KR20220106079A - Genome replacement and insertion technology using reverse transcriptase based on Francisella novicida Cas9 module - Google Patents

Abstract

The present invention relates to a genome replacement and insertion technology using reverse transcriptase based on the Francisella novicida Cas9 module. Since the gene replacement and insertion technology according to the present invention has a different nick position formed in a non-target strand inside a target DNA, the technology has an advantage capable of expanding the existing insertion and replacement technology.

Description

{Genome replacement and insertion technology using reverse transcriptase based on Francisella novicida Cas9 module}

The present invention relates to a genome replacement and insertion technology using a reverse transcriptase based on a Francisella novicida Cas9 module.

The CRISPR-Cas9 system can be easily utilized by replacing only the guide RNA sequence without the need to create a new DNA binding domain according to the target gene sequence every time.

The recently proposed RT (reverse-transcriptase) enzyme-based target DNA external gene insertion technology ( prime editing) works based on the S. There is a lot of room for improvement.

In the case of prime editing technology, which combines reverse transcriptase (RT) with the CRISPR-SpCas9 module and inserts an external gene into the target DNA, pathogenic mutations (pyrimidine(C,T) or purine(A,G) ) or purine (A, G) to pyrimidine (C, T) transversion), since it has the advantage of being able to correct, it is being investigated that more than 90% of the pathogenic mutations reported so far can be recovered.

However, among many disease-causing mutations, polygenic disease is more common than singe-gene disorder disease.

In other words, with the current SpCas9-based Prime editing technology, when the target site exists in multiple locus, multiple locus targeting is practically difficult due to non-target correction issues and guide duplication issues. In addition, since SpCas9-based Prime editing technology allows gene insertion and substitution at a distance of 3bp from the PAM (NGG) site on the target DNA, it is judged that there will be significant limitations in its application unless the limitation of PAM is resolved.

Under this background, the present inventors have completed the present invention by extending the existing prime editing technology and presenting a new FnCas9-based prime editing technology to develop a new technology that reduces off-target specificity and improves safety when developing gene therapy for human subjects. did.

The present inventors found that FnCas9(H969A) has a different nick-forming position on the target DNA compared to SpCas9(H840A) (6bp away from PAM for FnCas9(H969A), whereas 3bp away from PAM for SpCas9(H840A)) , completed the present invention by confirming that genome editing is easier (increasing genome editing scalability) than the existing correction technology. Moreover, since the guide RNA scaffold structure recognized by FnCas9 is different from that of SpCas9, by simultaneously applying FnCas9-based prime editing technology with existing SpCas9-based prime editing technology, each nucleotide sequence within multiple genes is subjected to prime editing without interfering with each other. Insertion or substitution may be made possible. In addition, multiplexed prime editing technology that can be applied to the treatment of polygenic diseases and simultaneous correction of multiple genes can be performed through prime editing based on the novel FnCas9 module.

Accordingly, the present invention is Francisella novicida Cas9 protein;

reverse transcriptase protein; and

A prime editing complex comprising a prime editing guide RNA (PEgRNA), wherein the prime editing guide RNA (PEgRNA) aims to provide a prime editing complex having an extension arm.

The present invention also relates to Francisella novicida Cas9 protein; reverse transcriptase protein; and one or more polynucleotides encoding a prime editing complex comprising a prime editing guide RNA (PEgRNA).

Another object of the present invention is to provide a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein.

Another object of the present invention is to provide a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein.

The present invention also aims to provide a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

The present invention also relates to a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; And it aims to provide a prime editing complex system comprising a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

The present invention also relates to a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; And it aims to provide a composition for gene editing comprising a prime editing complex comprising a vector comprising a polynucleotide encoding the prime editing guide RNA (PEgRNA).

The present invention also relates to a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; It is an object of the present invention to provide a pharmaceutical composition comprising a prime editing complex comprising a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

The present invention also relates to Francisella novicida Cas9 protein; reverse transcriptase protein; And it aims to provide a cell comprising a prime editing complex comprising a prime editing guide RNA (PEgRNA).

The invention also provides a method of providing a substitution, deletion, insertion or combination thereof in a nucleic acid sequence comprising contacting the nucleic acid sequence with a prime editing complex, the method comprising:

The method comprises (i) nicking a double-stranded DNA sequence onto a target strand (or PAM strand) and generating free single-stranded DNA having a 3' end;

(ii) priming the polymerase by hybridizing the 3' end of the free single-stranded DNA to the primer binding site of the guide RNA;

(iii) polymerizing a strand of DNA from the 3' end, thereby creating a single-stranded DNA flap comprising a nucleotide change; and

(iv) replacing the endogenous DNA strand immediately downstream of the cleavage site on the target strand (or PAM strand) with a single stranded DNA flap, thereby providing the desired nucleotide change in the double stranded DNA sequence. An object of the present invention is to provide a method for providing substitution, deletion, insertion or a combination thereof.

The present invention also aims to provide a method for correcting a gene comprising the step of contacting a nucleic acid sequence with a prime editing complex.

The present invention also aims to provide a method for altering the expression of a gene product comprising the step of contacting the nucleic acid sequence with a prime editing complex.

The present invention also aims to provide a method of treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) comprising the step of contacting the nucleic acid sequence with a prime editing complex.

The present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and

It is an object to provide a pharmaceutical composition comprising a prime editing complex comprising a prime editing guide RNA (PEgRNA), or one or more polynucleotides encoding the prime editing complex, or one or more vectors or cells comprising these polynucleotides do it with

The present invention relates to (1) Francisella novicida Cas9 protein; reverse transcriptase protein; and

a prime editing complex comprising a prime editing guide RNA (PEgRNA), or one or more polynucleotides encoding the prime editing complex, or one or more vectors or cells comprising these polynucleotides, and (2) a pharmaceutically acceptable carrier An object of the present invention is to provide a pharmaceutical composition comprising a.

The present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and

A mutant or single-nucleotide polymorphism comprising a prime editing complex comprising a prime editing guide RNA (PEgRNA), or one or more polynucleotides encoding the prime editing complex, or one or more vectors or cells comprising these polynucleotides ( An object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to SNP).

Hereinafter, the present invention will be described in detail.

The technology according to the invention has the following features:

1. It corresponds to a technology that can insert or substitute a foreign gene into the target DNA sequence by binding reverse transcriptase using the FnCas9 ( Franicella novicida Cas9 ) module, one of the CRISPR-Cas9 orthologs (prime editing). .

2. External gene insertion and substitution technology (prime editing) using FnCas9 ( Franicella novicida Cas9 ) module is different from the existing SpCas9 in that the nick position formed on the non-target strand inside the target DNA is different, so it is possible to expand the existing genome insertion and substitution technology. has the advantage of being able to

3. External gene insertion and substitution technology (prime editing) using the FnCas9 ( Franicella novicida Cas9 ) module has less non-target binding than the existing prime editing technology using SpCas9, so it has higher target specificity (safety), so more accurate prime editing can be performed. have.

4. When using the FnCas9 ( Francicella novicida Cas9 ) prime editor (reverse transcriptase linkage) and the existing SpCas9 prime editor in combination, there is no cross-interference, so it has the advantage of easy insertion and substitution of external genes of multiple genes.

5. By engineering the FnCas9 module, it is possible to recognize various PAM sequences or to improve the properties to develop an effective FnCas9 prime editor (FnCas9-PE).

6. FnCas9(H969A)-PE is used simultaneously with SpCas9 nickase (H840A) and the target sequence to generate nicks in NTS (non-target strand) and TS (target strand), respectively, and foreign genes are inserted by FnCas9-PE efficiency can be amplified.

The present invention is Francisella novicida Cas9 protein;

reverse transcriptase protein; and

A prime editing complex comprising a prime editing guide RNA (PEgRNA), wherein the prime editing guide RNA (PEgRNA) provides a prime editing complex having an extension arm.

According to one embodiment of the present invention, the present invention relates to a Francisella novicida Cas9 protein;

reverse transcriptase protein; and

A prime editing complex comprising a prime editing guide RNA (PEgRNA), comprising:

The prime editing guide RNA (PEgRNA) provides that it includes a primer binding site (PBS), an editing template (Scaffold) and a homology arm (extension arm).

Prime Edit

"Prime editing" is a versatile, accurate genome editing method that directly records new genetic information into a specified DNA site using the Cas9 protein as a platform for genome editing, wherein the prime editing system specifies the target site and also on the guide RNA. Prime editing (PE) to be the template for the synthesis of the desired editing in the form of an alternative DNA strand by means of an engineered extension (DNA or RNA) (e.g., at the 5' or 3' end or on the inner portion of the guide RNA) ) is programmed with a guide RNA (“PEgRNA”). The replacement strand containing the desired edit (eg, a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited. Through DNA repair and/or replication machinery, the endogenous strand of the target site is replaced by a newly synthesized replacement strand containing the desired edit. Specifically, a Cas9 protein-reverse transcriptase protein is used to target a specific DNA sequence by a guide RNA, generate a single-stranded nick at the target site, and convert the nicked DNA into a guide RNA for reverse transcription of the integrated reverse transcriptase protein. The method used as a primer is used.

Prime editing works by contacting a target DNA molecule with the Francisella novicida Cas9 protein complexed with a prime editing guide RNA (PEgRNA).

A prime editing guide RNA (PEgRNA) comprises an extension at the 3' or 5' end of the guide RNA or at an intramolecular location within the guide RNA and encodes a desired nucleotide change (e.g., a single nucleotide change, insertion or deletion). do. The Francisella novicida Cas9 protein/extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the Francisella novicida Cas9 protein to bind to the target locus.

A nick is then introduced into one of the strands of the DNA of the target locus, making a 3' end available for one of the strands of the target locus. Next, the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA to prime reverse transcription (ie, “target-primed RT”). In certain embodiments, the 3' end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., a “reverse transcriptase priming sequence” or “primer binding site” on the PEgRNA. Next, a reverse transcriptase enzyme is introduced that synthesizes a single strand of DNA from the 3' end of the primed site towards the 5' end of the prime editing guide RNA. It contains the desired nucleotide changes (eg, single base changes, insertions or deletions, or combinations thereof) and otherwise forms a single-stranded DNA flap homologous to endogenous DNA at or adjacent to the nick site. Then, Francisella novicida Cas9 and guide RNA are released. Finally, it relates to the degradation of single-stranded DNA flaps that allow for the incorporation of desired nucleotide changes into the target locus. This process can be driven towards the desired product formation by eliminating the corresponding 5' endogenous DNA flap, which is formed when a 3' single-stranded DNA flap invades and hybridizes to an endogenous DNA sequence. This process may also be driven towards product formation by "second strand nicking. This process may introduce at least one or more of the aforementioned conversions, transfers, deletions and insertions.

"Prime Edit (PE) System", "Prime Edit (PE)" or "PE System"

A complex comprising Francisella novicida Cas9 protein, reverse transcriptase, and prime editing guide RNA, as well as auxiliary elements to help drive the prime editing process towards edited product formation, such as the target DNA target-strand (TS) Compositions involved in genome editing methods including, but not limited to, forming a second strand nicking component (e.g., second strand sgRNA) and a 5' endogenous DNA flap removal endonuclease (e.g., FEN1) refers to

By way of example, the PE system may be configured as follows:

[NLS]-[ Francisella novicida Cas9 (H969A)]-[Linker]-[MMLV_RT(wt)] + PEgRNA of interest

[NLS]-[ Francisella novicida Cas9 (H969A)]-[Linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + PEgRNA of interest

In the present invention, when the Francisella novicida Cas9 protein and the reverse transcriptase protein are complexed with a prime editing guide RNA (PEgRNA), they can bind to a target DNA sequence.

according to the invention

The PE system may be one in which the target or complementary non-target strand is nicked to have a priming sequence with a free 3' end. This nick site may be a PE system, 6 bp upstream (-6) from the 5' end of the PAM sequence.

The prime editor (PE) according to the present invention means that it includes a Francisella novicida Cas9 protein and a reverse transcriptase and can be used separately or as a fusion construct such as a fusion protein. It can perform prime editing on the target nucleotide sequence in the presence of PEgRNA (or "extended guide RNA").

Cas9

The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain or fragment thereof (eg, a protein comprising an active or inactive DNA cleavage domain of Cas9 and/or a gRNA binding domain of Cas9). refers to cleavage.

Francisella novicida Cas9 protein may be Francisella novicida- derived Cas9 having nickase activity or a functional equivalent thereof, and may be Francisella novicida- derived nuclease-active Cas9, nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9).

The nuclease-inactivated Cas9 domain may be interchangeably referred to as a “dCas9” protein (for nuclease-“dysfunctional” Cas9). Methods for generating a Cas9 domain (or fragment thereof) having an inactive DNA cleavage domain are known.

For example, in Francisella novicida , mutation H969A completely inactivates the nuclease activity of Francisella novicida Cas9 . Accordingly, Francisella novicida according to the present invention may be Francisella novicida Cas9 (H969A) including the mutation H969A from the parent sequence.

More specifically, Cas9 derived from Francisella novicida may have the amino acid sequence of SEQ ID NO: 1.

As a "dCas9" protein, Francisella novicida Cas9 (H969A) may have, for example, the amino acid sequence of SEQ ID NO: 2. Francisella novicida Cas9 according to the present invention has altered PAM specificity. Specifically, the scalability of gene editing is greatly increased because the nick-forming site on the target DNA occurs 6-8bp away from the PAM.

According to the present invention, for Francisella novicida Cas9 , its full-length protein sequence or a fragment thereof may be used. Proteins comprising Francisella novicida Cas9 or fragments thereof may be referred to as " Francisella novicida Cas9 variants" and share homology to the above-mentioned proteins.

For example, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to Francisella novicida Cas9 (H969A) of SEQ ID NO: 2 or at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical.

Francisella novicida Cas9 (H969A) according to the present invention may have a deactivating mutation in the HNH nuclease domain.

"Nickase" refers to Cas9 in which one of the two nuclease domains is inactivated. This enzyme can only cleave one strand of the target DNA

reverse transcriptase (RT) protein

"Reverse transcriptase" describes a class of polymerases that are characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template.

Reverse transcriptase has been mainly used to transcribe mRNA into cDNA, which can then be cloned into a vector for further manipulation. The enzyme has 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-directed DNA polymerase activity and RNase H activity.

During the synthesis of a single-stranded DNA flap based on an RT template integrated with a guide RNA, error-causing reverse transcriptase introduces one or more nucleotides that are mismatched with the RT template sequence, through error polymerization of the single-stranded DNA flap. Changes can be introduced in the nucleotide sequence. These errors introduced during the synthesis of single-stranded DNA flaps then refer to the properties of certain reverse transcriptases that are error-prone in activity and through hybridization to the corresponding endogenous target strand, removal of the endogenous replaced strand, ligation. .

Such exemplary reverse transcriptase enzymes may use, for example, any naturally occurring mutant RT, engineered mutant RT, or other variant RT, including truncated variants that retain function.

More specifically, reverse transcriptases are multifunctional enzymes that typically have three enzymatic activities, including RNA- and DNA-dependent DNA polymerization activities, and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Some mutants of reverse transcriptase incapacitate the RNaseH moiety, preventing unintended damage to mRNA. These enzymes that synthesize complementary DNA (cDNA) using mRNA as a template were first identified in RNA viruses.

Subsequently, reverse transcriptase was isolated and purified directly from viral particles, cells or tissues.

(See, e.g., Kacian et al., 1971, Biochim. Biophys. Acta 46: 365-83; Yang et al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11; Gerard et al. , 1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch. Virol. 55 187-200; Kato et al., 1984, J. Virol. Methods 9: 325-39; Luke et al ., 1990, Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol. 65: 7004-07, each of which is incorporated by reference). More recently, mutants and fusion proteins have been generated in the search for improved properties such as thermostability, fidelity and activity.

Any of the wild-type, variant, and/or mutant forms of a reverse transcriptase known in the art or that can be prepared using methods known in the art are contemplated herein.

The reverse transcriptase (RT) gene (or the genetic information contained therein) can be obtained from a number of different sources. For example, a gene can be obtained from a eukaryotic cell infected with a retrovirus, or from multiple plasmids containing part or all of the retroviral genome. In addition, messenger RNA-like RNA containing the RT gene can be obtained from a retrovirus. Examples of sources for RT include Moloney Murine Leukemia Virus (MMLV or MLVRT); human T-cell leukemia virus type 1 (HTLV-1); bovine leukemia virus (BLV); roux sarcoma virus (RSV); human immunodeficiency virus (HIV); Yeasts such as Saccharomyces, Neurospora, Drosophila; primates; and rodents. See, eg, Weiss, et al., US Pat. No. 4,663,290 (1987); Gerard, G. R., DNA:271-79 (1986); Kotewicz, M. L., et al., Gene 35:249-58 (1985); Tanese, N., et al., Proc. Natl. Acad. Sci. (USA):4944-48 (1985);

Roth, M. J., et al., J. Biol. Chem. 260:9326-35 (1985); Michel, F., et al., Nature 316:641-43 (1985); Akins, R. A., et al., Cell 47:505-16 (1986), EMBO J. 4:1267-75 (1985); and Fawcett, D. F., Cell 47:1007-15 (1986), each of which is incorporated herein by reference in its entirety.

wild-type RT

M-MLV reverse transcriptase and RSV reverse transcriptase. Enzymes with reverse transcriptase activity are commercially available. In certain embodiments, the reverse transcriptase is provided in trans to another component of the prime editor (PE) system. That is, the reverse transcriptase is expressed or otherwise provided as a separate component, ie not as a fusion protein with napDNAbp.

Those of ordinary skill in the art are skilled in the art of Moloney murine leukemia virus (M-MLV); Human immunodeficiency virus (HIV) reverse transcriptase and avian sarcoma-leukemia virus (ASLV) reverse transcriptase such as, but not limited to, Rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, avian erythroblastosis virus (AEV) helper virus MCAV reverse transcriptase, avian myelocytosis virus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian Wild-type reverse transcriptases including, but not limited to, sarcoma virus Y73 helper virus YAV reverse transcriptase, Rous-associated virus (RAV) reverse transcriptase, and myeloblastosis-associated virus (MAV) reverse transcriptase are suitable for the subject methods and compositions described herein. will recognize that it can be used

Variant and error-prone RT

Reverse transcriptase is essential for synthesizing a complementary DNA (cDNA) strand from an RNA template. Reverse transcriptases are enzymes composed of distinct domains that exhibit different biochemical activities. The enzyme catalyzes the synthesis of DNA from the RNA template as follows: In the presence of annealed primers, the reverse transcriptase binds to the RNA template and initiates the polymerization reaction. RNA-dependent DNA polymerase activity synthesizes complementary DNA (cDNA) strands to incorporate dNTPs. RNase H activity degrades the RNA template of the DNA:RNA complex. Thus, reverse transcriptases include (a) binding activity that recognizes and binds to RNA/DNA hybrids, (b) RNA-dependent DNA polymerase activity, and (c) RNase H activity. In addition, reverse transcriptases are generally considered to have various properties including their thermostability, processability (rate of dNTP incorporation), and fidelity (or error rate). Reverse transcriptase variants contemplated herein have their enzymatic activity (e.g., RNA-dependent DNA polymerase activity, RNase H activity, or DNA/RNA hybrid-binding activity) or enzymatic properties (e.g., thermostability, processing sex, or fidelity); Such variants are available in the public domain, commercially available in the art, or can be prepared using known mutagenesis methods, including directed evolutionary processes (eg, PACE or PANCE).

In various embodiments, the reverse transcriptase may be a variant reverse transcriptase. As used herein, "variant reverse transcriptase" refers to any naturally occurring or genetically engineered variants. RT naturally has several activities, including RNA-dependent DNA polymerase activity, ribonuclease H activity, and DNA-dependent DNA polymerase activity. Collectively, these activities enable enzymes to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then be integrated into the host genome, from which a new RNA copy can be made via host-cell transcription. Variant RTs can include mutations that affect one or more of these activities (either reducing or increasing these activities, or eliminating them all together). In addition, a variant RT may contain one or more mutations that make the RT more or less stable, lower the tendency to aggregation, facilitate purification and/or detection, and/or other modification of a property or characteristic.

Those of ordinary skill in the art are skilled in the art of Moloney murine leukemia virus (M-MLV); Human immunodeficiency virus (HIV) reverse transcriptase and avian sarcoma-leukemia virus (ASLV) reverse transcriptase such as, but not limited to, Rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, avian erythroblastosis virus (AEV) helper virus MCAV reverse transcriptase, avian myelocytosis virus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian Variant reverse transcriptases derived from other reverse transcriptases including, but not limited to, sarcoma virus Y73 helper virus YAV reverse transcriptase, Rous-associated virus (RAV) reverse transcriptase, and myeloblastosis-associated virus (MAV) reverse transcriptase are described herein. It will be appreciated that the subject methods and compositions may be suitably used.

RTs in the prime editor can be "error-causing" reverse transcriptase variants. Error-causing reverse transcriptases known and/or available in the art may be used. Reverse transcriptases do not have any corrective function in nature; It will therefore be appreciated that the error rate of reverse transcriptase is generally higher than that of DNA polymerases with corrective activity. The error rate of any particular reverse transcriptase is a property of the enzyme's "fidelity" indicative of the accuracy of the template-directed polymerization of DNA to its RNA template. RTs with high fidelity have a low error rate. Conversely, RTs with low fidelity have high error rates. The fidelity of M-MLV-based reverse transcriptase is reported to have an error rate ranging from 1 error to 15,000 to 27,000 nucleotides synthesized.

Thus, for the purposes of this application, a reverse transcriptase that is considered "error-prone" or has "error-prone fidelity" is one that has an error rate of less than one error in the 15,000 nucleotides synthesized.

Also consider reverse transcriptases with mutations in the RNaseH domain. As mentioned above, one of the intrinsic properties of reverse transcriptase is its RNase H activity, which simultaneously cleaves the RNA template of an RNA:cDNA hybrid. RNase H activity may be undesirable for the synthesis of long cDNAs as the RNA template may be degraded prior to completion of full-length reverse transcription. RNase H activity may also reduce reverse transcription efficiency, possibly due to its competition with the enzyme's polymerase activity. Accordingly, the present disclosure contemplates any reverse transcriptase variant comprising altered RNaseH activity.

The disclosure herein also contemplates a reverse transcriptase having a mutation in the RNA-dependent DNA polymerase domain. As mentioned above, one of the intrinsic properties of reverse transcriptase is its RNA-dependent DNA polymerase activity, which incorporates a nucleobase into the emerging cDNA strand as encoded by the template RNA strand of the RNA:cDNA hybrid. RNA-dependent DNA polymerase activity can be increased or decreased (ie, in terms of its rate of incorporation) to increase or decrease the processability of the enzyme. Accordingly, the present disclosure contemplates any reverse transcriptase variant comprising an RNA-dependent DNA polymerase activity modified to increase or decrease the processing power of the enzyme compared to an unmodified version.

Also contemplated herein are reverse transcriptase variants with altered thermostability characteristics. The ability of reverse transcriptases to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperature helps to denature RNA with strong secondary structure and/or high GC content, allowing reverse transcriptase to read along the sequence. As a result, reverse transcription at higher temperatures enables full-length cDNA synthesis and higher yields, which may lead to improved production of 3' flap ssDNA as a result of the prime editing process. Wild-type M-MLV reverse transcriptase typically has an optimum temperature in the range of 37-48 °C; 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C Mutations can be introduced that allow for reverse transcription activity at higher temperatures above 48°C, including , 66°C, and above.

According to the present invention, the reverse transcriptase has any one sequence selected from the group consisting of SEQ ID NOs: 3 to 14. More specifically, the reverse transcriptase is Moloney murine leukemia virus (M-MLV) reverse transcriptase and has SEQ ID NO:3.

The following mutations at the corresponding amino acid positions in the wild-type M-MLV RT of SEQ ID NO: 3 or in another wild-type RT polypeptide sequence: P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, variant RT comprising one or more of F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N.

More specifically, the sequence according to the present invention may be a mutant sequence of wild-type M-MLV RT, and specifically may have the nucleotide sequence of SEQ ID NO: 15.

fusion protein

Francisella novicida Cas9 protein according to the present invention; and the reverse transcriptase protein may also be provided in the form of a fusion protein.

The prime editor (PE) system described herein comprises the Francisella novicida Cas9 protein; and the reverse transcriptase protein may optionally be provided in the form of a fusion protein linked by a linker.

Combinations of any of the examples of the Francisella novicida Cas9 protein and reverse transcriptase protein previously described herein are contemplated, and they may be combined via any linker sequence.

Linker "Linker" refers to a chemical group or molecule that connects two molecules or moieties, eg, the cleavage domain and the binding domain of a nuclease.

In some embodiments, the linker is an amino acid or a plurality of amino acids (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, 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 in length. Longer or shorter linkers are also contemplated.

Exemplary linkers in this regard include GGS (SEQ ID NO:16); GGSGGS (SEQ ID NO: 17);

GGSGGSGGS (SEQ ID NO: 18); SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 19); SGSETPGTSESATPES (SEQ ID NO: 20) can be exemplified.

In addition, if necessary, the fusion protein according to the present invention may include a nuclear localization signal (NLS) for promoting the translocation of the protein to the cell nucleus.

The NLS can be any known NLS sequence in the art. The NLS may also be any later-discovered NLS for nuclear localization. The NLS can also be any naturally occurring NLS, or any non-naturally occurring NLS (eg, an NLS with one or more desired mutations).

Exemplary such sequences include, but are not limited to, PKKKRKV (SEQ ID NO: 21), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 22), and the like.

Prime Editing Guide RNA (PEgRNA)

A guide RNA is a specific type of guide nucleic acid that most commonly associates with a Cas9 protein and directs the Cas9 protein to a specific sequence within a DNA molecule that includes complementarity to the protospacer sequence of the guide RNA.

As used herein, a “prime editing guide RNA” (or “PEgRNA”) may have an extension arm.

The extension arm comprises a primer binding site and a DNA synthesis template sequence encoding a single-stranded DNA flap containing the genetic change of interest via a polymerase (eg, reverse transcriptase) at the 3' end of the PEgRNA or A single stranded extension at the 5' end, which can then be integrated into endogenous DNA by replacing the corresponding endogenous strand, to provide the desired genetic change.

In particular, the extension arm may have a primer binding sequence, a homology arm, an editing template. The primer binding site binds to a primer sequence formed from the endogenous DNA strand of the target site when nicked by the prime editor complex, exposing the 3' end on the endogenous nicked strand.

The editing template may be as small as a single nucleotide substitution, or it may be an insertion or inversion of DNA. Additionally, the editing template may also contain deletions, which may be engineered by encoding the homology arms containing the desired deletions.

"Homology arm" refers to the portion of the extension arm that encodes the portion of the resulting reverse transcriptase-encoded single-stranded DNA flap that will be integrated into the target DNA site by replacing the endogenous strand. The portion of the single-stranded DNA flap encoded by the homology arms is complementary to the non-edited strand of the target DNA sequence, which facilitates replacement of the endogenous strand and annealing of the single-stranded DNA flap in situ. to install This component is further defined elsewhere. The homology arms are by definition part of the DNA synthesis template because they are encoded by the polymerase of the prime editor described herein.

As used herein, a “prime editing guide RNA” (or “PEgRNA”) may further comprise, in addition to an extension arm, at least one or more of the following components:

Spacer sequence - a sequence in the PEgRNA that binds to a protospacer in the target DNA (about 20 nt in length).

gRNA core (or gRNA scaffold or backbone sequence) - refers to the sequence within the gRNA responsible for Cas9 binding, which does not contain the 20 bp spacer/targeting sequence used to guide Cas9 to the target DNA.

Transcription Terminator - A guide RNA or PEgRNA may contain a transcription termination sequence 3' of the molecule.

Prime editing guide RNA” or “PEgRNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and compositions described herein.

As described herein, a prime editing guide RNA comprises one or more “extended regions” of a nucleic acid sequence. The extended region may include, but is not limited to, single stranded RNA or DNA. Additionally, extended regions may occur at the 3' end of the traditional guide RNA. In another arrangement, the extended region may occur at the 5' end of the traditional guide RNA. In another arrangement, the extended region may occur in the intramolecular region of a traditional guide RNA, eg, in the gRNA core region that associates and/or binds Cas9. The extended region is (a) designed to be homologous to the endogenous target DNA to be edited, and (b) contains at least one desired nucleotide change (e.g., a transition, transformation, deletion or insertion) to be introduced or integrated into the endogenous target DNA. "DNA synthesis template" that encodes (by a polymerase of a prime editor) comprising single-stranded DNA. The extended region may also include other functional sequence elements such as, but not limited to, “primer binding sites” and “spacer or linker” sequences, or other structural elements such as, but not limited to, aptamers, stem loops, hairpins, toe loops (e.g., , 3' to loop) or an RNA-protein recruitment domain (eg, MS2 hairpin). As used herein, "primer binding site" includes a sequence that hybridizes to a single stranded DNA sequence having a 3' end generated from the nicked DNA of an R-loop.

In certain embodiments, the PEgRNA exhibits a PEgRNA having a 5' extension arm, a spacer and a gRNA core. The 5' extension further comprises a reverse transcriptase template, a primer binding site and a linker in the 5' to 3' direction.

In certain other embodiments, the PEgRNA exhibits a PEgRNA having a 5' extension arm, a spacer and a gRNA core. The 5' extension further comprises a reverse transcriptase template, a primer binding site and a linker in the 5' to 3' direction.

In another embodiment, the PEgRNA shows a PEgRNA having a spacer (1), a gRNA core (2) and an extension arm (3) in the 5′ to 3′ direction. The extension arm (3) is at the 3' end of the PEgRNA. The extension arm (3) further comprises a "primer binding site" (A), a "editing template" (B) and a "homology arm" (C) in the 5' to 3' direction. The extension arm 3 may also comprise, at the 3' and 5' ends, an optional modifier region, which may be the same sequence or a different sequence. Additionally, the 3' end of the PEgRNA may comprise a transcription terminator sequence. These sequence elements of PEgRNA are further described and defined herein.

In another embodiment, the PEgRNA shows a PEgRNA having extending arms (3), a spacer (1) and a gRNA core (2) in the 5′ to 3′ direction. The extension arm (3) is at the 5' end of the PEgRNA. The extension arm (3) further comprises in the 3' to 5' direction a "primer binding site" (A), a "editing template" (B) and a "homology arm" (C). The extension arm 3 may also comprise, at the 3' and 5' ends, an optional modifier region, which may be the same sequence or a different sequence. The PEgRNA may also include a transcription terminator sequence at the 3' end. These sequence elements of PEgRNA are further described and defined herein.

According to one embodiment of the present invention, the guide RNA comprises a -20 nt protospacer sequence and a gRNA core region that binds Cas9. The guide RNA comprises an RNA fragment extending at the 5' end, i.e., a 5' extension. In this embodiment, the 5' extension comprises a reverse transcription template sequence, a reverse transcription primer binding site, and an optional 5-20 nucleotide linker sequence. The RT primer binding site hybridizes to the free 3' end formed after the nicking of the non-target strand of the R-loop, priming DNA polymerization in the 5'-3' direction of the reverse transcriptase.

According to another embodiment of the present invention, the guide RNA comprises a -20 nt protospacer sequence and a gRNA core that binds Cas9. In this embodiment, the guide RNA comprises an RNA segment extending at the 3' end, ie a 3' extension. In this embodiment, the 3' extension comprises a reverse transcription template sequence, and a reverse transcription primer binding site.

The RT primer binding site hybridizes to the free 3' end formed after the nicking of the non-target strand of the R-loop, priming DNA polymerization in the 5'-3' direction of the reverse transcriptase.

According to another embodiment of the present invention, the guide RNA comprises a -20 nt protospacer sequence and a gRNA core that binds Cas9. Guide RNAs comprise RNA segments extending at intermolecular locations within the gRNA core, ie, intramolecular extensions. The intramolecular extension includes a reverse transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybridizes to the free 3' end formed after the nicking of the non-target strand of the R-loop, priming DNA polymerization in the 5'-3' direction of the reverse transcriptase.

The length of the RNA extension can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

The RT template sequence may also be of any suitable length. For example, the RT template sequence may be at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

In another embodiment, the reverse transcription primer binding site sequence is at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

In other embodiments, the optional linker or spacer sequence is at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides. nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

The RT template sequence encodes a single-stranded DNA molecule that is homologous to the non-target strand (and thus complementary to the corresponding site of the target strand), but contains one or more nucleotide changes. The at least one nucleotide change may comprise one or more single-base nucleotide changes, one or more deletions, and one or more insertions.

The synthesized single-stranded DNA product of the RT template sequence is homologous to the non-target strand and contains one or more nucleotide changes. The single-stranded DNA product of the RT template sequence hybridizes in equilibrium with the complementary target strand sequence, displacing the homologous endogenous target strand sequence. The replaced endogenous strand may in some embodiments be referred to as a 5' endogenous DNA flap species. This 5' endogenous DNA flap species can be removed by a 5' flap endonuclease (e.g., FEN1), and the single-stranded DNA product now hybridized to the endogenous target strand is ligated and synthesized de novo with the endogenous sequence. Mismatches can be created between the strands. Mismatches can be resolved by the cell's innate DNA repair and/or replication processes.

In various embodiments of the extended guide RNA, cellular repair of the single stranded DNA flap results in the installation of the desired nucleotide change to form the desired product.

In another embodiment, the desired nucleotide change is between about -5 and about +5 of the nick site, or between about -10 and about +10 of the nick site, or between about -50 and about +50 of the nick site To install Windows and edit it.

In particular, the prime editing technique according to the present invention can be used for editing by taking advantage of the large advantage of the nicking effect 6-8bp ahead compared to the existing prime editor.

The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell.

PEgRNA comprises three major component elements aligned in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm can be further divided into the following structural elements in the 5' to 3' direction: the primer binding site (A), the editing template (B), and the homology arm (C).

PEgRNA comprises three major component elements aligned in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm can be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C).

Prime editing guide RNA (PEgRNA) may include the Francisella novicida Cas9 (H969A) binding sequence of SEQ ID NO: 23. This Francisella novicida Cas9 (H969A) binding sequence is at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or at least about 99.5% identical based on SEQ ID NO:23, above. Sequences that are identical, at least about 99.8% identical, or at least about 99.9% identical, exhibit Francisella novicida Cas9 (H969A) binding can be used.

The present invention also relates to Francisella novicida Cas9 protein; reverse transcriptase protein; and one or more polynucleotides encoding a prime editing complex comprising a prime editing guide RNA (PEgRNA).

More specifically, a polynucleotide encoding a Francisella novicida Cas9 protein is provided. According to one embodiment of the present invention, such a sequence may have, for example, the nucleotide sequence of SEQ ID NO: 24.

A polynucleotide encoding a reverse transcriptase protein is provided. According to one embodiment of the present invention, such a sequence may have, for example, the nucleotide sequence of SEQ ID NO: 25.

A polynucleotide encoding a prime editing guide RNA (PEgRNA) is provided. Francisella novicida Cas9 protein; and a polynucleotide encoding a fusion protein comprising a reverse transcriptase protein. According to one embodiment of the present invention, such a sequence may have, for example, the nucleotide sequence of SEQ ID NO: 26.

In addition, the present invention also provides a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein.

The present invention also aims to provide a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA) comprising a primer binding site, an editing template and a homology arm. A schematic schematic diagram of a vector according to an embodiment of the present invention is shown in FIG. 2 . Specifically, SV40 NLS-FnCas9(H969A) nickase-linker-M-MLV RT was followed to prepare a vector capable of preparing a fusion protein.

The invention also provides a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

The recombinant vector may comprise a promoter operably linked to said nucleotide.

As used herein, the term "promoter" is a DNA regulatory region capable of binding to a polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.

As used herein, the term "operably linked" refers to a functional linkage (cis) between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be at least one selected from the group consisting of a replication origin, a promoter, a transcription terminator, and the like.

The promoter of the present invention is one of the transcriptional regulatory sequences that regulate the initiation of transcription of a specific gene, and may be a polynucleotide fragment typically of about 100 bp to about 2500 bp in length.

프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터, 백시니아 바이러스 7.5K 프로모터, HSV의 tk 프로모터, SV40E1 프로모터, 호흡기 세포융합 바이러스(Respiratory syncytial virus; RSV) 프로모터, 메탈로티오닌 프로모터(metallothionin promoter), β-액틴 프로모터, 유비퀴틴 C 프로모터, 인간 IL-2(human interleukin-2) 유전자 프로모터, 인간 림포톡신(human lymphotoxin) 유전자 프로모터, 인간 GM-CSF(human granulocyte-macrophage colony stimulating factor) 유전자 프로모터 등으로 이루어진 군에서 선택된 1종 이상일 수 있으나, 이에 한정되는 것은 아니다. 일 예에서, 상기 프로모터는 CMV immediate-early 프로모터, U6 프로모터, EF1-alpha(elongation factor 1-a) 프로모터, EF1-alpha short(EFS) 프로모터 등으로 이루어진 군에서 선택된 것일 수 있다. 상기 전사 종결 서열은 폴리아데닐화 서열(pA) 등일 수 있다. 상기 복제 원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점, BBV 복제원점 등일 수 있다.The promoter can be used without limitation, as long as it can regulate transcription initiation in a cell, for example, a eukaryotic cell (eg, a plant cell, or an animal cell (eg, a mammalian cell such as a human, a mouse, etc.), etc.) do. For example, the promoter is a CMV promoter (cytomegalovirus promoter; for example, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter , SV40 promoter, adenovirus promoter (major late promoter), pL

promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, HSV tk promoter, SV40E1 promoter, Respiratory syncytial virus (RSV) promoter, metallotionin promoter ), β-actin promoter, ubiquitin C promoter, human IL-2 (human interleukin-2) gene promoter, human lymphotoxin gene promoter, human GM-CSF (human granulocyte-macrophage colony stimulating factor) gene promoter, etc. It may be one or more selected from the group consisting of, but is not limited thereto. In one example, the promoter may be selected from the group consisting of CMV immediate-early promoter, U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, and the like. The transcription termination sequence may be a polyadenylation sequence (pA) or the like. The origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, or a BBV origin of replication.

The vector of the present invention may be selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as the recombinant vector include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1). , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (eg, adeno-associated virus (AAV) vectors, etc.) It may be manufactured on the basis of, but is not limited thereto.

The production of the recombinant expression vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. have.

Another aspect of the present invention provides a method for producing a transformant comprising the step of introducing the composition for genome editing into an isolated cell or organism. wherein the organism may exclude a human.

The composition for genome editing of the present invention may be introduced into a cell or organism by a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ, and as is known in the art, suitable according to the host cell This can be done by selecting standard techniques. Such methods include, for example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic The liposome method and the lithium acetate-DMSO method may be included, but are not limited thereto.

The present invention also relates to Francisella novicida Cas9 protein; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA).

Cells that may contain any of the compositions described herein include prokaryotic and eukaryotic cells. The methods described herein are used to deliver a Cas9 protein, a prime editor, and a prime editing guide RNA (PEgRNA) or prime editing complex system into a eukaryotic cell (eg, a mammalian cell, such as a human cell). In some embodiments, the cells are in vitro (eg, cultured cells). In some embodiments, the cell is in vivo (eg, in a subject, such as a human subject). In some embodiments, the cell is ex vivo (eg, isolated from a subject and capable of being re-administered to the same or a different subject).

Mammalian cells of the present disclosure include human cells, primate cells (eg, Vero cells), rat cells (eg, GH3 cells, OC23 cells) or mouse cells (eg, MC3T3 cells). Without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell line (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 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from myeloma) A variety of human cell lines exist, including Saos-2 (bone cancer) cells. In some embodiments, the rAAV vector is delivered into human embryonic kidney (HEK) cells (eg, HEK 293 or HEK 293T cells). In some embodiments, the rAAV vector is a stem cell (e.g., a human stem cell), such as, e.g., a pluripotent stem cell (e.g., a human pluripotent stem cell, including a human induced pluripotent stem cell (hiPSC)) transmitted to me Stem cells refer to cells that, in culture, have the ability to divide for an indefinite period of time and produce specialized cells. Pluripotent stem cells refer to a type of stem cell capable of differentiating into all tissues of an organism, but not capable of sustaining complete organism development by themselves. Human induced pluripotent stem cells express genes and factors important for maintaining the defining characteristics of embryonic stem cells.

Refers to a somatic cell (eg, mature or adult) that has been reprogrammed to an embryonic stem cell-like state by being forced to do so. Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cellular features of all three germ layers (ectoderm, endoderm, mesoderm).

The present invention also relates to a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; and a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

The present invention also relates to a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; and a prime editing complex system comprising a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA).

A prime editing complex system according to the present invention comprises delivering a polynucleotide, such as one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom to a host cell. provides In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editing agent as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.

Provided delivery methods include nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. .

The invention also provides a method of providing a substitution, deletion, insertion or combination thereof in a nucleic acid sequence comprising contacting the nucleic acid sequence with a prime editing complex, the method comprising:

The method comprises (i) nicking a double-stranded DNA sequence onto a target strand (or PAM strand) and generating free single-stranded DNA having a 3' end;

(ii) priming the polymerase by hybridizing the 3' end of the free single-stranded DNA to the primer binding site of the guide RNA;

(iii) polymerizing a strand of DNA from the 3' end, thereby creating a single-stranded DNA flap comprising a nucleotide change; and

(iv) replacing the endogenous DNA strand immediately downstream of the cleavage site on the target strand (or PAM strand) with a single stranded DNA flap, thereby providing the desired nucleotide change in the double stranded DNA sequence. An object of the present invention is to provide a method for providing substitution, deletion, insertion or a combination thereof.

The use of prime editing complexes as above reveals the overall mechanism of prime editing, which can achieve goals such as changes, transformations, transfers, deletions or insertions of one or more genes.

In addition, depending on the purpose of use, mutagenesis using prime editing with error-prone RT can be used, prime editing can be used to treat triplet expansion disorders, prime editing can be used for peptide tagging, Prime editing can be used for RNA tagging, prime editing can be used for generation of sophisticated gene libraries, prime editing can be used for the insertion of immunoepitopes, and can be used in an off-axis manner in an unbiased manner. Prime edits can be used to confirm target edits, prime edits can be used for insertion of inducible dimerization domains, prime edits can be used for cellular data recording, prime edits can be used to tune biomolecular activity can be used, and its use can be aimed at according to various utilization methods, such as using prime editing for inserting a recombinase target site.

Such examples include examples of utilization methods listed in WO 2020/191234.

The present invention also aims to provide a method for correcting a gene comprising the step of contacting a nucleic acid sequence with a prime editing complex.

The gene editing of the present invention can be applied to eukaryotic organisms. The eukaryotic organism is a eukaryotic cell (e.g., a fungus such as yeast, eukaryotic and/or eukaryotic plant-derived cells (e.g., embryonic cells, stem cells, somatic cells, germ cells, etc.), eukaryotic cells (e.g., For example, vertebrates or invertebrates, more specifically humans, primates such as monkeys, mammals including dogs, pigs, cattle, sheep, goats, mice, rats, etc.), and eukaryotic plants (eg, green algae, etc.) may be selected from the group consisting of monocots or dicotyledons such as algae, corn, soybeans, wheat, and rice), but is not limited thereto.

The present invention also aims to provide a method for altering the expression of a gene product comprising the step of contacting the nucleic acid sequence with a prime editing complex.

The present invention also aims to provide a method of treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) comprising the step of contacting the nucleic acid sequence with a prime editing complex.

Methods of treating diseases associated with such mutations or single-nucleotide polymorphisms (SNPs) are associated with or by point mutations or other mutations (eg, deletions, insertions, inversions, duplications, etc.) that can be corrected by an editing system. It may mean providing a method of treating a subject diagnosed with the induced disease.

Virtually any disease-causing genetic defect can be repaired by using prime editing, which involves selecting an appropriate prime editor fusion protein, and (a) targeting the appropriate target DNA containing the editing site, ( b) designing an appropriate PEgRNA designed to provide a template for the synthesis of a single strand of DNA from the 3' end of the nick site, comprising the desired editing to replace and replace the endogenous strand immediately downstream of the nick site do.

In addition, the present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and

It provides a prime editing complex comprising a prime editing guide RNA (PEgRNA), or a composition for editing a gene comprising one or more polynucleotides encoding the prime editing complex.

In addition, the present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and

Provided is a prime editing complex comprising a prime editing guide RNA (PEgRNA), or a pharmaceutical composition comprising one or more polynucleotides encoding the prime editing complex.

In addition, the present invention (1) Francisella novicida Cas9 protein; reverse transcriptase protein; and

It aims to provide a pharmaceutical composition comprising a prime editing complex comprising a prime editing guide RNA (PEgRNA), or one or more polynucleotides encoding the prime editing complex, and (2) a pharmaceutically acceptable carrier. .

The present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and

A pharmaceutical composition for the prophylaxis or treatment of a disease associated with a prime editing complex comprising prime editing guide RNA (PEgRNA), or a mutation or single-nucleotide polymorphism (SNP) comprising one or more polynucleotides encoding the prime editing complex provides

Diseases for preventing, ameliorating, or treating diseases associated with mutations or single-nucleotide polymorphisms (SNPs) in such subjects include, for example, genetic diseases, non-hereditary diseases, viral infections, bacterial infections, cancer, or autoimmune diseases. do.

Herein, "genetic disease" refers to a disease caused in part or wholly, directly or indirectly, by one or more abnormalities in the genome, in particular a condition present at birth. The abnormality may be a mutation, insertion or deletion. It can affect the coding sequence of gene or its regulatory sequence.The genetic disease is DMD, hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, Congenital hepatic porphyria, hereditary disorders of liver metabolism, Lesch Nyhan syndrome, sickle cell anemia, thalassemia, dry skin pigmentation, Fanconi's anemia, retinitis pigmentosa, telangiectasia, Bloom syndrome (Bloom's syndrome), retinoblastoma and Tay-Sachs disease.

The non-hereditary disease target treats the disease by regulating normal genes other than the mutated gene, and may typically be age-related macular degeneration (AMD), but is not limited thereto.

Here, the virus, bacterial infection, or a disease caused by them includes, but is not limited to, AIDS, avian flu, flu, CMV-infected disease, tuberculosis or leprosy.

Here, the cancer is bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or It includes any one selected from the group consisting of cervical cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor, liver cancer, and colorectal cancer, but is not limited thereto.

Here, the autoimmune disease is type 1 diabetes, rheumatoid arthritis, celiac disease-sprue, IgA deficiency, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, skin Sclerosis, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Grave's disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia reduced purpura, liver cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's syndrome, pemphigoid bullae, lupus erythematosus, ulcerative colitis or dense deposit disease, and the like.

In the context of the above, treatment provides a positive therapeutic response to the disease or condition. A “positive therapeutic response” is intended to ameliorate a disease or condition, and/or ameliorate symptoms associated with the disease or condition.

The improvement of the symptoms includes administration of an effective amount or a therapeutically effective amount of the composition for genome editing. An “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce beneficial or desired results. A therapeutically effective amount may vary depending on one or more of the following: the subject and the disease state being treated, the weight and age of the subject, the severity of the disease state, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. .

The formulation of the pharmaceutical composition of the present invention may be for parenteral use. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. In particular, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition of the present invention can be administered parenterally, and can be administered by intratumoral administration, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraarterial, intraventricular, intralesional, intrathecal, topical, and combinations thereof. It may be administered by any one route selected from the group consisting of.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, and may be appropriately selected by those skilled in the art. can For a desirable effect, the pharmaceutical composition of the present invention may be administered at 0.01 ug/kg to 100 mg/kg per day, specifically 1 ug/kg to 1 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. Accordingly, the above dosage does not limit the scope of the present invention in any way.

Another object of the present invention is to provide a pharmaceutical use using the pharmaceutical composition.

Here, the pharmaceutical use may be for preventing, ameliorating or treating a disease associated with a mutation or a single-nucleotide polymorphism (SNP) in a subject. More specifically, for preventing, ameliorating or treating diseases associated with mutations or single-nucleotide polymorphisms (SNPs), including genetic diseases, non-hereditary diseases, viral infections, bacterial infections, cancer, or autoimmune diseases. can

Another object of the present invention for use in genome editing, Francisella novicida Cas9 protein; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA).

Another object of the present invention is for use in the prophylaxis or treatment of diseases associated with mutations or single-nucleotide polymorphisms (SNPs), including genetic diseases, non-hereditary diseases, viral infections, bacterial infections, cancer, or autoimmune diseases. Francisella novicida Cas9 protein for; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA).

Another object of the present invention is Francisella novicida Cas9 protein; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA).

Another object of the present invention is to prepare a medicament for use in the prevention or treatment of a disease associated with mutation or single-nucleotide polymorphism (SNP), comprising: Francisella novicida Cas9 protein; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA).

It is a technology that can insert or substitute foreign genes into target DNA sequences by combining reverse transcriptase using the Francicella novicida Cas9 ( FnCas9 ) module, one of the CRISPR-Cas9 orthologs (prime editing).

This technology extends the existing genome insertion and replacement technology because the nick position formed on the non-target strand inside the target DNA is different from that of the existing SpCas9 in the external gene insertion and replacement technology (prime editing) using the FnCas9 ( Franicella novicida Cas9 ) module. There are advantages to doing.

In addition, when using the FnCas9 ( Francicella novicida Cas9 ) prime editor (reverse transcriptase linkage) and the existing SpCas9 prime editor in combination, there is no cross-interference, so it has the advantage of easy insertion and substitution of external genes of multiple genes. It is possible to develop an effective FnCas9 prime editor (FnCas9-PE) by recognizing various PAM sequences or improving properties.

In addition, FnCas9(H969A)-PE is used simultaneously with SpCas9 nickase (H840A) and the target nucleotide sequence to generate nicks on the NTS (non-target strand) and TS (target strand) respectively, and the foreign gene is inserted by FnCas9-PE It can also amplify the efficiency.

1 shows a schematic diagram of comparison of prime editing using the FnCas9 nickase module and the SpCas9 nickase module. 1A shows a method of inserting an external gene (XX) using a SpCas9 nickase (H840A) type reverse transcriptase (RT) coupled SpCas9 prime editor that causes only non-target strand cleavage, FIG. 1B shows a method of inserting a foreign gene (XX) using the FnCas9 prime editor coupled with a reverse transcriptase (RT: reverse transcriptase) type of FnCas9 nickase (H969A) that only cuts non-target strands. At this time, after nick occurs between the PBS (primer binding site) of the pegRNA and the RT template (reverse transcription template), as the base pairing with the complementary part, the reverse transcriptase starts synthesis along the pegRNA, and the insertion of the part corresponding to XX (prime editing) occurs (RT(reverse transcriptase): reverse transcriptase, arrow (cleavage point): nick part, green: PAM (NGG) sequence, XX: external sequence to be inserted, yellow: PBS)
Figure 2 shows a schematic diagram of a CMV promoter-based FnCas9 prime editior expression vector in one embodiment of the present invention. It is designed to be expressed in higher animal cells based on the CMV promoter, and indicates that the vector is designed so that it can be expressed simultaneously by linking reverse transcriptase to the existing wild type and H969A mutant (nickase) FnCas9 C-terminus. .
3 shows a schematic diagram of a pegRNA (prime editing guide RNA) expression plasmid for FnCas9-PE based on the U6 promoter in one embodiment of the present invention. It indicates that pegRNA can be expressed based on the U6 promoter and designed to have a target sequence in the downstream.
4 shows a schematic diagram of a pegRNA for FnCas9-PE operation in an embodiment of the present invention (a target gene insertion site and a priming sequence insertion site diagram). Specifically, the PBS (primer binding site) and the reverse transcription template sequence were designed and manufactured to be located downstream of the FnCas9 scaffold, and as the target sequence was changed, the target sequence and the corresponding PBS The +RT sequence was inserted using a restriction enzyme to make FnCas9-PE workable.
5 is a HEK3 target site that corresponds to the HEK3 target site for checking whether targeting for editing is well using SpCas9 and FnCas9.
6 shows the NGS sequence analysis results obtained from the amplicons of the HEK3 site treated with SpCas9 and FnCas9. The blue bar histogram shows indels (%) analyzed at the HEK3 site of SpCas9-treated cells, and the orange bar histogram shows indels (%) analyzed at the HEK3 site of FnCas9-treated cells.
7 shows the nucleotide sequence analysis results of c-Myc, EMX1 and HEK3 target positions for confirming the FnCas9 cleavage point and the cleavage point of the SpCas9 system. The red line indicates the point where the sequencing was finished, indicating the DNA fragment cut by FnCas9 or SpCas9 (TS: target strand, NTS: non-target strand).
Figure 8 shows the expected nucleotide sequence and pegRNA design for HEK3 site prime editing using SpCas9 (shows the target DNA nucleotide sequence after being edited by 'CTT' sequence insertion using the SpCas9 prime editor, and the length of PBS and RT inside pegRNA is optimized according to the target).
Figure 9 shows the HEK3 site prime editing predicted nucleotide sequence and pegRNA design using FnCas9 (predicted DNA sequence after TT insertion and editing using FnCas9 prime editor. The targets of SpCas9 and FnCas9 are the same, but the prime editing guide Attempts to optimize efficiency by diversifying the length of the primer binding site (PBS) and RT (reverse transcription template) in RNA).
10 shows the NGS results analyzed after prime editing was performed using SpCas9 and FnCas9 at the HEK3 target site (SpCas9 prime editor was used as a control, and prime editing was performed so that bases were inserted at different positions of the same target). As a result of NGS, it can be confirmed that CTT insertion (marked in red box) occurred at the correct position in the case of SpCas9, and it indicates that TT insertion (marked in red box) occurs in the correct position also in the case of FnCas9 prime editor. In addition, in the case of the FnCas9 prime editor, it was confirmed that the insertion occurred in front of the SpCas9 prime editor, which can be corrected from 3bp in front of the PAM (indicated in yellow) 6bp.
11 shows the expected nucleotide sequence and pegRNA design for NRAS site prime editing using SpCas9 (shows the target DNA nucleotide sequence after editing with 'TT' sequence insertion using the SpCas9 prime editor, and the length of PBS and RT inside pegRNA is optimized according to the target).
12 shows the results of confirming that TT insertion occurred 3bp before PAM (yellow box) as a result of selecting the NRAS gene as a target and confirming it through NGS after prime editing with the existing SpCas9 prime editor
13 shows the predicted nucleotide sequence and pegRNA design for NRAS site prime editing using FnCas9 (shows the predicted DNA sequence after TT insertion and editing using FnCas9 prime editor, SpCas9 and FnCas9 have the same target, but FnCas9 cleavage The insertion position according to the point is set differently).
14 shows the results of confirming that TT insertion occurred at 6bp before PAM (indicated by yellow box) as a result of NGS after prime editing using FnCas9 prime editor by selecting the NRAS gene as a target.
Figure 15 shows the analysis of the results of NGS after prime editing using SpCas9 and FnCas9 on the NRAS target site (red box: insertion, yellow box: PAM).
16 shows a schematic schematic diagram of the expanded target range using the FnCas9 prime editor.
17 shows the results of confirming target-specific nucleotide insertion into c-Myc and NRAS genes of prime editing based on SpCas9 nickase (H840A) and FnCas9 (H969A) nickase. (Yellow: PAM, italic TT: insertion sequence )
18 shows the result of confirming the change in accuracy of insertion when a linker sequence is added between FnCas9 and reverse transcriptase sequence.
19 shows a schematic diagram of a triple or quadruple plasmid delivery system for priming editing according to the present invention.
20 shows target sequence information for confirming the range extension of prime editing using FnCas9(H969A)-RT.
21 shows the results of comparing the AA insertion efficiency with respect to the c-Myc gene. (underline AA: insertion sequence, CGG: PAM)
22 shows the results of confirming that point mutations and insertions at desired positions are made quite freely when using the FnCas9 prime editor.
23 shows the results of confirming the excellent insertion effect through FnCas9(H969A)-RT.
24 is a result confirming that when FnCas9(H969A)-RT is used, the off-target correction effect is less and exhibits excellent properties in target specificity.

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. FnCas9-based prime editor (FnCas9-PE) production

The existing prime editor was developed based on SpCas9 nickase (H840A) to nick the non-target strand in the target gene and then insert the foreign gene by the linked reverse transcriptase (RT).

A prime editor (FnCas9-PE) based on FnCas9 (H969A) nickase was developed to operate in a similar manner, and a specific schematic diagram thereof is shown in FIG. 1 .

Specifically, as the difference in Prime editing technology based on SpCas9 nickase (H840A) and FnCas9 (H969A) nickase, A in FIG. 1 shows SpCas9 nickase (H840A) based Prime editing technology, and B in FIG. 1 is FnCas9 (H969A) Represents nickase-based Prime editing technology.

After nick occurs between the PBS (primer binding site) of pegRNA and the RT template (reverse transcription template), as the base pairing with the complementary part, the reverse transcriptase starts synthesis along the pegRNA, and insertion of the part corresponding to XX (prime) editing) takes place. Unlike the use of SpCas9 nickase, the use of FnCas9(H969A) nickase increased the scalability of genome editing compared to the existing editing technology by forming a nick 6bp away from the PAM. In addition, since the guide RNA scaffold structure recognized by FnCas9 is different from that of SpCas9, multiple external gene insertion or substitution is possible through prime editing without interfering with each other by simultaneously applying the new FnCas9-based prime editing technology with the existing SpCas9-based prime editing technology. .

An FnCas9-PE expression vector was prepared so that a prime editor (FnCas9-PE) based on FnCas9 (H969A) nickase that operates through the above method can be operated in higher animal cells.

Specifically, human codon optimization was performed to optimize the expression of reverse transcriptase (RT) linked to FnCas9 (H969A) nickase in human-derived cells, and a vector capable of expression based on the CMV promoter was prepared.

A schematic diagram of the FnCas9-PE expression vector prepared above is shown in FIG. 2 .

Meanwhile, pegRNA (prime editing guide RNA) including a nucleotide sequence for inserting an external gene was designed based on the U6 promoter.

Specifically, it was designed so that pegRNA could be expressed based on the U6 promoter (FIG. 3). In particular, the position where the PBS (primer binding site) and reverse transcription template sequence can be inserted is designed to be located downstream of the FnCas9 scaffold, and as the target sequence is changed, the target sequence and the corresponding PBS + RT sequence are It was inserted using a restriction enzyme to operate FnCas9-PE (FIG. 4). A specific manufacturing method for pegRNA (prime editing guide RNA) including a nucleotide sequence for inserting an external gene will be described in more detail in Examples below.

Example 2. Confirmation of operation of FnCas9 as a CRISPR-Cas9 effector

In order to directly compare whether the gene sequence targeting of the existing SpCas9-based CRISPR-Cas9 module is targeted, sgRNA (single-guide RNA) expression capable of inducing a mutation (indel) in the target sequence ( HEK3 site) for SpCas9 and FnCas9, respectively Vector was designed and produced.

Specifically, the FnCas9-PE expression vector prepared in Example 1 was used.

The SpCas9-PE expression vector is basically homologous to the FnCas9-PE expression vector, and was prepared by using the SpCas9 nickase (H840A) gene instead of the FnCas9 nickase (H969A) gene.

The target location of the HEK3 site was selected with reference to the existing literature (Nature, Anzalone et al, 2019.). An exemplary target position thereof is shown in FIG. 5 (SEQ ID NO: 27: GGCCCAGACTGAGCACGTGATGG) .

An FnCas9 pegRNA expression vector and an SpCas9 pegRNA expression vector including a target position of the HEK3 site were prepared so that pegRNA could be expressed.

Specifically, a vector capable of expressing pegRNA using the U6 promoter was used, and the target nucleotide sequence information of the HEK3 site (SEQ ID NO: 27: GGCCCAGACTGAGCACGTGATTGG ) and the foreign gene insertion sequence (SEQ ID NO: 28: CATCACGTAAGCTCAGTCTG (PBS +) RT)) was connected.

To test the activation of the FnCas9 module before running the prime editor

The CRISPR-Cas9 effector (SpCas9 and FnCas9) and the sgRNA expression vector prepared above were simultaneously transfected.

Specifically, tansfection was performed through an electroporator method.

Then, NGS analysis was performed based on the target amplicon using an illumina sequencing device, and the target-specific ( HEK3 site) genome editing result (indel pattern) was confirmed.

The results are shown in FIG. 6 .

As can be seen from FIG. 6 , it was confirmed that an excellent correction effect was exhibited when FnCas9 was used as the CRISPR-Cas9 effector.

Then, in-vitro cleavage assay was performed to confirm the location where nick occurred on the NTS (non-target strand) before prime editing was performed using each of the FnCas9 module and the SpCas9 module.

That is, after cloning the target sites of HEK3 site, NRAS site, AAVS1 site, EMX1 site, and c-Myc site into T-vector, in-vitro cleavage was performed with FnCas9 or SpCas9, and then the cut fragments were analyzed by run-off sequencing. Through this, the cleavage point of FnCas9 was confirmed.

The target sequence information is shown in Table 1 below.

target gene CRISPR-FnCas9 (FnCas9)
target sequence (5' to 3') sequence for FnCas9
(5'' to 3'') sgRNA sequence for SpCas9 (5′′-3′′) c-Myc AGGCAGAGGGAGCGAGCGGG CGG
(SEQ ID NO: 29) AGGCAGAGGGAGCGAGCGGGGUUUCAGUUGCUGAAAAUGCUCUGUAAUCAUUUAAAAGUAUUUUGAACGGACCUCUGUUUGACACGUCUGAAUAACUAAAAA
(SEQ ID NO: 30) AGGCAGAGGGAGCGAGCGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
(SEQ ID NO: 31) AAVS1 TCTAACCCCCACCTCCTGTT AGG (SEQ ID NO: 32) UCUAACCCCCACCUCCUGUUGUUUCAGUUGCUGAAAAUGCUCUGUAAUCAUUUAAAAGUAUUUUGAACGGACCUCUGUUUGACACGUCUGAAUAACUAAAAA
(SEQ ID NO: 33) UCUAACCCCCACCUCCUGUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
(SEQ ID NO: 34) EMX1 TGGTTGCCCACCCTAGTCAT TGG (SEQ ID NO: 35) UGGUUGCCCACCCUAGUCAUGUUUCAGUUGCUGAAAAUGCUCUGUAAUCAUUUAAAAGUAUUUUGAACGGACCUCUGUUUGACACGUCUGAAUAACUAAAAA
(SEQ ID NO: 36) UGGUUGCCCACCCUAGUCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
(SEQ ID NO: 37) HEK3 GGCCCAGACTGAGCACGTGA TGG (SEQ ID NO: 38) GGCCCAGACUGAGCACGUGAGUUUCAGUUGCUGAAAAUGCUCUGUAAUCAUUUAAAAGUAUUUUGAACGGACCUCUGUUUGACACGUCUGAAUAACUAAAAA
(SEQ ID NO: 39) GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
(SEQ ID NO: 40) NRAS GGTAAGGGGGCAGGGAGGGA GGG (SEQ ID NO: 41) GGUAAGGGGGCAGGGAGGGAGUUUCAGUUGCUGAAAAUGCUCUGUAAUCAUUUAAAAGUAUUUUGAACGGACCUCUGUUUGACACGUCUGAAUAACUAAAAA
(SEQ ID NO: 42) GGUAAGGGGGCAGGGAGGGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
(SEQ ID NO: 43)

PAM sequences (NGG) are underlined. Specifically, the cleavage points of FnCas9 and SpCas9 were compared and analyzed through Sanger sequencing.

The results are shown in FIG. 7 . 7, it was confirmed that cleavage occurred in FnCas9 with 6-8nt 5'-overhang on the non-target strand, and it was confirmed that cleavage occurred with 3nt 5'-overhang of SpCas9. .

Through the above results, it was confirmed that when FnCas9 was used as the CRISPR-Cas9 effector, the position for forming the nick on the target DNA was different compared to the case of using the existing SpCas9.

Example 3. Identification of FnCas9-based prime editing (HEK3 target sequence)

In order to directly compare the SpCas9-based prime editing system and the FnCas9-based prime editing system, the SpCas9(H840A)-RT, FnCas9(H969A)-RT type prime editor was inserted into the target sequence ( HEK3 site) of the foreign gene (CTT), respectively. or TT), a pegRNA (prime editing-guide RNA) expression vector that can be inserted was designed.

Specifically, a schematic diagram of the expected nucleotide sequence and pegRNA design for HEK3 site prime editing using SpCas9 is shown in FIG. 8 .

As shown in FIG. 8, the 'CTT' sequence insertion was designed to be edited using the SpCas9 prime editor, and the lengths of PBS and RT inside the pegRNA were optimized according to the target.

Specifically, the FnCas9-PE expression vector prepared in Example 1 was used.

The SpCas9-PE expression vector is basically homologous to the FnCas9-PE expression vector, and was prepared by using the SpCas9 nickase (H840A) gene instead of the FnCas9 nickase (H969A) gene.

The target position of the HEK3 site was selected as a target for easy prime editing by referring to the existing literature, and an exemplary target position thereof is shown in FIG. 5 (SEQ ID NO: 27: GGCCCAGACTGAGCACGTGATTGG).

On the other hand, a schematic diagram of the expected nucleotide sequence and pegRNA design for HEK3 site prime editing using FnCas9 is shown in FIG. 9 .

As shown in Fig. 9, using the FnCas9 prime editor, the TT insertion was designed to be edited. Although the targets of SpCas9 and FnCas9 mentioned above are the same, efficiency optimization was performed by diversifying the lengths of the primer binding site (PBS) and reverse transcription template (RT) in the prime editing guide RNA, and the sequence information is shown in Table 2 below. It was.

Preparation of the vector for the above experiment was carried out similarly to Examples 1 and 2 above.

Target gene location and PBS+RTT
pegRNA by length
(PBS length - RTT length) target sequence
(SEQ ID NO: 27) pegRNA sequence (5'-3')
HEK3 FnCas9_pegRNA_10-10 GGCCCAGACTGAGCACGTGA TGG
SEQ ID NO: 44

5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA CATCACGTAAGCTCAGTCTG TTTTTTT
3' HEK3 FnCas9_pegRNA_10-12 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 45

5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA GCCATCACGTaaGCTCAGTCTG TTTTTTT
3' HEK3 FnCas9_pegRNA_10-15 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 46

5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA TCTGCCATCACGTaaGCTCAGTCTG TTTTTTT
3' HEK3 FnCas9_pegRNA_11-10 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 47
5'
G GGCCCAGACTGAGCACGTGA GTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA CATCACGTaaGCTCAGTCTGG TTTTTTT
3' HEK3 FnCas9_pegRNA_11-12 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 48
5'
G GGCCCAGACTGAGCACGTGA GTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA GCCATCACGTaaGCTCAGTCTGG TTTTTTT
3' HEK3 FnCas9_pegRNA_11-15 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 49
5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA TCTGCCATCACGTaaGCTCAGTCTGG TTTTTTT
3' HEK3 FnCas9_pegRNA_13-10 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 50
5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA CATCACGTaaGCTCAGTCTGGGC TTTTTTT
3' HEK3 FnCas9_pegRNA_13-12 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 51
5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA GCCATCACGTaaGCTCAGTCTGGGC TTTTTTT
3' HEK3 FnCas9_pegRNA_13-15 GGCCCAGACTGAGCACGTGA TGG SEQ ID NO: 52
5'
G GGCCCAGACTGAGCACGTGA GTTTCAGTTGCTGAATTATTTGGTAAACAGTACCAAATAATTAATGCTCTGTAATCATTTAAAAGTATTTTGAACGGACCTCTGTTTGACACGTCTGAATAACTAAAAA TCTGCCATCACGTaaGCTCAGTCTGGGC TTTTTTT
3'

(TGG: PAM, Target: underlined, priming template(PBS+RTT): italic, FnCas9 scaffold: underlined+italic.)

Similar to Example 2 above, the prepared vector systems were transfected with a prime editor and a pegRNA expression vector into HEK293FT cells at the same time, and then the target-specific ( HEK3 site) foreign gene insertion result was confirmed through NGS analysis.

That is, the candidate pegRNA and prime editor of Table 2 were transfected into HEK293 cells,

After 72 hours, genomic DNA (gDNA) was extracted, and the target amplicon was obtained from the gDNA and targeted amplicon sequencing (NGS) was performed using this to analyze the efficiency of external gene insertion.

The results are shown in FIG. 10 .

As can be seen from FIG. 10 , when using the FnCas9 prime editor, it was confirmed that the foreign gene 'TT' was inserted 6bp ahead of the PAM in the target DNA sequence on the genomic DNA. On the other hand, in the case of using the SpCas9 prime editor, it was confirmed that the insertion was performed at a position 3bp ahead of the PAM.

Example 4. Identification of FnCas9-based prime editing (NRAS target sequence)

A comparative experiment was performed between SpCas9-based prime editing system and FnCas9-based prime editing for NRAS target sequences.

Specifically, exemplary schematic diagrams of SpCas9-based prime editing system and FnCas9-based prime editing are shown in FIGS. 11 and 13, respectively, and sequence information is shown in Table 3 below.

The experiment proceeded similarly to the methods of Examples 2 and 3 above, and the results of confirming the insertion of the sequence through NGS are shown in FIGS. 12 and 14, respectively.

As can be seen through FIGS. 12 and 14 , when using the FnCas9 prime editor, it was confirmed that the foreign gene 'TT' was inserted 6bp ahead of the PAM exactly in the target DNA sequence on the genomic DNA. On the other hand, in the case of using the SpCas9 prime editor, it was confirmed that the insertion was performed at a position 3bp ahead of the PAM.

In addition, as can be seen in FIG. 15 , results showing a sufficient editing rate were confirmed even though the scalability of the insertion position was doubled using the FnCas9 prime editor. In particular, the secondary unwanted indel formation was 2.5% induced in the case of SpCas9(H840A)-RT, whereas 1.3% in the case of FnCas9(H969A)-RT was induced, confirming that accurate correction was possible.

Example 5. FnCas9-based prime editing optimization performed

A schematic diagram of the expanded target range using the FnCas9 prime editor identified through Examples 2 to 4 is shown in FIG. 16 . SpCas9(H840A) formed a nick 3-4bp upstream in PAM (NGG), and FnCas9(H969A) formed a nick 6-8bp upstream in PAM(NGG) It was confirmed through previous experiments.

Accordingly, target-specific nucleotide insertion into c-Myc and NRAS genes was performed with reference to the methods of Examples 3 and 4, and the results are shown in FIG. 17 .

As can be seen from FIG. 17 , it was confirmed that the insertion position for the target sequence was different depending on the type of Cas9 used in both c-Myc and NRAS.

Then, by adding a linker sequence between the FnCas9 and reverse transcriptase sequence to improve accuracy, it was checked whether the improvement in accuracy of its insertion was improved.

As the linker sequence, the experiment was performed by setting the sequence of SEQ ID NO: 19 to 0.5X, 1X, and 2X.

After constructing a sequence as shown in FIG. 18 to include the above sequence, an experiment was performed as in Example 4 using this.

As a result, as can be seen in FIG. 18 , it was confirmed that prime editing efficiency was optimized when the linker sequence was included as 1X.

Example 6. Preparation of FnCas9(H969A)-RT and pegRNA expression vector for intracellular prime editing by FnCas9(H969A)-RT

Using FnCas9(H969A)-RT, A system including nicking guide RNA (ngRNA) for the purpose of prime editing and nicking of the target sequence was prepared.

In the case of nicking guideRNA, it is used for the purpose of improving prime editing efficiency, and it was confirmed that it is preferable to insert at the -6 to +3 position at the nicking point for accurate and extended insertion.

A schematic diagram of the triple or quadruple plasmid delivery system according to the present invention is shown in FIG. 19 .

Similar to Example 2, CMV promoter-based SpCas9(H840A)-RT, FnCas9(H969A)-RT expression vectors were constructed. Then, a U6 promoter-based pegRNA (prime editing-guide RNA) expression vector was designed to insert a foreign gene (TT) into the target sequence. Then, PE2 (no ngRNA), PE3 triple (FnCas9(H969A)-RT with ngRNA for FnCas9), PE3 triple (comparison) (SpCas9(H840A)-RT with ngRNA for SpCas9), PE3-quadruple (FnCas9( It was confirmed that gene correction by expression of the prime editor was possible through intracellular delivery of H969A)-RT and ngRNA for SpCas9) method.

Example 7. Expansion of the scope of prime editing using FnCas9(H969A)-RT

A review of the prime range extension range of FnCas9(H969A)-RT and SpCas9(H840A)-RT was performed.

Since the FnCas9 nickase (H969A) module has a different nick-inducing position on the non-target strand side of the target gene compared to the SpCas9 nickase (H840A) module, the base sequence recognized as PAM compared to the SpCas9 nickase (H840A)-based prime editing system. From (NGG) to the farther away (SpCas9: 3-4 bp, FnCas9: 7-8 bp), the reverse transcription start point by reverse transcriptase changes. Using this point, the range that can induce prime editing based on the FnCas9 nickase (H969A) module compared to the SpCas9 nickase (H969A) module was investigated based on the target sequence of FIG. 20 for the same target sequence.

The prime editing efficiency of each of the 293FT cells was compared for the same target nucleotide sequence in the gene. (-6 to +3bp point based on the point at which nicking is formed).

The results of performing an experiment on the C-myc gene are shown in FIG. 21 .

As can be seen from FIG. 21 , as a result of comparing the AA insertion efficiency, in the case of SpCas9 nickase(H969A)-RT, it showed 10-38% correction efficiency from -1 to +3 position based on the nick position. On the other hand, in the case of FnCas9 nickase (H969A)-RT, it showed 3-8% calibration efficiency from -6 to +3 position based on the nick position, and FnCas9 nickase (H969A) based on the PAM sequence (NGG) within the same target. -RT greatly expanded the range of gene editing.

In addition, it could be confirmed from the results of FIG. 22 that point mutations and insertions at desired positions can be made quite freely. Specifically, Figure 22 shows the next-generation sequencing result of the site-specific (c-Myc, NRAS) base insertion induced by FnCas9(H969A)-RT, and it was confirmed that the efficiency of one base substitution or base insertion was excellent.

Example 8. Confirmation of off-target specificity of FnCas9(H969A)-RT

The non-target specificity of FnCas9 nickase(H969A)-RT was confirmed rather than SpCas9 nickase(H840A)-RT.

Specifically, PE3 triple (using FnCas9(H969A)-RT and ngRNA for FnCas9) of Example 5, PE3 triple (using SpCas9(H840A)-RT and ngRNA for SpCas9), PE3-quadruple (FnCas9(H969A)-RT and ngRNA for SpCas9) were used to confirm the target specificity for NRAS and c-Myc, and the results are shown in FIG. 23 .

As can be seen from FIG. 23 , an excellent insertion effect was confirmed through FnCas9(H969A)-RT, and it was confirmed that non-target targeting was reduced.

The results of confirming the specificity for the non-target are shown in FIG. 24 by comparing them with SpCas9(H840A)-RT and e SpCas9(H840A)-RT, respectively. As can be seen through Figure 24, the gene (NRAS, FANCF, HBB, c-Myc, HEK3)-specific nucleotide insertion results induced by FnCas9(H969A)-RT were compared with SpCas9(H840A)-RT. In this case, the off-target correction effect of FnCas9(H969A)-RT on the nucleotide sequence target of each gene (NRAS, FANCF, HBB, c-Myc, HEK3) was less, indicating excellent properties in target specificity.

Up to now, the present invention has been looked at focusing on the embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Genome replacement and insertion technology using reverse transcriptase based on Francisella novicida Cas9 module <130> P21219-KRIBB-PA <150> KR 10-2021-0008674 <151> 2021-01-21 <160> 52 <170> KoPatentIn 3.0 <210> 1 <211> 1628 <212> PRT <213> Artificial Sequence <220> <223> Francisella novicida cas 9 <400> 1 Met Asn Phe Lys Ile Leu Pro Ile Ala Ile Asp Leu Gly Val Lys Asn 1 5 10 15 Thr Gly Val Phe Ser Ala Phe Tyr Gln Lys Gly Thr Ser Leu Glu Arg 20 25 30 Leu Asp Asn Lys Asn Gly Lys Val Tyr Glu Leu Ser Lys Asp Ser Tyr 35 40 45 Thr Leu Leu Met Asn Asn Arg Thr Ala Arg Arg His Gln Arg Arg Gly 50 55 60 Ile Asp Arg Lys Gln Leu Val Lys Arg Leu Phe Lys Leu Ile Trp Thr 65 70 75 80 Glu Gln Leu Asn Leu Glu Trp Asp Lys Asp Thr Gln Gln Ala Ile Ser 85 90 95 Phe Leu Phe Asn Arg Arg Gly Phe Ser Phe Ile Thr Asp Gly Tyr Ser 100 105 110 Pro Glu Tyr Leu Asn Ile Val Pro Glu Gln Val Lys Ala Ile Leu Met 115 120 125 Asp Ile Phe Asp Asp Tyr Asn Gly Glu Asp Asp Leu Asp Ser Tyr Leu 130 135 140 Lys Leu Ala Thr Glu Gln Glu Ser Lys Ile Ser Glu Ile Tyr Asn Lys 145 150 155 160 Leu Met Gln Lys Ile Leu Glu Phe Lys Leu Met Lys Leu Cys Thr Asp 165 170 175 Ile Lys Asp Asp Lys Val Ser Thr Lys Thr Leu Lys Glu Ile Thr Ser 180 185 190 Tyr Glu Phe Glu Leu Leu Ala Asp Tyr Leu Ala Asn Tyr Ser Glu Ser 195 200 205 Leu Lys Thr Gln Lys Phe Ser Tyr Thr Asp Lys Gln Gly Asn Leu Lys 210 215 220 Glu Leu Ser Tyr Tyr His His Asp Lys Tyr Asn Ile Gln Glu Phe Leu 225 230 235 240 Lys Arg His Ala Thr Ile Asn Asp Arg Ile Leu Asp Thr Leu Leu Thr 245 250 255 Asp Asp Leu Asp Ile Trp Asn Phe Asn Phe Glu Lys Phe Asp Phe Asp 260 265 270 Lys Asn Glu Glu Lys Leu Gln Asn Gln Glu Asp Lys Asp His Ile Gln 275 280 285 Ala His Leu His His Phe Val Phe Ala Val Asn Lys Ile Lys Ser Glu 290 295 300 Met Ala Ser Gly Gly Arg His Arg Ser Gln Tyr Phe Gln Glu Ile Thr 305 310 315 320 Asn Val Leu Asp Glu Asn Asn His Gln Glu Gly Tyr Leu Lys Asn Phe 325 330 335 Cys Glu Asn Leu His Asn Lys Lys Tyr Ser Asn Leu Ser Val Lys Asn 340 345 350 Leu Val Asn Leu Ile Gly Asn Leu Ser Asn Leu Glu Leu Lys Pro Leu 355 360 365 Arg Lys Tyr Phe Asn Asp Lys Ile His Ala Lys Ala Asp His Trp Asp 370 375 380 Glu Gln Lys Phe Thr Glu Thr Tyr Cys His Trp Ile Leu Gly Glu Trp 385 390 395 400 Arg Val Gly Val Lys Asp Gln Asp Lys Lys Asp Gly Ala Lys Tyr Ser 405 410 415 Tyr Lys Asp Leu Cys Asn Glu Leu Lys Gln Lys Val Thr Lys Ala Gly 420 425 430 Leu Val Asp Phe Leu Leu Glu Leu Asp Pro Cys Arg Thr Ile Pro Pro 435 440 445 Tyr Leu Asp Asn Asn Asn Arg Lys Pro Pro Lys Cys Gln Ser Leu Ile 450 455 460 Leu Asn Pro Lys Phe Leu Asp Asn Gln Tyr Pro Asn Trp Gln Gln Tyr 465 470 475 480 Leu Gln Glu Leu Lys Lys Leu Gln Ser Ile Gln Asn Tyr Leu Asp Ser 485 490 495 Phe Glu Thr Asp Leu Lys Val Leu Lys Ser Ser Lys Asp Gln Pro Tyr 500 505 510 Phe Val Glu Tyr Lys Ser Ser Asn Gln Gln Ile Ala Ser Gly Gln Arg 515 520 525 Asp Tyr Lys Asp Leu Asp Ala Arg Ile Leu Gln Phe Ile Phe Asp Arg 530 535 540 Val Lys Ala Ser Asp Glu Leu Leu Leu Asn Glu Ile Tyr Phe Gln Ala 545 550 555 560 Lys Lys Leu Lys Gln Lys Ala Ser Ser Glu Leu Glu Lys Leu Glu Ser 565 570 575 Ser Lys Lys Leu Asp Glu Val Ile Ala Asn Ser Gln Leu Ser Gln Ile 580 585 590 Leu Lys Ser Gln His Thr Asn Gly Ile Phe Glu Gln Gly Thr Phe Leu 595 600 605 His Leu Val Cys Lys Tyr Tyr Lys Gln Arg Gln Arg Ala Arg Asp Ser 610 615 620 Arg Leu Tyr Ile Met Pro Glu Tyr Arg Tyr Asp Lys Lys Leu His Lys 625 630 635 640 Tyr Asn Asn Thr Gly Arg Phe Asp Asp Asp Asn Gln Leu Leu Thr Tyr 645 650 655 Cys Asn His Lys Pro Arg Gln Lys Arg Tyr Gln Leu Leu Asn Asp Leu 660 665 670 Ala Gly Val Leu Gln Val Ser Pro Asn Phe Leu Lys Asp Lys Ile Gly 675 680 685 Ser Asp Asp Asp Leu Phe Ile Ser Lys Trp Leu Val Glu His Ile Arg 690 695 700 Gly Phe Lys Lys Ala Cys Glu Asp Ser Leu Lys Ile Gln Lys Asp Asn 705 710 715 720 Arg Gly Leu Leu Asn His Lys Ile Asn Ile Ala Arg Asn Thr Lys Gly 725 730 735 Lys Cys Glu Lys Glu Ile Phe Asn Leu Ile Cys Lys Ile Glu Gly Ser 740 745 750 Glu Asp Lys Lys Gly Asn Tyr Lys His Gly Leu Ala Tyr Glu Leu Gly 755 760 765 Val Leu Leu Phe Gly Glu Pro Asn Glu Ala Ser Lys Pro Glu Phe Asp 770 775 780 Arg Lys Ile Lys Lys Phe Asn Ser Ile Tyr Ser Phe Ala Gln Ile Gln 785 790 795 800 Gln Ile Ala Phe Ala Glu Arg Lys Gly Asn Ala Asn Thr Cys Ala Val 805 810 815 Cys Ser Ala Asp Asn Ala His Arg Met Gln Gln Ile Lys Ile Thr Glu 820 825 830 Pro Val Glu Asp Asn Lys Asp Lys Ile Ile Leu Ser Ala Lys Ala Gln 835 840 845 Arg Leu Pro Ala Ile Pro Thr Arg Ile Val Asp Gly Ala Val Lys Lys 850 855 860 Met Ala Thr Ile Leu Ala Lys Asn Ile Val Asp Asp Asn Trp Gln Asn 865 870 875 880 Ile Lys Gln Val Leu Ser Ala Lys His Gln Leu His Ile Pro Ile Ile 885 890 895 Thr Glu Ser Asn Ala Phe Glu Phe Glu Pro Ala Leu Ala Asp Val Lys 900 905 910 Gly Lys Ser Leu Lys Asp Arg Arg Lys Lys Ala Leu Glu Arg Ile Ser 915 920 925 Pro Glu Asn Ile Phe Lys Asp Lys Asn Asn Arg Ile Lys Glu Phe Ala 930 935 940 Lys Gly Ile Ser Ala Tyr Ser Gly Ala Asn Leu Thr Asp Gly Asp Phe 945 950 955 960 Asp Gly Ala Lys Glu Glu Leu Asp His Ile Ile Pro Arg Ser His Lys 965 970 975 Lys Tyr Gly Thr Leu Asn Asp Glu Ala Asn Leu Ile Cys Val Thr Arg 980 985 990 Gly Asp Asn Lys Asn Lys Gly Asn Arg Ile Phe Cys Leu Arg Asp Leu 995 1000 1005 Ala Asp Asn Tyr Lys Leu Lys Gln Phe Glu Thr Thr Asp Asp Leu Glu 1010 1015 1020 Ile Glu Lys Lys Ile Ala Asp Thr Ile Trp Asp Ala Asn Lys Lys Asp 1025 1030 1035 1040 Phe Lys Phe Gly Asn Tyr Arg Ser Phe Ile Asn Leu Thr Pro Gln Glu 1045 1050 1055 Gln Lys Ala Phe Arg His Ala Leu Phe Leu Ala Asp Glu Asn Pro Ile 1060 1065 1070 Lys Gln Ala Val Ile Arg Ala Ile Asn Asn Arg Asn Arg Thr Phe Val 1075 1080 1085 Asn Gly Thr Gln Arg Tyr Phe Ala Glu Val Leu Ala Asn Asn Ile Tyr 1090 1095 1100 Leu Arg Ala Lys Lys Glu Asn Leu Asn Thr Asp Lys Ile Ser Phe Asp 1105 1110 1115 1120 Tyr Phe Gly Ile Pro Thr Ile Gly Asn Gly Arg Gly Ile Ala Glu Ile 1125 1130 1135 Arg Gln Leu Tyr Glu Lys Val Asp Ser Asp Ile Gln Ala Tyr Ala Lys 1140 1145 1150 Gly Asp Lys Pro Gln Ala Ser Tyr Ser His Leu Ile Asp Ala Met Leu 1155 1160 1165 Ala Phe Cys Ile Ala Ala Asp Glu His Arg Asn Asp Gly Ser Ile Gly 1170 1175 1180 Leu Glu Ile Asp Lys Asn Tyr Ser Leu Tyr Pro Leu Asp Lys Asn Thr 1185 1190 1195 1200 Gly Glu Val Phe Thr Lys Asp Ile Phe Ser Gln Ile Lys Ile Thr Asp 1205 1210 1215 Asn Glu Phe Ser Asp Lys Lys Leu Val Arg Lys Lys Ala Ile Glu Gly 1220 1225 1230 Phe Asn Thr His Arg Gln Met Thr Arg Asp Gly Ile Tyr Ala Glu Asn 1235 1240 1245 Tyr Leu Pro Ile Leu Ile His Lys Glu Leu Asn Glu Val Arg Lys Gly 1250 1255 1260 Tyr Thr Trp Lys Asn Ser Glu Glu Ile Lys Ile Phe Lys Gly Lys Lys 1265 1270 1275 1280 Tyr Asp Ile Gln Gln Leu Asn Asn Leu Val Tyr Cys Leu Lys Phe Val 1285 1290 1295 Asp Lys Pro Ile Ser Ile Asp Ile Gln Ile Ser Thr Leu Glu Glu Leu 1300 1305 1310 Arg Asn Ile Leu Thr Thr Asn Asn Ile Ala Ala Thr Ala Glu Tyr Tyr 1315 1320 1325 Tyr Ile Asn Leu Lys Thr Gln Lys Leu His Glu Tyr Tyr Ile Glu Asn 1330 1335 1340 Tyr Asn Thr Ala Leu Gly Tyr Lys Lys Tyr Ser Lys Glu Met Glu Phe 1345 1350 1355 1360 Leu Arg Ser Leu Ala Tyr Arg Ser Glu Arg Val Lys Ile Lys Ser Ile 1365 1370 1375 Asp Asp Val Lys Gln Val Leu Asp Lys Asp Ser Asn Phe Ile Ile Gly 1380 1385 1390 Lys Ile Thr Leu Pro Phe Lys Lys Glu Trp Gln Arg Leu Tyr Arg Glu 1395 1400 1405 Trp Gln Asn Thr Thr Ile Lys Asp Asp Tyr Glu Phe Leu Lys Ser Phe 1410 1415 1420 Phe Asn Val Lys Ser Ile Thr Lys Leu His Lys Lys Val Arg Lys Asp 1425 1430 1435 1440 Phe Ser Leu Pro Ile Ser Thr Asn Glu Gly Lys Phe Leu Val Lys Arg 1445 1450 1455 Lys Thr Trp Asp Asn Asn Phe Ile Tyr Gln Ile Leu Asn Asp Ser Asp 1460 1465 1470 Ser Arg Ala Asp Gly Thr Lys Pro Phe Ile Pro Ala Phe Asp Ile Ser 1475 1480 1485 Lys Asn Glu Ile Val Glu Ala Ile Ile Asp Ser Phe Thr Ser Lys Asn 1490 1495 1500 Ile Phe Trp Leu Pro Lys Asn Ile Glu Leu Gln Lys Val Asp Asn Lys 1505 1510 1515 1520 Asn Ile Phe Ala Ile Asp Thr Ser Lys Trp Phe Glu Val Glu Thr Pro 1525 1530 1535 Ser Asp Leu Arg Asp Ile Gly Ile Ala Thr Ile Gln Tyr Lys Ile Asp 1540 1545 1550 Asn Asn Ser Arg Pro Lys Val Arg Val Lys Leu Asp Tyr Val Ile Asp 1555 1560 1565 Asp Asp Ser Lys Ile Asn Tyr Phe Met Asn His Ser Leu Leu Lys Ser 1570 1575 1580 Arg Tyr Pro Asp Lys Val Leu Glu Ile Leu Lys Gln Ser Thr Ile Ile 1585 1590 1595 1600 Glu Phe Glu Ser Ser Gly Phe Asn Lys Thr Ile Lys Glu Met Leu Gly 1605 1610 1615 Met Lys Leu Ala Gly Ile Tyr Asn Glu Thr Ser Asn 1620 1625 <210> 2 <211> 1628 <212> PRT <213> Artificial Sequence <220> <223> Francisella novicida Cas9 (H969A) <400> 2 Met Asn Phe Lys Ile Leu Pro Ile Ala Ile Asp Leu Gly Val Lys Asn 1 5 10 15 Thr Gly Val Phe Ser Ala Phe Tyr Gln Lys Gly Thr Ser Leu Glu Arg 20 25 30 Leu Asp Asn Lys Asn Gly Lys Val Tyr Glu Leu Ser Lys Asp Ser Tyr 35 40 45 Thr Leu Leu Met Asn Asn Arg Thr Ala Arg Arg His Gln Arg Arg Gly 50 55 60 Ile Asp Arg Lys Gln Leu Val Lys Arg Leu Phe Lys Leu Ile Trp Thr 65 70 75 80 Glu Gln Leu Asn Leu Glu Trp Asp Lys Asp Thr Gln Gln Ala Ile Ser 85 90 95 Phe Leu Phe Asn Arg Arg Gly Phe Ser Phe Ile Thr Asp Gly Tyr Ser 100 105 110 Pro Glu Tyr Leu Asn Ile Val Pro Glu Gln Val Lys Ala Ile Leu Met 115 120 125 Asp Ile Phe Asp Asp Tyr Asn Gly Glu Asp Asp Leu Asp Ser Tyr Leu 130 135 140 Lys Leu Ala Thr Glu Gln Glu Ser Lys Ile Ser Glu Ile Tyr Asn Lys 145 150 155 160 Leu Met Gln Lys Ile Leu Glu Phe Lys Leu Met Lys Leu Cys Thr Asp 165 170 175 Ile Lys Asp Asp Lys Val Ser Thr Lys Thr Leu Lys Glu Ile Thr Ser 180 185 190 Tyr Glu Phe Glu Leu Leu Ala Asp Tyr Leu Ala Asn Tyr Ser Glu Ser 195 200 205 Leu Lys Thr Gln Lys Phe Ser Tyr Thr Asp Lys Gln Gly Asn Leu Lys 210 215 220 Glu Leu Ser Tyr Tyr His His Asp Lys Tyr Asn Ile Gln Glu Phe Leu 225 230 235 240 Lys Arg His Ala Thr Ile Asn Asp Arg Ile Leu Asp Thr Leu Leu Thr 245 250 255 Asp Asp Leu Asp Ile Trp Asn Phe Asn Phe Glu Lys Phe Asp Phe Asp 260 265 270 Lys Asn Glu Glu Lys Leu Gln Asn Gln Glu Asp Lys Asp His Ile Gln 275 280 285 Ala His Leu His His Phe Val Phe Ala Val Asn Lys Ile Lys Ser Glu 290 295 300 Met Ala Ser Gly Gly Arg His Arg Ser Gln Tyr Phe Gln Glu Ile Thr 305 310 315 320 Asn Val Leu Asp Glu Asn Asn His Gln Glu Gly Tyr Leu Lys Asn Phe 325 330 335 Cys Glu Asn Leu His Asn Lys Lys Tyr Ser Asn Leu Ser Val Lys Asn 340 345 350 Leu Val Asn Leu Ile Gly Asn Leu Ser Asn Leu Glu Leu Lys Pro Leu 355 360 365 Arg Lys Tyr Phe Asn Asp Lys Ile His Ala Lys Ala Asp His Trp Asp 370 375 380 Glu Gln Lys Phe Thr Glu Thr Tyr Cys His Trp Ile Leu Gly Glu Trp 385 390 395 400 Arg Val Gly Val Lys Asp Gln Asp Lys Lys Asp Gly Ala Lys Tyr Ser 405 410 415 Tyr Lys Asp Leu Cys Asn Glu Leu Lys Gln Lys Val Thr Lys Ala Gly 420 425 430 Leu Val Asp Phe Leu Leu Glu Leu Asp Pro Cys Arg Thr Ile Pro Pro 435 440 445 Tyr Leu Asp Asn Asn Asn Arg Lys Pro Pro Lys Cys Gln Ser Leu Ile 450 455 460 Leu Asn Pro Lys Phe Leu Asp Asn Gln Tyr Pro Asn Trp Gln Gln Tyr 465 470 475 480 Leu Gln Glu Leu Lys Lys Leu Gln Ser Ile Gln Asn Tyr Leu Asp Ser 485 490 495 Phe Glu Thr Asp Leu Lys Val Leu Lys Ser Ser Lys Asp Gln Pro Tyr 500 505 510 Phe Val Glu Tyr Lys Ser Ser Asn Gln Gln Ile Ala Ser Gly Gln Arg 515 520 525 Asp Tyr Lys Asp Leu Asp Ala Arg Ile Leu Gln Phe Ile Phe Asp Arg 530 535 540 Val Lys Ala Ser Asp Glu Leu Leu Leu Asn Glu Ile Tyr Phe Gln Ala 545 550 555 560 Lys Lys Leu Lys Gln Lys Ala Ser Ser Glu Leu Glu Lys Leu Glu Ser 565 570 575 Ser Lys Lys Leu Asp Glu Val Ile Ala Asn Ser Gln Leu Ser Gln Ile 580 585 590 Leu Lys Ser Gln His Thr Asn Gly Ile Phe Glu Gln Gly Thr Phe Leu 595 600 605 His Leu Val Cys Lys Tyr Tyr Lys Gln Arg Gln Arg Ala Arg Asp Ser 610 615 620 Arg Leu Tyr Ile Met Pro Glu Tyr Arg Tyr Asp Lys Lys Leu His Lys 625 630 635 640 Tyr Asn Asn Thr Gly Arg Phe Asp Asp Asp Asn Gln Leu Leu Thr Tyr 645 650 655 Cys Asn His Lys Pro Arg Gln Lys Arg Tyr Gln Leu Leu Asn Asp Leu 660 665 670 Ala Gly Val Leu Gln Val Ser Pro Asn Phe Leu Lys Asp Lys Ile Gly 675 680 685 Ser Asp Asp Asp Leu Phe Ile Ser Lys Trp Leu Val Glu His Ile Arg 690 695 700 Gly Phe Lys Lys Ala Cys Glu Asp Ser Leu Lys Ile Gln Lys Asp Asn 705 710 715 720 Arg Gly Leu Leu Asn His Lys Ile Asn Ile Ala Arg Asn Thr Lys Gly 725 730 735 Lys Cys Glu Lys Glu Ile Phe Asn Leu Ile Cys Lys Ile Glu Gly Ser 740 745 750 Glu Asp Lys Lys Gly Asn Tyr Lys His Gly Leu Ala Tyr Glu Leu Gly 755 760 765 Val Leu Leu Phe Gly Glu Pro Asn Glu Ala Ser Lys Pro Glu Phe Asp 770 775 780 Arg Lys Ile Lys Lys Phe Asn Ser Ile Tyr Ser Phe Ala Gln Ile Gln 785 790 795 800 Gln Ile Ala Phe Ala Glu Arg Lys Gly Asn Ala Asn Thr Cys Ala Val 805 810 815 Cys Ser Ala Asp Asn Ala His Arg Met Gln Gln Ile Lys Ile Thr Glu 820 825 830 Pro Val Glu Asp Asn Lys Asp Lys Ile Ile Leu Ser Ala Lys Ala Gln 835 840 845 Arg Leu Pro Ala Ile Pro Thr Arg Ile Val Asp Gly Ala Val Lys Lys 850 855 860 Met Ala Thr Ile Leu Ala Lys Asn Ile Val Asp Asp Asn Trp Gln Asn 865 870 875 880 Ile Lys Gln Val Leu Ser Ala Lys His Gln Leu His Ile Pro Ile Ile 885 890 895 Thr Glu Ser Asn Ala Phe Glu Phe Glu Pro Ala Leu Ala Asp Val Lys 900 905 910 Gly Lys Ser Leu Lys Asp Arg Arg Lys Lys Ala Leu Glu Arg Ile Ser 915 920 925 Pro Glu Asn Ile Phe Lys Asp Lys Asn Asn Arg Ile Lys Glu Phe Ala 930 935 940 Lys Gly Ile Ser Ala Tyr Ser Gly Ala Asn Leu Thr Asp Gly Asp Phe 945 950 955 960 Asp Gly Ala Lys Glu Glu Leu Asp Ala Ile Ile Pro Arg Ser His Lys 965 970 975 Lys Tyr Gly Thr Leu Asn Asp Glu Ala Asn Leu Ile Cys Val Thr Arg 980 985 990 Gly Asp Asn Lys Asn Lys Gly Asn Arg Ile Phe Cys Leu Arg Asp Leu 995 1000 1005 Ala Asp Asn Tyr Lys Leu Lys Gln Phe Glu Thr Thr Asp Asp Leu Glu 1010 1015 1020 Ile Glu Lys Lys Ile Ala Asp Thr Ile Trp Asp Ala Asn Lys Lys Asp 1025 1030 1035 1040 Phe Lys Phe Gly Asn Tyr Arg Ser Phe Ile Asn Leu Thr Pro Gln Glu 1045 1050 1055 Gln Lys Ala Phe Arg His Ala Leu Phe Leu Ala Asp Glu Asn Pro Ile 1060 1065 1070 Lys Gln Ala Val Ile Arg Ala Ile Asn Asn Arg Asn Arg Thr Phe Val 1075 1080 1085 Asn Gly Thr Gln Arg Tyr Phe Ala Glu Val Leu Ala Asn Asn Ile Tyr 1090 1095 1100 Leu Arg Ala Lys Lys Glu Asn Leu Asn Thr Asp Lys Ile Ser Phe Asp 1105 1110 1115 1120 Tyr Phe Gly Ile Pro Thr Ile Gly Asn Gly Arg Gly Ile Ala Glu Ile 1125 1130 1135 Arg Gln Leu Tyr Glu Lys Val Asp Ser Asp Ile Gln Ala Tyr Ala Lys 1140 1145 1150 Gly Asp Lys Pro Gln Ala Ser Tyr Ser His Leu Ile Asp Ala Met Leu 1155 1160 1165 Ala Phe Cys Ile Ala Ala Asp Glu His Arg Asn Asp Gly Ser Ile Gly 1170 1175 1180 Leu Glu Ile Asp Lys Asn Tyr Ser Leu Tyr Pro Leu Asp Lys Asn Thr 1185 1190 1195 1200 Gly Glu Val Phe Thr Lys Asp Ile Phe Ser Gln Ile Lys Ile Thr Asp 1205 1210 1215 Asn Glu Phe Ser Asp Lys Lys Leu Val Arg Lys Lys Ala Ile Glu Gly 1220 1225 1230 Phe Asn Thr His Arg Gln Met Thr Arg Asp Gly Ile Tyr Ala Glu Asn 1235 1240 1245 Tyr Leu Pro Ile Leu Ile His Lys Glu Leu Asn Glu Val Arg Lys Gly 1250 1255 1260 Tyr Thr Trp Lys Asn Ser Glu Glu Ile Lys Ile Phe Lys Gly Lys Lys 1265 1270 1275 1280 Tyr Asp Ile Gln Gln Leu Asn Asn Leu Val Tyr Cys Leu Lys Phe Val 1285 1290 1295 Asp Lys Pro Ile Ser Ile Asp Ile Gln Ile Ser Thr Leu Glu Glu Leu 1300 1305 1310 Arg Asn Ile Leu Thr Thr Asn Asn Ile Ala Ala Thr Ala Glu Tyr Tyr 1315 1320 1325 Tyr Ile Asn Leu Lys Thr Gln Lys Leu His Glu Tyr Tyr Ile Glu Asn 1330 1335 1340 Tyr Asn Thr Ala Leu Gly Tyr Lys Lys Tyr Ser Lys Glu Met Glu Phe 1345 1350 1355 1360 Leu Arg Ser Leu Ala Tyr Arg Ser Glu Arg Val Lys Ile Lys Ser Ile 1365 1370 1375 Asp Asp Val Lys Gln Val Leu Asp Lys Asp Ser Asn Phe Ile Ile Gly 1380 1385 1390 Lys Ile Thr Leu Pro Phe Lys Lys Glu Trp Gln Arg Leu Tyr Arg Glu 1395 1400 1405 Trp Gln Asn Thr Thr Ile Lys Asp Asp Tyr Glu Phe Leu Lys Ser Phe 1410 1415 1420 Phe Asn Val Lys Ser Ile Thr Lys Leu His Lys Lys Val Arg Lys Asp 1425 1430 1435 1440 Phe Ser Leu Pro Ile Ser Thr Asn Glu Gly Lys Phe Leu Val Lys Arg 1445 1450 1455 Lys Thr Trp Asp Asn Asn Phe Ile Tyr Gln Ile Leu Asn Asp Ser Asp 1460 1465 1470 Ser Arg Ala Asp Gly Thr Lys Pro Phe Ile Pro Ala Phe Asp Ile Ser 1475 1480 1485 Lys Asn Glu Ile Val Glu Ala Ile Ile Asp Ser Phe Thr Ser Lys Asn 1490 1495 1500 Ile Phe Trp Leu Pro Lys Asn Ile Glu Leu Gln Lys Val Asp Asn Lys 1505 1510 1515 1520 Asn Ile Phe Ala Ile Asp Thr Ser Lys Trp Phe Glu Val Glu Thr Pro 1525 1530 1535 Ser Asp Leu Arg Asp Ile Gly Ile Ala Thr Ile Gln Tyr Lys Ile Asp 1540 1545 1550 Asn Asn Ser Arg Pro Lys Val Arg Val Lys Leu Asp Tyr Val Ile Asp 1555 1560 1565 Asp Asp Ser Lys Ile Asn Tyr Phe Met Asn His Ser Leu Leu Lys Ser 1570 1575 1580 Arg Tyr Pro Asp Lys Val Leu Glu Ile Leu Lys Gln Ser Thr Ile Ile 1585 1590 1595 1600 Glu Phe Glu Ser Ser Gly Phe Asn Lys Thr Ile Lys Glu Met Leu Gly 1605 1610 1615 Met Lys Leu Ala Gly Ile Tyr Asn Glu Thr Ser Asn 1620 1625 <210> 3 <211> 677 <212> PRT <213> Artificial Sequence <220> <223> moloney murine leukemia virus RT <400> 3 Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 Glu Asn Ser Ser Pro 675 <210> 4 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> moloney murine leukemia virus RT <220> <221> MISC_FEATURE <222> (229) <223> Xaa can be any naturally occurring amino acid <400> 4 Ala Phe Pro Leu Glu Arg Pro Asp Trp Asp Tyr Thr Thr Gln Ala Gly 1 5 10 15 Arg Asn His Leu Val His Tyr Arg Gln Leu Leu Leu Ala Gly Leu Gln 20 25 30 Asn Ala Gly Arg Ser Pro Thr Asn Leu Ala Lys Val Lys Gly Ile Thr 35 40 45 Gln Gly Pro Asn Glu Ser Pro Ser Ala Phe Leu Glu Arg Leu Lys Glu 50 55 60 Ala Tyr Arg Arg Tyr Thr Pro Tyr Asp Pro Glu Asp Pro Gly Gln Glu 65 70 75 80 Thr Asn Val Ser Met Ser Phe Ile Trp Gln Ser Ala Pro Asp Ile Gly 85 90 95 Arg Lys Leu Gly Arg Leu Glu Asp Leu Lys Ser Lys Thr Leu Gly Asp 100 105 110 Leu Val Arg Glu Ala Glu Lys Ile Phe Asn Lys Arg Glu Thr Pro Glu 115 120 125 Glu Arg Glu Glu Arg Ile Arg Arg Glu Thr Glu Glu Lys Glu Glu Arg 130 135 140 Arg Arg Thr Val Asp Glu Gln Lys Glu Lys Glu Arg Asp Arg Arg Arg 145 150 155 160 His Arg Glu Met Ser Lys Leu Leu Ala Thr Val Val Ile Gly Gln Glu 165 170 175 Gln Asp Arg Gln Glu Gly Glu Arg Lys Arg Pro Gln Leu Asp Lys Asp 180 185 190 Gln Cys Ala Tyr Cys Lys Glu Lys Gly His Trp Ala Lys Asp Cys Pro 195 200 205 Lys Lys Pro Arg Gly Pro Arg Gly Pro Arg Pro Gln Thr Ser Leu Leu 210 215 220 Thr Leu Gly Asp Xaa Gly Gly Gln Gly Gln Asp Pro Pro Glu Pro 225 230 235 240 Arg Ile Thr Leu Lys Val Gly Gly Gln Pro Val Thr Phe Leu Val Asp 245 250 255 Thr Gly Ala Gln His Ser Val Leu Thr Gln Asn Pro Gly Pro Leu Ser 260 265 270 Asp Lys Ser Ala Trp Val Gln Gly Ala Thr Gly Gly Lys Arg Tyr Arg 275 280 285 Trp Thr Thr Asp Arg Lys Val His Leu Ala Thr Gly Lys Val Thr His 290 295 300 Ser Phe Leu His Val Pro Asp Cys Pro Tyr Pro Leu Leu Gly Arg Asp 305 310 315 320 Leu Leu Thr Lys Leu Lys Ala Gln Ile His Phe Glu Gly Ser Gly Ala 325 330 335 Gln Val Val Gly Pro Met Gly Gln Pro Leu Gln Val Leu Thr Leu Asn 340 345 350 Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val 355 360 365 Ser Leu Gly Phe Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu 370 375 380 Ser Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro 385 390 395 400 Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser 405 410 415 Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp 420 425 430 Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu 435 440 445 Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu 450 455 460 Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn 465 470 475 480 Pro Tyr Asn Leu Leu Ser Gly Leu Pro Ser His Gln Trp Tyr Thr 485 490 495 Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr 500 505 510 Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile 515 520 525 Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser 530 535 540 Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg 545 550 555 560 <210> 5 <211> 669 <212> PRT <213> Artificial Sequence <220> <223> Feline leukemia virus RT <400> 5 Thr Leu Gln Leu Glu Glu Glu Tyr Arg Leu Phe Glu Pro Glu Ser Thr 1 5 10 15 Gln Lys Gln Glu Met Asp Ile Trp Leu Lys Asn Phe Pro Gln Ala Trp 20 25 30 Ala Glu Thr Gly Gly Met Gly Thr Ala His Cys Gln Ala Pro Val Leu 35 40 45 Ile Gln Leu Lys Ala Thr Ala Thr Pro Ile Ser Ile Arg Gln Tyr Pro 50 55 60 Met Pro His Glu Ala Tyr Gln Gly Ile Lys Pro His Ile Arg Arg Met 65 70 75 80 Leu Asp Gln Gly Ile Leu Lys Pro Cys Gln Ser Pro Trp Asn Thr Pro 85 90 95 Leu Leu Pro Val Lys Lys Pro Gly Thr Glu Asp Tyr Arg Pro Val Gln 100 105 110 Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr Val 115 120 125 Pro Asn Pro Tyr Asn Leu Leu Ser Thr Leu Pro Ser His Pro Trp 130 135 140 Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His 145 150 155 160 Ser Glu Ser Gln Leu Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Ile 165 170 175 Gly Leu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys 180 185 190 Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Ser Asp Leu Ala Asp 195 200 205 Phe Arg Val Arg Tyr Pro Ala Leu Val Leu Leu Gln Tyr Val Asp Asp 210 215 220 Leu Leu Leu Ala Ala Ala Thr Arg Thr Glu Cys Leu Glu Gly Thr Lys 225 230 235 240 Ala Leu Leu Glu Thr Leu Gly Asn Lys Gly Tyr Arg Ala Ser Ala Lys 245 250 255 Lys Ala Gln Ile Cys Leu Gln Glu Val Thr Tyr Leu Gly Tyr Ser Leu 260 265 270 Lys Asp Gly Gln Arg Trp Leu Thr Lys Ala Arg Lys Glu Ala Ile Leu 275 280 285 Ser Ile Pro Val Pro Lys Asn Ser Arg Gln Val Arg Glu Phe Leu Gly 290 295 300 Thr Ala Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Leu Ala 305 310 315 320 Ala Pro Leu Tyr Pro Leu Thr Arg Pro Gly Thr Leu Phe Gln Trp Gly 325 330 335 Thr Glu Gln Gln Leu Ala Phe Glu Asp Ile Lys Lys Ala Leu Leu Ser 340 345 350 Ser Pro Ala Leu Gly Leu Pro Asp Ile Thr Lys Pro Phe Glu Leu Phe 355 360 365 Ile Asp Glu Asn Ser Gly Phe Ala Lys Gly Val Leu Val Gln Lys Leu 370 375 380 Gly Pro Trp Lys Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Thr 385 390 395 400 Val Ala Ser Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala 405 410 415 Ile Leu Val Lys Asp Ala Gly Lys Leu Thr Leu Gly Gln Pro Leu Thr 420 425 430 Ile Leu Thr Ser His Pro Val Glu Ala Leu Val Arg Gln Pro Pro Asn 435 440 445 Lys Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Met Leu Leu 450 455 460 Asp Ala Glu Arg Val His Phe Gly Pro Thr Val Ser Leu Asn Pro Ala 465 470 475 480 Thr Leu Leu Pro Leu Pro Ser Gly Gly Asn His His Asp Cys Leu Gln 485 490 495 Ile Leu Ala Glu Thr His Gly Thr Arg Pro Asp Leu Thr Asp Gln Pro 500 505 510 Leu Pro Asp Ala Asp Leu Thr Trp Tyr Thr Asp Gly Ser Ser Phe Ile 515 520 525 Arg Asn Gly Glu Arg Glu Ala Gly Ala Ala Val Thr Thr Glu Ser Glu 530 535 540 Val Ile Trp Ala Ala Pro Leu Pro Pro Gly Thr Ser Ala Gln Arg Ala 545 550 555 560 Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys 565 570 575 Leu Thr Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Thr His Val 580 585 590 His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys 595 600 605 Glu Ile Lys Asn Lys Asn Glu Ile Leu Ala Leu Leu Glu Ala Leu Phe 610 615 620 Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly 625 630 635 640 Asp Ser Pro Gln Ala Lys Gly Asn Arg Leu Ala Asp Asp Thr Ala Lys 645 650 655 Lys Ala Ala Thr Glu Thr His Ser Ser Leu Thr Val Leu 660 665 <210> 6 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> artificial sequence (RT) <220> <221> MISC_FEATURE <222> (280) <223> Xaa can be any naturally occurring amino acid <400> 6 Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met 1 5 10 15 Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys 20 25 30 Ala Leu Val Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser 35 40 45 Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys 50 55 60 Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Leu 65 70 75 80 Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His 85 90 95 Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val Gly 100 105 110 Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr 115 120 125 Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr 130 135 140 Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe 145 150 155 160 Gln Ser Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro 165 170 175 Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp 180 185 190 Leu Glu Ile Gly Gln His Arg Thr Lys Ile Glu Glu Leu Arg Gln His 195 200 205 Leu Leu Arg Trp Gly Leu Thr Thr Pro Asp Lys Lys His Gln Lys Glu 210 215 220 Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr 225 230 235 240 Val Gln Pro Ile Val Leu Pro Glu Lys Asp Ser Trp Thr Val Asn Asp 245 250 255 Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Pro 260 265 270 Gly Ile Lys Val Arg Gln Leu Xaa Lys Leu Leu Arg Gly Thr Lys Ala 275 280 285 Leu Thr Glu Val Ile Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala 290 295 300 Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Val Tyr Tyr Asp 305 310 315 320 Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln 325 330 335 Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly 340 345 350 Lys Tyr Ala Arg Met Arg Gly Ala His Thr Asn Asp Val Lys Gln Leu 355 360 365 Thr Glu Ala Val Gln Lys Ile Thr Thr Glu Ser Ile Val Ile Trp Gly 370 375 380 Lys Thr Pro Lys Phe Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Thr 385 390 395 400 Trp Trp Thr Glu Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe 405 410 415 Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu 420 425 430 Pro Ile Val Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn Arg 435 440 445 Glu Thr Lys Leu Gly Lys Ala Gly Tyr Val Thr Asn Arg Gly Arg Gln 450 455 460 Lys Val Val Thr Leu Thr Asp Thr Thr Asn Gln Lys Thr Glu Leu Gln 465 470 475 480 Ala Ile Tyr Leu Ala Leu Gln Asp Ser Gly Leu Glu Val Asn Ile Val 485 490 495 Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp Gln 500 505 510 Ser Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gln Leu Ile Lys Lys 515 520 525 Glu Lys Val Tyr Leu Ala Trp Val Pro Ala His Lys Gly Ile Gly Gly 530 535 540 Asn Glu Gln Val Asp Lys Leu Val Ser Ala Gly Ile Arg Lys Val Leu 545 550 555 560 <210> 7 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> artificial sequence (RT) <400> 7 Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met 1 5 10 15 Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys 20 25 30 Ala Leu Val Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser 35 40 45 Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys 50 55 60 Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Leu 65 70 75 80 Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His 85 90 95 Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val Gly 100 105 110 Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr 115 120 125 Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr 130 135 140 Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe 145 150 155 160 Gln Ser Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro 165 170 175 Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp 180 185 190 Leu Glu Ile Gly Gln His Arg Thr Lys Ile Glu Glu Leu Arg Gln His 195 200 205 Leu Leu Arg Trp Gly Leu Thr Thr Pro Asp Lys Lys His Gln Lys Glu 210 215 220 Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr 225 230 235 240 Val Gln Pro Ile Val Leu Pro Glu Lys Asp Ser Trp Thr Val Asn Asp 245 250 255 Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Pro 260 265 270 Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala 275 280 285 Leu Thr Glu Val Ile Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala 290 295 300 Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Val Tyr Tyr Asp 305 310 315 320 Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln 325 330 335 Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly 340 345 350 Lys Tyr Ala Arg Met Arg Gly Ala His Thr Asn Asp Val Lys Gln Leu 355 360 365 Thr Glu Ala Val Gln Lys Ile Thr Thr Glu Ser Ile Val Ile Trp Gly 370 375 380 Lys Thr Pro Lys Phe Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Thr 385 390 395 400 Trp Trp Thr Glu Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe 405 410 415 Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu 420 425 430 Pro Ile Val Gly Ala Glu Thr Phe 435 440 <210> 8 <211> 858 <212> PRT <213> Artificial Sequence <220> <223> rous sarcoma virus RT <400> 8 Thr Val Ala Leu His Leu Ala Ile Pro Leu Lys Trp Lys Pro Asn His 1 5 10 15 Thr Pro Val Trp Ile Asp Gln Trp Pro Leu Pro Glu Gly Lys Leu Val 20 25 30 Ala Leu Thr Gln Leu Val Glu Lys Glu Leu Gln Leu Gly His Ile Glu 35 40 45 Pro Ser Leu Ser Cys Trp Asn Thr Pro Val Phe Val Ile Arg Lys Ala 50 55 60 Ser Gly Ser Tyr Arg Leu Leu His Asp Leu Arg Ala Val Asn Ala Lys 65 70 75 80 Leu Val Pro Phe Gly Ala Val Gln Gln Gly Ala Pro Val Leu Ser Ala 85 90 95 Leu Pro Arg Gly Trp Pro Leu Met Val Leu Asp Leu Lys Asp Cys Phe 100 105 110 Phe Ser Ile Pro Leu Ala Glu Gln Asp Arg Glu Ala Phe Ala Phe Thr 115 120 125 Leu Pro Ser Val Asn Asn Gln Ala Pro Ala Arg Arg Phe Gln Trp Lys 130 135 140 Val Leu Pro Gln Gly Met Thr Cys Ser Pro Thr Ile Cys Gln Leu Ile 145 150 155 160 Val Gly Gln Ile Leu Glu Pro Leu Arg Leu Lys His Pro Ser Leu Arg 165 170 175 Met Leu His Tyr Met Asp Asp Leu Leu Leu Ala Ala Ser Ser His Asp 180 185 190 Gly Leu Glu Ala Ala Gly Glu Glu Val Ile Ser Thr Leu Glu Arg Ala 195 200 205 Gly Phe Thr Ile Ser Pro Asp Lys Val Gln Lys Glu Pro Gly Val Gln 210 215 220 Tyr Leu Gly Tyr Lys Leu Gly Ser Thr Tyr Ala Ala Pro Val Gly Leu 225 230 235 240 Val Ala Glu Pro Arg Ile Ala Thr Leu Trp Asp Val Gln Lys Leu Val 245 250 255 Gly Ser Leu Gln Trp Leu Arg Pro Ala Leu Gly Ile Pro Pro Arg Leu 260 265 270 Arg Gly Pro Phe Tyr Glu Gln Leu Arg Gly Ser Asp Pro Asn Glu Ala 275 280 285 Arg Glu Trp Asn Leu Asp Met Lys Met Ala Trp Arg Glu Ile Val Gln 290 295 300 Leu Ser Thr Thr Ala Ala Leu Glu Arg Trp Asp Pro Ala Leu Pro Leu 305 310 315 320 Glu Gly Ala Val Ala Arg Cys Glu Gin Gly Ala Ile Gly Val Leu Gly 325 330 335 Gln Gly Leu Ser Thr His Pro Arg Pro Cys Leu Trp Leu Phe Ser Thr 340 345 350 Gln Pro Thr Lys Ala Phe Thr Ala Trp Leu Glu Val Leu Thr Leu Leu 355 360 365 Ile Thr Lys Leu Arg Ala Ser Ala Val Arg Thr Phe Gly Lys Glu Val 370 375 380 Asp Ile Leu Leu Leu Pro Ala Cys Phe Arg Asp Glu Leu Pro Leu Pro 385 390 395 400 Glu Gly Ile Leu Leu Ala Leu Arg Gly Phe Ala Gly Lys Ile Arg Ser 405 410 415 Ser Asp Thr Pro Ser Ile Phe Asp Ile Ala Arg Pro Leu His Val Ser 420 425 430 Leu Lys Val Arg Val Thr Asp His Pro Val Pro Gly Pro Thr Val Phe 435 440 445 Thr Asp Ala Ser Ser Ser Thr His Lys Gly Val Val Val Trp Arg Glu 450 455 460 Gly Pro Arg Trp Glu Ile Lys Glu Ile Ala Asp Leu Gly Ala Ser Val 465 470 475 480 Gln Gln Leu Glu Ala Arg Ala Val Ala Met Ala Leu Leu Leu Trp Pro 485 490 495 Thr Thr Pro Thr Asn Val Val Thr Asp Ser Ala Phe Val Ala Lys Met 500 505 510 Leu Leu Lys Met Gly Gin Glu Gly Val Pro Ser Thr Ala Ala Ala Phe 515 520 525 Ile Leu Glu Asp Ala Leu Ser Gln Arg Ser Ala Met Ala Ala Val Leu 530 535 540 His Val Arg Ser His Ser Glu Val Pro Gly Phe Phe Thr Glu Gly Asn 545 550 555 560 Asp Val Ala Asp Ser Gln Ala Thr Phe Gln Ala Tyr Pro Leu Arg Glu 565 570 575 Ala Lys Asp Leu His Thr Ala Leu His Ile Gly Pro Arg Ala Leu Ser 580 585 590 Lys Ala Cys Asn Ile Ser Met Gln Gln Ala Arg Glu Val Val Gln Thr 595 600 605 Cys Pro His Cys Asn Ser Ala Pro Ala Leu Glu Ala Gly Val Asn Pro 610 615 620 Arg Gly Leu Gly Pro Leu Gln Ile Trp Gln Thr Asp Phe Thr Leu Glu 625 630 635 640 Pro Arg Met Ala Pro Arg Ser Trp Leu Ala Val Thr Val Asp Thr Ala 645 650 655 Ser Ser Ala Ile Val Val Thr Gln His Gly Arg Val Thr Ser Val Ala 660 665 670 Ala Gln His His Trp Ala Thr Val Ile Ala Val Leu Gly Arg Pro Lys 675 680 685 Ala Ile Lys Thr Asp Asn Gly Ser Cys Phe Thr Ser Lys Ser Thr Arg 690 695 700 Glu Trp Leu Ala Arg Trp Gly Ile Ala His Thr Thr Gly Ile Pro Gly 705 710 715 720 Asn Ser Gln Gly Gln Ala Met Val Glu Arg Ala Asn Arg Leu Leu Lys 725 730 735 Asp Lys Ile Arg Val Leu Ala Glu Gly Asp Gly Phe Met Lys Arg Ile 740 745 750 Pro Thr Ser Lys Gln Gly Glu Leu Leu Ala Lys Ala Met Tyr Ala Leu 755 760 765 Asn His Phe Glu Arg Gly Glu Asn Thr Lys Thr Pro Ile Gln Lys His 770 775 780 Trp Arg Pro Thr Val Leu Thr Glu Gly Pro Pro Val Lys Ile Arg Ile 785 790 795 800 Glu Thr Gly Glu Trp Glu Lys Gly Trp Asn Val Leu Val Trp Gly Arg 805 810 815 Gly Tyr Ala Ala Val Lys Asn Arg Asp Thr Asp Lys Val Ile Trp Val 820 825 830 Pro Ser Arg Lys Val Lys Pro Asp Ile Ala Gln Lys Asp Glu Val Thr 835 840 845 Lys Lys Asp Glu Ala Ser Pro Leu Phe Ala 850 855 <210> 9 <211> 675 <212> PRT <213> Artificial Sequence <220> <223> cauliflower mosaic virus RT <400> 9 Met Met Asp His Leu Leu Gln Lys Thr Gln Ile Gln Asn Gln Thr Glu 1 5 10 15 Gln Val Met Asn Ile Thr Asn Pro Asn Ser Ile Tyr Ile Lys Gly Arg 20 25 30 Leu Tyr Phe Lys Gly Tyr Lys Lys Ile Glu Leu His Cys Phe Val Asp 35 40 45 Thr Gly Ala Ser Leu Cys Ile Ala Ser Lys Phe Val Ile Pro Glu Glu 50 55 60 His Trp Ile Asn Ala Glu Arg Pro Ile Met Val Lys Ile Ala Asp Gly 65 70 75 80 Ser Ser Ile Thr Ile Asn Lys Val Cys Arg Asp Ile Asp Leu Ile Ile 85 90 95 Ala Gly Glu Ile Phe His Ile Pro Thr Val Tyr Gln Gln Glu Ser Gly 100 105 110 Ile Asp Phe Ile Ile Gly Asn Asn Phe Cys Gln Leu Tyr Glu Pro Phe 115 120 125 Ile Gln Phe Thr Asp Arg Val Ile Phe Thr Lys Asp Arg Thr Tyr Pro 130 135 140 Val His Ile Ala Lys Leu Thr Arg Ala Val Arg Val Gly Thr Glu Gly 145 150 155 160 Phe Leu Glu Ser Met Lys Lys Arg Ser Lys Thr Gln Gln Pro Glu Pro 165 170 175 Val Asn Ile Ser Thr Asn Lys Ile Ala Ile Leu Ser Glu Gly Arg Arg 180 185 190 Leu Ser Glu Glu Lys Leu Phe Ile Thr Gln Gln Arg Met Gln Lys Ile 195 200 205 Glu Glu Leu Leu Glu Lys Val Cys Ser Glu Asn Pro Leu Asp Pro Asn 210 215 220 Lys Thr Lys Gln Trp Met Lys Ala Ser Ile Lys Leu Ser Asp Pro Ser 225 230 235 240 Lys Ala Ile Lys Val Lys Pro Met Lys Tyr Ser Pro Met Asp Arg Glu 245 250 255 Glu Phe Asp Lys Gln Ile Lys Glu Leu Leu Asp Leu Lys Val Ile Lys 260 265 270 Pro Ser Lys Ser Pro His Met Ala Pro Ala Phe Leu Val Asn Asn Glu 275 280 285 Ala Glu Lys Arg Arg Gly Lys Lys Arg Met Val Val Asn Tyr Lys Ala 290 295 300 Met Asn Lys Ala Thr Val Gly Asp Ala Tyr Asn Leu Pro Asn Lys Asp 305 310 315 320 Glu Leu Leu Thr Leu Ile Arg Gly Lys Lys Ile Phe Ser Ser Phe Asp 325 330 335 Cys Lys Ser Gly Phe Trp Gln Val Leu Leu Asp Gln Asp Ser Arg Pro 340 345 350 Leu Thr Ala Phe Thr Cys Pro Gin Gly His Tyr Glu Trp Asn Val Val 355 360 365 Pro Phe Gly Leu Lys Gln Ala Pro Ser Ile Phe Gln Arg His Met Asp 370 375 380 Glu Ala Phe Arg Val Phe Arg Lys Phe Cys Cys Val Tyr Val Asp Asp 385 390 395 400 Ile Leu Val Phe Ser Asn Asn Glu Glu Asp His Leu Leu His Val Ala 405 410 415 Met Ile Leu Gln Lys Cys Asn Gln His Gly Ile Ile Leu Ser Lys Lys 420 425 430 Lys Ala Gln Leu Phe Lys Lys Lys Ile Asn Phe Leu Gly Leu Glu Ile 435 440 445 Asp Glu Gly Thr His Lys Pro Gin Gly His Ile Leu Glu His Ile Asn 450 455 460 Lys Phe Pro Asp Thr Leu Glu Asp Lys Lys Gln Leu Gln Arg Phe Leu 465 470 475 480 Gly Ile Leu Thr Tyr Ala Ser Asp Tyr Ile Pro Lys Leu Ala Gln Ile 485 490 495 Arg Lys Pro Leu Gln Ala Lys Leu Lys Glu Asn Val Pro Trp Lys Trp 500 505 510 Thr Lys Glu Asp Thr Leu Tyr Met Gln Lys Val Lys Lys Asn Leu Gln 515 520 525 Gly Phe Pro Pro Leu His His Pro Leu Pro Glu Glu Lys Leu Ile Ile 530 535 540 Glu Thr Asp Ala Ser Asp Asp Tyr Trp Gly Gly Met Leu Lys Ala Ile 545 550 555 560 Lys Ile Asn Glu Gly Thr Asn Thr Glu Leu Ile Cys Arg Tyr Ala Ser 565 570 575 Gly Ser Phe Lys Ala Ala Glu Lys Asn Tyr His Ser Asn Asp Lys Glu 580 585 590 Thr Leu Ala Val Ile Asn Thr Ile Lys Lys Phe Ser Ile Tyr Leu Thr 595 600 605 Pro Val His Phe Leu Ile Arg Thr Asp Asn Thr His Phe Lys Ser Phe 610 615 620 Val Asn Leu Asn Tyr Lys Gly Asp Ser Lys Leu Gly Arg Asn Ile Arg 625 630 635 640 Trp Gln Ala Trp Leu Ser His Tyr Ser Phe Asp Val Glu His Ile Lys 645 650 655 Gly Thr Asp Asn His Phe Ala Asp Phe Leu Ser Arg Glu Phe Asn Arg 660 665 670 Val Asn Ser 675 <210> 10 <211> 366 <212> PRT <213> Artificial Sequence <220> <223> Klebsiella pneuomiae RT <400> 10 Met Lys Glu Lys Ile Ser Lys Ile Asp Lys Asn Phe Tyr Thr Asp Ile 1 5 10 15 Phe Ile Lys Thr Ser Phe Gln Asn Glu Phe Glu Ala Gly Gly Val Ile 20 25 30 Pro Pro Ile Ala Lys Asn Gln Val Ser Thr Ile Ser Asn Lys Asn Lys 35 40 45 Thr Phe Tyr Ser Leu Ala His Ser Ser Pro His Tyr Ser Ile Gln Thr 50 55 60 Arg Ile Glu Lys Phe Leu Leu Lys Asn Ile Pro Leu Ser Ala Ser Ser 65 70 75 80 Phe Ala Phe Arg Lys Glu Arg Ser Tyr Leu His Tyr Leu Glu Pro His 85 90 95 Thr Gln Asn Val Lys Tyr Cys His Leu Asp Ile Val Ser Phe Phe His 100 105 110 Ser Ile Asp Val Asn Ile Val Arg Asp Thr Phe Ser Val Tyr Phe Ser 115 120 125 Asp Glu Phe Leu Val Lys Glu Lys Gln Ser Leu Leu Asp Ala Phe Met 130 135 140 Ala Ser Val Thr Leu Thr Ala Glu Leu Asp Gly Val Glu Lys Thr Phe 145 150 155 160 Ile Pro Met Gly Phe Lys Ser Ser Pro Ser Ile Ser Asn Ile Ile Phe 165 170 175 Arg Lys Ile Asp Ile Leu Ile Gln Lys Phe Cys Asp Lys Asn Lys Ile 180 185 190 Thr Tyr Thr Arg Tyr Ala Asp Asp Leu Leu Phe Ser Thr Lys Lys Glu 195 200 205 Asn Asn Ile Leu Ser Ser Thr Phe Phe Ile Asn Glu Ile Ser Ser Ile 210 215 220 Leu Ser Ile Asn Lys Phe Lys Leu Asn Lys Ser Lys Tyr Leu Tyr Lys 225 230 235 240 Glu Gly Thr Ile Ser Leu Gly Gly Tyr Val Ile Glu Asn Ile Leu Lys 245 250 255 Asp Asn Ser Ser Gly Asn Ile Arg Leu Ser Ser Ser Lys Leu Asn Pro 260 265 270 Leu Tyr Lys Ala Leu Tyr Glu Ile Lys Lys Gly Ser Ser Ser Lys His 275 280 285 Ile Cys Ile Lys Val Phe Asn Leu Lys Leu Lys Arg Phe Ile Tyr Lys 290 295 300 Lys Asn Lys Glu Lys Phe Glu Ala Lys Phe Tyr Ser Ser Gln Leu Lys 305 310 315 320 Asn Lys Leu Leu Gly Tyr Arg Ser Tyr Leu Leu Ser Phe Val Ile Phe 325 330 335 His Lys Lys Tyr Lys Cys Ile Asn Pro Ile Phe Leu Glu Lys Cys Val 340 345 350 Phe Leu Ile Ser Glu Ile Glu Ser Ile Met Asn Arg Lys Phe 355 360 365 <210> 11 <211> 263 <212> PRT <213> Artificial Sequence <220> <223> Escerichia coli RT <400> 11 Met Lys Ile Thr Ser Asn Asn Val Thr Ala Val Ile Asn Gly Lys Gly 1 5 10 15 Trp His Ser Ile Asn Trp Lys Lys Cys His Gln His Val Lys Thr Ile 20 25 30 Gln Thr Arg Ile Ala Lys Ala Ala Cys Asn Gln Gln Trp Arg Thr Val 35 40 45 Gly Arg Leu Gln Arg Leu Leu Val Arg Ser Phe Ser Ala Arg Ala Leu 50 55 60 Ala Val Lys Arg Val Thr Glu Asn Ser Gly Arg Lys Thr Pro Gly Val 65 70 75 80 Asp Gly Gln Ile Trp Ser Thr Pro Glu Ser Lys Trp Glu Ala Ile Phe 85 90 95 Lys Leu Arg Arg Lys Gly Tyr Lys Pro Leu Pro Leu Lys Arg Val Phe 100 105 110 Ile Pro Lys Ser Asn Gly Lys Lys Arg Pro Leu Gly Ile Pro Val Met 115 120 125 Leu Asp Arg Ala Met Gln Ala Leu His Leu Leu Gly Leu Glu Pro Val 130 135 140 Ser Glu Thr Asn Ala Asp His Asn Ser Tyr Gly Phe Arg Pro Ala Arg 145 150 155 160 Cys Thr Ala Asp Ala Ile Gln Gln Val Cys Asn Met Tyr Ser Ser Arg 165 170 175 Asn Ala Ser Lys Trp Val Leu Glu Gly Asp Ile Lys Gly Cys Phe Glu 180 185 190 His Ile Ser His Glu Trp Leu Leu Glu Asn Ile Pro Met Asp Lys Gln 195 200 205 Ile Leu Arg Asn Trp Leu Lys Ala Gly Ile Ile Glu Lys Ser Ile Phe 210 215 220 Ser Lys Thr Leu Ser Gly Thr Pro Gln Gly Gly Ile Ile Ser Pro Val 225 230 235 240 Leu Ala Asn Met Ala Leu Asp Gly Leu Glu Arg Leu Leu Gln Asn Arg 245 250 255 Phe Gly Arg Asn Arg Leu Ile 260 <210> 12 <211> 438 <212> PRT <213> Artificial Sequence <220> <223> Bacillus subtilis RT <400> 12 Met Ser Lys Ile Lys Ile Asn Tyr Glu Lys Tyr His Ile Lys Pro Phe 1 5 10 15 Pro His Phe Asp Gln Arg Ile Lys Val Asn Lys Lys Val Lys Glu Asn 20 25 30 Leu Gln Asn Pro Phe Tyr Ile Ala Ala His Ser Phe Tyr Pro Phe Ile 35 40 45 His Tyr Lys Lys Ile Ser Tyr Lys Phe Lys Asn Gly Thr Leu Ser Ser 50 55 60 Pro Lys Glu Arg Asp Ile Phe Tyr Ser Gly His Met Asp Gly Tyr Ile 65 70 75 80 Tyr Lys His Tyr Gly Glu Ile Leu Asn His Lys Tyr Asn Asn Thr Cys 85 90 95 Ile Gly Lys Gly Ile Asp His Val Ser Leu Ala Tyr Arg Asn Asn Lys 100 105 110 Met Gly Lys Ser Asn Ile His Phe Ala Ala Glu Val Ile Asn Phe Ile 115 120 125 Ser Glu Gln Gln Gln Ala Phe Ile Phe Val Ser Asp Phe Ser Ser Tyr 130 135 140 Phe Asp Ser Leu Asp His Ala Ile Leu Lys Glu Lys Leu Ile Glu Val 145 150 155 160 Leu Glu Glu Gln Asp Lys Leu Ser Lys Asp Trp Trp Asn Val Phe Lys 165 170 175 His Ile Thr Arg Tyr Asn Trp Val Glu Lys Glu Glu Val Ile Ser Asp 180 185 190 Leu Glu Cys Thr Lys Glu Lys Ile Ala Arg Asp Lys Lys Ser Arg Glu 195 200 205 Arg Tyr Tyr Thr Pro Ala Glu Phe Arg Glu Phe Arg Lys Arg Val Asn 210 215 220 Ile Lys Ser Asn Asp Thr Gly Val Gly Ile Pro Gln Gly Thr Ala Ile 225 230 235 240 Ser Ala Val Leu Ala Asn Val Tyr Ala Ile Asp Leu Asp Gln Lys Leu 245 250 255 Asn Gln Tyr Ala Leu Lys Tyr Gly Gly Ile Tyr Arg Arg Tyr Ser Asp 260 265 270 Asp Ile Ile Met Val Leu Pro Met Thr Ser Asp Gly Gln Asp Pro Ser 275 280 285 Asn Asp His Val Ser Phe Ile Lys Ser Val Val Lys Arg Asn Lys Val 290 295 300 Thr Met Gly Asp Ser Lys Thr Ser Val Leu Tyr Tyr Ala Asn Asn Asn 305 310 315 320 Ile Tyr Glu Asp Tyr Gln Arg Lys Arg Glu Ser Lys Met Asp Tyr Leu 325 330 335 Gly Phe Ser Phe Asp Gly Met Thr Val Lys Ile Arg Glu Lys Ser Leu 340 345 350 Phe Lys Tyr Tyr His Arg Thr Tyr Lys Lys Ile Asn Ser Ile Asn Trp 355 360 365 Ala Ser Val Lys Lys Glu Lys Lys Val Gly Arg Lys Lys Leu Tyr Leu 370 375 380 Leu Tyr Ser His Leu Gly Arg Asn Tyr Lys Gly His Gly Asn Phe Ile 385 390 395 400 Ser Tyr Cys Lys Lys Ala His Ala Val Phe Glu Gly Asn Lys Lys Ile 405 410 415 Glu Ser Leu Ile Asn Gln Gln Ile Lys Arg His Trp Lys Lys Ile Gln 420 425 430 Lys Arg Leu Val Asp Val 435 <210> 13 <211> 426 <212> PRT <213> Artificial Sequence <220> <223> Eubacterium rectale RT <400> 13 Asp Thr Ser Asn Leu Met Glu Gln Ile Leu Ser Ser Asp Asn Leu Asn 1 5 10 15 Arg Ala Tyr Leu Gln Val Val Arg Asn Lys Gly Ala Glu Gly Val Asp 20 25 30 Gly Met Lys Tyr Thr Glu Leu Lys Glu His Leu Ala Lys Asn Gly Glu 35 40 45 Thr Ile Lys Gly Gln Leu Arg Thr Arg Lys Tyr Lys Pro Gln Pro Ala 50 55 60 Arg Arg Val Glu Ile Pro Lys Pro Asp Gly Gly Val Arg Asn Leu Gly 65 70 75 80 Val Pro Thr Val Thr Asp Arg Phe Ile Gln Gln Ala Ile Ala Gln Val 85 90 95 Leu Thr Pro Ile Tyr Glu Glu Gln Phe His Asp His Ser Tyr Gly Phe 100 105 110 Arg Pro Asn Arg Cys Ala Gln Gln Ala Ile Leu Thr Ala Leu Asn Ile 115 120 125 Met Asn Asp Gly Asn Asp Trp Ile Val Asp Ile Asp Leu Glu Lys Phe 130 135 140 Phe Asp Thr Val Asn His Asp Lys Leu Met Thr Leu Ile Gly Arg Thr 145 150 155 160 Ile Lys Asp Gly Asp Val Ile Ser Ile Val Arg Lys Tyr Leu Val Ser 165 170 175 Gly Ile Met Ile Asp Asp Glu Tyr Glu Asp Ser Ile Val Gly Thr Pro 180 185 190 Gln Gly Gly Asn Leu Ser Pro Leu Leu Ala Asn Ile Met Leu Asn Glu 195 200 205 Leu Asp Lys Glu Met Glu Lys Arg Gly Leu Asn Phe Val Arg Tyr Ala 210 215 220 Asp Asp Cys Ile Ile Met Val Gly Ser Glu Met Ser Ala Asn Arg Val 225 230 235 240 Met Arg Asn Ile Ser Arg Phe Ile Glu Glu Lys Leu Gly Leu Lys Val 245 250 255 Asn Met Thr Lys Ser Lys Val Asp Arg Pro Ser Gly Leu Lys Tyr Leu 260 265 270 Gly Phe Gly Phe Tyr Phe Asp Pro Arg Ala His Gln Phe Lys Ala Lys 275 280 285 Pro His Ala Lys Ser Val Ala Lys Phe Lys Lys Arg Met Lys Glu Leu 290 295 300 Thr Cys Arg Ser Trp Gly Val Ser Asn Ser Tyr Lys Val Glu Lys Leu 305 310 315 320 Asn Gln Leu Ile Arg Gly Trp Ile Asn Tyr Phe Lys Ile Gly Ser Met 325 330 335 Lys Thr Leu Cys Lys Glu Leu Asp Ser Arg Ile Arg Tyr Arg Leu Arg 340 345 350 Met Cys Ile Trp Lys Gln Trp Lys Thr Pro Gln Asn Gln Glu Lys Asn 355 360 365 Leu Val Lys Leu Gly Ile Asp Arg Asn Thr Ala Arg Arg Val Ala Tyr 370 375 380 Thr Gly Lys Arg Ile Ala Tyr Val Cys Asn Lys Gly Ala Val Asn Val 385 390 395 400 Ala Ile Ser Asn Lys Arg Leu Ala Ser Phe Gly Leu Ile Ser Met Leu 405 410 415 Asp Tyr Tyr Ile Glu Lys Cys Val Thr Cys 420 425 <210> 14 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> Geobacillus stearothermophilus <400> 14 Ala Leu Leu Glu Arg Ile Leu Ala Arg Asp Asn Leu Ile Thr Ala Leu 1 5 10 15 Lys Arg Val Glu Ala Asn Gln Gly Ala Pro Gly Ile Asp Gly Val Ser 20 25 30 Thr Asp Gln Leu Arg Asp Tyr Ile Arg Ala His Trp Ser Thr Ile His 35 40 45 Ala Gln Leu Leu Ala Gly Thr Tyr Arg Pro Ala Pro Val Arg Arg Val 50 55 60 Glu Ile Pro Lys Pro Gly Gly Gly Thr Arg Gln Leu Gly Ile Pro Thr 65 70 75 80 Val Val Asp Arg Leu Ile Gln Gln Ala Ile Leu Gln Glu Leu Thr Pro 85 90 95 Ile Phe Asp Pro Asp Phe Ser Ser Ser Ser Phe Gly Phe Arg Pro Gly 100 105 110 Arg Asn Ala His Asp Ala Val Arg Gln Ala Gln Gly Tyr Ile Gln Glu 115 120 125 Gly Tyr Arg Tyr Val Val Asp Met Asp Leu Glu Lys Phe Phe Asp Arg 130 135 140 Val Asn His Asp Ile Leu Met Ser Arg Val Ala Arg Lys Val Lys Asp 145 150 155 160 Lys Arg Val Leu Lys Leu Ile Arg Ala Tyr Leu Gln Ala Gly Val Met 165 170 175 Ile Glu Gly Val Lys Val Gln Thr Glu Glu Gly Thr Pro Gln Gly Gly 180 185 190 Pro Leu Ser Pro Leu Leu Ala Asn Ile Leu Leu Asp Asp Leu Asp Lys 195 200 205 Glu Leu Glu Lys Arg Gly Leu Lys Phe Cys Arg Tyr Ala Asp Asp Cys 210 215 220 Asn Ile Tyr Val Lys Ser Leu Arg Ala Gly Gln Arg Val Lys Gln Ser 225 230 235 240 Ile Gln Arg Phe Leu Glu Lys Thr Leu Lys Leu Lys Val Asn Glu Glu 245 250 255 Lys Ser Ala Val Asp Arg Pro Trp Lys Arg Ala Phe Leu Gly Phe Ser 260 265 270 Phe Thr Pro Glu Arg Lys Ala Arg Ile Arg Leu Ala Pro Arg Ser Ile 275 280 285 Gln Arg Leu Lys Gln Arg Ile Arg Gln Leu Thr Asn Pro Asn Trp Ser 290 295 300 Ile Ser Met Pro Glu Arg Ile His Arg Val Asn Gln Tyr Val Met Gly 305 310 315 320 Trp Ile Gly Tyr Phe Arg Leu Val Glu Thr Pro Ser Val Leu Gln Thr 325 330 335 Ile Glu Gly Trp Ile Arg Arg Arg Leu Arg Leu Cys Gln Trp Leu Gln 340 345 350 Trp Lys Arg Val Arg Thr Arg Ile Arg Glu Leu Arg Ala Leu Gly Leu 355 360 365 Lys Glu Thr Ala Val Met Glu Ile Ala Asn Thr Arg Lys Gly Ala Trp 370 375 380 Arg Thr Thr Lys Thr Pro Gln Leu His Gln Ala Leu Gly Lys Thr Tyr 385 390 395 400 Trp Thr Ala Gln Gly Leu Lys Ser Leu Thr Gln Arg 405 410 <210> 15 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> moloney murine leukemia virus mutant RT <400> 15 Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asn Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Lys Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Trp Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 Glu Asn Ser Ser Pro Ser Gly 675 <210> 16 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 16 Gly Gly Ser One <210> 17 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 17 Gly Gly Ser Gly Gly Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 18 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 19 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 19 Ser Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 Ser <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 20 Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1 5 10 15 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 21 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 22 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 22 Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys 1 5 10 15 Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys 20 25 30 <210> 23 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> FnCas9 scaffold <400> 23 gtttcagttg ctgaattatt tggtaaacag taccaaataa ttaatgctct gtaatcattt 60 aaaagtattt tgaacggacc tctgtttgac acgtctgaat aactaaaaa 109 <210> 24 <211> 4884 <212> DNA <213> Artificial Sequence <220> <223> Francisella novicida Cas9 (H969A) <400> 24 atgaacttca agatcctgcc tatcgccatc gacctgggcg tgaagaacac cggcgtgttc 60 agcgccttct accagaaggg caccagcctg gaacggctgg acaacaagaa cggcaaggtg 120 tacgagctga gcaaggacag ctacaccctg ctgatgaaca accggaccgc cagacggcac 180 cagcggagag gcatcgacag aaagcagctc gtgaagcggc tgttcaagct gatctggacc 240 gagcagctga acctggaatg ggacaaggac acccagcagg ccatcagctt tctgttcaac 300 agacggggct tcagcttcat caccgacggc tacagccccg agtacctgaa catcgtgccc 360 gaacaagtga aggccatcct gatggacatc ttcgacgact acaacggcga ggacgacctg 420 gactcctacc tgaagctggc caccgagcag gaaagcaaga tcagcgaaat ctacaacaag 480 ctgatgcaga agatcctgga gtttaagctg atgaagctgt gcaccgacat caaggacgat 540 aaggtgtcca ccaagaccct gaaagagatc accagctacg agttcgagct gctggccgac 600 tacctggcca actacagcga gagcctgaaa acccagaagt tctcctacac cgacaagcag 660 ggcaatctga aagagctgag ctactaccac cacgacaagt acaacatcca ggaatttctg 720 aagcggcacg ccaccatcaa cgacagaatc ctggacacac tgctgaccga cgatctggac 780 atctggaact tcaacttcga gaagttcgac ttcgacaaga acgaggaaaa gctgcagaac 840 caggaagata aggaccacat ccaggcccat ctgcaccact tcgtgttcgc cgtgaacaag 900 atcaagagcg agatggccag cggcggcaga cacagaagcc agtacttcca ggaaatcacc 960 aacgtgctgg acgagaacaa ccaccaggaa ggctatctga agaatttctg cgagaacctg 1020 cacaacaaaa agtacagcaa cctgtccgtg aagaatctcg tgaacctgat cggcaacctg 1080 agcaacctgg aactgaagcc tctgcgcaag tacttcaacg acaagatcca cgccaaggcc 1140 gaccactggg acgagcagaa gttcaccgag acatactgcc actggattct gggcgagtgg 1200 cgcgtgggag tgaaggacca ggacaagaag gacggcgcca agtactccta caaggacctg 1260 tgcaacgagc tgaaacagaa agtgaccaag gccggactgg tggacttcct gctggaactg 1320 gacccctgca ggaccatccc cccctacctg gataacaaca atcggaagcc ccccaagtgc 1380 cagagcctga tcctgaaccc caagttcctg gacaaccagt accccaactg gcagcagtat 1440 ctgcaggaac tgaaaaaact gcagagcatc cagaactacc tggacagctt cgagacagac 1500 ctgaaggtgc tgaagtccag caaggaccag ccctacttcg tggagtacaa gagcagcaac 1560 cagcagatcg cctccggcca gcgggactac aaggatctgg atgcccgcat cctgcagttc 1620 atcttcgata gagtgaaagc ctccgatgag ctgctgctga acgaaatcta ttttcaggcc 1680 aagaaactga agcagaaggc cagctccgaa ctggaaaagc tggaaagcag caagaaactg 1740 gacgaagtga tcgccaacag ccagctgtcc cagatcctga aaagccagca caccaacggc 1800 atcttcgagc agggcacctt cctgcacctc gtgtgcaagt actacaagca gcggcagcgg 1860 gccagagaca gcagactgta catcatgccc gagtacagat acgacaagaa gctgcacaag 1920 tataacaaca ccggccgctt cgacgatgac aaccagctgc tgacctactg caaccacaag 1980 ccccggcaga agcggtatca gctgctgaat gatctggccg gcgtgctgca ggtgtccccc 2040 aacttcctga aggacaagat cggctccgac gacgacctgt tcatctccaa gtggctggtg 2100 gaacacatcc gcggcttcaa gaaagcctgc gaggacagcc tgaagattca gaaggacaac 2160 cggggcctgc tgaaccacaa aatcaatatc gcccggaaca ccaagggcaa gtgcgagaaa 2220 gagatttta acctgatctg caagatcgag ggatccgagg ataagaaggg caattacaag 2280 cacggcctgg cctacgagct gggagtgctg ctgttcggcg agcccaatga ggccagcaag 2340 cccgagttcg accggaagat taagaagttc aacagcatct acagctttgc ccagatccag 2400 cagattgctt tcgccgagcg gaagggcaac gccaatacct gcgccgtgtg cagcgccgac 2460 aacgcccata gaatgcagca gatcaagatc accgagcccg tggaagataa caaggataag 2520 atcatcctgt ctgccaaggc ccagcggctg cctgccatcc ctaccagaat tgtggatggc 2580 gccgtgaaaa agatggccac catcctggcc aagaacatcg tggacgacaa ctggcagaac 2640 atcaaacagg tgctgagcgc caagcaccag ctgcacatcc ccatcatcac cgagagcaac 2700 gccttcgagt tcgagcccgc cctggccgat gtgaagggca agagcctgaa ggaccggcgg 2760 aagaaggccc tggaaagaat cagccccgag aacatcttca aggacaagaa caaccggatc 2820 aaagagttcg ccaagggcat cagcgcctac agcggcgcca atctgaccga cggcgatttc 2880 gacggcgcca aagaggaact ggacgccatc atccccagaa gccacaagaa gtacggcacc 2940 ctgaacgacg aggccaacct gatctgcgtg accagaggcg acaacaaaaa caagggcaac 3000 aggatcttct gcctgcggga cctggccgac aactacaagc tgaagcagtt cgagacaacc 3060 gacgacctgg aaatcgagaa gaagatcgcc gacaccatct gggacgccaa caagaaggac 3120 ttcaagttcg gcaactaccg cagcttcatc aacctgaccc cccaggaaca gaaagccttc 3180 cggcacgccc tgtttctggc cgacgagaac cctatcaagc aggccgtgat ccgggccatc 3240 aacaacagaa accggacctt cgtgaacggc acccagcggt actttgccga ggtgctggcc 3300 aacaatatct acctgcgggc caagaaagag aacctgaaca ccgacaagat cagcttcgac 3360 tacttcggca tccccaccat cggcaacggc agaggaatcg ccgagatccg gcagctgtac 3420 gagaaggtgg acagcgacat ccaggcttat gccaagggcg acaagcccca ggccagctac 3480 agccacctga tcgacgccat gctggctttc tgcattgccg ccgacgagca cagaaacgac 3540 ggcagcatcg gcctggaaat tgacaaaaac tacagcctgt accccctgga taagaacacc 3600 ggcgaggtgt tcaccaagga catcttcagc cagatcaaga tcaccgacaa cgagttcagc 3660 gacaagaaac tcgtgcgcaa gaaggccatc gagggcttca acacccaccg gcagatgacc 3720 agggacggca tctacgccga gaactacctg cccatcctga tccacaaaga actgaacgaa 3780 gtgcggaagg gctacacctg gaagaacagc gaggaaatca agattttcaa ggggaagaaa 3840 tacgatatcc agcagctgaa caacctggtg tactgcctga agttcgtgga caagcccatc 3900 agcatcgaca ttcagatcag caccctggaa gaactgcgga acatcctgac caccaacaac 3960 attgccgcta ccgccgagta ctactacatc aatctgaaaa cccagaagct gcacgagtac 4020 tatatcgaga attacaacac cgccctgggc tacaagaaat actccaaaga gatggagttc 4080 ctgcggagcc tggcctacag atccgagaga gtgaagatca agagcatcga cgacgtgaaa 4140 caggtgctgg acaaggacag caacttcatc atcggcaaga tcacactgcc ctttaagaaa 4200 gagtggcagc ggctgtaccg cgagtggcag aataccacca tcaaggacga ctacgagttc 4260 ctgaagagtt tcttcaacgt gaagtccatc acaaagctgc acaagaaagt gcgcaaggat 4320 ttcagcctgc ctatctctac caacgagggc aagttcctcg tgaagagaaa gacctgggac 4380 aacaatttca tctaccagat cctgaacgat agcgacagca gagccgacgg caccaagccc 4440 ttcatccccg ccttcgacat cagcaagaac gagatcgtgg aagccatcat cgacagcttt 4500 accagcaaga atatcttctg gctgcccaaa aacatcgagc tgcagaaagt ggacaacaag 4560 aacattttcg ccatcgacac ctccaagtgg ttcgaggtgg aaacccccag cgacctgaga 4620 gacatcggaa tcgccacaat ccagtacaag atcgacaaca acagccggcc caaagtgcgc 4680 gtgaagctgg actacgtgat cgacgatgac agcaagatca actacttcat gaaccacagc 4740 ctgctgaagt ccagataccc cgacaaagtg ctggaaatcc tgaagcagag cacaatcatc 4800 gagtttgaga gcagcgggtt caacaagacc atcaaagaaa tgctgggcat gaagctggcc 4860 gggatctaca acgagacaag caac 4884 <210> 25 <211> 2038 <212> DNA <213> Artificial Sequence <220> <223> moloney murine leukemia virus RT mutant <400> 25 accctaaata tagaagatga gtatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttaat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggaaggc aggcttctgt cgcctcttca tccctgggtt tgcagaaatg 960 gcagcccccc tgtaccctct caccaaaccg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggtggc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcatagaaa attcatcacc ctctggcg 2038 <210> 26 <211> 7021 <212> DNA <213> Artificial Sequence <220> <223> FnCas9-RT fusion <400> 26 atgaacttca agatcctgcc tatcgccatc gacctgggcg tgaagaacac cggcgtgttc 60 agcgccttct accagaaggg caccagcctg gaacggctgg acaacaagaa cggcaaggtg 120 tacgagctga gcaaggacag ctacaccctg ctgatgaaca accggaccgc cagacggcac 180 cagcggagag gcatcgacag aaagcagctc gtgaagcggc tgttcaagct gatctggacc 240 gagcagctga acctggaatg ggacaaggac acccagcagg ccatcagctt tctgttcaac 300 agacggggct tcagcttcat caccgacggc tacagccccg agtacctgaa catcgtgccc 360 gaacaagtga aggccatcct gatggacatc ttcgacgact acaacggcga ggacgacctg 420 gactcctacc tgaagctggc caccgagcag gaaagcaaga tcagcgaaat ctacaacaag 480 ctgatgcaga agatcctgga gtttaagctg atgaagctgt gcaccgacat caaggacgat 540 aaggtgtcca ccaagaccct gaaagagatc accagctacg agttcgagct gctggccgac 600 tacctggcca actacagcga gagcctgaaa acccagaagt tctcctacac cgacaagcag 660 ggcaatctga aagagctgag ctactaccac cacgacaagt acaacatcca ggaatttctg 720 aagcggcacg ccaccatcaa cgacagaatc ctggacacac tgctgaccga cgatctggac 780 atctggaact tcaacttcga gaagttcgac ttcgacaaga acgaggaaaa gctgcagaac 840 caggaagata aggaccacat ccaggcccat ctgcaccact tcgtgttcgc cgtgaacaag 900 atcaagagcg agatggccag cggcggcaga cacagaagcc agtacttcca ggaaatcacc 960 aacgtgctgg acgagaacaa ccaccaggaa ggctatctga agaatttctg cgagaacctg 1020 cacaacaaaa agtacagcaa cctgtccgtg aagaatctcg tgaacctgat cggcaacctg 1080 agcaacctgg aactgaagcc tctgcgcaag tacttcaacg acaagatcca cgccaaggcc 1140 gaccactggg acgagcagaa gttcaccgag acatactgcc actggattct gggcgagtgg 1200 cgcgtgggag tgaaggacca ggacaagaag gacggcgcca agtactccta caaggacctg 1260 tgcaacgagc tgaaacagaa agtgaccaag gccggactgg tggacttcct gctggaactg 1320 gacccctgca ggaccatccc cccctacctg gataacaaca atcggaagcc ccccaagtgc 1380 cagagcctga tcctgaaccc caagttcctg gacaaccagt accccaactg gcagcagtat 1440 ctgcaggaac tgaaaaaact gcagagcatc cagaactacc tggacagctt cgagacagac 1500 ctgaaggtgc tgaagtccag caaggaccag ccctacttcg tggagtacaa gagcagcaac 1560 cagcagatcg cctccggcca gcgggactac aaggatctgg atgcccgcat cctgcagttc 1620 atcttcgata gagtgaaagc ctccgatgag ctgctgctga acgaaatcta ttttcaggcc 1680 aagaaactga agcagaaggc cagctccgaa ctggaaaagc tggaaagcag caagaaactg 1740 gacgaagtga tcgccaacag ccagctgtcc cagatcctga aaagccagca caccaacggc 1800 atcttcgagc agggcacctt cctgcacctc gtgtgcaagt actacaagca gcggcagcgg 1860 gccagagaca gcagactgta catcatgccc gagtacagat acgacaagaa gctgcacaag 1920 tataacaaca ccggccgctt cgacgatgac aaccagctgc tgacctactg caaccacaag 1980 ccccggcaga agcggtatca gctgctgaat gatctggccg gcgtgctgca ggtgtccccc 2040 aacttcctga aggacaagat cggctccgac gacgacctgt tcatctccaa gtggctggtg 2100 gaacacatcc gcggcttcaa gaaagcctgc gaggacagcc tgaagattca gaaggacaac 2160 cggggcctgc tgaaccacaa aatcaatatc gcccggaaca ccaagggcaa gtgcgagaaa 2220 gagatttta acctgatctg caagatcgag ggatccgagg ataagaaggg caattacaag 2280 cacggcctgg cctacgagct gggagtgctg ctgttcggcg agcccaatga ggccagcaag 2340 cccgagttcg accggaagat taagaagttc aacagcatct acagctttgc ccagatccag 2400 cagattgctt tcgccgagcg gaagggcaac gccaatacct gcgccgtgtg cagcgccgac 2460 aacgcccata gaatgcagca gatcaagatc accgagcccg tggaagataa caaggataag 2520 atcatcctgt ctgccaaggc ccagcggctg cctgccatcc ctaccagaat tgtggatggc 2580 gccgtgaaaa agatggccac catcctggcc aagaacatcg tggacgacaa ctggcagaac 2640 atcaaacagg tgctgagcgc caagcaccag ctgcacatcc ccatcatcac cgagagcaac 2700 gccttcgagt tcgagcccgc cctggccgat gtgaagggca agagcctgaa ggaccggcgg 2760 aagaaggccc tggaaagaat cagccccgag aacatcttca aggacaagaa caaccggatc 2820 aaagagttcg ccaagggcat cagcgcctac agcggcgcca atctgaccga cggcgatttc 2880 gacggcgcca aagaggaact ggacgccatc atccccagaa gccacaagaa gtacggcacc 2940 ctgaacgacg aggccaacct gatctgcgtg accagaggcg acaacaaaaa caagggcaac 3000 aggatcttct gcctgcggga cctggccgac aactacaagc tgaagcagtt cgagacaacc 3060 gacgacctgg aaatcgagaa gaagatcgcc gacaccatct gggacgccaa caagaaggac 3120 ttcaagttcg gcaactaccg cagcttcatc aacctgaccc cccaggaaca gaaagccttc 3180 cggcacgccc tgtttctggc cgacgagaac cctatcaagc aggccgtgat ccgggccatc 3240 aacaacagaa accggacctt cgtgaacggc acccagcggt actttgccga ggtgctggcc 3300 aacaatatct acctgcgggc caagaaagag aacctgaaca ccgacaagat cagcttcgac 3360 tacttcggca tccccaccat cggcaacggc agaggaatcg ccgagatccg gcagctgtac 3420 gagaaggtgg acagcgacat ccaggcttat gccaagggcg acaagcccca ggccagctac 3480 agccacctga tcgacgccat gctggctttc tgcattgccg ccgacgagca cagaaacgac 3540 ggcagcatcg gcctggaaat tgacaaaaac tacagcctgt accccctgga taagaacacc 3600 ggcgaggtgt tcaccaagga catcttcagc cagatcaaga tcaccgacaa cgagttcagc 3660 gacaagaaac tcgtgcgcaa gaaggccatc gagggcttca acacccaccg gcagatgacc 3720 agggacggca tctacgccga gaactacctg cccatcctga tccacaaaga actgaacgaa 3780 gtgcggaagg gctacacctg gaagaacagc gaggaaatca agattttcaa ggggaagaaa 3840 tacgatatcc agcagctgaa caacctggtg tactgcctga agttcgtgga caagcccatc 3900 agcatcgaca ttcagatcag caccctggaa gaactgcgga acatcctgac caccaacaac 3960 attgccgcta ccgccgagta ctactacatc aatctgaaaa cccagaagct gcacgagtac 4020 tatatcgaga attacaacac cgccctgggc tacaagaaat actccaaaga gatggagttc 4080 ctgcggagcc tggcctacag atccgagaga gtgaagatca agagcatcga cgacgtgaaa 4140 caggtgctgg acaaggacag caacttcatc atcggcaaga tcacactgcc ctttaagaaa 4200 gagtggcagc ggctgtaccg cgagtggcag aataccacca tcaaggacga ctacgagttc 4260 ctgaagagtt tcttcaacgt gaagtccatc acaaagctgc acaagaaagt gcgcaaggat 4320 ttcagcctgc ctatctctac caacgagggc aagttcctcg tgaagagaaa gacctgggac 4380 aacaatttca tctaccagat cctgaacgat agcgacagca gagccgacgg caccaagccc 4440 ttcatccccg ccttcgacat cagcaagaac gagatcgtgg aagccatcat cgacagcttt 4500 accagcaaga atatcttctg gctgcccaaa aacatcgagc tgcagaaagt ggacaacaag 4560 aacattttcg ccatcgacac ctccaagtgg ttcgaggtgg aaacccccag cgacctgaga 4620 gacatcggaa tcgccacaat ccagtacaag atcgacaaca acagccggcc caaagtgcgc 4680 gtgaagctgg actacgtgat cgacgatgac agcaagatca actacttcat gaaccacagc 4740 ctgctgaagt ccagataccc cgacaaagtg ctggaaatcc tgaagcagag cacaatcatc 4800 gagtttgaga gcagcgggtt caacaagacc atcaaagaaa tgctgggcat gaagctggcc 4860 gggatctaca acgagacaag caactctgga ggatctagcg gaggatcctc tggcagcgag 4920 acaccaggaa caagcgagtc agcaacacca gagagcagtg gcggcagcag cggcggcagc 4980 agcaccctaa atatagaaga tgagtatcgg ctacatgaga cctcaaaaga gccagatgtt 5040 tctctagggt ccacatggct gtctgatttt cctcaggcct gggcggaaac cggggggcatg 5100 ggactggcag ttcgccaagc tcctctgatc atacctctga aagcaacctc tacccccgtg 5160 tccataaaac aataccccat gtcacaagaa gccagactgg ggatcaagcc ccacatacag 5220 agactgttgg accagggaat actggtaccc tgccagtccc cctggaacac gcccctgcta 5280 cccgttaaga aaccagggac taatgattat aggcctgtcc aggatctgag agaagtcaac 5340 aagcgggtgg aagacatcca ccccaccgtg cccaaccctt acaacctctt gagcgggctc 5400 ccaccgtccc accagtggta cactgtgctt gatttaaagg atgccttttt ctgcctgaga 5460 ctccacccca ccagtcagcc tctcttcgcc tttgagtgga gagatccaga gatgggaatc 5520 tcaggacaat tgacctggac cagactccca cagggtttca aaaacagtcc caccctgttt 5580 aatgaggcac tgcacagaga cctagcagac ttccggatcc agcacccaga cttgatcctg 5640 ctacagtacg tggatgactt actgctggcc gccacttctg agctagactg ccaacaaggt 5700 actcgggccc tgttacaaac cctagggaac ctcgggtatc gggcctcggc caagaaagcc 5760 caaatttgcc agaaacaggt caagtatctg gggtatcttc taaaagaggg tcagagatgg 5820 ctgactgagg ccagaaaaga gactgtgatg gggcagccta ctccgaagac ccctcgacaa 5880 ctaagggagt tcctagggaa ggcaggcttc tgtcgcctct tcatccctgg gtttgcagaa 5940 atggcagccc ccctgtaccc tctcaccaaa ccggggactc tgtttaattg gggcccagac 6000 caacaaaagg cctatcaaga aatcaagcaa gctcttctaa ctgccccagc cctggggttg 6060 ccagatttga ctaagccctt tgaactcttt gtcgacgaga agcagggcta cgccaaaggt 6120 gtcctaacgc aaaaactggg accttggcgt cggccggtgg cctacctgtc caaaaagcta 6180 gacccagtag cagctgggtg gcccccttgc ctacggatgg tagcagccat tgccgtactg 6240 acaaaggatg caggcaagct aaccatggga cagccactag tcattctggc cccccatgca 6300 gtagaggcac tagtcaaaca accccccgac cgctggcttt ccaacgcccg gatgactcac 6360 tatcaggcct tgcttttgga cacggaccgg gtccagttcg gaccggtggt agccctgaac 6420 ccggctacgc tgctcccact gcctgaggaa gggctgcaac acaactgcct tgatatcctg 6480 gccgaagccc acggaacccg acccgaccta acggaccagc cgctcccaga cgccgaccac 6540 acctggtaca cggatggaag cagtctctta caagagggac agcgtaaggc gggagctgcg 6600 gtgaccaccg agaccgaggt aatctgggct aaagccctgc cagccgggac atccgctcag 6660 cgggctgaac tgatagcact cacccaggcc ctaaagatgg cagaaggtaa gaagctaaat 6720 gtttatactg atagccgtta tgcttttgct actgcccata tccatggaga aatatacaga 6780 aggcgtgggt ggctcacatc agaaggcaaa gagatcaaaa ataaagacga gatcttggcc 6840 ctactaaaag ccctctttct gcccaaaaga cttagcataa tccattgtcc aggacatcaa 6900 aagggacaca gcgccgaggc tagaggcaac cggatggctg accaagcggc ccgaaaggca 6960 gccatcacag agactccaga cacctctacc ctcctcatag aaaattcatc accctctggc 7020 g 7021 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HEK3 target site <400> 27 ggcccagact gagcacgtga tgg 23 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEK3 target (PBS + RT) <400> 28 catcacgtaa gctcagtctg 20 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> c-Myc target site <400> 29 aggcagaggg agcgagcggg cgg 23 <210> 30 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for FnCas9 <400> 30 aggcagaggg agcgagcggg guuucaguug cugaaaaugc ucuguaauca uuuaaaagua 60 uuuugaacgg accucuguuu gacacgucug aauaacuaaa aa 102 <210> 31 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for SpCas9 <400> 31 aggcagaggg agcgagcggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 target site <400> 32 tctaaccccc acctcctgtt agg 23 <210> 33 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for FnCas9 <400> 33 ucuaaccccc accuccuguu guuucaguug cugaaaaugc ucuguaauca uuuaaaagua 60 uuuugaacgg accucuguuu gacacgucug aauaacuaaa aa 102 <210> 34 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for SpCas9 <400> 34 ucuaaccccc accuccuguu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> EMX1 target site <400> 35 tggttgccca ccctagtcat tgg 23 <210> 36 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for FnCas9 <400> 36 ugguugccca cccuagucau guuucaguug cugaaaaugc ucuguaauca uuuaaaagua 60 uuuugaacgg accucuguuu gacacgucug aauaacuaaa aa 102 <210> 37 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for SpCas9 <400> 37 ugguugccca cccuagucau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HEK3 target site <400> 38 ggcccagact gagcacgtga tgg 23 <210> 39 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for FnCas9 <400> 39 ggcccagacu gagcacguga guuucaguug cugaaaaugc ucuguaauca uuuaaaagua 60 uuuugaacgg accucuguuu gacacgucug aauaacuaaa aa 102 <210> 40 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for SpCas9 <400> 40 ggcccagacu gagcacguga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NRAS target site <400> 41 ggtaaggggg cagggaggga ggg 23 <210> 42 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for FnCas9 <400> 42 gguaaggggg cagggaggga guuucaguug cugaaaaugc ucuguaauca uuuaaaagua 60 uuuugaacgg accucuguuu gacacgucug aauaacuaaa aa 102 <210> 43 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> sgRNA sequence for SpCas9 <400> 43 gguaaggggg cagggaggga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102 <210> 44 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_10-10 <400> 44 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa catcacgtaa gctcagtctg ttttttt 157 <210> 45 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_10-12 <400> 45 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa gccatcacgt aagctcagtc tgttttttt 159 <210> 46 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_10-15 <400> 46 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa tctgccatca cgtaagctca gtctgttttt tt 162 <210> 47 <211> 158 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_11-10 <400> 47 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa catcacgtaa gctcagtctg gttttttt 158 <210> 48 <211> 160 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_11-12 <400> 48 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa gccatcacgt aagctcagtc tggttttttt 160 <210> 49 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_11-15 <400> 49 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa tctgccatca cgtaagctca gtctggtttt ttt 163 <210> 50 <211> 160 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_13-10 <400> 50 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa catcacgtaa gctcagtctg ggcttttttt 160 <210> 51 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_13-12 <400> 51 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa gccatcacgt aagctcagtc tgggcttttt tt 162 <210> 52 <211> 165 <212> DNA <213> Artificial Sequence <220> <223> HEK3 FnCas9_pegRNA_13-15 <400> 52 gggcccagac tgagcacgtg agtttcagtt gctgaattat ttggtaaaca gtaccaaata 60 attaatgctc tgtaatcatt taaaagtatt ttgaacggac ctctgtttga cacgtctgaa 120 taactaaaaa tctgccatca cgtaagctca gtctgggctt ttttt 165

Claims (21)

Francisella novicida Cas9 protein;
reverse transcriptase protein; and
A prime editing complex comprising a prime editing guide RNA (PEgRNA), comprising:
wherein the prime editing guide RNA (PEgRNA) comprises a primer binding site, an editing template and a homology arm. The prime editing complex according to claim 1, wherein the Francisella novicida Cas9 protein is at least 98% identical to Francisella novicida Cas9 (H969A) of SEQ ID NO: 2. The prime editing complex according to claim 2, wherein the Francisella novicida Cas9 protein has the amino acid sequence of SEQ ID NO: 2, Francisella novicida Cas9 (H969A) of SEQ ID NO: 2. The prime editing complex according to claim 1, wherein the Francisella novicida Cas9 protein has a position that forms a nick in the target DNA 6bp away from the PAM. The prime editing complex according to claim 1, wherein the reverse transcriptase has the sequence of SEQ ID NO: 15. The prime editing complex according to claim 1, wherein the Francisella novicida Cas9 protein and the reverse transcriptase protein are in the form of a fusion protein. The prime editing complex according to claim 1, wherein the Francisella novicida Cas9 protein and the reverse transcriptase protein are capable of binding to a target DNA sequence when complexed with a prime editing guide RNA (PEgRNA). The prime editing complex of claim 1 , wherein the spacer sequence of the prime editing guide RNA (PEgRNA) is a sequence that binds to a protospacer in the target DNA. The prime editing complex of claim 1 , wherein the gRNA core of the prime editing guide RNA (PEgRNA) is a sequence within the gRNA responsible for Cas9 binding. The single stranded DNA of claim 1 , wherein the extension arm of the prime editing guide RNA (PEgRNA) comprises a DNA synthesis template sequence encoding a primer binding site and a single stranded DNA flap containing the genetic change of interest via reverse transcriptase. A strand extension, the prime editing complex. The prime editing complex of claim 1 , wherein the prime editing guide RNA (PEgRNA) comprises the sequence of SEQ ID NO:23. The prime editing complex of claim 1 , wherein the target or complementary non-target strand is nicked to form a priming sequence having a free 3′ end. The prime editing complex of claim 1 , wherein the nick site is -6 to the 5' end of the PAM sequence. Francisella novicida Cas9 protein; and a polynucleotide encoding a fusion protein comprising a reverse transcriptase protein. A vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein. A vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA) comprising a primer binding site, an editing template and a homology arm. Francisella novicida Cas9 protein; reverse transcriptase protein; and a prime editing complex comprising a prime editing guide RNA (PEgRNA). A composition for gene editing comprising the prime editing complex according to any one of claims 1 to 13. a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; and a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA) comprising a primer binding site, an editing template, and a homology arm. A pharmaceutical composition for preventing or treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) comprising the prime editing complex according to any one of claims 1 to 13. a vector comprising a polynucleotide encoding a Francisella novicida Cas9 protein and a reverse transcriptase protein; and a vector comprising a polynucleotide encoding a prime editing guide RNA (PEgRNA) comprising a primer binding site, an editing template and a homology arm for the prevention or treatment of a disease associated with a mutation or single-nucleotide polymorphism (SNP) pharmaceutical composition for use.

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