas veronii WP_021140819. 1; Aeromonas salmonicida fig|1094342. 5. peg. 1611; Alcanivorax xenomutans fig|1932666. 4. peg. 1886; Marinaria WP_087148323. 1; Ferrobacterium polysporum WP_064022638. 1; Methylomonas DH-1 PIY64876. 1; Shewanella CG_4_10_14_0_8_um_filter_42_13 WP_006710190. 1; Vibrio piscicola WP_045040928. 1; Photorhabditis iliacis WP_054543201. 1; Vibrio scintillans WP_080540293. 1; Vibrio vulnificus fig|2032624. 3. peg. 2540; Halomonas WN018 KJT50308. 1; Enteric Salmonella enterica subsp. Heidelberg strain RI-11-014588 retrotransposon-Sen1 (Se72) WP_005761319. 1; ODQ05744. 1; Shigella FC130 KKW01006. 1; Candidate Saccharibacteria bacteria GW2011_GWC2_48_9 KMZ12260. 1; candidate Burkholderia oleracea SFQ04394. 1; Ralstonia NFACC01 WP_025373922. 1;Advenella mimigardefordensis WP_093341200. 1; PDC80 of the genus Pseudomonas WP_091453700. 1; Giesbergeria anulus SAY51889. 1; Neisseria welshii WP_065255232. 1; Moraxella lacunosus WP_049330876. 1; Neisseria fig|1196095. 197. peg. 151; Bee Gillie's fungus WP_072956843. 1; Vibrio aerogenes fig|857087. 3. peg. 3286; Methylomonas MC09 fig|1952222. 3. peg. 1307; Methylococcaceae UBA3127 WP_039486261. 1; Vibrio Sinaloa WP_065545234. 1; Vibrio maxima WP_033094845. 1; Cold red Colwellia WP_057552475. 1; Vibrio cholerae WP_004726393. 1; Vibrio furnissii fig|2020862. 3. peg. 1934; Halophilic Vibrio spp. fig|624. 1260. peg. 1437; Sonneshiga WP_011516221. 1; Burkholderiales fig|1947370. 3. peg. 1923; Brachymycetes UBA4517 WP_038400955. 1; Yersinia pseudotuberculosis fig|1951903. 3. peg. 117; Halieaceae bacteria UBA3099 WP_024914507. 1;Chania multitudinisentens WP_042893228. 1; Enterobacteriaceae WP_038238211. 1; Nematophila pathogenic bacteria EXI65661. 1; Candidate Accumulibacter genus SK-12 WP_016452106. 1; Delftia WP_013517170. 1; Lipocyclilla denitrificans OXC73828. 1; Caballeronia sordidicola AIO65205. 1; Burkholderia oklahomaensis WP_013234866. 1; Spirillum vesiculosus WP_082884385. 1; Fish Rickettsiaceae NZ-RLO1 fig|2006849. 4. peg. 371; Xanthomonadales bacteria WP_074262787. 1; Paraburkholderia phenazinium WP_009906786. 1; Burkholderia thailandensis WP_022524328. 1; WP_081817450. 1; Halomonas HL-48 WP_020312233. 1; Pseudomonas syringae KPY75916. 1; Pseudomonas amygdaloids pathogenic variant nicotianae fig|1793966. 3. peg. 180; Pseudomonas aeruginosa fig|1891229. 16. peg. 2033; Pseudomonadales bacteria WP_099454886. 1; Pseudomonas faecalis WP_092400423. 1; Pseudomonas NFACC39-1 WP_012315430. 1; Pseudomonas faecalis WP_020799819. 1; Pseudomonas G5 (2012) WP_004574016. 1; fig|1435425. 3. peg. 787; Pseudomonas QTF5 WP_045490543. 1; Pseudomonas StFLB209 WP_011506503. 1; requires salt-colored salt bacteria fig|1609967. 3. peg. 3047; Halomonas HG01 fig|1492738. 3. peg. 2698; Flavobacterium seoulense WP_092849245. 1; Pectinophaga algae WP_025835957. 1; Pseudomonas aeruginosa fig|2025877. 3. peg. 668; Parabacterium AT13 fig|246787. 6. peg. 2081; Cellulose pseudobacteria fig|1339287. 3. peg. 1113; Pseudomonas fragilis strain 3986 T(B)9 fig|1946017. 3. peg. 1516; another genus UBA940 WP_038655380. 1; Leech-eating slime mold WP_093669272. 1; Myxobacterium MAR_2009_124 WP_073241067. 1; Flavobacterium inland sea WP_096193803. 1; Cellulophagoales TFI 002 WP_076357635. 1; WP_073238193. 1; Sewage soil bacteria WP_076451370. 1; WP_091906542. 1; Porphyromonadaceae KH3R12 WP_051365712. 1; Saltcap Flavobacterium fig|1938609. 3. peg. 1765; Flavobacterium LM4 SDJ72221. 1; Non-centrifuged Flavobacterium fig|1985174. 3. peg. 2584; Chitinophages IBVUCB2 WP_092737749. 1; Morchella in pigeon throat fig|192149. 3. peg. 42; Murina fig|418630. 3. peg. 1685; Rhodopseudomonas grandis fig|1915314. 3. peg. 3469; Bordetella sulphurea DLFJ5-1 fig|2030815. 3. peg. 2725; Sulfonylureas martensii fig|2035451. 3. peg. 4632; Rhizobium L18 WP_043872258. 1; Indian Ocean Fast-growing Bacillus WP_055683826. 1; Nassauria rubrum fig|1947537. 3. peg. 498; Sphingomonas sp.UBA6198 WP_069065961. 1; Sphingolipids RAC03 WP_084280100. 1; Neosphingobacteria B1 fig|1895845. 3. peg. 487; Sphingolipids 66-54 GAK73419. 1; Agrobacterium truncatum TR3 = NBRC 13261 WP_090966398. 1; Goldenomonas phyllostomonas WP_091860144. 1; Bosella robiniae WP_085092006. 1; Rice Azospirillum fig|1528100. 4. peg. 28; Methylomagnum ishizawai fig|32057. 3. peg. 9515; PCC 7103 fig|103690. 10. peg. 3571; Nostoc PCC 7120 = FACHB-418 fig|1137095. 11. peg. 15; Pseudocladens HK-05 CDZ48826. 1; Neorhizobium galegae bv. officinalis) WP_072340070. 1; Devosulia enhydra OYR18277. 1; Thiophene candida WP_093509439. 1; Sphingomonas YR583 WP_081799025. 1; Neosphingolipids feeding on tree resin PIY55545. 1;ζ-Proteobacteria CG_4_10_14_0_8_um_filter_49_80 SDT44912. 1; Bradyrhizobium canariensis WP_096350346. 1; WP_074962594. 1; Nassauria rubrum WP_038724888. 1; Burkholderia pseudomallei WP_012217410. 1; Burkholderia multiphaga WP_100428762. 1; Janssenella 67 WP_082161008. 1; Candidate denitrifying competitive Bacillus AFL73219. 1; Thiocystis purpurogenum DSM 198 WP_014427842. 1; fig|364030. 3. peg. 3554; Thiomonas elaborata KGW20495. 1; Burkholderia pseudomallei MSHR2451 SFE83076. 1; Coprophagnum OK212 WP_013028226. 1;Sideroxydans lithotrophicus WP_080311424. 1; Burkholderia pseudomallei fig|337. 13. peg. 3872; Burkholderia vesiculata WP_082643860. 1; Pseudomonas CKH90039. 1; Pseudomonas aeruginosa WP_083287254. 1; Unclassified Johnson bacteria WP_122648546. 1; Burkholderia pseudomallei WP_082706753. 1; Unclassified Pseudomonas WP_080936076. 1; Klebsiella pneumoniae WP_000746343. 1; Enterobacteriaceae EMX54653. 1; Escherichia coli MP020980. 2 WP_053270700. 1; Escherichia coli fig|1736224. 3. peg. 3731; Serratia Leaf51 fig|1175299. 4. peg. 709; Dictyophora maydis ZJU1202 WP_001461245. 1; Enterobacteriaceae fig|617145. 3. peg. 3535; Vibrio scindens 1F-157 fig|1440054. 3. peg. 3851; Vibrio OY15 fig|617135. 3. peg. 594; Vibrio fischeri ZF-211 WP_023267764. 1; Shewanella decoloris fig|1481663. 36. peg. 3628; Meteorological Vibrio fig|670. 893. peg. 2716; Vibrio parahaemolyticus fig|680. 33. peg. 5391; Vibrio campbeni fig|298386. 8. peg. 4344; Deep-sea Photorhabdus SS9 fig|663. 73. peg. 714; Vibrio alginolyticus fig|1333511. 3. peg. 3208; Pseudoalteromonas maritima TAB23 WP_064574154. 1; Hafnia paraalvei WP_064645509. 1; Proteobacteria fig|630. 105. peg. 4248; Yersinia coli fig|400673. 7. peg. 1969; Legionella pneumophila strain Corby WP_092678546. 1; Rosenbergiella nectarea WP_069476513. 1; Raoultia oxyornithine fig|1267535. 3. peg. 2394; Bryobacterales bacteria KBS 96 WP_000446053. 1; Acinetobacter baumanni fig|1948587. 3. peg. 786; Gammaproteobacteria UBA1902 WP_014949305. 1; Alteromonas macledy fig|1797397. 3. peg. 2386; Bdellovibrioles bacteria RIFOXYC1_FULL_54_43 fig|1386968. 3. peg. 847; Francisella tularensis subsp. PA10-7858 WP_074900850. 1; fig|1975705. 3. peg. 898; Psychrobacterium FDAARGOS_221 WP_066184577. 1; Toxoplasma fig|1780380. 4. peg. 4010; Fungal bacteria CHKCI004 fig|556261. 3. peg. 2546; Clostridium D5 fig|1193534. 6. peg. 2375; Uncultured Fusobacteria fig|1042163. 3. peg. 3771; Brevibacterium brevisporum LMG 15441 WP_062492190. 1; Paenibacillus 32O-W WP_081674606. 1; Harbin Lactobacillus WP_050781686. 1; Lactobacillus rod-shaped bacteria WP_021109137. 1; Enterococcus faecium WP_046309803. 1; Staphylococcus CBL03706. 1; Pamela Gordonia 7-10-1-b WP_090944285. 1; Pelosinus propionicus WP_077305443. 1; Clostridium beijerinckii fig|410072. 5. peg. 40; Accompanying Pseudomonas aeruginosa WP_011669870. 1; Leptospira poppatersonii WP_015565235. 1; Pseudomonas aeruginosa CUO23478. 1; Pseudomonas aeruginosa WP_085748688. 1; Rhizobium Glucophilum WP_093270014. 1; Psychrobacterium OK032 SHE86352. 1; Atopostipes suicloacalis DSM 15692 WP_000346292. 1; Unclassified Streptococcus WP_080465410. 1; Lactobacillus plantarum WP_080662531. 1; Lactobacillus brevis WP_093131554. 1; Bacillus koningii WP_093336905. 1; Halotoxin-tolerant Bacillus fig|1974627. 3. peg. 386; Candidate Levenomyces bacteria CG_4_9_14_0_2_um_filter_35_21 fig|1802603. 3. peg. 453; Candidate Woykebacteria RIFCSPHIGHO2_12_FULL_ 45_10 fig|392734. 5. peg. 3006; K. rosea AGL61879. 1; Candidate Saccharimonas aalborgensis fig|319224. 16. peg. 2726; Shewanella putrefaciens CN-32 fig|1720343. 3. peg. 1263; Pseudoalteromonas 1_2015MBL_MicDiv fig|1136158. 3. peg. 3691; Cyclovibrio 1F97 fig|666. 3017. peg. 1000; Vibrio cholerae fig|1909458. 3. peg. 2277; Halovibrio ML198 fig|1638949. 3. peg. 831; Vibrio spp.ECSMB14106 fig|493915. 3. peg. 158; Pseudoalteromonas NJ631 BAC94535. 1; Vibrio vulnificus YJ016 fig|1191313. 3. peg. 1135; Vibrio scindens 1S-124 fig|670. 1244. peg. 3807; Vibrio parahaemolyticus fig|1659714. 3. peg. 4264; Citrobacter brunneri fig|1192730. 4. peg. 1976; Enteric Salmonella enterica subspecies Kintambo serotype fig|550. 1216. peg. 4296; Enterobacter vulvae WP_072269713. 1; Serratia WP_053898075. 1; Escherichia coli fig|624. 1264. peg. 1635; Songnei Zhiheba fig|1181777. 3. peg. 78; Escherichia coli KTE233 fig|1802256. 3. peg. 310; Thiomonas RIFOXYB12_FULL_35_9 PHR73342. 1; Toxoplasma fig|2014260. 3. peg. 3813; Bacteria (candidate Blackallbacteria) CG13_big_fil_rev_8_21_14_ 2_50_49_14 WP_042497590. 1; Vibrio marineus WP_063522799. 1; Vibrio HI00D65 WP_004186757. 1; Enterobacteriaceae WP_040122746. 1; Vibrio WP_086046550. 1; Vibrio harveyi group WP_063849005. 1; Enterobacter vulvae WP_023486614. 1; Enterobacteriaceae WP_070992278. 1; Pseudoalteromonas fig|1005665. 3. peg. 2532; Coxsackia oryzendophytica fig|1219066. 3. peg. 3636; Vibrio parahaemolyticus NBRC 12711 fig|1225184. 4. peg. 1222; Pantoea A4 fig|675814. 3. peg. 1256; Vibrio corallii ATCC BAA-450 SFR59865. 1; Pseudomonas butyricivibrio NOR37 fig|853. 16. peg. 1112; Pseudomonas aeruginosa fig|1965572. 3. peg. 1423; Pseudoclosporin An176 fig|588581. 3. peg. 3589; Clostridium papulolyticum DSM 2782 fig|1396. 1409. peg. 4169; Bacillus cereus fig|1428. 538. peg. 4047; Bacillus thuringiensis fig|1465. 16. peg. 946; Brevibacterium brevisporum WP_087385137. 1; AIF42417. 1; Cladosporium SK37 WP_076543941. 1; Halophilic anaerobic bacteria fig|1121093. 3. peg. 3089; Bacillus spondylodis subtilis DSM 19096 fig|29367. 3. peg. 2029; Clostridium punicatum WP_089719707. 1; Congo halophilic anaerobic bacteria fig|307249. 3. peg. 3585; Uncultured Mycosporium WP_072949666. 1; Ruminococcus flavus CCX81854. 1; Ruminococcus CAG:108 fig|1491. 669. peg. 2217; Clostridium botulinum fig|1872455. 3. peg. 401; Alkaliphila fig|576117. 5. peg. 4005; Halophilic fast-growing Bacillus fig|1225647. 3. peg. 1829; Pseudomonas 11ANDIMAR09 fig|1380380. 4. peg. 1574; Arensella 13_GOM-1096m fig|293. 7. peg. 2956; Brevundimonas diminuta WP_095437634. 1; Rhizobium 11515TR fig|1912891. 7. peg. 702; Sphingolipids fig|1736574. 3. peg. 4024; Pseudomonas Root630 fig|227946. 13. peg. 4105; Xanthomonas translucentus poae pathogenic variant fig|1761791. 3. peg. 4793; Lysobacterium yr284 fig|1560195. 5. peg. 485; Janssenella BJB301 fig|1503054. 43. peg. 6257; Burkholderia stagnans fig|1207504. 10. peg. 4279; Burkholderia pseudopolyphaga WP_092172515. 1; Unclassified Pseudomonas WP_074815429. 1; Pseudomonas syringae ffig|150146. 3. peg. 3162; Yellow bacterium of Gili fig|76832. 8. peg. 3775; pseudo-scented scent fungi fig|1202724. 3. peg. 994; Akkaya flavonoids fig|1805473. 3. peg. 3678; Flavour bacillus timonianum fig|253. 33. peg. 3826; Indole-producing Aureobacterium WP_076561634. 1; Aureobacterium inertum fig|2024823. 3. peg. 95; Altibacter genus fig|1250278. 4. peg. 3462; Halobacterium Hel_I_6 fig|1797342. 3. peg. 689; Pseudomonas GWF2_33_38 WP_084184261. 1; Urolyticum aureobacterium fig|1948560. 3. peg. 3003; Delta-Proteobacteria UBA6106 fig|1392. 364. peg. 2564; Bacillus anthracis fig|872970. 3. peg. 1713; Marine amphibian bacteria fig|1385514. 3. peg. 313; Yanchenghai Bacillus Y32 fig|76853. 4. peg. 2614; Silver Bacillus fig|1423774. 3. peg. 1262; Lactobacillus nantes DSM 16982 fig|1410670. 3. peg. 2844; Ruminococcus aureus MA2007 fig|169435. 7. peg. 1348; Anaerobic club bacteria fig|1946597. 3. peg. 2104;Hungatella genus UBA4568 fig|1948087. 3. peg. 796; Firmicutes bacteria UBA6113 fig|642492. 3. peg. 2638; Cellulosilyticum lentocellum DSM 5427 fig|1950841. 3. peg. 2383; Clostridiales UBA2436 fig|555512. 3. peg. 1251; Marine Salipiger fig|383381. 3. peg. 2538; Rhodobacterium JL475 WP_081629462. 1; fig|1736258. 3. peg. 3392; Methylobacterium Leaf112 fig|1950192. 3. peg. 426; Anaerobic Rhizobacteriales bacterium UBA2232 fig|170623. 6. peg. 4661; Azotobacter beijer fig|170623. 7. peg. 704; Azotobacter beijer fig|1981099. 3. peg. 513;Niveispirillum lacus fig|1250539. 3. peg. 3491; Deep-sea olive fungus fig|1947582. 3. peg. 2979; Sulfatium spp. UBA1132 fig|1909294. 17. peg. 3456; Rhizobium bacteria fig|1735583. 3. peg. 1657; Pseudovibrio W64 fig|670. 1220. peg. 4688; Vibrio parahaemolyticus fig|1004786. 3. peg. 925; Alteromonas mediterranea DE1 fig|2013797. 3. peg. 2109;γ-Proteobacteria HGW-γ-Proteobacteria-15 fig|1948580. 3. peg. 3400; Gammaproteobacteria UBA1012 fig|1714300. 3. peg. 306; Deep-sea sea bacillus fig|1961547. 3. peg. 1371; Desulfobacteriaceae UBA2273 fig|441162. 10. peg. 6621; Burkholderia oklahomaensis C6786 fig|615. 307. peg. 4666; Serratia marcescens fig|631. 3. peg. 1883; Yarrowia intermedia fig|1763535. 3. peg. 1547; Hydrogenophage crassostreae fig|43263. 5. peg. 2702; Alkali-producing Pseudomonas fig|244366. 46. peg. 3595; Klebsiella varicella fig|1224150. 8. peg. 3856; Dickinia banana NCPPB 2511 fig|61645. 10. peg. 2019; Enterobacter aegypti fig|1948706. 3. peg. 2225; Bacteria fengyouensis UBA1333 fig|2026771. 13. peg. 1697; Fungal bacteria fig|2026771. 11. peg. 1955; Fungal bacteria fig|2026772. 5. peg. 424; Fengyou bacteria fig|2026801. 20. peg. 1798; Verrucomicrobia bacteria fig|2026801. 14. peg. 1176; Verrucomicrobia bacteria fig|1951369. 3. peg. 1157; Akkermansiaceae UBA6946 fig|1977087. 12. peg. 1918; Proteobacteria fig|2026779. 14. peg. 4171; Fungi of the family Planctomycetes fig|2026779. 28. peg. 3264; Fungi of the family Planctomycetes fig|2026779. 30. peg. 3181; Fungi of the family Planctomycetes fig|2026779. 29. peg. 2310; Fungi of the family Planctomycetes fig|1797235. 3. peg. 3; Aerithromyces RIFCSPHIGHO2_12_41_5 fig|316. 284. peg. 937; Pseudomonas stutzeri fig|296. 11. peg. 442; Pseudomonas fragilis fig|1981714. 3. peg. 993; Pseudomonas B5 (2017) fig|50340. 44. peg. 6020; Pseudomonas sphaeroides fig|1761897. 3. peg. 509; Pseudomonas ok272 fig|1402514. 3. peg. 154; Pseudomonas aeruginosa BWHPSA014 fig|1938440. 3. peg. 5997; Pseudomonas T fig|1566250. 3. peg. 959; Pseudomonas NFACC02 fig|316. 357. peg. 479; Pseudomonas stutzeri fig|287. 4433. peg. 2945; Green Pseudomonas aeruginosa fig|1970515. 3. peg. 709; Hydrogenophiles 12-61-10 fig|95486. 85. peg. 1748; Burkholderia neocypris fig|292. 61. peg. 8104; Burkholderia cepacia fig|1408450. 3. peg. 3766; Methylobacterium tundripaludum 21/22 fig|157910. 3. peg. 5727; Uplift Vice Burkholder ig|279058. 16. peg. 4239; Monospora arenae fig|1537272. 3. peg. 1916; Janssenella HH100 fig|1218081. 3. peg. 1751; Kururi Subburkholderia sulphur-oxidizing subsp. NBRC 107107 fig|573. 14059. peg. 3113; Klebsiella pneumoniae fig|40324. 192. peg. 51; Oligotrophomonas maltophilia fig|1219041. 3. peg. 4613; Sphingomonas azotobacter NBRC 15497 fig|1561196. 3. peg. 560; Burkholderia E7m39 fig|1882750. 3. peg. 1035; Burkholderia GAS332 fig|1736266. 3. peg. 1145; Durum Leaf126 fig|2015350. 3. peg. 1640; Burkholderia AU18528 fig|58133. 4. peg. 815; Nitrosospira NpAV fig|1691980. 3. peg. 1912; Rhodotorulaceae Paddy-1 fig|305. 393. peg. 1023; Ralstonia solanacearum fig|56449. 3. peg. 3604; Bromoxanthomonas fig|1281282. 5. peg. 1894; Xanthomonas campestris pathogenic variant CN14 fig|40324. 334. peg. 1103; Oligotrophomonas maltophilia fig|1349793. 3. peg. 2529; Spiral Hydrophage NBRC 102512 fig|1842727. 3. peg. 1491; Korean red bacteria fig|1619952. 3. peg. 5158; Burkholderiaceae 16 fig|1970380. 3. peg. 1914; Halostaurum 14-55-98 fig|2015568. 3. peg. 2963; Burkholderia PBB6 fig|1752215. 3. peg. 2312; Gammaproteobacteria Ga0077554 fig|1706231. 5. peg. 3125; Janssenella CG23_2 fig|2013716. 3. peg. 2169; β-Proteobacteria HGW-β-Proteobacteria-4 fig|1946997. 3. peg. 3049; Nitrospira UBA7655 fig|765913. 3. peg. 2527; Rhodococcus delbrueckii AZ1 fig|1743159. 3. peg. 1891; Polymyxin Bacillus fig|1597955. 3. peg. 3923; Haemophilus DM1 fig|1184267. 3. peg. 1626; Bdellovibrio exotrophicus JSS fig|101571. 190. peg. 3007; Uboburkholderia fig|123899. 5. peg. 1710; Bordetella woundis fig|463035. 3. peg. 3900; Bordetella genotype 12 fig|1395608. 4. peg. 211; Bordetella genotype 5 fig|1947379. 3. peg. 2784; Rhodophyte UBA5149 WP_074294985. 1; Paraburkholderia phenazinium fig|1324617. 3. peg. 820; Vice Burkholder aspalathi fig|80868. 3. peg. 3458; Acidophage bacterium Carterii fig|1388764. 3. peg. 1840; Pseudomonas ferrooxidans EGD-HP2 fig|251747. 15. peg. 4695; Chromobacteria under Hemlock fig|670. 1020. peg. 382; Vibrio parahaemolyticus fig|1055803. 3. peg. 1434; Pseudoalteromonas TB51 fig|1201036. 3. peg. 177; Pseudocalbuminae AO18b fig|1220581. 4. peg. 1434; Agrobacterium NBRC 13257 fig|398. 6. peg. 6695; Tropical Rhizobium fig|931866. 6. peg. 8184; Bradyrhizobium ottawa fig|142585. 3. peg. 1658; Bradyrhizobium C9 fig|1082933. 13. peg. 1537; Rhizobium in Amorpha fruticosa CCNWGS0123 fig|1768789. 3. peg. 791; Methylobacterium CCH7-A2 fig|1381123. 3. peg. 3819;Aliihoeflea 2WW fig|1297570. 3. peg. 1970; Mesorhizobium STM 4661 fig|935546. 3. peg. 3816; Rhizobium NZP2037 in Rhizobium radix fig|1128253. 3. peg. 1960; Bradyrhizobium japonicum CCBAU 15354 fig|1444315. 4. peg. 3983; Capsicum azetiliensis AZ78 fig|1185327. 3. peg. 1608; Xanthomonas cassava wilt pathogenic variant strain Xam668 fig|1881043. 3. peg. 2597; Pseudomonas GM95 ALN84423. 1; Capsicum lysate fig|56460. 15. peg. 1977; Xanthomonas vesicatoria fig|1317116. 6. peg. 2759; Marine Bacteria 22II-s10i fig|564137. 3. peg. 4320; Antarctic rose-colored lemon fungus fig|1952800. 3. peg. 3583; Rhodobacteriaceae UBA2553 fig|218673. 12. peg. 3041; Suspected Sulfatium fig|1912092. 3. peg. 2119; Sediment National Institute of Oceanography Bacteria fig|1736558. 3. peg. 5006; Sword mushroom Root558 fig|91360. 5. peg. 3717; Desulfurizing Clavatechus singaporeans fig|1948756. 3. peg. 2576; Borrelia spirochetes UBA2205 fig|1855322. 3. peg. 103; Bradyrhizobium Rc3b fig|1437360. 11. peg. 2429; Bradyrhizobium erythrocytosis fig|1871052. 3. peg. 1026; Aphidonia fig|1038860. 3. peg. 8756; Bradyrhizobium elsdenii WSM2783 fig|1898112. 54. peg. 3758; Rhodospirillaceae bacteria fig|1660129. 3. peg. 4854; Pseudomonas SCN 70-31 fig|1482074. 3. peg. 4109; Hartmannii nitrogen-fixing bacteria fig|1970306. 3. peg. 552; Acidobacterium 35-58-6 fig|1686310. 5. peg. 1409; Bartonella apis fig|1798192. 3. peg. 1953; Conch genus KO164 fig|1235461. 17. peg. 11; Sinorhizobium lucerne GR4 fig|442. 12. peg. 222; Gluconobacter oxydans fig|1938607. 3. peg. 1954; Sphingomonas LM7 fig|1231624. 3. peg. 39; Bogor Asai bacteria NBRC 16594 fig|1121271. 3. peg. 4112; Armillaria mellea DSM 15620 fig|33059. 16. peg. 1690; Thermothorax acidothiobacil fig|502025. 10. peg. 925; Halobacterium ochraceum DSM 14365 fig|1734406. 3. peg. 691; α-Proteobacteria BRH_c36 fig|1979207. 3. peg. 4304; Brachymetra fig|1953057. 3. peg. 74; UBA4496 of the family Brachymectomycetaceae fig|858423. 3. peg. 10004; Bradyrhizobium oleraceum fig|267128. 3. peg. 2015; Granulosphingomonas fig|582667. 3. peg. 5553; Methylobacterium sicouracifolium fig|1187852. 3. peg. 2712; Methylobacterium tahanianum fig|582675. 3. peg. 1247; Methylobacterium gesipekola fig|1951640. 3. peg. 515; Deferiorhizium bacterium UBA6799 fig|1948417. 4. peg. 1606; α-Proteobacteria UBA6187 fig|45074. 5. peg. 981; Legionella of the Holy Cross fig|1434232. 4. peg. 2927; Magnetofaba australis IT-1 fig|1945950. 3. peg. 3568; Acinetobacter spp. UBA6526 fig|106654. twenty two. peg. 994; Hospital Acinetobacter fig|1977883. 3. peg. 3023; Acinetobacter ANC 3903 fig|1945948. 3. peg. 700; Acinetobacter spp. UBA5984 fig|1226327. 3. peg. 2796; Acinetobacter kutschii fig|1879049. 4. peg. 5949; Acinetobacter WCHAc010034 fig|1945955. 3. peg. 1951; Acinetobacter spp. UBA7614 fig|1675530. 3. peg. 2149; Acinetobacter genotype 33YU fig|1310638. 3. peg. 1006; Aegilops baumanni 1437282 fig|1400001. 4. peg. 34; Marseilles bacillus fig|1132496. 5. peg. 136; Pasteurella multocida subsp. multocida strain HN06 fig|1908263. 4. peg. 2604; Trehalose saccharomyces fig|375432. 4. peg. 200; Haemophilus influenzae R3021 fig|400668. 8. peg. 3776; Marinomonas MWYL1 fig|1913989. 193. peg. 841; Gammaproteobacteria fig|856793. 5. peg. 1975; Microvibrio cupreus ARL-13 SBW23286. 1; European Bacillus citrate fig|1736225. 3. peg. 985; Evansella Leaf53 fig|29486. 12. peg. 818; Yersinia ruckeri fig|914128. 3. peg. 2502;Commensal Serratia strain Tucson fig|1796497. 3. peg. 952;Grimontia celer fig|1095649. 3. peg. 3298; Vibrio cholerae O1 strain EM-1676A fig|137584. 4. peg. 1627;Deep-sea monas viridans fig|173990. 3. peg. 1773; Rheinheimia pacifica fig|1720343. 3. peg. 3189; Pseudoalteromonas 1_2015MBL_MicDiv fig|1202962. 4. peg. 1481; M. maritima ATCC 15381 fig|669. 50. peg. 2993; Vibrio harveyi fig|691. 32. peg. 1517; Vibrio natrigensis fig|156578. 3. peg. 2521; Alteromonadales bacteria TW-7 fig|661. 14. peg. 380; Pseudomonas aeruginosa fig|654. 94. peg. 1733; Aeromonas veronii fig|703. 9. peg. 319; Shigamonas fig|589873. 36. peg. 1971; Alteromonas australis fig|28107. 3. peg. 3571; Pseudoalteromonas espegia fig|1547444. 3. peg. 4264; Pseudoalteromonas PLSV fig|629266. 7. peg. 847; Pseudomonas syringae var. kiwifruit strain M302091 fig|251722. 19. peg. 4059; Pseudomonas aesculi pathogenic variant fig|587851. 4. peg. 1470; Pseudomonas chlororaphis subsp. aurea fig|1265490. 3. peg. 2330; Pseudomonas URMO17WK12:I8 fig|316. 101. peg. 3534; Pseudomonas stutzeri fig|1916993. 3. peg. 4917; Pseudomonas foetida fig|1628833. 3. peg. 2448; Pseudomonas ES3-33 fig|1283291. 4. peg. 1991; Pseudomonas URMO17WK12:I11 fig|83963. 5. peg. 3885; Pseudomonas syringae herpes simplex virus variant fig|1206777. 3. peg. 4334; Pseudomonas Lz4W fig|113268. 3. peg. 3785; Deep-sea mussel methane-trophic gill symbiosis fig|1131284. 3. peg. 1562;ζ-Proteobacterium SCGC AB-137-C09 fig|2026807. 7. peg. 2258;ζ-Proteobacteria fig|281689. 4. peg. 2060; Desulfomonas acetyloxidans DSM 684 fig|1188231. 4. peg. 1200; Ferrous oxide deep-sea bacteria M34 fig|1367489. 3. peg. 682; Vibrio fischeri SA1G fig|1873135. 3. peg. 4249; Shewanella spp. SACH fig|663. 73. peg. 2465; Vibrio alginolyticus fig|1588629. 3. peg. 1134; Aeromonas L_1B5_3 fig|1121922. 3. peg. 3454;Glaciecola pallidula DSM 14239 = ACAM 615 fig|351745. 9. peg. 2506; Shewanella W3-18-1 fig|29497. 20. peg. 3798; Vibrio scintillans fig|1367486. 3. peg. 187; Vibrio fischeri CB37 fig|511062. 4. peg. 1890; Marine Mononas GK1 fig|654. 12. peg. 188; Aeromonas veronii fig|29497. twenty one. peg. 4482; Vibrio scintillans fig|1659713. 3. peg. 560; Enterobacter baumannii fig|1124991. 3. peg. 3617; Morganella morganii subsp. morganii KT fig|104623. 3. peg. 1381; Serratia ATCC 39006 fig|1256989. 3. peg. 902; Providencia alkali-producing R90-1475 fig|1125694. 3. peg. 1143; Proteobacterium mirabilis WGLW6 fig|574096. 6. peg. 2693; Pantotrichum garlici fig|1095774. 3. peg. 2623; Pantoea pineapple PA13 fig|869692. 4. peg. 2910; Escherichia coli 3003 WP_140159440. 1; Escherichia coli retrotransposons-Eco2 (Ec67) fig|550. 437. peg. 1444; Enterobacter vulvae fig|573. 13605. peg. 2600; Klebsiella pneumoniae fig|550. 285. peg. 3783; Enterobacter vulvae fig|1265672. 3. peg. 3869; Enteric Salmonella enterica subspecies Agona serotype 70. E. 05 fig|573. 10028. peg. 542; Klebsiella pneumoniae fig|749537. 3. peg. 218; Escherichia coli MS 115-1 ANK06786. 1; Escherichia coli O25b:H4 fig|670. 880. peg. 975; Vibrio parahaemolyticus fig|1192730. 4. peg. 3; Enteric Salmonella enterica subspecies Kintambo serotype fig|1224144. 4. peg. 4030; Dickinsonella CSL RW240 fig|568766. 10. peg. 2937; Dickie's bacterium NCPPB 3274 fig|1076549. 3. peg. 4260; Pantoea rhoda fig|548. 102. peg. 3401; Klebsiella aerogenes fig|630. 90. peg. 1795; Yersinia coli fig|79883. 5. peg. 266; Bacillus horikoshii fig|180861. 3. peg. 3762; Bacillus thuringiensis serovar Sumiyoshi fig|1390. 157. peg. 339; Bacillus starch-solubilizing fig|293386. 15. peg. 304; Stratospheric Bacillus fig|1053181. 3. peg. 3820; Bacillus cereus BAG2X1-3 fig|1884375. 3. peg. 681; Paenibacillus PDC88 fig|334735. 5. peg. 923; Korean spore-forming cocci fig|79884. 3. peg. 1120; Bacillus pseudoalkalinophilus fig|1628206. 3. peg. 4802; Bacillus LK2 fig|1396. 1605. peg. 6235; Bacillus cereus fig|182710. 3. peg. 317; Yihai Ocean Bacillus fig|860. 10. peg. 486; Clostridium periodontalis fig|1855308. 3. peg. 1467; Trichococcus ellipsis fig|931626. 3. peg. 151; Acetobacterium wustii DSM 1030 fig|1965575. 3. peg. 2547; Intestinal core bacteria genus An181 fig|1352. 2757. peg. 71; Enterococcus faecium fig|1299895. 3. peg. 900; Listeria monocytogenes CFSAN002349 fig|53346. 29. peg. 1591; Enterococcus mansoni fig|1649188. 10. peg. 1545; Listeria monocytogenes indica fig|158847. 6. peg. 432; Super Megamonas fig|1121289. 3. peg. 2775; Eat less Clostridiisalibacter DSM 22131 fig|1950885. 3. peg. 858; Clostridiales UBA4693 fig|1965576. 3. peg. 1978; Pseudoclosporium An184 fig|1952416. 3. peg. 1629; Ruminococcaceae bacteria UBA642 fig|1262803. 3. peg. 8; Clostridium CAG:413 fig|28037. 216. peg. 60; Mild Streptococcus fig|1074052. 3. peg. 33; Streptococcus tc-9 fig|1304. 207. peg. 1536; Streptococcus salivarius fig|1154859. 3. peg. 955; Streptococcus agalactiae LMG 14609 fig|1080071. 3. peg. 332; Streptococcus oris fig|1139219. 3. peg. 2194; Enterococcus dispar ATCC 51266 fig|1834176. 3. peg. 811; Enterococcus 3G1_DIV0629 fig|1622. 15. peg. 947; Lactobacillus murinus fig|565651. 6. peg. 1942; Enterococcus faecalis ARO1/DG fig|1473546. 3. peg. 703; Lysine Bacillus sp. BF-4 fig|37734. 13. peg. 137; Enterococcus carinii fig|492670. 92. peg. 623; Bacillus velesiae fig|1639. 1907. peg. 2641; Listeria monocytogenes fig|1123489. 3. peg. 170; Large Weilmannella DSM 19857 fig|1280687. 3. peg. 1880; Vibrio fibronectin YRB2005 fig|1262889. 3. peg. 680; Fungus CAG:38 fig|1235800. 3. peg. 2226; Lachnospiraceae bacteria 10-1 fig|1897035. 3. peg. 445; Firmicutes CAG:552_39_19 fig|199. 588. peg. 774; Simple Bend Fungus fig|1111133. 4. peg. 219; Bacteroides BV3AC2 fig|936589. 3. peg. 875; Veronella AS16 WP_070600378. 1; fig|1896998. 3. peg. 1750; Pseudomonas CAG:131 related_45_246 fig|41170. 3. peg. 3013; Microbacterium acetyltransferase fig|59620. 44. peg. 897; Uncultured Clostridium spp. fig|1262843. 3. peg. 313; Clostridium CAG:813 fig|1262834. 3. peg. 1287; Clostridium CAG:715 fig|1256219. 3. peg. 760; Lactobacillus paracasei subsp. paracasei Lpp230 fig|115778. 31. peg. 1994; Leuconostoc pyralis subsp. fig|29385. 174. peg. 531; Saprophytic Staphylococcus fig|1295. twenty one. peg. 75; Staphylococcus schleife fig|148814. 13. peg. 1360; Lactobacillus quinquefolius fig|1282. 1242. peg. 673; Staphylococcus epidermidis fig|1581078. 3. peg. 1186; Staphylococcus HMSC10C03 fig|1891097. 3. peg. 280; Macrococcus gattii WP_080703103. 1; fig|1214184. 3. peg. 1129; Streptococcus suis 22083 fig|1154771. 3. peg. 209; Streptococcus agalactiae FSL C1-487 fig|1415765. 3. peg. 1578; Mild Streptococcus 21/39 fig|1581074. 3. peg. 720; Granular Streptozoa HMSC31F03 fig|1349. 233. peg. 712; Streptococcus uberis fig|1946281. 3. peg. 392; Catarrhalis spp. UBA5893 fig|1328309. 5. peg. 1889; Lactobacillus plantarum IPLA88 fig|1214190. 3. peg. 2034; Streptococcus suis YS17 fig|29385. 135. peg. 2098; Saprophytic Staphylococcus fig|1715184. 3. peg. 1265; Aerococcus HMSC035B07 fig|1881068. 3. peg. 2940; Sphingomonas OV641 fig|1522072. 3. peg. 3829; Sphingolipids ba1 fig|1802172. 3. peg. 237; Sphingomonas RIFCSPHIGHO2_12_FULL_65_19 fig|1128204. 3. peg. 2189; Bradyrhizobium elsdenii CCBAU 43297 fig|1708715. 5. peg. 4517; Sword mushroom aridi fig|195105. 3. peg. 2062; Haemobacterium masaiense fig|1283312. 3. peg. 4182; Sphingomonas vermifuss DC-6 fig|1120654. 4. peg. 406; Scutellaria LC499 fig|529. 36. peg. 3144; Candida albicans fig|1194716. 3. peg. 4774; Sinorhizobium lucerne AK75 fig|1660088. 4. peg. 2967; Agrobacterium SCN 61-19 fig|1951259. 3. peg. 2515; Sphingomonasales bacterium UBA6174 fig|1912891. 5. peg. 2102; Sphingolipids fig|1670800. 3. peg. 1844; Rhizobia in the ocean fig|2032658. 3. peg. 157; α-Proteobacteria WMHbin7 fig|1819565. 5. peg. 2208; Marine Flavimaricola fig|1245469. 3. peg. 1160; Oligotrophic Bradyrhizobium S58 fig|1615890. 4. peg. 173; Bradyrhizobium LTSP849 fig|56454. 3. peg. 3464; Xanthomonas hortorum fig|40324. 384. peg. 1060; Oligotrophomonas maltophilia fig|1801972. 3. peg. 1832; Fungi RBG_19FT_COMBO_48_8 fig|1978765. 3. peg. 3488; Nitrospira ST-bin5 fig|2009322. 3. peg. 2770; Ophanophyta ohadii IS1 fig|1325564. 3. peg. 3733; Nitrospira japonica fig|43662. 9. peg. 1688; Pseudoalteromonas fish-killing fig|670. 134. peg. 4439; Vibrio parahaemolyticus fig|998520. 3. peg. 3325; Pseudoalteromonas agaricus fig|1723759. 3. peg. 401; Pseudoalteromonas P1-26 fig|672. 133. peg. 585; Vibrio vulnificus fig|1324960. 19. peg. 585; Aeromonas salmonis subsp. pectinolyticus 34mel fig|196024. 16. peg. 3965; Aeromonas hydrophila fig|654. 27. peg. 4266; Aeromonas veronii fig|1802253. 3. peg. 1045; Thiomonas RIFCSPLOWO2_12_36_12 fig|636. 16. peg. 3905;Edwardii tarda fig|1124958. 3. peg. 5012; Enteric Salmonella enterica subsp. Muenster serotype 0315 fig|573. 10007. peg. 225; Klebsiella pneumoniae fig|1946737. 3. peg. 4002; Luxenbergia spp. UBA1284 fig|1398203. 3. peg. 3712; Bacillus kraussei Quebec fig|615. 247. peg. 2151; Serratia marcescens fig|52441. 3. peg. 3752; Estuarine Nitrosomonas fig|1951948. 3. peg. 242; Mycelium spp. UBA2389 fig|165186. 29. peg. 27; Uncultured Ruminococcus spp. fig|2013842. 3. peg. 1881; HGW-Synergistetes-1 fig|411484. 7. peg. 436; Clostridium SS2/1 fig|460384. 4. peg. 447; Clostridium lavarroni fig|1761781. 3. peg. 2961; Clostridium DSM 8431 fig|1451. 25. peg. 614; Bacillus starch-degrading bacteria fig|1776378. 3. peg. 2009; Fukuoka Bacillus fig|1866315. 3. peg. 2122; Bacillus sp. N35-10-4 fig|1034836. 4. peg. 4077; Bacillus starch-solubilizing XH7 fig|1397. 14. peg. 5097; Bacillus annuli fig|1497681. 5. peg. 772; Listeria monocytogenes fig|1053224. 3. peg. 4333; Bacillus cereus VD021 fig|1374. 4. peg. 2798; Pinococcus kucuri fig|458233. 11. peg. 419; Macrococcus casei JCSC5402 fig|417368. 6. peg. 944; Enterococcus thailandensis fig|1353. 16. peg. 736; Enterococcus quail fig|1639. 1307. peg. 2578; Listeria monocytogenes fig|1649188. 4. peg. 450; Listeria monocytogenes indica fig|333990. 5. peg. 1279; Sarcobacterium AT7 fig|1121085. 3. peg. 4805; Bacillus edinii DSM 18341 fig|659243. 6. peg. 1163; Bacillus siamensis fig|1965645. 3. peg. 1428; another genus An54 fig|1950664. 3. peg. 363; Pseudomonas aeruginosa UBA5918 fig|681398. 3. peg. 1596; Jiangxi Parudibacillum fig|1947481. 3. peg. 1596; Sphingobacterium UBA1498 fig|1946424. 3. peg. 2345; Dysgonomonas genus UBA4861 fig|188932. 3. peg. 968; Low-temperature soil bacteria fig|505249. 7. peg. 1802; Marine Toxoplasma fig|1802259. 3. peg. 374; Thiomonas RIFOXYD12_FULL_33_39 fig|1872629. 13. peg. 663; Toxoplasma fig|497650. 4. peg. 949; Sulfur Bacteria enrichment culture pure line C5 fig|1981711. 3. peg. 707; Pseudomonas B8 (2017) fig|287. 926. peg. 3808; Green bacillus fig|157782. 3. peg. 183; Pseudomonas paraxanthinus fig|1225174. 5. peg. 576; Pseudomonas mendocina S5. 2 fig|237610. 8. peg. 4301; Psychrotolerant Pseudomonas fig|1116369. 3. peg. 182; Hefflera 108 WP_080858354. 1; fig|1679460. 3. peg. 2715; Deep-sea marine bacillus fig|1811547. 3. peg. 510; Redsea-S28_B5 fig|93684. 8. peg. 518; Salt-tolerant rose-colored bright fungus EMZ69714. 1; Escherichia coli 174900 fig|103796. 87. peg. 3165; Pseudomonas syringae var. kiwifruit WP_078828851. 1; Pantoea pineapple fig|2018067. 3. peg. 1734; Pseudomonas FDAARGOS_380 fig|294. 255. peg. 5151; Pseudomonas fluorescens fig|287. 4271. peg. 5445; Green Pseudomonas aeruginosa fig|46677. 3. peg. 3237; Pseudomonas chrysanthemi fig|83964. 10. peg. 849; Pseudomonas porri pathogenic variant fig|1932113. 4. peg. 2793; Pseudomonas PA1 (2017) fig|1712677. 3. peg. 189; Pseudomonas 2822-15 fig|1479235. 3. peg. 2741; Halomonas HL-48 fig|227946. 12. peg. 3247; Xanthomonas translucentus poae pathogenic variant fig|40324. 220. peg. 2801; Oligotrophomonas maltophilia fig|227946. 13. peg. 35; Xanthomonas translucentus poae pathogenic variant fig|487909. 15. peg. 4212; Xanthomonas translucentus pathogenic variant vimentin fig|40324. 145. peg. 2120; Oligotrophomonas maltophilia fig|1182783. 3. peg. 8; Xanthomonas campestris JX fig|1736581. 3. peg. 4144; Lysobacterium Root667 fig|470. 4256. peg. 2128; Acinetobacter baumanni fig|1804984. 3. peg. 4735; Burkholderia OLGA172 fig|1882792. 3. peg. 5959; Burkholderia CF145 fig|1458357. 5. peg. 7849;Caballeronia jiangsuensis fig|674703. 3. peg. 3992; Rhodozoa Z2-YC6860 fig|1230476. 3. peg. 595; Bradyrhizobium DFCI-1 fig|1752222. 3. peg. 1730; Rhizobiales bacteria Ga0077525 fig|1948848. 3. peg. 320; Patellar flora bacteria UBA6220 fig|1860092. 3. peg. 3966; α-Proteobacteria MedPE-SWcel fig|398. 7. peg. 3301; Tropical rhizobia fig|418630. 3. peg. 960; Giant Rhododendron fig|56. 40. peg. 5712; Soilbergia cellulose fig|1660160. 3. peg. 2510; Acidobacterium spp. SCN 69-37 fig|1661042. 3. peg. 2224; Pseudomonas NBRC 111127 fig|1712678. 3. peg. 4198; Pseudomonas 2822-17 fig|1736561. 3. peg. 128; Pseudomonas Root562 fig|76760. 8. peg. 1730; Pseudomonas rhodesianus fig|1295133. 4. peg. 7170; Pseudomonas faecalis JCM 18798 fig|1718917. 3. peg. 3132; Pseudomonas ICMP 460 fig|237306. 3. peg. 591; Pseudomonas syringae pathogenic variant peach fig|1079060. 3. peg. 1479; Pseudomonas savarroa v. bean pathogenic variant 1644R fig|1981714. 3. peg. 1068; Pseudomonas B5 (2017) fig|1419583. 3. peg. 4516; Pseudomonas mandellii PD30 fig|1718918. 3. peg. 4166; Pseudomonas ICMP 561 fig|64988. 7. peg. 76; Alkanophage fig|1961564. 3. peg. 685; Desulfovibrioideae bacterium UBA5546 fig|2004648. 3. peg. 1747; Acinetobacter WCHA39 fig|1080187. 3. peg. 399;Cupricobacterium UYPR2. 512 fig|76114. 8. peg. 258; Aromatic aromatic bacteria EbN1 fig|196367. 9. peg. 6286;Caballeronia sordidicola fig|1217418. 3. peg. 694; Copperophilus HPC(L) fig|1752216. 3. peg. 4007; Nitrosomonasales Ga0074132 fig|1249621. 3. peg. 3614; Cupricobacterium HMR-1 fig|179879. 8. peg. 6514; Burkholderia anthina fig|1246301. 3. peg. 4482; Controversy over bacteriophage B4 WP_092746164. 1; Acidobacterium valerianum fig|536. 30. peg. 857; Chromobacterium violaceum fig|1961112. 3. peg. 115; UTPLA1 of the Phylum Planctomycetes fig|44574. 5. peg. 4575; Nitrosomonas vulgaris fig|265901. 4. peg. 190; Photorhabdus J15 fig|80852. twenty one. peg. 1289; Vibrio votansalis fig|1136159. 3. peg. 2534; Cyclovibrio 1F111 fig|24. 6. peg. 4539; Shewanella putrefaciens fig|888433. 3. peg. 1974; Pseudoalteromonas GutCa3 fig|196024. 6. peg. 3651; Aeromonas hydrophila fig|1352943. 3. peg. 5028; Vibrio harveyi E385 WP_088124663. 1; Vibrio cholerae fig|29497. 15. peg. 1220; Vibrio scintillans fig|670. 413. peg. 1227; Vibrio parahaemolyticus fig|584. 91. peg. 3337; Proteobacterium mirabilis fig|263819. 5. peg. 201; Yersinia alexei fig|1656094. 3. peg. 1449; Alteromonas synapomorpha fig|634. 5. peg. 607; Yersinia burnetii fig|630. 85. peg. 4080; Yersinia coli fig|1212491. 3. peg. 1847; Legionella fallopian tuberculosis LLAP-10 fig|1498499. 3. peg. 2812; Legionella nolanii fig|1844092. 4. peg. 3143; Pseudomonas 8 R 14 fig|1441629. 3. peg. 2119; Pseudomonas chicory JBC1 WP_092369835. 1; Pseudomonas selenogenum fig|477228. 3. peg. 2040; Pseudomonas stutzeri TS44 fig|317. 249. peg. 4299; Pseudomonas syringae fig|1597. 16. peg. 2055; Lactobacillus paracasei fig|1184720. 6. peg. 2708; Anhui Rhizobium fig|1566263. 3. peg. 185; Rhizobium NFR03 fig|1951216. 3. peg. 1622; Rhizobium bacteria UBA1909 fig|1219052. 3. peg. 3572; Sphingomonas pallidum NBRC 15498 fig|376620. 8. peg. 122; Gluconobacter japonicus fig|1736587. 3. peg. 2638; Devospasella Root685 WP_011269850. 1; Xanthomonas campestris fig|1195246. 3. peg. 467; Shewanella spp. BL06 fig|1931276. 3. peg. 1294; Halocystis UPWRP_2 fig|1931204. 4. peg. 12; Syngenetic microorganisms fig|2052957. 3. peg. 3497; Pseudorabacterium MZDSW-24AT fig|1952825. 3. peg. 2772; UBA4205 of the family A. cerevisiae fig|1189622. 3. peg. 1716; Pseudomonas amygdaloids pathogenic variant 6605 fig|294. 173. peg. 2741; Pseudomonas fluorescens fig|294. 122. peg. 3307; Pseudomonas fluorescens fig|1198456. 3. peg. 4053; Pseudomonas glutenin fig|1855380. 3. peg. 1951; Pseudomonas Z003-0. 4C(8344-21) fig|1144330. 3. peg. 3879; Pseudomonas GM48 fig|86265. 3. peg. 2799; Pseudomonas thievery fig|1881035. 3. peg. 3817; Songjiang bacteria PDC51 fig|511. 8. peg. 774; fecal alkali-producing bacteria fig|1095552. 3. peg. 2955; Methylobacterium luteum IMV-B-3098 fig|1690268. 3. peg. 1172; Acidophage SD340 fig|871652. 3. peg. 1451; Sedimentation Poseidonocella fig|1946868. 3. peg. 175; Methylophaga UBA1490 fig|1924940. 3. peg. 1147; Marine microbial colonies fig|1912891. 7. peg. 5370; Sphingolipids fig|1236503. 3. peg. 1539; Acetobacterium serrata JCM 25330 fig|1745182. 3. peg. 1942; Paracoccus MKU1 fig|1112. 5. peg. 2875; Porphyromonas neolithospermum WP_051585410. 1; Sphingomonas paucimobilis fig|1082931. 4. peg. 3584; Halophilic Farbrachiatus B2 fig|1907665. 3. peg. 5475; Agrobacterium DSM 25558 fig|1841652. 4. peg. 3782; Agrobacterium 13-626 fig|1736312. 3. peg. 3441; Rhizobium Leaf262 fig|1768770. 3. peg. 4687; Pseudomonas CCH5-E12 fig|355591. 9. peg. 1867; Haemophilus versicolor fig|1869214. 4. peg. 1848; Rheinheimia fig|1946470. 3. peg. 3546; Rhodobacterium UBA2510 fig|1860090. 3. peg. 231; Roseobacillus MedPE-SWde fig|2020902. 8. peg. 1814; Genus Ponticaulis fig|940286. 3. peg. 3612; Bacillus 174Bp2 fig|1736380. 3. peg. 1842; Rhizobium Leaf453 fig|665126. 3. peg. 2283; Micrococcidiales hirschii fig|2029410. 3. peg. 1956; Mesorhizobium WSM4311 WP_003169203. 1; Brevundimonas diminuta fig|1884373. 3. peg. 3317; Mesorhizobium YR577 fig|989436. 3. peg. 3203; Pseudomycin Ad5 fig|1736359. 3. peg. 3976; Rhizobium Leaf386 fig|104102. 12. peg. 3797; Tropical Acetobacter fig|1500305. 3. peg. 4736; Rhizobium OK665 fig|1842535. 30. peg. 6; Gemmatimonas RAC04 fig|70775. 16. peg. 91; Pseudomonas aeruginosa fig|287. 4262. peg. 2063; Pseudomonas aeruginosa WP_017702484. 1; Pseudomonas syringae fig|1357292. 3. peg. 4700; Pseudomonas syringae pathogenic variant pea strain PP1 fig|287. 2436. peg. 3554; Green bacillus fig|76758. 3. peg. 4722; Pseudomonas orientalis fig|1904755. 3. peg. 3469; Pseudomonas 43NM1 fig|47879. 37. peg. 693; Pseudomonas undulatus fig|1771311. 3. peg. 1935; Pseudomonas ATCC PTA-122608 fig|2008975. 3. peg. 1976; Pseudomonas Irchel 3E13 fig|1259798. 3. peg. 1121; Pseudomonas LAMO17WK12:I2 fig|1736487. 3. peg. 2103; Neospirillum Root189 fig|1706231. 5. peg. 1557; Janssenella CG23_2 fig|1804984. 3. peg. 4700; Burkholderia OLGA172 fig|54067. 3. peg. 2925; Staphylococcus aureus fig|40324. 357. peg. 1426; Oligotrophomonas maltophilia fig|1967657. 4. peg. 913; Enteric Salmonella enterica subspecies Telelkebir serotype fig|573. 14330. peg. 816; Klebsiella pneumoniae fig|615. 357. peg. 16; Serratia marcescens fig|1122616. 3. peg. 231; Ocelothionemus beijerii DSM 7166 fig|314276. 4. peg. 1389; Baltic Sea-derived bacteria OS145 fig|1038921. 4. peg. 2790; Pseudomonas chlororaphis subsp. aurea 30-84 fig|292. 72. peg. 3280; Burkholderia cepacia fig|1899355. 18. peg. 947; Marine Spirillaceae bacteria fig|2015356. 3. peg. 5401; Burkholderia AU33647 fig|206665. 3. peg. 1731;Desulfurization bacteria on the seafloor fig|1987165. 3. peg. 2564; Sphingolipids GW456-12-10-14-TSB1 fig|1283312. 3. peg. 2182; Sphingomonas vermifuss DC-6 fig|1223566. 3. peg. 1810; Bradyrhizobium CCGE-LA001 fig|76761. 16. peg. 744; Pseudomonas veronii PIY00499. 1; Hydrophilales bacteria CG_4_10_14_3_um_filter_58_23 fig|305. 94. peg. 4778; Ralstonia solanacearum fig|1758178. 5. peg. 2546; Ethanol fast-growing Bacillus fig|1354263. 4. peg. 2524; Hafnia paraalvei ATCC 29927 fig|1125979. 3. peg. 1941; Rhizobium PDO1-076 fig|1338032. 3. peg. 3393; Vibrio parahaemolyticus O1:K33 strain CDC_K4557 fig|1898112. 54. peg. 3344; Rhodospirillaceae bacteria fig|1432558. 3. peg. 4265; Klebsiella pneumoniae ISC21 fig|333962. 3. peg. 2767; Providencia hirae fig|60552. 10. peg. 2414; Burkholderia vietnamense WP_011808964. 1; Helminbacter essenii fig|1844107. 4. peg. 2966; Pseudomonas 58 R 12 fig|1952916. 3. peg. 906; Intertrophobacteriaceae UBA5549 fig|458817. 8. peg. 542; Shewanella halifax HAW-EB4 fig|1674859. 3. peg. 1291; Spiro_03 of the order Spirochiales fig|1121434. 3. peg. 22; Desulfovibrio eleri DSM 10141 fig|1262899. 3. peg. 286; Fusobacterium CAG:439 fig|57320. 3. peg. 1084; Pseudodesulfovibrio deep fig|1736444. 3. peg. 3753;Acinetobacter Root1280 fig|1310670. 3. peg. 2122; Acinetobacter 907131 fig|505345. 6. peg. 150; Carinii genotype 3 fig|670. 887. peg. 4391; Vibrio parahaemolyticus fig|196024. 16. peg. 2750; Aeromonas hydrophila fig|663. 48. peg. 1321; Vibrio alginolyticus fig|28141. 133. peg. 4717; Cronobacter sakazakii fig|1117315. 3. peg. 3; Pseudoalteromonas marinum ATCC 14393 fig|1917164. 4. peg. 2739; Shewanella spp. UCD-KL21 fig|2006083. 3. peg. 3222; Photorhabdus CECT 9192 fig|584. 227. peg. 2146; Proteobacterium mirabilis fig|1792834. 4. peg. 1793; Sedimentary Marinella fig|1333513. 3. peg. 3775; Pseudoalteromonas maritima TAE56 fig|1305826. 3. peg. 1246; Streptomyces Amel2xC10 WP_048809063. 1; Microbacterium ginseng fig|1987376. 3. peg. 4246; Pseudonocardia N23 fig|164115. 3. peg. 6832; Nivea fungus fig|285676. 33. peg. 4994; Micromonospora sarellisii SFF52649. 1; Alnicotinella fig|1100822. 3. peg. 6408; Streptomyces AmelKG-E11A WP_098467790. 1; fig|1190417. 3. peg. 2916; Geoderma telluris SDS16714. 1; Carbonaceous agrobacteria fig|692370. 5. peg. 1108; Dongtan Alternaria alternata fig|1736370. 3. peg. 1383; Sphingomonas Leaf412 fig|1759074. 3. peg. 2615; Sphingomonas genus HIX fig|1120928. 3. peg. 922; Acinetobacter gingerii DSM 14971 = CIP 107465 fig|470. 4268. peg. 2032; Acinetobacter baumanni fig|1217627. 3. peg. 995; Acinetobacter baumanni NIPH 67 fig|28450. 149. peg. 5786; Burkholderia mallei fig|2032650. 3. peg. 3543; Magnetococcales bacteria HCHbin5 fig|101571. 169. peg. 3909; Uboburkholderia fig|396597. 7. peg. 2128; Burkholderia bifida MEX-5 fig|869212. 3. peg. 3514;Tuneriella parva DSM 21527 fig|1196083. 80. peg. 1885; Alveus Nordgrassella fig|1304886. 3. peg. 1257; Desulfurized Corynebacterium DSM 7044 fig|555. 16. peg. 1362; Carrot fungus subsp. carotenoids fig|1421338. 3. peg. 151; Enterobacter aegypti L1 fig|443144. 3. peg. 785; Geobacter M21 fig|1265503. 3. peg. 1271; Corveria piezophila ATCC BAA-637 fig|55601. 100. peg. 1601; Vibrio anguillarum fig|299766. 9. peg. 4890; Enterobacter steigerwaltii subsp. fig|243231. 5. peg. 1360; Geobacter sulfadioxidans PCA fig|1263083. 3. peg. 558; Klebsiella varicella CAG:634 fig|57706. 9. peg. 1106; Citrobacter brunneri fig|1619244. 3. peg. 1323; Enterobacter baumannii SHO56340. 1; Vibrio pentatus fig|688. 15. peg. 906; Vibrio rosenbergii fig|663. 75. peg. 4800; Vibrio alginolyticus fig|1967612. 3. peg. 4508; Enteric Salmonella Houghton subspecies 50:z4,z23:-serotype fig|82985. 3. peg. 3508; Spring Prague bacteria fig|55207. 5. peg. 1840;β-angiophore fig|582. 25. peg. 388; Morganella morganii fig|1006598. 5. peg. 80; Serratia marcescens RVH1 fig|82977. 3. peg. 3268; Butyrospermum vulgare CRY53703. 1; Yarrowia intermedia fig|595494. 3. peg. 706; Toluenemonas australis DSM 9187 fig|1217694. 3. peg. 3300; Acinetobacter sp.CIP 64. 2 fig|470. 2679. peg. 715; Acinetobacter baumanni fig|1879050. 4. peg. 2797; Acinetobacter wuhouensis fig|2004650. 3. peg. 1818; Acinetobacter chinensis fig|648. 80. peg. 1405; Aeromonas caviae fig|1217722. 3. peg. 1866; Pseudomonas S13. 1. 2 fig|294. 88. peg. 4234; Pseudomonas fluorescens fig|629262. 5. peg. 1917; Pseudomonas syringae Japanese pathogenic variant strain M301072 WP_053932309. 1; Pseudomonas coronatus fig|1844101. 3. peg. 4702; Pseudomonas 31 R 17 fig|380021. 13. peg. 6149; Protect Pseudomonas fig|287. 3716. peg. 2163; Green Pseudomonas aeruginosa fig|287. 3208. peg. 1464; Green bacillus fig|1952221. 3. peg. 555; Methylococcaceae UBA2780 fig|1869214. 3. peg. 3542; Rheinheimia fig|375286. 7. peg. 830; Janssenella Marseille fig|536. 26. peg. 4616; Chromobacterium violaceum fig|983548. 3. peg. 2977; Dokshima bacteria 4H-3-7-5 fig|307480. 5. peg. 1780; Aureobacterium frostii fig|1262921. 3. peg. 2213; Prevotella CAG:1185 fig|1965649. 3. peg. 4193; Butyrivibrio An62 fig|1951558. 3. peg. 3731; Chitinophage bacterium UBA4411 fig|1950669. 3. peg. 2035; Pseudomonas aeruginosa UBA6192 fig|1869230. 3. peg. 3025; Aureobacterium CBo1 fig|1500294. 3. peg. 2814; Aureobacterium YR485 fig|1756149. 11. peg. 2545; Elizabethella bruuniana fig|1137281. 3. peg. 1436; Jiemingjiao yellow seawater bacteria fig|1964365. 5. peg. 2525; Schnidella fig|28450. 428. peg. 5049; Burkholderia pseudomallei fig|1628751. 3. peg. 813; Lin's Nostoc z16 fig|60137. 10. peg. 1138; Pontiacillus sulfatide fig|1580596. 3. peg. 2701; Fish brown rod fungus fig|1041141. 4. peg. 4741; Rhizobium vulgaris biotype 128C53 WP_063290764. 1; Unclassified Pseudomonas fig|1816219. 4. peg. 1873; Corveria spp.PAMC 21821 fig|651. 3. peg. 13; Aeromonas intermedius fig|134375. 17. peg. 3222; Achromobacterium WP_011296194. 1; Copper-eating bacteria pinatubonensis fig|1513890. 4. peg. 2322; Pseudomonas chlororaphis fish subspecies fig|294. 193. peg. 980; Pseudomonas fluorescens fig|287. 2516. peg. 114; Green Pseudomonas aeruginosa fig|2006083. 3. peg. 3219; Photorhabditis spp. CECT 9192 fig|458817. 8. peg. 538; Shewanella halifax HAW-EB4 fig|1073383. 3. peg. 1289; Aeromonas veronii AMC34 fig|190893. 14. peg. 2110; Vibrio corallii fig|663. 144. peg. 2659; Vibrio alginolyticus fig|55601. 106. peg. 926; Vibrio anguillarum fig|669. 51. peg. 5531; Vibrio harveyi fig|1250059. 5. peg. 3511; Myxobacterium MAR_2009_124 fig|906888. 6. peg. 2449;Nonlabens ulvanivorans WP_042276051. 1;SedimentNonlabens fig|1953167. 3. peg. 1254; Pseudomonas aeruginosa UBA6221 fig|991. 14. peg. 629; Echinococcus fig|1121890. 3. peg. 5; Flavopsora frigida DSM 17623 fig|387094. 4. peg. 1115; Flavobacterium hussifolium fig|1946558. 3. peg. 2413; Flavobacterium UBA7665 fig|253. 27. peg. 2882; Indole-producing Aureobacterium fig|1685010. 5. peg. 4376; Ice goldenrod fig|1500289. 3. peg. 4103; Aureobacterium OV705 fig|1500298. 3. peg. 2823; Aureobacterium YR561 fig|1797331. 3. peg. 2180; Pseudomonas GWE2_29_8 fig|1947498. 3. peg. 1366; Sphingobacterium spp. UBA4616 WP_074239321. 1; Niab Chitinophage fig|192149. 7. peg. 174; Murina fig|718222. 3. peg. 4924; Bacillus cerevisiae TIAC219 fig|1053210. 3. peg. 211; Bacillus cereus HuB4-10 fig|2026089. 3. peg. 6077; Bacillus XY044 fig|1938610. 3. peg. 3678; Flavobacterium LM5 fig|1947482. 3. peg. 632; Sphingobacterium UBA1575 fig|1948844. 3. peg. 823; Patellar flora bacteria UBA6130 fig|986. 7. peg. 83; Flavobacterium johnsonii fig|1950382. 3. peg. 461; Pseudomonas aeruginosa UBA1181 fig|1947145. 3. peg. 376; Prevotella UBA3765 fig|1122989. 3. peg. 367; Prevotella oralis DSM 18711 = JCM 12252 fig|1896974. 3. peg. 2001; Pseudomonas 43_108 fig|2014804. 3. peg. 4306; Levinellaceae SD302 fig|1428. 517. peg. 2380; Bacillus thuringiensis fig|1428. 574. peg. 3351; Bacillus thuringiensis fig|1428. 590. peg. 5685; Bacillus thuringiensis fig|720554. 3. peg. 188; Clostridium forskohlii DSM 19732 fig|1122203. 4. peg. 2283; Halococcus halophilus DSM 16375 fig|1462525. 3. peg. 3489; Deep-sea Bacillus TM-1 fig|1395513. 3. peg. 363; Lactobacillus lactis DSM 442 fig|1262834. 3. peg. 1229; Clostridium CAG:715 SCI87282. 1; Uncultured Roseburia fig|1952116. 3. peg. 2349; Lachnospiraceae bacterium UBA6480 WP_069150959. 1; Lachnospiraceae fig|1265. 10. peg. 3100; yellow Ruminococcus fig|1120998. 3. peg. 2858; Anaerovorax odorimutans DSM 5092 WP_072702499. 1; Butyrivibrio hungarianensis fig|1232453. 3. peg. 2795; Clostridiales VE202-21 fig|39485. 11. peg. 251; Lachnospiraceae WP_072832325. 1; fig|1509. twenty four. peg. 2487; Clostridium sporogenes SCI88558. 1; Uncultured Clostridium spp. fig|1490. 6. peg. 2635; Bifermentative Clostridium fig|1947399. 3. peg. 1322; Clostridium hengenii UBA3548 fig|1953138. 3. peg. 796; Pseudomonas aeruginosa UBA1312 fig|1950875. 3. peg. 364; Clostridiales UBA4139 fig|1396. 1518. peg. 4860; Bacillus cereus fig|1305675. 3. peg. 2174; Bacillus solimangrovi fig|1423. 436. peg. 4365; Bacillus subtilis fig|361277. 6. peg. 1539; Geobacter saccharophilus fig|1392. 356. peg. 4724; Bacillus anthracis fig|1053189. 3. peg. 4219; Bacillus cereus BAG5X1-1 WP_079442297. 1; Chromium-reducing Clostridium fig|1953262. 3. peg. 783; Candidate Omnitrophica bacteria UBA1562 fig|1797955. 3. peg. 2304; RIFOXYA12_FULL_51_18 fig|1953111. 3. peg. 2436; Acidobacterium spp. UBA7540 WP_099010551. 1; Escherichia coli retrotransposon-Eco1 (Ec86) fig|1005565. 3. peg. 1153; Escherichia coli 3006 fig|158822. 8. peg. 1905;Sindiaceae neislui fig|1444060. 3. peg. 4830; Escherichia coli 4-203-08_S1_C1 fig|29484. twenty two. peg. 4571; Yersinia freundii fig|529823. 3. peg. 332; Vibrio OA-2007 fig|48296. 218. peg. 3142; Acinetobacter pitei fig|550. 518. peg. 3445; Enterobacter vulvae fig|204773. 6. peg. 4; Arsenic-oxidizing Bacillus serrata fig|670. 190. peg. 4348; Vibrio parahaemolyticus fig|44577. 7. peg. 209; Urea Nitrosomonas fig|1125747. 3. peg. 1; Algae-degrading water-dwelling bacteria NO2 fig|1338034. 3. peg. 2437; Vibrio parahaemolyticus O1:Kuk strain FDA_R31 fig|1952844. 3. peg. 2619; Rhodotorulaceae UBA5533 fig|1288788. 3. peg. 2384; Vibrio parahaemolyticus 3631 fig|644. 31. peg. 975; Aeromonas hydrophila fig|498292. 3. peg. 28; Swinging yellow bacillus fig|1948088. 3. peg. 4515; Firmicutes bacteria UBA6132 fig|1408433. 3. peg. 3094; Saffron yellow thread fungus catalasitica ATCC 23190 WP_074236572. 1; Aureobacterium zeae fig|1127353. 3. peg. 1738; Enteric Salmonella enterica subsp. Newport serotype #11-4 fig|1881110. 4. peg. 120; Pantoea sesame fig|34038. 6. peg. 27; Aquatic Rhabditis elegans fig|630. 95. peg. 4256; Yersinia coli fig|149387. 11. peg. 1139; Enteric Salmonella enterica subspecies Brandenburg serotype fig|1343738. 3. peg. 2232; Vibrio cholerae 2012EL-1759 retrotransposon-Vch3 (Vc137) fig|1423. 175. peg. 4339; Bacillus subtilis fig|2021695. 3. peg. 3399; Bacillus 7894-2 fig|189426. 10. peg. 597; Odor-like Bacillus fig|2020949. 3. peg. 856; Rombutsiella weinsteinii fig|1243664. 3. peg. 1004; Bacillus martensii fig|1855345. 3. peg. 2971; Bacillus sp.RRD69 fig|1946358. 3. peg. 2514; Clostridium UBA4108 fig|1520. 90. peg. 2502; Clostridium beijerinckii fig|79672. 3. peg. 288; Bacillus suyungensis serotype Medellin fig|189426. 19. peg. 3973; Odor-like Bacillus fig|1497. 3. peg. 4049; Clostridium formoaceticum fig|169760. 4. peg. 4269; Bacillus asterospora WP_073588670. 1;Anaerocolumna xylanovorans fig|1950815. 3. peg. 1585; Clostridiales UBA1341 fig|1897004. 3. peg. 2166; Fungus 45_250 fig|1946293. 3. peg. 290; Catarrhalis spp. UBA7571 fig|1796620. 3. peg. 3489; Acute bacillus in mice fig|76857. 53. peg. 2245; Fusobacterium nucleatum polymorphic subsp. fig|2013784. 3. peg. 1260; Firmicutes bacteria HGW-Firmicutes-3 fig|79884. 3. peg. 1106; Bacillus pseudoalkalinophilus fig|135461. 47. peg. 1552; Bacillus subtilis subsp. subtilis WP_016122013. 1; Bacillus cereus fig|1499688. 3. peg. 3214; Bacillus LF1 fig|1318. 8. peg. 1495; Streptococcus parahaemolyticus fig|1381091. 3. peg. 1173; Streptococcus equi subspecies zooepidemicus SzAM60 fig|1409369. 3. peg. 815; Staphylococcus aureus AMMC6050 fig|1497681. 5. peg. 3014; Listeria monocytogenes fig|1095727. 3. peg. 409; Streptococcus SK643 fig|1318. 12. peg. 313; Streptococcus parahaemolyticus fig|1681184. 3. peg. 4899; Lysine Bacillus ZYM-1 fig|561879. 29. peg. 3569; Bacillus safari fig|1884359. 3. peg. 2978; Psychrobacterium OK028 fig|29367. 3. peg. 1112; Clostridium pomegranate fig|225345. 3. peg. 1103; Chromium-reducing Clostridium fig|1345695. 10. peg. 2438; Clostridium saccharobutylicum DSM 13864 fig|119641. 3. peg. 784; Clostridium uliginosum fig|1761781. 3. peg. 88; Clostridium DSM 8431 fig|1946346. 3. peg. 2999; Clostridium UBA1056 fig|1492. 48. peg. 3952; Clostridium butyricum fig|1121302. 3. peg. 4511; Clostridium cavendishi DSM 21758 fig|398512. 4. peg. 5301; Pseudomonas aeruginosa ATCC 35603 = DSM 2933 fig|1946357. 3. peg. 500; Clostridium UBA3947 fig|642492. 3. peg. 3338; Cellulosilyticum lentocellum DSM 5427 fig|1946690. 3. peg. 1553; Intestinal core bacteria genus UBA3320 fig|397290. 3. peg. 150; Lachnospiraceae A2 fig|97138. 3. peg. 1713; Clostridium ASF356 fig|1965545. 3. peg. 499; Theileria An114 fig|1047063. 3. peg. 240; WS1 bacteria JGI 0000059-K21 fig|1192034. 3. peg. 1508; Chondroitinib spiculate DSM 436 AAA88323. 1; Myxococcus flavus retrotransposons-Mxa2 (Mx65) fig|33. 8. peg. 8196; Myxococcus aurantiacus fig|215803. 3. peg. 1485; Salt water slime bacteria fig|1406225. 3. peg. 2150; Purple Protothecoides Cb vi76 fig|1952931. 3. peg. 5137; Verrucomicrobia 3 bacteria UBA6082 fig|1972460. 3. peg. 279; Anaerobic bacteria 4572_78 fig|1950201. 3. peg. 3297; Anaerobic Rhizobacteriales bacterium UBA2796 WP_015247705. 1; fig|1799658. 3. peg. 2777; SCGC AG-212-F19 of the family Planctomycetes fig|214688. 26. peg. 6659; Cryptococcus UQM 2246 fig|2023130. 3. peg. 4250; Pyricularia MGV fig|52. 7. peg. 9046; Chondroitinibacterium safflower fig|448385. 16. peg. 2914; Soilbergia cellulose So ce56 fig|888845. 4. peg. 14202; Microcystis rosea fig|1752210. 3. peg. 1275; δ-Proteobacteria Ga0077539 fig|1391654. 3. peg. 3562;Labilithrix luteola fig|1752218. 3. peg. 3670; Fungus family Ga0077529 WP_006981058. 1; Xanthomonas flavus fig|1952939. 3. peg. 2584; Verrucomicrobiaceae UBA1938 fig|2024858. 3. peg. 3711; Genus Sandaracinus WP_009096166. 1; Pyrrophyta SWK7 fig|595453. 3. peg. 1506; Pyricularia SM50 fig|1263868. 3. peg. 4100; European red pear-shaped fungus SH398 fig|167547. 3. peg. 303; Marine Prochlorococcus strain MIT 9311 fig|1499501. 3. peg. 459; Prochlorococcus SS52 fig|1905359. 3. peg. 4335; Marine bacteria AO1-C WP_002700020. 1; Microtremorum marineum fig|1913989. 145. peg. 263; Gammaproteobacteria WP_073154989. 1; Seinosporium philum fig|46223. 3. peg. 3648; Biqi high-temperature yellow micrococcus fig|1329796. 3. peg. 1947; Marseille Risungbinella fig|1123252. 3. peg. 3225; Kribissoma shimamotoi DSM 45090 fig|714067. 3. peg. 3719; Kloppensteadia ivory fig|1341151. 3. peg. 628; Leucocephalus saccharus 1-1 fig|2026763. 3. peg. 3089; Myxococcal bacteria fig|1797895. 3. peg. 2518;δ-Proteobacteria RIFOXYA12_FULL_58_15 fig|373672. 4. peg. 4082; Aureobacterium gambryanum fig|1416778. 5. peg. 4374; Peanut goldenrod fig|1603293. 4. peg. 829; Flavobacterium 316 CCB70859. 1; Flavobacterium gillophilum FL-15 fig|1986952. 3. peg. 951; Sphingobacteriaceae GW460-11-11-14-LB5 fig|1476464. 3. peg. 4304; Xixi soil bacteria fig|1761785. 3. peg. 3112; Flavobacterium ov086 fig|1664068. 3. peg. 3690; Bacteria 336/3 fig|880071. 3. peg. 3500;Bernardetia litoralis DSM 6794 fig|1121902. 3. peg. 2906; Eisenbergia elegans DSM 3317 fig|1509483. 4. peg. 1923; Bacillus spp. BAB-3569 fig|1166018. 3. peg. 5312;Fibrella aestuarina BUZ 2 fig|634771. 3. peg. 967; Chitinophaga essenii fig|29529. 3. peg. 396; Chitinophages arvensis fig|1891659. 3. peg. 6032; Chitinophage CB10 fig|2033437. 3. peg. 3442; Chitinophage MD30 fig|1004. 4. peg. 1677; Chitinobacterium ginseng fig|1881041. 3. peg. 3698; Chitinophage YR627 fig|1123078. 3. peg. 2010; Archaeotrophic fungus DSM 19591 fig|354355. 3. peg. 1846; Rongzhou Agricultural Research Institute Mycobacterium fig|1951546. 3. peg. 1574; Chitinophage bacterium UBA1946 fig|1812911. 3. peg. 633; Flavobacterium CACIAM 22H1 fig|477680. 4. peg. 4668; Lineomonas imperfecta fig|221126. 7. peg. 3781; Select algae bacteria fig|342954. 4. peg. 734; Algae lake bacteria fig|1871037. 5. peg. 2180; Flavobacteriaceae fig|669041. 3. peg. 843; Myxobacterium bicentrum WP_074538568. 1; Baltic fiber phage fig|1248440. 3. peg. 1511; Franzmannii ATCC 700399 fig|1121007. 3. peg. 1574; Musselsia marinum DSM 19832 fig|688867. 3. peg. 2332; Ohtaekwangia, South Korea fig|926565. 3. peg. 717; Myxococcosis fibrosus DSM 11118 fig|1257021. 3. peg. 5265; Pyrochromocytaceae bacteria 311 fig|2044937. 5. peg. 2350; Candidate division KSB3 bacteria fig|1499966. 3. peg. 180; Candidate Moduliflexus fig|1948269. 3. peg. 1254; Verrucomicrobia UBA6053 fig|694433. 3. peg. 3103; Giant saprophytic spirochetes DSM 2844 fig|2008677. 3. peg. 3337; Songjiang nodosa fig|946333. 3. peg. 3570; Rhizobium gloeosporum fig|1736433. 3. peg. 5559; Rhizobium Root1221 fig|1500265. 3. peg. 5760; Methylobacterium YR605 fig|1121349. 4. peg. 2836; Composting Commissurotomus DSM 21721 fig|1082851. 3. peg. 91; serinivorans fig|1121480. 5. peg. 5468; Pseudomonas pseudomallei DSM 15887 fig|2045208. 3. peg. 1247; Purple-black Marseilles fig|1736455. 3. peg. 3692; Marsella Root133 fig|34073. 25. peg. 8569; Debate on phages fig|1884311. 3. peg. 7121; Coprophagnum OK202 fig|1123487. 3. peg. 1763;Uliginosibacterium gangwonense DSM 18521 fig|2029111. 3. peg. 3032; Combretum bacterium NML120219 fig|1977087. 20. peg. 1226; Proteobacteria fig|754436. 4. peg. 4454; Non-luminescent Photorhabditis spp. fig|265726. 7. peg. 1038; Halotoxinia fig|1121867. 3. peg. 59; Enterobacter calvei DSM 14347 fig|1238431. 3. peg. 2655; Vibrio nigromaculata BLFn1 fig|1384589. 3. peg. 2721; [Evansella] Oil gourd fig|1261127. 3. peg. 2947; Citrobacter malonic acid-free Y19 fig|349521. 8. peg. 3588;Hahella chejuensis KCTC 2396 fig|525918. 3. peg. 1501; Caldifontis fig|1737490. 4. peg. 4974; Agrobacterium spp. fig|251229. 3. peg. 427; PCC 7203 of Pseudochromococcus pyogenes fig|1245923. 3. peg. 9587; Mikimoto's pseudoclade VB511283 fig|1503470. 5. peg. 10896; Cyanobacteria TDX16 fig|2005460. 3. peg. 1118; Chondrocystis NIES-4102 fig|179408. 3. peg. 4679; PCC 7112 fig|1612423. 3. peg. 5384; Lin's Nostoc z1 fig|63737. 11. peg. 472; Nostoc punctata PCC 73102 fig|224013. 5. peg. 7163; Pond-grown Nostoc CENA21 fig|1932621. 3. peg. 7363; Nostoc T09 fig|373994. 3. peg. 3383; PCC 7116 fig|2005463. 3. peg. 257; NIES-4105 fig|2005459. 3. peg. 7019; Monophyllium NIES-4075 fig|184925. 3. peg. 2602; Pseudomonas flexneri PCC 9212 fig|454136. 5. peg. 3127; Suspicious phyllophyta IAM M-71 fig|203124. 6. peg. 2732; Red Sea Trichodesmium IMS101 fig|2040638. 3. peg. 3067; Buchnerella FEM_GT703 Fig|1880991. 4. peg. 2927; Cyanobacteria tremuloides USR001 fig|1173028. 3. peg. 7115; Pseudomonas PCC 10802 fig|568701. 4. peg. 2073;Moorea bouillonii PNG fig|927677. 3. peg. 4187; Synechocystis PCC 7509 fig|179408. 3. peg. 6267; Dark Green Algae PCC 7112 fig|1710894. 3. peg. 2079; Aphanizomenon LD13 fig|1947888. 3. peg. 4484; Cyanobacteria UBA6047 fig|1705388. 3. peg. 1178; Pseudohylostomium SR001 fig|454136. 5. peg. 4162; Suspicious phyllophyta IAM M-71 fig|2005458. 3. peg. 378; Nostoc NIES-4103 fig|1947874. 3. peg. 4590; Cyanobacteria UBA1583 fig|1781255. 3. peg. 802; Desertifilum genus IPPAS B-1220 fig|1128427. 4. peg. 2346; ESFC-1 fig|1946321. 3. peg. 3928; Chlorobacterium UBA7656 fig|118173. 3. peg. 1030; Pseudoanabaena PCC 6802 fig|1922337. 4. peg. 4802; Genus Hensonii fig|927668. 3. peg. 2766; Pseudoanabaena biceps PCC 7429 fig|1173020. 3. peg. 6191; Microcystis PCC 6605 fig|329726. 14. peg. 4440; Marine Acaryochloris MBIC11017 fig|215803. 3. peg. 1649; Salt water slime bacteria fig|1920190. 3. peg. 9548; Protothecoides Cb G35 fig|1961464. 3. peg. 5181; Myxococcal bacteria UBA2376 fig|765913. 3. peg. 336; Rhodococcus delbrueckii AZ1 fig|1396141. 3. peg. 2891; Halostyra BvORR071 fig|1961463. 3. peg. 5253; Myxococcales UBA1671 AAL40743. 1; Nephrolepis erossima retrotransposons-Nex2 (Ne144) fig|54. 3. peg. 4123; Erosion fungus fig|53367. 3. peg. 3417; Asanoella rustaflora fig|460265. 11. peg. 3882; Methylobacterium nodosa ORS 2060 fig|298794. 3. peg. 462; Methylobacterium mutans fig|190148. 4. peg. 3492; Bradyrhizobium paxllaeri fig|1075417. 3. peg. 445; Catalinimonas alkaloidigena fig|1429438. 4. peg. 7505; Candidate endothelial factor fig|1977087. 12. peg. 1756; Proteobacteria fig|92487. 3. peg. 3972; Sulfur fungus eikelboomii fig|1977087. 20. peg. 1473; Proteobacteria fig|1123400. 3. peg. 3276; Flexible Thiofilum DSM 14609 fig|34062. 8. peg. 73; Oslo Moraxella fig|1699623. 3. peg. 1502; Psychrobacterium P11G3 fig|1123509. 3. peg. 848; Zooshikella ganghwensis DSM 15267 fig|2026735. 3. peg. 2222;ζ-Proteobacteria fig|1977087. 12. peg. 2982; Proteobacteria fig|2026763. 4. peg. 1195; Myxococcal bacteria fig|1977087. 12. peg. 510; Proteobacteria fig|1123508. 3. peg. 7252; Zavarzinella formosa DSM 19928 fig|214688. 26. peg. 3091; Cryptococcus UQM 2246 fig|1908690. 5. peg. 1204; Fimbriiglobus ruber fig|1805126. 3. peg. 4431;δ-Proteobacteria CG2_30_63_29 fig|1882752. 4. peg. 1962; Singulisphaera genus GP187 fig|1636152. 3. peg. 5364; Fungus SH-PL62 APR75442. 1; Microcystis rosea fig|54. 3. peg. 8798; Erosion fungus fig|980254. 4. peg. 4083;Roseimaritima ulvae fig|1856297. 3. peg. 3627; Gammaproteobacteria 45_16_T64 fig|1219077. 3. peg. 1945; Vibrio fargesii NBRC 104587 fig|1334629. 3. peg. 167; Myxococcus aurantiacus 124B02 AAA25405. 1; Myxococcus flavus retrotransposons - Mxa1 (Mx162) fig|378806. 16. peg. 4444; Orange-colored pile fungus DW4/3-1 WP_002615305. 1; Auricularia aurantiaca retrotransposase-Sau1 (Sa163) fig|48. 3. peg. 757; Transitional Protothecoides fig|448385. 16. peg. 2083; Soilbergia cellulose So ce56 fig|52. 7. peg. 5100; Chondroitinibacterium safflower fig|1752210. 3. peg. 5621; δ-Proteobacteria Ga0077539 fig|2024858. 3. peg. 6345; Genus Sandaracinus WP_012826728. 1; Halobacterium ochraceum fig|927083. 3. peg. 3408;Sandaracinus WP_006977315. 1; Paracystis pacifica fig|1400863. 5. peg. 627; Candidate denitrifying competitive Bacillus Run_A_D11 fig|1961463. 3. peg. 4303; Myxococcal bacteria UBA1671 fig|1898731. 3. peg. 3099; Competitive Bacillus MCBA15_001 fig|1279028. 3. peg. 3374; Competitive Bacillus 314Chir4. 1 fig|1898733. 3. peg. 2056; Competitive Bacillus MCBA15_004 fig|1795630. 3. peg. 3476; Pseudomonas PAMC 28766 fig|2033654. 3. peg. 3461; Competitive Bacillus 'Ferrero' fig|1736329. 5. peg. 1436; Leaf304 fig|1736292. 3. peg. 1083; Lassa Leaf185 fig|1736327. 3. peg. 206; Lassa Leaf296 fig|1736311. 3. peg. 3668; Competitive Bacillus Leaf261 fig|1736308. 3. peg. 2333; Psoraleaf254 fig|656366. 8. peg. 2905; Alpine arborvitae fig|494023. 3. peg. 138; Antarctic Glutamibacterium ASN40093. 1; Arthrobacter 7749 fig|1494608. 3. peg. 465; Arthrobacter PAMC 25486 fig|656366. 4. peg. 2620; Alpine arborvitae fig|656366. 3. peg. 1944; Alpine arborvitae fig|1132441. 3. peg. 1888; Arthrobacter 35W fig|1704044. 3. peg. 520; Arthrobacter ERGS1:01 fig|1496689. 3. peg. 681; Arthrobacter L77 fig|1681197. 3. peg. 149; Arthrobacter RIT-PI-e fig|37921. 12. peg. 1481; Agile rod fungus fig|1736303. 3. peg. 982; Arthrobacterium Leaf234 fig|1312978. 3. peg. 1472; Arthrobacter H41 fig|1348338. 3. peg. 1472;Rafsonia rubber CMS 76R fig|1452536. 3. peg. 1955; Microbacterium Cr-K20 fig|1736525. 3. peg. 446; Revsonella Root4 fig|1529318. 3. peg. 434; Thermobacterium MLB-32 fig|1267973. 3. peg. 3479; Arthrobacter H5 fig|150121. 3. peg. 1900; Agrobacterium prausnitzii fig|123316. 3. peg. 955; Agrelia VKM Ac-2052 fig|1052260. 3. peg. 3617; Soil Klenkia fig|1566299. 3. peg. 3962; Marine Klenkia fig|1736356. 3. peg. 3150; Modena Leaf380 fig|1736354. 3. peg. 1787; Geoderma Leaf369 fig|479431. 6. peg. 3115; Nakamurella multipartita DSM 44233 fig|1090615. 3. peg. 2397; Nakamurella panacicegetis fig|1306174. 4. peg. 4778; Aurantiacum JCM 3230 fig|546871. 3. peg. 1120;Flemingella flavum fig|630515. 4. peg. 525; Soil Pleurotus eryngii fig|546874. 3. peg. 1181; Freidensis sagamiharensis BAK35674. 1; Phosphate-accumulating NM-1 fig|1380390. 4. peg. 72; Soil Rhodococcales bacteria URHD0059 fig|1283299. 3. peg. 2784; Bindella woodi Iso977N fig|929712. 3. peg. 3165; DSM 18081 of DSM 18081 fig|1123262. 3. peg. 3125; Soil Rhodobacterium DSM 22325 fig|1861. 4. peg. 5240; Dark-striped dermatophyte fig|1137993. 4. peg. 1318; African dermatophytes fig|1070870. 3. peg. 778; Dermatophagoides nigromaculata fig|1190417. 3. peg. 1785; Geodermatophytes telluris fig|477641. 3. peg. 1023; Marine Mordecai bacteria fig|1798228. 3. peg. 3756; Staphylococcus DSM 46838 WP_091929708. 1; Staphylococcus DSM 46786 SHH20361. 1; Endophytes of leprosy tree fig|1844. 3. peg. 1151; Nocardia lutea fig|748909. 6. peg. 1418; Nocardia alpina fig|402596. 3. peg. 987; Nocardia albicans fig|1736322. 3. peg. 1963; Nocardia Leaf285 fig|1445613. 3. peg. 3490; Marine South China Sea Institute of Bacteria SE31 fig|543632. 4. peg. 9742; Subtropical zoospermic actinomycetes fig|1036182. 3. peg. 2958; dark orange motile actinomycetes fig|1246995. 3. peg. 737; Actinozoa friuliensis DSM 7358 fig|56427. 3. peg. 3052; Coccidioides cyanobacteria subsp. cyanobacteria fig|1710355. 3. peg. 2225; Actinomycetes TFC3 fig|649831. 3. peg. 2352; Actinomycetes N902-109 fig|35754. 4. peg. 6321; Dactylocystis citrinum fig|1881. 4. peg. 2703; Micromonospora chlororaphis fig|47863. 3. peg. 3975; Micromonospora sphaeroides fig|285665. 4. peg. 2050; Micromonospora spp. fig|1192034. 3. peg. 4668; Chondroitinib spicata DSM 436 fig|1198133. 3. peg. 2442; Myxococcus flavus DZ2 fig|33. 8. peg. 521; Myxococcus aurantiacus fig|394193. 3. peg. 7794; Amycota saalfeldensis fig|369932. 4. peg. 5621; Niigata pseudomycotic acid bacteria fig|1238180. 3. peg. 5340; Amycolatopsis coelicolor DSM 43854 fig|589385. 3. peg. 7940; Xylan-producing Amycolatopsis fig|1068980. 3. peg. 1527; Amycolatopsis nigromaculata CSC17Ta-90 fig|1854586. 3. peg. 2100; Antarctic pseudomycotic bacteria fig|587909. 3. peg. 3086; Desert Jade Bacteria fig|2030. 3. peg. 3051; Xerocystis fig|1382595. 4. peg. 3164; Saccharopolyspora rubrum D WP_013675061. 1; Pseudonocardia spp. fig|1660131. 3. peg. 2805; Pseudonocardia SCN 72-86 fig|366584. 3. peg. 4349; Pseudonocardia oxyphylla fig|1885031. 4. peg. 5241; Pseudonocardia Ae331_Ps2 fig|1690815. 5. peg. 5350; Pseudonocardia HH130630-07 fig|1123023. 3. peg. 3229; Pseudonocardia acacia DSM 45401 fig|1449976. 3. peg. 8114; Kutzneria albida DSM 43870 WP_007238159. 1; fig|1220583. 3. peg. 1582; Gordonia aichiensis NBRC 108223 GAB07179. 1; Gordonia sludge NBRC 15530 fig|1223545. 3. peg. 704; Soil Gordonia NBRC 108243 fig|1223540. 3. peg. 3237;Gordonella desulfuricans NBRC 100010 fig|1112204. 3. peg. 4913; Gordonia polyisoprene-feedingensis VH2 AFR49048. 1; Gordonia KTR9 fig|402289. 3. peg. 1558; Rhodococcus HA99 fig|1077144. 3. peg. 224; Dietzella alimentaria 72 fig|1344003. 3. peg. 1864; Pinellia fig|1463823. 3. peg. 3407; Microbial spores NRRL B-24597 fig|1903117. 3. peg. 1566; William's genus 1138 fig|1603258. 4. peg. 1828; Williamsella herbipolensis fig|644548. 3. peg. 652; Clouded Leopard Feces Gordonia NRRL B-59395 fig|1136941. 3. peg. 1310; Gordonia phthalatica fig|1223542. 3. peg. 3439; Sewage Gordonia NBRC 108250 fig|47312. 10. peg. 4232; Pulmonaria fig|57704. 14. peg. 421; Tyrosine-resistant T. Tsukamura fig|521096. 6. peg. 2443; slightly changed Tsukamura fungus DSM 20162 fig|1123241. 3. peg. 3642; Nakamurella lactea DSM 19367 fig|1210073. 4. peg. 1031; Nocardia salinarum NBRC 100378 fig|1206740. 4. peg. 4617;Thai Nocardia NBRC 100428 fig|1210064. 4. peg. 2434; Nocardia altamilis NBRC 108246 fig|1123258. 3. peg. 1651; Neocystis DSM 44881 = NBRC 103563 fig|1443888. 3. peg. 2891; Rhodococcus fasciatus 02-815 fig|1517936. 4. peg. 882; Rhodococcus CUA-806 fig|398843. 6. peg. 4214; Rhodococcus kyonii fig|1813677. 3. peg. 4031; Rhodococcus EPR-157 WP_008711873. 1; Unnamed organism fig|1381122. 3. peg. 6103; Rhodococcus erythropolis DN1 fig|1736210. 3. peg. 2766; Rhodococcus Leaf7 fig|1736300. 3. peg. 1279; Rhodococcus Leaf225 fig|1219012. 3. peg. 1705; Rhodococcus NBRC 14404 fig|1219023. 3. peg. 2791; Rhodococcus spp. NBRC 100604 fig|616997. 3. peg. 2548; Altamira fig|1303689. 4. peg. 2934; Korean Rhodococcus JCM 10743 = NBRC 100607 fig|644. 85. peg. 4392; Aeromonas hydrophila. Using sequence listings to identify homologous RT/ncRNA pairs

By identifying that the ncRNA sequences in Table B and the RT sequences in Table A have the same genome accession number indicated for each sequence in the sequence listing , the homologous pairing of ncRNA and RT (amino acid or nucleotide sequence) can be easily identified from the information presented/included in the sequence listing. In various other aspects, the retrotranscript RT component and the ncRNA component can be from different bacterial species, that is, not found together as a homologous pair in nature. In other embodiments, the retrotranscript RT component and the ncRNA component can be from the same lineage at the same time to generate functional types (e.g., type IA, type I-B1, type I-B2, type IC, etc.). For example, a recombinant retrotranscript-based genome editing system may comprise a retrotranscript RT from type IA (i.e., SEQ ID Nos: 3980-4178 for AA and SEQ ID Nos: 11231-11429 for NT - see Table A) and a retrotranscript ncRNA also from type IA (i.e., SEQ ID Nos: 16886-17078 - see Table B).

The sequence listing filed with this PCT application forms part of the specification of the initial application. As described above, the sequence listing can be searched to identify homologous pairs between retrotranscript RT (Table A - AA or NT) and retrotranscript ncRNA (Table B). The following steps can be followed: Step 1. Select RT sequence. Select any RT sequence from Table A by its sequence identifier (e.g., SEQ ID NO: 4178). Step 2. Locate the sequence entry in the sequence listing. Use any text search function in a suitable text editing software to identify the selected sequence identifier (e.g., SEQ ID NO: 4178) in the sequence listing, which is used to read the sequence listing file (e.g., Microsoft Word) submitted with this specification at the time of application and forming part of the specification of the initial application. Considering that the sequence listing originally included in this specification is in the form of an XML document, the search will be directed to the following text (in {brackets}): {sequenceIDNumber = "4178"}. Step 3. Determine which organism and/or which NCBI sequence reference number is associated with the sequence entry. Once step 2 locates the text being searched, check the lines of text in the sequence entry that reference "</INSDQualifier_value>" and note the NCBI reference sequence number presented to the left of the identified line. The indicator "</INSDQualifier_value>" also indicates the source organism. For example, for the entry {sequenceIDNumber="4178"}, the organism identified at the first "</INSDQualifier_value>" is "Shewanell sp." and the NCBI reference number identified at the second "</INSDQualifier_value>" is "Related to NZ_JWGX01000025.1" Therefore, for SEQ ID NO: 4178, the NCBI reference number is NZ_JWGX01000025.1 and the organism is Shewanella sp. Step 4. Find homologous retrotranscript ncRNA sequences. Perform a text search in the sequence listing for the NCBI reference number identified in step 3. Check the entry for the related SEQ ID NO:. In the case of SEQ ID NO: 4178, the NCBI reference number is "NZ_JWGX01000025.1" and the related ncRNA sequence is SEQ ID NO: 17078.

This is further illustrated as follows: Step 1. Pick any RT sequence from Table A (e.g., SEQ ID NO: 4178). Step 2. Search and identify SEQ ID NO: 4178 in the sequence table file by searching the text in brackets as follows: {sequenceIDNumber="4178"}. Check the hit region, which is imaged as the following computer screen image and shows in bold (a) the amino acid sequence of SEQ ID NO: 4178, (b) the related organism is "Shewanella sp.", and (c) the related NCBI reference number is "NZ_JWGX01000025.1". Step 3. Identify the NCBI reference number. In this example, the NCBI reference number is NZ_JWGX01000025.1. Step 4. The NCBI reference number is then used to locate the homologous retrotranscript ncRNA sequence in the sequence listing. In this case, the homologous ncRNA sequence is SEQ ID NO: 17078, as shown in the following computer screen image of the ncRNA sequence entry for SEQ ID NO: 17078, which, upon inspection, shares the NCBI reference number "NZ_JWGX010000025.1" and the organism "Shewanella sp.".

Therefore, for embodiments involving retrotranscript editing systems, the homologous retrotranscript RT from Table A and the retrotranscript ncRNA from Table B can be easily identified, wherein the retrotranscript editing system comprises a retrotranscript RT that is paired with its homologous retrotranscript ncRNA.

The following paragraphs describe exemplary and non-limiting embodiments of the present disclosure.

Paragraph 1. An engineered retrotranscript comprising: a) an msr locus (which encodes the msr RNA portion of msDNA); b) an msd locus encoding the msd RNA portion of the msDNA; c) a retrotranscriptase (RT), wherein the msd RNA is capable of being reverse transcribed by the retrotranscriptase (RT) to form the msDNA; d) a heterologous nucleic acid inserted at the msd locus, upstream of the msr locus, upstream or downstream of the msd locus, or within the msd locus; and e) a sequence modification ( e.g. , insertion, deletion, and/or substitution of one or more nucleotides) in the msr locus and/or the msd locus, wherein the sequence modification: i) modulates ( e.g. , enhances) the reverse transcription, processability, accuracy/fidelity, and/or production ( e.g. , in mammalian cells) of the msDNA; ii) modulate ( e.g. , reduce) the immunogenicity of the ncRNA encoded by the engineered retrotranscript ( e.g. , msr locus and/or msd locus) in a host ( e.g. , a host comprising the mammalian cell); and/or iii) comprise a nucleotide sequence that modulates ( e.g. , permanently or temporarily inhibits) the function of the msDNA; and/or iv) modulates ( e.g. , improves) the efficiency of targeted genome editing/engineering.

Paragraph 2. The engineered retrotranscript of paragraph 1, wherein the engineered retrotranscript is based on and/or engineered to be similar to the secondary structure of a wild-type or consensus retrotranscript encoding a wild-type or consensus retrotranscript ncRNA covered below: 1) any sequence and/or structure as depicted in SEQ ID NO: 1-21 and/or Figures 2-22; or 2) a variant of 1) having: A) up to 1, 2 or 3 ( e.g. , up to 1) nucleotide changes per 10 red-letter nucleotides; B) up to 4, 5 or 6 ( e.g. , up to 1 or 2) nucleotide changes per 10 black-letter nucleotides; and/or C) up to 7, 8 or 9 ( e.g. , up to 3 or 4) nucleotide changes per 10 grey-letter nucleotides; and/or further comprising, as appropriate: a) There are 7, 8, 9 or 10 ( e.g. , 9 or 10) nucleotides for every 10 red circled nucleotides; b) there are 6, 7, 8, 9 or 10 ( e.g. , 8, 9 or 10) nucleotides for every 10 black circled nucleotides; c) there are 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 6, 7, 8, 9 or 10) nucleotides for every 10 grey circled nucleotides; and/or d) there are 2, 3, 4, 5, 6, 7, 8, 9 or 10 ( e.g. , 4, 5, 6, 7, 8, 9 or 10) nucleotides for every 10 white circled nucleotides.

Paragraph 3. The engineered retrotranscript of any of paragraphs 1-2, wherein the engineered retrotranscript is engineered by introducing the sequence modification into a wild-type retrotranscript encoding a wild-type ncRNA or into a synthetic/artificial retrotranscript encoding a common retrotranscript ncRNA.

Paragraph 4. The engineered retrotransposons of any of paragraphs 1-3, wherein in the encoded retrotransposons ncRNA, the sequence modification comprises one or more of the following: (i) a modified ( e.g. , mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii) an extension or shortening of a1, a2, or both a1 and a2; (iii) an extension or shortening of the spacer sequence between the hairpin loops ( e.g. , S1, S2, S3, S4, S5, and/or S6); (iv) a modified ( e.g. , mutated or eliminated) bulge in the hairpin loop ( e.g. , L1, L2, L3, L4, L5, and/or L6); ( e.g. , by removing unpaired bases in the protrusion, or by replacing unpaired bases with an equal number of base pairs); (v) modified ( e.g. , extended or shortened) hairpin length ( e.g. , L1, L2, L3, L4, L5 and/or L6); (vi) substituted L1 and/or L2 with complement, inverted or inverted complement sequences; (vii) modified ( e.g. , increased) number of unpaired bases at the tip of the hairpin ( e.g. , L1, L2, L3, L4, L5 and/or L6); (viii) modified ( e.g. , increased or decreased) GC content in the hairpin ( e.g. , L1, L2, L3, L4, L5 and/or L6); (ix) Insertion of the heterologous nucleic acid in the spacer sequence between the hairpin loops ( e.g. , S1, S2, S3, S4, S5 and/or S6) or at the tip of the hairpin loop ( e.g. , L1, L2, L3, L4, L5 and/or L6); (x) Deletion of the hairpin loop ( e.g. , L1, L2, L3, L4, L5 and/or L6); (xi) Addition of a new loop in the spacer sequence between the hairpin loops ( e.g. , S1, S2, S3, S4, S5 and/or S6); (xii) Circularization of the ncRNA, wherein the 5' end and the 3' end of the ncRNA are linked directly or via the spacer sequence; (xiii) Repositioning of branched guanosine capable of initiating reverse transcription initiation; (xiv) (xv) an antisense sequence complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes with and inhibits the gRNA in the encoded retrotranscript ncRNA, and wherein the antisense sequence is removed after reverse transcription of the msDNA.

Paragraph 5. The engineered retroviral transcript of any one of paragraphs 1-4, comprising: (i) a heterologous nucleic acid (such as a coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as in the S region ( e.g. , S1, S2, S3, S4, S5 and/or S6), or the tip of the L region ( e.g. , L1, L2, L3, L4, L5 and/or L6)), or upstream or downstream of the msr locus or the msd locus; or (ii) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a CRISPR/Cas guide RNA (gRNA).

Paragraph 6.   The engineered retrotransposons of any of paragraphs 1-5, wherein the retrotransposons reverse transcriptase (RT) is a homologous RT, a retrotransposon RT from a species within the same evolutionary branch of the homologous RT, or a retrotransposon RT not within the same evolutionary branch of the homologous RT (such as an unrelated RT or an engineered RT).

Paragraph 7. The engineered retrotransposons of any of paragraphs 1-6, wherein the heterologous nucleic acid encodes a protein or peptide of interest, or wherein the heterologous nucleic acid comprises or encodes a donor/template sequence ( e.g. , a donor for correcting/repairing/removing a mutation at a target genome site (e.g., a mutant exon in a disease gene); a functional DNA element (e.g., a promoter, an enhancer, a protein binding sequence, a methylation site, a homologous region for assisting gene editing, etc.); or a coding sequence for a functional RNA element (ncRNA, etc.)).

Paragraph 8. The engineered retroviral transcript of Paragraph 7, wherein the related protein or peptide comprises a therapeutic protein that can be used to treat a disease (such as a wt protein that is defective in diseased cells, or a therapeutic antibody or an antigen-binding fragment thereof).

Paragraph 9. The engineered retrotransposons of Paragraph 7, wherein the donor/template sequence corrects/repairs/removes a mutation at a target genomic site (e.g., a mutant exon in a disease gene).

Paragraph 10. The engineered retrotranscript of any of paragraphs 1-9, further comprising or encoding a sequence-specific nuclease (such as a CRISPR/Cas effector enzyme, ZFN, TALEN, a meganuclease, TnpB, IscB or a restriction endonuclease (RE)) and/or a DNA repair regulatory biomolecule.

Paragraph 11. The engineered retrotransposon of paragraph 10, wherein the sequence-specific nuclease is fused to the RT via a flexible linker ( e.g. , a flexible linker comprising Gly and Ser-rich sequences such as G4S repeats or GS repeats) or by a generally disordered protein sequence (e.g., an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer), as appropriate.

Paragraph 12. The engineered retrotransposon of paragraph 10 or 11, wherein the nuclease is a CRISPR/Cas effector enzyme that forms a complex with a guide RNA (gRNA) that recognizes a target sequence, wherein the gRNA is linked to the ncRNA and/or the msDNA directly or via a linker/spacer polynucleotide.

Paragraph 13. The engineered retrotransposons of paragraph 10, wherein the DNA repair regulatory biomolecule is a regulatory protein that regulates ( e.g. , enhances) HDR, and the regulatory protein is fused to the RT or the sequence-specific nuclease via a flexible linker ( e.g. , a flexible linker comprising Gly and Ser-rich sequences (such as G4S repeats or GS repeats)) or by a generally disordered protein sequence (such as an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer), as the case may be.

Paragraph 14. A vector system comprising a vector comprising the engineered retrotransposon of any one of Paragraphs 1-13.

Paragraph 15. The vector system of paragraph 14, wherein the msr locus, the msd locus and the RT gene are provided by the same vector.

Paragraph 16. The vector system of paragraph 14 or 15, wherein the same vector comprises a promoter operably linked to the msr locus and/or the msd locus.

Paragraph 17. The vector system of any one of paragraphs 14-16, wherein the promoter is further operably linked to the RT gene.

Paragraph 18. The vector system of paragraph 14, wherein the msr locus, the msd locus and the RT gene are provided by at least two different vectors.

Paragraph 19. The vector system of paragraph 14 or 18, wherein the RT gene is provided in trans relative to the msr gene and/or the msd gene.

Paragraph 20. The vector system of any one of paragraphs 14-19, wherein the one or more vectors comprise viral vectors and/or non-viral vectors.

Paragraph 21. The vector system of paragraph 20, wherein the non-viral vector comprises a plasmid.

Paragraph 22. The vector system of any one of paragraphs 14-21, comprising a vector encoding a sequence-specific nuclease.

Paragraph 23. The vector system of paragraph 22, wherein the sequence-specific nuclease comprises an RNA-guided sequence-specific nuclease ( e.g. , a CRISPR/Cas effector enzyme, an engineered RNA-guided FokI-nuclease ( e.g., dCas-FokI), an RNA-guided DNA endonuclease, TnpB, IscB, or a translocon-associated nuclease) or a non-RNA-guided sequence-specific nuclease ( e.g. , a meganuclease, a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), or a restriction endonuclease (RE)).

Paragraph 24. The vector system of paragraph 23, wherein the Cas effector enzyme is class 1, type I, type II or type III Cas; class 2, type II Cas ( e.g., Cas9); or class 2, type V Cas ( e.g. , Cpf1).

Paragraph 25. The vector system of paragraph 23, wherein: 1) the RNA-guided sequence-specific nuclease comprises the CRISPR/Cas effector enzyme, the engineered RNA-guided FokI nuclease ( e.g., dCas-FokI), the RNA-guided DNA endonuclease, TnpB, IscB, IsrB, or a translocon-associated nuclease; or, 2) the non-RNA-guided sequence-specific nuclease comprises the meganuclease, the zinc finger nuclease (ZFN), the TALE nuclease (TALEN), or the restriction endonuclease (RE).

Paragraph 26. The vector system of any one of paragraphs 14-25, further comprising a vector encoding a homologous recombination enhancer protein.

Paragraph 27. An isolated host cell comprising the engineered retrotransposon of any one of paragraphs 1-13 or the vector system of any one of paragraphs 14-26.

Paragraph 28. The host cell of Paragraph 27, wherein the host cell is a prokaryotic, archeon or eukaryotic host cell.

Paragraph 29. The host cell of Paragraph 28, wherein the eukaryotic host cell is a mammalian host cell.

Paragraph 30. The host cell of paragraph 28, wherein the eukaryotic host cell is a non-human host cell.

Paragraph 31. The host cell of Paragraph 29, wherein the mammalian host cell is a human host cell.

Paragraph 32. The host cell of any one of paragraphs 28-31, wherein the host cell is an artificial cell or a genetically modified cell.

Paragraph 33. A pharmaceutical composition comprising the engineered retrotransposon of any one of Paragraphs 1-13.

Paragraph 34. A delivery vehicle comprising an engineered retrotransposon of any of paragraphs 1-13 or an ncRNA encoded by an engineered retrotransposon of any of paragraphs 1-13, a vector of any of paragraphs 14-26, a host cell of any of paragraphs 27-32, or a pharmaceutical composition of paragraph 33.

Paragraph 35. The delivery medium of Paragraph 34, which is lipid nanoparticles.

Paragraph 36. The delivery medium of Paragraph 35, wherein the lipid nanoparticles comprise at least one cationic lipid selected from the group consisting of lipids in Table (I), lipids having a structure of formula (I), lipids having a structure of formula (II), lipids having a structure of formula (III), lipids having a structure of formula (IV), lipids having a structure of formula (V), lipids having a structure of formula (VI), and combinations thereof.

Paragraph 37. A kit comprising an engineered retrotranscript of any of paragraphs 1-13 or an ncRNA encoded by an engineered retrotranscript of any of paragraphs 1-13, a vector or vector system of any of paragraphs 14-26, a host cell of any of paragraphs 27-32, a pharmaceutical composition of paragraph 33, or a delivery vehicle of any of paragraphs 34-36, and instructions for genetically modifying cells using the engineered retrotranscript, the ncRNA, the vector/vector system, the host cell, the pharmaceutical composition, or the delivery vehicle.

Paragraph 38. A method for modifying a target DNA sequence in a host ( e.g. , mammal) cell, the method comprising introducing an engineered retrotranscript of any of paragraphs 1-13 or an ncRNA encoded by an engineered retrotranscript of any of paragraphs 1-13, a vector or vector system of any of paragraphs 14-26, a pharmaceutical composition of paragraph 33, or a delivery vehicle of any of paragraphs 34-36 into the mammalian cell to allow production of the msDNA in the host ( e.g. , mammal) cell, wherein the encoded heterologous nucleic acid in the msDNA is integrated into the target DNA sequence in the genome of the host ( e.g. , mammal) cell.

Paragraph 39. Use of an engineered retrotransposons of any of paragraphs 1-13 or ncRNA encoded by an engineered retrotransposons of any of paragraphs 1-13, a vector or vector system of any of paragraphs 14-26, a pharmaceutical composition of paragraph 33, or a delivery vehicle of any of paragraphs 34-36 for achieving modification of a target DNA sequence in a host ( e.g. , mammalian) cell. Exemplary Embodiment Group B

The following paragraphs further describe exemplary and non-limiting embodiments of the present disclosure.

Paragraph 1. An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus, wherein the ncRNA comprises: (I) ncRNAs listed in Table B, or ncRNAs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with the ncRNAs listed in Table B; and/or (II) ncRNAs having a conservative structure of any of the ncRNA structures of Figures 2-27; and wherein the ncRNAs exclude any ncRNAs associated with any of the retrotransposons in Table X in nature, as the case may be.

Paragraph 2. The engineered nucleic acid construct of Paragraph 1, further comprising a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT or a portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

Paragraph 3.   The engineered nucleic acid construct of Paragraph 2, wherein the second polynucleotide comprises: III)       A polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polynucleotide listed in Table A; and/or IV)    Encoding a consensus amino acid sequence of Table C, or encoding an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with an amino acid sequence listed in Table C; and/or wherein the second polynucleotide encodes: V)    A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table A; and/or VI)     A polypeptide comprising a consensus sequence of the polypeptides listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with an amino acid sequence listed in Table C; and/or wherein the second polynucleotide does not encode an amino acid sequence listed in Table X as the case may be.

Paragraph 4. An engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: 1) an msr locus encoding the msr RNA portion of multiple copies of single-stranded DNA (msDNA); and 2) an msd locus encoding the msd RNA portion of the msDNA; b) one or more heterologous nucleic acids inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT or a portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the non-coding RNA (ncRNA) of the first polynucleotide optionally has a conserved structure of any one of the ncRNA structures of Figures 2-27; wherein the second polynucleotide comprises: 1) The polynucleotides listed in Table A, or polynucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the polynucleotides listed in Table A; and/or wherein the second polynucleotide encodes: II) A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or IV) A polypeptide comprising a consensus sequence of a polypeptide listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table C; and wherein the second polynucleotide optionally does not encode the amino acid sequence of Table X.

Paragraph 4a. An engineered nucleic acid construct comprising: 1) an msr locus (which encodes the msr RNA portion of the msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retrotranscriptase (RT), wherein the msd RNA can be reverse transcribed by the retrotranscriptase (RT) to form the msDNA; and 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct has, as the case may be, (a) the secondary structure of the wild-type ncRNA of any one of Figures 2-27, or b) a variant of a), having: i) up to 1, 2 or 3 (e.g., up to 1) nucleotide changes for every 10 red letter nucleotides; ii) Up to 4, 5 or 6 (eg, up to 1 or 2) nucleotide changes per 10 black lettered nucleotides; and/or iii) up to 7, 8 or 9 (eg, up to 3 or 4) nucleotide changes per 10 grey lettered nucleotides.

Paragraph 5. An engineered nucleic acid construct of any one of paragraphs 1 to 4a, comprising one or more sequence modifications (e.g., insertion, deletion and/or substitution of one or more nucleotides) in the msr locus and/or msd locus, wherein the one or more sequence modifications: a) modulate (e.g., enhance) the reverse transcription, processability, accuracy/fidelity and/or production (e.g., in mammalian cells) of the msDNA; b) modulate (e.g., reduce) the immunogenicity of the ncRNA encoded by the engineered retrotranscript (e.g., msr locus and/or msd locus) in a host (e.g., a host comprising the mammalian cell); c) modulate (e.g., permanently or temporarily inhibit) the function of the msDNA; and/or d) modulate (e.g., improve) the efficiency of targeted genome editing/engineering.

Paragraph 6.   An engineered nucleic acid construct of any of paragraphs 1 to 4, wherein the engineered nucleic acid construct has a secondary structure of a wild-type retrotranscript encoding a wild-type retrotranscript ncRNA covered below: a)  Any structure as depicted in Figure 2-27, or b)  A variant of a) having: i)   Up to 1, 2 or 3 (e.g., up to 1) nucleotide changes per 10 red-letter nucleotides; ii)  Up to 4, 5 or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black-letter nucleotides; and/or iii) Up to 7, 8 or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey-letter nucleotides; and/or further comprising, as the case may be: i)  7, 8, 9 or 10 (e.g., 9 or 10) nucleotides are present for every 10 red circled nucleotides; ii) 6, 7, 8, 9 or 10 (e.g., 8, 9 or 10) nucleotides are present for every 10 black circled nucleotides; iii) 4, 5, 6, 7, 8, 9 or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides are present for every 10 grey circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present for every 10 white circled nucleotides.

Paragraph 7. The engineered nucleic acid construct of any of paragraphs 1-6, wherein the nucleic acid construct is engineered by introducing the one or more sequence modifications into a wild-type retrotranscript of a wild-type ncRNA listed in Coding Table B.

Paragraph 8.   The engineered nucleic acid construct of any of paragraphs 1-7, wherein the one or more sequence modifications in the ncRNA include one or more of the following: (i)          A modified (e.g., mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii)          An extension or shortening of a1, a2, or both a1 and a2; (iii)        An extension or shortening of the spacer sequence between the hairpin loops (e.g., S1, S2, S3, and/or S4); (iv)        An additional or modified (e.g., mutated or eliminated) bulge in the hairpin loop (e.g., L2 and/or L3 (e.g., by removing unpaired bases in the protrusion, or by replacing unpaired bases with an equal number of base pairs); (v)         the modified (e.g., extended or shortened) length of the hairpin (e.g., L1, L2, L3 and/or L4); (vi)        replacing L1 and/or L2 with complement, reverse or reverse complement sequences; (vii)       the modified (e.g., increased) number of unpaired bases at the tip of the hairpin (e.g., L1, L2, L3 and/or L4); (viii)      the modified (e.g., increased or decreased) GC content in the hairpin (e.g., L1, L2, L3 and/or L4); (ix) Insertion of the heterologous nucleic acid in the spacer sequence between the hairpin loops (e.g., S1, S2, S3 and/or S4) or at the tip of the hairpin loop (e.g., L1, L2, L3 and/or L4); (x)         Deletion of one or more hairpin loops (e.g., L1, L2, L3 and/or L4); (xi)        Addition of new loops in the spacer sequence between the hairpin loops (e.g., S1, S2, S3 and/or S4); (xii)       Circularization of the ncRNA, wherein the 5' end and the 3' end of the ncRNA are directly or via a spacer sequence; (xiii)      Repositioned branched guanosine capable of initiating reverse transcription initiation; (xiv)     a staggered end sequence that reduces the immunogenicity of the retrotranscript ncRNA, generated by, for example, adding or removing 5' a1 nucleotides and/or 3' a2 nucleotides; and/or, (xv)       an antisense sequence that is complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes with and inhibits the gRNA in the encoded retrotranscript ncRNA, and wherein the antisense sequence is removed after reverse transcription of the msDNA.

Paragraph 9. The engineered nucleic acid construct of any one of paragraphs 1-8, wherein the one or more heterologous nucleic acid sequences comprise: a) a heterologous nucleic acid (such as a coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as the tip of the S region (e.g., S1, S2, S3 and/or S4), or the L region (e.g., L1, L2, L3 and/or L4), or upstream or downstream of the msr locus or the msd locus; or b) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a guide RNA.

Paragraph 10.     The engineered nucleic acid construct of any one of paragraphs 1-9, wherein the heterologous nucleic acid encodes: (a) a protein or peptide of interest, or wherein the heterologous nucleic acid comprises; (b) a DNA donor template sequence; (c) a functional DNA element selected from a promoter, an enhancer, a protein binding sequence, a methylation site, a homologous region for assisting gene editing, and the like; or (d) a coding sequence for a functional RNA element selected from a guide RNA and a ncRNA.

Paragraph 11. The engineered nucleic acid construct of Paragraph 10, wherein the related protein or peptide comprises a therapeutic protein that can be used to treat a disease.

Paragraph 12. The engineered nucleic acid construct of Paragraph 10, wherein the DNA donor template sequence corrects/repairs/removes a mutation at a target genomic site.

Paragraph 13. The engineered nucleic acid construct of any of paragraphs 1-12, further comprising or encoding a sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)) and/or a DNA repair regulatory biomolecule.

Paragraph 13b. The engineered nucleic acid construct of paragraphs 1-13, wherein the engineered nucleic acid is an all-RNA component system.

Paragraph 13c. The engineered nucleic acid construct of paragraphs 1-13, wherein the engineered nucleic acid is a fully DNA molecule system.

Paragraph 14. The engineered nucleic acid construct of paragraph 13, wherein the sequence-specific nuclease is fused to the RT via a flexible linker (e.g., a flexible linker comprising Gly and Ser-rich sequences such as G4S repeats or GS repeats) or by a generally disordered protein sequence (e.g., an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer), as appropriate.

Paragraph 15. The engineered nucleic acid construct of paragraph 13 or 14, wherein the nuclease is a CRISPR/Cas effector enzyme that forms a complex with a guide RNA (gRNA) that recognizes a target sequence, wherein the gRNA is linked to the ncRNA and/or the msDNA directly or via a linker/spacer polynucleotide.

Paragraph 16. The engineered nucleic acid construct of paragraph 13, wherein the DNA repair regulatory biomolecule is a regulatory protein that regulates (e.g., enhances) HDR, and the regulatory protein is fused to the RT or the sequence-specific nuclease, as the case may be, via a flexible linker (e.g., a flexible linker comprising Gly and Ser-rich sequences (such as G4S repeats or GS repeats)) or by a generally disordered protein sequence (such as an unstructured hydrophilic, biodegradable protein polymer, such as an XTEN peptide polymer).

Paragraph 17. A vector system comprising one or more vectors comprising the engineered nucleic acid construct of any one of paragraphs 1-16, wherein the vector system is optionally all-RNA.

Paragraph 18. The vector system of paragraph 17, wherein the msr locus, the msd locus and the polynucleotide encoding the RT are contained in the same vector.

Paragraph 19. The vector system of paragraph 17 or 18, wherein the same vector further comprises a promoter operably linked to the msr locus and/or the msd locus.

Paragraph 20. The vector system of paragraph 19, wherein the promoter is further operably linked to the polynucleotide encoding the RT.

Paragraph 21. A vector system comprising one or more vectors, comprising the engineered nucleic acid construct of paragraph 1 or 2, wherein the vector system further comprises a second polynucleotide encoding a reverse transcriptase (RT) or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the msr locus, the msd locus and the second polynucleotide encoding the RT are provided by at least two different vectors.

Paragraph 22. The vector system of paragraph 21, wherein: a) the second polynucleotide comprises: i)   a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polynucleotide listed in Table A; and/or b) the second polynucleotide encodes: i)  A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table A; and/or ii) A polypeptide listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with a polypeptide listed in Table C; and wherein the second polynucleotide does not encode a polypeptide listed in Table X, as the case may be.

Paragraph 23. The vector system of paragraph 21 or 22, wherein the polynucleotide encoding the RT is provided in trans relative to the msr gene and/or the msd gene.

Paragraph 24. The vector system of any of paragraphs 17-23, wherein the one or more vectors comprise a viral vector.

Paragraph 25. The vector system of Paragraph 24, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a poxvirus vector or a herpes simplex virus vector.

Paragraph 26. The vector system of any of paragraphs 17-23, wherein the one or more vectors comprise a non-viral vector.

Paragraph 27. The vector system of paragraph 26, wherein the non-viral vector comprises a plasmid.

Paragraph 28. The vector system of Paragraph 26, wherein the non-viral vector comprises a liposome, a lipid nanoparticle (LNP), a cationic polymer, a vesicle or a gold nanoparticle.

Paragraph 29. The vector system of any one of paragraphs 17-28, comprising a vector encoding a sequence-specific nuclease.

Paragraph 30. The vector system of paragraph 29, wherein the sequence-specific nuclease comprises an RNA-guided sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme, an engineered RNA-guided FokI-nuclease (e.g., dCas-FokI), an RNA-guided DNA endonuclease, TnpB, IscB, or a translocon-associated nuclease) or a non-RNA-guided sequence-specific nuclease (e.g., a meganuclease, a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), or a restriction endonuclease (RE)).

Paragraph 31. The vector system of paragraph 30, wherein the Cas effector enzyme is class 1, type I, type II or type III Cas; class 2, type II Cas (e.g., Cas9); or class 2, type V Cas (e.g., Cpf1).

Paragraph 32. The vector system of paragraph 30, wherein: 1)      the RNA-guided sequence-specific nuclease comprises the CRISPR/Cas effector enzyme, the engineered RNA-guided FokI nuclease (e.g., dCas-FokI), the RNA-guided DNA endonuclease, TnpB, IscB, IsrB, or a translocon-associated nuclease; or, 2)      the non-RNA-guided sequence-specific nuclease comprises the meganuclease, the zinc finger nuclease (ZFN), the TALE nuclease (TALEN), or the restriction endonuclease (RE).

Paragraph 33. The vector system of any one of paragraphs 17-32, further comprising a vector encoding a homologous recombination enhancer protein.

Paragraph 34. An RNA molecule encoded by the engineered nucleic acid construct of any of Paragraphs 1-16.

Paragraph 35. A genome editing system based on recombinant retrotranscripts, comprising: a) non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a sequence encoding a reverse transcriptase (RT) or a domain thereof, comprising: i) A polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) The polypeptides listed in Table C, or polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the polypeptides listed in Table C; and wherein the RT optionally does not comprise a polypeptide listed in Table X.

Paragraph 36. A genome editing system based on recombinant retrotranscripts, comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding a msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding a msd RNA portion of the msDNA; wherein the ncRNA comprises: i) ncRNAs listed in Table B, or ncRNAs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the ncRNAs listed in Table B; and/or ii) ncRNAs having a conservative structure of any of the ncRNA structures of Figures 2-27; and wherein the ncRNA excludes any ncRNA associated with any of the retrotransposons in Table X in nature, as the case may be; b) a heterologous nucleic acid inserted at or within a position selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT) or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

Paragraph 37. A genome editing system based on recombinant retrotranscripts, comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding a msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding a msd RNA portion of the msDNA; wherein the ncRNA comprises: i) ncRNAs listed in Table B, or ncRNAs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the ncRNAs listed in Table B; and/or ii) ncRNAs having a conservative structure of any ncRNA structure of Figures 2-27; and wherein the ncRNA excludes any ncRNA associated with any retrotransposase of Table X in nature as the case may be; b) A heterologous nucleic acid inserted at or within a position selected from the group consisting of: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in Table A, or an RT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an RT listed in Table A; and/or ii) The consensus sequence listed in Table C, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence listed in Table C; and wherein the RT optionally does not include the RT listed in Table X.

Paragraph 38. An isolated host cell comprising the engineered nucleic acid construct of any of paragraphs 1-16, the vector system of any of paragraphs 17-33, the RNA molecule of paragraph 34, or the recombinant retroviral transcript-based genome editing system of any of paragraphs 35-37.

Paragraph 39. The isolated host cell of paragraph 38, wherein the host cell is a prokaryotic, archeon or eukaryotic host cell.

Paragraph 40. The isolated host cell of paragraph 38, wherein the eukaryotic host cell is a mammalian host cell.

Paragraph 41. The isolated host cell of Paragraph 39, wherein the eukaryotic host cell is a non-human host cell.

Paragraph 42. The isolated host cell of Paragraph 40, wherein the mammalian host cell is a human host cell.

Paragraph 43. The isolated host cell of any one of paragraphs 38-42, wherein the host cell is an artificial cell or a genetically modified cell.

Paragraph 44. A pharmaceutical composition comprising: a)  An engineered nucleic acid construct of any one of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, a genome editing system based on a recombinant retrotransposon of any one of paragraphs 35-37, and/or an isolated host cell of any one of paragraphs 38-43; and b)  A pharmaceutically acceptable carrier.

Paragraph 45. A pharmaceutical composition comprising: a)  Lipid nanoparticles (LNP); and b)  An engineered nucleic acid construct of any one of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, and/or a genome editing system based on a recombinant retrotransposon of any one of paragraphs 35-37.

Paragraph 46. The pharmaceutical composition of Paragraph 45, wherein the LNP encapsulates the engineered nucleic acid construct, the ncRNA, the vector system, the RNA molecule and/or the engineered nucleic acid-enzyme construct.

Paragraph 47.     The pharmaceutical composition of paragraph 45 or 46, wherein the lipid nanoparticles comprise: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.

Paragraph 48. The pharmaceutical composition of Paragraph 47, wherein the one or more ionizable lipids are selected from the group consisting of those disclosed in Table 2.

Paragraph 49.     The pharmaceutical composition of paragraph 47 or 48, wherein the one or more structured lipids are selected from the group consisting of: cholesterol, natriuretic acid, β-myristol, sterol, ergosterol, campesterol, stigmasterol, sterosterol, tomatine, tomatine, ursolic acid, α-tocopherol, prednisolone, dexamethasone, prednisolone and hydrocortisone.

Paragraph 50.     The pharmaceutical composition of any of paragraphs 47-49, wherein the one or more PEGylated lipids are selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

Paragraph 51.     The pharmaceutical composition of any one of paragraphs 47-50, wherein the one or more phospholipids are selected from the group consisting of: 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC ), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycero) sodium salt (DOPG) and sphingomyelin.

Paragraph 52.     The pharmaceutical composition of any of paragraphs 47-51, wherein the lipid nanoparticles comprise about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 40 mol% structural lipids and about 1.5 mol% PEG lipids.

Paragraph 53.     The pharmaceutical composition of any of paragraphs 47-52, wherein the lipid nanoparticles comprise approximately 48.5 mol% ionizable lipids, approximately 10 mol% phospholipids, approximately 39 mol% structural lipids and approximately 2.5 mol% PEG lipids.

Paragraph 54.     The pharmaceutical composition of any of Paragraphs 47-53, wherein the LNP further comprises a targeting portion operably linked to the LNP.

Paragraph 55.     The pharmaceutical composition of any of paragraphs 47-54, wherein the LNP further comprises one or more additional components selected from the group consisting of: DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP and C12-200.

Paragraph 56. The pharmaceutical composition of Paragraph 45, wherein the lipid nanoparticles comprise at least one cationic lipid selected from the group consisting of lipids in Table 2, lipids having a structure of formula (I), lipids having a structure of formula (II), lipids having a structure of formula (III), lipids having a structure of formula (IV), lipids having a structure of formula (V), lipids having a structure of formula (VI), and combinations thereof.

Paragraph 57.     A kit comprising an engineered nucleic acid construct of any of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any of paragraphs 1-16, a vector system of any of paragraphs 17-33, an RNA molecule of paragraph 34, a genome editing system based on a recombinant retrotransposon of any of paragraphs 35-37, a host cell of any of paragraphs 38-43, or a pharmaceutical composition of any of paragraphs 44-56, and instructions for genetically modifying cells using the engineered nucleic acid construct, the ncRNA, the vector system, the host cell, or the pharmaceutical composition.

Paragraph 58.     A method for modifying a target DNA sequence in a host (e.g., mammal) cell, the method comprising introducing an engineered nucleic acid construct of any of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any of paragraphs 1-16, a vector system of any of paragraphs 17-33, an RNA molecule of paragraph 34, a recombinant retroviral transcript-based genome editing system of any of paragraphs 35-37, or a pharmaceutical composition of any of paragraphs 44-56 into the mammalian cell to allow production of the msDNA in the host (e.g., mammal) cell, wherein the heterologous nucleic acid in the msDNA modifies the genome of the host.

Paragraph 59. The method of paragraph 58, wherein the modification comprises introducing insertions, deletions and/or substitutions into the target DNA sequence.

Paragraph 60.     A method for treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an engineered nucleic acid construct of any one of paragraphs 1-16, an ncRNA encoded by an engineered nucleic acid construct of any one of paragraphs 1-16, a vector system of any one of paragraphs 17-33, an RNA molecule of paragraph 34, a recombinant retrotranscript-based genome editing system of any one of paragraphs 35-37, a host cell of any one of paragraphs 38-43, or a pharmaceutical composition of any one of paragraphs 44-56, thereby treating the disease or condition in the individual.

Paragraph 61. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a host cell of any one of paragraphs 38-43, thereby treating the disease or condition in the subject.

Paragraph 62. The method of Paragraph 61, wherein the host cell is autologous to the individual.

Paragraph 63. The method of Paragraph 61 , wherein the host cell is allogeneic to the individual.

The following paragraphs also describe exemplary and non-limiting embodiments of the present disclosure.

Paragraph 1. A gene editing system comprising one or more delivery vehicles, wherein: The delivery vehicle(s) comprises an RNA cargo, The RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscript reverse transcriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the programmable nuclease, Each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), and (a)(i), (a)(ii), (b) and (c) are delivered by one delivery vehicle or more than one delivery vehicle.

Paragraph 2. The gene editing system of Paragraph 1, wherein (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retrotransposase.

Paragraph 3. The gene editing system of Paragraph 2, wherein (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.

Paragraph 4. The gene editing system of Paragraph 3, wherein the fusion protein comprises the C-terminus of the nucleic acid programmable nuclease fused to the N-terminus of the retroviral reverse transcriptase (nuclease:RT fusion).

Paragraph 5. The gene editing system of Paragraph 3, wherein the fusion protein comprises the N terminus of the nucleic acid programmable nuclease fused to the C terminus of the reverse transcriptase (RT: nuclease fusion).

Paragraph 6. The gene editing system of Paragraph 1, wherein (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retrotransposase.

Paragraph 7. The gene editing system of Paragraph 1, wherein (c) is separated from (a)(i), (a)(ii) and (b) or provided in trans .

Paragraph 8. The gene editing system of Paragraph 1, wherein (b) the engineered retrotran ncRNA and (c) the guide RNA are fused or provided in cis form .

Paragraph 9. The gene editing system of Paragraph 8, wherein the guide RNA is fused to the 5' end of the retrotranscript ncRNA.

Paragraph 10. The gene editing system of Paragraph 8, wherein the guide RNA is fused to the 3' end of the retrotranscript ncRNA.

Paragraph 11. The gene editing system of Paragraph 8, wherein the engineered ncRNA comprises a first guide RNA fused to the 5' end of the retrotranscript ncRNA and a second guide RNA fused to the 3' end of the retrotranscript ncRNA, and the first and second guide RNAs target different sequences.

Paragraph 12. The gene editing system of Paragraph 1, wherein the one or more delivery vehicles comprise liposomes or lipid nanoparticles (LNPs).

Paragraph 13. The gene editing system of Paragraph 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in the same delivery medium.

Paragraph 14. The gene editing system of Paragraph 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in a separate delivery medium.

Paragraph 15. The gene editing system of Paragraph 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery medium.

Paragraph 16. The gene editing system of Paragraph 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.

Paragraph 17. The gene editing system of Paragraph 1, wherein the engineered retrotranscript ncRNA comprises a related sequence encoding a donor polynucleotide comprising the intended edit at a target sequence to be integrated into a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at the 5' position of the target sequence and a 3' homology arm hybridized to a sequence at the 3' position of the target sequence.

Paragraph 18. The gene editing system of Paragraph 1, wherein the nucleic acid programmable nuclease comprises Cas9 nuclease, TnpB nuclease or Cas12a nuclease.

Paragraph 19. The gene editing system of Paragraph 18, wherein the nucleic acid programmable nuclease comprises a Cas9 nuclease.

Paragraph 20. The gene editing system of Paragraph 1, wherein the retrovirus reverse transcriptase has an amino acid sequence of Table A, or an amino acid sequence having at least 90%, 95%, 99% or 100% sequence identity with any amino acid sequence of Table A.

Paragraph 21. The gene editing system of paragraph 1, wherein the engineered retrotransposon ncRNA comprises: A) a leading msr sequence having a first complementary region of the retrotransposon ncRNA; B) an msr sequence comprising an msr stem-loop structure; C) an msd sequence comprising an msd stem-loop structure and related sequences, wherein the msd sequence templates a single-stranded DNA product (RT-DNA) in the presence of the retrotransposon reverse transcriptase; and D) an msd post-sequence having a second complementary region, wherein the first and second complementary regions form the a1/a2 duplex region of the retrotransposon ncRNA, wherein the msr stem-loop structure, the msd stem-loop structure or the a1/a2 duplex comprises a modification that results in increased editing efficiency in the presence of a nucleic acid programmable nuclease associated with one or more guide RNAs, and wherein, as the case may be, one or more guide RNAs of (c) are coupled to the leading msr sequence, the msd post-sequence, or both the leading msr sequence and the msd post-sequence.

Paragraph 22. The gene editing system of paragraph 21, wherein the sequence of interest encodes a donor polynucleotide comprising the desired edit at a target sequence to be integrated into a cell, wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at the 5' position of the target sequence and a 3' homology arm hybridized to a sequence at the 3' position of the target sequence.

Paragraph 23. The gene editing system of Paragraph 21, wherein the ncRNA has a nucleotide sequence of any sequence in Table B, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity with any sequence in Table B.

Paragraph 24. An isolated cell comprising the gene editing system of Paragraph 1.

Paragraph 25. The isolated cell of Paragraph 24, wherein the isolated cell is a mammalian cell.

Paragraph 26. The isolated cell of Paragraph 25, wherein the mammalian cell is a human cell.

Paragraph 27. A composition comprising: a) the gene editing system of Paragraph 1; and b) a pharmaceutically or veterinarily acceptable carrier.

Paragraph 28. The composition of paragraph 27, wherein the delivery medium is a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structured lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.

Paragraph 29. The composition of Paragraph 28, wherein the one or more ionizable lipids comprise the ionizable lipids set forth in Table 2.

Paragraph 30. A method for genetically modifying a cell, the method comprising: contacting the gene editing system of paragraph 17 with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein the guide RNA guides the complex to the target sequence, the nucleic acid programmable nuclease produces a double-strand break in the target sequence, the retrotranscriptase and the engineered retrotranscript ncRNA produce RT DNA comprising the donor polynucleotide, and the donor polynucleotide is integrated into the target sequence. Examples Example 1: Retrotranscript engineering

This example demonstrates that an engineered (or recombinant) retrotranscript as described herein can be engineered based on existing sequence information in a sequence database.

Specifically, retrotranscript-like reverse transcriptases (RTs) are first identified from various genome or metagenomic sequence databases. The identified ncRNA regions of the retrotranscripts are then determined by computer prediction and/or empirically, such as by reconstructing putative retrotranscript systems in living cells, to analyze msDNA production.

Once a particular wild-type retrotranscript is identified and confirmed, one or more sequence elements of the retrotranscript are modified based on the methods described herein, and/or the associated RT is modified or engineered to enhance the overall activity and/or processability of the retrotranscript. For example, a wild-type retrotranscript can be engineered by one or more of the following methods: (a) adding relevant heterologous nucleic acid sequences (e.g., nucleotide sequences encoding HDR donor templates) to various parts and structures of the msd locus; (b) performing any/all structural modifications described herein; (c) optionally linking the engineered retrotranscript ncRNA to one or more CRISPR gRNAs, e.g., a gRNA linked to the 3' end of the retrotranscript ncRNA, or a gRNA linked to the 5' end of the retrotranscript ncRNA, or a pair of gRNAs, one linked to the 3' end and one linked to the 5' end of the retrotranscript ncRNA.

The engineered retrotranscript or its encoded ncRNA is optionally linked to a sequence-specific nuclease, such as a CRISPR/Cas enzyme and/or gRNA, ZFN, TALEN, TnpB or IscB and the like.

For example, RT is fused to a CRISPR/Cas enzyme (such as Cas9 or Cpf1) as an N-terminal or C-terminal fusion, and optionally further fused to a nuclear localization signal (NLS) at the N-terminal and/or C-terminal end of the fusion. The ncRNA or msDNA obtained after reverse transcription is also linked to a guide RNA (gRNA) at the 5' or 3' end and can be used together with the cognate CRISPR/Cas enzyme in the method of the present invention.

In another example, RT is linked to a DNA repair regulatory biomolecule as described above, such as an HDR promoter and/or an NHEJ peptide inhibitor. Example 2A: Genome Engineering

This example demonstrates that engineered retrotransposons can be used to introduce heterologous nucleic acid sequences ( eg , targeting DNA donors or templates) into the genome of host cells ( eg , human cells).

First, a targeting DNA is introduced into the msd portion of an engineered retrotranscript, as shown in Figure 3, as a "marker". The heterologous nucleic acid sequence is designed so that it is flanked by 10-100 or more base pairs of homologous sequences that are substantially identical/homologous to the genome sequence at the target site. Thus, the desired edits on the "marker" are between the homologous sequence arms and include insertions, deletions, and/or other mutations.

A sequence-specific nuclease, such as the Cas9 nuclease, forms a complex with a guide RNA that specifically targets a location on a genomic sequence at or near the desired editing site. The nuclease is designed so that once the edit is correctly installed, it does not cleave the target. In this experiment, the Cas9 nuclease was linked to a retrotranscriptase (RT) as a fusion protein. The Cas9 gRNA was also linked to ncRNA or msDNA produced by the engineered retrotranscript.

Next, the engineered retrotransposons encoding the sequence-specific nuclease (and fused RT) and the ncRNA (and linked gRNA) are introduced into host cells, such as human cells.

The engineered retroviral vector is introduced as part of a plasmid for transfection into cells, or as part of a viral vector (such as an AAV vector) for infecting cells.

Alternatively, the transcribed ncRNA (and linked gRNA) can be formulated in vitro , for example, in a lipid nanoparticle or delivery system of the present invention, for direct delivery to a host cell. The sequence-specific nuclease (and fused RT) can be delivered to a host cell alone (using plasmid transfection or AAV infection, etc.), or delivered together with the ncRNA in a lipid nanoparticle. For example, the coding sequence ( e.g. , mRNA) of the Cas9-RT fusion is formulated together with the ncRNA in the same lipid nanoparticle, or formulated separately into lipid nanoparticles for simultaneous or sequential delivery.

Once inside the cell, the Cas9-RT fusion is translated by the host cell translation machinery, and the ncRNA is transcribed from the engineered retrotranscript (if the ncRNA was not delivered directly to the cell). The fused RT portion continues to reverse transcribe the ncRNA and convert it into msDNA, which includes the heterologous nucleic acid sequence that serves as cargo/donor/template. At the same time, the CRISPR/Cas9 nuclease generates a double-strand break (DSB) at the target site. Then, after the Cas9 nuclease forms the DSB, the cargo/donor/template sequence is integrated into the host genome at the target site through host cell DNA repair ( e.g. , HDR).

The relevant cells are then analyzed for correct installation of the edit in the heterologous nucleic acid sequence, either by phenotypic changes due to the edit, by direct DNA sequencing of the target site, or both . Example 2B. Precise retrotranscript editing levels in mammalian cells are comparable between plastid-delivered and RNA-delivered retrotranscripts

Although CRISPR/Cas technology has revolutionized genome editing technology, precise editing of pathological mutations or insertion of large DNA fragments at specific locations to restore cell health is still in its infancy. Delivery of donor DNA for precise editing is a bottleneck for current therapeutic applications. Current therapeutic ex vivo and in vivo delivery of donor DNA relies on AAV (adeno-associated virus) transduction. However, AAV is complex and expensive to manufacture. In addition, AAV has raised safety concerns in some gene therapy clinical trials. The inventors attempted to develop a method for delivering DNA donors that avoids the shortcomings of AAV-based DNA delivery.

Instead, the inventors sought to develop an RNA-based method for delivering DNA donors into cells for precise editing.

Retrotranscriptons have been found in many bacterial species. These genetic elements are defined by their unique ability to produce unusual satellite DNA, called msDNA (multi-copy single-stranded DNA). The DNA includes a reverse transcriptase (RT) encoding gene ( ret ) and two consecutive inverted non-coding sequences (named msr and msd ). The ret gene and the non-coding RNA (ncRNA, msr and msd ) are transcribed into a single RNA, which is folded into a specific secondary structure. Once translated, RT binds to the RNA template downstream of the msd locus, thereby initiating reverse transcription of the RNA towards its 5' end with the help of the 2'OH group present in the conserved branch-chain guanosine residues that act as a primer. Retrotranscription stops before reaching the msr locus, and the resulting DNA (msDNA) remains covalently linked to the RNA template via 2'-5' phosphodiester bonds and base pairing between the msDNA and the 3' end of the RNA template. In this example, these unique features of retrotranscriptors were exploited to generate donor DNA for precise genomic editing by a validated RNA delivery system, and were shown to produce similar levels of Cas9-based precise editing in mammalian cells as compared to plastid-delivered retrotranscriptors.

Figures 1L and 1M show the results of the first set of experiments. Three different types of retrotranscripts (Eco1-R1, Eco3-R2, Eco5-R3) were evaluated for their ability to insert 16 base pairs of insertions at a specific genomic site (EMX1 gene). The mRNA of each reverse transcriptase and the homologous non-coding reverse transcriptase RNA were electroporated into HEK293T mammalian cells expressing Cas9. The guide RNA of Cas9 was fused to the non-coding reverse transcriptase RNA to ensure that the guide RNA and the single- stranded donor DNA generated in situ (i.e., the reverse transcriptase product of the ncRNA template) were co-localized at the editing target site. As a control, the previously established plasmid system encoding Eco1 reverse transcriptase and its non-coding RNA was lipid-transfected into the same cell line. The edited sequences were PCR amplified from extracted genomic DNA, and NGS libraries were made and analyzed by sequencing. The results suggest that retrotranscript components delivered in RNA form can provide donor DNA templates for precise editing as effectively as those delivered via plasmid transfection.

In a second set of experiments (data not shown), the ratio between the reverse transcriptase mRNA and its non-coding RNA template was optimized. Increasing amounts of non-coding RNA template were combined with a given amount of reverse transcriptase mRNA (tested at two different fixed concentrations of reverse transcriptase mRNA). The results indicate that the ratio between the reverse transcriptase mRNA and its non-coding RNA template can be optimized in order to increase editing efficiency. For example, the data showed that increasing the non-coding RNA template to a given amount of RT enzyme improved the integration rate of 16 bp inserts. Example 3: Computational discovery of novel retrotranscriptors and phylogenetic analysis to increase retrotranscript diversity for genome editing applications

Reverse transcriptases (RTs), also known as RNA-directed DNA polymerases, are enzymes capable of synthesizing DNA using RNA as a template. Although they are found in all three domains of life and viruses; prokaryotic RTs have historically been less explored compared to their eukaryotic counterparts. Prokaryotic RTs can be divided into six major groups: (1) Group II introns, (2) CRISPR-associated RTs, (3) diversity-generating retrotranscript elements, (4) retrotranscripts, (5) Abi (abortive infection) RTs, and (6) the unknown group of RTs (UG). Over the past five years, a large number of studies have increased the understanding of prokaryotic RTs, resulting in the discovery of novel putative systems with potential anti-phage properties, including retrotranscripts. In this example, a systematic search of public databases was performed with the goal of increasing the number and diversity of known retrotranscripts for potential use in genome editing applications. As a result, new types of retrotranscripts have been identified, and the increase in data has allowed the identification of new related ncRNAs.

As a first step in this work, a set of known retrotranscript RTs was manually curated, pruned, and aligned to create suitable inputs, which were then used to train an HMM model to identify new retrotranscript RTs.

This model is then applied to existing protein sequence databases (e.g., the nr database from NCBI) to identify potential candidate retrotran RTs.

Next, the identified candidates were grouped by sequence identity and individual representative sequences were selected from each group. The RT domains of these representative candidates were then aligned and an initial phylogenetic tree was constructed.

Next, using information about other known RT classes, the complete germline occurrence is divided into true retrotranscript RT candidates and RTs that may belong to other classes (e.g., group II introns, DGRs, CRISPR-Cas RTs, etc.). New alignments and germline occurrence trees are then built from these validated candidates, as shown in Figure 28. For all sequences in the new alignment, a protein neighborhood matrix is built to indicate which proteins are present near the candidate retrotranscript RT. Thus, types and subtypes of retrotranscripts are defined based on the RT germline occurrence and the identity of the associated effector proteins. The candidate RT sequences are presented in Table A.

In order to predict ncRNAs close to candidate retrotranscript RTs, an iterative convergent model was used to extract, align, and analyze the structural covariance of genomic regions close to candidate retrotranscript RTs. ncRNA classification method:

In order to identify which type of ncRNA a sequence may belong to, the sequence can be checked against covariance models for existing ncRNA types. The Infernal package (http://eddylab.org/infernal/) provides tools for doing this. In short, covariance models can be built based on structural alignments of known ncRNAs of each type and then organized into a CM database. From this, new sequences can be searched in the database to verify whether the sequence fits into any of the represented families.

The resulting retrotranscript RT sequences are provided in Table A herein. The resulting and corresponding retrotranscript ncRNA sequences are provided in Table B herein. Example 4: Design and demonstration of a retrotranscript-based editing system in cells

Current genome editing methods can effectively disrupt target genes by inserting and deleting genetic information using programmable nucleases. However, gene disruption that does not distinguish between mutant and wild-type alleles damages not only the mutant allele in the pathogenic mechanism, but also the wild-type allele in the normal physiology of the organism. For example, in transthyretin (TTR)-induced amyloidosis, the mutant TTR allele causes the disease, but the non-pathogenic wild-type allele plays a role in neuroprotection and injury response (MF, 2021). Since single nucleotide exchanges are the major variant in TTR (J, 2021), the vast majority of pathogenic alleles in humans differ from their non-pathogenic alleles by small modifications that require much more precise editing techniques to correct. Homology-directed repair (HDR) stimulated by nuclease-mediated DNA breaks has been widely used to install precise edits. However, HDR relies on the delivery of exogenous donor DNA, which can elicit a strong immune response and has been shown to be inefficient in delivering high abundances in recipients (D, synthetic DNA delivery systems, 2000). Current therapeutic ex vivo and in vivo delivery of donor DNA relies on AAV (adeno-associated virus) transduction. However, AAV is complex and expensive to manufacture. Furthermore, AAV has raised safety concerns in 30% of the 255 completed or ongoing clinical trials examined in the meta-analysis (W, 2022). In contrast, RNA delivery has successfully demonstrated safety, ease of manufacture, and efficacy in two SARS-Covid2 vaccines. Based on this adaptability of RNA as cargo, we searched for systems in which donor DNA for precise editing could be generated by RNA delivered within cells.

Retrotranscripts are defined by their unique ability to generate unusual satellite DNA, called msDNA (multi-copy single-stranded DNA), from RNA inside the cell (S, 1989). These bacterial elements are involved in phage defense (A, 2020) and consist of a non-coding RNA and a reverse transcriptase (RT) that reverses the non-coding RNA (ncRNA) into msDNA. Its strictly defined retrotranscription site enables the insertion of donor DNA sequences into the non-coding RNA substrate. In addition, its compact size is suitable for delivery with programmable nucleases in the form of all-RNA. These features make retrotranscripts attractive tools for precise genome editing in therapeutic applications.

An independent process for generating new DNA sequences in cells by retrotranscription is prime editing and its virally derived RT can achieve insertions of up to 44 bp in length (AV, 2019). Retrotranscripts in natural sequences can generate single-stranded DNA of up to 163 nucleotides (T, 1987), indicating that longer insertions are feasible in retrotranscript-mediated editing. Consistent with this, the newly identified retrotranscript could insert 100 bp with a 6 bp deletion into the EMX1 locus of HEK293T cells in the Streptococcus pyocyaneus Cas9 nuclease system. A. Evidence for retrotranscripts from different evolutionary branches in precise editing

In the first step, retrotranscript/retrotranscript-like RTs were searched in bacterial genomes. Phylogenetic analysis of over 7,000 retrotranscript/retrotranscript-like sequences identified from searches against bacterial genomes yielded the phylogenetic tree of Figure 28. Noncoding RNA genes were searched near the genomes of retrotranscript/retrotranscript-like RT sequences to find conserved RNA secondary structures similar to characteristic msr-msd transcripts: terminal self-hybridizing inverted repeats, hairpin structures in msr and msd, such as those represented in Figures 2-27. ncRNAs were identified with high confidence in some RTs using covariance modeling and consensus structure detection. To evaluate the suitability of retrotranscripts for gene editing in different lineages, Eco1, Eco3, Eco5, Aco1, RTX3-2042, 6083 v1, and 6943 were selected for further analysis (Figure 30). Eco1, Eco3, and Eco5 are retrotranscripts that have been previously experimentally validated to show msDNA production (9). Aco1 has recently been annotated in the literature but has not been experimentally validated to produce msDNA (10). RTX3-2042, 6083 v1, and 6943 are novel retrotranscripts.

Gene editing analysis was first performed in the form of plastid DNA. Retrotranscript elements were assembled in plastids, where RT was transcribed under the CAG promoter, and ncRNA was fused at its 3' end with the 5' end of the single guide RNA (sgRNA) of the Cas9 nuclease under the U6 or H1 RNA polymerase III promoter (see 31A and 31B). The desired sequence inserted at the EMX1 targeting site flanked by homologous arms on both sides was inserted into the msD stem loop of the ncRNA of each retrotranscript. As described in Figures 32 and 33, plastids were transfected into human embryonic kidney 293T (HEK293T) cells expressing Cas9 via lipid transfection. Three days after transfection, the EMX1 target gene locus was amplified by PCR, and the sequence was analyzed by next generation sequencing (NGS). The precise edits analyzed were characterized by 10 bp insertions and 6 bp substitutions, and their percentages were calculated by CRISPResso 2 analysis along with other editing results.

Representative editing results are shown in Figure 35. Eco1 (Figure 36), Aco1 (Figure 37), RTX3-2042 (Figure 38), RTX3-6083 v1 and 6943 (Figure 39A) showed precise editing efficiencies of 0.3%, 0.1%, 0.25%, 0.06% and 0.05% (left panels in Figures 36, 37, 38 and 39, respectively). Unwanted editing results were defined as indels that incorporated random nucleotides or deleted sequences near the Cas9 cut site, reaching 50%, 3%, 5%, 2% and 4% (right panels in Figures 36, 37, 38 and 39A, respectively). Subsequent experiments using the same assay indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 times more precise edits than Eco1, while indels generated by these two retrotranscripts were 2-3 times lower (Figures 39B and 39C). RTX3_2042 showed a similar frequency of precise edits as Eco1, but with greater variability than the other samples (Figures 39B and 39C). These data suggest that in addition to the previously characterized Eco1, the recently reported (Aco1) and three novel retrotranscripts (RTX3-2042, RTX6083 v1, RTX3-6943) also enable precise gene editing in human cells (11).

Subsequent experiments using the same analysis indicated that RTX3-6342S and RTX3-6342L, as well as RTX-1262, generated precise edits 10-20 times more frequently than Eco1 and approximately 5-10 times more frequently than RTX3-2042, 6083v1, 6943, and 0637 (see Figure 39E - left). In addition, indels were generated two times less frequently using RTX3-6342S, 6342L, 1262, 0637, and 6083v1 compared to Eco1 (Figure 39E - right). Indels were generated 80% less frequently using RTX3-2042 compared to Eco1 (Figure 39E - right). Additionally, all of these retrotranscripts generated precise edits more frequently than another retrotranscript validated in the literature, Eco3, but with similar or higher indel frequencies (Figure 39E). A small set of retrotranscripts were also modified with deletions in the msd stem to assess whether precise editing could be improved. We noted that in six retrotranscripts, precise editing increased after shortening the msd stem, while in the remaining three retrotranscripts, precise editing decreased or remained unchanged (Figures 57 and 58).

These data indicate that, in addition to Eco1 and Eco3, which have been previously characterized in gene editing, the recently reported (Aco1) and six novel retrotranscripts (RTX3-0637, RTX3-1262, RTX3-2042, RTX6083 v1, RTX3-6342, RTX3-6943) also enable precise gene editing in human cells. In addition, modifying the msd stem length can alter precise editing, but the effect is likely retrotranscript-specific. B. Evidence for retrotranscript-mediated gene editing in a whole RNA system

To establish an RNA-based editing system, gene editing assays were first performed in HEK293T cells that constitutively express the Cas9 nuclease with two RNA components (RT mRNA and ncRNA fused to the sgRNA of the Cas9 nuclease). With the RT and ncRNA isolated in RNA form, by optimizing the ratio between the RT enzyme and its substrate, it may be easier to identify conditions for higher precision editing and lower indels than when they are together in the same plastid. Three previously known retrotranscripts, EcoI, Eco3, and Eco5, were selected for this first set of experiments using the all-RNA format.

RT mRNA of each retrotransposons and its cognate ncRNA fused to sgRNA of Cas9 nuclease were generated by in vitro transcription. The experimental scheme in Figure 40 was followed. In HEK293T cells expressing Cas9, the gene editing activities mediated by Eco1, Eco3 and Eco5 were compared at two different ratios of RT mRNA and ncRNA-sgRNA fusion. More ncRNA than RT mRNA was transfected to maximize msDNA production for precise gene editing. The results showed that the precise editing for Eco3 was as high as 0.4% and the indel for Eco3 was as low as 10% (Figure 41). The precise editing frequency in RNA-based analysis can be comparable to that in plastid-based analysis.

Given that Eco3 produced the highest edits, RNA loading and RT mRNA: ncRNA-sgRNA fusion ratios were further optimized in the Eco3 retrotranscript system. ncRNA-sgRNA fusions were added to 0.2 or 0.5 µg RT mRNA at 1:2, 1:3, 1:4, 1:5, 1:8, 1:10 ratios of RT mRNA to ncRNA. 0.5 μg RT mRNA produced more precise edits than 0.2 ug at any ratio, and increased ncRNA correlated with higher precision edits (Figure 42, left). Indels were as low as 6% (Figure 42, right). With both RNA components, precise edits were achieved in up to 1% of the cell population.

Next, Cas9 mRNA was added to the RT mRNA and ncRNA-sgRNA fusions to create a full RNA editing system in HEK293T cells (Figure 43). The amount of Cas9 mRNA was titrated with the Eco3 retrotranscript at optimized RNA loading and ratios of RT mRNA to ncRNA-sgRNA fusions (Figure 44). Precise editing was observed in up to 0.1% of the cell population using 0.2 µg of Cas9 mRNA, and adding more did not increase editing efficiency. Although the frequency of precise editing was orders of magnitude lower than the 2-RNA system, editing occurred through the specific action of the Cas9 nuclease and the retrotranscript, as the absence of either abolished precise editing.

The whole RNA system was also delivered to cells via lipofection using the MessengerMAX reagent (Figure 45). This approach is more similar to the in vivo delivery of therapeutic RNA loaded in lipid nanoparticles (LNPs) in terms of RNA/lipid complex formation and cellular uptake mechanisms. When applied to the Aco1 retrotranscript system composed of Aco1 RT mRNA, Aco1 ncRNA-sgRNA fusion, and Cas9 mRNA, precise editing of 56 bp insertions and 6 bp deletions was observed in 0.1% of the cell population, and the insertion-deletion frequency was approximately 1.5% (Figure 46). The Aco1 retrotranscript has recently been annotated but has not yet been experimentally validated (MR, 2020). Aco1-mediated precise gene editing strongly suggests that the Aco1 retrotranscriptome may generate msDNA inside cells. Furthermore, the insert lengths in the precise editing exceed those produced by editing mediated by other reverse transcriptases (AV, 2019). C. Enhancement of retrotranscriptome-mediated gene editing in an all-RNA system by sgRNA incorporation

In all of the above experiments, a retrotranscriptomic ncRNA fused to the sgRNA of the Cas9 nuclease was used. This fusion RNA served as a template for the RT, while the sgRNA portion was likely complexed with the Cas9 nuclease. Two enzymes acting on a single RNA fragment could produce spatial effects that affect the activity of either enzyme. Consistent with this hypothesis, the indel frequency suggests that the overall Cas9 nuclease activity is only about 1.5% at 400 ng of ncRNA-sgRNA fusion (Figure 46, right).

To systematically test this hypothesis, Cas9 nuclease activity as indicated by indel frequency was compared between equimolar amounts of ncRNA-sgRNA fusions and isolated sgRNAs. When electroporated with 200 ng of Cas9 mRNA, 1 µg (7.5 pmol) of ncRNA-sgRNA fusions showed 20-fold lower activity than 0.266 µg (7.5 pmol) of isolated sgRNA alone (Figure 47). The results suggest that ncRNA-sgRNA fusions may inhibit the formation of a complex with the Cas9 protein or inhibit the activity of the Cas9-sgRNA complex, or both. In addition, chemically modified sgRNAs showed 6-fold higher activity than unmodified sgRNAs. These results suggest that Cas9 cleavage activity achieved by ncRNA-sgRNA fusions may limit precise editing and that addition of isolated sgRNAs may compensate for this. Modification of the isolated sgRNAs is expected to further enhance precise editing.

Figure 48 summarizes the analysis of sgRNA spike-in in the all-RNA system, and Figure R. shows the results using the Eco3 retrotransposons. At 0.2 μg Cas9 mRNA and 0.5 μg RT mRNA, the amount of sgRNA spike-in was titrated at 50, 100, and 200 ng (Figure 41, left panel). Titration was performed at two different RT mRNA: ncRNA-sgRNA fusion ratios = 1:6 or 1:8. By adding 50 ng of sgRNA spike-in to a 1:6 ratio, precise editing reached 40-fold and gradually increased with more sgRNA, reaching up to a 50-fold increase at 200 ng. The 1:8 ratio of RT mRNA:ncRNA-sgRNA fusion responded similarly to sgRNA addition and achieved slightly higher activity than 1:6, with up to 13% precise editing. In the right panel, the higher precision edits achieved by sgRNA addition were accompanied by higher indels.

An orthogonal delivery method, lipofection, was used to confirm the effect of sgRNA incorporation in precise editing (Figure 50). Although the total amount of RNA transfected was five-fold lower than electroporation, 3.4% precise editing was obtained without sgRNA incorporation, which is 7.5-fold higher than the precise editing achieved by electroporation (Figures 49 and 51, left). With the addition of as little as 2 ng of sgRNA, precise editing increased further by up to 3.5-fold, reaching 12% efficiency (Figure 51, left). Increasing the amount of sgRNA to 10 ng did not further change the efficiency. The observed precise editing was dependent on the retrotranscript, as the loss of retrotranscript components (RT mRNA and ncRNA-sgRNA fusion) completely abolished editing.

These data support that physical fusion of the ncRNA and the sgRNA of the Cas9 nuclease may be a limiting factor for Cas9 activity and thus precise editing, and that sgRNA incorporation significantly enhances editing by complementing it. D. Isolation of ncRNA and sgRNA

The effect of separation of sgRNA and ncRNA on editing was tested in the Eco3 retrotranscript system, and editing efficiency was compared between ncRNA-sgRNA fusions and juxtaposed separated ncRNA and sgRNA (Figure 52). Increasing amounts of sgRNA were added to 300 ng ncRNA, which is an equimolar amount of 400 ng ncRNA-sgRNA fusion. In the absence of added sgRNA, precise editing was not observed as expected. At 10 ng sgRNA, precise editing peaked at 2.23%, compared to 1.78% obtained with ncRNA-sgRNA fusions (Figure 52, left). Increasing the amount of sgRNA beyond 10 ng did not further improve precise editing. The frequency of indels (Fig. 52, right) shows a trend similar to that of precise edits.

These data suggest that separation of ncRNA and sgRNA may enable editing with comparable or even greater precision than ncRNA-sgRNA fusions. E. Leader retrotranscripts from different evolutionary clades display precise editing activity in alternative cell types

These examples have shown robust precise editing activity in 293T cells. To determine whether these results can be extended to other cell types, plasmids encoding the leader retrotransposon reverse transcriptase and ncRNA/EMX1 sgRNA fusion were co-electroporated with plasmids encoding Cas9-mCherry into K562 cells (an erythroblastic leukemia cell line) (Figure 87). Figure 88 (left panel) shows that RTX3_2042, 6083v1, 6943, 1262, 6342L and 6342S can generate precise editing frequencies of 1.3%, 1.2%, 1%, 2.9%, 5% and 6.7%, respectively. By including the EMX1 gRNA expression cassette on the Cas9-mCherry plasmid, the precise editing frequency of RTX3_6342S could be increased to 10.5%. This system also generated robust indel frequencies in all samples. These frequencies varied between retrotranscripts and did not correlate with precise editing frequencies (Figure 88, right). The lack of correlation between indels and precise editing may be explained by spatial effects in the ncRNA/sgRNA fusion that affect the interaction between Cas9 and gRNA or between the reverse transcriptase and ncRNA.

Precise editing efficiencies using the novel retrotranscripts were also compared to several retrotranscripts that have been experimentally validated in this system using Cas9-mCherry EMX1 gRNA plasmids (MR, 2020). Retrotranscripts Eco1, Eco3, Aco1, Sau1, Sen1, RTX3_2781, RTX3_1262, RTX3_6342L, and RTX3_6342S were tested and observed to have precise editing efficiencies of 0.1%, 0.8%, 14%, 3.4%, 0.1%, 14.2%, 19%, and 19.5%, respectively (Figures 89 and 90, left). Indels also varied between samples and were not associated with precise editing levels, which may be explained by the same spatial effects mentioned previously (Figures 89 and 90, right). These data indicate that RTX3_1262, 6342L, and 6342S generate precise edits at the same or greater frequency than previously documented retrotranscripts. F. Lead retrotranscripts can robustly generate precise edits using RNA-guided nicking enzymes

Previous experiments have shown that retrotranscripts can use double-strand breaks (DSBs) as recombination initiation events to install precise edits. However, using DSBs to initiate repair can lead to indel formation and chromosomal rearrangements at off-target sites, which are undesirable effects for therapeutic development (Schiorli, 2019). To avoid indels, ssDNA nicking can be used instead of DSBs to initiate recombination (13). To determine whether the Cas9 D10A nickase can initiate retrotranscript-mediated precise edits, a Cas9 D10A-mCherry EMX1 gRNA plasmid was co-electroporated into K562 cells with a plasmid encoding a retrotranscript RT and a ncRNA/EMX1 sgRNA fusion. No significant editing was observed when retrotranscripts Eco1, Eco3, RTX3_6083v1, or RTX3_6943 were used (Figure 91, left). However, precise editing frequencies were found to be about 0.2%, about 0.6%, about 0.2%, and about 0.4% for RTX3_2042, RTX3_1262, RTX3_6342L, and RTX3_6342S, respectively (Figure 91, left). As expected, the precise editing frequencies initiated by nicks were >10-fold lower than those initiated using DSBs, and the indel frequencies were much lower than those seen using Cas9 nuclease (Figure 91, right). These data demonstrate that RNA-guided nicking can be used in vitro to install editing encoded by novel retrotranscripts using a plastid-based system. G. Retrotran-mediated gene editing is compatible with LbCas12a and TnpB nucleases

These examples have used Cas9 nuclease or nickase to perform retrotranscript-mediated gene editing. However, alternative nucleases (such as but not limited to LbCas12a (Zetsche B, 2015) and TnpB (Karvelis, 2021)) can be used in retrotranscript-based gene editing systems to replace Cas9 nuclease or nickase. To determine whether Cas12a or TnpB can induce retrotranscript-mediated gene editing, plasmids encoding LbCas12a, TnpB or Cas9 and their corresponding EMX1 gRNA and plasmids encoding the retrotranscript RTX3_6083v1 reverse transcriptase and ncRNA templates for each nuclease were co-electroporated into K562 cells. Using this method, it can be noted that LbCas12a and RTX3_6083v1 may mediate about 0.6% of precise editing (Figure 92, left), with about 30% fewer indels than the Cas9 comparator (Figure 92, right). However, the precise editing frequency generated by TnpB and RTX3_6083v1 was about 0.04% in one guide and could not be detected in the other (Figure 93, left). This may be attributed to the limited cutting activity of gRNA#2, as indels were not detected above background. Although similar indel frequencies were seen using Cas12a with sgRNA#1 (Figure 93, right), the precise editing frequency was substantially lower than those seen using Cas12a (Figure 92, left). Differences in precise editing frequency may be due to differences in DSB formation, DSB resolution, or protein expression of nucleases. In addition, it should be noted that precise editing frequencies using separated Cas9 gRNA and retrotranscript ncRNA resulted in precise editing efficiencies similar to those seen in previous experiments when ncRNA/sgRNA fusions were co-electroporated with plasmids encoding Cas9 and EMX1 gRNA expression cassettes (Figure 91, left). In summary, retrotranscript-mediated gene editing can be achieved by Cas12a-like and TnpB-like nucleases, and it is not necessary to fuse gRNA to retrotranscript ncRNA to facilitate precise editing using plastid-based retrotranscript gene editors. H. Retrotranscript-mediated gene editing can be performed using truncated RT proteins

Retrotranscripts contain cooperating domains that may be critical for physiological phage defense functions but are dispensable for reverse transcriptase activity (A, 2020). RTX3_6342 contains an N-terminal region composed of several α-helices that are predicted not to align with the reverse transcriptase domain (Figure 94) (Jumper, 2021). To determine whether this region is required for precise editing, N-terminal truncations of varying lengths were created and precise editing activity was assessed by NGS. It should be noted that deletions of residues 1-49 AA, 1-99 AA, and 1-144 AA did not impair the precise editing activity of RTX3_6342 (Figure 95). As expected, indels were unaffected (Figure 95). These data indicate that the non-RT domains in 6342S are not required for reverse transcription. I. Retrotranscript-mediated installation of exon inserts 200-400 bp in size

To determine whether retrotransposon-mediated gene editing could install edits greater than 10 bp, attempts were made to edit 10, 25, 205, 305, and 405 bp at the EMX1 locus. Using RTX3_6342, precise editing efficiencies of about 35%, about 40%, about 30%, about 35%, and about 40% were observed for 10 bp, 25 bp, 205 bp, 305 bp, and 405 bp, respectively (Figure 96, left). RTX3_1262 was also tested in a similar manner and precise editing efficiencies of about 22%, about 35%, about 28%, about 2%, and about 10% were observed for 10 bp, 25 bp, 205 bp, 305 bp, and 405 bp, respectively. It should be noted that the frequency of indels was similar between samples, and thus nuclease activity was not limiting the installation of these edits (Figure 96, right). Together, these data indicate that inserts >205 bp can be efficiently installed using retrotranscript-mediated editing, which exceeds the size installed by previous reverse transcriptase-based gene editors (AV, 2019). In addition, RTX3_6342S tends to install longer edits at a more robust frequency than RTX3_1262.

Reverse transcriptases (RTs) tend to be more error prone compared to replicative DNA polymerases due to the lack of a proofreading domain and structural differences in the enzyme active site (Oscorbin IP, 2021). To determine whether longer edits were accurately installed using the RTX3_6342S reverse transcriptase, previous data were reanalyzed using the CRISPResso2 parameter that quantifies substitutions in the edited allele. It should be noted that when substitutions were quantified using CRISPResso2, the percentage of accurate edits decreased as the insert became longer (Figure 97, left panel, compare circles to triangles). This trend was not observed when indels were quantified (Figure 97, right, compare circles to triangles), indicating that reverse transcriptase fidelity is responsible for the mutations seen in the precise edits installed by RTX3_6342S. This suggests that further optimization of reverse transcriptase fidelity can be performed to accurately install large edits. J. Rational mutation induction of retrotranscript RT to improve processability and fidelity

The biochemical properties of various RTs, such as processability and fidelity, have been extensively characterized in the literature (Oscorbin IP, 2021). In addition, some RTs have been extensively engineered for high processability and fidelity for applications such as RNA sequencing library preparation and prime editing (Oscorbin IP, 2021) ( Yarnall MTN, 2023). However, the fidelity and processability of retrotranscript RTs have not been characterized, and no attempts have been made to improve these properties. The palm, finger, and thumb domains within most DNA polymerases interact with the template molecule and guide the incoming nucleotide to the catalytic core for efficient DNA synthesis (Oscorbib IP, 2020). Here, selected residues in these domains were mutated based on structural comparison of the Alphafold predicted structure of RTX3_6342S with the crystal structures of MMLV or Marathon RT and Eco1 (Table J1 below). Table J1. Mutations of selected residues in RTX3_6342S.

These residues were prioritized based on their proximity to the RNA/DNA template present in the MMLV/Eco1 structure and previously engineered MMLV or Marathon variants cited in the literature (Oscorbin IP, 2021) (Grünewald, 2023).

As a proof of concept, 32 RT variants were engineered in 9 residues in the RTX3_6342 RT thumb domain. Specifically, the following 32 RT variants are built based on RTX3_6342S: (1) E466K, E466N, E466Q, E466R; (2) K475N, K475Q, K475R; (3) M470K, M470N, M470Q, M470R; (4) N465K, N465Q, N465R; (5) S468K, S468N, S468Q, S468R; (6) S472G, S472P; (7) S476K, S476N, S476Q, S476R; (8) V477F, V477I, V477L, V477W; and (9) W473F, W473K, W473R, W473Y.

These constructs were then co-electroporated into K562 cells with plasmids encoding Cas9, EMX1 sgRNA, and RTX3_6342S ncRNA with 10 bp or 305 bp inserts. After 72 h, cells were harvested for amplicon sequencing analysis and edits were quantified using CRISPResso. Most rationally designed mutations moderately reduced the precise editing to indel ratio (PEIR) using 10 bp or 305 bp inserts (Figure 98, top). However, it should be noted that N465K increased PEIR when a 305 bp insertion was installed, indicating that the mutation increased the processivity of the RTX_6342 reverse transcriptase or total ssDNA production (Figure 98, bottom). It was observed that the literature mutations (T306K and W313F) that improved MMLV activity decreased the PEIR of 10 bp and 305 bp inserts when using heterologous mutants of RTX3_6342 (M470K and V477F) as single mutants and in combination (Figures 98 and 99). It was also noted that RTX3_6342 V265P (a heterolog of MMLV L139P) decreased the PEIR of 305 bp inserts, had no effect on 205 bp inserts, and increased the ratio when using 405 bp inserts (Figure 99). These results may be due to the presence of different sequence features or secondary structures in each individual insert. A second set of mutations was also screened based on previously engineered marathon RT variants or favorable Rosetta scores after modeling the 6342 RT. It was observed that mutations E238R, E479K, and K255P all resulted in an increase in PEIR relative to WT 6342 RT (Figure 100). An additional N-terminal truncation was constructed that retained the α-helix that could contact the retrotranscript RNA (Figure 101). It should be noted that when this partial truncation was used, rather than completely eliminating the N-terminal domain, the exact edit:indel ratio was increased when longer edits were installed (Figure 102). K. Protein Fusions to Improve Retrotranscript RT Processability

DNA polymerase processivity has not only been optimized by mutation induction of the polymerase itself, but domain fusions are also known to improve processivity (Oscorbib IP, 2020). The small DNA binding proteins Sso7d and Sac7d have been fused to MMLV RT to improve processivity nearly eightfold during PCR reactions on DNA and RNA templates. Additionally, attempts were made to improve the editing efficiency of the lead editor by fusing these proteins to MMLV RT, but no significant improvement in editing efficiency was observed (Yarnall MTN, 2023). However, the edits installed in these experiments were small, < 90 bp, and the native MMLV has a processivity of 69 ± 14 bp/binding event, suggesting that much longer edits would need to be installed to observe improvements (Yarnall MTN, 2023) (Oscorbib IP, 2020). This publication teaches and demonstrates that retrotran-mediated genome editing can be used to install edits up to 400 bp long. bp editing. To determine whether fusion of Sso7d or Sac7d might improve the efficiency of retrotranscript-mediated genome editing, two variants of each of Sac7d or Sso7d were fused at the N- or C-terminus to retrotranscript RTs from RTX3_1262 and RTX2_6342. The installation of the 405 bp insertion in the EMX1 locus was then evaluated. It was observed that fusion of these domains to the N-terminus of either RT tended to be more robust than C-terminal fusions. In addition, these N-terminal fusions resulted in an approximately tenfold increase in the frequency of installation of the 405 bp insert using RTX3_1262, and an approximately two-fold increase when using RTX3_1262 (Figure 103). We believe that this difference in magnitude may be due to the difference in the frequency of installation of the 405 bp insert in RTX3_6342 (approximately 4%) and RTX3_1262. The differences in baseline processability or ssDNA generation of these fusions (approximately 0.4%) are not statistically significant, and the effects of these fusions on RTs like RTX3_6342 may be more pronounced with longer inserts. L. Identification of alternative retrotransposons from the RTX3_6342 family

In addition to engineering retrotransposons, screening additional retrotransposons in the same family as certain highly efficient retrotransposons (e.g., RTX3_6342, 6943, 6083, and 1262) may also identify novel retrotransposons with more desirable gene editing characteristics. An additional 99 unique retrotransposons were selected from the initial dataset, with at least 70% similarity to RTX3_6342, 6943, 6083, or 1262 RTs or their corresponding ncRNAs. These retrotransposons were cloned into a plasmid vector expressing RT and ncRNA from a CAG promoter as ncRNA-gRNA fusions designed to insert 305 bp into the hEMX1 locus. This plasmid was co-electroporated into K562 cells with a second plasmid containing Cas9 and the same h EMX1 sgRNA to increase editing efficiency. After 72 h, the genomic DNA was isolated and the h EMX1 locus was amplified for sequencing. It was observed that all retrotranscripts in this dataset capable of precise editing frequencies > 1% were similar to the lead retrotranscript RTX3_6342, indicating that this family is particularly enriched for RTs compatible with retrotranscript-mediated gene editing (Figure 104). However, none of these RTs showed a precise editing frequency comparable to RTX3_6342. Example 5: Improved ncRNA generated by transcription from a linearized plasmid with a template having blunt ends shows higher precise editing efficiency

The previously performed in vitro transcription experiments to generate ncRNA used double-stranded DNA templates containing 3' overhangs (on the same strand as the T7 promoter sequence). Design and test new templates with blunt ends. See Figure 52. As shown in Figure 53, four RNAs (cas9 mRNA, Eco3 RT mRNA, gRNA targeting the EMX1 locus, and ncRNA) were transfected into 293T cells using MessengerMAX lipid transfection reagent. Cells were harvested 3 days later, genomic DNA was isolated, the EMX1 locus was amplified, an Illumina library was generated, and sequencing was performed on NextSeq. As compared with the precise editing of ncRNA generated using the overhang template, the precise editing percentage of ncRNA generated using the blunt-end template was improved by more than 5 times. Example 6: Improved ncRNA due to improved in vitro template optimization and ncRNA end protection of MS2 hairpin

By encoding additional elements on the plasmid template, these elements can be added to the ncRNA. Figure 53 shows a modified Eco3 ncRNA that includes an MS2 stem-loop hairpin structure added to its 3' end. As shown in Figure 54, accurate editing of the ncRNA containing the 3' MS2 structure resulted in a nearly 15-fold increase in accurate editing compared to ncRNA generated from the overhanging template, and a nearly 3-fold increase in accurate editing compared to ncRNA generated from the blunt-end template.

Figure 53 schematically depicts modifications performed to improve RNA quality. The first approach concerns how to linearize plasmid DNA templates for in vitro transcription (Figure 53, A). Two identical plasmids encoding Eco3 ncRNA were prepared, except for the restriction enzyme site at the 3' end of the ncRNA: one produced a 3' overhang and the other produced a blunt end. Templates with 3' overhangs can cause RNA polymerase to extend on opposite strands, resulting in longer transcripts containing complementary strand sequences (13). The second consideration is to add an MS2 hairpin at the 3' end of the ncRNA (Figure 53, B). Retrotranscript ncRNAs contain mutually complementary sequences at the 5' and 3' ends, which form a stem-loop structure. However, these sequences may also mediate intermolecular interactions, thereby producing higher-order structures. Adding an MS2 hairpin at one end may reduce these molecular interactions. The MS2 sequence is a bacteriophage-derived RNA aptamer that does not interact with endogenous proteins in mammalian cells.

Figure 54 shows testing of these new RNAs in a gene editing assay. The four components of the total RNA system (RT mRNA, Cas9 mRNA, ncRNA, sgRNA) were delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected in fixed amounts as shown. Using RNA with a template containing a 3' overhang produced 1.35% precise edits. Using a template containing a blunt end increased precise edits to 5.94%. Adding an MS2 stem loop to the 3' end of the ncRNA further increased precise edits to 12.39%. In the right figure, the indel frequency under the respective conditions is shown. In summary, precise edits were increased nine (9) times by optimizing the in vitro transcription template and 3' end protection of the MS2 hairpin. Example 7: Modified ncRNA with a cap and/or tail

ncRNA potency can be altered by adding a cap structure at the 5' end and/or a poly(A) tail at the 3' end, as depicted in Figure 55. As shown in Figure 56, the use of ncRNAs with either or both protections provided by a cap and a tail reduces indels compared to uncapped/tailed ncRNAs. In this experiment, a 4-component total RNA system (RT mRNA + Cas9 mRNA + ncRNA-sgRNA + sgRNA) was delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected at fixed amounts of RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusions were capped (+cap–tail) or poly(A) tailed (-cap+tail), or both capped and poly(A) tailed (+cap+tail). Using RNA without end protection (-cap-tail) resulted in approximately 4.5% precise editing, and editing was dependent on the retrotranscript, as RT deficiency abolished precise editing. Using RNA with either or both cap and tail protection resulted in less precise editing (left), but reduced indels (right), compared to the case without cap and tail. Example 8: Precise editing using Cas9 nickase

Wild-type (WT) Cas9 nucleases generate double-strand breaks (DSBs) that can potentially increase genomic instability by generating larger deletions or chromosomal translocations. In addition, DSBs induce the p53-mediated DNA damage response pathway, which is usually impairing the physiology of the edited cell. Single-point mutations in either Cas9 nuclease domain can generate a nickase that only cleaves one strand of the target site. The D10A variant uses the complete HNH nuclease domain to cleave the target strand and the H840A variant uses the complete RuvC domain to cleave the non-target strand. Nicking can initiate homology-directed repair (HDR) and causes less mutation-inducing end joining than DSBs. These features have generated considerable interest in the use of nickases for precise retrotran-mediated editing.

Figure 73 shows the results of testing Cas9 nickase activity in a 4-component total RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. D10A and H840A nickases were used in parallel with WT Cas9 and tested with isolated ncRNA or ncRNA-sgRNA fusions. Total RNA was transfected with fixed amounts of RT mRNA 100 ng, ncRNA/ncRNA-sgRNA 400 ng, Cas9 variant mRNA 100 ng, and sgRNA 5 ng. When ncRNA or ncRNA-sgRNA was used with additional sgRNA, the precise editing of Cas9 WT (wild type) was about 9%. For the Cas9 H480A mutant, which nicks the off-target strand, little activity above background was seen under any condition. Using the Cas9 D10A mutant, which nicks the on-target strand, precise edits were observed at a frequency of approximately 0.1% in both isolated and fused ncRNA and sgRNA formats. The right panel shows indels under the respective conditions in the left panel. As expected, indels were extremely low for either nicking enzyme in the presence or absence of additional sgRNA, and only WT Cas9 generated significant indels.

Figure 74 shows negative controls lacking one component (Cas9 variant or ncRNA/ncRNA-sgRNA or RT) in the all-RNA system. They were tested with or without additional sgRNAs. All controls in the top panel showed no precise editing activity above background levels, supporting that precise editing achieved by Cas9 WT or Cas9 nickase depends on each component of the all-RNA system. Example 9: Dual sgRNAs achieve higher precise editing than single sgRNAs

It is speculated that the use of multiple sgRNAs near the target site may generate more free DNA ends after cutting and recruit more repair mechanisms, which will provide a higher chance of accurate editing. Two pairs of sgRNAs were found to test this hypothesis, sgRNA1 inserted immediately on the left side and sgRNA2 or sgRNA3 inserted on the right side of the EMX1 gene (schematic diagram on the right side of Figure 68). The test results of double sgRNA and single sgRNA are shown in Figure 37. The RTX2042 retrotransposons in the all-RNA system were delivered to HEK293T cells by lipofection. Additional sgRNAs were added to the given amount of RT mRNA, Cas9 mRNA and ncRNA-sgRNA. Under all conditions, the same ncRNA-sgRNA was used, and its template encoded a 25 bp insertion on the non-target strand of the Cas9 sgRNA. When one additional sgRNA (#1) was used, precise editing was 0.27%, and in the presence of a second sgRNA, precise editing increased up to 5-fold to 1.33% (#2) or 1.13% (#3). In the bottom graph, the indel frequencies under the respective conditions are shown.

These data suggest that dual sgRNAs targeting nearby sites may enhance precise editing mediated by retrotranscripts. Example 10. Editing using larger insert sizes

To date, most experiments have tested 10 bp insertions at the 3’ of the EMX1 gene by retrotranscript-mediated precise editing. The feasibility of longer inserts was examined by designing new ncRNAs with increasing insert lengths: 25, 50, 75 and 100 bp for Eco3, Aco1, RTX-2042 and RTX-6943 retrotranscripts, respectively. The inserted sequences were randomly selected, sharing no homology with the human genome, and all inserts had a GC content of 50%, generating no bias. At given amounts of RT mRNA (100 ng) and Cas9 mRNA (100 ng), 400 ng of ncRNA-sgRNA fusions with different template lengths ranging from 10 to 100 bp were added and delivered to HEK 293T cells by lipofection. In summary, the efficiency of all retrotranscripts was comparable across insert sizes. For 100 bp inserts, precise editing reached 1.04% for Eco3, 1.52% for Aco1, 1.37% for RTX2042, and 0.05% frequency for RTX6943 retrotranscript ( FIG. 59A ). In the bottom row of each figure, the indel frequencies of the individual conditions showed very similar trends to precise editing ( FIG. 59B ).

When sgRNA spike-in was added to the above conditions (Figures 60A and 60B), overall activity increased and activity was not compromised by longer inserts. For 100 bp inserts, precise editing increased 1.8-8 fold compared to no sgRNA spike-in, with precise editing of 1.82% for Eco3, 4.50% for Aco1, 4.40% for RTX-2042, and 0.4% for RTX-6943. In the bottom graph, the frequency of indels under the respective conditions also increased.

Combined with Cas9 nuclease, retrotransposons can precisely insert 100 bp into the target site of the genome with an efficiency of up to 4.5%. Example 11. Insertion of an entire gene by precise editing mediated by retrotransposons

Correction of large mutation hotspots or precise insertion of medium-sized genes into the genome will broaden the applicability of retrotranscript-mediated gene editing. To explore this point, the GFP gene (1267 bp, Figure 61A) with its own promoter and poly A signal was integrated into the ncRNA of the retrotranscript, and gene editing analysis was performed with three retrotranscripts Eco3, Aco1 and RTX6943. As a readout of insertion, the 5' and 3' junctions between the EMX1 host and the insertion sequence were amplified by qPCR. ΔCt (Ct of samples without RT - Ct of samples with RT) values are used for relative quantification of integration. The larger the ΔCt value, the higher the insertion frequency in the indicated sample. For all three retrotranscripts, amplicon at the 5' and 3' junctions were detected significantly above background levels (Figure 61B). The amplicon at the 3' junction was more abundant than the amplicon at the 5' junction, probably because the 5' end of the msDNA produced by the retrotranscription was enriched, promoting insertion at the 3'. Figure 61D shows the PCR product of the Aco1 retrotranscript, analyzed by Agilent Tapestation High Sensitivity D1000 screentape. In the presence of RT (+RT samples, left on the gel), amplicon of the expected size was detected at both the 5' and 3' junctions of the edited allele, as indicated by the arrows. In the absence of RT (-RT samples, right side of the gel), the edited allele was not detected. The triangle indicates the expected amplicon of the unedited allele, which is present in both -RT and +RT samples. In addition, Figure 61C shows qPCR data, depicting the insertion of GFP at the 5' or 3' junction of two retrotransposons in the EMX1 locus. The ncRNA-sgRNA engineered to contain the GFP gene insert was transfected into HEK293T cells with/without additional sgRNA together with the RT mRNA and Cas9 mRNA of each retrotransposon. The genome DNA was analyzed by the primers indicated in the table to detect the GFP gene insertion at the 5' or 3' junction on the edited allele. ΔCt was obtained by subtracting the junction Ct value from the EMX1 Ct value for the relative quantification of GFP insertion at the EMX1 locus. For both retrotranscripts, both 5' and 3' spliced amplicon were detected significantly above background levels. The 3' spliced amplicon was slightly more abundant than the 5' spliced amplicon, probably because the 5' end of the msDNA generated by retrotranscription is more enriched, promoting insertion at the 3' end. Thus, two different retrotranscripts have been shown to insert entire genes by retrotran editing.

Together, these results demonstrate the feasibility of inserting entire genes via retrotranscript-mediated precise editing. Example 12. Sequence of sgRNA and ncRNA in fusion RNA

Next, the order of sgRNA and ncRNA in the fusion was resolved. This order may play an important role in ensuring the activity of two enzymes (Cas9 and retrotranscript RT) on one fusion substrate. Comparison of A. ncRNA-sgRNA and B. sgRNA-ncRNA orders for precise editing with Eco3, Aco1 and RTX-2042 retrotranscripts (Figure 62A). For Eco3 and RTX-2042, the sgRNA-ncRNA order allowed significantly higher activity in precise editing (6-fold and 3-fold, respectively) and indels (3-fold and 2-fold, respectively) compared to the ncRNA-sgRNA order. The same order provided higher indels (2-fold) for Aco1, indicating that the sgRNA at the 5' of the fusion allows higher Cas9 activity. However, precise editing with Aco1 was comparable between the two orders.

When sgRNA spike-in was added to the above conditions, overall activity increased slightly (Figure 62B). The addition of sgRNA spike-in compensated for the poor Cas9 activity when using ncRNA-sgRNA (compared to Cas9 activity when using sgRNA-ncRNA), so that precise editing and indels became comparable in reverse order for all retrotranscripts.

Precise editing depends on the presence of RT and Cas9 (Figure 63). When either of these components is removed, precise editing is undetectable or at background levels in any sequence of ncRNA and sgRNA fusions (Figure 63 top panel). This is consistent with all three retrotranscripts. In the bottom panel, the indel frequency under each condition is shown (Figure 63 bottom panel). Example 13. Isolation of ncRNA and sgRNA

Adding sgRNA incorporation in either order of ncRNA and sgRNA fusion increased precise editing and indels (Figures 62A, 62B and Figure 63). These data suggest that physical fusion of ncRNA and sgRNA of the Cas9 nuclease may be a limiting factor for Cas9 activity and therefore precise editing, and therefore separation of ncRNA and sgRNA may be a better strategy for precise editing.

The effect of separation of sgRNA and ncRNA on editing was tested in the Eco3, Aco1, RTX-2042 and RTX-6943 retrotranscript system, and editing efficiency was compared between A. ncRNA-sgRNA or B. sgRNA-ncRNA fusions and C. isolated ncRNA and sgRNA with or without additional sgRNA (Figure 64). When using Eco3, the separation effect was significantly better (about 5-fold) in precise editing than any fusion order of ncRNA and sgRNA. In the case where no sgRNA was added to the isolated ncRNA, precise editing was not observed as expected. On the other hand, the isolated forms of Aco1 and RTX-2042 ncRNAs achieved a level of precise editing comparable to that of the fusion RNA. The isolated ncRNA of RTX-6943 showed lower activity than the fusion form, but the overall activity of this retrotran was an order of magnitude lower than other retrotranscripts.

These data suggest that separation of ncRNA and sgRNA may enable comparable or even higher precision editing than fusion of ncRNA and sgRNA, depending on the retrotranscript type. Example 14. Retrotranscript-mediated precise editing occurs at another genomic locus, AAVS1

All previous gene editing analyses were performed at the EMX1 locus. The AAVS1 locus was targeted for precise editing using the Eco3, Aco1, RTX2042, and RTX6943 retrotranscripts. The AAVS1 locus in intron 1 of the PPP1R12C (protein phosphatase 1 regulatory subunit 12C) gene is referred to as a "safe harbor" because its disruption does not adversely affect the cell, and robust transcription of the locus allows for stable expression of exogenously inserted genes. Within the msD stem loop of the ncRNA from each retrotranscript, a 25 bp insertion donor sequence was incorporated, and the sgRNA targeting AAVS1 was fused to the 3' end of the engineered ncRNA.

Figure 65 shows the results of testing four retrotranscripts to edit the AAVS1 locus. For all four retrotranscripts, precise editing was enhanced in the presence of additional sgRNA and ranged from 0.73% to 2.05%. On the right, the indel frequencies under the respective conditions had similar trends for precise editing. Removing RT, ncRNA-sgRNA, or Cas9 mRNA abolished precise editing, showing retrotranscript and Cas9 editing specificity (Figure 66), while indels were robust in the bottom graph. Example 15. Testing template strand bias for precise editing

Cas9 uses different nuclease domains to cleave on the sgRNA-targeted strand or the non-targeted strand. The time at which each nuclease domain of Cas9 is released from the DNA substrate after cleavage is different, which may affect how the single-stranded donor interacts with the broken DNA and the repair pathway (Richardson, 2016).

Results of testing donor templates on opposite strands relative to the Cas9 sgRNA targeted strand are shown in Figure 67. Eco3 and Aco1 retrotranscripts in the all-RNA system were delivered to HEK293T cells by lipofection. A given amount of RT mRNA, Cas9 mRNA, additional sgRNA targeting the EMX1 locus, and the appropriate ncRNA-sgRNA described below the figure were added. All donor templates tested contained a 25 bp insert. All ncRNAs used in previous experiments encode donor templates for the strand targeted by the Cas9 sgRNA and represented by the letter T. ncRNAs containing reverse complementary sequences of the Cas9 targeted strand are represented as NT. For Eco3, in the presence of additional sgRNA, the precise edits were 1.67% when the template was on the targeted strand and 0.85% when the template was on the non-targeted strand. For Aco1, in the presence of additional sgRNA, the precise edits were 2.96% when the template was on the targeted strand and 3.40% when the template was on the non-targeted strand. In the bottom column of each figure, the indel frequencies under the respective conditions are shown.

At least in the EMX1 locus, retrotranscript ncRNAs encoding either the Cas9 targeting strand or the non-targeting strand produced similar levels of precise editing. Example 16. Improvement of retrotranscript-mediated Cas9 nickase precise editing by combining dual sgRNAs

In previous studies, retrotranscript-mediated precise editing using the Cas9 nickase was observed, but the efficiency was low (about 0.1%). This is consistent with other studies that a single nick does not efficiently induce gene editing because the induced single-strand break is very efficiently repaired by the base excision repair pathway (Nakajuma K, 2018) (Ran FA, 2013). In contrast, exporting sgRNA with a pair of PAMs (protospacer adjacent motifs) near the Cas9 nickase results in efficient gene editing (precise editing and indels) (Ran FA, 2013). This double-nicking approach induces site-specific double-strand breaks, resulting in indels of the target gene, but off-targets are significantly reduced by 50-1000 times compared to Cas9 WT.

Regardless of whether the double nicking approach supports retrotranscript-mediated precise editing, three sgRNAs were selected at the 3' of the last exon of EMX1 (Figure 70). Combinations of sgRNA 1+3, 2+3, and 1+2 were tested with Cas9 D10A nickase, while Cas9 WT was tested using sgRNA1. All RNA was transfected with a fixed amount of RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 variant mRNA 100 ng, and sgRNA 5 ng. The sgRNA 1+3 combination gave the highest precise editing with an efficiency of 3% when using nickase (Figure 70, upper left). The efficiency when using dual sgRNAs was approximately 30 times higher than that achieved using a single sgRNA, but was still lower than Cas9 WT guided by sgRNA1 (13% editing efficiency). Editing is dependent on the retrotranscript system, as RT deficiency abolishes editing. The exact editing is not a ratio, but rather correlates with the indel frequency (Figure 54, bottom left). Example 17. The novel retrotranscript R6083 is able to insert up to 100 bp in the all-RNA system

Figure 71 shows the results of testing different template lengths/ncRNA and sgRNA separation or fusion with R6083 retrotransposons in a full RNA system. Full RNA was delivered to HEK293T cells by lipofection. Under a given amount of RT mRNA and Cas9 mRNA, ncRNA-sgRNA fusions with different template lengths ranging from 25 to 100 bp or ncRNAs with 25 bp insertions were added. It was observed that the precise insertion of 100 bp into the EMX1 locus was about 2% efficient (Figure 71 upper left). The separated ncRNA exhibited lower activity than the ncRNA-sgRNA fusion with 25 bp insertion. Accurate editing depends on the presence of RT and ncRNA, as shown in the top right figure. In the bottom row of each figure, the insertion and deletion frequencies under the respective conditions are shown.

Figure 72 shows that the R6083 retrotranscript is able to insert a 25 bp template into the AAVS1 locus in an all-RNA system. Using additional sgRNAs, 3% editing efficiency was observed, and activity was completely dependent on the presence of RT or ncRNA (Figure 72 left). Example 18. Cas9-RT fusion and isolated Cas9 and RT

In all previous studies, Cas9 and RT mRNA were delivered separately. Physically fused Cas9-RT may be beneficial for the colocalization of the two enzymes in the cellular compartment, and is harmful by interfering with the activity of each enzyme. To address this, Cas9 and Eco3 RT were fused by SGGSx2-XTEN-SGGSx2 (SEQ ID NO: 19932) flexible linker, and precise gene editing was tested in parallel with the separated form. Separation was performed using the same molar concentration of Cas9 (150 ng) and RT mRNA (50 ng) corresponding to 200 ng Cas9-RT fusion, and the editing efficiency of the fused or separated enzymes was compared for single ncRNA (left side of the left figure of Figure 73) or ncRNA-sgRNA fusion (right side of the right figure of Figure 73). Separated Cas9 and RT achieved higher editing efficiencies than when they were fused when both ncRNA (3-fold) and ncRNA-sgRNA (1.5-fold) were used. The higher editing of the separated enzyme was more pronounced when the ncRNA was separated from the sgRNA, suggesting that the separated enzymes searched the respective targets more flexibly. Consistent with precise editing, the fusion enzymes had lower indels in both ncRNA and ncRNA-sgRNA fusions. These results suggest that Cas9 and RT in fusion form can constrain each other, even with a flexible linker in between. Example 19. Retrotranscript ncRNA end protection by cap and tail (new experiment)

The 5’ and 3’ ends of eukaryotic mRNAs are modified by capping and tailing, respectively. Both caps and tails protect the transcript from exonucleases and aid in its export from the nucleus and translation on the ribosome. Recently, long noncoding RNAs (>200 nt) were shown to be commonly capped and tailed (L Statello, 2021).

In an attempt to increase the half-life of retrotranscript ncRNAs in cells, caps or tails, as well as both caps and tails, were added to the ncRNAs (Figure 55 schematic). Figure 74 shows the results of end protection of ncRNA-sgRNA fusions by caps and tails in a 4-component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All-RNAs were transfected with fixed amounts of RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusions were capped (+cap–tail) or poly A-tailed (-cap+tail), or both capped and poly A-tailed (+cap+tail). When additional sgRNAs were added, ncRNAs without end protection (-cap-tail) produced approximately 4.5% precise editing, and editing was dependent on the presence of RT. While capping alone did not alter editing efficiency, tailing alone slightly increased editing, and both capping and tailing significantly improved editing efficiency by approximately 2-fold (left panel). In the right panel, indels under all conditions are comparable as shown.

These results support that cap and tail protection of ncRNA ends may promote accurate retrotranscript-mediated editing by enhancing ncRNA stability in cells. Example 20. ncRNA circularization

We sought alternative ways to stabilize ncRNA. Circular RNAs form covalently closed continuous loops and this endless form confers durability for extension. Figure 75 describes how circular ncRNAs are made. Circularization utilizes the group I self-splicing intron method, which is reported to be more suitable for long RNA circularization and only requires the addition of GTP and Mg2 ⁺ as cofactors (22). The ncRNA is surrounded by self-splicing introns and complementary homologous sequences (Figure 75, step 1). External and internal homologous sequences bring the two introns into proximity (step 2). Exogenously added GTP initiates a series of transesterifications, thereby excising the intron and joining the ncRNA into a circular form (step 3).

Figure 76 summarizes the results of testing modified ncRNAs for precise editing at the EMX1 locus in HEK293T cells by the Eco3 whole RNA system. Whole RNA was transfected with fixed amounts of RT mRNA 100 ng, ncRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. When the MS2 stem loop was added to the 3' end of the ncRNA, the activity was almost doubled relative to the unmodified ncRNA (approximately 8%), with an efficiency of 15%. Circularization of the ncRNA achieved a further increase in efficiency, reaching 22%. Circularization of the ncRNA was performed as described in Figure 75. Without sgRNA, precise editing and indels could not be detected as expected. The right figure shows indels under the respective conditions of the left figure. Example 21. RT and its cognate ncRNA from the same retrotranscript are required to support accurate editing

Retrotranscript RT does not require exogenous primers, but instead utilizes specific branched chain G residues within the folded secondary structure of the template ncRNA to initiate retrotranscription (Lampson BC, 2005). Some early discovered RTs were shown to specifically recognize secondary structures and initiate msD synthesis in G in their homologous ncRNAs (Shimamoto T, 1993). This RT-ncRNA specificity was tested in a gene editing system using the recently characterized Aco1 retrotranscript and one of the early retrotranscripts, Eco3. Consistent with previous studies, Aco1 only supports precise editing when paired with its homologous ncRNA, and abolishes activity when paired with Eco3 ncRNA (Figure 77, left). The same observations were made for the Eco3 retrotranscript and Eco3, and only their homologous ncRNAs promoted precise editing. The right panel shows the indels under different conditions in the left panel. Example 22. Accurate insertion of exon-sized sequences

Exon-sized long insertions have clinical value in replacing mutation-rich exons that cause genetic diseases. For example, exon 8 is a mutation hotspot in Wilson's disease patients, with approximately 500 mutations that vary in region and ethnicity (Liu L, 2019). We tested Aco1, Eco3, and a de novo R1262 retrotransposons of varying sizes (up to 305 bp) inserted at the EMX1 locus in HEK293T cells. Aco1 achieved precise insertions of 10, 100, and 205 bp at 8, 4, and 1.3% (Figure 78, top left). Eco3 performed precise insertions of 10 and 205 bp at 12 and 2.3%, respectively (Figure 78, top center). The novel retrotranscript R1262 obtained 25, 205, and 305 bp insertions with efficiencies of 20, 15, and 8.8% (Figure 78, top panel, right). Compared with Aco1 or Eco3, the activity of R1262 was very robust, with no significant decrease in activity when the size increased from 25 to 205 bp. In addition, the ratio of precise editing to indels was minimal (1:2) when using R1262 (indels in the bottom panel of Figure 78). In the absence of RT or sgRNA, precise editing was abolished, indicating that the system is dependent on the retrotranscript and site-specific nuclease.

In addition to the EMX1 locus, the R1262 retrotranscript also supported a 205 bp precise insertion at the AAVS1 locus with an efficiency of 11% (Figure 79). These data expand the potential of retrotranscript-mediated gene editing as a genetic medicine. Example 23. Optimization of RT:ncRNA ratios for >200 bp long inserts using the R1262 retrotranscript

ncRNA is a source of single-stranded DNA that serves as a repair template, and the production of more single-stranded DNA may induce higher editing. However, among RT, Cas9 mRNA and sgRNA, ncRNA may have the shortest survival time in cells and is a limiting factor in retrotranscript-based editing systems because unchemically modified ncRNA is susceptible to cellular nucleases and, after retrotranscription, ncRNA is degraded by cellular RNase H (Palka C, 2022). Based on these assumptions, the all-RNA system is prepared with the ncRNA that accounts for the highest ratio in the mixture, which is consistent with previous studies using the Eco3 retrotranscript. Figure 80 shows the results of testing the RT: ncRNA ratio for >200 bp long inserts at the EMX1 locus using the novel retrotranscript R1262. RT: ncRNA ratios were tested at 1:12.5, 1:16.7, and 1:25 molar ratios. The 1:16.7 ratio corresponds to the 1:4 mass ratio used in the remaining studies. When using ncRNA (right side in left figure), optimal editing was observed at a 1:12.5 ratio for 205 and 305 bp inserts. When using ncRNA-sgRNA fusions (left side in left figure), all ratios tested produced comparable edits, but a weak trend of increased editing was seen with increasing amounts of ncRNA-sgRNA. Example 24. Novel retrotranscript R2781 is capable of inserting up to 100 bp in an all-RNA system

Figure 105 shows the results of testing ncRNAs of different template lengths with the novel retrotransposons R2781 in the full RNA system. Full RNA was delivered to HEK293T cells by lipofection. Under a given amount of RT mRNA and Cas9 mRNA, ncRNAs with different template lengths were added. In Figure 16, R2781 demonstrated 10 bp insertion at the EMX1 locus, with similar activity to other hits R1262, R6342S, and 6342L. Here, two different homology arm (HA) lengths (double-arm 30 bp or 49 bp left arm/65 bp right arm) were used to assess the activity of R2781 inserting 25 bp, 205 bp, and 405 bp at the EMX1 locus. Accurate insertion of 25 bp and 30 bp homology arms was achieved with 11% efficiency (left figure). When using longer homology arms (49/65 bp), the efficiency of insertion of the same length is reduced to half, indicating that this retrotranscript reverse transcriptase may have limited enzymatic processivity. In contrast, the activity of insertions of 205 or 405 bp is significantly reduced. The right figure shows the indels under the respective conditions of the left figure. Example 25. Precise editing with Cas9 nickase and retrotranscript R6342S

Wild-type (WT) Cas9 nucleases generate double-strand breaks (DSBs) that can potentially increase genomic instability by generating larger deletions or chromosomal translocations or chromosomal losses (Nahmad AD, 2022). Single-point mutations in either Cas9 nuclease domain can generate a nickase that cleaves only one strand of the target site. The D10A variant uses the full HNH nuclease domain to cleave the target strand and the H840A variant uses the full RuvC domain to cleave the non-target strand. Nicks can initiate homology-directed repair (HDR) and cause less mutation-inducing end joining than DSBs (Maizels N, 2018). These features have generated great interest in the use of nickases in retrotranscript-based precision editing.

Previously, the Eco3 retrotranscript was used in combination with the D10A nickase to achieve retrotranscript-based precise insertion of 10 bp at 0.1% efficiency (Figure 76). Here, D10A wild type and D10A nickase with R221K/N394K mutations, which had previously shown improved Cas9 nuclease activity (Spencer JM, 2017), were tested with one of the leading retrotranscripts, R6342S. The precise editing activity of R6342S was observed to be 3-4 times higher than that of Eco3 using the wild-type nickase, and was further enhanced (7 times higher) when the R221K/N394K nickase mutant was used (Figure 106). Example 26. Retrotranscript-mediated precise deletion of genomic DNA

Many genetic diseases can potentially be corrected by simply deleting abnormal genomic sequences. The dystrophin gene in muscle disease (Nelson CE, 2016) and the Col7A1 gene in skin disease (Bonafont J, 2019) as well as repeat expansion disorders (Meijboom KE, 2022) are good examples of deleting sequences containing unwanted mutations. Here, precise deletions mediated by retrotranscript R1262 were tested in a total RNA system. In Figure 107, two deletion strategies are depicted in the top figure. Retrotranscript ncRNAs were designed by juxtaposing left and right homology arm sequences to delete intervening sequences. Del1 deleted 214 bp upstream of the Cas9 cleavage site at the EMX1 locus and del2 deleted 248 bp downstream of the Cas9 cleavage site. Retrotranscript-mediated deletions were compared with direct delivery of increasing amounts of single-stranded DNA (ssDNA) donors (150 and 300 ng). In the bottom figure, the left figure shows that R1262 can delete 248 bp (del2) from the EMX1 locus with activity similar to that of the ssDNA donor. The right figure shows the indels under the respective conditions of the left figure. It should be noted that the accidental indel activity caused by retrotranscript-mediated deletions is about 6 times lower than that mediated by ssDNA donors. No 214 bp (del1) deletion activity of R1262 was detected.

This data suggests that retrotranscripts may be used to precisely delete genomic DNA with activity similar to that of ssDNA donors, but unexpectedly with lower activity. Example 27. NHEJ-based insertion of retrotranscript-derived double-stranded DNA at the target site

Current retrotranscript-mediated gene editing methods rely on the homology-dependent repair (HDR) mechanism, which is only effective in dividing cells. Most retrotranscripts generate single-stranded DNA (ssDNA), which can be used as HDR templates. In order to apply retrotranscript-mediated editing to non-dividing cells (for example, in non-dividing cells) where HDR is ineffective, alternative methods can be considered. One such alternative method is to integrate double-stranded DNA (dsDNA) at the target cleavage site using non-homologous DNA end joining (NHEJ). NHEJ-based dsODN/dsDNA integration has been demonstrated many times in the literature and works in cells and in vivo. This method can be used for small insertions as well as large insertions, such as exon replacements and safe harbor knock-ins.

A dsDNA template can be formed from a retrotranscript by using two ncRNAs of any retrotranscript, the encoded RT ssDNA products of which complement each other and form a duplex to become dsDNA (Figure 108, schematic diagram on the left). These dsDNAs contain msR and msD spacer sequences at both ends, which do not hybridize with each other and may inhibit end joining. Including sgRNA recognition sequences (same as the genome target sequence) in series at both ends of the insert will result in blunt-ended double-stranded DNA, and one insertion direction is more stable than the other because it prevents Cas9 from re-cutting. Insertion in the other direction allows Cas9 to re-cut, so the edited sequence is less stable. Using this strategy, the R1262 retrotranscript with two complementary ncRNAs inserted approximately 120 bp of dsDNA at the EMX1 locus with an efficiency of approximately 1%, and the efficiency was higher when the inserted sequence was flanked by two tandem sgRNA sequences (Figure 108, upper right). The basal insertion activity of R6342 without tandem sgRNA sequences was slightly higher than that of R1262. The right side of the bottom graph shows the insertions and deletions under the respective conditions of the top graph. This data suggests that retrotranscript-derived dsDNA may be directly integrated into the target site through the NHEJ mechanism and does not require homology to the integration site. Example 28. Retrotranscript RNA does not induce an obvious immune response

The potential immunogenicity of retrotranscript-mediated editing was tested in a total RNA system using human peripheral blood mononuclear cells (hPBMCs). PBMCs contain innate and adaptive immune cells, and these cells are equipped with sensors to detect foreign nucleic acids. RNA quality and the presence of 5' triphosphates in RNA generated by ex vivo transcription may activate immune sensors. In addition, single-stranded DNA prepared by retrotranscript RT using ncRNA as a template may potentially induce an immune response. To reduce the immune response, uridine in RT mRNA was replaced by the less immunogenic m1Ψ (Nace KD, 2021), and the 5' triphosphate of ncRNA was capped with m7G. These modified forms of RNA were electroporated into hPBMCs alone or in combination, along with GFP mRNA (TriLink) without the base modification as a control. After overnight culture, the supernatants were analyzed for interleukin production. Of the 25 interleukins and chemokines examined, those above the detection limit are shown in Figure 109. Type I interferons (a marker of immune response to foreign nucleic acids) were not detected in any of the retrotranscript RNA transfected cells, and low levels of some inflammatory interleukins and chemokines were detected in retrotranscript RNA transfected cells, but the levels were much lower than the GFP mRNA control except for the RT mRNA without the U modification. It should be noted that the ncRNA was not modified for U as a GFP mRNA control and was electroporated four times more than RT mRNA or GFP mRNA, reflecting the conditions used for gene editing. Despite this similarity and difference, ncRNA-transfected cells produced much less cytokines/interleukins than the GFP mRNA control. Example 29. Evidence for precise retrotranscript-mediated editing in primary cells

First, bone marrow-derived CD34+ human stem cells (HSCs) were used to evaluate the efficacy of retrotransposons-mediated gene editing in primary cells. Due to their long lifespan and their ability to generate the entire hematopoietic system, HSC transplantation has been used for decades to treat hematopoietic disorders such as genetic diseases or leukemias. Although transplantation therapy has a supportive effect on patients, it lacks clear curative results. In contrast, directly correcting mutations present in the genome of a patient's HSCs has the potential to produce a curative effect. This view will ultimately be confirmed by HSC gene editing therapy in a clinical setting.

In Figure 110, the results of testing retrotranscript-mediated gene editing in a total RNA system in human stem cells are shown. Frozen HSCs were thawed to expand for three days in the presence of hSCF, hFLT3-L, hTPO cytokines to prevent differentiation. A total of 3 or 5 μg of Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA, and ncRNA were mixed at the mass ratios indicated in each label in the left figure and electroporated into HSCs. After another three days of culture, genomic DNA was extracted and sequenced to measure the frequency of editing. Using a total of 5 μg of RNA (split into 1: 0.3: 4: 1 = Cas9: gRNA: ncRNA: RT), a precise insertion of 25 bp at the AAVS1 locus was observed at a frequency of 0.1% (left). The right panel shows the indels under their respective conditions.

Secondly, T cells are cells in the immune system that seek out and destroy unhealthy cells. These unhealthy cells are usually cells infected with harmful pathogens, but some T cells can also naturally recognize and kill cancer cells. Genetic engineering of T cell genomes enhances T cell function and immunotherapy to enhance T cell function has achieved remarkable clinical success in treating B cell acute lymphoblastic leukemia (Ellis GI, 2021). Based on these successes, extensive clinical development for cancer, infectious diseases and autoimmune diseases has utilized T cell genome engineering.

In Figure 111, the results of testing retrotranscript-mediated gene editing in a total RNA system in human T cells are shown. Human pan T cells from peripheral blood were thawed and activated for two days in the presence of anti-CD3/anti-CD28-conjugated magnetic beads and IL-2 cytokines. After culture, anti-CD3/anti-CD28 beads were removed from the cells with a magnet. A total of 3 or 5 μg of Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA, and ncRNA were mixed at the mass ratio indicated in each label in the left figure and electroporated into T cells by Neon or Lonza electroporators. After another three days of culture, genomic DNA was extracted and sequenced to measure the frequency of editing. Using a total of 3 µg RNA (split 1: 0.3: 3: 1 = Cas9: gRNA: ncRNA: RT), precise insertions of 25 bp at the AAVS1 locus were observed with a frequency of up to 1.7% using the Neon machine (left). The right panel shows indels under their respective conditions. Example 30. Retrotran-based gene editing in vivo using LNP

In certain embodiments, retrotranscript-mediated gene editing can be achieved with an all-RNA system without the need for a viral DNA donor or an exogenous DNA donor, because retrotranscripts can generate donor DNA from ncRNA templates inside cells. As proposed herein, this feature is particularly suitable for lipid nanoparticle (LNP)-based platforms. According to the methods described herein, the efficacy of retrotranscript-based gene editing in vivo is studied. Retrotranscript-based gene editing systems may be formulated in one or more LNPs.

Exemplary and non-limiting LNP formulation systems can be prepared for in vivo delivery of retrotranscript-based editing systems. LNP formulations for in vivo delivery of retrotranscript-based editing systems

Ionizable lipids, phospholipids, cholesterol, and PEG-lipids were dissolved in pure ethanol at defined molar ratios (exemplary formulations are shown below as formulation molar ratios A, B, C, and D), with a total lipid concentration of approximately 7.2 mM. A polynucleotide solution (exemplary concentration of 0.067 mg/mL) was prepared using an acidic buffer (pH 4.0-5.0) containing a gene editing system (e.g., Cas9 mRNA/gRNA/ncRNA/RT at a ratio of 1:0.3:4:1). Nucleotide and lipid solutions were mixed at a 3:1 volume ratio using a NanoAssemblr microfluidic system at a total flow rate of 12 mL/min, resulting in rapid mixing and self-assembly of LNPs. The formulation was further dialyzed against PBS (pH 7.4) at 4°C overnight, concentrated and filtered (0.2 µm pore size) using centrifugal filtration. The particle size and polydispersity index (PDI) of the formulation were measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen analysis. Formulation molar ratio A: Ionizable lipids DSPC Cholesterol DMG PEG-2k 48.5 10 39 2.5 Formulation molar ratio B: Ionizable lipids DSPC Cholesterol DMG PEG-2k 55 10 32.5 2.5 Formulation molar ratio C: Ionizable lipids DSPC Cholesterol DMPE PEG-2k 55 10 32.5 2.5 Preparation molar ratio D: Ionizable lipids DSPC Cholesterol DSPE PEG-2k 48.5 10 40 1.5 In vivo delivery solution based on LNP retrotranscript editing system

CD-1 female mice ranging in age from newborn to adult (6-10 weeks) were used in each study. LNP was administered via the caudal vein in a volume of approximately 5 mL/kg of body weight. The animals were observed regularly for side effects for at least 24 hours after dosing. Mice were dosed at 2 mpk. Five animals were dosed for each formulation. After 7 days, animals were euthanized by exsanguination via cardiac puncture under isoflurane anesthesia. On-target/off-target tissues were collected from each animal for DNA extraction and analysis. Editing was measured in mouse colonies by next generation sequencing (NGS). The overall health and well-being of the animals was recorded to determine whether delivery of the gene editing payload resulted in deleterious off-target editing. Example 31. Optimization of retrotranscript ncRNA sequences by high-throughput screening of ncRNA variant libraries

Retrotranscripts are native to prokaryotes and therefore, ncRNA sequences can be further engineered to adapt their applications and maximize their efficiency in human cells.

To understand and optimize the editing efficiency of retrotranscript ncRNA in human cells, a pooled ncRNA library was developed with 191 variants of different lengths and sequences in several key elements (sequences provided in Table 31A below) plus 2 controls (positive and negative controls) (such as, but not limited to, modifications of the length of the a1:a2 stem, msR stem-loop). Each of these variants is associated with a unique barcode designed in the donor region, and the variant library is synthesized into a pooled oligo library. The synthesized oligo library is cloned into a pooled DNA library, from which pooled ncRNAs are generated by in vitro transcription. The RNA library was then transfected into 293T cell lines and cells were harvested at different time points after transfection. Next generation sequencing was then applied to the unique barcodes to measure the abundance of each RNA variant, ssDNA donor production, and precise insertion of the edited loci. By comparing these datasets, the stability and efficacy of each ncRNA variant can be scored. From the perspective of the scores, enhanced ncRNAs can be optimized/engineered by integrating the most effective modified features. Methods:

191 ncRNA variants were designed, in which mutations were introduced into the following ncRNA elements: (1) a1a2; (2) msD stem; (3) msR stem; (4) msR loop; (5) terminator sequence (within the msR spacer); (6) msD, msR spacer base deletion (minimal variant). In addition, there is also a positive control (WT R6342S ncRNA) and a negative control with a disrupted reverse transcription start site. Each of these variants is associated with several unique barcodes designed in the donor region targeting the human EMX1 gene (e.g., "ACCTATCATTCANNNNNNNN"). The variant library was synthesized as a pooled oligo library. The synthetic oligo library was assembled into a pooled DNA library from which pooled ncRNAs were generated by in vitro transcription. ncRNA variant sequences and control sequences are provided in Table 31A. Representative library ncRNA constructs are depicted in Figure 112. The variant types in the library are depicted in Figure 113. Table 31A - ncRNA variant sequences and barcodes Mutant DNA sequence (variable regions are marked with lowercase letters) SEQ ID NO: Barcode SEQ ID NO: Type Positive control (WT) CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG ACCTATCATTCANNNNNNNN Positive control Negative control CATAGATTTCTTatCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG ACCTATCATTCANNNNNNNN Negative control Var0001 tataaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGttata 19543 ACCTATCATTCANNNNNNNN 19735 a1a2 variant Var0002 tgataGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGtatca 19544 ACCTATCATTCANNNNNNNN 19736 a1a2 variant Var0003 tctacGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGgtaga 19545 ACCTATCATTCANNNNNNNN 19737 a1a2 variant Var0004 gatccGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGgggatc 19546 ACCTATCATTCANNNNNNNN 19738 a1a2 variant Var0005 acgcgGCCTTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGcgcgt 19547 ACCTATCATTCANNNNNNNN 19739 a1a2 variant Var0006 cgcgcGCCTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGgcgcg 19548 ACCTATCATTCANNNNNNNN 19740 a1a2 variant Var0007 tattaaatttGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGaaatttaata 19549 ACCTATCATTCANNNNNNNN 19741 a1a2 variant Var0008 tataatacgaGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGtcgtattata 19550 ACCTATCATTCANNNNNNNN 19742 a1a2 variant Var0009 ttctgccaatGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGattggcagaa 19551 ACCTATCATTCANNNNNNNN 19743 a1a2 variant Var0010 tctcctcgagGCCTTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGctcgaggaga 19552 ACCTATCATTCANNNNNNNN 19744 a1a2 variant Var0011 ccgggttcgcGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGgcga acccgg 19553 ACCTATCATTCANNNNNNNN 19745 a1a2 variant Var0012 ggccgggcccGCCTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGgggcccggcc 19554 ACCTATCATTCANNNNNNNN 19746 a1a2 variant Var0013 aaatttaattataaaGCCTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGtttataatta aattt 19555 ACCTATCATTCANNNNNNNN 19747 a1a2 variant Var0014 atattctattacttgGCCTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGcaagtaataga atat 19556 ACCTATCATTCANNNNNNNN 19748 a1a2 variant Var0015 gcgtatagtaatctgGCCTTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGcagatt actatacgc 19557 ACCTATCATTCANNNNNNNN 19749 a1a2 variant Var0016 aacgcgaaacgctggGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGccag cgtttcgcgtt 19558 ACCTATCATTCANNNNNNNN 19750 a1a2 variant Var0017 gaggcgggtgccgcaGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGtg cggcacccgcctc 19559 ACCTATCATTCANNNNNNNN 19751 a1a2 variant Var0018 gcggccgcgggcgggGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGc ccgcccgcggccgc 19560 ACCTATCATTCANNNNNNNN 19752 a1a2 variant Var0019 aatattatattaaatattatGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGataatat ttaatataatatt 19561 ACCTATCATTCANNNNNNNN 19753 a1a2 variant Var0020 gaacaaactaaatataaatcGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTA Ggattttatatttagtttgttc 19562 ACCTATCATTCANNNNNNNN 19754 a1a2 variant Var0021 cgatttctaagacttcggtaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG taccgaagtcttagaaatcg 19563 ACCTATCATTCANNNNNNNN 19755 a1a2 variant Var0022 gtcagggaactccaggaggaGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGt cctcctggagttccctgac 19564 ACCTATCATTCANNNNNNNN 19756 a1a2 variant Var0023 cgtgggccgcgcttgagccgGCCTTTTATGCTGTGGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATT GTTAGcggctcaagcgcggcccacg 19565 ACCTATCATTCANNNNNNNN 19757 a1a2 variant Var0024 cggccgccggcgccggcgcgGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGT TAGcgcgccggcgccggcggccg 19566 ACCTATCATTCANNNNNNNN 19758 a1a2 variant Var0025 attaatatataaatttaattatataGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGt atataattaaatttatatattaat 19567 ACCTATCATTCANNNNNNNN 19759 a1a2 variant Var0026 gttatactaataagaattatctgaaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG ttcagataattctttattagtataac 19568 ACCTATCATTCANNNNNNNN 19760 a1a2 variant Var0027 ttaagatagaggcacttctagtgagGCCTTTTATGCTGTGGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGT TAGctcactagaagtgcctctatcttaa 19569 ACCTATCATTCANNNNNNNN 19761 a1a2 variant Var0028 caggcagcacgcacacagccgaaatGCCTTTTATGCTGTGGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATT GTTAGatttcggctgtgtgcgtgctgcctg 19570 ACCTATCATTCANNNNNNNN 19762 a1a2 variant Var0029 caccactggcgccgccagcaggcggGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGT TAGccgcctgctggcggcgccagtggtg 19571 ACCTATCATTCANNNNNNNN 19763 a1a2 variant Var0030 cgcggccgcgccgggcgcgcgcccgGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAA TATAAGAATTGTTAGcgggcgcgcgcccggcgcggccgcg 19572 ACCTATCATTCANNNNNNNN 19764 a1a2 variant Var0031 aataattattatataataatatattaataaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG ttattaatatattattatataataattatt 19573 ACCTATCATTCANNNNNNNN 19765 a1a2 variant Var0032 aataataatcccttagtctattagtttaGCCTTTTATGCTGTGGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGT TAGtaaactaatagactaagggattatattatt 19574 ACCTATCATTCANNNNNNNN 19766 a1a2 variant Var0033 taatttacaggacagggactttactcgatcGCCTTTTATGCTGTGGTGTTGCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAA GAATTGTTAGgatcgagtaaagtccctgtcctgtaaatta 19575 ACCTATCATTCANNNNNNNN 19767 a1a2 variant Var0034 tatctggcaacggccagagagagcgcctgaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATA AGAATTGTTAGtcaggcgctctctctggccgttgccagata 19576 ACCTATCATTCANNNNNNNN 19768 a1a2 variant Var0035 gccgtcccgcgcggcctgcccaaacgccctGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGagggcgtttgggcaggccgcgcgggacggc 19577 ACCTATCATTCANNNNNNNN 19769 a1a2 variant Var0036 cgccgccggccgggcgcgggcgccggcggcGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGgccgccggcgcccgcgcccggccggcggcg 19578 ACCTATCATTCANNNNNNNN 19770 a1a2 variant Var0037 aatatataaatattaataataataataaatGCCTTTTATGCTGTGGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATT GTTAGatttattaatattattattaatatttatatatatt 19579 ACCTATCATTCANNNNNNNN 19771 a1a2 variant Var0038 ttaatatctaacaattattgaagttgctttatttcGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGgaaataaagcaacttcaataattgttagatattaa 19580 ACCTATCATTCANNNNNNNN 19772 a1a2 variant Var0039 cagttatagcgcgaactagtttgacctgatttgtaGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGtacaaatcaggtcaaactagttcgcgctataactg 19581 ACCTATCATTCANNNNNNNN 19773 a1a2 variant Var0040 atagactggccgcccgtaggaacttgaggacgcacGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAAT ATAAGAATTGTTAGgtgcgtcctcaagttcctacgggcggccagtctat 19582 ACCTATCATTCANNNNNNNN 19774 a1a2 variant Var0041 gcgagcgcgaggacgccacgagcacgcgccctgccGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTC ACACAACCAAATATAAGAATTGTTAGggcagggcgcgtgctcgtggcgtcctcgcgctcgc 19583 ACCTATCATTCANNNNNNNN 19775 a1a2 variant Var0042 cggccggcgcccgggccgggcgggcccgcgggcggGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTC ACACAACCAAATATAAGAATTGTTAGccgcccgcgggcccgcccggcccgggcgccggccg 19584 ACCTATCATTCANNNNNNNN 19776 a1a2 variant Var0043 catagagGCCTTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGctctatg 19585 ACCTATCATTCANNNNNNNN 19777 a1a2 variant Var0044 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcaattcgctacgctaccaatactgtgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGA TGTCAcacagtattggtgctacgctgaagtgtcacaaccAAATATAAGAATTGTTAGCAAGAAATCTATG 19586 ACCTATCATTCANNNNNNNN 19778 msD stem variant Var0045 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcaattcgctacgctaccaataACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAttatt ggtgctacgctgaagtgtcacaaccAAATATAAGAATTGTTAGCAAGAAATCTATG 19587 ACCTATCATTCANNNNNNNN 19779 msD stem variant Var0046 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcaattcgctacgctacACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgtg ctacgctgaagtgtcacaaccAAATATAAGAATTGTTAGCAAGAAATCTATG 19588 ACCTATCATTCANNNNNNNN 19780 msD stem variant Var0047 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcaattcgctacACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtacgctgaagt gtcacaaccAAATATAAGAATTGTTAGCAAGAAATCTATG 19589 ACCTATCATTCANNNNNNNN 19781 msD stem variant Var0048 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcaattcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgaagtgtcacaacc AAATATAAGAATTGTTAGCAAGAAATCTATG 19590 ACCTATCATTCANNNNNNNN 19782 msD stem variant Var0049 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgcgcgcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgtgtcacaaccAAATATA AGAATTGTTAGCAAGAAATCTATG 19591 ACCTATCATTCANNNNNNNN 19783 msD stem variant Var0050 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggttgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcaaccAAATATAAGAATTGTTAGCAAGAAATC TATG 19592 ACCTATCATTCANNNNNNNN 19784 msD stem variant Var0051 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtataaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAttataAAATATAAGAATTGTTAGCAAGAAATCTATG 19593 ACCTATCATTCANNNNNNNN 19785 msD stem variant Var0052 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtgataACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtatcaAAATATAAGAATTGTTAGCAAGAAATCTATG 19594 ACCTATCATTCANNNNNNNN 19786 msD stem variant Var0053 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtctacACAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgtagaAAATATAAGAATTGTTAGCAAGAAATCTATG 19595 ACCTATCATTCANNNNNNNN 19787 msD stem variant Var0054 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgatccACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAggatcAAATATAAGAATTGTTAGCAAGAAATCTATG 19596 ACCTATCATTCANNNNNNNN 19788 msD stem variant Var0055 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAacgcgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcgcgtAAATATAAGAATTGTTAGCAAGAAA TCTATG 19597 ACCTATCATTCANNNNNNNN 19789 msD stem variant Var0056 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcgcgcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgcgcgAAATATAAGAATTGTTAGCAA GAAATCTATG 19598 ACCTATCATTCANNNNNNNN 19790 msD stem variant Var0057 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtattaaatttACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAaaatttaataAAATATAAGAATTGTTAGCAA GAAATCTATG 19599 ACCTATCATTCANNNNNNNN 19791 msD stem variant Var0058 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtataatacgaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtcgtattataAAATATAAGAATTGTTAGCAA GAAATCTATG 19600 ACCTATCATTCANNNNNNNN 19792 msD stem variant Var0059 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAttctgccaatACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAattggcagaaAAATATAAGAATTGTTA GCAAGAAATCTATG 19601 ACCTATCATTCANNNNNNNN 19793 msD stem variant Var0060 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtctcctcgagACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCActcgaggagaAAATATAAGAATTGTTA GCAAGAAATCTATG 19602 ACCTATCATTCANNNNNNNN 19794 msD stem variant Var0061 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAccgggttcgcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgcgaacccggAAATATAAGAATT GTTAGCAAGAAATCTATG 19603 ACCTATCATTCANNNNNNNN 19795 msD stem variant Var0062 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAggccgggcccACAAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgggcccggccAAATATAAGAATTGTTA GCAAGAAATCTATG 19604 ACCTATCATTCANNNNNNNN 19796 msD stem variant Var0063 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaaatttaattataaaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtttataattaaatttAAATATAAGAATT GTTAGCAAGAAATCTATG 19605 ACCTATCATTCANNNNNNNN 19797 msD stem variant Var0064 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAatattctattacttgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcaagtaatagaatatAAATATAAGAATT GTTAGCAAGAAATCTATG 19606 ACCTATCATTCANNNNNNNN 19798 msD stem variant Var0065 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgcgtatagtaatctgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcagattactatacgcAAATATAA GAATTGTTAGCAAGAAATCTATG 19607 ACCTATCATTCANNNNNNNN 19799 msD stem variant Var0066 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaacgcgaaacgctggACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAccagcgtttcgc gttAAATATAAGAATTGTTAGCAAGAAATCTATG 19608 ACCTATCATTCANNNNNNNN 19800 msD stem variant Var0067 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgaggcgggtgccgcaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtgcggcacccgcctc AAATATAAGAATTGTTAGCAAGAAATCTATG 19609 ACCTATCATTCANNNNNNNN 19801 msD stem variant Var0068 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgcggccgcgggcgggACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcccgcccgcggccg cAAATATAAGAATTGTTAGCAAGAAATCTATG 19610 ACCTATCATTCANNNNNNNN 19802 msD stem variant Var0069 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaatattatattaaatattatACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAataatatttaataataatattAAATATA AGAATTGTTAGCAAGAAATCTATG 19611 ACCTATCATTCANNNNNNNN 19803 msD stem variant Var0070 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgaacaaactaaatataaatcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgatttatatttagtt tgttcAAATATAAGAATTGTTAGCAAGAAATCTATG 19612 ACCTATCATTCANNNNNNNN 19804 msD stem variant Var0071 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcgatttctaagacttcggtaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtaccgaagtcttaga aatcgAAATATAAGAATTGTTAGCAAGAAATCTATG 19613 ACCTATCATTCANNNNNNNN 19805 msD stem variant Var0072 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgtcagggaactccaggaggaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtcctcctggagttcc ctgacAAATATAAGAATTGTTAGCAAGAAATCTATG 19614 ACCTATCATTCANNNNNNNN 19806 msD stem variant Var0073 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcgtgggccgcgcttgagccgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcggctcaagc gcggcccacgAAATATAAGAATTGTTAGCAAGAAATCTATG 19615 ACCTATCATTCANNNNNNNN 19807 msD stem variant Var0074 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcggccgccggcgccggcgcgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAcgcgccggc gccggcggccgAAATATAAGAATTGTTAGCAAGAAATCTATG 19616 ACCTATCATTCANNNNNNNN 19808 msD stem variant Var0075 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAattaatatataaatttaattatataACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAttataattaaatttatatattaat AAATATAAGAATTGTTAGCAAGAAATCTATG 19617 ACCTATCATTCANNNNNNNN 19809 msD stem variant Var0076 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgttatactaataagaattatctgaaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAttcagataattctttat tagtataacAAATATAAGAATTGTTAGCAAGAAATCTATG 19618 ACCTATCATTCANNNNNNNN 19810 msD stem variant Var0077 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAttaagatagaggcacttctagtgagACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCActcactagaagtg cctctatcttaaAAATATAAGAATTGTTAGCAAGAAATCTATG 19619 ACCTATCATTCANNNNNNNN 19811 msD stem variant Var0078 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcaggcagcacgcacacagccgaaatACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAatttcggct gtgtgcgtgctgcctgAAATATAAGAATTGTTAGCAAGAAATCTATG 19620 ACCTATCATTCANNNNNNNN 19812 msD stem variant Var0079 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcaccactggcgccgccagcaggcggACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAccgcctgct ggcggcgccagtggtgAAATATAAGAATTGTTAGCAAGAAATCTATG 19621 ACCTATCATTCANNNNNNNN 19813 msD stem variant Var0080 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcgcggccgcgccgggcgcgcgcccgACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAc gggcgcgcgcccggcgcggccgcgAAATATAAGAATTGTTAGCAAGAAATCTATG 19622 ACCTATCATTCANNNNNNNN 19814 msD stem variant Var0081 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaataattattatataataatatattaataaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAttattatatattata ataattattAAATATAAGAATTGTTAGCAAGAAATCTATG 19623 ACCTATCATTCANNNNNNNN 19815 msD stem variant Var0082 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaataatataatcccttagtctattagtttaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtaaactaatag actaagggattatattattAAATATAAGAATTGTTAGCAAGAAATCTATG 19624 ACCTATCATTCANNNNNNNN 19816 msD stem variant Var0083 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtaatttacaggacagggactttactcgatcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAgatcga gtaaagtccctgtcctgtaaattaAAATATAAGAATTGTTAGCAAGAAATCTATG 19625 ACCTATCATTCANNNNNNNN 19817 msD stem variant Var0084 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAtatctggcaacggccagagagagcgcctgaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAtcaggc gctctctctggccgttgccagataAAATATAAGAATTGTTAGCAAGAAATCTATG 19626 ACCTATCATTCANNNNNNNN 19818 msD stem variant Var0085 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgccgtcccgcgcggcctgcccaaacgccctACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGT CAagggcgtttgggcaggccgcgcgggacggcAAATATAAGAATTGTTAGCAAGAAATCTATG 19627 ACCTATCATTCANNNNNNNN 19819 msD stem variant Var0086 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcgccgccggccgggcggggcgccggcggcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGT CAgccgccggcgcccgcgcccggccggcggcgAAATATAAGAATTGTTAGCAAGAAATCTATG 19628 ACCTATCATTCANNNNNNNN 19820 msD stem variant Var0087 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAaatatataaaatattaataataatataataaatACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAatttattaattatt attattaatatttatatatattAAATATAAGAATTGTTAGCAAGAAATCTATG 19629 ACCTATCATTCANNNNNNNN 19821 msD stem variant Var0088 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAttaatatctaacaattattgaagttgctttatttcACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA gaaataaagcaacttcaataattgttagatattaaAAATATAAGAATTGTTAGCAAGAAATCTATG 19630 ACCTATCATTCANNNNNNNN 19822 msD stem variant Var0089 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcagttatagcgcgaactagtttgacctgatttgtaACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGT CAtacaaatcaggtcaaactagttcgcgctataactgAAATATAAGAATTGTTAGCAAGAAATCTATG 19631 ACCTATCATTCANNNNNNNN 19823 msD stem variant Var0090 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAatagactggccgcccgtaggaacttgaggacgcacACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCAg tgcgtcctcaagttcctacgggcggccagtctatAAATATAAGAATTGTTAGCAAGAAATCTATG 19632 ACCTATCATTCANNNNNNNN 19824 msD stem variant Var0091 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAgcgagcgcgaggacgccacgagcacgcgccctgccACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACAT CGATGTCAggcagggcgcgtgctcgtggcgtcctcgcgctcgcAAATATAAGAATTGTTAGCAAGAAATCTATG 19633 ACCTATCATTCANNNNNNNN 19825 msD stem variant Var0092 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAcggccggcgcccgggccgggcgggcccgcgggcggACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACAT CGATGTCAccgcccgcgggcccgcccggcccgggcgccggccgAAATATAAGAATTGTTAGCAAGAAATCTATG 19634 ACCTATCATTCANNNNNNNN 19826 msD stem variant Var0093 CATAGATTTCTTGGCCTTTATGCTGTGGTGtgtCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19635 ACCTATCATTCANNNNNNNN 19827 msR cyclomutant Var0094 CATAGATTTCTTGGCCTTTATGCTGTGGTGgttCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19636 ACCTATCATTCANNNNNNNN 19828 msR cyclomutant Var0095 CATAGATTTCTTGGCCTTTATGCTGTGGTGttcCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19637 ACCTATCATTCANNNNNNNN 19829 msR cyclomutant Var0096 CATAGATTTCTTGGCCTTTATGCTGTGGTGtctCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19638 ACCTATCATTCANNNNNNNN 19830 msR cyclomutant Var0097 CATAGATTTCTTGGCCTTTATGCTGTGGTGcttCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19639 ACCTATCATTCANNNNNNNN 19831 msR cyclomutant Var0098 CATAGATTTCTTGGCCTTTATGCTGTGGTGtttCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19640 ACCTATCATTCANNNNNNNN 19832 msR cyclomutant Var0099 CATAGATTTCTTGGCCTTTATGCTGTGGTGttttCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19641 ACCTATCATTCANNNNNNNN 19833 msR cyclomutant Var0100 CATAGATTTCTTGGCCTTTATGCTGTGGTGttgcCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19642 ACCTATCATTCANNNNNNNN 19834 msR cyclomutant Var0101 CATAGATTTCTTGGCCTTTATGCTGTGGTGcttgCGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19643 ACCTATCATTCANNNNNNNN 19835 msR cyclomutant Var0102 CATAGATTTCTTGCCTTTAttataaTTGttataGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19644 ACCTATCATTCANNNNNNNN 19836 msR stem variant Var0103 CATAGATTTCTTGCCTTTTATtgataTTGtatcaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19645 ACCTATCATTCANNNNNNNN 19837 msR stem variant Var0104 CATAGATTTCTTGGCCTTTATctacTTGgtagaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19646 ACCTATCATTCANNNNNNNN 19838 msR stem variant Var0105 CATAGATTTCTTGGCCTTTATgatccTTGggatcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19647 ACCTATCATTCANNNNNNNN 19839 msR stem variant Var0106 CATAGATTTCTTGGCCTTTATacgcgTTGcgcgtGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19648 ACCTATCATTCANNNNNNNN 19840 msR stem variant Var0107 CATAGATTTCTTGGCCTTTATcgcgcTTGgcgcgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTA TG 19649 ACCTATCATTCANNNNNNNN 19841 msR stem variant Var0108 CATAGATTTCTTGGCCTTTATattaaatttTTGaaatttaataGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19650 ACCTATCATTCANNNNNNNN 19842 msR stem variant Var0109 CATAGATTTCTTGCCTTTATataatacgaTTGtcgtattataGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19651 ACCTATCATTCANNNNNNNN 19843 msR stem variant Var0110 CATAGATTTCTTGCCTTTTATttctgccaatTTGattggcagaaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19652 ACCTATCATTCANNNNNNNN 19844 msR stem variant Var0111 CATAGATTTCTTGCCTTTATctcctcgagTTGctcgaggagaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19653 ACCTATCATTCANNNNNNNN 19845 msR stem variant Var0112 CATAGATTTCTTGGCCTTTATccgggttcgcTTGgcgaacccggGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG CAAGAAATCTATG 19654 ACCTATCATTCANNNNNNNN 19846 msR stem variant Var0113 CATAGATTTCTTGGCCTTTATggccgggcccTTGgggcccggccGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19655 ACCTATCATTCANNNNNNNN 19847 msR stem variant Var0114 CATAGATTTCTTGGCCTTTAaatttaattataaaTTGtttataattaaatttGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG CAAGAAATCTATG 19656 ACCTATCATTCANNNNNNNN 19848 msR stem variant Var0115 CATAGATTTCTTGGCCTTTATattattctattacttgTTGcaagtaagaatatGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAG CAAGAAATCTATG 19657 ACCTATCATTCANNNNNNNN 19849 msR stem variant Var0116 CATAGATTTCTTGGCCTTTATgcgtatagtaatctgTTGcagattactatacgcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATT GTTAGCAAGAAATCTATG 19658 ACCTATCATTCANNNNNNNN 19850 msR stem variant Var0117 CATAGATTTCTTGGCCTTTATaacgcgaaacgctggTTGccagcgtttcgcgttGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAA TATAAGAATTGTTAGCAAGAAATCTATG 19659 ACCTATCATTCANNNNNNNN 19851 msR stem variant Var0118 CATAGATTTCTTGGCCTTTATgaggcgggtgccgcaTTGtgcggcacccgcctcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAAT ATAAGAATTGTTAGCAAGAAATCTATG 19660 ACCTATCATTCANNNNNNNN 19852 msR stem variant Var0119 CATAGATTTCTTGGCCTTTTATgcggccgcgggcgggTTGcccgcccgcggccgcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAA TATAAGAATTGTTAGCAAGAAATCTATG 19661 ACCTATCATTCANNNNNNNN 19853 msR stem variant Var0120 CATAGATTTCTTGGCCTTTATaatattatattaaatattatTTGataatatttaataatattGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTA GCAAGAAATCTATG 19662 ACCTATCATTCANNNNNNNN 19854 msR stem variant Var0121 CATAGATTTCTTGCCTTTATgaacaaactaaatataaatcTTGgatttatatttagtttgttcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACA ACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19663 ACCTATCATTCANNNNNNNN 19855 msR stem variant Var0122 CATAGATTTCTTGGCCTTTATcgatttctaagacttcggtaTTGtaccgaagtcttagaaatcgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGCAAGAAATCTATG 19664 ACCTATCATTCANNNNNNNN 19856 msR stem variant Var0123 CATAGATTTCTTGGCCTTTATgtcagggaactccaggaggaTTGtcctcctggagttccctgacGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACC AAATATAAGAATTGTTAGCAAGAAATCTATG 19665 ACCTATCATTCANNNNNNNN 19857 msR stem variant Var0124 CATAGATTTCTTGGCCTTTATcgtgggccgcgcttgagccgTTGcggctcaagcgcggcccacgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATG TCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19666 ACCTATCATTCANNNNNNNN 19858 msR stem variant Var0125 CATAGATTTCTTGGCCTTTATcggccgccggcgccggcgcgTTGcgcgccggcgccggcggccgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATG TCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19667 ACCTATCATTCANNNNNNNN 19859 msR stem variant Var0126 CATAGATTTCTTGCCTTTTattaataatatataaatttaattatataTTGtatataattaaatttatatattaatGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAAT ATAAGAATTGTTAGCAAGAAATCTATG 19668 ACCTATCATTCANNNNNNNN 19860 msR stem variant Var0127 CATAGATTTCTTGCCTTTTATgttatactaataagaattatctgaaTTGttcagataattcttattagtataacGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACA CAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19669 ACCTATCATTCANNNNNNNN 19861 msR stem variant Var0128 CATAGATTTCTTGCCTTTTATTttaagatagaggcacttctagtgagTTGctcactagaagtgcctctatcttaaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATG TCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19670 ACCTATCATTCANNNNNNNN 19862 msR stem variant Var0129 CATAGATTTCTTGGCCTTTATcaggcagcacgcacacagccgaaatTTGatttcggctgtgtgcgtgctgcctgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATG GGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19671 ACCTATCATTCANNNNNNNN 19863 msR stem variant Var0130 CATAGATTTCTTGGCCTTTTATcaccactggcgccgccagcaggcggTTGccgcctgctggcggcgccagtggtgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGG ACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19672 ACCTATCATTCANNNNNNNN 19864 msR stem variant Var0131 CATAGATTTCTTGGCCTTTATcgcggccgcgccgggcgcgcgcccgTTGcgggcgcgcgcccggcgcggccgcgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACG AAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19673 ACCTATCATTCANNNNNNNN 19865 msR stem variant Var0132 CATAGATTTCTTGGCCTTTAataataattattatataataatatattaataaTTGttattaatatattattatataataattattGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAAT ATAAGAATTGTTAGCAAGAAATCTATG 19674 ACCTATCATTCANNNNNNNN 19866 msR stem variant Var0133 CATAGATTTCTTGGCCTTTATaataataatcccttagtctattagtttaTTGtaaactaatagactaagggattatattattGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTC ACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19675 ACCTATCATTCANNNNNNNN 19867 msR stem variant Var0134 CATAGATTTCTTGGCCTTTTATtaatttacaggacagggactttactcgatcTTGgatcgagtaaagtccctgtcctgtaaattaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATG GGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19676 ACCTATCATTCANNNNNNNN 19868 msR stem variant Var0135 CATAGATTTCTTGGCCTTTATtatctggcaacggccagagagcgcctgaTTGtcaggcgctctctctggccgttgccagataGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATG GGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19677 ACCTATCATTCANNNNNNNN 19869 msR stem variant Var0136 CATAGATTTCTTGGCCTTTATgccgtcccgcgcggcctgcccaaacgccctTTGagggcgtttgggcaggccgcgcgggacggcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCA CGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19678 ACCTATCATTCANNNNNNNN 19870 msR stem variant Var0137 CATAGATTTCTTGGCCTTTATcgccgccggccgggcgcgggcgccggcggcTTGgccgccgcgcccgcgcccggccggcggcgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCA CGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19679 ACCTATCATTCANNNNNNNN 19871 msR stem variant Var0138 CATAGATTTCTTGGCCTTTATaatatataaatattaataataataataaatTTGatttattaatattattattaatatttatatatattGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGA TGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19680 ACCTATCATTCANNNNNNNN 19872 msR stem variant Var0139 CATAGATTTCTTGGCCTTTATttaatatctaacaattattgaagttgctttatttcTTGgaaataaagcaacttcaataattgttagatattaaGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAG GCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19681 ACCTATCATTCANNNNNNNN 19873 msR stem variant Var0140 CATAGATTTCTTGGCCTTTATcagttatagcgcgaactagtttgacctgatttgtaTTGtacaaaatcaggtcaaactagttcgcgctataactgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGC CACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19682 ACCTATCATTCANNNNNNNN 19874 msR stem variant Var0141 CATAGATTTCTTGGCCTTTATatagactggccgcccgtaggaacttgaggacgcacTTGgtgcgtcctcaagttcctacgggcggccagtctatGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGC CACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19683 ACCTATCATTCANNNNNNNN 19875 msR stem variant Var0142 CATAGATTTCTTGCCTTTATgcgagcgcgaggacgccacgagcacgcgccctgccTTGggcagggcgcgtgctcgtggcgtcctcgcgctcgcGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCAT CACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19684 ACCTATCATTCANNNNNNNN 19876 msR stem variant Var0143 CATAGATTTCTTGGCCTTTATcggccggcgcccgggccgggcgggcccgcgggcggTTGccgcccgcgggcccgcccggcccgggcgccggccgGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACC GGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19685 ACCTATCATTCANNNNNNNN 19877 msR stem variant Var0144 CATAGATTTCTTGgcatGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19686 ACCTATCATTCANNNNNNNN 19878 Minimal variant Var0145 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCGCCACGGTggtgtcaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19687 ACCTATCATTCANNNNNNNN 19879 Minimal variant Var0146 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacactaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19688 ACCTATCATTCANNNNNNNN 19880 Minimal variant Var0147 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19689 ACCTATCATTCANNNNNNNN 19881 Minimal variant Var0148 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaataGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19690 ACCTATCATTCANNNNNNNN 19882 Minimal variant Var0149 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggcatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19691 ACCTATCATTCANNNNNNNN 19883 Minimal variant Var0150 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagattttaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19692 ACCTATCATTCANNNNNNNN 19884 Minimal variant Var0151 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggtaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19693 ACCTATCATTCANNNNNNNN 19885 Minimal variant Var0152 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCaaagaattgttagCAAGAAATCTATG 19694 ACCTATCATTCANNNNNNNN 19886 Minimal variant Var0153 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCaaataagaaagCAAGAAATCTATG 19695 ACCTATCATTCANNNNNNNN 19887 Minimal variant Var0154 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCaatgttagCAAGAAATCTATG 19696 ACCTATCATTCANNNNNNNN 19888 Minimal variant Var0155 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCaaatatagCAAGAAATCTATG 19697 ACCTATCATTCANNNNNNNN 19889 Minimal variant Var0156 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCaaagCAAGAAATCTATG 19698 ACCTATCATTCANNNNNNNN 19890 Minimal variant Var0157 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacacataattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19699 ACCTATCATTCANNNNNNNN 19891 Stop variant Var0158 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacacattattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19700 ACCTATCATTCANNNNNNNN 19892 Stop variant Var0159 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacaaatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19701 ACCTATCATTCANNNNNNNN 19893 Stop variant Var0160 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacatatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19702 ACCTATCATTCANNNNNNNN 19894 Stop variant Var0161 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacaaataattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19703 ACCTATCATTCANNNNNNNN 19895 Stop variant Var0162 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacaaattattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19704 ACCTATCATTCANNNNNNNN 19896 Stop variant Var0163 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacatataattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19705 ACCTATCATTCANNNNNNNN 19897 Stop variant Var0164 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacatattattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19706 ACCTATCATTCANNNNNNNN 19898 Stop variant Var0165 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaataaacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19707 ACCTATCATTCANNNNNNNN 19899 Stop variant Var0166 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19708 ACCTATCATTCANNNNNNNN 19900 Stop variant Var0167 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaataaaaatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19709 ACCTATCATTCANNNNNNNN 19901 Stop variant Var0168 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaataaatatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19710 ACCTATCATTCANNNNNNNN 19902 Stop variant Var0169 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaataaaatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19711 ACCTATCATTCANNNNNNNN 19903 Stop variant Var0170 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatatatatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19712 ACCTATCATTCANNNNNNNN 19904 Stop variant Var0171 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtaaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19713 ACCTATCATTCANNNNNNNN 19905 Stop variant Var0172 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgttaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19714 ACCTATCATTCANNNNNNNN 19906 Stop variant Var0173 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtaaaataaacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19715 ACCTATCATTCANNNNNNNN 19907 Stop variant Var0174 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtaaaatacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19716 ACCTATCATTCANNNNNNNN 19908 Stop variant Var0175 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgttaaataaacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19717 ACCTATCATTCANNNNNNNN 19909 Stop variant Var0176 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgttaaatatacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19718 ACCTATCATTCANNNNNNNN 19910 Stop variant Var0177 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttatcaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19719 ACCTATCATTCANNNNNNNN 19911 Stop variant Var0178 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttttcaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19720 ACCTATCATTCANNNNNNNN 19912 Stop variant Var0179 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttataaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19721 ACCTATCATTCANNNNNNNN 19913 Stop variant Var0180 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttattaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19722 ACCTATCATTCANNNNNNNN 19914 Stop variant Var0181 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttttaaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19723 ACCTATCATTCANNNNNNNN 19915 Stop variant Var0182 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagattttttaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19724 ACCTATCATTCANNNNNNNN 19916 Stop variant Var0183 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacacatccttaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTTCCCCATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19725 ACCTATCATTCANNNNNNNN 19917 Stop variant Var0184 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcaaatacacatcgttaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19726 ACCTATCATTCANNNNNNNN 19918 Stop variant Var0185 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggaggtttgtcaaatacacaccattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19727 ACCTATCATTCANNNNNNNN 19919 Stop variant Var0186 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagctttgtcaaatacacagcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCAAATATAAGAATTGTTAGCAAG AAATCTATG 19728 ACCTATCATTCANNNNNNNN 19920 Stop variant Var0187 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagattggtcaaatacccatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG 19729 ACCTATCATTCANNNNNNNN 19921 Stop variant Var0188 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagattcgtcaaatacgcatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACATCACCAAATATAAGAATTGTTAGCAAGAAA TCTATG 19730 ACCTATCATTCANNNNNNNN 19922 Stop variant Var0189 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtccaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19731 ACCTATCATTCANNNNNNNN 19923 Stop variant Var0190 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgtcgaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19732 ACCTATCATTCANNNNNNNN 19924 Stop variant Var0191 CATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTggagatttgccaaatacacatcattaGGTTGCGACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACCTGCAGGACCTATCATTCANNNNNNNNAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACACAACCAAATATAAGAATTGTTAGCAAGAAATC TATG 19733 ACCTATCATTCANNNNNNNN 19925 Stop variant

The screening method is depicted in Figure 114. Essentially, the ncRNA library was co-transfected into 293T cells with Cas9 mRNA, reverse transcriptase mRNA, and guide RNA targeting the EMX1 site. Total nucleic acid including RNA, ssDNA was collected 16 hours after transfection, and genomic DNA (gDNA) was harvested 96 hours after transfection. Barcode abundance was measured by in-house high-throughput sequencing of ncRNA, ssDNA templates, and genomic EMX1 sites. First, the barcode counts in ssDNA and gDNA were normalized to the input ncRNA level of the initial ncRNA library. Then, the ssDNA template generation level or gDNA insertion level for each variant was calculated by averaging the unique barcodes associated with this type of variant. Results:

The results are shown in the scatter plot of Figure 115. The level of genomic insertion correlates with the level of ssDNA template production (Figure), and the negative control ncRNA exhibits low efficiency in both ssDNA production and gDNA insertion. ncRNA variants that exhibit higher activity than WT R6342S ncRNA can be identified in Figure 115 as those data points belonging to the upper right quadrant. Table 31B below lists the 13 best performing ncRNA variants, which show more than 1.5-fold higher activity in both gDNA insertion and ssDNA production levels. Table 31B - Best performing ncRNA variants Variants with improved potency Variant Type Var0015 a1a2 variant Var0020 a1a2 variant Var0026 a1a2 variant Var0031 a1a2 variant Var0044 msD stem variant Var0060 msD stem variant Var0061 msD stem variant Var0062 msD stem variant Var0065 msD stem variant Var0066 msD stem variant Var0070 msD stem variant Var0071 msD stem variant Var0076 msD stem variant Example: Materials and Methods

Mammalian cell culture HEK293T (ATCC CRF-3216) or 293T-Cas9 (Genecopoeia SL502) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Gibco). Cells were maintained at 37°C and 5% CO2.

Genomic DNA extraction After incubation, the medium was removed from the cells and genomic DNA was extracted by adding prepGEM reagent (Thomas Scientific: PUN0050) directly to each well of the tissue culture plate. Lysed cells were transferred to a 96-well PCR plate and incubated at 72°C for 10 minutes, followed by an enzyme inactivation step at 95°C for 2 minutes.

High-throughput DNA sequencing of genomic DNA samples The human EMX1 gene or AAVS1 locus was amplified from genomic DNA samples and sequenced on an Illumina NextSeq. Briefly, amplification primers containing Illumina forward and reverse adapters were used in the first round of PCR (PCR1) to amplify the EMX1 target site. A 25 µl PCR1 reaction was performed with 0.3 µM each of the forward and reverse primers, 1 µl of genomic DNA extract, and 12.5 µl of KAPA HIFI HOTSTART PCR Master Mix. The PCR reaction was performed as follows: 95°C for 1 min, followed by 25 cycles of [98°C for 20 sec, 65°C for 15 sec, and 72°C for 15 sec], followed by a final extension at 72°C for 2 min. PCR reactions were purified using Ampure XP beads (Beckman Coulter) and eluted in 20 µl H20. In the secondary PCR reaction (PCR2), unique Illumina barcoded primer pairs were added to each sample. Specifically, 25 µl PCR2 reactions were performed with 5 µl IDT for Illumina UDI primers (Illumina), 1 µl purified PCR1 reaction, and 12.5 µl KAPA HIFI HOTSTART PCR Master Mix. PCR reactions were performed as follows: 95°C for 3 min, followed by 10 cycles of [95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec], followed by a final extension at 72°C for 5 min. PCR2 reactions were purified by SequalPrep normalization plate kit (Thermo Fisher Scintific) and pooled. Size and purity were assessed by Tape station D1000 analysis (Agilent). DNA concentration was measured by fluorescence quantification (Qubit, Thermo Fisher Scientific), and libraries were sequenced on an Illumina NextSeq 2000 instrument with 30% PhiX sequencing controls using either the P1 or P2 300 cycle kit. Sequencing reads were decoded, and amplicon sequences were aligned to a reference sequence using CRISPresso2. CRISPresso2 was run in HDR mode using the desired sequence as the expected allele, and the accurate edit yield was calculated as the number of HDR aligned reads divided by the total aligned reads.

Junction qPCR for GFP integration qPCR was used to determine the presence of GFP at the EMX1 locus after editing. Primers were designed to flank the 5' and 3' junctions of the GFP insertion at the EMX1 locus. Briefly, a 25 µl qPCR reaction was performed with 12.5 µl KAPA HiFi HotStart ReadyMix (Roche), 0.3 µM each forward and reverse primers, 2 µl gDNA, 1X SYBR Green (Thermo Fisher Scientific: S7563), and 1X Rox reference dye (Thermo Fisher Scientific:54881). The qPCR reaction was performed as follows: 50°C for 2 minutes, 95°C for 3 minutes, followed by 40 cycles of [98°C for 30 seconds, 67°C for 30 seconds, and 72°C for 30 seconds], 72°C extension for 2 minutes, followed by melting curve analysis with 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 1 second. The ΔCt was obtained by subtracting the Ct value of the PCR perijunction from the Ct value of the PCR perijunction cut to relative quantify the GFP insertion at the EMX1 locus. In addition, qPCR samples were run on a Tapestation 4200 (Agilent) with high sensitivity D1000 reagent (Agilent) to confirm the expected band size.

Plasmid-based gene editing. Retroviral encoding plasmids were synthesized by Twist Bioscience. Plasmids containing Cas12a and TnpB were synthesized by Azenta Life Sciences. Cas9-mCherry and Cas9-mCherry EMX1 sgRNA plasmids were synthesized by Vector Builder Inc. The deletion variant of RTX3_6342S was synthesized by Quintara Biosciences. For 293T-Cas9 transfection, 25,000 293T-Cas9 cells (Genecopoeia: SL502) were transfected using Lipofectamine 3000 (Thermofisher: L3000001) according to the manufacturer's protocol. Transfected cells were incubated at 37°C for 72 h and then lysed in prepGEM reagent (Thomas Scientific: PUN0050) according to the manufacturer's protocol. For K562 cells, Neon or Lonza electroporation systems were used. 100,000 K562 cells were electroporated in OPTI-MEM (Neon) or SF buffer (Lonza) using the following settings: 1450 V with three 10 ms pulses (Neon) or EH120 (Lonza) with 500 ng total plasmid DNA (250 ng Retron RT + ncRNA/EMX1 sgRNA fusion, and 250 ng Cas9-mCherry, 250 ng Cas9-mCherry EMX1 sgRNA, 250 ng Cas12a EMX1, or 250 ng TnpB EMX1. Cells were incubated at 37°C for 72 h. 1 A 1 µL aliquot of the crude lysate was used as template to amplify the EMX1 target site by PCR. Illumina adapters and UDI were added in a second round of PCR followed by loading onto the Illumina NextSeq. The resulting FASTQ files were used as input to CRISPResso2, which quantified precise edits and indels at the EMX1 locus.

In vitro transcription Plasmid templates were synthesized by Twist Bioscience. T7 promoter was inserted upstream of RT or ncRNA sequence. Plasmids were linearized by Sbf1-HF enzyme (NEB, R3642S) or EcoR V (NEB, R0195L) at 37°C for 16 h, and complete linearization was checked by agarose gel electrophoresis. Linearized templates were purified by GeneRT PCR Purification Kit (K0701, Thermo Fisher Scientific) and quantified by Nanodrop. mRNA was synthesized using HiScribe T7 mRNA Kit and CleanCap Reagent AG (NEB, E2080S) at 37°C for 16 h, and ncRNA was synthesized using HiScribe T7 High Yield RNA Synthesis Kit (NEB, 2040S) according to the manufacturer's protocol. After DNAse1 treatment, RNA was purified using the Qiagen RNeasy midi kit (Qiagen, 75144). RNA concentration was measured by Nanodrop (Thermo Fisher), and purity and size were assessed by RNA ScreenTape analysis on Tape station (Agilent). RT mRNA was further poly(A)-tailed by E. coli poly(A) polymerase (NEB, M0276) and purified by the Qiagen RNeasy midi kit. Tail length was estimated by RNA ScreenTape analysis by comparing before and after poly(A) adenylation.

RNA transfection by Neon electroporation system HEK293T (ATCC CRF-3216) or 293T-Cas9 (Genecopoeia SL502) cells were harvested and resuspended in R buffer. 50,000 cells containing 1 µl RNA mixture were electroporated using a 10 µl Neon tip at 1150 volts for 20 pulses. The electroporated cells were gently transferred to a 96-well plate (VWR) containing pre-warmed medium and incubated for 72 hours before genomic DNA extraction.

RNA transfection by Lipofectamine MessengerMAX 20,000 HEK293T (ATCC CRF-3216) cells were seeded in 96-well plates. 120,000 cells were seeded in 24-well plates. 16-24 hours after seeding, cells were transfected with 0.3 µl (or 0.1 µl Lipofectamine MessengerMax/100 ng RNA) Lipofectamine MessengerMax (Thermo Fisher Scientific) and the appropriate amount of RNA mixture. Cells were cultured for 72 hours and genomic DNA was then extracted.

ncRNA CircularizationTemplates were designed using the strategy and sequences described in (27). In vitro transcription was performed similarly to that described above, except that 7.5 mM NTP was used instead of 10 mM concentration. RNA was purified using the Qiagen RNeasy midi kit (Qiagen, 75144) after DNAse1 treatment. RNA was treated with RNAse R (Biosearch, RNR0725) to enrich for circular RNAs and purified using the Qiagen RNeasy midi kit. RNA concentration was measured by Nanodrop (Thermo Fisher), and purity and size were assessed by RNA ScreenTape analysis on a Tape station (Agilent). Example: ReferencesA, M. (2020). Bacterial retrons funciton in anti-pahge defense.183. AJ, S. (2019). Retrons and their applications in genome engineering.47(21). AV, a. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA.Nature, 149-157. Bonafont J, M. A. (2019). 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Example: Sequence Selection of RT and ncRNA sequences - Overview describe SEQ ID NO: EMX1 gRNA sequence (nt) 19109 Eco1 ncRNA (nt) 19110 AcoI ncRNA (nt) 19111 RTX_1262 RT (aa) 1262 RTX_1262 ncRNA (nt) 15327 RTX3_2042 RT (aa) 2042 or 19112 RTX_2042 ncRNA (nt) 19113 RTX_2781 RT (aa) 2781 RTX_2781 ncRNA (nt) 16411 RTX_6083v1 RT (aa) 6083 or 19114 RTX3_6083v1 ncRNA (nt) 18547 RTX_6342 RT (aa) 6342 RTX_6342 ncRNA (nt) 18731 RTX_6342L ncRNA (nt) 19927 RTX_6342S ncRNA (nt) 19928 Engineered 6342S ncRNA + HDR template 19929 Engineered 6342L ncRNA + HDR template 19930 RTX_6943 RT (aa) 6943 or 19116 RTX_6943 ncRNA (nt) 19053 Engineered RTX_6943 ncRNA 19117 Eco1 RT (nt) 19118 Eco1 ncRNA (nt) 19119 Eco3 RT (nt) 19120 Eco3 ncRNA (nt) 19121 Eco5 RT – (nt) 19122 Eco5 ncRNA (nt) 19123 EMX1 – sgRNA modified (nt) 19124 EMX1 sgRNA unmodified (nt) 19125 Select RT and ncRNA sequencesEMX1 s.p Cas9 gRNA GAGTCCGAGCAGAAGAAGAA (SEQ ID NO: 19109) Eco1 ncRNA +HDR_ templateTGATAAGATTCCGTATGCGCACCCTTAGCGAGAGGTTTATCATTAAGGTCAACCTCTGGATGTTGTTTCGGCATCCTGCATTGAATCTGAGTTACTGTCTGTTTTCCT ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA AGGAAACCCGTTTCTTCTGACGTAAGGGTGCGCATACGGAATCTTATCA (SEQ ID NO: 19110) Aco1 ncRNA +HDR templateCCGTAGTGGGAGCCTCAGGCGAGGGTGTGTATCATGCCCGTTCTGCCAAGACCCACCAAAGAAGGGCACCGTGGAGG ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAACAGAAGTGCAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA CCTCCACGGTGCAATGCGAAAGCAACTTGAGGCTTTGCTTAGTATGAGGCTCCCACTACGG (SEQ ID NO: 19111) Reverse Recorder 1262 RTMISFSEIKSRNDFADALQIPRSVLTHVLYIAKPESFYESFTIPKKNGEDRIIMAPKGTLKSIQTKLSKQLVEYRASISQKGQEKSNISHGFEREKSIITNAQIHRNKRYVINYDLKDFFDSFHFGRVVGFFEKNKHFLLPYEVAVIIAQLTCYNGRLPQGAPTSPVITNLICEILDYRVLKIAKRYKLDYTRYADDLTFSTNYSRFLEVFDSFAKE LLQEISNSGFTINQSKTRLLYRDSRQEVTGLVVNKKIGVNREYVKSTRAMAQALYSTGEFTINGIPG TIKQLEGRFGFIDQLDHYNNVIDDAKHDAYSLNGREKQFQEFLFFKTFFFNEYPLVITEGKTDIRYLKAALKSLHQKYPELICKEDDGTFRFKISFFRRSKRWKYFFGISKDGADAMKLLYRFFTGQKGVKNYYRLFAEKYKAVQRNPVIMLFDNEMESKRPLNKFISEEVKIPSSEQQLFKEQLYYHLIPGSKTYLMTHPLPPGK TEAEIEDLFPTEVLGVKLDGKSFSTKDKFDTSKFYGKDIFSSYVYEHWKSIDFSGFIPLLDKINMLVQNEKKPGLNT (SEQ ID NO: 1262)ncRNAGAGCGTGTGACAAGATTTTGGGCTTGTGTTTCGCAAGCTTTGATTAAAATGGTAGATGGATTTGCTATCTATCGTCATTTCCAGTACTGTTATGTTTATGTTCTGTTTACCCGTATGCACCATCCGCATAATGAGTTGTCACACGCTC (SEQ ID NO: 15327) Reverse Recorder 2042 RTMKDDQYSQWKKYYESRGILPEIQDKLLNYAKIHIDNNTPVIFNFEHLTLLLLGREKNYLSSVVNSPDSHYRKFKIKKRSGGEREITAPYLSLLEMQYWIYRNILINVKIHYAAHGFAQDKSIITNSRNHLGQKHLLKMDLKDFFPSIKLNRIIYIFKSLGYPNIIAFYLASICSYKGHLPQGSPTSPILSNIVSITLDNRLV KFARKMKLRYSRYADDLTFSGDKIPTNYIKYITDIINDEGFEVNDTKTKLYLKAGKRIVTGISVIGNDPKLPREYKRKLKQELHYIFTYGIGSHMAKKKIKKINYLYRIIGKVNFWLNIEPDNEYARNAKAKLLLLIDN* (SEQ ID NO: 19112 or SEQ ID NO: 2042)Homologous ncRNAAGGTGGTTATATTCTAGTATTTATGAAGTGTAGTCGCTTCGATCGTTAAGGCTGATTTTAACCTCTGCATAATAATATCGGTAGATATTATTATGCACGCTCCCTTTAGCAGAGCTAAGAATCGCTCACTCAGGCACAAGCTTTGAGGAGCGACCTCCTCAAAGCTTGTGCCTGAGTGAGAGCTAAAGAAAAGAAAAGTAGAATAAGCCACCT (SEQ ID NO: 15833) 204 2 ncRNA: RTX3_2042 ncRNA +HDR_ templateGTTAAGGTGGTTATATTCTAGTATTTATGAAGTGTAGTCGCTTCGATCGTTAAGGCTGATTTTAACCTCTGCATAATAATATCGGTAGATATTATTATGCACGCTCCCTTTAGCAGAGCTAAGAATCGCTCACTCAGGCACAAGCTTTGAGG ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAATCCTAAGAGAAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA CCTCAAAGCTTGTGCCTGAGTGAGAGCTAAAGAAAAGAAAGTAGAATAAGCCACCTTAAC (SEQ ID NO: 19113) Reverse Recorder 2781 RTMSGRLWKYLTPQQEREFILSLNLLPASCRKRMSGEAQLALLYAISNHVERHYRKVRIPKKDGKSRRLLVPDGLLKGVQRNILRHCLDGRSVSEYACAYRRGLSAADNAAPHAGGGKDKLLLKLDIRDFFDSILFWQVYGAAFPGSLFPPAAAGLLTHLCCCGDRLPQGAPTSPAISNLVMKPFDEFMGAWCRQRQIVYTRYCDDLTFSGAFDPKAV YHKARHLLEAMGFALNAEKTALRNRGMRQSVTGLVVNERVRTGVPYRRRIRQEMYYCRKYGVGEHLQAVGRLTGTETQEEAAEAERAFLCALLGKIGYVLLLEPENREFLEYREICRGMQAEQEKASCPPACGEREQKRRGGKEK (SEQ ID NO: 2781)Homologous ncRNAAAGAGCAACTAGATTGAGGCGATTCGCCTCCTTGGAAAAGGGTACTAAGTTTCTGTCGCACACCAATTTATAAGCTTATAAATTGGTGTGCGACAGAAATGAAATAAATAGTAGTTGCTCTT (SEQ ID NO: 16411) Reverse Recorder 6083 RTMSNPQPTRAEIFERIKQSSKQEVILEEMQRLGFWPRSEGQPEVAADLIQREGELQRELAELNKKLAVKRNPERALREMRKQRMKDARDKREVTKRAQAQQRYDKALLWHEKRASHVAYLGPGVSASLHENSSATQEQGDKGKPKRARDRAVPDLQRLTLNGLPALISAAQLAESMGVSVAELRFLSFHREVARTNHYHSFTLPKKTGGERLISAPMPRLK RAQYWVLDNVLAKMPAHDAAHGFLAGRSIISNAKPHAGQDVVINLDVKDFFPSIAFGRIKGVFRQLGYGESIATVFALLCSENRAQAWQVDGERLFVGGKARERVLPQGAPTSPML TNLLCRRMDRRLLGLAKQLGFVYTRYADDLTFSASGEPARDNVGKLLSRVRWILRDEGFTPHPDKERVMRKGRRQEVTGLVVNSDTPSVSRETRRRLRAALHRASQPDAASKPAHWQGHTAQPSQLLGLATFVHQIDPKQGKTLLADAQQLMRSPIDRANDAAKSASRADAAQQSFRVLAAAGKPPVLADGKNWWQPAPPATPVLEK TDQQRREERQATRRQQAAAAAPPPSSTRRNERPQQAAHEQQGDAQPQNEAPPRFDPDQYAPPPRNVMTYWAQIAISFFLGSILHNRLITIFAMVAVIALYYMRRQRWDVFMGILVVATLLGYLVRGMG* (SEQ ID NO: 19114 or SEQ ID NO: 6083)Homologous ncRNACCGGAGCAATGAGCAGGCTCTTGCAATCCGGGCGGTGTTTCGCCGCCCTTGTGAACTGCCGTTCATGCACCACGGGCCCGTTTTCACTGTGCCACCCCAGCCACGGTAGTGCTTTTCCTGAGTGGCTTGCCAGCGAAGCAGCACTACCGTGGCGGGGTGGCGTCGAGCGAACAGCTCCCGTCCCGTGAGCCCTACAGGCTCTTCGACGAGATGCACATTGCTCCG (SEQ ID NO: 18547) Engineered 6083 ncRNA: RTX3_6083v1 ncRNA +HDR templateGCTCCGGAGCAATGAGCAGGCTCTTGCAATCCGGGCGGTGTTTCGCCGCCCTTGTGAACTGCCGTTTCATGCACCACGGGCCGCCGTTTTCACTGTGCCACCCCAGCCACGGTAGTGCT ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACTACAAGGTTAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA AGCACTACCGTGGCGGGGTGGCGTCGAGCGAACAGCTCCCGTCCCGTGAGCCCTACAGGCTCTTCGACGAGATGCACATTGCTCCGGAGC (SEQ ID NO: 19115) Reverse Recorder 6342 RTMMENQENNMNNRKNYREAVNTMGKKEFTLIKMQEYGFWPKNLPTPYERQESETEEQYKERKCLLEKYEKVIDEISKLYEEKDKINLKLRELQKKYDETWDYERIRLDVSKTIMQESIERRAERKRQRELEKEQRSEEWKKEKENKIVFIGKGYSSLLYDKETDENKLLLQELPIIKDDKELANFLGIEYKKLRFLVYHRDVISV DNYHRYTIPKKKGGVRNIAAPKSILKNSQRIILEEILSKIPTS NYSHGFLKGKSVVSAAKVHVKKPDLLINIDLEDFFPTITTFERVRGMFKSFGYSGYVASMLAMICTYCERMKVEVRGEEKYVKISDRILPQGSPASPMITNIICVKLDKRLNGLSTKYDFIYTRYADDMSFSFTGDINELSVGSFMGLVSKIVKEEGFNINKDKTKFLRKNNRQCITGIVINNEEIGVPKKWIKILRAAIYNANKVKNSGEIL SNKVINEISGMTSWVKSVNEERYKDIINDAMNLINN (SEQ ID NO: 6342)Homologous ncRNAACATAGATTTCTTGGCCTTTATGCTGTGGTGTTGCCACGGTGGAGATTTGTCAAATACACATCATTAGGTTGCGCGCAATTCGCTACGCTACCAATACTGTGGGCATTAAAAATCGAGCCTTCGGTTATGCCACAGTATTGGTGCTACGCTGAAGTGTCACAACCAAATATAAGAATTGTTAGCAAGAAATCTATG (SEQ ID NO: 18731) RTX_6342S ncRNA CAUAGAUU UCUUGGCCUUUAUGCUGGUGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCG_Place_INSERT_HERE_CACA ACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUG (SEQ ID NO: 19927) RTX_6342L ncRNA CAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCGCGCAAUUCGCUACGCUACCAAUACUGUGGGCAU_placement_insert_here_AUGCCACAGUAUUGGU GCUACGCUGAAGUGUCACAACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUG (SEQ ID NO: 19928) RTX_6342S ncRNA + HDR template insert CAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCG ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAATCGTCAGTGCAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA CACAACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUG (SEQ ID NO: 19929) RTX_6342L ncRNA + HDR template insert CAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCGCGCAAUUCGCUACGCUACCAAUACUGUGGGCAU ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACGTAATCCGGAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA AUGCCACAGUAUUGGUGCUACGCUGAAGUGUCACAACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUG (SEQ ID NO: 19930) Reverse Recorder 6943 RTMEESTNYKLLVWGLSVIQPATPNEVLNYLTSTLNDNGLLPDVEKMIHYFELLDQLGYIHQVSKRNNLYSLTPRGNERLTPALKRLRDKIRLFMLDNCHSISKLGVLASTDTENMGGDSPSLQLRHNLKEVPHPSLSWAAGTLPSSPRQAWVRIYEQLNIGSMSSDEASTPTTARNAPLSFVGRLGFSLNYYSFNKIDEPLFNNDGVTAIASCIGISPGL ITAMVKSPKRYYRTFNLRKKSGGFRSILAPR KFIKTIQYWLKDHVLNRLKIHSSCYSYRSGVSIKDNAINHVKKKFVASIDISDYFGSINKKMVKDCFYKNNIPDHIVNTISGIVTYNDVLPQGAPTSPIISNAILFEFDEEMTAHALTLDCIYTRYSDDISISSDYKENIAILINIAEANLLSAGFTLNRQKQRIASDNSRQVVTGILVNESIRPTRCYRKKIRSAFDHALKEQDGSQLTINKLRG YLNYLKSFETYGFKFNEKKYKETLDFLIALKQS* (SEQ ID NO: 19116 or SEQ ID NO: 6943))ncRNAACTGATACAGAGAATATGGGCGGTGATTCGCCGTCTTTACAGTTAAGGCACAATTTAAAAGAGGTTCCGCACCCAAGCCTGTCTTGGGCTGCAGGGACCCTGCCCAGCAGCCCAAGACAGGCTTGGGTGCGGATCTACGAGCAATTAAATATTGGTTCGATGTCCAGT (SEQ ID NO: 19053) Engineered ncRNA: RTX3_6943 ncRNA +HDR templateGCGGAGTGCTGGCCTCAACTGATACAGAGAATATGGGCGGTGATTCGCCGTCTTTACAGTTAAGGCACAATTTAAAAGAGGTTCCGCACCCAAGCCTGTCTTGGGCTGC ACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAACGTATGGCCTAAAGTTCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCA GCAGCCCAAGACAGGCTTGGGTGCGGATCTACGAGCAATTAAATATTGGTTCGATGTCCAGTGATGAGGCCAGCACCCGC (SEQ ID NO: 19117)Eco1 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and encoded poly A of approximately 120 bases are added. Completely replaced with 1N-methyl-pseudouridine) AUGAAAUCUGCAGAGUAUCUGAAUACGUUCCGCCUUAGGAAUUUGGGCCUCCCCGUGAUGAACAAUCUCCACGAUAUGAGCAAGGCGACUCGAAUAUCCGUGGAAACGCUGAGACUGCUCAUCUAUACAGCAGACUUUCGGUACAGGAUCUACACGGUCGAAAAGAAGGGGCCUGAGAAACGCAUGCGAACAAUUUAUCAACCUAGCCGAGAGCUCAAGGCGUUGCAGGGCUGGGUUCUUCGAAAC AUCCUUGACAAACUCUCAUCAUCACCCUUUAGUAUUGGGUUUGAAAAGCACCAAAGCAUCCUUAACAACGCGACGCCACACAUAGGUGCCAAUUUCAUAUGAACAUCGACUUGGAGGAUUUUUUCCGAGCCUCACAGCCAAUAAAGUGUUCGGUGUUUUUCACAGUCUUGGGUACAAUCGCCUUAUUAGUUCCGUUCUUACCAAGAUUUGUUGUUACAAGAAUCUUGCCC CAGGGAGCACCCA GCAGUCCGAAAUUGGCGAAUUUGAUUUGUUCCAAGCUCGAUUAUCGAAUACAAGGGUACGCGGGCAGCCGGGGACUCAUCUACCCGCUACGCAGACGAUCUUACGCUGUCUGCCCAAUCAAUGAAGAAGGUCGUAAAGGCGCGGGAUUUCUUGUUUUCUAUCCAUCCCGAGGGCUUGGUAAUUAAUUCCAAAAAGACUUGUAUCCAGGACCACGAUCUCAGCGAAAAGUGACAGG ACUCGU CAUUUCUCAAGAAAAAGUCGGUAAGGGAGAGAAGUAUAAGGAAAUCCGCGCGAAGAUCCACCACAUAUUCUGUGGCAAGAGCAGCGAGAUAGAACACGUCCGAGGCUGGUUGUCCUUCAUACUGAGCGUGGACUCAAAAAGCCACCGCCGGUUGAUCACCUAUAUUUCAAAACUGGAAAAGAAAUAUGGAAAGAACCCACUCAACAAAGCUAAAACACCACCAAAGAAGAAAAGAAAGGUCUGA (SEQ ID NO: 19118) Eco1 ncRNA ncRNA-sgRNA fusion (6 bp substitution in EMX1 gene) GAAAUGAUAAAGAUUCCGUAUGCGCACCCUUAGCGAGAGGUUUAUCAUUAAGGUCAACCUCUGGAUGUUGUUUCGGCAUCCUGCAUUGAAUCUGAGUUACUGUCUGUUUUCCUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAAGGAAACCC Eco3 RTCoding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and encoded poly A of approximately 120 bases are added. Completely replaced with 1N-methyl-pseudouridine AUGCGCAUUUACUCUCUGAUCGACAGCCAAACCUUAAUGACCAAAGGGUUCGCAUCCGAGGUCAUGAGGAGCCCAGAACCCCCUAAGAAGUGGGACAUUGCGAAGAAGAAGGGCGGAAUGCGUACGAUAUACCAUCCCUCUUCUAAGGUGAAGCUGAUACAGUACUGGCUGAUGAACAACGUGUUCUCCAAAUUGCCGAUGCACAACGCCGCGUACGCUUUCGUGAAGAAUAGAUCUAUCAAG UCUAACGCACUGCUGCACGCAGAGAGUAAGAACAAAUACUACGUUAAGAUUGACCUGAAGGACUUCUUUCCAAGCAUCAAGUUCACAGACUUCGAAUAUGCCUUUACCCGGUACCGUGACAGAAUAGAGUUCACGACCGAGUACGACAAAGAACUGCUUCAGCUGAUUAAGACCAUUUGUUUCAUUUCUGACUCUACACUGCCAAUAGGCUUCCCCACUUCCCCUUAUAGCCAAUUUCGU CG CCAGGGAGCUGGACGAAGCUCACUCAGAAGCUGAACGCUAUAGACAAGCUCAACGCUACGUACACUCGCUACGCAGACGACAUAAUCGUGAGCACGAACAUGAAGGGCGCCUCUAAGCUGAUCUUAGACUGCUUCAAGCGGACCAUGAAGGAAAUCGGACCCGAUUUCAAGAUCAAUAUCAAGAAGUUCAAAAUAUGCUCUGCCAGUGGCGGCUCAAUUGUCGUGACGGGGCCUUAAGGUCUG UCAUGACUUCCACAUAACUCUGCACCGGUCUAUGAAGGACAAGAUCCGCCUGCACCUCUCUCUCCUGUCCAAAGGUAUUCUGAAGGACGAGGACCACAACAAGCUGUCCGGGUACAUCGCCUACGCUAAGGACAUCGAUCCACACUUCUACACCAAGCUCAAUAGGAAGUACUUCCAGGAGAUCAAGUGGAUACAAAACCUGCAUAAUAAGGUGGAGCCACCAAAGAAGAAAAGAAAGGUCUGA (SEQ ID NO. : 19120) Eco3 ncRNA ncRNA-sgRNA fusion (10 bp insertion in EMX1 gene) GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUC GAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUUUUUU (SEQ ID NO: 19121) Eco5 RTCoding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and encoded poly A of approximately 120 bases are added. Completely replaced with 1N-methyl-pseudouridine AUGGACGCUACCAGAACGACUCUCCUUGCAUUGGAUCUCUUCGGAUCUCCAGGUUGGUCCGCCGAUAAAGAAAUUCAGAGGCUUCAUGCGCUCAGUAAUCAUGCUGGAAGGCAUUACAGAAGGAUUAUAUUAAGUAAAAGGCACGGCGGACAGCGUCUUGUGCUUGCACCUGAUUACUUGUUAAAGACCGUUCAGCGCAACAUUUUUGAAGAACGUUUUGAGUCAAUUUCCACUGUCACCAUUUGCU ACAGCCUACAGACCGGGAUGCCCAAUCGUGUCUAACGCGCAGCCACACUGCCAACAGCCACAGAUCUUGAAACUCGAUAGAAAACUUCUUCGAUUCUAUUAGUUGGUUGCAGGUGUGGCGGGUGUUUCGCCAGGCCCAGUUGCCCCGAAAUGUCGUAAACGAUGCUCACUUGGAUAUGUUGUUAUAACGACGCACUUCCGCAGGGGUCCCCCUACAUCCCCUGCAAUUUCCAAUCUCGUCAU GAGA AGGUUUGAUGAACGGAUUGGAGAAUGGUGUCAGGCUCGAGGGAUUACCUACACUCGCUACUGCGAUGACAUGACGUUUAGUGGACACUUCAAUGCAAGCAGGUCAAGAAUAAAGUCUGCGGUCUCUUAGCUGAGCUGGGCCUUUCCCUGAAUAAACGGAAAGGCUGCCUCAUAGCGGCUUGUAAGCGCCAGCAAGUCACCGGCAUUGUUGUGAAUCACAAGCCACAGCUUGCCCGAGAAGCC AGG CGUGCCCUGCGUCAGGAAGUGCCACCUGUGCCAGAAAUAUGGAGUUAUCUCUCUCUCACAUAGAGGUGAACUGGAUCCUAGCGGAGAUCUGCACGCUCAGGCGACAGCGUAUCUCUAUGCACUCCAGGGGAGAAUUAACUGGCUUCUUCAAAUUAACCCUGAGGAUGAGGCGUUUCAACAGGCCCGGGAGUCCGUUAAGAGGAUGUUAGUUGCCUGGCCACCAAAGAAGAAAAGAAAGGU CUGA (SEQ ID NO: 19122) Eco5 ncRNAncRNA-sgRNA fusion (10 bp insertion in EMX1 gene) GAAAUGAUAAGAUUCCGUACGCCAGCAGUGGCAAUAGCGUUUCCGGCCUUUUGUGCCGGGAGGGUCGGCGAGUCGCUGACUUAACGCCAGUAGUAUGUCCAUAUACCCAAAGUCGCUUCAUUGUAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUCCUUCGAGCAAAGUUCUCCCAUCACAUCA ACCGGUGGCGCAUU GCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUACAGUUACGCGCCUUCGGGAUGGUUUAAUGGUAUUGCCGCUGUUGGCGUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUUUUU (SEQ ID NO: 19123) EMX1 sgRNA modifiedContains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU (SEQ ID NO: 19124) EMX1 sgRNA is unmodified Unmodified GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU (SEQ ID NO: 19125)RNA Sequence - Overview Sequence Name describe SEQ ID NO: Eco1 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine 19126 Eco1 ncRNA-sgRNA ncRNA-sgRNA fusion (6 bp substitution in EMX1 gene) 19127 Eco3 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine 19128 Eco3 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) 19129 Eco5 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine 19130 Eco5 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) 19131 Cas9 All U are N-1-methylpseudouridine. 19132 EMX1 sgRNA modified Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate 19133 EMX1 sgRNA unmodified No modification 19134 Eco3 ncRNA Eco3 ncRNA, with only 10 bp insertion 19135 Eco3 ncRNA-MS2 Eco3 ncRNA, with only a 10 bp insertion and a 3' MS2 stem loop 19136 Aco1 RT Aco1 RT RNA sequence, including 5' and 3' UTR 19137 Aco1 ncRNA-sgRNA_50 Aco1 ncRNA-sgRNA with 50 bp insert 19138 Aco1 sgRNA-ncRNA_50 Aco1 sgRNA-ncRNA with 50 bp insert 19139 Eco3_EMX1 gRNA_ncRNA_25 Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end 19140 Eco3_ncRNA only Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene 19141 Eco3_ncRNA_AAVS1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end 19142 Eco3_ncRNA_EMX1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19143 Eco3_ncRNA_EMX1 gRNA_50 Eco3 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19144 Eco3_ncRNA_EMX1 gRNA_75 Eco3 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19145 Eco3_ncRNA_EMX1 gRNA_100 Eco3 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19146 Eco3_ncRNA_EMX1 gRNA_GFP gene Eco3 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. 19147 Eco3_NT_ncRNA_EMX gRNA_25 The Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end 19148 Aco1_EMX1 gRNA_ncRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end 19149 Aco1_ncRNA only Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene 19150 Aco1_ncRNA_AAVS1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end 19151 Aco1_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19152 Aco1_ncRNA_EMX1 gRNA_50 The Aco1 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19153 Aco1_ncRNA_EMX1 gRNA_75 The Aco1 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19154 Aco1_ncRNA_EMX1 gRNA_100 Aco1 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19155 Aco1_ncRNA_EMX1 gRNA_GFP gene Aco1 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. 19156 Aco1_NT_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end 19157 R2042_EMX1 gRNA_ncRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end 19158 R2042_ncRNA_25 only R2042 ncRNA contains a 25 bp insertion in the EMX1 gene 19159 R2042_ncRNA_AAVS1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the AAVS1 sgRNA at the 3' end 19160 R2042_ncRNA_EMX1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19161 R2042_ncRNA_EMX1 gRNA_50 The R2042 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19162 R2042_ncRNA_EMX1 gRNA_75 The R2042 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19163 R2042_ncRNA_EMX1 gRNA_100 The R2042 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19164 R2042_ncRNA_NT_EMX1 gRNA_25_Double guide The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) that is fused to the EMX1 sgRNA at the 3' end 19165 R2042_RT R2042 RT mRNA sequence, coding sequence only 19166 R6943 RT R6943 RT mRNA sequence, coding sequence only 19167 R6943_ncRNA_EMX1gRNA_GFP gene The R6943 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. 19168 R6943_ncRNA_EMX1gRNA_100bp The R6943 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19169 R6943_ncRNA_EMX1gRNA_75bp The R6943 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19170 R6943_ncRNA_EMX1gRNA_50bp The R6943 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19171 R6943_ncRNA_EMX1gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end 19172 R6943_ncRNA_AAVS1 gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end 19173 R6943_ncRNA only_25bp R6943 ncRNA contains a 25 bp insertion in the EMX1 gene 19174 AAVS1 sgRNA Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate 19175 R1262_RT-1 (R1262 RT) R1262 retrotranscript RT mRNA 19176 R1262_nc-5 (R1262_ncRNA-EMX1_505) R1262 ncRNA contains a 505 bp insertion in the EMX1 locus 19177 R1262_nc-3 (R1262_ncRNA-EMX1_305) R1262 ncRNA contains a 305 bp insertion in the EMX1 locus 19178 R1262_nc-1 (R1262_ncRNA-EMX1_25) R1262 ncRNA contains a 25 bp insertion in the EMX1 locus 19179 R1262_nc-4 (R1262_ncRNA-EMX1_405) R1262 ncRNA contains a 405 bp insertion in the EMX1 locus 19180 R1262_nc-2 (R1262_ncRNA-EMX1_205) R1262 ncRNA contains a 205 bp insertion in the EMX1 locus 19181 R1262_nc-19 (R1262_ncRNA-sgEMX1_205) The R1262 ncRNA contains a 205 bp insertion in the EMX1 locus, which is combined with the EMX1 sgRNA at the 3' end 19182 R1262_nc-20 (R1262_sgEMX1-ncRNA_205) The R1262 ncRNA contains a 205 bp insertion in the EMX1 locus, which is combined with the EMX1 sgRNA at the 5' end 19183 R1262_nc-13 (R1262_ncRNA-AAVS1_25) R1262 ncRNA contains a 25 bp insertion in the AAVS1 locus 19184 R1262_nc-14 (R1262_ncRNA-AAVS1_205) R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus 19185 R1262_nc-15 (R1262_ncRNA-AAVS1_505) R1262 ncRNA contains a 505 bp insertion in the AAVS1 locus 19186 R1262_nc-18 (R1262_ncRNA-AAVS1-MS2_505) The R1262 ncRNA contains a 505 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end 19187 R1262_nc-17 (R1262_ncRNA-AAVS1-MS2_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end 19188 R1262_nc-16 (R1262_ncRNA-AAVS1-MS2_25) The R1262 ncRNA contains a 25 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end 19189 R1262_nc-9 (R1262_ncRNA-EMX1-MS2_305) R1262 ncRNA contains a 305 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19190 R1262_nc-7 (R1262_ncRNA-EMX1-MS2_25) R1262 ncRNA contains a 25 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19191 R1262_nc-6 (R1262_ncRNA-EMX1_P2A-GFP) R1262 ncRNA contains a P2A-GFP insertion in the EMX1 locus 19192 R1262_nc-22 (R1262_sgAAVS1-ncRNA_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus, which is combined with the sgAAVS1 at the 5' end. 19193 R1262_nc-8 (R1262_ncRNA-EMX1-MS2_205) R1262 ncRNA contains a 205 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19194 R1262_nc-21 (R1262_ncRNA-sgAAVS1_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus, which is combined with the sgAAVS1 at the 3' end. 19195 R1262_nc-10 (R1262_ncRNA-EMX1-MS2_405) R1262 ncRNA contains a 405 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19196 R1262_nc-11 (R1262_ncRNA-EMX1-MS2_505) R1262 ncRNA contains a 505 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19197 R1262_nc-12 (R1262_ncRNA-EMX1-MS2_P2A-GFP) R1262 ncRNA contains a P2A-GFP insertion in the EMX1 locus and an MS2 stem loop at the 3' end 19198 R1262_nc-23 (R1262_ncRNA-EMX1_P2A-GFP_LongHA) Same as R1262_nc-6, but with longer homology arms 19199 R1262_nc-24 (R1262_ncRNA-EMX1-MS2_P2A-GFP_long HA) Same as R1262_nc-12, but with longer homology arms 19200 6083 v1 RT 6083 Retrotranscript RT mRNA 19201 6083 v1_ncRNA_AAVS1 gRNA_25bp The R6083 retrotransposons contain a 25 bp insertion at the AAVS1 locus 19202 6083 v1_ncRNA_only_25bp The R6083 retrotranscript contains a 25 bp insertion at the EMX1 locus 19203 6083 v1_ncRNA_EMX1 gRNA_25bp The R6083 retrotranscript contains a 25 bp insertion at the EMX1 locus and sgEMX1 at the 3' end 19204 6083 v1_ncRNA_EMX1 gRNA_GFP gene The R6083 retrotranscript contains a GFP gene inserted at the EMX1 locus and sgEMX1 at the 3' end 19205 6083 v1_ncRNA_EMX1 gRNA_50bp The R6083 retrotranscript contains a 50 bp insertion at the EMX1 locus and sgEMX1 at the 3' end 19206 6083 v1_ncRNA_EMX1 gRNA_100bp The R6083 retrotranscript contains a 100 bp insertion at the EMX1 locus and sgEMX1 at the 3' end 19207 6083 v1_ncRNA_EMX1 gRNA_75bp The R6083 retrotranscript contains a 75 bp insertion at the EMX1 locus and sgEMX1 at the 3' end 19208 Aco1_ncRNA-EMX1_100 Aco1 ncRNA contains a 100 bp insertion in the EMX1 gene 19209 Aco1_ncRNA-EMX1_205 Aco1 ncRNA contains a 205 bp insertion in the EMX1 gene 19210 Eco3_ncRNA-EMX1_205 Eco3 ncRNA contains a 205 bp insertion in the EMX1 gene 19211 RNA sequence Sequence Name describe sequence Eco1 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGAAAUCUGCAGAGUAUCUGAAUACGUUCCGCCUUAGGAAUUUGGGCCUCCCCGUGAUGAACAAUCUCCACGAUAUGAGCAAGGCGACUCGAAUAUCCGUGGAAACGCUGAGACUGCUCAUCUAUACAGCAGACUUUCGGUACAGGAUCUACACGGUCGAAAAGAAGGGGCCUGAGAAACGCAUGCGAACAAUUUAUCAACCUAGCCGAGAGCUCAAGGCGUUGCAGGGCUGGGUUCUUCG AAAC AUCCUUGACAAACUCUCAUCAUCACCCUUUAGUAUUGGGUUUGAAAAGCACCAAAGCAUCCUUAACAACGCGACGCCACACAUAGGUGCCAAUUUCAUAUGAACAUCGACUUGGAGGAUUUUUUCCGAGCCUCACAGCCAAUAAAGUGUUCGGUGUUUUUCACAGUCUUGGGUACAAUCGCCUUAUUAGUUCCGUUCUUACCAAGAUUUGUUGUUACAAGAAUCUUGCCC CAGGGAGCACCCA GCAGUCCGAAAUUGGCGAAUUUGAUUUGUUCCAAGCUCGAUUAUCGAAUACAAGGGUACGCGGGCAGCCGGGGACUCAUCUACCCGCUACGCAGACGAUCUUACGCUGUCUGCCCAAUCAAUGAAGAAGGUCGUAAAGGCGCGGGAUUUCUUGUUUUCUAUCCAUCCCGAGGGCUUGGUAAUUAAUUCCAAAAAGACUUGUAUCCAGGACCACGAUCUCAGCGAAAAGUGACAGG ACUCGU CAUUUCUCAAGAAAAAGUCGGUAAGGGAGAGAAGUAUAAGGAAAUCCGCGCGAAGAUCCACCACAUAUUCUGUGGCAAGAGCAGCGAGAUAGAACACGUCCGAGGCUGGUUGUCCUUCAUACUGAGCGUGGACUCAAAAAGCCACCGCCGGUUGAUCACCUAUAUUUCAAAACUGGAAAAGAAAUAUGGAAAGAACCCACUCAACAAAGCUAAAACACCACCAAAGAAGAAAAGAAAGGUCUGA (SEQ ID NO: 19126) Eco1 ncRNA-sgRNA ncRNA-sgRNA fusion (6 bp substitution in EMX1 gene) GAAAUGAUAAAGAUUCCGUAUGCGCACCCUUAGCGAGAGGUUUAUCAUUAAGGUCAACCUCUGGAUGUUGUUUCGGCAUCCUGCAUUGAAUCUGAGUUACUGUCUGUUUUCCUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAAGGAAACCC GUUUCUUCUGACGUAAGGGUGCGCAUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUUUUU (SEQ ID NO: 19127) Eco3 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGCGCAUUUACUCUCUGAUCGACAGCCAAACCUUAAUGACCAAAGGGUUCGCAUCCGAGGUCAUGAGGAGCCCAGAACCCCCUAAGAAGUGGGACAUUGCGAAGAAGAAGGGCGGAAUGCGUACGAUACCAUCCCUCUUCUAAGGUGAAGCUGAUACAGUACUGGCUGAUGAACAACGUGUUCUCCAAAUUGCCGAUGCACAACGCCGCGUACGCUUUCGUGAAGAAUAGAUCUAUCAAG UCUAACGCACUGCUGCACGCAGAGAGUAAGAACAAAUACUACGUUAAGAUUGACCUGAAGGACUUCUUUCCAAGCAUCAAGUUCACAGACUUCGAAUAUGCCUUUACCCGGUACCGUGACAGAAUAGAGUUCACGACCGAGUACGACAAAGAACUGCUUCAGCUGAUUAAGACCAUUUGUUUCAUUUCUGACUCUACACUGCCAAUAGGCUUCCCCACUUCCCCUUAUAGCCAAUUUCGU CG CCAGGGAGCUGGACGAAGCUCACUCAGAAGCUGAACGCUAUAGACAAGCUCAACGCUACGUACACUCGCUACGCAGACGACAUAAUCGUGAGCACGAACAUGAAGGGCGCCUCUAAGCUGAUCUUAGACUGCUUCAAGCGGACCAUGAAGGAAAUCGGACCCGAUUUCAAGAUCAAUAUCAAGAAGUUCAAAAUAUGCUCUGCCAGUGGCGGCUCAAUUGUCGUGACGGGGCCUUAAGGUCUG UCAUGACUUCCACAUAACUCUGCACCGGUCUAUGAAGGACAAGAUCCGCCUGCACCUCUCUCUCCUGUCCAAAGGUAUUCUGAAGGACGAGGACCACAACAAGCUGUCCGGGUACAUCGCCUACGCUAAGGACAUCGAUCCACACUUCUACACCAAGCUCAAUAGGAAGUACUUCCAGGAGAUCAAGUGGAUACAAAACCUGCAUAAUAAGGUGGAGCCACCAAAGAAGAAAAGAAAGGUCUGA (SEQ ID NO. : 19128) Eco3 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUC GAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUUUUUU (SEQ ID NO: 19129) Eco5 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGGACGCUACCAGAACGACUCCUUGCAUUGGAUCUCUUCGGAUCUCCAGGUUGGUCCGCCGAUAAAGAAAUUCAGAGGCUUCAUGCGCUCAGUAAUCAUGCUGGAAGGCAUUACAGAAGGAUUAUAUUAAGUAAAAGGCACGGCGGACAGCGUCUUGUGCUUGCACCUGAUUACUUGUUAAAGACCGUUCAGCGCAACAUUUUGAAGAACGUUUUGAGUCAAUUUCCACU GUCACCAUUUGCU ACAGCCUACAGACCGGGAUGCCCAAUCGUGUCUAACGCGCAGCCACACUGCCAACAGCCACAGAUCUUGAAACUCGAUAGAAAACUUCUUCGAUUCUAUUAGUUGGUUGCAGGUGUGGCGGGUGUUUCGCCAGGCCCAGUUGCCCCGAAAUGUCGUAAACGAUGCUCACUUGGAUAUGUUGUUAUAACGACGCACUUCCGCAGGGGUCCCCCUACAUCCCCUGCAAUUUCCAAUCUCGUCAU GAGA AGGUUUGAUGAACGGAUUGGAGAAUGGUGUCAGGCUCGAGGGAUUACCUACACUCGCUACUGCGAUGACAUGACGUUUAGUGGACACUUCAAUGCAAGCAGGUCAAGAAUAAAGUCUGCGGUCUCUUAGCUGAGCUGGGCCUUUCCCUGAAUAAACGGAAAGGCUGCCUCAUAGCGGCUUGUAAGCGCCAGCAAGUCACCGGCAUUGUUGUGAAUCACAAGCCACAGCUUGCCCGAGAAGCC AGG CGUGCCCUGCGUCAGGAAGUGCCACCUGUGCCAGAAAUAUGGAGUUAUCUCUCUCUCACAUAGAGGUGAACUGGAUCCUAGCGGAGAUCUGCACGCUCAGGCGACAGCGUAUCUCUAUGCACUCCAGGGGAGAAUUAACUGGCUUCUUCAAAUUAACCCUGAGGAUGAGGCGUUUCAACAGGCCCGGGAGUCCGUUAAGAGGAUGUUAGUUGCCUGGCCACCAAAGAAGAAAAGAAAGGU CUGA (SEQ ID NO: 19130) Eco5 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) GAAAUGAUAAGAUUCCGUACGCCAGCAGUGGCAAUAGCGUUUCCGGCCUUUUGUGCCGGGAGGGUCGGCGAGUCGCUGACUUAACGCCAGUAGUAUGUCCAUAUACCCAAAGUCGCUUCAUUGUAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUCCUUCGAGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUU GCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUACAGUUACGCGCCUUCGGGAUGGUUUAAUGGUAUUGCCGCUGUUGGCGUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUUUUUU (SEQ ID NO: 19131) Cas9 All U are N-1-methylpseudouridine. GGGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUCGUUGCAGGCCUUAUUCGGAUCCGCCACCAUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGCAUCAA GAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAA CAGCAGAAGCAACAAGACUGAAGAGAAC AGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAU GGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCA CGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCA CACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGAC AAGCUGUUCAUCCAGCUGGUCCAGACAAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAG CUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAAC UUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGC AUGAUCAAGAGAUACGACGAACCACCAGGACCUGACACUGCUGAA GGCACUGGUCAGACAGCAGCUG CCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGA GCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUG CUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGAC AACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGA AACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUC ACACCGUGGAACUUCGAAGAAGUC GUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAAC GAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGAC CUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAA UGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAG AAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUG UUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAG CUGAUCAACGGAAUCAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUC GCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAG AAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAA GGUCAUGGGAAGACACAAGCCGGAA AACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAC ACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAG GAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACG ACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCG AAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC AAAGCUGAUCACACAGAGAA AGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCG ACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAU ACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGG AAAUCGGAAAGGCAACAG CAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACU UCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAUCGUCAAGAAGACAGAAGUCCAGACAG GAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACU GGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGA UCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCC ACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGC ACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAAGCACAGAGACAAGCCGAUCA GAGAACAGGCAGAAAACAUCA UCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAAUCGACAG AAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUA CGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGU CUAGCUAGCCAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAAGAGAAAGAAAAUGAAGAUCAA UAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUC UUUAAUCAUUUUGCCUCUUUUCUCUG UGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG (SEQ ID NO: 19132) EMX1 sgRNA modified Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU (SEQ ID NO: 19133) EMX1 sgRNA unmodified No modification GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU (SEQ ID NO: 19134) Eco3 ncRNA Eco3 ncRNA has only a 10 bp insertion GGGAUAAUUGAUAAGAAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGA GGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAGAU (SEQ ID NO: 19135) Eco3 ncRNA-MS2 Eco3 ncRNA has only a 10 bp insertion and a 3' MS2 stem loop GGGAUAAUUGAUAAGAAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGA GGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAACAUGAGGAUCACCCAUGUGAU (SEQ ID NO: 19136) Aco1 RT Aco1 RT RNA sequence, including 5' and 3' UTR AGGGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGCCCAAUGACUACGUAAAUAGGUUGCGACAUGCUAUGGAAAUAAGUGAAAACCCGCGCUUUAGCCCUGAGUACAUUGCUCAAUGUUGUACUUACGCCGAGAAUCUCCUCAAGCAAGGGCUGCCAGUUCUGUUCGAUCAAACGCAUAUUCGGAAAGUUCUGGGGAUGGCGGCACCUCGAUUGUGUGAUUAUCACAGA UUCACAAUCCCAAAGCACAACGGAUCUAGAAUUAUCACGGCCCCAUCUA GGAAGCUGAAGCUUCGACAACAAUGGAUAUACCAGAAUAUCCUUAUACGAAAGGAGGCUUCACCGUACACGCACGGAUUUGUUCCUGAACGCAGCAUCGUGACUAACGCAAUCCUCCAUAUAGGAUACGCAUACACCUACUGCGUGGAUAUCACGGAUUUCUUUCCUAGCAUCACUAAGAAGCAGGUCUUGCCUAUAUUCCGAAAUAUGGGCUAUAGUGGUUCUGCUGCAAAUACUCUGC GACCUCUGUUGCUAUGACGGGGUCCUCCCCCAGGGGGCGCCUACUAGCCCAUACCUCA GUAACAUGAUUUGUCGCGAUCUUGAUGACGAAUUGGGGGCUAUGGCCGGCGGUUCCGGGGGAUUUUUACACGGUAUGCGGAUGACAUAGCUAUCCACAAACCAACAACAGCCGCAACUUUUGGAUGCCUUGGGACUUAUCCUCGGGAAGCACGGAUUUCUUAUGAAUCUCGAUAAGUGUCGAGUCUAUAAUCCUGGACAGCCCAAAAGAAUUACUGGAUUGACCGUUCACAAUA GAGUAUCAGUUCCGAAAACCUUUAAGCGAAAUUGCGGCAGAAAUACAUUACUGUCAGAAGU UCGGAGUGACUGCACAUUUGGAAAACACGAAGGCUGCACGAUCCAUCCACUAUAGGGAACAUCUGUAUGGAAAGGCAUACUAUGUUAAAAUGGUUGAGCCCUGAGCUCGGGGCGCACUUCCUCGAUGAGUUGUCAAAGGUAGACUGGCCAGAGCCACCAAAGAAGAAAAGAAAGGUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCCAGCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCCCUGCA (SEQ ID NO: 19137) Aco1 ncRNA-sgRNA_50 Aco1 ncRNA-sgRNA with 50 bp insert GGGGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCC ACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGCACGGCAAACAGACAGAUCCAUUAUUAUUCACAAUUUAUUUAGUGAUCGUUCCCGGUUUAUACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUCCUGCA (SEQ ID NO: 19138) Aco1 sgRNA-ncRNA_50 Aco1 sgRNA-ncRNA with 50 bp insert GGGGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGUGCUUUUUGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGA AGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAAAGUUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGCACGGCAAACAGACAGAUCCAUUAUUAUACAAUUUAUUUAGUGAUCGUUCCCGGUUUUAUACCCUGCA (SEQ ID NO: 191 39) Eco3_EMX1 gRNA_ncRNA_25 Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUAAUAACAACGAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGA ACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCA(SEQ ID NO: 19140) Eco3_ncRNA only Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAAGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGA UGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCA(SEQ ID NO: 19141) Eco3_ncRNA_AAVS1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGACUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCCUCCUUCCUAGUCUCCUGAUAUUGGG UCUAACCCCCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGGGGCCCACUAGGGACAGGAUGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU(SEQ ID NO: 19142) Eco3_ncRNA_EMX1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAG CAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUU(SEQ ID NO: 19143) Eco3_ncRNA_EMX1 gRNA_50 Eco3 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguuCUCCCAUCACA UCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU(SEQ ID NO: 1 9144) Eco3_ncRNA_EMX1 gRNA_75 Eco3 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCC UAUAaag uuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU (SEQ ID NO: 19145) Eco3_ncRNA_EMX1 gRNA_100 Eco3 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAU GGGGCCCAUGGUUGAAUG ACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUU(SEQ ID NO: 19146) Eco3_ncRNA_EMX1 gRNA_GFP gene Eco3 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. (SEQ ID NO: 19147) Eco3_NT_ncRNA_EMX gRNA_25 The Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccgguugaugugaugggagaacuuUAUAGGAGUCAUUCAGAGCUCGAGUuuucuucugcggacuca ggcccuuccuccuccagcuucugccguuuguUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU(SEQ ID NO: 19148) Aco1_EMX1 gRNA_ncRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end gaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuAAUAACAACguauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccgcgcgugcguacaaacggcagaagcuggagg aggaagggccugaguccgagcagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauac (SEQ ID NO: 19149) Aco1_ncRNA only Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcggcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacg cacgcacggcaaacagacagauccauuauuauuacaauuuauuuagugaucguucccgguuuuauac(SEQ ID NO: 19150) Aco1_ncRNA_AAVS1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccgcggcguUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAacgcacgcacggcaa acagacagauccauuauuauuacaauuuauuagugaucguucccgguuuuauacAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO: 19151) Aco1_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaac gcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu (SEQ ID NO: 19152) Aco1_ncRNA_EMX1 gRNA_50 Aco1 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaac gcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO: 19153) Aco1_ncRNA_EMX1 gRNA_75 The Aco1 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguucucccaucacaucaacc gguggc gcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauauuacaauuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgaguc ggugcuuuuu(SEQ ID NO: 19154) Aco1_ncRNA_EMX1 gRNA_100 Aco1 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcggcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUA aaguucuccacaca ucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaag uggcaccgagucggugcuuuuu(SEQ ID NO: 19155) Aco1_ncRNA_EMX1 gRNA_GFP gene Aco1 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. (SEQ ID NO: 19156) Aco1_NT_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccgguugaugugaugggagaacuuUAUAGGAGUCAUUCAGAGCUCGAGUuuucuucugcucggacucaggcccuuccuccuccagcuucugccguuuguac gcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO: 19157) R2042_EMX1 gRNA_ncRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end gaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuAAUAACAACGCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCA CGCUCCCUUUAGCAGAGCUA AGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGC(SEQ ID NO: 19158) R2042_ncRNA_25 only R2042 ncRNA contains a 25 bp insertion in the EMX1 gene GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAAAGACUCCUAUAAAGUUC UCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGC(SEQ ID NO: 19159) R2042_ncRNA_AAVS1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the AAVS1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAUGACAGAAAAGCCC CAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu (SEQ ID NO: 19160) R2042_ncRNA_EMX1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAAAGU UCUCCCAUCA CAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuu uu(SEQ ID NO: 19161) R2042_ncRNA_EMX1 gRNA_50 The R2042 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAUCAGUAUCAUGGGG CCCAUGGUUGAAUGACUCCUAUAa aguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagu cggugcuuuuu(SEQ ID NO: 19162) R2042_ncRNA_EMX1 gRNA_75 The R2042 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end (SEQ ID NO: 19163) R2042_ncRNA_EMX1 gRNA_100 The R2042 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end (SEQ ID NO: 19164) R2042_ncRNA_NT_EMX1 gRNA_25_Double guide The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) that is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccuauaguuaguguacugcaacuuUAUAGG AGUCAUUCAGAGC UCGAGUuuucuucugcucggacucaggcccuuccuccuccagcuucugccguuuguCCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgaguc ggugcuuuuu(SEQ ID NO: 19165) R2042_RT R2042 RT mRNA sequence, coding sequence only (SEQ ID NO: 19166) R6943 RT R6943 RT mRNA sequence, coding sequence only (SEQ ID NO: 19167) R6943_ncRNA_EMX1gRNA_GFP gene The R6943 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. (SEQ ID NO: 19168) R6943_ncRNA_EMX1gRNA_100bp The R6943 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end (SEQ ID NO: 19169) R6943_ncRNA_EMX1gRNA_75bp The R6943 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaaaaagu ucucccaucacaucaa ccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcuuuuu(SEQ ID NO: 19170) R6943_ncRNA_EMX1gRNA_50bp The R6943 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguucucccaucacaaccggugg cgcau ugccacgaagcaggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcu uuuu(SEQ ID NO: 19171) R6943_ncRNA_EMX1gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggagggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucuccccaucacaucaaccgguggcgcauugccacgaagcag gccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO: 19172) R6943_ncRNA_AAVS1 gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCU GAUAUUGGGUCUAACCCCCAGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu(SEQ ID NO: 19173) R6943_ncRNA only_25bp R6943 ncRNA contains a 25 bp insertion in the EMX1 gene GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucuccccaucacaucaaccgguggcgcauugccacgaagcaggccaaugg ggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGC(SEQ ID NO: 19174) AAVS1 sgRNA Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate GGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu (SEQ ID NO: 19175) Error! Not a valid link.Primer sequence describe sequence AAVS1 NGS_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCTG GGACCACCTTATATTCC(SEQ ID NO: 19212) AAVS1 NGS_R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTCT GGCAAGGAGAGAGATG (SEQ ID NO: 19213) EMX1_NGS_F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGaggtgaag gtgtggttccag(SEQ ID NO: 19214) EMX1_NGS_R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGtagtcatt ggaggtgacatcg(SEQ ID NO: 19215) EMX1_NGS_F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGaggtgaag gtgtggttccag(SEQ ID NO: 19216) EMX1_NGS_R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGgccaga gtccagcttggg(SEQ ID NO: 19217) qPCR primers qPCR Primers - Non-NGS EMX1 forward primer aggtgaaggtgtggttccag(SEQ ID NO: 19218) EMX1 reverse primer tagtcattggaggtgacatcg(SEQ ID NO: 19219) 5' junction GFP reverse primer ccccttgagcatctgacttc(SEQ ID NO: 19220) 3' junction GFP forward primer catcactttcccagtttaccc(SEQ ID NO: 19221) RT variant (coding sequence) RT variant ORF sequence SEQ ID NO: 6342S_N_465_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAAGGTGATCAAGGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19222 6342S_N_465_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAAGGTGATCCAGGAGA TCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19223 6342S_N_465_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAAGGTGATCCGGGAGA TCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19224 6342S_E_466_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAAGATC AGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19225 6342S_E_466_N ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACATCA GCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19226 6342S_E_466_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACCAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19227 6342S_E_466_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACCGGAT CAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19228 6342S_S_468_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAAGGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19229 6342S_S_468_N ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAACGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19230 6342S_S_468_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CCAGGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19231 6342S_S_468_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CCGGGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19232 6342S_M_470_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCAAGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19233 6342S_M_470_N ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCAACACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19234 6342S_M_470_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCCAGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19235 6342S_M_470_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCCGGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19236 6342S_S_472_G ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCGGCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19237 6342S_S_472_P ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCCCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19238 6342S_W_473_F ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTTCGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19239 6342S_W_473_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCAAGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19240 6342S_W_473_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCCGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19241 6342S_W_473_Y ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTACGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19242 6342S_K_475_N ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAACTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19243 6342S_K_475_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGCAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19244 6342S_K_475_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGCGGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19245 6342S_S_476_K ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGAAGGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19246 6342S_S_476_N ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGAACGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19247 6342S_S_476_Q ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGCAGGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19248 6342S_S_476_R ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGCGGGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19249 6342S_V_477_F ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCTTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19250 6342S_V_477_I ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCATCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19251 6342S_V_477_L ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCCTGAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19252 6342S_V_477_W ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCTGGAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19253 6342S_V_265_P ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGCCTCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGAT CAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACccaccaaagaagaaaagaaaggtctga SEQ ID NO: 19254 6342S_M470K_V477W ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCC AGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAATAAGGTTAAGAATAGTGGAGAAATTCTAAGTAATAAAGTCATAAACGAG ATCAGCGGCAAGACCTCCTGGGTGAAGTCCTTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACCCACCAAAGAAGAAAAGAAAGGTCTGA SEQ ID NO: 19255 6342S_M470K_V477W_V265P ATGATGGAGAATCAGGAGAACAACATGAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGA AGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAG CGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATACACCAT CCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGC TTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGCCCCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAG GGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGC TTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAATAAGGTTAAGAATAGTGGAGAAATTCTAAGTAATAAAGTCATAAACGAG ATCAGCGGCAAGACCTCCTGGGTGAAGTCCTTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAACCCACCAAAGAAGAAAAGAAAGGTCTGA SEQ ID NO: 19256 6342S_ntermtrunc_whelix ATGGAGAGGAGAGCTGAAAGAAAGCGCCAGCGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAGAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAACTGAGATTCCTGGTGTACCACCGGGACGTG ATCTCCGTGGACAACTACCACAGATACACCATCCCC AAGAAGAAGGGCGGCGTGAGAAACATCGCCGCTCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGCTTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCTTCGGCT ACAGCGGATACGGTGGCCAGCATGCTGGCCATGATC TGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGCTTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAG CAAGATCGTGAAGGAAGAGGGCTTCAACATCAAC AAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAATAAGGTGATCAACGAGATCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAAGAGAGATACAAGGACaTTATCAACGATGCCATGAACCTGATCAACAA CccaccaaagaagaaaCGGaaggtctga SEQ ID NO: 19257 Sso7d-PASTE (v2)_WT 6342S ATGACCGTCAAGTTCAAGTACAAGGGTGAGGAACTTGAAGTTGATATTAGCAAAATCAAGAAGGTTTGGCGCGTTGGTAAAATGATATCTTTTACTTATGACGACAACGGCAAGACAGGTAGAGGGGCAGTGTCTGAGAAAGACGCCCCCAAGGAGCTGTTGCAAATGTTGGAAAAGTCTGGGAAAAAGTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCGGGACAGGTGGC GGTGGTGTCGAGAATCAGGAGAACAACATGAACAACAGAAAGAACTACAGAGAGGCCGTGAACACATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTAC AAGGAGAGAAAGTGCCTGCTCGAGAAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGAAGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAGCGGGAGCTGGAGAAGGAGCCGCTCCGAGGAGTGGAAGAAGGA GAAGGAGAACAAGATCGTCTTCATCGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCC GTGGACAACTACCACAGATACACCATCCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGTCCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGCTTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGT GAGAGGCATGTTCAAGAGCTTCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATC TGCGTGAAGCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGCTTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAA GAAGTGGATCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGATCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAAC SEQ ID NO: 19258 Sso7d-ncbiprotein (v1)_WT 6342S ATGACAGTGAAGTTCAAGTACAAGGGCGAGGAAAAGGAAGTCGACATCAGCAAGATCAAGAAAGTGTGGCGGGTGGGCAAGATGATCAGCTTCACCTACGACGAGGGCGGAGGAAAGACCGGCAGAGGCGCCGTGTCTGAGAAAGATGCCTCTAAGGAGCTGCTGCAGATGCTGGAAAAACAGAAGAAGCCCAAGAAGAAGAGGAAAGTCGGGACAGGTGGCGGTGGTGTCGAGAATCAGGAGAACAACATGAACAACA GAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAGAAGTACG AGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGAAGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAGCGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAGAAGGAGAACAAGATCGTCTTCATCGGCAA GGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACAGATAC ACCATCCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGTCCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGCTTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTTCAAGAGCT TCGGCTACAGCGGATACGTGGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAAGC TGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGCTTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGAT CAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGATCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAAC SEQ ID NO: 19259 Sac7d-ncbiprotein (v2)_WT 6342S ATGGTGAAGGTGAAATTCAAGTACAAGGGCGAGGAAAAAGAGGTGGATACAAGCAAGATCAAGAAAGTGTGGAGAGTGGGAAAGATGGTCAGCTTCACCTACGACGACAACGGCAAGACCGGCAGAGGCGCCGTGTCTGAGAAGGACGCCCCTAAGGAACTGCTGGATATGCTGGCTAGAGCCGAGCGGGAAAAGAAGCCCAAGAAGAAGAGGAAAGTCGGGACAGGTGGCGGTGGTGTCGAGAATCAGGAGAACAACAA CATGAACAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAG AAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGAAGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAGCGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAGAAGGAGAACAAGATCGTCTTCAT CGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACA GATACACCATCCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGTCCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGCTTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTT CAAGAGCTTCGGCTACAGCGGATACGTGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAA GCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGCTTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGA TCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGATCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAAC SEQ ID NO: 19260 Sac7d-ncbigene (v1)_WT 6342S ATGGTCAAGGTGAAGTTCAAGTACAAGGGCGAGGAAAAAGAGGTGGATACAAGCAAGATCAAGAAAGTGTGGAGAGTGGGAAAAATGGTGAGCTTCACCTACGACGACAACGGCAAGACCGGCAGAGGCGCCGTGTCTGAGAAGGACGCCCCTAAGGAACTGCTGGATATGCTGGCTAGAGCCGAGCGGGAAAAGAAGCCCAAGAAGAAGAGGAAAGTCGGGACAGGTGGCGGTGGTGTCGAGAATCAGGAGAA CAACATGAACAACAGAAAGAACTACAGAGAGGCCGTGAACACCATGGGCAAGAAGGAATTCACCCTGATCAAGATGCAGGAGTACGGCTTCTGGCCCAAGAACCTGCCTACTCCCTACGAGAGGCAGGAAAGCGAGACCGAGGAGCAGTACAAGGAGAGAAAGTGCCTGCTCGAG AAGTACGAGAAGGTGATCGACGAGATCAGCAAGCTGTACGAAGAGAAGGACAAGATCAACCTGAAGCTGAGGGAGCTCCAGAAGAAGTACGACGAGACCTGGGACTATGAGCGCATCCGGCTGGATGTGAGCAAGACCATCATGCAGGAGAGCATCGAGAGGAGAGCTGAAAGAAAGCGCCAGCGGGAGCTGGAGAAGGAGCAGCGCTCCGAGGAGTGGAAGAAGGAGAAGGAGAACAAGATCGTCTTCAT CGGCAAGGGGTACAGCAGCCTGCTGTACGACAAGGAGACCGACGAGAACAAGCTGCTGCTGCAGGAGCTCCCTATCATCAAGGATGATAAGGAGCTGGCCAACTTCCTGGGCATCGAGTACAAGAAGCTGAGATTCCTGGTGTACCACCGGGACGTGATCTCCGTGGACAACTACCACA GATACACCATCCCCAAGAAGAAGGGCGGCGTGAGAAACATCGCCGTCCCCAAGAGCATCCTGAAGAACTCCCAGAGGATTATCCTCGAGGAGATTCTGAGCAAGATCCCCACCTCCAACTACAGCCACGGCTTCCTGAAGGGGAAGTCCGTGGTGAGCGCCGCCAAGGTGCACGTGAAGAAACCCGACCTCCTGATCAACATCGATCTGGAGGACTTCTTCCCCACCATCACCTTCGAGAGGGTGAGAGGCATGTT CAAGAGCTTCGGCTACAGCGGATACGTGCCAGCATGCTGGCCATGATCTGCACCTACTGCGAGAGGATGAAGGTGGAGGTGAGGGGCGAGGAAGTACGTCAAGATCAGCGACAGAATTCTGCCCCAGGGCAGCCCTGCCAGCCCCATGATCACCAACATCATCTGCGTGAA GCTGGACAAGAGACTGAACGGCCTGAGCACCAAGTATGACTTCATTTATACTCGGTACGCTGACGACATGTCGTTCAGCTTCACCGGCGACATCAACGAACTGAGCGTCGGCAGCTTCATGGGCCTGGTGAGCAAGATCGTGAAGGAAGAGGGCTTCAACATCAACAAGGACAAGACCAAGTTCCTGAGAAAGAACAACAGACAGTGCATCACCGGCATCGTGATCAACAACGAGGAGATCGGCGTGCCCAAGAAGTGGA TCAAGATCCTGAGAGCCGCCATCTACAACGCCAACAAGGTGAAGAACAGCGGCGAGATCCTGAGCAACAAGGTGATCAACGAGATCAGCGGCATGACCTCCTGGGTGAAGTCCGTCAACGAGGAGAGATACAAGGACATCATCAACGATGCCATGAACCTGATCAACAAC SEQ ID NO: 19261 Plasmid sequence Name describe sequence SEQ ID NO: Eco1 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGAAAUCUGCAGAGUAUCUGAAUACGUUCCGCCUUAGGAAUUUGGGCCUCCCCGUGAUGAACAAUCUCCACGAUAUGAGCAAGGCGACUCGAAUAUCCGUGGAAACGCUGAGACUGCUCAUCUAUACAGCAGACUUUCGGUACAGGAUCUACACGGUCGAAAAGAAGGGGCCUGAGAAACGCAUGCGAACAAUUUAUCAACCUAGCCGAGAGCUCAAGGCGUUGCAGGGCUGGGUUCUUCG AAAC AUCCUUGACAAACUCUCAUCAUCACCCUUUAGUAUUGGGUUUGAAAAGCACCAAAGCAUCCUUAACAACGCGACGCCACACAUAGGUGCCAAUUUCAUAUGAACAUCGACUUGGAGGAUUUUUUCCGAGCCUCACAGCCAAUAAAGUGUUCGGUGUUUUUCACAGUCUUGGGUACAAUCGCCUUAUUAGUUCCGUUCUUACCAAGAUUUGUUGUUACAAGAAUCUUGCCC CAGGGAGCACCCA GCAGUCCGAAAUUGGCGAAUUUGAUUUGUUCCAAGCUCGAUUAUCGAAUACAAGGGUACGCGGGCAGCCGGGGACUCAUCUACCCGCUACGCAGACGAUCUUACGCUGUCUGCCCAAUCAAUGAAGAAGGUCGUAAAGGCGCGGGAUUUCUUGUUUUCUAUCCAUCCCGAGGGCUUGGUAAUUAAUUCCAAAAAGACUUGUAUCCAGGACCACGAUCUCAGCGAAAAGUGACAGG ACUCGU CAUUUCUCAAGAAAAAGUCGGUAAGGGAGAGAAGUAUAAGGAAAUCCGCGCGAAGAUCCACCACAUAUUCUGUGGCAAGAGCAGCGAGAUAGAACACGUCCGAGGCUGGUUGUCCUUCAUACUGAGCGUGGACUCAAAAAGCCACCGCCGGUUGAUCACCUAUAUUUCAAAACUGGAAAAGAAAUAUGGAAAGAACCCACUCAACAAAGCUAAAACACCACCAAAGAAGAAAAGGUCUGA SEQ ID NO: 19262 Eco1 ncRNA-sgRNA ncRNA-sgRNA fusion (6 bp substitution in EMX1 gene) GAAAUGAUAAAGAUUCCGUAUGCGCACCCUUAGCGAGAGGUUUAUCAUUAAGGUCAACCUCUGGAUGUUGUUUCGGCAUCCUGCAUUGAAUCUGAGUUACUGUCUGUUUUCCUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAAGGAAACCC GUUUCUUCUGACGUAAGGGUGCGCAUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUUUUUU SEQ ID NO: 19263 Eco3 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGCGCAUUUACUCUCUGAUCGACAGCCAAACCUUAAUGACCAAAGGGUUCGCAUCCGAGGUCAUGAGGAGCCCAGAACCCCCUAAGAAGUGGGACAUUGCGAAGAAGAAGGGCGGAAUGCGUACGAUACCAUCCCUCUUCUAAGGUGAAGCUGAUACAGUACUGGCUGAUGAACAACGUGUUCUCCAAAUUGCCGAUGCACAACGCCGCGUACGCUUUCGUGAAGAAUAGAUCUAUCAAG UCUAACGCACUGCUGCACGCAGAGAGUAAGAACAAAUACUACGUUAAGAUUGACCUGAAGGACUUCUUUCCAAGCAUCAAGUUCACAGACUUCGAAUAUGCCUUUACCCGGUACCGUGACAGAAUAGAGUUCACGACCGAGUACGACAAAGAACUGCUUCAGCUGAUUAAGACCAUUUGUUUCAUUUCUGACUCUACACUGCCAAUAGGCUUCCCCACUUCCCCUUAUAGCCAAUUUCGU CG CCAGGGAGCUGGACGAAGCUCACUCAGAAGCUGAACGCUAUAGACAAGCUCAACGCUACGUACACUCGCUACGCAGACGACAUAAUCGUGAGCACGAACAUGAAGGGCGCCUCUAAGCUGAUCUUAGACUGCUUCAAGCGGACCAUGAAGGAAAUCGGACCCGAUUUCAAGAUCAAUAUCAAGAAGUUCAAAAUAUGCUCUGCCAGUGGCGGCUCAAUUGUCGUGACGGGGCCUUAAGGUCUG UCAUGACUUCCACAUAACUCUGCACCGGUCUAUGAAGGACAAGAUCCGCCUGCACCUCUCUCUCCUGUCCAAAGGUAUUCUGAAGGACGAGGACCACAACAAGCUGUCCGGGUACAUCGCCUACGCUAAGGACAUCGAUCCACACUUCUACACCAAGCUCAAUAGGAAGUACUUCCAGGAGAUCAAGUGGAUACAAAACCUGCAUAAUAAGGUGGAGCCACCAAAGAAGAAAAGAAAGGUCUGA SEQ ID NO: 19264 Eco3 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUC GAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUUUUUUU SEQ ID NO: 19265 Eco5 RT Coding sequence only. RNA is generated by CRO and a proprietary 5' cap, 5' UTR, 3' UTR and approximately 120 bases of the encoded poly A are added. Completely replaced with 1N-methyl-pseudouridine AUGGACGCUACCAGAACGACUCCUUGCAUUGGAUCUCUUCGGAUCUCCAGGUUGGUCCGCCGAUAAAGAAAUUCAGAGGCUUCAUGCGCUCAGUAAUCAUGCUGGAAGGCAUUACAGAAGGAUUAUAUUAAGUAAAAGGCACGGCGGACAGCGUCUUGUGCUUGCACCUGAUUACUUGUUAAAGACCGUUCAGCGCAACAUUUUGAAGAACGUUUUGAGUCAAUUUCCACU GUCACCAUUUGCU ACAGCCUACAGACCGGGAUGCCCAAUCGUGUCUAACGCGCAGCCACACUGCCAACAGCCACAGAUCUUGAAACUCGAUAGAAAACUUCUUCGAUUCUAUUAGUUGGUUGCAGGUGUGGCGGGUGUUUCGCCAGGCCCAGUUGCCCCGAAAUGUCGUAAACGAUGCUCACUUGGAUAUGUUGUUAUAACGACGCACUUCCGCAGGGGUCCCCCUACAUCCCCUGCAAUUUCCAAUCUCGUCAU GAGA AGGUUUGAUGAACGGAUUGGAGAAUGGUGUCAGGCUCGAGGGAUUACCUACACUCGCUACUGCGAUGACAUGACGUUUAGUGGACACUUCAAUGCAAGCAGGUCAAGAAUAAAGUCUGCGGUCUCUUAGCUGAGCUGGGCCUUUCCCUGAAUAAACGGAAAGGCUGCCUCAUAGCGGCUUGUAAGCGCCAGCAAGUCACCGGCAUUGUUGUGAAUCACAAGCCACAGCUUGCCCGAGAAGCC AGG CGUGCCCUGCGUCAGGAAGUGCCACCUGUGCCAGAAAUAUGGAGUUAUCUCUCUCUCACAUAGAGGUGAACUGGAUCCUAGCGGAGAUCUGCACGCUCAGGCGACAGCGUAUCUCUAUGCACUCCAGGGGAGAAUUAACUGGCUUCUUCAAAUUAACCCUGAGGAUGAGGCGUUUCAACAGGCCCGGGAGUCCGUUAAGAGGAUGUUAGUUGCCUGGCCACCAAAGAAGAAAAGAAAGGU CUGA SEQ ID NO: 19266 Eco5 ncRNA-sgRNA ncRNA-sgRNA fusion (10 bp insertion in the EMX1 gene) GAAAUGAUAAGAUUCCGUACGCCAGCAGUGGCAAUAGCGUUUCCGGCCUUUUGUGCCGGGAGGGUCGGCGAGUCGCUGACUUAACGCCAGUAGUAUGUCCAUAUACCCAAAGUCGCUUCAUUGUAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUCCUUCGAGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUU GCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUACAGUUACGCGCCUUCGGGAUGGUUUAAUGGUAUUGCCGCUGUUGGCGUACGGAAUCUUUACAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUUUUUU SEQ ID NO: 19267 Cas9 Sequences were obtained from Intellia patients. All Us are N-1-methylpseudouridine. GGGUCCCGCAGUCGGCGUCCAGCGGCUGCUUGUUCGUGUGUGUCGUUGCAGGCCUUAUUCGGAUC CGCCACCAUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGGGCAGUCAU CACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGCAUCAA GAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAACAGCAGAAGCAACAAGACUGAAGAAC AGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAU GGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCA CGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCA CACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGAC AAGCUGUUCAUCCAGCUGGUCCAGACAAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAG CUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAAC UUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGC AUGAUCAAGAGAUACGACGAACCACCAGGACCUGACACUGCUGAA GGCACUGGUCAGACAGCAGCUG CCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGA GCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUG CUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGAC AACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGA AACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUC ACACCGUGGAACUUCGAAGAAGUC GUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAAC GAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGAC CUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAA UGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAG AAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUG UUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAG CUGAUCAACGGAAUCAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUC GCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAG AAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAA GGUCAUGGGAAGACACAAGCCGGAA AACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAC ACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAG GAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACG ACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCG AAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC AAAGCUGAUCACACAGAGAA AGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCG ACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAU ACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGG AAAUCGGAAAGGCAACAG CAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACU UCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAUCGUCAAGAAGACAGAAGUCCAGACAG GAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACU GGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGA UCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCC ACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGC ACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAAGCACAGAGACAAGCCGAUCA GAGAACAGGCAGAAAACAUCA UCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAAUCGACAG AAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUA CGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGU CUAGCUAGCCAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAAGAGAAAGAAAAUGAAGAUCAA UAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUC UUUAAUCAUUUUGCCUCUUUUCUCUG UGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG SEQ ID NO: 19268 EMX1 sgRNA modified Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU SEQ ID NO: 19269 EMX1 sgRNA unmodified No modification GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU SEQ ID NO: 19270 Eco3 ncRNA Eco3 ncRNA has only a 10 bp insertion GGGAUAAUUGAUAAGAAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGA GGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAGAU SEQ ID NO: 19271 Eco3 ncRNA-MS2 Eco3 ncRNA has only a 10 bp insertion and a 3' MS2 stem loop GGGAUAAUUGAUAAGAAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGA GGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19272 Eco3 circular ncRNA Eco3 ncRNA, with a 10 bp insertion only in circular form UACCGGCGAAACAAAAGAAAAAACCAAAAAAACAAAACACAUGAUAAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCCCAUCACAUCAACCGGUGGCGCAU UGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCG SEQ ID NO: 19273 Aco1 RT Aco1 RT RNA sequence, including 5' and 3' UTR AGGGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGCCCAAUGACUACGUAAAUAGGUUGCGACAUGCUAUGGAAAUAAGUGAAAACCCGCGCUUUAGCCCUGAGUACAUUGCUCAAUGUUGUACUUACGCCGAGAAUCUCCUCAAGCAAGGGCUGCCAGUUCUGUUCGAUCAAACGCAUAUUCGGAAAGUUCUGGGGAUGGCGGCACCUCGAUUGUGUGAUUAUCACAGA UUCACAAUCCCAAAGCACAACGGAUCUAGAAUUAUCACGGCCCCAUCUA GGAAGCUGAAGCUUCGACAACAAUGGAUAUACCAGAAUAUCCUUAUACGAAAGGAGGCUUCACCGUACACGCACGGAUUUGUUCCUGAACGCAGCAUCGUGACUAACGCAAUCCUCCAUAUAGGAUACGCAUACACCUACUGCGUGGAUAUCACGGAUUUCUUUCCUAGCAUCACUAAGAAGCAGGUCUUGCCUAUAUUCCGAAAUAUGGGCUAUAGUGGUUCUGCUGCAAAUACUCUGC GACCUCUGUUGCUAUGACGGGGUCCUCCCCCAGGGGGCGCCUACUAGCCCAUACCUCA GUAACAUGAUUUGUCGCGAUCUUGAUGACGAAUUGGGGGCUAUGGCCGGCGGUUCCGGGGGAUUUUUACACGGUAUGCGGAUGACAUAGCUAUCCACAAACCAACAACAGCCGCAACUUUUGGAUGCCUUGGGACUUAUCCUCGGGAAGCACGGAUUUCUUAUGAAUCUCGAUAAGUGUCGAGUCUAUAAUCCUGGACAGCCCAAAAGAAUUACUGGAUUGACCGUUCACAAUA GAGUAUCAGUUCCGAAAACCUUUAAGCGAAAUUGCGGCAGAAAUACAUUACUGUCAGAAGU UCGGAGUGACUGCACAUUUGGAAAACACGAAGGCUGCACGAUCCAUCCACUAUAGGGAACAUCUGUAUGGAAAGGCAUACUAUGUUAAAAUGGUUGAGCCCUGAGCUCGGGGCGCACUUCCUCGAUGAGUUGUCAAAGGUAGACUGGCCAGAGCCACCAAAGAAGAAAAGAAAGGUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCCAGCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCCCUGCA SEQ ID NO: 19274 Aco1 ncRNA_12 Aco1 ncRNA contains a 12 bp insertion in the EMX1 gene guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccgcggcguCAGAAGAAGAAGGGCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCCAAUGuucgaacgaucgGGUGGGCAACCACAAACCCACGAGGGCAGAGUGCUGCUUGCUGCUacgcacgc acggcaaacagacagauccauuauuauuacaauuuauuuagugaucguucccgguuuuauacAAUAAACGUCACCUCCAAUGACUAGGGguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19275 Aco1 ncRNA-sgRNA_50 Aco1 ncRNA-sgRNA with 50 bp insert GGGGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCC ACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGCACGGCAAACAGACAGAUCCAUUAUUAUUCACAAUUUAUUUAGUGAUCGUUCCCGGUUUAUACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUCCUGCA SEQ ID NO: 19276 Aco1 sgRNA-ncRNA_50 Aco1 sgRNA-ncRNA with 50 bp insert GGGGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGUGCUUUUUGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGA AGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGCACGGCAAACAGACAGAUCCAUUAUUAUUACAAUUUAUUUAGUGAUCGUUCCCGGUUUUAUACCCUGCA SEQ ID NO: 19277 Eco3_EMX1 gRNA_ncRNA_25 The Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUAAUAACAACGAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCU GAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCA SEQ ID NO: 19278 Eco3_ncRNA only Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGCAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUC GAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCA SEQ ID NO: 19279 Eco3_ncRNA_AAVS1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGACUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUC UAACCCCCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGGGGGCCACUAGGGACAGGAUGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU SEQ ID NO: 19280 Eco3_ncRNA_EMX1 gRNA_25 Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAG CAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUU SEQ ID NO: 19281 Eco3_ncRNA_EMX1 gRNA_50 Eco3 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguuCUCCCAUCA CAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU SEQ ID NO: 19282 Eco3_ncRNA_EMX1 gRNA_75 Eco3 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACU CCUUAa aguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUU SEQ ID NO: 19283 Eco3_ncRNA_EMX1 gRNA_100 Eco3 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAU GGGGCCCAUGGUUGAA UGACUCCUAUAaaguuCUCCCAUUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUU SEQ ID NO: 19284 Eco3_ncRNA_EMX1 gRNA_GFP gene Eco3 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAcccauauguccuuccgagugagagacacaaaaaauuccaacacaccuauugcaaugaaaauaa auuuccuuuuagccagaagucagaugcucaaggggcuucaugauguccccauaauuuuuggcagagggaaaaagaucucagugguauuugugagccagggcauuggccaccagccacccaccuugauaggcagccugcaccugaggagugcggccgcuuuacuuguacagcucguccaugccgaga gugaucccggcggcggucacgaacuccagcaggaccaugugaucgcgcuucucguuggggucuuugcucagggcggacugggugcucagguaguguugucgggcagcagcagcacggggccgucgccgaugggguguucugcugguaguggucggcgagcugcacgcugccguccucgauguuguggcggaucuugaaguucaccuugaugccguucuucugcuugu cggccaugauauagacguuguggcuguaguuguacuccagcuugugccccaggauguugccguccuccuugaagucgaugcccuucagcucgaugcgguucaccagggugucgccucgaacuucaccucggcgcgggucuuguaguugccgucguccuugaagaagauggugcgcuccuggacguagcc uucgggcauggcggacuugaagaagucgugcugcuucauguggucgggguagcggcugaagcacugcacgccguaggucaggguggucacgagggugggccagggcacgggcagcuugccgguggugcagaugaacuucagggucagcuugccguagguggcaucgcccucgcccucgccggacacgcugaacuuguggccguuuacgucgccguccagcucgaccag gaugggcaccacccggugaacagcuccucgcccuugcucaccaugguggcgaccgguggaucccgggcccgcgguaccgucgacugcagaauucgaagcuugagcucgagaucugaguccgguagcgcuagcgggaucugacgguucacuaaacccuguguucuggcggcaaacccguugcgaaaaagaa cguucacggcgacuacugcacuuauauacgguucucccccacccucggggaaaaggcggagccaguacacgacaucacuuucccaguuuaccccgcgccaccuucucuaggcaccgguucaauugccgaccccuccccccaacuucucggggacugugggcgaugugcgcucugcccguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGC CAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGCUUUUU SEQ ID NO: 19285 Eco3_NT_ncRNA_EMX gRNA_25 The Eco3 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccgguugaugugaugggagaacuuUAUAGGAGUCAUUCAGAGCUCGAGUuuucuucugcggacuca ggcccuuccuccuccagcuucugccguuuguUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCUUAUCAAAUAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUU SEQ ID NO: 19286 Aco1_EMX1 gRNA_ncRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end gaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuAAUAACAACguauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccgcgcgugcguacaaacggcagaagcugga ggaggaagggccugaguccgagcagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauac SEQ ID NO: 19287 Aco1_ncRNA only Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcggcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacg cacgcacggcaaacagacagauccauuauuauuacaauuuauuuagugaucguucccgguuuuauac SEQ ID NO: 19288 Aco1_ncRNA_AAVS1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcggcguUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAacgcacgcacggcaa acagacagauccauuauuauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19289 Aco1_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaac gcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19290 Aco1_ncRNA_EMX1 gRNA_50 The Aco1 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaac gcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19291 Aco1_ncRNA_EMX1 gRNA_75 The Aco1 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguucucccaucacaucaacc ggg gcgcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauuacaauuauuuagugaucguucccgguuuuuacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccga gucggugcuuuuu SEQ ID NO: 19292 Aco1_ncRNA_EMX1 gRNA_100 Aco1 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcggcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAa aguucuccaca caucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaacgcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaa aguggcaccgagucggugcuuuuu SEQ ID NO: 19293 Aco1_ncRNA_EMX1 gRNA_GFP gene Aco1 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaacccauauguccuuccgugagagagacacacaaaaaauuccaaccacacuauugcaaugaaaauaaauuuccuuuuagccagaagucagaug cucaaggggcuucaugaugucccccauaauuuuuggcagagggaaaaagaucucagugguauuugugagccagggcauuggccaccagccacccucugauaggcagccugcaccugaggagugcggccgcuuuacuuguacagcucguccaugccgagagugaucccggcggcggucacgaacuccagc aggaccaugugaucgcgcuucucguuggggucuuugcucagggcggacugggugcucagguagugguugucgggcagcagcacggggccgucgccgaugggguguucugcugguaguggucggcgagcugcacgcugccguccucgauguuguggcggaucuugaaguucaccuugaugccguucuucugcuugucggccaugauauagacguuguggcuguguagu uguacuccagcuuggccccaggauguugccguccuccuugaagucgaugcccuucagcucgaugcgguucaccaggggugucgcccucgaacuucaccucggcgcgggucuuguaguugccgucguccuugaagaagauggugcgcuccuggacguagccuucgggcauggcggacuugaagaag ucggcugcuucauguggucgggguagcggcugaagcacugcacgccguaggucaggguggucacgagggugggccagggcacgggcagcuugccgguggugcagaugaacuucagggucagcuugccguagguggcaucgccuccgcccucgccggacacgcugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccacccggugaac agcuccucgcccuugcucaccaugguggcgaccgguggaucccgggcccgcgguaccgucgacugcagaauucgaagcuugagcucgagaucugaguccgguagcgcuagcggaucugacgguucacuaaacccuguguucuggcggcaaacccguugcgaaaaagaacguucacggcgacuacugcac uuauauacgguucucccccacccucgggaaaaaggcggagccaguacacgacaucacuuucccaguuuaccccgcgccaccuucuaggcaccgguucaauugccgaccccuccccccaacuucucggggacugugggcgaugugcgcucugcccguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugu caacgcacgcacggcaaacagacagauccauuauauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19294 Aco1_NT_ncRNA_EMX1 gRNA_25 The Aco1 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) which is fused to the EMX1 sgRNA at the 3' end guauaaaaccgggaacgaucagaccggggugaauucgcccccuugaucaaacggcacuaaccacuguuugccggcgugcguugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccgguugaugugaugggagaacuuUAUAGGAGUCAUUCAGAGCUCGAGUuuucuucugcucggacucaggcccuuccuccuccagcuucugccguuugu acgcacgcacggcaaacagacagauccauuauuacaauuuauuuagugaucguucccgguuuuauacAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19295 R2042_EMX1 gRNA_ncRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 5' end gaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuAAUAACAACGCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCA CGCUCCCUUUAGCAGAGC UAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGC SEQ ID NO: 19296 R2042_ncRNA_25 only R2042 ncRNA contains a 25 bp insertion in the EMX1 gene GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAUGACUCCUAUAAAGU UCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGC SEQ ID NO: 19297 R2042_ncRNA_AAVS1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the AAVS1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAUGACAGAAAAGC CCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19298 R2042_ncRNA_EMX1 gRNA_25 The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAAAAGACUCCUAUAAAGUUC UCCCAU CACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuu uuu SEQ ID NO: 19299 R2042_ncRNA_EMX1 gRNA_50 The R2042 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAUCAGUAUCAUGGGGCCC AUGGUUGAAUGACUCCUAU AaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcuuuuu SEQ ID NO: 19300 R2042_ncRNA_EMX1 gRNA_75 The R2042 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACCGCAGCUUGCC AGCACUUUCAGUAUCAUGGGGCCCAUGGUUG AAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuuga aaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19301 R2042_ncRNA_EMX1 gRNA_100 The R2042 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAaACUCGAGCUCUGAGCCCCACUGUCGAGAA GUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUG GGGCCCAUGGUUGAAUGACUCCUAUAaaguuCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCAAAGCUUGUGCCUGAGUGAGAGGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccgu uaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19302 R2042_ncRNA_NT_EMX1 gRNA_25_Dual guide The R2042 ncRNA contains a 25 bp insertion in the EMX1 gene (on the opposite strand to that cut by the sgRNA) that is fused to the EMX1 sgRNA at the 3' end GCGUUAAGGUGGUUAUAUUCUAGUAUUUAUGAAGUGUAGUCGCUUCGAUCGUUAAGGCUGAUUUUAACCUCUGCAUAAUAAUAUCGGUAGAUAUUAUUAUGCACGCUCCCUUUAGCAGAGCUAAGAAUCGCUCACUCAGGCACAAGCUUUGAGGugacaucgauguccuccccauuggccugcuucguggcaaugcgccaccuauaguuaguguacugcaacuuUAUAGGAG UCAUUCAGA GCUCGAGUuuucuucugcucggacucaggcccuuccuccaccagcuucugccguuuguCCUCAAAGCUUGUGCCUGAGUGAGAGCUAAAGAAAAGAAAAGUAGAAUAAGCCACCUUAACGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagu cggugcuuuuu SEQ ID NO: 19303 R2042_RT R2042 RT mRNA sequence, coding sequence only AUGAAGGACGACCAGUACUCUCAGUGGAAGAAGUACUACGAGAGCAGGGGCAUCCUGCCCGAGAUCCAGGACAAGCUCCUGAACUACGCCAAGAUCCACAUCGACAACAACACCCCGGUGAUCUUCAACUUCGAGCACCUGACCCUGCUGCUGGGCAGGGAGAAGAACUACCUGUCCAGCGUGGUGAACAGCCCCGACAGCCACUACAGAAAGUUCAAGAUCAAGAAGAGAUCCGGAGGCGAGAGGGAGAUC ACCGCUCCC UAUCUCAGCCUCCUGGAGAUGCAGUACUGAUCUACAGGAACAUCCUGAUCAACGUGAAGAUCCACUACGCCGCUCACGGCUUCGCUCAGGAUAAGAGCAUUAUCACCAACUCCAGGAACCACCUGGGGCAGAAACAUCUGCUCAAGAUGGAUCUGAAGGAUUUCUUCCCCAGCAUCAAGCUGAACAGAAUCAUCUACAUCUUCAAGAGCCUGGGCUACCCCAUAAUCAUCGCCUUCUACCUGGC CAGCAUCUGCAGCUACA AGGGCCACCUGCCCCAGGGCAGCCCCACAAGCCCUAUCCUGAGCAACAUCGUGAGCAUCACCCUGGACAACAGACUGGUGAAGUUCGCCAGAAAGAUGAAGCUGAGAUCAGCAGGUACGCCGACGACCUGACGUUCAGCGGGGACAAGAUCCCCACCAACUACAUCAAGUACAUCACCGACAUCAUCAAUGAUGAGGGCUUCGAGGUGAACGACACCAAGACCAAGCUCUACCUGAAGGCCGGGAAGAGAAUCGUGACG GG CAUUUCCGUGAUCGGAAAUGACCCGAAGCUUCCGCGGGAAUACAAGCGGAAGCUGAAGCAGGAGCUGCACUACAUCUUCACCUACGGCAUCGGCAGCCACAUGGCCAAGAAGAAGAUCAAGAAGAUCAACUACCUACAGGAUCAUCGGCAAGGUGAACUUCUGGCUGAACAUCGAGCCCGACAACGAGUACGCCAGAAACGCCAAGGCCAAGCUGCUGCUGCUCAUCGACAACccaccaaagaaaaaa agaaagucuga SEQ ID NO: 19304 R6943 RT R6943 RT mRNA sequence, coding sequence only AUGGAGGAGAGCACCAACUACAAGCUGCUCGGUGUGGGGACUGAGCGUGAUCCAACCCGCUACCCCCAACGAGGUGCUGAACUACCUCACCAGCACCCUGAACGAUAACGGGCUGCUGCCCGACGUGGAGAAGAUGAUCCACUACUUUGAGCUGCUGGACCAGCUGGGCUACAUCCACCAGGUGAGCAAGAGGAACAACCUCUACUCACUGACCCCCAGGGGCAACGAAAGGUUGACCCCUGCCCUGAAGAGACU CAGGGACAAGAUCAGACUGUUCAUGCUGGACAACUGCCACAGCAUCAGCAAGCUGGGCGUGCUGGCCAGCACAGAUACAGAGAACAUGGGGGGCGACAGCCCCUCACUCCAGCUGAGGCACAACCUC AAGGAGGUGCCUCCAUCCUUAGCUGGGCUGCUGGAACCCUGCCUAGCUCCUAGGCAGGCUUGGGUUCGGAUCUACGAACAGCUGAACAUCGGCAGCAUGAGCAGCGACGAGGCUAGCACACCCACCACCGCCAGAAAUGCCCCCCUGAGCUUCGUGGGCAGGCUGGGCUUCAGCCUGAACUACUACAGCUUCAACAAGAUCGACGAGCCCCUGUUCAACAACGAUGGCGUGACCGCCAUCGCCAGCUG CAUCGGCAUCAGCCCCGGGCUGAUUACCGCUAUGGUGAAGUCACCAAAGCGGUACUACAGGACCUUCAACCUGAGAAAGAAGUCCGGGGGCUUCAGAUCCAUUCUGGCCCCCAGAAAGUUCAUCAAG ACCAUCCAGUACUGGCUGAAGGAUCAUGUGCUGAACAGGCUCAAGAUCCACAGCUCCUGUUACAGCUACAGGAGCGGCGUGUCCAUCAAGGACAACGCCAUCAACCACGUGAAGAAGAAGUUCGUGGCCAGCAUCGACAUUUCCGAUUACUUCGGAAGCAUCAACAAGAAGAUGGUGAAGGACUGCUUUUACAAGAACAAUAUUCCCGAUCACAUCGUGAAUACCAUCAGCGGCAUCGUGACCUACAA CGACGUGCUGCCUCAGGGCGCUCCCACCAGCCCUAUCAUUAGCAACGCCAUCCUGUUCGAGUUCGACGAGGAGAUGACGGCUCAUGCCCUCACUCUCGACUGUAUCUACACCAGAUACAGCGACGACAUCUCG AUAUCCUCCGACUAUAAGGAGAAUAUCGCCAUCCUGAUCAACAUCGCCGAGGCCAACCUGUUGAGCGCUGGAUUCACGCUCAACAGACAGAAGCAAAGGAUUGCUUCUGACAACAGCCGCCAGGUUGUGACCGGCAUCCUGGUGAACGAGCAUCAGACCCACCAGAUGCUACAGAAAGAAGAUCAGAAGCGCCUUUGAUCACGCCCUGAAGGAGCAGGACGGCUCCCAGCUGACAAUCAACAAGUU GAGGGGCUACCUCAACUACCUGAAGUCCUUCGAGACCUACGGCUUCAAGUUCAACGAGAAGAAGUAUAAGGAGACCCUGGAUUUCCUGAUCGCUCUGAAGCAGAGCccaccaaagaagaaaagaaaggucuga SEQ ID NO: 19305 R6943_ncRNA_EMX1gRNA_GFP gene The R6943 ncRNA contains a GFP gene inserted into the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end. The entire GFP cassette is in antisense orientation and contains a miniature EF1a promoter and a β-globin poly A signal. GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaacccauauguccuuccgugagagacacacaaaaaauuccaacacacuauugcaaugaaaauaauuuccuu uauuagccagaagucagaugcucaaggggcuucaugauguccccuaaauuuuuggcagagggaaaaagaucucagugguauuugugagccagggcauuggccaccagccaccaccuugauaggcagccugcaccugaggagugcggccgcuuuacuuguacagcucguccaugccgagagugaucccggcg gcggucacgaacuccagcaggaccaugugaucgcgcuucucguuggggucuuugcucagggcggacuggggcucagguaguguugucgggcagcagcacggggccgucgccgauggggguguucugcugguaguggucggcgagcugcacgcugccguccucgauguuguggcggaucuugaaguucaccuugaugccguucuucugcuugucggccaugau auagacguuguggcuguuguaguuguacuccagcuuggccccaggauguugccguccuccuugaagucgaugcccuucagcucgaugcgguucaccaggggugucgccucgaacuucaccucggcgcgggucuuguaguugccgucguccuugaagaagauggugcgcuccuggacguagccuucgggcauggcggac uugaagaagucgugcugcuucauguggucgggguagcggcugaagcacugcacgccguaggucaggguggucacgagggugggccagggcacgggcagcuugccgguggugcagaugaacuucagggucagcuugccguagguggcaucgcccucgcccucgccggacacgcugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccacccc ggugaacagcuccucgcccuugcuccaugguggcgaccgguggaucccgggcccgcgguaccgucgacugcagaauucgaagcuugagcucgagaucugaguccgguagcgcuagcgggaucugacgguucacuaaacccuguguucuggcggcaaacccguugcgaaaaagaacguucacggcgacuacugcac uuauauacgguucucccccacccucgggaaaaaggcggagccaguacacgacaucacuuucccaguuuaccccgcgccaccuucuaggcaccgguucaauugccgaccccuccccccaacuucucggggacugugggcgaugugcgcucugcccguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugu caGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19306 R6943_ncRNA_EMX1gRNA_100bp The R6943 ncRNA contains a 100 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAU GGUUGAAUGACUCCUAUAaaguu cucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuuga aaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19307 R6943_ncRNA_EMX1gRNA_75bp The R6943 ncRNA contains a 75 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggagggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaaaaagu ucucccaucacauc aaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggca ccgagucggugcuuuuu SEQ ID NO: 19308 R6943_ncRNA_EMX1gRNA_50bp The R6943 ncRNA contains a 50 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguucucccaucacaaccggugg cgc auugccacgaagcaggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucgg cuuuuu SEQ ID NO: 19309 R6943_ncRNA_EMX1gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the EMX1 gene, which is fused to the EMX1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggagggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaagc aggccaauggggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19310 R6943_ncRNA_AAVS1 gRNA_25bp The R6943 ncRNA contains a 25 bp insertion in the AAVS1 gene, which is fused to the AAVS1 sgRNA at the 3' end GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCCGCUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUC CUGAUAUUGGGUCUAACCCCCAGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGCAAUAACAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19311 R6943_ncRNA only_25bp R6943 ncRNA contains a 25 bp insertion in the EMX1 gene GCGGAGUGCUGGCCUCAACUGAUACAGAGAAUAUGGGCGGUGAUUCGCCGUCUUUACAGUUAAGGCACAAUUUAAAAGAGGUUCCGCACCCAAGCCUGUCUUGGGCUGCacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucuccccaucacaucaaccgguggcgcauugccacgaagcaggccaaugg ggaggacaucgaugucaGCAGCCCAAGACAGGCUUGGGUGCGGAUCUACGAGCAAUUAAAUAUUGGUUCGAUGUCCAGUGAUGAGGCCAGCACCCGC SEQ ID NO: 19312 AAVS1 sgRNA Contains chemical modifications from Synthego: 2'-F, 2'-O-methyl, phosphorothioate GGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19313 R1262_RT-1 (R1262 RT) R1262 retrotranscript RT mRNA AGGAUAAUGGGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAUCAGCUUCAGCGAGAUCAAGAGCAGAAACGAUUUUGCAGACGCUCUGCAGAUUCCUCGGAGCGUGCUGACGCACGUUCUGUACAUUGCCAAGCCCGAGAGCUUCUACGAGAGCUUCACCAUCCCCAAGAAGAAUGGGGAGGACAGAAUCAUCAUGGCCCCCAAGGGCACCCUGAAGU CCAUCCAGACCAAGCUGAGCAAGCAGCUGGUGGAGUACAGAGCCUCCAUCAGCCAGAAAGGCCAGGAGAAGUCCAACAUCUCCCAUGGCUUCGAGAGGGAGAAGUCCAUCAUCACCAAUGCUCAGAUACACCGCAACAAGCGGUAUGUCAUCAACUACGACCUGAAGGACUUCUUCGACUCCUUCCACUUCGGCAGAGUGGUGGGAUUCUUCGAGAAGAACAAGCACUUCCUGCUGC CCUACGAGGUGGCCGUGAUCAUCGCCCAGCUGACCUGCUAUAAUGGCAGGCUGCCCCAGGGCGCCCCCACAAGCCCUGUGAUCACCAACCUGAUCUGCGAGAUCCUGGACUACAGGGUGCUCAAGAUCGCCAAGAGAUACAAGCUGGACUACACCAGGUACGCUGACGACCUGACCUUCAGCACCAACUACUCCAGAUUCCUGGAGGUGUUCGACAGCUUCGCCAAGGAGCUGCUC CAGGAGAUCAGCAACUCUGGUUUUACCAUAAACCAGAGCAAGACCAGGCUGCUGUACAGAGACAGCCGCCAGGAGGUCACAGGCCUCGUGGUGAAUAAGAAGAUCGGCGUGAACAGAGAGUACGUCAAGAGCACUAGGGCGAUGGCCCAGGCUCUGUACAGCACCGGCGAGUUCACCAUCAACGGCAUCCCCGGCACCAUCAAACAGCUGGAGGGCAGGUUCGGCUUCAUCGACCAG CUGGACCACUACAACAACGUGAUCGACGAUGCCAAGCACGACGCCUACUCCCUGAACGGCAGGGAGAAGCAGUUCCAGGAGUUCCUGUUCUUCAAGACAUUCUUCAACGAGUACCCCCUGGUGAUCACCGAGGGCAAGACCGACAUCAGAUACCUGAAGGCCGCCCUCAAGAGCCUGCACCAGAAGUACCCCGAGCUGAUCUGCAAGGAGGACGACGGAACCUUUCGGUUCAA GAUCAGCUUCUUCAGAAGAUCCAAGAGAUGGAAGUACUUCUGGCAUCAGCAAGGAUGGCGCUGAUGCCAUGAAGCUGCUGUACCGGUUCUUUACCGGACAGAAGGGCGUGAAGAACUACUACAGGCUGUUCGCCGAGAAGUACAAGGCCGUGCAGAGAAACCCCGUGAUCAUGCUGUUUGACAACGAGAUGGAGAGCAAGAGACCCCUCAACAAGUUCAUCUCCGAGGAGGUGAA GAUUCCCUCCUCGGAGCAGCAGCUGUUCAAGGAGCAGCUGUACUACCACCUGAUCCCCGGCAGCAAGACCUACCUGAUGACCCACCCCUUGCCUCCUGGCAAGACAGAGGCCGAGAUCGAGGACCUGUUCCCUACCGAGGUGUUGGGCGUGAAGCUCGACGGCAAGAGCUUCAGCACCAAGGACAAGUUCGACACCAGCAAGUUCUACGGCAAGGACAUCUUCAGCAGCUACGUGUA CGAGCACUGGAAGUCCAUCGACUUCAGCGGCUUCAUCCCCCUGCUGGACAAGAUCACAUGCUCGUGCAGAACGAGAAGAAGCCCGGGCUGAACACACCACCAAAGAAGAAAAGAAAGGUCUGAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGAU SEQ ID NO: 19314 R1262_nc-5 (R1262_ncRNA-EMX1_505) R1262 ncRNA contains a 505 bp insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAACUUCAGCUAAGGAAGCUACCAAUAUUUAGUUUCUGAGUCUCACGACAGACCUCGCGCGUAGAUUGCCAUGCGUAGAGCUAAC GAGCCAGCGGAAAGCGUGAGGCGCUUUUAAGCAUGGCGAGUAAGUGAUCCAACGCUUCGGAUAUGACUAUAUACUUAGGUUCGAUCUCGUCCCGAGAAUUCUAAGCCUCAACAUCUAUGAGUUAUGAGGUUAGCCGAAAAAG CACGUGGUGGCGCCCACCGACUGUUCCCAGACUGUAGCUCUUUGUUCUGUCAAGGCCCGACCUUCAUCGCGGCCGAUUCCUUCUGCGGACCAUACCGUCCUGAUACUUUGGUCAUGUUUCCGUUGUAGGAGUGAACCCACUUGCCUUUGCGUCUUAAUACCAAUGAAAAACCUAUGCACUUUGUACAGGGUACCAUCGGGAUUCUGAACCCUCAGAUAGUGGGGAUCCCGGGUAUAGACC UUUAUCUGCGGUCCAACUUAGGCAUAAACCUCCAUGCUACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19315 R1262_nc-3 (R1262_ncRNA-EMX1_305) R1262 ncRNA contains a 305 bp insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAACGACUACCAAAUCCGCAUGUUACGGGACUUCUUAUUAAUUCUUUUUUCGUGAGGAGCAGCGGAUCUUAAUGGAUGGCC GCAGGUGGUAUGGAAGCUAAUAGCGCGGGUGAGAGGGUAAUCAGCCG UGUCCACCAACACAACGCUAUCGGGCGAUUCUAUAAGAUUCCGCAUUGCGUCUACUUAUAAGAUGUCUCAACGGUAUCCGCAACUUGCGAUGUGCCUGCUAUCCUUAAAUGCAUAUCUCGCCCAGUAGCUUCCCAAUAUGAGAGCAUCAAUUGUAGAUCGGGCCGGGAUAGUCAUGUCGUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGA UGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19316 R1262_nc-1 (R1262_ncRNA-EMX1_25) R1262 ncRNA contains a 25 bp insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUC GAUGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19317 R1262_nc-4 (R1262_ncRNA-EMX1_405) R1262 ncRNA contains a 405 bp insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAACUGCUAAAUCCGCGUGAUAGGGGAUUUGAAGUUUAAUCUUCUAUCGCAAGGAACUGCCGAUCUUAAUGGAUGGCCGGAGGUGG UAUGGAAGCUAUAAGCGCGGGUGAGAGGGUAAUUAGGCGUGUUCACCUACGCUACGCUAACGGGCGAUUCUAUAAGAUUGCACAUUGCGUCA ACUCAUAAGAUGUCUCAACGGCAUGCGCAACUUGUGAAGUGUCUACUAUCCUUAAACGCAUAUCUCGCACAGUAACUCCCGAAUAUGUCCGGCAUCAUGUUGCCCGGGCCGAGUUAGUGUUGAGCUCACGGAACUUAUUGUAUGAGUAGUGAUUUGUAAGAGUUGUCAGUUAGCUCGUUCAGGUAAUAGUUGCCCACACAACGUCAAAAUAAGAGAACGGUCGUAACAUAAAGUUCCCAUC ACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19318 R1262_nc-2 (R1262_ncRNA-EMX1_205) R1262 ncRNA contains a 205 bp insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCA AGCCGAGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19319 R1262_nc-19 (R1262_ncRNA-sgEMX1_205) The R1262 ncRNA contains a 205 bp insertion in the EMX1 locus, which is combined with the EMX1 sgRNA at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCCAGUGUACCUGCAAGCCGAGAUG GCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCA CCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCAAUAACAACGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUGAU SEQ ID NO: 19320 R1262_nc-20 (R1262_sgEMX1-ncRNA_205) The R1262 ncRNA contains a 205 bp insertion in the EMX1 locus, which is combined with the EMX1 sgRNA at the 5' end GGGAUAAUGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUAAUAACAACGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGA AGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGG UCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUG GGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19321 R1262_nc-13 (R1262_ncRNA-AAVS1_25) R1262 ncRNA contains a 25 bp insertion in the AAVS1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCCUCCACCCACAGUGGGGCCACUAGGGACAGAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCG AGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19322 R1262_nc-14 (R1262_ncRNA-AAVS1_205) R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCCUCCACCCACAGUGGGGCCACUAGGGACAGAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCG AGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19323 R1262_nc-15 (R1262_ncRNA-AAVS1_505) R1262 ncRNA contains a 505 bp insertion in the AAVS1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCUCCACCCCACAGUGGGGCCACUAGGGACAGACUUCAGCUAAGGAAGCUACCAAUAUUUAGUUUCUGAGUCUCACGACAGACCUCGCGCGUAGAUUGCCAUGCGUAGAGCUAACGAGCCAGCGG AAAGCGUGAGGCGCUUUUAAGCAUGGCGAGUAAGUGAUCCAACGCUUCGGAUAUGACUAUAUACUUAGGUUCGAUCUCGUCCCGAGAAUUCUAAGCCUCAACAUCUAUGAGUUAUGAGGUUAGCCGAAAAAGCACGU GGUGGCGCCCACCGACUGUUCCCAGACUGUAGCUCUUUGUUCUGUCAAGGCCCGACCUUCAUCGCGGCCGAUUCCUUCUGCGGACCAUACCGUCCUGAUACUUUGGUCAUGUUUCCGUUGUAGGAGUGAACCCACUUGCCUUUGCGUCUUAAUACCAAUGAAAAACCUAUGCACUUUGUACAGGGUACCAUCGGGAUUCUGAACCCUCAGAUAGUGGGGAUCCCGGGUAUAGACCU UAUCUGCGGUCCAACUUAGGCAUAAACCUCCAUGCUACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19324 R1262_nc-18 (R1262_ncRNA-AAVS1-MS2_505) The R1262 ncRNA contains a 505 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCUCCACCCCACAGUGGGGCCACUAGGGACAGACUUCAGCUAAGGAAGCUACCAAUAUUUAGUUUCUGAGUCUCACGACAGACCUCGCGCGUAGAUUGCCAUGCGUAGAGCUAACGAGCCAGCGG AAAGCGUGAGGCGCUUUUAAGCAUGGCGAGUAAGUGAUCCAACGCUUCGGAUAUGACUAUAUACUUAGGUUCGAUCUCGUCCCGAGAAUUCUAAGCCUCAACAUCUAUGAGUUAUGAGGUUAGCCGAAAAAGCACGUGGUGGCGCC CACCGACUGUUCCCAGACUGUAGCUCUUUGUUCUGUCAAGGCCCGACCUUCAUCGCGGCCGAUUCCUUCCGGACCAUACCGUCCUGAUACUUUGGUCAUGUUUCCGUUGUAGGAGUGAACCCACUUGCCUUUGCGUCUUAAUACCAAUGAAAAACCUAUGCACUUUGUACAGGGUACCAUCGGGAUUCUGAACCCUCAGAUAGUGGGGAUCCCGGGUAUAGACCUUUAUCUGC GGUCCAACUUAGGCAUAAACCUCCAUGCUACAAUUAAUGACAGAAAAGCCCCAUCCUUAAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19325 R1262_nc-17 (R1262_ncRNA-AAVS1-MS2_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCCUCCACCCCACAGUGGGGGCCACUAGGGACAGAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUG UGCUUGGAGUCAAUCGCAUGUAGGAUGGUCUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19326 R1262_nc-16 (R1262_ncRNA-AAVS1-MS2_25) The R1262 ncRNA contains a 25 bp insertion in the AAVS1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCCUCCACCCAGUGGGGCCACUAGGGACAGAACUCGAGCUCUGAAUGACUCCUAUAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCCUGAUAUUGGGUCUAACCCCCAG CAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19327 R1262_nc-9 (R1262_ncRNA-EMX1-MS2_305) R1262 ncRNA contains a 305 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAACGACUACCAAAUCCGCAUGUUACGGGACUUCUUAUUAAUUCUUUUUUCGUGAGGAGCAGCGGAUCUUAAUGGAUGGCC GCAGGUGGUAUGGAAGCUAAUAGCGCGGGUGAGAGGGUAAUCAGCCGUGUCCACCA ACACAACGCUAUCGGGCGAUUCUAUAAGAUUCCGCAUUGCGUCUACUUAUAAGAUGUCUCAACGGUAUCCGCAACUUGCGAUGUGCCUGCUAUCCUUAAAUGCAUAUCUCGCCCAGUAGCUUCCCAAUGAGAGCAUCAAUUGUAGAUCGGGCCGGGAUAGUCAUGUCGUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAG CAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19328 R1262_nc-7 (R1262_ncRNA-EMX1-MS2_25) R1262 ncRNA contains a 25 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACA UCGAUGUCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19329 R1262_nc-6 (R1262_ncRNA-EMX1_P2A-GFP) R1262 ncRNA contains a P2A-GFP insertion in the EMX1 locus GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAGGAAGCGGAGCCACUAACUUCUCCCUGUUGAAACAAGCAGGGGAUGUCGAAGAGAAUCCCGGGCCAGUGAGCAAGGGCGAGGAGCU GUUCACCGGGGUGGUGCCCA UCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCU UCU UCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACA CCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCGGCAUGGACGAGCUACAAGUAGAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCG AU SEQ ID NO: 19330 R1262_nc-22 (R1262_sgAAVS1-ncRNA_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus, which is combined with the sgAAVS1 at the 5' end. GGGAUAAUGGGGCCACUAGGGACAGGAUGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUUUAAUAACAACGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCUCCC ACCCCACAGUGGGGCCACUAGGGACAGAAAGCGGUCUUACGGUCAGU CGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCCUCCUUCCUAGUCCUGAAUAUUG GGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19331 R1262_nc-8 (R1262_ncRNA-EMX1-MS2_205) R1262 ncRNA contains a 205 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAU GGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19332 R1262_nc-21 (R1262_ncRNA-sgAAVS1_205) The R1262 ncRNA contains a 205 bp insertion in the AAVS1 locus, which is combined with sgAAVS1 at the 3' end. GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUCUGUCCCCUCCACCCCACAGUGGGGGCCACUAGGGACAGAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUG GAGUCAAUCGCAUGUAGGAUGGUCUCCAGACACCGGGGCACCAGU UUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCUCCUUCCUAGUCUCCUGAUAUUGGGUCUAACCCCCAGCAUAAUGAGUUGUCACACGCUCAAUAACAACGGGGCCACUAGGGACAGGAUGUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACU UGAAAAGUGGCACCGAGUCCGGUGCUUUUUGAU SEQ ID NO: 19333 R1262_nc-10 (R1262_ncRNA-EMX1-MS2_405) R1262 ncRNA contains a 405 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAACUGCUAAAUCCGCGUGAUAGGGGAUUUGAAGUUUAAUCUUCUAUCGCAAGGAACUGCCGAUCUUAAUGGAUGGCCGGAGGUGGUA UGGAAGCUAUAAGCGCGGGUGAGAGGGUAAUUAGGCGUGUUCACCUACGCUACGCUAACGGGCGAUUCUAUAAGAUUGCACAUUGCGUCAACUCAUAAG AUGUCUCAACGGCAUGCGCAACUUGUGAAGUGUACUAUCCUUAAACGCAUAUCUCGCACAGUAACUCCCGAAUAUGUCGCAUCUGAUGUUGCCCGGGCCGAGUUAGUGUUGAGCUCACGGAACUUAUUGUAUGAGUAGUGAUUUGUAAGAGUUGUCAGUUAGCUCGUUCAGGUAAUAGUUGCCCACACAACGUCAAAAUAAGAGAACGGUCGUAACAUAAAGUUCCCCAUCACAUCA ACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19334 R1262_nc-11 (R1262_ncRNA-EMX1-MS2_505) R1262 ncRNA contains a 505 bp insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAACUUCAGCUAAGGAAGCUACCAAUAUUUAGUUUCUGAGUCUCACGACAGACCUCGCGCGUAGAUUGCCAUGCGUAGAGCUAAC GAGCCAGCGGAAAGCGUGAGGCGCUUUUAAGCAUGGCGAGUAAGUGAUCCAACGCUUCGGAUAUGACUAUACUUAGGUUCGAUCUCGUCCCGAGAAUUCUAAGCCUCAACAUCUAUGAGUUAUGAGGUUAGCCGAAAAAGCACGUGGUG GCGCCCACCGACUGUUCCCAGACUGUAGCUCUUUGUUCUGUCAAGGCCCGACCUUCAUCGCGGCCGAUUCCUUCUGCGGACCAUACCGUCCUGAUACUUUGGUCAUGUUUCCGUUGUAGGAGUGAACCCACUUGCCUUUGCGUCUUAAUACCAAUGAAAAACCUAUGCACUUUGUACAGGGUACCAUCGGGAUUCUGAACCCUCAGAUAGUGGGGAUCCCGGGUAUAGACCUUUAUCU GCGGUCCAACUUAGGCAUAAACCUCCAUGCUACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19335 R1262_nc-12 (R1262_ncRNA-EMX1-MS2_P2A-GFP) R1262 ncRNA contains a P2A-GFP insertion in the EMX1 locus and an MS2 stem loop at the 3' end GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAGGAAGCGGAGCCACUAACUUCUCCCUGUUGAAACAAGCAGGGGAUGUCGAAGAGAAUCCCGGGCCAGUGAGCAAGGGCGAGGAGCU GUUCACCGGGGUGGUGCCCAUCCU GGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCU UCAAGGACG ACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGC GAC GGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCGGCAUGGACGAGCUGUACAAGUAGAAAGUUCUCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACC CAUGUGAU SEQ ID NO: 19336 R1262_nc-23 (R1262_ncRNA-EMX1_P2A-GFP_LongHA) Same as R1262_nc-6, but with longer homology arms GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUGAGGCCCCAGUGGCUGCUCUGGGGGCCUCCUGAGUUUCUCAUCUGUGCCCCUCCCUCCCUGGCCCAGGUGAAGGGUGGUUCCAGAACCGGAGGACAAAGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGA GUCCGAGCAGAAGAAGGAAGCGGAGCCACUAACUUCUCCCUGUUGAAACAAGCAGGGGAUGUCGAA GAGAAUCCCGGGCCAGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGGGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGC ACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCAC CAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCGCCGACCA CUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCCGCCCGACAAC CACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCGGCAUGGACGAGCUGUACAAGUAGAAAGUUCUCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCCAAUGACUAGGGUGGGCAACCACAAACCCACGAGGGCAGAGUGCUGCUUGCUGCUG GCCAGGCCCCUGCGUGGGCCCAAGCGCAUAAUGAGUUGUCACACGCUCGAU SEQ ID NO: 19337 R1262_nc-24 (R1262_ncRNA-EMX1-MS2_P2A-GFP_long HA) Same as R1262_nc-12, but with longer homology arms GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUGAGGCCCCAGUGGCUGCUCUGGGGGCCUCCUGAGUUUCUCAUCUGUGCCCCUCCCUCCCUGGCCCAGGUGAAGGGUGGUUCCAGAACCGGAGGACAAAGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGU CCGAGCAGAAGAAGGAAGCGGAGCCACUAACUUCUCCCUGUUGAAACAAGCAGGGGAUGUCGAAGAGAA UCCCGGGCCAGUGAGCAAGGGCGAGGAGCUGUGUACCGGGGUGGGGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACU UCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUU CAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAAC ACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCAC CCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCGGCAUGGACGAGCUGUACAAGUAGAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCCAAUGACUAGGGUGGGCAACCACAAACCACGAGGGCAGAGUGCUGCUUGCUGCUGGCCAGGCCCCUGC GUGGGCCCAAGCGCAUAAUGAGUUGUCACACGCUCACAUGAGGAUCACCCAUGUGAU SEQ ID NO: 19338 6083 v1 RT 6083 Retrotranscript RT mRNA AUGAGCAACCCUCAGCCCACCAGGGCUGAGAUCUUCGAGCGCAUCAAACAGAGCUCGAAGCAGGAGGUGAUCCUGGAGGAGAUGCAGAGACUGGGCUUCUGGCCAAGGUCAGAAGGCCAGCCUGAGGUCGCUGCCGACCUCAUCCAGAGAGAAGGCGAGCUCCAAAGGGAGCUCGCCGAACUCAACAAGAAGUUGGCGGUCAAACGCAACCCCGAGAGGGCCCUCAGAGAAAUGAGGAAGCAGAGAAUGAAGGAC GCCAGAGACAAGAGAGAGGUGACCAAGAGAGCCCAGGCUCAGCAGAUACGACAAGGCCCUGCUGUGGCACGAGAAGAGGGCCAGCCACGUGGCCUAUCUUGGCCCUGGUGUGAGCGCCAGCUUACACGAGAACAGCUCUGCUACCCAGGAGCAGGGCGACAAGGGGAAACCCAAGAGCCCGAGAUCGGGCUGUGCCAGACCUGCAGAGGCUGACACUGAACGGGCUGCCUGCUGAUUAGCGCAG CCCAA CUCGCUGAGUCCAUGGGAGUCAGCGUGGCUGAGCUGAGAUUCCUCAGCUUCCACAGGGAGGUGGCUAGGACAAACCACUACCACAGCUUCACGCUCCCCAAGAAGACAGGUGGGGAGCGUCUGAUCAGGCCCCCAUGCCCAGACUGAAGAGGCCCAGUACUGGGUGCUGGACAACGUGCUGGCAAAGAUGCCAGCGCACGAUGCGGCUCACGGCUUCCUGGCCGGCAGAAGCAUCAUCAAGCAAUG CCAAGCCC CAUGCGGGGCAGGAUGUGGUCAUUAACUUGGACGUUAAGGACUUCUUCCCCAGCAUCGCCUUCGGCAGAAUCAAGGGCGUGUUCAGACAGCUGGGCUACGGCGAAAGCAUCGCCACAGUGUUCGCCCUGCUGGCAGCGAGAACAGAGCCCAGGCCUGGCAAGUGGACGGAGAGAGACUCUUCGUCGGCGGCAAGGCCAGAGAAAGAGUGCUGCCUCAGGGCGCUCCUACCAGCCCCAUGCCAGACCAACC UGCUG UGCCGGCGGAUGGAUAGACGUCUGCUGGGUCUCGCGAAGCAGCUGGGCUUCGUGUACACCAGAUACGCCGACGACCUGACCUUCAGCGCCUCCGGCGAACCCGCAAGGGAUAACGUCGGAAAGCUGCUGAGCAGGGUCCGUGGAUCCUGAGGGAUGAGGGGUUCACCCCUCAUCCCGAUAAGGAGAGAGUGAUGAGAAAGGGCAGAAGACAGGAGGUGACCGGCCUUGUGGUCAACUCCGACACUCCC AGCGUG AGCAGGGAGACCAGAAGAAGGCUGAGAGCCGCUCUGCACAGAGCCUCGCAGCCUGACGCUGCGAGCAAACCUGCACAUUGGCAGGGCCAUACGGCCCAGCCAAGUCAGCUGCUGGGCCUGGCCACAUUCGUGCAUCAGAUCGACCCCAAGCAGGGCAAGACCCUGCUCGCUGACGCUCAGCAACUGAUGCGCAGCCCUAUCGACCGCGCUAAUGACGCGGCGAAGUCUGCUAGCAGGGCUGACGCUGCU CAGCAG AGCUUCAGAGUGCUGGCUGCUGCCGGCAAGCCACCAGUUCUUGCCGACGGCAAGAACUGGUGGCAACCUGCUCCUCGCCACACCUGUGCCGGAGAAGACCGACCAGCAGAGAAGAGAGGAAAGGCAGGCUACCAGAAGGCAGCAGGCCGCUGCUGCUGCUCCUCCUCCUAGCAGCACAAGAAGAAACGAGCGGCCGCAACAGGCCGCUCAUGAGCAGCAGGGAGAUGCCCAGCCUCAGAACGAGGC CCCUCCU AGAUUCGACCCCGACCAGUACGCUCCUCCCCCGAGGAACGUGAUGACCUACUGGGCCCAGAUCGCCAUCAGCUUCUUUCUGGGCAGCAUCCUGCACAACAGACUGAUCACGAUCUUCGCCAUGGUGGCGGUGAUCGCUCUGUACUACAUGAGAAGACAGAGAUGGGAUGUCUUCAUGGGCAUCCUGGUGGUGGCCACCCUGCUGGGAUACCUGGUCAGGGGCAUGGGCccaccaaagaaaaaga aaggucuga SEQ ID NO: 19339 6083 v1_ncRNA_AAVS1 gRNA_25bp The R6083 retrotranscript contains a 25 bp insertion at the AAVS1 locus GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUUCUGUCCCCUCCACCCCACAGUGGGGCCACUAGGGACAGAAAUGACAGUGGUUGGUGCUCUAAAAAUUAAUGACAGAAAAGCCCCAUCCUUAGGCCUCCCUCCUUCCUAGUCUC CUGAUAUUGGGUCUAACCCCCAAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACGGGGCCACUAGGGACAGGAUguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19340 6083 v1_ncRNA_only_25bp The R6083 retrotranscript contains a 25 bp insertion at the EMX1 locus GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaag caggccaauggggaggacaucgaugucaAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGC SEQ ID NO: 19341 6083 v1_ncRNA_EMX1 gRNA_25bp The R6083 retrotranscript contains a 25 bp insertion at the EMX1 locus and sgEMX1 at the 3' end GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAAUGACUCCUAUAaaguucucccaucacaucaaccgguggcgcauugccacgaag c aggccaauggggaggacaucgaugucaAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuu uu SEQ ID NO: 19342 6083 v1_ncRNA_EMX1 gRNA_GFP gene The R6083 retrotranscript contains a GFP gene inserted at the EMX1 locus and sgEMX1 at the 3' end GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaccauauguccuuccgugagagacacaaaaaauuccaacacaccuauugcaaugaaaauaa auuuccuuuuuagccagaagucagaugcucaaggggcuucaugauguccccauaauuuuuggcagagggaaaaagaucucagugguauuugugagccagggcauuggccaccagccacccaccuucugauaggcagccugcaccugaggagugcggccgcuuuacuuguacagcucguccaugccgagagugauccc ggcggcggucacgaacuccagcaggaccaugugaucgcgcuucucguuggggucuuugcucagggcggacugggugcucagguaguguugucgggcagcagcacggggccgucgccgauggggguguucugcugguaguggucggcgagcugcacgcugccguccucgauguuguggcggaucuugaaguucaccuugaugccguucuucugcuugcggc caugauauagacguuguggcuguaguaguuguacuccagcuuggccccaggauguugccguccuccuugaagucgaugcccuucagcucgaugcgguucaccaggggugucgcccucgaacuucaccucggcgcgggucuuguaguugccgucguccuugaagaagauggugcgcuccuggacguagccuucgggcauggcggacu ugaagaagucgugcugcuucauguggucgggguagcggcugaagcacugcacgccguaggucaggguggucacgagggugggccagggcacgggcagcuugccgguggugcagaugaacuucagggucagcuugccguagguggcaucgcccucgcccucgccggacacgcugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccacccc ggugaacagcuccucgcccuugcucaccaugguggcgaccgguggaucccgggcccgcgguaccgucgacugcagaauucgaagcuugagcucgagaucugaguccgguagcgcuagcgggaucugacgguucacuaaacccuguguucuggcggcaaacccguugcgaaaaagaacguucacggcgacuacugcacuuauau acgguucucccccacccucggggaaaaaggcggagccaguacacgacaucacuuucccaguuuaccccgcgccaccuucucuaggcaccgguucaauugccgaccccuccccccaacuucucggggacugugggcgaugugcgcucugcccguucucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaAG CACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACgaguccgagcagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19343 6083 v1_ncRNA_EMX1 gRNA_50bp The R6083 retrotranscript contains a 50 bp insertion at the EMX1 locus and sgEMX1 at the 3' end GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggagggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguucucca caucaaccgguggcgca uugccacgaagcaggccaauggggaggacaucgaugucaAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagu cggugcuuuuu SEQ ID NO: 19344 6083 v1_ncRNA_EMX1 gRNA_100bp The R6083 retrotranscript contains a 100 bp insertion at the EMX1 locus and sgEMX1 at the 3' end GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUA UCAUGGGGCCCAUGGUUGAAUGACUCCUAUAaaguuc ucccaucacaucaaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuauca acuugaaaaaguggcaccgagucggugcuuuuu SEQ ID NO: 19345 6083 v1_ncRNA_EMX1 gRNA_75bp The R6083 retrotranscript contains a 75 bp insertion at the EMX1 locus and sgEMX1 at the 3' end GCUCCGGAGCAAUGAGCAGGCUCUUGCAAUCCGGGCGGUGUUUCGCCGCCCUUGUGAACUGCCGUUUCAUGCACCACGGGCGCCGUUUUCACUGUGCCACCCCAGCCACGGUAGUGCUacaaacggcagaagcuggaggaggaagggccugaguccgagcagaagaaaACUCGAGCUCUGAGCCCCACCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGA CUCCUAUAaaguucucccaucacauc aaccgguggcgcauugccacgaagcaggccaauggggaggacaucgaugucaAGCACUACCGUGGCGGGGUGGCGUCGAGCGAACAGCUCCCGUCCCCUGAGCCCUACAGGCUCUUGGACGAGAUGCACAUUGCUCCGGAGCAAUAACAACgaguccgagcagaagaagaaguuucagagcuaugcuggaaacagcauagcaaguugaaauaaggcuaguccguuaucaacuugaaa aaguggcaccgagucggugcuuuuu SEQ ID NO: 19346 Aco1_ncRNA-EMX1_100 Aco1 ncRNA contains a 100 bp insertion in the EMX1 gene GGGAUAAUGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAAAGACUCCUAUAAAGUUCUC CCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGCACGGCAAACAGACAGAUCCAUUAUUAUACAAUUUAUUUAGUGAUCGUUCCCGGUUUUAUACGAU SEQ ID NO: 19347 Aco1_ncRNA-EMX1_205 Aco1 ncRNA contains a 205 bp insertion in the EMX1 gene GGGAUAAUGUAUAAAACCGGGAACGAUCAGACCGGGGUGAAUUCGCCCCCUUGAUCAAACGGCACUAACCACUGUUUGCCGUGCGUGCGUACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUGGAGUCAAUCG CAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAACGCACGGCAAACAGACAGAUCCAUUAUUAUUACAAUUUAUUUAGUGAUCGUUCCCGGUUU UAUACGAU SEQ ID NO: 19348 Eco3_ncRNA-EMX1_205 Eco3 ncRNA contains a 205 bp insertion in the EMX1 gene GGGAUAAUUGAUAAAGAUUCCGUAAGAGCCAAACCUAGCAUUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCAUGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUUCAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGU GUACCUGCAAGCCGAG AUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUGGCUCUUACGGAAUCU UAUCAGAU SEQ ID NO: 19349 R2781_RT-1 R2781 Retrotranscript AGGAUAAUGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGGAUCCGCCACCAUGGGUUACAACUACGAGUACACCAUCAGGCUGUGUGAGACGCUCAAGUCCUUGAGCGCUGACGACGUGUACAUCAGCAAGUGCUGCAACUACGCCGAGGGACUCCUGGAUAAGGAGCUCCCUGUGAUCUUCGACCCCACCCACCUGAAGCAGAUCCUGAGGCUGGACGACAUCAGCCUCGACGAGUACCACAUCU UCUAUAUCGACAAGAAGAACGGGGGCAGCAGAGAGAUCAAUGC CCCCAGCGAGGAGCUGAAGAAGCGCCAACGCUGGAUUCUCAAGAACAUCCUUGAGAAGAUCAGCAUCAGCCACAACGUGCACGGCUUCAUCAAGGGCAAGAGCAUUGUCAGCAAUGCUCGCAAGCACCUGAACAAGGAGUAUGUGCUGAACAUCGACAUCAAGGACUUCUUCCCCAGCGUCACCAAAUACUCCGUGGAGAAGAUCUUCCGGAGAAUGGGCUACUGCAACAGCGUGGCCCAGCUGCUUGCCCGC GUCUGUUGUUACAGAGGCGGGCUGCCUCAGGGAGCUCCUACAAGCCC CUACCUGGCCAACCUCGCUUUUGACGAGGUUGACCAGGAGAUCAUCAACGUGGUGAGAAACCGGGACAUCACCUACACCAGAUACGCCGACGACAUGACCUUCAGCGCCAACUACGACCUGAGCACCUUCAAGAAGGAGGUGUACAAGAGCCUGGGCAAAUACAGAUUCAGCCCCAACAUCAUGAAGACUCACCAGAUGUCUGGCGAGAAGAGAAAGCUGGUGACCGGCCUGAUCGUGGACGACAAGG UGAAGGUGUGCAAGAAGUACAAGAGAAAGCUGCGGCAGGAGAUCUACUACUG CAAGAAGUUCGGCGUGACCAACCACCUGAGAAACUGCCACAGCGAGAAGUCCAUCAACUACAAGGAGUACCUGUACGGCAAGGCCUACUUCAUCAAGAUGGUCGAGGAGAUCGUCGGCGAAGUUCCUGGCCGACCUGGACAGCAUUGACUGGUACCCACCAAAGAAGAAAAGAAAGGUCUGACUCGAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCCAGCCCUCCUCCCCUUCCU GCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC SEQ ID NO: 19350 R2781_nc-01_25bp_49-65HA R2781 ncRNA contains a 25 bp insertion in the EMX1 gene with 49 and 69 bp homology arms GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGACAGAAAUGAAAUAAAUAGUUGCUCUU SEQ ID NO: 19351 R2781_nc-02_205bp_49-65HA R2781 ncRNA contains a 205 bp insertion in the EMX1 gene with 49 and 69 bp homology arms GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCGAGUUCCGUCCAGUUGAGCGUGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACC GGGGCACCAGUUUUCACGCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGACAGAAAUGAAAUAAAUAGUAGUUGCUCUU SEQ ID NO: 19352 R2781_nc-03_405bp_49-65HA R2781 ncRNA contains a 405 bp insertion in the EMX1 gene with 49 and 69 bp homology arms GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCACAAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAACUGCUAAAUCCGCGUGAUAGGGGAUUUGAAGUUUAAUCUUCUAUCGCAAGGAACUGCCGAUCUUAAUGGAUGGCCGGAGGUGGUAUGGAAGCUAUAAGCGCGGGUGAGAGGGUAUUAGGCGUGU UCACCUACGCUACGCUAACGGGCGAUUCUAUAAGAUUGCACAUUGCGUCAACUCAUAAGAUGUCUCAACGG CAUGCGCAACUUGUGAAGUGUCUACUAUCCUUAAACGCAUAUCUCGCACAGUAACUCCCGAAUAUGUCCGGCAUCUGAUGUUGCCCGGGCCGAGUUAGUGUUGAGCUCACGGAACUUAUUGUAUGAGUAGUGAUUUGUAAGAGUUGUCAGUUAGCUCGUUCAGGUAAUAGUUGCCCACACAACGUCAAAAUAAGAGAACGGUCGUAACAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUG CCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCAGACAGAAAUGAAAUAAAUAGUAGUUGCUCUU SEQ ID NO: 19353 R2781_nc-04_25bp_30HA R2781 ncRNA contains a 25 bp insertion in the EMX1 gene with 30 bp homology arms on each side GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAACUCGAGCUCUGAAUGACUCCUAUAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCGACAGAAAUGAAAUAAAUAGUAGUUGCUCUU SEQ ID NO: 19354 R2781_nc-05_205bp_30HA R2781 ncRNA contains a 205 bp insertion in the EMX1 gene with 30 bp homology arms on each side GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAAAAGCGGUCUUACGGUCAGUCGUAUUCCUUCUCGAGUUCCGUCCAGUUGAGCGUCACUCCCAGUGUACCUGCAAGCCGAGAUGGCUGUGCUUGGAGUCAAUCGCAUGUAGGAUGGUCCAGACACCGGGGCACCAGUUUUCAC GCCUAAAGCAUAAACGACGAGCAGUCAUGAAAGUCUUAGUACUGGACGUGCCGUUUCACAAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUGCGACAGAAAUGAAAUAAAUAGUAGUUGCUCUU SEQ ID NO: 19355 R2781_nc-06_405bp_30HA R2781 ncRNA contains a 405 bp insertion in the EMX1 gene with 30 bp homology arms on each side GGGAUAAUAAGAGCAACUAGAUUGAGGCGAUUCGCCUCCUUGGAAAAGGGUACUAAGUUUCUGUCGGAGGAAGGGCCCUGAGUCCGAGCAGAAGAACUGCUAAAUCCGCGUGAUAGGGGAUUUGAAGUUUAAUCUUCUAUCGCAAGGAACUGCCGAUCUUAAUGGAUGGCCGGAGGUGGUAUGGAAGCUAUAAGCGCGGGUGAGAGGGUAAUUAGGCGUGUUCACCUACGCUACGCUA ACGGCGAUUCUAUAAGAUUGCACAUUGCGUCAACUCAUAAGAUGU CUCAACGGCAUGCGCAACUUGUGAAGUGUCUACUAUCCUUAAACGCAUAUCUCGCACAGUAACUCCCGAAUAUGUCCGGCAUCUGAUGUUGCCCGGGCCGAGUUAGUGUUGAGCUCACGGAACUUAUUGUAUGAGUAGUGAUUUGUAAGAGUUGUCAGUUAGCUCGUUCAGGUAAUAGUUGCCCACACAACGUCAAAAUAAGAGAACGGUCGUAACAUAAAGUUCUCCCAUCACAUCAACCGGUG GCGCAUUGCGACAGAAAUGAAAUAAAUAGUAGUUGCUCUU SEQ ID NO: 19356 R1262_nc-46 (ncRNA-EMX1 site 1-deletion) R1262 ncRNA contains EMX1 deletion (del1) GAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUUAACCCUAUGUAGCCUCAGUCUUCCCAUUCAGGCUCAGCUCAGCCUGAGUGUUGAGGCCCCAGUGGCUGCUCUGGGUGGGCAACCACAAAACCCACGAGGGCAGAGUGCUGCUUGCUGCUGCAUAAUGAGUUGUCACA CGCUC SEQ ID NO: 19357 R1262_nc-47 (ncRNA-EMX1 site 2-deletion) R1262 ncRNA contains EMX1 deletion (del2) GAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUCAGAAGAAGAAGGGCUCCCAUCACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUCACCUCCAAUGACCGCAGCCUCCCAGCUGCUCUCCGUGUCUCCAAUCUCCCUUUUGUUUUGAU GCAGCAUAAUGAGUUGUCACACGCUC SEQ ID NO: 19358 R1262_nc-61 (ncRNA-EMX1 sgRNA tandem_sense)-001 The R1262 ncRNA contains a sense sequence with a tandem sgRNA target for insertion into EMX1 GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUgaguccgagcagaagaagaagggAUAGGAGUCAUUCAACCAUGGGCCCCAUGAUACUGAAAGUGCUGGCAAGCUGCGGGAGCGAGAUACAUACUUCUCGACAGUGGGGCUCAGAGCUCGAGUugaguccgagcaga agaagaagggGCAUAAUGAGUUGUCACACGCUC SEQ ID NO: 19359 R1262_nc-62 (ncRNA-EMX1 sgRNA tandem_antisense)-001 The R1262 ncRNA contains an antisense sequence with a tandem sgRNA target for insertion into EMX1 GGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUgaguccgagcagaagaagaagggAUAGGAGUCAUUCAACCAUGGGCCCCAUGAUACUGAAAGUGCUGGCAAGCUGCGGGAGCGAGAUACAUACUUCUCGACAGUGGGGCUCAGAGCUCGAGUugaguccgagcaga agaagaagggGCAUAAUGAGUUGUCACACGCUC SEQ ID NO: 19360 R1262_nc-63 (ncRNA_NHEJ_sense)-001 R1262 ncRNA contains a sense sequence to insert into EMX1 GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUAAGGAGUCAUUCAACCAUGGGCCCCAUGAUACUGAAAGUGCUGGCAAGCUGCGGGAGCGAGAUACAUACUUCUCGACAGUGGGGCUCAGAGCUCGAGUuGCAAUGAGUUGUCACACGCUC SEQ ID NO: 19361 R1262_nc-64 (ncRNA_NHEJ_antisense)-001 R1262 ncRNA contains an antisense sequence to insert into EMX1 GGGAUAAUGAGCGUGUGACAAGAUUUUGGGCUUGUGUUUCGCAAGCUUUGAUUAAAAUGGUAGAUGGAUUUGCUAUCUAUCGUCAUUUCCAGUACUGUUAUGUaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUGCAUAAUGAGUUGUCACACGCUC SEQ ID NO: 19362 R6342S_nc-63 (ncRNA_NHEJ_antisense)-001 R6342S ncRNA contains a sense sequence to insert into EMX1 GGGAUAAUCAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCGaACUCGAGCUCUGAGCCCCACUGUCGAGAAGUAUGUAUCUCGCUCCCGCAGCUUGCCAGCACUUUCAGUAUCAUGGGGCCCAUGGUUGAAUGACUCCUAUCACAACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUGACAUGAGGAUCACCCAUGU SEQ ID NO: 19363 R6342S_nc-64 (ncRNA_NHEJ_sense)-001 R6342S ncRNA contains an antisense sequence to insert into EMX1 GGGAUAAUCAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGGUGGAGAUUUGUCAAAUACACAUCAUUAGGUUGCGAUAGGAGUCAUUCAACCAUGGGCCCCAUGAUACUGAAAGUGCUGGCAAGCUGCGGGAGCGAGAUACAUACUUCUCGACAGUGGGGCUCAGAGCUCGAGUuCACAACCAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUGACAUGAGGAUCACCCAUGU SEQ ID NO: 19364

The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present disclosure, which aspects may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A is a schematic diagram depicting a naturally occurring retrotranscript obtained from the genomic DNA stage by generating msDNA chimeric microsatellite molecules. Retrotranscripts are encoded in the bacterial genome and include a noncoding RNA (ncRNA) portion and a portion encoding a specific reverse transcriptase (RT). ncRNA and RT are initially transcribed from the retrotranscript DNA as a single polycistronic message. The initial transcript is processed, resulting in the removal or separation of the transcript encoding the retrotranscript RT. The remaining transcript is ncRNA, which undergoes folding to form a secondary structure having several characteristic stem-loops and a duplex formed between the 5' and 3' regions of the ncRNA (i.e., a1/a2 duplex). The folded ncRNA is recognized by the accompanying RT, which is translated separately and provided in trans . The translated RT usually recognizes certain secondary structures in the ncRNA and binds to the RNA template downstream of the msd region. RT starts from the 2' end of the conserved guanosine (G) residue found immediately after the double-stranded RNA structure (a1/a2 region) within the ncRNA and initiates reverse transcription of the RNA toward its 5' end. A portion of the ncRNA (i.e., the msd region) serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the ncRNA segment that serves as a template, but does not degrade other parts of the ncRNA. The result of reverse transcription, msDNA (lower right of the schematic), remains covalently linked to the RNA template via a 2'-5' phosphodiester bond, and the 3' end of the msDNA is used for base pairing with the RNA template. Figure 1B is a schematic diagram depicting one embodiment of the recombinant retrotranscript considered in the present disclosure. In this embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the ncRNA region (msr/msd region) of the retrotranscript, as depicted in the upper left corner. The msr region has been modified by introducing one or more nucleotide modifications (e.g., nucleotide substitutions, deletions, or insertions). For example, it may be necessary to introduce one or more nucleotide substitutions in the msr to enhance functionality (e.g., binding of the corresponding ncRNA to RT, improved stability, improved folding, etc.). The modified msr is referred to as msr'. In addition, the msd has been modified by introducing a heterologous nucleotide sequence encoding an HDR donor template. Finally, the retroviral DNA has been modified to introduce a nucleotide sequence encoding a guide RNA at the 3' end of the retroviral DNA sequence, and the retroviral DNA is configured on a DNA vector (e.g., a plasmid). The DNA is shown to be transcribed as a polycistronic message, which includes a msr'/msd' region (forming a ncRNA) that is fused to the guide RNA at its 3' end (in other embodiments, the guide RNA may be fused to the 5' end of the retroviral ncRNA). This intermediate is shown to form a complex with a reverse transcriptase provided in trans (e.g., by means of a separate expression vector or delivered mRNA). The schematic diagram on the upper right shows the formation of a complex between the recombinant ncRNA and RT and the initiation of reverse transcription from a covalently linked conserved guanosine (G) (i.e., "activating G" or "activating guanosine") using msd RNA as a template sequence. After the template RNA sequence is reverse transcribed and degraded by RNaseH, a recombinant msDNA is formed, which includes three modifications as shown below: (a) a guide RNA attached to the 3' end of the msDNA, (b) nucleotide changes in msr', and (c) the reverse transcribed single-stranded DNA includes a region that serves as an HDR donor template. This type of recombinant msDNA may then promote a variety of genome modification applications in cells, including genome editing using RNA-guided nucleases provided to cells in trans . Figure 1C is a schematic diagram depicting the genome editing system based on recombinant retrotranscripts described herein. In the case of genome editing involving RNA-guided nucleases, the components of such systems may include (a) guide RNA provided in cis (e.g., fused to recombinant retrotranscript msDNA) and/or in trans (e.g., expressed alone in cells), (b) recombinant ncRNA (including at least a sequence encoding an HDR donor template and, optionally, a guide RNA fused to the ncRNA), (c) a reverse transcriptase, and (d) a programmable nuclease. These components are provided to cells in the form of DNA and/or RNA and/or protein by delivery means (e.g., LNP, liposomes, virus-based delivery, or passive/active transport). Once inside the cell, recombinant msDNA is formed. The msDNA and the programmable nuclease translocate to the nuclease to perform gene editing at the target DNA site, thereby generating an edited DNA target. Figure 1D provides a simplified schematic diagram of the natural life cycle of a retrotranscript. A retrotranscript typically comprises a reverse transcriptase (RT) and two non-coding continuous reverse sequences ( msr and msd ) transcribed into a single RNA, which is folded into a specific secondary structure. The conserved NAXXH motif and VTG triplet in the retrotranscript RT are indicated. RT binds to the RNA downstream of the msd locus, thereby initiating reverse transcription of the RNA template toward its 5' end with the assistance of the 2'OH group present in the conserved branch chain G residue acting as a primer. Reverse transcription stops before reaching the msr locus, and the resulting msDNA remains covalently linked to the RNA template via 2'-5' phosphodiester bonds and base pairing at the 3' end of the molecules. Figure 1E provides a detailed representation of the natural biological pathway of the retrotransposons in the cell, ending with the production of msDNA satellite molecules. This figure is parallel to Figure 1D, but more completely depicts the stages of msDNA production. (1) Depiction of the retrotransposons locus, which includes an ncRNA locus with an msr locus and an msd locus (both of which are non-coding) and a reverse transcriptase (RT) locus. The ncDNA locus and RT locus are transcribed into a single RNA transcript, which is depicted in (2). The colors representing each component in (1) run through each of stages (2) to (6). Stages (3) and (4) depict the ncRNA portion folding into a series of stem loops, wherein the 5' and 3' ends of the ncRNA form a duplex. In addition, the position of the conserved branched-chain guanosine residue with a 2'OH group is also shown. The branched-chain guanosine serves as the future start site of the reverse transcriptase. Stage (4) further shows that the transcription region encoding the reverse transcriptase is removed and translated alone to produce the reverse transcriptase. In stage (5), the reverse transcriptase binds to the folded ncRNA and starts from the primer site (i.e., the conserved branched-chain guanosine residue with a 2'OH end) and uses the msd RNA sequence as a template to polymerize single-stranded DNA (i.e., the reverse transcriptase product). Reverse transcription terminates at the msr region. The msd RNA template is removed by exonucleolytic exotomy, thereby generating a chimeric molecule comprising a msr RNA region, which is covalently linked to the ssDNA transcript by covalently linking to a conserved guanosine primer residue. A short duplex region is also formed between the 3' end of the msr RNA and the 3' end of the ssDNA reverse transcript. The complete molecule is called "msDNA". Figure 1F is a schematic diagram depicting that the genome modification system based on the recombinant retrotranscriptor disclosed herein can be implemented as (a) a cell recorder system, (b) a genome editing system, and (c) a recombinant engineering system. These uses are not intended to be limiting. Figure 1G is a schematic diagram depicting various configurations considered for the recombinant retrotranscriptor disclosed herein. (1) shows the operator structure of a wild-type retrotranscript; (2) shows the operator structure of a recombinant retrotranscript, which is configured to encode the HDR donor template in the final msDNA molecule; (3) shows (2), but is further modified to encode the guide RNA at the 3' end of the retrotranscript; (4) shows (2), but is further modified to encode the trans- guide RNA. Figure 1H is a schematic diagram that emphasizes that any suitable configuration for presenting the components of the recombinant retrotranscript-based genome modification system disclosed herein to a cell is contemplated, including the case where RT and/or programmable nuclease are provided in trans relative to the retrotranscript ncRNA. In some embodiments, RT and programmable nuclease can be provided as fusion proteins. Figure 1I depicts that RT and programmable nucleases can be provided as fusion proteins (top, middle) or separately from each other. Figure 1J depicts that nuclear localization signals can be engineered into polypeptides of the present disclosure (e.g., RNA-guided nucleases) to facilitate translocation of the nuclease into the cell in which editing occurs. Figure 1K is a schematic (not to scale) depicting an embodiment of genome editing in which a double-strand break (DSB) generated by an appropriate nuclease (such as a CRISPR/Cas effector enzyme, ZFN, TALEN, meganuclease, TnpB, IscB, or restriction enzyme (Res)) facilitates insertion of a donor or template sequence (shown here as a "marker," flanked by homologous sequences that match those sequences flanking the DSB provided on the "donor vector"). FIG. 1L depicts the ability of three different types of retrotranscripts (Eco1-R1, Eco3-R2, Eco5-R3) to insert 16 base pair insertions at a specific genome site (EMX1 gene). FIG. 1M summarizes the procedure used to evaluate the ability of retrotranscripts to produce genome insertions. FIG. 2 (SEQ ID NO: 19970) is a schematic diagram of the consensus secondary structure of the retrotranscript ncRNA msr / msd of a type IA/IIA1 retrotranscript, generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 3 (SEQ ID NO: 19971) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IB1 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present in 97% of cases, black represents a base present in 90-97% of cases, gray represents a base present in 75-90% of cases, and white represents a base present in 50-75% of cases), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 4 (SEQ ID NO: 19972) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IB2 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 5 (SEQ ID NO: 19973) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IC retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present in 97% of cases, black represents a base present in 90-97% of cases, gray represents a base present in 75-90% of cases, and white represents a base present in 50-75% of cases), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 6 (SEQ ID NO: 19974) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of other retrotranscripts of type IIA, which were generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 7 (SEQ ID NO: 19975) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIA2 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 8 (SEQ ID NO: 19976) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIA3 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 9 (SEQ ID NO: 19977) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIA4 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 10 (SEQ ID NO: 19978) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIA5 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 11 (SEQ ID NO: 19979) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIIA1 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 12 (SEQ ID NO: 19980) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIIA2 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 13 (SEQ ID NO: 19981) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of the IIIA3 retrotranscript, which were generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 14 (SEQ ID NO: 19982) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIIA4 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 15 (SEQ ID NO: 19983) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIIA5 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 16 (SEQ ID NO: 19984) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IIIunk retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 17 (SEQ ID NO: 19985) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IV retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 18 (SEQ ID NO: 19986) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type IX retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 19 (SEQ ID NO: 19987) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of V-type retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present in 97% of cases, black represents a base present in 90-97% of cases, gray represents a base present in 75-90% of cases, and white represents a base present in 50-75% of cases), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 20 (SEQ ID NO: 19988) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type VI retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 21 (SEQ ID NO: 19989) is a schematic representation of the consensus secondary structure of retrotranscript ncRNA msr/msd of type XI group 1 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 22 (SEQ ID NO: 19990) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type XI group 2 retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 23 (SEQ ID NO: 19991) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type XII retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 24 (SEQ ID NO: 19992) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type XIII retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 25 (SEQ ID NO: 19993) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of type XIV retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 26 (SEQ ID NO: 19994) is a schematic diagram of the consensus secondary structure of the retrotranscript ncRNA msr/msd of the Ec107 retrotranscript, which was generated by computational structural alignment of the ncRNA sequences from Table B as described in Example 3. The colored dots represent the probability of a base being at that position ( e.g. , a red circle indicates that the base is present 97% of the time, black indicates that the base is present 90-97% of the time, gray indicates that the base is present 75-90% of the time, and white indicates that the base is present 50-75% of the time), as opposed to a gap (no base), and the colored letters represent bases with varying degrees of conservation ( e.g. , where red indicates 97%+ conservation, black is 90%+ conservation, and gray indicates at least 75% conservation). Each highlighted base pair represents a significantly covariant base pair. Figure 27 (SEQ ID NO: 19995) is a schematic diagram of the consensus secondary structure of retrotranscript ncRNA msr/msd of outgroup A retrotranscripts generated by computational structural alignment of ncRNA sequences from Table B as described in Example 3. Colored dots represent the probability of a base being at that position ( e.g. , a red circle represents a base present 97% of the time, black represents a base present 90-97% of the time, gray represents a base present 75-90% of the time, and white represents a base present 50-75% of the time), as opposed to a gap (no base), and colored letters represent bases of varying degrees of conservation ( e.g. , where red represents 97%+ conservation, black represents 90%+ conservation, and gray represents at least 75% conservation). Each highlighted base pair represents a significant covariant base pair. Figure 28 is a germline tree of the RT sequence constructed according to Example 3. Figure 29 is a structural representation of the retrotransposon locus associated with each retrotransposon type in Figure 28. Figure 30 shows the position of some retrotransposon (EcoI, Eco3, Eco5, AcoI, RTX003_2042, RTX003_6083v1 and RTX003_6943) in the retrotransposon germline tree of Figure 28. Figure 31A is a plastid map of an exemplary retrotransposon (EcoI) tested in Examples herein. Figure 31B is a linear representation of the 5' to 3' direction of the plastid map of Figure 31A. Figure 31C is a plastid map of an exemplary retrotransposons (RTX3_6083v1) tested in the examples herein. Figure 31D is a linear representation of the 5' to 3' direction of the plastid map of Figure 31C. Figure 32 represents a plastid-based analysis for measuring precise editing and insertion and deletion of retrotransposons, as performed in the examples. In step 1, a plastid (e.g., a plasmid of Figure 31A or Figure 31C) is transfected into a human cell (e.g., HEK293t cell) engineered to express Cas9. Editing is allowed to proceed at 37°C for 72 hours. In step 2, genomic DNA is extracted from the cells and used to prepare a next generation sequencing (NGS) library for sequencing. The library is sequenced at the target site of the edit (e.g., EMX1) to generate sequence reads. In step 3, the sequencing reads are analyzed to obtain the frequency of sequence reads containing the desired edit (percentage of exact edits) and the frequency of indels at the desired edit site (percentage of indels). Figure 33 is an equivalent representation of Figure 32. Figure 34 represents a method for transfecting HEK293T cells. One day before transfection, cells are seeded in 24-well plates. An appropriate amount of plasmid and transfection reagent (e.g., Lipofectamine 3000) are mixed and transferred to the cells. After 72 hours of incubation, the genomic DNA is extracted and the target edited region is expanded into the sequencing library. Sequencing data was analyzed by CRISPResso2 and the percentage of accurate edits and indels was calculated. Figure 35 (SEQ ID NO: 19996-20003) (top to bottom) is an example of a reference sequence of the Eco3 retrotransposons at the EMX1 genome site and the desired editing results. The editing results were analyzed using the CRISPresso2 pipeline. In this example, the editing template inserted a 10 bp insertion into the EMX1 gene (TTACGTCTGC) (SEQ ID NO: 19931) and simultaneously inserted a 6 bp substitution to mutate the PAM sequence (GAAGGG>AAAGTT) (SEQ ID NO: 19954). Figure 36 shows the results of plastid-based analysis (e.g., according to Figure 33), demonstrating that the use of Eco1 retrotransposons in HEK293T cells expressing Cas9 achieves precise editing up to about 0.3% and indels as low as 40%. Plasmids encoding Eco1 RT and Eco1 ncRNA-sgRNA fusions targeting EMX1 were transfected by lipid transfection using two different amounts of Lipofectamine. Figure 37 shows the results of plastid-based analysis (e.g., according to Figure 33), demonstrating that the use of AcoI achieves precise editing up to about 0.1% and indels as low as 3%. Aco1 retrotransposons have not been experimentally verified to produce msDNA. The precise editing activity observed in this experiment strongly supports that Aco1 retrotransposons can generate msDNA in human cells. Figure 38 shows the results of a plastid-based analysis (e.g., based on Figure 33), demonstrating that precise editing up to about 0.3% and indels as low as 5% were achieved using RTX003_2042. This retrotranscript can achieve precise editing comparable to Eco1, but with significantly lower indels (10-fold). RTX003_2042 is a novel retrotranscript, and the precise editing activity observed in this experiment strongly supports that the RTX003_2042 retrotranscript may generate msDNA in human cells. Figure 39A shows the results of a plastid-based analysis, demonstrating that precise editing up to about 0.05-0.08% and indels as low as 2.5-4% were achieved using RTX003_6083v1 and 6943. Both are novel retrotranscripts, and the precise editing activity observed in this experiment strongly supports that the RTX003_6083v1 and 6943 retrotranscripts may generate msDNA in human cells. Figure 39B shows a follow-up experiment using the same analysis of Figure 39A, indicating that RTX3_6083v1 and RTX3_6943 generate 3-4 times more precise edits than Eco1, while the indels generated by these two retrotranscripts are 2-3 times lower. RTX3_2042 shows a similar frequency of precise edits as Eco1, but with greater variability than other samples. Figure 39C shows a follow-up experiment using the same analysis of Figure 39A, indicating that RTX3_6083v1 and RTX3_6943 generate 3-4 times more precise edits than Eco1, while the indels generated by these two retrotransposons are 2-3 times lower. RTX3_2042 shows a similar frequency of precise edits as Eco1, but has greater variability than other samples. Figure 39D shows the results of a plasmid-based analysis, demonstrating that precise edits as high as about 0.7% and indels as low as about 4% are achieved using RTX003_0637, RTX003_1262, and RTX003_6342 compared to empty vector and EcoI. RTX003_0637, RTX003_1262, and RTX003_6342 are novel retrotransposons, and the precise editing activity observed in this experiment strongly supports that these retrotransposons can generate msDNA in cells. Figure 39E shows the results of plastid-based analysis, demonstrating precise editing and indel generation for an array of retrotransposons including EcoI, Eco3, RTX3_2042_RT_inactive, RTX3_2042, RTX3_6083v1, RTX3_6943, RTX3_6943, RTX3_1262, RTX3_6342S, and RTX3_6342L. Compared to empty vector and EcoI as controls, accurate editing up to about 2.5% and indels as low as about 4% were achieved using EcoI, Eco3, RTX3_2042_RT_inactive, RTX3_2042, RTX3_6083v1, RTX3_6943, RTX3_6943, RTX3_1262, RTX3_6342S, and RTX3_6342L. Figure 40 shows the 2-RNA editing assay used in the examples, which uses electroporation-based delivery of two RNA components in HEK293T cells to measure the relative editing efficiency of exemplary retrotransposons. Appropriate amounts of RT mRNA and ncRNA-sgRNA fusions were mixed and electroporated into cells. After 72 hours of incubation, genomic DNA was extracted and the targeted region was expanded into the sequencing library. Sequencing data were analyzed by CRISPResso2 and precise edits and indels were calculated. Figure 41 shows the results of a 2-RNA system (RT mRNA + ncRNA-sgRNA fusion) delivered by electroporation to HEK293T cells expressing Cas9. Eco1, Eco3 and Eco5 retrotranscripts were tested. The results showed that precise editing for Eco3 (left figure) was as high as 0.4% and indels for Eco3 were as low as 10% (right figure). Eco3-mediated precise editing increased with increasing amounts of ncRNA-sgRNA fusions, with a ratio between RT mRNA and ncRNA-sgRNA fusions of 1:2 to 1:4. Figure 42 shows the titration results of a two-component Eco3 RNA system (RT mRNA + ncRNA-sgRNA fusion) delivered to 293T cells expressing Cas9 by electroporation. RT mRNA and ncRNA were mixed at ratios of 1:2, 1:3, 1:4, 1:5, 1:8, and 1:10, respectively, and two different amounts of RT mRNA (0.2 or 0.5 µg) were delivered. The left data show that 0.5 µg Eco3 produced the highest percentage of accurate edits at 1:3 and 1:5 RT mRNA:ncRNA ratios. The right data further show that the more equal the RT mRNA:ncRNA ratio, the lower the trend of indel percentage. Figure 43 represents a three-RNA retrotranscript editing system involving the delivery of three RNA components (RT mRNA, retrotranscript ncRNA-sgRNA fusion, and Cas9 mRNA) to HEK293T cells by electroporation. Appropriate amounts of RT mRNA, ncRNA-sgRNA fusion, and Cas9 mRNA were mixed and electroporated into cells. After 72 hours of incubation, genomic DNA was extracted and the targeted region was expanded into a sequencing library. Sequencing data was analyzed by CRISPResso2 and precise edits and indels were calculated. Figure 44 shows the Cas9 mRNA titration results of the three-component Eco3 RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA) delivered to 293T cells by electroporation. RT mRNA and ncRNA-sgRNA fusion were mixed in the amounts given in the figure, and the amount of Cas9 mRNA was titrated. At 0.2 µg Cas9 mRNA, precise edits of up to 0.1% were observed. Although the editing efficiency was orders of magnitude lower than the 2-RNA system, the editing occurred through the specific action of Cas9 and the retrotransposons, as the absence of either abolished the editing. Figure 45 depicts the lipofection process in HEK293T cells using the three-RNA system. One day before transfection, cells were seeded in 96-well plates. Appropriate amounts of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA and Lipofectamine reagent were mixed and transferred to the cells. After 72 hours of incubation, the genomic DNA was extracted and the targeted region was expanded into a sequencing library. Sequencing data were analyzed by CRISPResso2 and precise edits and indels were calculated. Figure 46 shows the results of a three-component Aco1 RNA system (RT mRNA + ncRNA + Cas9 mRNA) delivered to HEK293T cells by lipofection. RT mRNA, ncRNA, and Cas9 mRNA were mixed and transfected into HEK293T cells in the amounts indicated in the figure. 56 bp insertions and 6 bp deletions at the EMX1 locus were scored as precise edits, and approximately 0.1% of the cell population in the left figure had undergone precise edits. Editing is dependent on the Cas9 nuclease, as its absence cancels editing. The frequency of indels in the right figure is approximately 1.5%. Figure 47 shows the results of minimal Cas9 nuclease activity when sgRNA is fused to the ncRNA of the Eco3 retrotransposons. Cas9 activity was assessed by indel frequency. 1 µg of ncRNA-sgRNA fusion showed 20-fold lower activity than the sgRNA isolated alone at the same mole. At the same time, the activity of chemically modified to unmodified sgRNA was compared and under the conditions described in the figure, the former showed 6-fold higher activity than the latter. Figure 48 shows the whole RNA editing analysis used in the examples to measure the relative editing efficiency of sample retrotransposons in whole RNA form, which were modified with a trans- guide RNA addition step. Electroporation was performed in HEK293T cells using the three-RNA system + sgRNA trans-addition. Appropriate amounts of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA and sgRNA were mixed and electroporated into cells. After 72 hours of incubation, genomic DNA was extracted and the targeted region was expanded into the sequencing library. Sequencing data were analyzed by CRISPResso2 and precise edits and indels were calculated. Figure 49 shows the results of guide RNA addition in the full RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA + sgRNA) delivered to HEK293T cells by electroporation. At the given amount of Cas9 and RT mRNA in the figure, the amount of guide RNA addition was titrated at 50, 100 and 200 ng. Titration was performed at two different RT mRNA: ncRNA-sgRNA fusion ratios = 1:6 or 1:8. Guide RNA addition in the all-RNA system increases precise editing by up to about 50-fold. Increasing the amount of guide RNA gradually increases precise editing and the effect of 1:8 RT mRNA:ncRNA-sgRNA fusion is slightly better than 1:6, achieving 13% precise editing. The right figure shows the insertion and deletion frequency under various conditions. Figure 50 shows the lipid transfection process using the three-RNA system + gRNA trans-addition in HEK293T cells. The day before transfection, cells were inoculated in 96-well plates. The appropriate amount of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA, sgRNA and Lipofectamine reagent were mixed and transferred to the cells. After 72 hours of incubation, the genomic DNA was extracted and the targeted region was expanded to the sequencing library. Sequencing data were analyzed by CRISPResso2 and precise edits and indels were calculated. Figure 51 shows the results of guide RNA addition in the whole RNA system (RT mRNA + ncRNA-sgRNA fusion + Cas9 mRNA + sgRNA) delivered to HEK293T cells by lipid transfection (i.e., a single molecular cluster of guide RNA was delivered. At the given amount of RT mRNA, ncRNA-sgRNA fusion and Cas9 mRNA in the figure, at 2, 5 and 10 The amount of guide RNA added was titrated at 0, 2, 5, 10, 50 and 100 ng. The addition of guide RNA in the all-RNA system increased precise editing by up to 3.5-fold and the efficiency by 12%. Increasing the amount of guide RNA within this range did not further increase precise editing. Precise editing is completely dependent on the presence of the retrotranscriptome machinery. The right graph shows the insertion and deletion frequencies under the respective conditions. Figure 52 shows the results of ncRNA-sgRNA fusion separation in the all-RNA system (RT mRNA + Cas9 mRNA + ncRNA-sgRNA fusion or ncRNA + sgRNA alone) delivered to HEK293T cells by lipid transfection. At the given amounts of RT mRNA and Cas9 mRNA in the figure, the amount of guide RNA added was titrated at 0, 2, 5, 10, 50 and 100 ng. At 10 ng guide RNA, accurate editing peaked at 2.23%, compared to 1.78% for ncRNA-sgRNA fusions. Increasing the amount of guide RNA within this range did not further increase accurate editing. The right panel shows the indel frequency under each condition. Figure 53 is a graph used for in vitro transcription to generate ncRNA Schematic diagram of improved templates (left side or A) and ncRNA modifications (right side or B). (A) is about optimizing RNA production by in vitro transcription. Previous in vitro transcription experiments to produce RNA used double-stranded DNA templates containing a 3' overhang (on the same strand as the T7 promoter sequence). New templates with blunt ends were designed and tested and resulted in increased precision editing efficiency as shown in Figure 54. (B) is about modified ncRNA, which was modified by adding an MS2 stem loop hairpin to the 3' end of the ncRNA. Without being bound by theory, the MS2 loop helps stabilize the ncRNA and results in significantly improved precision editing efficiency as shown in Figure 54. Figure 54 shows the optimization of RNA production by Lipofectamine Results of ncRNA-sgRNA fusion isolation in a 4-component total RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by MessengerMAX. Total RNA was transfected at fixed amounts as shown. Using RNA generated from a linearized plastid template containing a 3' overhang, 1.35% precise edits were produced. Using RNA generated from a modified linearized plastid template containing blunt ends increased precise edits to 5.94%. Adding an MS2 stem loop to the 3' end (blunt end) of the ncRNA further increased precise edits to 12.39%. The right panel shows the indel frequencies under the respective conditions. Figure 55 (SEQ ID NO: 19969) provides a schematic diagram of end protection of RNA from cellular nuclease activity by capping and tailing. In (A), 7-methylguanosine cap0 is added to the 5' triphosphate of the RNA. In (B), a poly A tail is added to the 3' end by enzymatic addition. The tail length is estimated to be over 50 nucleotides. In (C), RNA containing both a 5' cap and a 3' tail is shown. The results are shown in Figure 56. Figure 56 shows the results of end protection of ncRNA-sgRNA fusions by caps and tails in a 4-component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected with fixed amounts of RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. The ncRNA-gRNA fusions were capped (+cap-tail) or poly-A-tailed (-cap+tail), or both capped and poly-A-tailed (+cap+tail). Using RNA without end protection (-cap-tail) resulted in approximately 4.5% precise editing, and editing was retrotranscript-dependent, as RT deficiency abolished precise editing. Using RNA with either or both cap and tail protection resulted in lower precise editing (left panel), but reduced indels (right panel), compared to the case without cap and tail. Figure 57 shows the results that shortening the msd stem can regulate precise editing in a retrotranscript-specific manner. The modified retrotranscripts include RTX3_4536 (long (L) and short (S) forms), RTX3_6279 (long (L) and short (S) forms), RTX3_6342 (long (L) and short (S) forms), RTX3_6438 (long (L) and short (S) forms), RTX3_6549 (long (L) and short (S) forms) and RTX3_6605 (long (L) and short (S) forms). Figure 58 shows that shortening the msd stem can regulate the results of precise editing in a retrotranscript-specific manner. The modified retrotranscripts include RTX3_5752 (long (L) and short (S) forms), RTX3_6221 (long (L) and short (S) forms) and RTX3_6034 (long (L) and short (S) forms). Figure 59A demonstrates that four retrotransposons from multiple evolutionary branches can insert up to 100 bp into the EMX1 locus. The figure shows the test results of different template lengths of four different retrotransposons in a total RNA system delivered to HEK293T cells by lipofection. Under a given amount of RT mRNA and Cas9 mRNA, ncRNA-sgRNA fusions with different template lengths ranging from 10 to 100 bp were added. For 100 bp insertion, the precise editing of Eco3 was 1.04%, the precise editing of Aco1 was 1.52%, the precise editing of R2042 was 1.37%, and the precise editing of R6943 was 0.05%. In the bottom row of each figure, the insertion and deletion frequencies under the respective conditions are shown. Figure 59B demonstrates that four retrotransposons from multiple evolutionary branches can insert up to 100 bp into the EMX1 locus. The figure shows the indel results corresponding to the constructs of Figure 59A, illustrating the indel frequencies under individual conditions. Figure 60A demonstrates that additional sgRNAs targeting the same genome locus increase indel and insertion frequencies. This figure reports the same conditions as Figures 59A-59B, but with the addition of sgRNAs, which increase the frequency of precise editing and indels. For 100 bp insertions, the precise editing of Eco3 was 1.82%, the precise editing of Aco1 was 4.50%, the precise editing of R2042 was 4.40%, and the precise editing of R6943 was 0.38%. In the bottom row of each figure, the insertion and deletion frequency under each condition is shown. Figure 60B proves that additional sgRNA targeting the same genome locus will increase the insertion and deletion and insertion frequency. This figure reports the insertion and deletion frequency associated with the retrotransposons of Figure 60A. Figure 61A is a schematic diagram showing unedited alleles and alleles edited by inserting the GFP gene at the EMX1 locus using the retrotransposons editing system. The primers for EMX1 forward and reverse only cross over the 5' and 3' homologous arms and extend 169 bp on the unedited allele. On the edited allele, it extends 1433 bp across the GFP gene insertion. The primers for detecting 5' and 3' junctions are indicated on the edited allele. The 5' junction on the edited allele was amplified by the following primer pair: EMX1 forward and 5' junction GFP reverse, and the 3' junction was amplified by the following primer pair: 3' junction GFP forward and EMX1 reverse. Neither the 5' junction GFP reverse primer nor the 3' junction GFP forward primer binds to the unedited allele. Figure 61B demonstrates that three retrotransposons from multiple evolutionary branches are capable of GFP integration at the EMX1 locus. The figure provides qPCR data depicting the insertion of GFP at the 5' or 3' junction of the three retrotransposons in the EMX1 locus. The ncRNA-sgRNA engineered to contain the GFP gene insert was transfected into HEK293T cells with/without additional sgRNAs, together with the RT mRNA and Cas9 mRNA of each retrotransposon. The genomic DNA was analyzed by the indicated primer pairs to detect the GFP gene insertion at the 5' or 3' junction on the edited allele. The ΔCt was obtained by subtracting the Ct value of the +RT sample from the Ct value of the -RT sample for relative quantification of GFP insertion at the EMX1 locus. The larger the ΔCt value, the higher the insertion frequency in the indicated sample. For all three retrotransposons, 5' and 3'-joined amplicon were detected to be significantly higher than the background level. The 3'-joined amplicon is more abundant than the 5'-joined amplicon, probably because the 5' end of the msDNA produced by retrotransposition is more enriched, which promotes the insertion at the 3'. Figure 61C proves that the two retrotransposons are able to integrate GFP at the EMX1 locus. The figure provides qPCR data depicting the insertion of GFP at the 5' or 3' junction in the EMX1 locus by two retrotranscripts. ncRNA-sgRNAs engineered to contain GFP gene inserts were transfected into HEK293T cells along with the RT mRNA and Cas9 mRNA of each retrotranscript with/without additional sgRNA. Genomic DNA was analyzed by the primer pairs indicated in the table to detect GFP gene insertion at the 5' or 3' junction on the edited allele. ΔCt was obtained by subtracting the junction Ct value from the EMX1 Ct value for relative quantification of GFP insertion at the EMX1 locus. For all two retrotranscripts, amplicon at the 5' and 3' junctions was detected significantly above background levels. The amplicon at the 3' junction is slightly more abundant than the amplicon at the 5' junction, probably because the 5' end of the msDNA generated by reverse transcription is more enriched, promoting insertion at the 3'. Figure 61D shows that the PCR product of Aco1 was run on an Agilent Tapestation high sensitivity D1000 screentape. In the presence of RT (+RT sample, left side of gel), amplicon of the expected size was detected at both the 5' and 3' junctions of the edited allele, as shown by the arrows. In the absence of RT (-RT sample, right side of gel), the edited allele was not detected. The triangle indicates the expected amplicon of the unedited allele, which is present in both -RT and +RT samples. Figure 62A demonstrates that sgRNA and ncRNA sequences in fusion RNAs where the sgRNA is located upstream of the ncRNA are more active in terms of precise editing and indels. Results of testing different ncRNA-sgRNA fusion sequences of three different retrotransposons in a whole RNA system delivered to HEK293T cells by lipofection. At a given amount of RT mRNA and Cas9 mRNA, ncRNA fusions with different sequences were added. For Eco3, Aco1, and R2042, the increase in precise editing was higher when sgRNA-ncRNA was used compared to ncRNA-sgRNA. In the bottom row of each figure, the indel frequency under the respective conditions is shown. Figure 62B demonstrates that in the case where the sgRNA is located downstream of the ncRNA, the additional sgRNA compensates for the lower editing activity of the configuration. Same as the previous figure, but with the addition of sgRNA. The difference between ncRNA-sgRNA and sgRNA-ncRNA in the presence of additional sgRNA depends on each retrotranscript. For Eco3 and R2042, sgRNA-ncRNA is more effective than ncRNA-sgRNA. For Aco1, ncRNA-sgRNA is slightly more effective than sgRNA-ncRNA. In the bottom row of each figure, the insertion and deletion frequency under the respective conditions is shown. Figure 63 demonstrates that RT and Cas9 are required for precise editing. Test results of negative controls in a whole RNA system delivered to HEK293T cells by lipid transfection. RT and Cas9 are required for precise editing. When any of these components is removed, precise editing cannot be detected or is at background levels. In the bottom graph, the insertion and deletion frequencies under the respective conditions are shown. Figure 64 demonstrates that the separation of ncRNA and gRNA does not lead to a decrease in the frequency of precise editing. Results of testing ncRNA-sgRNA fusions of four different retrotranscripts against single ncRNAs in a whole RNA system delivered to HEK293T cells by lipofection. ncRNA/ncRNA-sgRNA/sgRNA-ncRNA were added with a given amount of RT mRNA, Cas9 mRNA, and additional sgRNA as described below the figure. All inserted templates tested were 25 bp. The optimal ncRNA format depends on each retrotranscript. For Eco3, the highest precise editing (9.88%) was observed when using single ncRNA + additional sgRNA. For Aco1, the highest precise editing was observed when ncRNA-sgRNA + additional sgRNA was used (2.96%). For R2042, the highest precise editing was observed when ncRNA alone + additional sgRNA was used (7.4%). For R6943, the highest precise editing was observed when ncRNA-sgRNA fusion + additional sgRNA was used (0.39%). In the bottom row of each figure, the indel frequency under each condition is shown. Figure 65 demonstrates that retrotranscripts can achieve precise editing at the AAVS1 locus. Test results of four different retrotranscripts inserting a 25 bp template into the AAVS1 locus. Retrotranscripts were delivered as whole RNA to HEK293T cells by lipofection. For all four retrotranscripts, the most precise editing was observed in the presence of additional sgRNA and ranged from 0.73% to 2.05%, depending on the retrotranscript used. On the right, the indel frequencies under the respective conditions are shown. Figure 66 demonstrates that retrotranscripts can achieve precise editing at the AAVS1 locus. Negative control results for the AAVS1 experiment in the previous figure. Removing RT, ncRNA-sgRNA, or Cas9 mRNA abolishes precise editing. In the bottom row of each figure, the indel frequencies under the respective conditions are shown. Figure 67 demonstrates that templates of either the target strand or the non-target strand can be used for precise editing. Results of testing templates on different strands of two different retrotransposons versus the Cas9 sgRNA target strand in an all-RNA system delivered to HEK293T cells by lipofection. ncRNA-sgRNA was added at given amounts of RT mRNA, Cas9 mRNA, and additional sgRNA. All insert templates tested were 25 bp. All previous figures encode insert templates on the same strand targeted by the Cas9 sgRNA, indicated by the letter T. For Eco3, in the presence of additional sgRNA, precise editing was 1.67% when the template was on the target strand and 0.85% when the template was on the non-target strand. For Aco1, in the presence of additional sgRNA, precise editing was 2.96% when the template was on the target strand and 3.40% when the template was on the non-target strand. In the bottom row of each figure, the indel frequency under each condition is shown. Figure 68 demonstrates that retrotranscript-mediated precise editing is observed using Cas9 nickase in the 4-component all-RNA system. Cas9 nickase activity in the 4-component all-RNA system (RT mRNA + Cas9 mRNA + ncRNA/ncRNA-sgRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected with fixed amounts of RT mRNA 100 ng, ncRNA/ncRNA-sgRNA 400 ng, Cas9 variant mRNA 100 ng, and sgRNA 5 ng. Isolated ncRNA or ncRNA-sgRNA fusion was used. When ncRNA or ncRNA-sgRNA was used with additional sgRNA, the precise editing of Cas9 WT (wild type) was about 9%. Using the Cas9 H480A mutant that nicks the non-target strand, almost no activity above background was seen under any condition. Using the Cas9 D10A mutant that nicks the target strand, precise editing was observed at a frequency of about 0.1% in both isolated and fused ncRNA and sgRNA formats. The right panel shows the indels under the respective conditions of the left panel. As expected, indels were extremely low for any nickase in the presence or absence of additional sgRNAs, and only WT Cas9 generated significant indels. Figure 69 demonstrates that precise retrotranscript-mediated editing observed by Cas9 nickase depends on each component of the all-RNA system. Same as the previous figure, but negative controls were tested that lacked one component of the all-RNA system with or without additional sgRNAs (Cas9 or ncRNA/ncRNA-sgRNA or RT). In the top figure, all control conditions tested showed background precise editing activity, indicating that precise editing achieved by Cas9 WT or Cas9 nickase depends on each component of the all-RNA system. In the bottom figure, significant indels were detected when only Cas9 WT was used. Figure 70 demonstrates that dual sgRNAs increase precise editing but not indels. The figure shows the results of testing dual sgRNAs of R2042 in a whole RNA system delivered to HEK293T cells by lipofection. Additional sgRNAs were added at given amounts of RT mRNA, Cas9 mRNA, and ncRNA-sgRNA. Under all conditions, the same ncRNA-sgRNA was used, whose template encoded a 25 bp insertion on the non-target strand of the Cas9 sgRNA. When one additional sgRNA (#1) was used, precise editing was 0.27%, which increased to 1.33% (#2) or 1.13% (#3) in the presence of a second sgRNA. In the bottom figure, the indel frequency under the respective conditions is shown. See Example 9. FIG. 71 Precise editing and indel results in HEK293T cells administered by lipid transfection of the R6083-based all-RNA retrotranscript editing system in various configurations. Configurations included testing of different template lengths (25 bp – 100 bp) using fusion ncRNA-sgRNA constructs (with and with spiked sgRNA) as well as separated ncRNA and sgRNA constructs. With given amounts of RT mRNA and Cas9 mRNA, ncRNA-sgRNA fusions with different template lengths ranging from 25 to 100 bp or ncRNA with a 25 bp insertion were added. Precise insertion of 100 bp was observed with an efficiency of approximately 2%. Separated ncRNAs showed lower activity than ncRNA-sgRNA fusions with a 25 bp insertion. Precise editing depends on the presence of RT and ncRNA, as shown in the top right figure. In the bottom row of each figure, the insertion and deletion frequencies under the respective conditions are shown. Figure 72 Results of testing the R6083 retrotranscript to insert a 25 bp template into the AAVS1 locus in HEK293T cells. The retrotranscript (ncRNA-sgRNA fusion), retrotranscript RT and Cas9 components were delivered to HEK293T cells as whole RNA by lipofection. Using additional sgRNA, an editing efficiency of 3% was observed, and the activity was completely dependent on the presence of RT or ncRNA. On the right, the insertion and deletion frequencies under the respective conditions are shown. Figure 73 demonstrates that the separation of Cas9 and RT enzymes shows higher editing efficiency than when they are fused to Eco3 in retrotranscript editing. Results of precise editing of Eco3 against Cas9 and RT fusions in an all-RNA system delivered to HEK293T cells by Lipofectamine MessengerMAX. Cas9 or RT mRNAs were isolated at the same molar concentration as the Cas9-RT fusion format, and the editing efficiency of the fused or isolated enzymes was compared against Eco3 ncRNA (left side on the left figure) or Eco3 ncRNA-sgRNA fusion (right side on the right figure). When both ncRNA and ncRNA-sgRNA were used, isolated Cas9 and RT achieved higher editing efficiency than when they were fused. The right figure shows the indels under the respective conditions of the left figure. Figure 74 demonstrates that both cap and tail modifications of the ncRNA significantly increased the editing efficiency by about 2-fold. Results of end protection of ncRNA-sgRNA fusions by capping and tailing in a 4-component total RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected with fixed amounts of RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusions were capped (+cap–tailing) or poly-A-tailed (-cap+tailing), or both capped and poly-A-tailed (+cap+tailing). Using RNA without end protection (-cap–tailing) resulted in approximately 4.5% precise editing, and editing was dependent on the retrotranscript, as RT deficiency abolished precise editing. Although capping alone does not change editing efficiency, tailing alone slightly increases editing, and both capping and tailing significantly increase editing efficiency by about 2-fold (left figure). In the right figure, indels under all conditions are comparable. Figure 75 Schematic diagram of ncRNA circularization, as demonstrated in Figure 76, ncRNA circularization increases editing efficiency. Step 1. The ncRNA is flanked by internal homologous sequences, introns, and another homologous sequence toward both ends. Step 2. The two homologous sequences bring the two ends closer. Exogenous GTP initiates a series of transesterification Step 3. Introns are removed and the ncRNA is joined into a circular form. FIG. 76 Results of testing for precise editing of modified ncRNAs (3'MS2 modified and circular ncRNA) in a 4-component total RNA system (RT mRNA + Cas9 mRNA + ncRNA + sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. Total RNA was transfected with fixed amounts of 100 ng RT mRNA, 400 ng ncRNA, 100 ng Cas9 mRNA, and 5 ng sgRNA. When the MS2 stem loop was added to the 3' end of the ncRNA, the activity was almost doubled relative to the unmodified ncRNA (approximately 8%), with an efficiency of 15%. Circularization of the ncRNA achieved a further increase in efficiency, resulting in an editing efficiency of 22%. Circularization of the ncRNA was performed as described in FIG. 75. Without sgRNA, precise editing and indels cannot be detected as expected. The right figure shows indels under the respective conditions of the left figure. The results of Figure 77 show that the pairing between RT and its homologous ncRNA is required for precise editing. The pairing results of RT and ncRNA from the same or different retrotransposons are used for precise editing of the EMX1 locus in HEK293T cells by the all-RNA system. All RNA was transfected with a fixed amount of RT mRNA 100 ng, ncRNA 400 ng, Cas9 mRNA 100 ng and sgRNA 5 ng. In the left figure, Aco1 RT is paired with Eco3 ncRNA or its homologous Aco1 ncRNA (left figure). Eco3 RT is paired with Aco1 ncRNA or its homologous Eco3 ncRNA (right figure). The system supports precise editing only when the RT is paired with its cognate ncRNA. The right figure shows the indels under the respective conditions of the left figure. Figure 78 Retrotranscript editing supports the insertion of exon-sized long inserts up to 305 bp. Test results of exon-sized long inserts at the EMX1 locus using Aco1, Eco3 and R1262 retrotranscripts. Aco1 achieved precise insertions of 10, 100 and 205 bp with 8, 4 and 1.3% (left figure). Eco3 performed precise insertions of 10 and 205 bp with 12 and 2.3%, respectively. The novel retrotranscript R1262 obtained 25, 205 and 305 bp insertions with efficiencies of 20, 15 and 8.8%. The bottom figure shows the indels under the respective conditions of the top figure. Figure 79 demonstrates that the novel retrotranscript R1262 inserts a 205 bp insertion at AAVS1 with over 11% efficiency. Test results of a 205 bp insertion at the AAVS1 locus using the novel retrotranscript R1262. R1262 obtained 25 and 205 bp insertions with 25% and 11.1% efficiency, respectively. As a control, accurate editing depends on the presence of RT and sgRNA. The right figure shows the indels under the respective conditions of the left figure. Figure 80 Optimization of the RT: ncRNA ratio to install 205-305 bp insertions with accurate editing by R1262. Test results of the RT: ncRNA ratio for >200 bp long insertions at the EMX1 locus using the novel retrotranscript R1262. RT: ncRNA ratios were tested at 1:12.5, 1:16.7, and 1:25 molar ratios. The 1:16.7 ratio corresponds to the 1:4 mass ratio used in the rest of the study. When using ncRNA (right side in the left figure), the best editing was observed at a 1:12.5 ratio for 205 and 305 bp insertions. When using ncRNA-sgRNA fusions (left side in the left figure), all ratios tested produced comparable edits, but a slight trend of increased editing was seen as the amount of ncRNA-sgRNA increased. The right figure shows the indels under the respective conditions of the left figure. Figure 81 Double-stranded DNA made based on NHEJ insertion of the retrotransposon at target site A. Some retrotranscripts may use msR-msD hybrids as primers (e.g., Sen1) to generate double-stranded DNA. The desired sequence inserted into the genome by the flanking guide RNA recognition sequence is integrated into the msD of the ncRNA. The two ends of the double-stranded DNA generated by reverse transcription are trimmed by the nuclease-guide RNA complex and inserted into the target site cleaved by the nuclease-guide RNA complex. B. ncRNA is designed to contain a sequence inserted into the target site by the flanking guide RNA recognition sequence as in A. The second ncRNA includes the reverse complementary sequence of the first ncRNA so that the two form a duplex. This type of double-stranded DNA is trimmed by the nuclease-guide RNA complex and inserted into the target site cleaved by the nuclease-guide RNA complex. Figure 82A Non-coding RNA optimization A. There are multiple structural components in the retrotranscript ncRNA, 1. a1/a2 inverted repeat sequence 2. msR stem loop 3. msD stem loop 4. msR DNA. Fine-tuning the strength of these structural elements by changing the GC content, length and number of the stem loop may enhance msD DNA production and subsequent gene editing. Figure 82B Non-coding RNA optimization B. Separation of msR RT primer and msD RT template allows chemical modification of msR RT primer. In combination with end protection of msD RT template by cap and tail, the stability of ncRNA may be enhanced. The use of reverse complementary sequences at the 3' end of the RT primer and the 5' end of the msD RT template (depicted in dotted lines) will stabilize the primer/template complex. Figure 83A provides a schematic outline of a modified retrotranscript gene editing system comprising engineered variant reverse transcriptases (RTs) with improved fidelity and/or processability. The engineered RTs are generated by introducing selective amino acid substitutions at residue positions in the retrotranscript RT sequence that are heterologous to those altered residues in the murine leukemia virus (MMLV) RT, thereby improving the fidelity and processability of the MMLV RT. In step (a), a structural alignment is constructed between the retrotranscript RT and an engineered MMLV RT having one or more amino acid substitutions associated with improved fidelity and processability in the context of the MMLV RT. In step (b), the alignment is checked to identify heterologous homologous amino acid residues in the retrotranscript RT that correspond to the substituted MMLV RT amino acid. Once identified, conventional recombinant engineering methods are used to construct an engineered retrotranscript RT comprising one or more corresponding heterologous homologous amino acid substitutions. In step (c), unnecessary residues may be deleted as appropriate and based on the structural alignment information. In one embodiment, the retrotranscript gene editing system may include ncRNA, a guide, a programmable nuclease (or an RNA encoding it), and an engineered RT (or an RNA encoding it) in (d). In step (e), the components (which may be in the form of whole RNA) are delivered to the cell (e.g., by LNP delivery), which results in (f) targeted single-strand (in the case of nickase) or double-strand cleavage, followed by (g) targeted repair at the cleavage site by the reverse transcriptase of the ncRNA. This results in (h) DNA with precise editing. Figure 83B provides an overview of the mutations installed in RTX3_1262 RT and RTX3_6242 and Eco1 RT, which are heterologous to known beneficial amino acid substitutions reported in the literature for MMLV. In addition, the table provides reported phenotypic changes associated with mutations and mutant domains in MMLV. Figure 83C provides a structural alignment between RTX3-6342 RT (yellow) and MMLV-RT (protein database 4MH8) (green), which shows that the N-terminus of RTX3-6342 RT can be truncated, for example at the proposed truncation point. Figure 83D shows the three-dimensional structure of MMLV RT near Q190 and the corresponding orthogonal position Q161 in Eco1. In the top picture, glutamine is conserved in both MMLV and Eco1. The middle picture shows that the Q161 position in Eco1 is involved in nucleic acid contacts. The bottom shows that the engineered MMLV variant with the Q190F mutation increases fidelity by about 2 times. The corresponding substitution at Q161 in Eco1 (i.e., the Q161F mutation) can also lead to increased fidelity of Eco1 RT. Figure 83E Mutations that improve processability in MMLV RT and Eco1. The three-dimensional structure of E302 of MMLV RT and the corresponding residue G283 in Eco1 is provided, both of which appear to be involved in binding to nucleic acids. Residues in the nucleic acid binding domain can be mutated to force stronger interactions. MMLV E302R generates a positive charge to interact with negatively charged templates. Eco1 G283 is in a conserved DNA binding alpha helix. Mutating G283 and heterologous retrotranscript residues to positively charged amino acids can improve processability. Figure 83F Mutations that improve processability in MMLV RT and Eco1. Provided are the three-dimensional structures of T306 of MMLV RT and the corresponding residue F287 in Eco1, both of which appear to be involved in binding to nucleic acids. Residues in nucleic acid binding domains can be mutated to force stronger interactions. MMLV T306K generates a positive charge to interact with negatively charged templates. Eco1 F287 is in a conserved DNA binding alpha helix. Mutating F287 and heterologous retrotranscript residues to positively charged amino acids can improve processability. Figure 84 describes the preparation of variant retrotranscriptor RTs by saturation mutagenesis induction of targeted domains involved in processability and fidelity (e.g., palm, finger, and thumb domains). Engineered RTs are generated by inducing saturation mutations into the palm, finger, and thumb domains in the retrotranscript RT, which domains are identified by structural alignment with the MMLV RT. In step (a), a structural alignment is constructed between the retrotranscript RT and the engineered MMLV RT, which has one or more amino acid substitutions associated with improved fidelity and processability in the context of the MMLV RT, particularly in the palm, finger, and thumb domains. In step (b), the palm, finger, and thumb domains are identified. In step (c), mutation induction is performed on the palm, finger and/or thumb domains using a saturation mutation induction method, and the resulting mutants are screened in an activity assay to select one or more lead variants with increased processibility and/or fidelity. In step (e), a component including the lead retrotranscript RT variant (which may be in the form of whole RNA) is delivered to the cell (e.g., by LNP delivery), which results in (f) targeted single-strand (in the case of nickase) or double-strand cleavage, followed by (g) targeted repair at the cleavage site by the retrotranscribed product of the ncRNA. This results in (h) DNA with precise editing. FIG. 85A depicts the preparation of variant retrotranscript RTs by constructing chimeric RT proteins that fuse the "Y region" of a retrotranscript RT (i.e., the region reported to be responsible for binding to the msr region of a retrotranscript ncRNA) to another RT (e.g., MMLV RT) or replace the "Y region" of one retrotranscript RT with the Y region of another retrotranscript RT. In this way, the chimeric protein may be engineered for any particular ncRNA to have a corresponding Y region that is expected to bind to that ncRNA. FIG. 85B (SEQ ID NO: 19955-19968) (top to bottom) provides a multiple sequence alignment of the amino acid sequences of various retrotranscript RTs (top). The boxed region indicates the location of the conserved VTG triplet, which marks the N-terminal side of the Y region (as reported in Anna J Simon, Andrew D Ellington, Ilya J Finkelstein, Retrons and their applications in genome engineering, Nucleic Acids Research , Volume 47, Issue 21, December 2, 2019, Pages 11007–11019, which is incorporated herein by reference), which is about 90 amino acids in length. The schematic diagram below is an enlarged version of the top figure. FIG. 86 depicts the preparation of a variant retrotranscript RT (e.g., POLE1, POLD1, POLG, Pfu, or KOD) by fusing one or more processability enhancers (e.g., Sso7d and Sac7d) and/or one or more fidelity enhancers (e.g., 3'>5' exonuclease domains) to increase proofreading activity. When selecting appropriate factors to enhance the processability and/or fidelity of the retrotranscript RT, refer to Oscorbin et al., "The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase," FEBS Lett, 594: 4338-4356 and Yarnall et al., "Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases," Nature Biotechnol, 2022. FIG87 Electroporation using plasmids in K562 cells. Appropriate plasmid mixtures were mixed and electroporated into cells using the Neon electroporation system. After 72 hours of incubation, genomic DNA was extracted and targeted regions were expanded into sequencing libraries. Sequencing data were analyzed by CRISPResso2 and precise edits and indels were calculated. FIG88 Results of plasmid-based analysis in K562 cells demonstrate precise edits up to approximately 1-10% and indels as low as 5-30% using RTX003_2042, 6083v1, 6943, 1262, 6342L, and 6342S. They are all novel retrotranscripts, and the precise editing activity observed in this experiment strongly supports that these novel retrotranscripts may generate msDNA in K562 cells. These data replicate the results previously seen in 293T cells, indicating that retrotranscript-mediated gene editing is not affected by cell type-specific effects. Figure 89 is based on the results of plastid analysis, which compares the retrotranscripts annotated in the literature with novel retrotranscripts in K562 cells. These data demonstrate that the novel retrotranscripts RTX3_1262, RTX3_6342L and RTX3_6342S can generate approximately 15-19% precise editing and as low as 20~35% indels. The novel retrotransposons performed as well or better than Aco1 and better than other retrotransposons validated in the literature: Eco1, Eco3, Sau1, and Sen1. Figure 90 Results of plastid-based analysis comparing RTX3_2781 to the leading retrotransposons RTX3_1262 and RTX3_6342S/L in K562 cells. These data demonstrate that RTX3_2781 installs precise edits with comparable efficiency to RTX3_1262 and 6342S/L. Figure 91 Results of plastid-based analysis in K562 cells demonstrate up to approximately 0.6% precise editing and as low as 5% indels using Cas9 D10A nickase, using RTX003_2042, 6083v1, 6943, 1262, 6342L, and 6342S. These are all novel retrotranscripts, and the precise editing activity observed in this experiment strongly supports that these novel retrotranscripts may generate msDNA in cells that can be used for nickase-mediated gene editing. Figure 92 Results of plastid-based analysis in K562 cells demonstrate up to 0.5% precise editing using LbCas12a nuclease. Precise editing in this system strongly suggests intracellular msDNA production and compatibility with Cas12a-like nucleases for gene editing. Figure 93 Results from plastid-based analysis in K562 cells demonstrate up to 0.5% precise editing using TnpB nuclease. Precise editing in this system suggests intracellular msDNA production and compatibility with TnpB-like nucleases for gene editing. Figure 94 RTX3_6342 (structure predicted from alpha-fold) (yellow) is aligned with MMLV-RT (PDB 4MH8) (green). The native RTX3_6342 RT is fused to a non-RT-related domain at the N-terminus, which may not be necessary for reverse transcription. Arrows mark potential relevant truncation points. Jumper et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). https://doi.org/10.1038/s41586-021-03819-2. Figure 95 Results from plastid-based analysis in K562 cells demonstrate up to ~20% precise editing and ~20% indels using RTX003_6342S and truncation variants. The precise editing activity observed in this experiment strongly supports that these truncations retain the ability to generate msDNA in cells. Figure 96 Results of plastid-based analysis in K562 cells demonstrate up to about 40% accurate editing and about 60% indels using RTX003_6342S and RTX3_1262 with different insert sizes. The accurate editing activity observed in this experiment shows that RTX3_6342S can insert sequences up to 405 bp into the EMX1 locus without reducing accurate editing. RTX3_1262 can insert up to 205 bp at the EMX1 locus before the editing efficiency decreases. During the CRISPResso2 analysis in this experiment, substitutions in the reads were ignored. Figure 97 Results from plastid-based assays in K562 cells demonstrate up to ~40% precise edits and ~40% indels when using RTX003_6342S and RTX3_1262 with different insert sizes while either ignoring or quantifying substitutions during the CRISPResso2 assay. The precise editing activity observed in this experiment suggests that RTX3_6342S can insert sequences up to 405 bp long into the EMX1 locus, but RT fidelity may limit accurate installation of intended edits (compare ignoring (circles) vs. counting (triangles) substitutions). FIG98 Results of plastid-based analysis in K562 cells screening for mutations in the RTX3_6342 reverse transcriptase that are predicted to interact with the ncRNA after structural alignment of the predicted alphafold RTX3_6342 structure and the crystal structure of the retrotranscript Eco1 with the ncRNA. Most mutations have a modest effect on the installation of shorter 10 p inserts, but only two (N465K and N465R) may increase the installation frequency of larger (305 p) inserts. FIG99 Results of plastid-based analysis in K562 cells demonstrate that heterologous homologous MMLV mutations in RTX3_6342S do not consistently increase the installation frequency of >205 bp inserts. MMLV (L139P) == RTX3_6342 (V265P), MMLV (T306K) == RTX3_6342 (M470K), MMLV (W313F) == RTX3_6342 (V477F). Figure 100 Results of a plastid-based analysis in K562 cells that screened for mutations in the RTX3_6342 reverse transcriptase that were predicted to interact with the ncRNA after structural alignment of the predicted alphafold RTX3_6342 structure and the crystal structure of the retrotranscript Eco1 with the ncRNA. E238R, E479K, and K255P are mutations that may increase the frequency of installation of the 305 bp insert at the EMX1 locus. Figure 101 Predicted structures of wild type and RTX3_6342 with different N-terminal truncations. The predicted structures were generated with Alphafold and aligned with the Eco1/CryoEM structure (79VU.pdb) using PyMoL alignment. Figure 102 Results of a plastid-based analysis in K562 cells evaluating the effect of partial or complete deletion of the RTX3_6342 N-terminal helix on the installation frequency of longer inserts. We observed that partial N-terminal truncations enhanced the precise editing/indel ratio of longer inserts. Figure 103 Results of a plastid-based analysis in K562 cells evaluating the effect of fusion of the DNA binding domain to RTX3_6342 on the installation frequency of 405 bp inserts. Fusion of the Sac7d or Sso7d DNA binding domain to the N-terminus of the retrotranscriptase resulted in a two-fold (RTX3_6342S) or ten-fold (RTX3_1262) increase in the frequency of installation of a 405 bp insert. FIG104 Results of a plastid-based assay in K562 cells that screened for retrotranscript family members similar to RTX3_6342, 6083, 6943, or 1262. It was noted that the majority of retrotranscripts with robust gene editing activity belonged to the RTX3_6342 family. FIG105 Results of testing the precise editing activity of the novel retrotranscript R2781 in an all-RNA system. In Figure 16, R2781 demonstrated 10 bp insertion at the EMX1 locus in the plastid system, with similar activity to other hits R1262, R6342S, 6342L. Here, two different homology arm (HA) lengths (double-arm 30 bp or 49 bp left arm/65 bp right arm) were used to assess the activity of R2781 inserting 25 bp, 205 bp and 405 bp at the EMX1 locus. Accurate insertion of 25 bp and 30 bp homology arms was achieved with 11% efficiency. When using longer homology arms (49/65 bp), the efficiency of insertion of the same length was reduced to half, indicating that this retrotransposons reverse transcriptase may have limited enzymatic processability. On the contrary, the activity of inserting 205 or 405 bp decreased significantly. The right figure shows the insertion and deletion under the respective conditions of the left figure. FIG. 106 Test results of the precise editing activity of the novel retrotranscript R6342S in an all-RNA system with Cas.9 D10A. Previously, it was demonstrated that retrotranscript R6342S could achieve precise editing by Cas9 WT. Here, the activity of a 25 bp insertion in the EMX1 locus was assessed in 293T cells using Cas9 D10A or Cas9 D10A R221K/N394K mutants and 10 or 50 ng sgRNA. Cas9 D10A demonstrated up to 0.4% editing, while Cas9 D10A R221K/N394K showed higher activity, up to 0.7% editing. These data indicate that retrotranscript-mediated genome insertion may be achieved using Cas9 nickase and Cas9 WT that produces double-strand breaks. The right figure shows the insertion and deletion under the respective conditions of the left figure. Figure 107 Test results of the precise deletion activity of the novel retrotranscript R1262 in the all-RNA system. In the top figure, two deletion strategies are shown. Retrotranscript ncRNAs are designed by juxtaposing left and right homologous arm sequences to delete intervening sequences. Del1 deletes 214 bp upstream of the Cas9 cleavage site at the EMX1 locus and del2 deletes 248 bp downstream of the Cas9 cleavage site. Retrotranscript-mediated deletions were compared with direct delivery of increasing amounts of single-stranded DNA (ssDNA) donors (150 and 300 ng). The bottom figure in the left figure shows that R1262 can delete 248 bp (del2) from the EMX1 locus with activity similar to that of the ssDNA donor. The right figure shows the insertion and deletion under the respective conditions of the left figure. It should be noted that the accidental insertion and deletion activity caused by the deletion mediated by the retrotranscript is about 6 times lower than that mediated by the ssDNA donor. The 214 bp (del1) deletion activity of R1262 was not detected. Figure 108 is based on the test results of non-homologous end joining (NHEJ) of the insertion activity of the novel retrotranscripts R1262 and R6342 in the all-RNA system. On the left, the strategy of deriving double-stranded DNA (dsDNA) is depicted, which is the substrate of the NHEJ mechanism. 1. Retrotranscript reverse transcriptase generates complementary single-stranded DNA from two ncRNAs containing sense or antisense insertion sequences. 2. Complementary single-stranded DNA forms a duplex 3. The Cas9-sgRNA complex removes the Y-shaped single-stranded parts (msR and msD spacer sequences) at both ends. 4. The blunt-ended double-stranded DNA trimmed by Cas9 is successfully inserted into the target site. Including sgRNA recognition sequences (the same as the genome target sequence) in tandem at both ends of the insert will result in blunt-ended double-stranded DNA, and only one insertion direction allows for more stable integration by preventing Cas9 from re-cutting. Using this strategy, R1262 with two complementary ncRNAs integrated approximately 120 bp dsDNA at the EMX1 locus with an efficiency of approximately 1%, and the efficiency was even higher when the inserted sequence was flanked by two tandem sgRNA sequences (right side of the top figure). The basal insertion activity of R6342 without the tandem sgRNA sequence is slightly higher than that of R1262. The right side of the bottom graph shows the insertions and deletions under the respective conditions of the top graph. Figure 109 Test results of immune response to retrotranscript RNA. Human peripheral blood mononuclear cells (hPBMCs) were used to assess immune responses because PBMCs contain innate and adaptive immune cells and are equipped with sensors to detect exogenous nucleic acids. Frozen hPBMCs were thawed and left overnight. As indicated by the labels, RT mRNAs with U or m1Ψ and ncRNAs with/without cap0 (m7G) at the 5' end were electroporated individually or together, while unmodified GFP mRNA from TriLink was used as a control. After overnight culture, the supernatant was analyzed for cytokine production. Of the 25 interleukins and chemokines examined, those detected above the detection limit were shown. Type I interferons (a marker of immune response to exogenous nucleic acids) were not detected in any retrotranscript RNA transfected cells, and low levels of some inflammatory interleukins and chemokines were detected in retrotranscript RNA transfected cells, but their levels were much lower than the control GFP mRNA except for the RT mRNA without U modification. Figure 110 Results of testing retrotranscript-mediated gene editing in human stem progenitor cells (HSPC) in the all-RNA system. Bone marrow-derived CD34+ human stem progenitor cells (HSPC) were used to evaluate the efficacy of retrotranscript-mediated gene editing in primary cells. Frozen HSPCs were thawed and expanded for three days in the presence of hSCF, hFLT3-L, and hTPO cytokines to prevent differentiation. Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA, and ncRNA were mixed at the mass ratio indicated in each tag and electroporated to HSPCs using two different programs using a Lonza electroporator. After another three days of culture, genomic DNA was extracted and sequenced to measure the frequency of editing. Using a total of 5 micrograms of RNA (separated by 1: 0.3: 4: 1 = Cas9: gRNA: ncRNA: RT), a precise insertion of 25 bp at the AAVS1 locus was observed at a frequency of 0.1% (left figure). The right figure shows the insertion and deletion under each condition. Figure 111 Test results of retrotranscript-mediated gene editing in human T cells in the all-RNA system. Human all-T cells from peripheral blood were used to evaluate the efficacy of retrotranscript-mediated gene editing in primary cells. Frozen T cells were thawed and activated for two days in the presence of anti-CD3/anti-CD28-conjugated magnetic beads and IL-2 cytokines. Cas9 mRNA, guide RNA (gRNA), R6342 RT mRNA and ncRNA were mixed at the mass ratio indicated in each label and electroporated into T cells by Neon or Lonza electroporator. After another three days of culture, genomic DNA was extracted and sequenced to measure the editing frequency. Using a total of 3 micrograms of RNA (separated by 1: 0.3: 3: 1 = Cas9: gRNA: ncRNA: RT), a precise insertion of 25 bp at the AAVS1 locus was observed with a frequency of up to 1.7% using the Neon machine (left figure). The right figure shows the insertion and deletion under their respective conditions. Figure 112 provides a schematic diagram of the retrotranscript ncRNA library. Variants of different elements in ncRNA (modified a1: a2 stem, msR, msD regions) are associated with unique barcodes, and the library is synthesized as an oligo library. By sequencing the barcode inserted at the genome locus, the efficiency of related ncRNA variants in human cells can be measured. Figure 113 summarizes the variant library of Example 31. Figure 114 provides a schematic diagram depicting the screening process for screening the ncRNA variant library described in Example 31. Figure 115 provides a scatter plot summarizing the results of Figure 31. In the scatter plot, each point represents a variant, where the relative genome insertion level is on the y-axis and the ssDNA production level is on the x-axis. The dotted line represents the genome insertion and ssDNA production levels of the WT R6342S control. Therefore, the ncRNA variants in the upper right portion of the dotted line are superior to the WT R6342S control on both levels. It can be seen that most of these variants have a1a2 or msD stem elements. We identified 13 such variants that showed >1.5-fold increases in both genomic insertion and ssDNA production levels compared to the WT R6342S control.

TW202426060A_112132199_SEQL.xml

Claims (48)

A gene editing system comprising one or more delivery vehicles, wherein: The delivery vehicle(s) comprises an RNA cargo, The RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retrotranscriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the nucleic acid programmable nuclease, Each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), and (a)(i), (a)(ii), (b) and (c) are delivered by one or more delivery vehicles. A gene editing system as claimed in claim 1, wherein the engineered retrovirus ncRNA comprises an HDR nucleotide sequence substituted into the retrovirus ncRNA; wherein the retrovirus reverse transcriptase has an amino acid sequence having at least 90% sequence identity with the retrovirus reverse transcriptase of Table A; wherein the retrovirus ncRNA has about 85% to 98% sequence identity with the retrovirus ncRNA of Table B. A gene editing system as claimed in claim 2, wherein the retrovirus ncRNA and the retrovirus reverse transcriptase are from the same evolutionary branch. A gene editing system as claimed in claim 2, wherein the retrotranscript ncRNA nucleotide sequence has approximately 85% to 98% sequence identity with SEQ ID NO: 15327, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type I-C retrotranscript reverse transcriptase. The gene editing system of claim 4, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:1262. The gene editing system of claim 2, wherein the retrotranscript ncRNA nucleotide sequence has about 85% to 98% sequence identity with SEQ ID NO: 16411, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type III retrotranscript reverse transcriptase. The gene editing system of claim 6, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:2781. The gene editing system of claim 2, wherein the retrotranscript ncRNA nucleotide sequence has about 55% to 90% sequence identity with SEQ ID NO: 18731, and the retrotranscript reverse transcriptase has at least 90% sequence identity with type XIII retrotranscript reverse transcriptase. The gene editing system of claim 8, wherein the engineered retrotran ncRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 19927 and an HDR template inserted therein. The gene editing system of claim 8, wherein the engineered retrotran ncRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 19928 and an HDR template inserted therein. The gene editing system of claim 8, wherein the retrovirus reverse transcriptase comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:6342. The gene editing system of claim 1, wherein the retrovirus reverse transcriptase comprises at least one amino acid substitution that increases processability and/or fidelity. The gene editing system of claim 12, wherein the retrovirus reverse transcriptase comprises an amino acid substitution in an amino acid residue corresponding to the following amino acid residue in Eco1 RT: Q190, E302 or T306. The gene editing system of claim 12, wherein the retrovirus reverse transcriptase comprises an amino acid substitution in an amino acid residue corresponding to the following amino acid substitution in Eco1 RT: Q190F, E302R or T306K. A gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprises an RNA cargo, the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered retrotranscript reverse transcriptase, (b) an engineered retrotranscript ncRNA, and (c) a guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), (a)(i), (a)(ii), (b) and (c) are delivered by one or more delivery vehicles, and The engineered retrotransposase comprises a processability-enhancing domain or a fidelity-enhancing domain. The gene editing system of claim 15, wherein the processability enhancing domain comprises Sso7d or Sac7d. A gene editing system as claimed in claim 15, wherein the fidelity enhancing domain comprises a 3’ to 5’ exonuclease domain. The gene editing system of claim 17, wherein the exonuclease domain comprises POLE1 POLD1, POLG, Pfu or KOD. A gene editing system as claimed in claim 15, wherein the engineered retrovirus ncRNA comprises an HDR nucleotide sequence substituted into the retrovirus ncRNA; wherein the retrovirus reverse transcriptase has an amino acid sequence having at least 90% sequence identity with the retrovirus reverse transcriptase of Table A; wherein the retrovirus ncRNA has about 85% to 98% sequence identity with the retrovirus ncRNA of Table B. A gene editing system as claimed in claim 15, wherein the retrovirus ncRNA and the retrovirus reverse transcriptase are from the same evolutionary branch. A gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprises an RNA cargo, the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) an engineered reverse transcriptase, (b) an engineered reverse transcriptase ncRNA, and (c) a guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), (a)(i), (a)(ii), (b) and (c) are delivered by one delivery vehicle or more than one delivery vehicle, and The engineered reverse transcriptase comprises a Y region domain from a reverse transcriptase RT corresponding to the engineered reverse transcriptase ncRNA. The gene editing system of claim 21, wherein the engineered reverse transcriptase is a chimera comprising MMLV RT fused to the Y region of the reverse transcriptase RT. The gene editing system of claim 1, wherein (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retrotransposase. The gene editing system of claim 23, wherein (a)(i) and (a)(ii) are encoded and expressed as a fusion protein. The gene editing system of claim 24, wherein the fusion protein comprises the C-terminus of the nucleic acid programmable nuclease fused to the N-terminus of the reverse transcriptase of the retrovirus (nuclease:RT fusion). A gene editing system as claimed in claim 24, wherein the fusion protein comprises the N-terminus of the nucleic acid programmable nuclease fused to the C-terminus of the reverse transcriptase (RT: nuclease fusion). The gene editing system of claim 1, wherein (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retrotransposase. The gene editing system of claim 1, wherein (c) is separated from (a)(i), (a)(ii) and (b) or provided in trans . The gene editing system of claim 1, wherein (b) the engineered retrotran ncRNA and (c) the guide RNA are fused or provided in cis form . A gene editing system as claimed in claim 29, wherein the guide RNA is fused to the 5’ end of the retrotranscript ncRNA. A gene editing system as claimed in claim 29, wherein the guide RNA is fused to the 3’ end of the retrotranscript ncRNA. A gene editing system as claimed in claim 29, wherein the engineered ncRNA comprises a first guide RNA fused to the 5’ end of the retrotranscript ncRNA and a second guide RNA fused to the 3’ end of the retrotranscript ncRNA, and the first and second guide RNAs target different sequences. A gene editing system as claimed in claim 1, wherein the one or more delivery vehicles comprise liposomes or lipid nanoparticles (LNPs). The gene editing system of claim 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in the same delivery medium. The gene editing system of claim 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retrotranscriptase and (b) the engineered retrotranscript ncRNA are in a separate delivery medium. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery medium. A gene editing system as claimed in claim 1, wherein the nucleic acid programmable nuclease and the retroviral reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery media. A gene editing system as claimed in claim 1, wherein the engineered retrotranscript ncRNA includes a related sequence encoding a donor polynucleotide, wherein the donor polynucleotide comprises the expected editing at a target sequence to be integrated into a cell, and wherein the donor polynucleotide is flanked by a 5' homology arm hybridized to a sequence at the 5' position of the target sequence and a 3' homology arm hybridized to a sequence at the 3' position of the target sequence. A gene editing system as claimed in claim 1, wherein the nucleic acid programmable nuclease comprises Cas9 nuclease, TnpB nuclease or Cas12a nuclease. A gene editing system as claimed in claim 1, wherein the nucleic acid programmable nuclease comprises a Cas9 nuclease. A gene editing system as claimed in claim 1, wherein the nucleic acid programmable nuclease comprises a Cas9 nickase. An isolated cell comprising the gene editing system of claim 1. The isolated cell of claim 42, wherein the isolated cell is a mammalian cell. The isolated cell of claim 43, wherein the mammalian cell is a human cell. A composition comprising: a)   the gene editing system of claim 1; and b)   a pharmaceutically or veterinarily acceptable carrier. A composition as claimed in claim 45, wherein the delivery medium is a lipid nanoparticle comprising: a)     one or more ionizable lipids; b)     one or more structured lipids; c)     one or more PEGylated lipids; and d)     one or more phospholipids. The composition of claim 46, wherein the one or more ionizable lipids comprise the ionizable lipids described in Table 2. A method for genetically modifying a cell, the method comprising: contacting the gene editing system of claim 1 with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein the guide RNA guides the complex to the target sequence, the nucleic acid programmable nuclease generates a double-strand break in the target sequence, the retrotransposase and the engineered retrotransposase ncRNA generate RT DNA containing the donor polynucleotide, and the donor polynucleotide is integrated into the target sequence, thereby editing the cell to be genetically modified.