WO2022103839A1 - Rna editing compositions and uses thereof - Google Patents

Abstract

Provided herein are engineered guide RNAs comprising structured loop stabilized scaffolds and engineered guide RNAs configured, upon hybridization to a target RNA, to form, at least in part, double stranded substrates with at least a portion of the target RNA and facilitate a chemical modification of a nucleotide base in the target RNA by recruitment of an RNA editing entity. Also provided herein are compositions, vectors, and cells comprising the engineered guide RNAs disclosed herein. Also provided herein are methods of introducing the engineered guide RNAs described herein into cells and methods of treating a disease or condition in a subject in need thereof comprising administering to the subject engineered guide RNAs described herein.

Description

RNA EDITING COMPOSITIONS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 63/112,312, filed November 11, 2020, U.S. Provisional Application No. 63/119,839, filed December 01, 2020, and U.S. Provisional Application No. 63/178,227 filed April 22, 2021, the disclosures of which are incorporated herein by reference in their entirety

SUMMARY

[002] Disclosed herein are engineered guide RNAs comprising: (a) a targeting domain that binds to a target RNA, and (b) a structural loop stabilized scaffold. In some embodiments, the engineered guide RNA can be configured, upon association with the target RNA, to facilitate a chemical transformation of a base of a nucleotide in the target RNA by an RNA editing entity. In some embodiments, the structural loop stabilized scaffold can comprise a 5’ end and a 3’ end that together form a secondary structure or a tertiary structure. In some embodiments, the structural loop stabilized scaffold can comprise a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof. In some embodiments, the structural loop stabilized scaffold can comprise at least 2 stem loop structures. In some embodiments, the structural loop stabilized scaffold can comprise a tRNA scaffold. In some embodiments, the engineered guide RNA can comprise an RNA editing entity recruiting domain. In some embodiments, the engineered guide RNA can be configured upon binding to the target RNA to form a guide-target RNA scaffold in conjunction with the target RNA. In some embodiments, the guide-target RNA scaffold can comprise a structural feature that recruits the RNA editing entity. In some embodiments, the target RNA can encode: ABCA4, AAT, HEXA, LRRK2, APP, CFTR, ALAS1, ATP7B, HFE, PCSK9, SCNN1A, SNCA, GBA, PINK1, Tau, or LIPA, a biological active fragment of any of these, or any combination thereof. In some embodiments, the engineered guide RNA can comprise a modified RNA nucleotide, a modified RNA base, an unmodified RNA base, an unmodified RNA nucleotide, or a combination thereof. In some embodiments, the targeting domain can comprise from about 20 nucleotides to about 200 nucleotides. In some embodiments, the guide-target RNA scaffold formed upon association of the targeting domain with the target RNA can comprise a nucleotide mismatch. In some embodiments, the mismatch can be: (a) an A to C mismatch; (b) a G to 5’ G mismatch; (c) a wobble base pair; or (d) or any combination of (a) to (c). In some embodiments, the target RNA can comprise a point mutation that is associated with a disease or a condition. In some embodiments, the point mutation can comprise a missense mutation. In some embodiments, the RNA editing entity can be: a) Adenosine deaminase acting on RNA (ADAR) or Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) enzyme; b) a catalytically active fragment of ADAR or APOBEC; c) fusion polypeptide comprising (a) or (b); or d) any combination of (a) to (c). In some embodiments, the RNA editing entity can be the ADAR, and the ADAR can comprise human ADAR (hADAR). In some embodiments, the RNA editing entity can be the ADAR, and the ADAR can comprise AD ARI , ADAR2, or a combination thereof. In some embodiments, the structural feature can comprise: (a) a hairpin loop; (b) an internal loop; (c) a polynucleotide loop; (d) a wobble base pair; (e) a bulge; or (g) or any combination of (a) to (e). In some embodiments, the engineered guide RNA can comprise from 1 to 50 structural features. In some embodiments, the structural feature can comprise a bulge. In some embodiments, the bulge can comprise from 2 to 4 nucleotides that are mismatched between the engineered guide RNA side and the target RNA in the guide-target RNA scaffold. In some embodiments, the bulge can comprise an asymmetric bulge. In some embodiments, the bulge can comprise a symmetric bulge. In some embodiments, the structural feature can comprise an internal loop. In some embodiments, the internal loop can comprise an asymmetric loop. In some embodiments, the internal loop can be a symmetric loop. In some embodiments, the structural feature can comprise a hairpin loop. In some embodiments, the engineered guide RNA can comprise from 1 to 10 hairpin loops. In some embodiments, the hairpin loop can be present at (i) 3’ of the engineered guide RNA, (ii) 5’ of the engineered guide RNA, or (iii) within the engineered guide RNA. In some embodiments, the hairpin loop can comprise a non-recruitment hairpin loop and the non-recruitment hairpin loop can be from a U7 snRNA. In some embodiments, the hairpin loop can comprise from about 10 to 500 nucleotides. In some embodiments, the structural feature can comprise a wobble base pair. In some embodiments, the wobble base pair can comprise a G paired with a U. In some embodiments, the structural loop stabilized scaffold can comprise at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with at least a portion of any one of SEQ ID NOs: 3-10, as determined by BLAST. In some embodiments, the engineered guide RNA can further comprise a spacer domain. In some embodiments, polynucleotides can encode an engineered guide RNA. In some embodiments, a delivery vector can comprise an engineered guide RNA or a polynucleotide. In some embodiments, the vector can comprise a viral vector. In some embodiments, the viral vector can comprise a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno-associated viral (AAV) vector, or a poxvirus vector. In some embodiments, the viral vector can comprise the AAV vector. In some embodiments, the AAV vector can be of a serotype selected from the group consisting of: AAV-1, AAV-2, AAV- 3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-rh74, AAV- rhlO, and AAV-2i8. In some embodiments, the AAV vector can comprise a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. In some embodiments, the delivery vector can be a non- viral delivery vector. In some embodiments, the non-viral delivery vector can comprise a microvesicle, a nanovesicle, a microparticle, or a nanoparticle. In some embodiments, a pharmaceutical composition in unit dose form can comprise: (a) an engineered guide RNA, a polynucleotide, or a delivery vector and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, a method of treating a disease or a condition in a subject in need thereof, can comprise administering to a subject, a therapeutic comprising an engineered guide RNA, a polynucleotide, or a delivery vector in a therapeutically effective amount to treat the disease or condition. In some embodiments, the administering of the therapeutic can result in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or the condition. In some embodiments, the target RNA can be encoded by the serpin family A member 1 (SERPINA 7) gene. In some embodiments, the SERPINA1 gene can comprise a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1. In some embodiments, the target RNA can be encoded by an ABCA4 gene, or a portion thereof. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2. In some embodiments, the administering can be intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above. In some embodiments, the disease or the condition can comprise Parkinson’s disease, Alzheimer’s disease, a dementia, liver cirrhosis, alpha- 1 antitrypsin deficiency (AAT deficiency), Stargardt disease, chronic obstructive pulmonary disease (COPD), or any combination thereof. In some embodiments, the chemical modification can be confirmed by an in vitro assay. [003] Also disclosed herein are polynucleotides encoding engineered guide RNAs. In some embodiments, a delivery vector can comprise an engineered guide RNA or a polynucleotide. In some embodiments, the vector can comprise a viral vector. In some embodiments, the viral vector can comprise a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno-associated viral (AAV) vector, or a poxvirus vector. In some embodiments, the viral vector can comprise the AAV vector. In some embodiments, the AAV vector can be of a serotype selected from the group consisting of: AAV-1, AAV-2, AAV- 3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-rh74, AAV- rhlO, and AAV-2i8. In some embodiments, the AAV vector can comprise a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. In some embodiments, the delivery vector can be a non- viral delivery vector. In some embodiments, the non-viral delivery vector can comprise a microvesicle, a nanovesicle, a microparticle, or a nanoparticle. In some embodiments, a pharmaceutical composition in unit dose form can comprise: (a) an engineered guide RNA, a polynucleotide, or a delivery vector and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, a method of treating a disease or a condition in a subject in need thereof, can comprise administering to a subject, a therapeutic comprising an engineered guide RNA, a polynucleotide, or a delivery vector in a therapeutically effective amount to treat the disease or condition. In some embodiments, the administering of the therapeutic can result in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or the condition. In some embodiments, the target RNA can be encoded by the serpin family A member 1 (SERPINA 7) gene. In some embodiments, the SERPINA1 gene can comprise a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1. In some embodiments, the target RNA can be encoded by an ABCA4 gene, or a portion thereof. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2. In some embodiments, the administering can be intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above. In some embodiments, the disease or the condition can comprise Parkinson’s disease, Alzheimer’s disease, a dementia, liver cirrhosis, alpha- 1 antitrypsin deficiency (AAT deficiency), Stargardt disease, chronic obstructive pulmonary disease (COPD), or any combination thereof. In some embodiments, the chemical modification can be confirmed by an in vitro assay.

[004] Also disclosed herein are delivery vectors comprising engineered guide RNAs or polynucleotides. In some embodiments, the vector can comprise a viral vector. In some embodiments, the viral vector can comprise a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno- associated viral (AAV) vector, or a poxvirus vector. In some embodiments, the viral vector can comprise the AAV vector. In some embodiments, the AAV vector can be of a serotype selected from the group consisting of: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV- 8, AAV-9, AAV-10, AAV-11, AAV-rh74, AAV-rhlO, and AAV-2i8. In some embodiments, the AAV vector can comprise a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. In some embodiments, the delivery vector can be a non-viral delivery vector. In some embodiments, the non-viral delivery vector can comprise a microvesicle, a nanovesicle, a microparticle, or a nanoparticle. Also disclosed herein are pharmaceutical compositions in unit dose form comprising: (a) engineered guide RNAs, polynucleotides, or delivery vectors and; (b) a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, a pharmaceutical composition in unit dose form can comprise: (a) an engineered guide RNA, a polynucleotide, or a delivery vector and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, a method of treating a disease or a condition in a subject in need thereof, can comprise administering to a subject, a therapeutic comprising an engineered guide RNA, a polynucleotide, or a delivery vector in a therapeutically effective amount to treat the disease or condition. In some embodiments, the administering of the therapeutic can result in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or the condition. In some embodiments, the target RNA can be encoded by the serpin family A member 1 (SERPINA 7) gene. In some embodiments, the SERPINA1 gene can comprise a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1. In some embodiments, the target RNA can be encoded by an ABCA4 gene, or a portion thereof. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2. In some embodiments, the administering can be intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above. In some embodiments, the disease or the condition can comprise Parkinson’s disease, Alzheimer’s disease, a dementia, liver cirrhosis, alpha- 1 antitrypsin deficiency (AAT deficiency), Stargardt disease, chronic obstructive pulmonary disease (COPD), or any combination thereof. In some embodiments, the chemical modification can be confirmed by an in vitro assay.

[005] Also disclosed herein are pharmaceutical compositions in unit dose form comprising: (a) engineered guide RNAs, polynucleotides, or delivery vectors and; (b) a pharmaceutically acceptable: excipient, carrier, or diluent.

[006] Also disclosed herein are methods of treating a disease or a condition in a subject in need thereof, the method comprising: administering to a subject, a therapeutic comprising an engineered guide RNA, a polynucleotide, or a delivery vector in a therapeutically effective amount to treat the disease or condition. In some embodiments, the administering of the therapeutic can result in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or the condition. In some embodiments, the target RNA can be encoded by the serpin family A member 1 (SERPINA 7) gene. In some embodiments, the SERPINA1 gene can comprise a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1. In some embodiments, the target RNA can be encoded by an ABCA4 gene, or a portion thereof. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2. In some embodiments, the ABCA4 gene can comprise a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2. In some embodiments, the administering can be intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above. In some embodiments, the disease or the condition can comprise Parkinson’s disease, Alzheimer’s disease, a dementia, liver cirrhosis, alpha- 1 antitrypsin deficiency (AAT deficiency), Stargardt disease, chronic obstructive pulmonary disease (COPD), or any combination thereof. In some embodiments, the chemical modification can be confirmed by an in vitro assay.

INCORPORATION BY REFERENCE

[007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[008] The novel features of the invention can be set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention can be utilized, and the accompanying drawings of which:

[009] FIG. 1A and FIG. IB shows non-limiting examples of target RNAs of engineered guide RNAs (gRNAs) of the present disclosure. FIG. 1A shows target pre-mRNAs. FIG. IB shows a target mRNA. The specific examples in these figures include a SERPINA1 pre-mRNA or mRNA carrying a mutation (E342K) in the exon 5 region.

[010] FIG. 2 shows the generation of an example of a structural loop stabilized scaffold, specifically a stabilized tRNA scaffold, with a gRNA (against SERPINA1) inserted into the anticodon region of the tRNA. The C indicates the mismatched nucleotide against the target adenosine in the target RNA.

[011] FIG. 3A shows a Sanger sequencing trace at top and a bar graph at bottom. The Sanger trace indicated an A at the expected position in SERPINA1 and validated that the cells used in RNA editing experiments carry the SERPINA1 mutation (E342K) to be targeted with the engineered gRNAs of the present disclosure. The bar graph at bottom indicated that SERPINA1 was expressed in cells. FIG. 3B is a Western blot showing expression of AD ARI in fibroblasts and a control (GAPDH). The fibroblasts do not express ADAR2.

[012] FIG. 4 shows, at left, a graph of percent editing of SERPINA1 pre-mRNA and mature mRNA using a gRNA placed in a tRNA structural loop stabilized scaffold (WT tRNA) versus placed in another engineered guide RNAs i (WT gRNA) or the control gRNA. The control gRNA at left is an engineered guide RNA against Rab7a. FIG. 4 shows, at right, Sanger sequencing traces showing editing of the target adenosine using various engineered gRNAs of the present disclosure, including preSERPINAl _tRNA gRNA (a gRNA placed in a tRNA structural loop stabilized scaffold targeting SERPINA1 pre-mRNA) and SERPINAl-tRNA gRNA (a gRNA placed in a RNA structural loop stabilized scaffold targeting SERPINA1 mRNA).

[013] FIG. 5, at left, shows guide RNAs in which additional mismatches were engineered by placing G in the targeting sequence opposite off-target As in the target sequence. FIG. 5, at right, shows a higher efficiency of editing of the mutation in target SERPENA1 pre-mRNA by using a gRNA placed in a structural loop stabilized scaffold (WT_tRNA) as compared to a control gRNA that is not in a stabilized scaffold or as compared to that gRNA not present in a stabilized scaffold.

[014] FIG. 6 shows a legend of various exemplary structural features present in guidetarget RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side)

DETAILED DESCRIPTION

OVERVIEW

[015] Aspects of the disclosure relate to a RNA editing approach for treating diseases, disorders, or conditions caused by mutations in a target RNA. The approach, in some cases, uses an engineered guide RNA that is a structured loop stabilized scaffold (SLS) guide RNA. An SLS gRNA can comprise a 5’ end and 3’ end that together form a secondary or tertiary structure, which is present in the absence of binding to a target RNA. Therefore, the SLS scaffold is a preformed structural feature that can be present in the SLS gRNA, and not a structural feature formed by latent structure provided in a targeting domain. For example, an SLS guide RNA utilizes a structured loop stabilized scaffold such as a tRNA scaffold, to impart beneficial properties onto the guide RNA. Without wishing to be bound by theory, an engineered guide comprising a SLS scaffold can provide greater stability, improved recruitment of RNA-editing entities (such as endogenous RNA editing enzymes), longer half-lives, and/or improved RNA- editing efficiency as compared to engineered guides without the SLS scaffold. A SLS guide RNA can comprise a stem-loop structure in which the 5’ end and the 3’ end together form part of the stem loop structure. A SLS scaffold can comprise a series of stem-loop structures. A SLS guide RNA can comprise a series of stem-loop structures. As disclosed herein, an engineered guide RNA comprising an SLS scaffold is referred to as “SLS guide RNA”, “SLS guide”, or “SLS gRNA”, which are used interchangeably.

[016] Such engineered guide RNAs (e.g., an SLS guide RNA) comprise a targeting region that can bind to a target RNA sequence, thus allowing for site-specific targeting of a specific target RNA sequence for editing. An engineered guide RNA can comprise an SLS scaffold as described herein (referred to as an “SLS guide”). Embodiments of an SLS guide include a tRNA scaffold in which a targeting sequence is inserted into an anticodon region of the tRNA scaffold. The targeting domain can include a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or fragment thereof and does not base pair, or does not fully base pair, with the base to be edited. This mismatch can help to localize editing of the RNA editing entity to the desired base of the target RNA.

[017] In some instances, additional functionality can be incorporated into the engineered guide (e.g., an SLS guide) to recruit RNA editing entities and/or provide enhanced editing efficiency and accuracy. For example, an RNA editing entity recruiting domain formed and present in the absence of binding to a target RNA can be included to recruit an RNA editing entity to the site of the desired edit. Additionally, latent structures that form only upon hybridization of the guide RNA to the target RNA can be designed to recruit the RNA editing entity. Latent structures when manifested become structural features described herein, including mismatches, bulges, internal loops, and hairpins. Without wishing to be bound by theory, the presence of structural features described herein that are produced upon hybridization of the guide RNA with the target RNA configure the engineered guide RNA to facilitate a specific, targeted edit of the target RNA via the RNA editing entity or biologically active fragment thereof.

Further, the structural features in combination with the mismatch described above generally have an increased amount of editing, fewer off target edits, or both, as compared to a engineered guide RNA that forms a mismatch with the target RNA in the absence of any other structural features. Either method of recruiting the RNA editing entity can be utilized in the engineered guide RNA in combination with the SLS scaffold to provide increases in editing and/or a reduction in off- target editing, relative to guide RNAs lacking these features. Further, beneficial properties imparted by the SLS scaffold, such as increased half-life, further improve editing of guide RNAs incorporating either recruiting technology.

[018] The chemical modification of a target nucleotide in a target RNA can be designed to correct a mutation in the target RNA. The correction of a mutation in the target RNA can correct a mutated protein sequence to a wild-type protein sequence, which can treat a disease.

[019] The chemical modification of a target nucleotide in a target RNA can be designed to modulate protein expression from the target RNA. Modulation can refer to altering the translation of a target RNA or portion thereof at one of various stages, with a view to alleviate a disease or condition associated with the expressed or a mutation in the protein. Modulation can be mediated post-transcriptionally. Modulating a target RNA can correct aberrant expression of splice variants generated by a mutation. In some cases, compositions and methods provided herein can be utilized to regulate translation of a target RNA. Modulation can refer to decreasing or knocking down the expression of an RNA or portion thereof by decreasing the abundance of the target RNA. The decreasing the abundance of the target RNA can be mediated by decreasing the processing, splicing, turnover or stability of the target RNA; or by decreasing the accessibility of the target RNA by translational machinery such as ribosome. In some cases, an engineered guide (e.g., SLS guide) described herein can facilitate a knockdown. A knockdown can reduce the expression of a target RNA. In some cases, a knockdown can be accompanied by editing of an mRNA. In some cases, a knockdown can occur with substantially little to no editing of an mRNA. In some instances, a knockdown can occur by targeting an untranslated region of the target RNA, such as a 3’ UTR, a 5’ UTR or both. In some cases, a knockdown can occur by targeting a coding region of the target RNA. In some instances, a knockdown can be mediated by an RNA editing enzyme (e.g., ADAR). In some instances, an RNA editing enzyme can cause a knockdown by hydrolytic deamination of multiple adenosines in an RNA. Hydrolytic deamination of multiple adenosines in an RNA can be referred to as hyper-editing. In some cases, hyper-editing can occur in cis (e.g., in an Alu element) or in trans (e.g., in a target RNA by an engineered guide). In some instances, an RNA editing enzyme can cause a knockdown by editing a target RNA to comprise a premature stop codon or prevent initiation of translation of the target RNA due to an edit in the target RNA.

[020] Given the inherent flexibility, changes to RNA can be relatively safe as they can in some cases be not permanently reflected in the DNA. In addition, alterations at the RNA level can be reworked when environmental conditions change. Thus, the methods and compositions in this disclosure present a safer, reversible and more flexible approach to treating genetic diseases in human and animal subjects, compared to DNA-based editing. The methods and compositions described herein can be used for treating a variety of conditions including Alpha- 1 antitrypsin deficiency (AATD), liver cirrhosis, Stargardt disease and neurodegenerative diseases.

Definitions

[021] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[022] Throughout this application, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.

Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[023] As used herein, the term “about” a number can refer to that number plus or minus 10% of that number.

[024] As disclosed herein, a “bulge” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair - a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a “mismatch.” Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an “internal loop.” A “symmetrical bulge” refers to a bulge where the same number of nucleotides is present on each side of the bulge. An “asymmetrical bulge” refers to a bulge where a different number of nucleotides are present on each side of the bulge.

[025] “Canonical amino acids” refer to those 20 amino acids that occur in nature, including for example, the amino acids shown in Table 1.

Table 1. Naturally occurring amino acids indicated with the three letter abbreviations, one letter abbreviations, structures, and corresponding codons

Positively charged residues

[026] The term “complementary” or “complementarity” refers to the ability of a nucleic acid to form one or more bonds with a corresponding nucleic acid sequence by, for example, hydrogen bonding (e.g., traditional Watson-Crick), covalent bonding, or other similar methods. In Watson-Crick base pairing, a double hydrogen bond forms between nucleobases T and A, whereas a triple hydrogen bond forms between nucleobases C and G. For example, the sequence A-G-T can be complementary to the sequence T-C-A. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson- Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” can mean that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein can refer to a degree of complementarity that can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions). Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” or “not specific” can refer to a nucleic acid sequence that contains a series of residues that may not be designed to be complementary to or can be only partially complementary to any other nucleic acid sequence. [027] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” can be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[028] The term “encode,” as used herein, refers to an ability of a polynucleotide to provide information or instructions sequence sufficient to produce a corresponding gene expression product. In a non-limiting example, mRNA can encode a polypeptide during translation, whereas DNA can encode an mRNA molecule during transcription.

[029] An “engineered latent guide RNA” refers to an engineered guide RNA that comprises a portion of sequence that, upon hybridization or only upon hybridization to a target RNA, substantially forms at least a portion of a structural feature, other than a single A/C mismatch feature at the target adenosine to be edited.

[030] As used herein, the term “facilitates RNA editing” by an engineered guide RNA refers to the ability of the engineered guide RNA when associated with an RNA editing entity and a target RNA to provide a targeted edit of the target RNA by the RNA edited entity. In some instances, the engineered guide RNA can directly recruit or position/ orient the RNA editing entity to the proper location for editing of the target RNA. In other instances, the engineered guide RNA when hybridized to the target RNA forms a guide-target RNA scaffold with one or more structural features as described herein, where the guide-target RNA scaffold with structural features recruits or positions/orients the RNA editing entity to the proper location for editing of the target RNA.

[031] A “guide-target RNA scaffold,” as disclosed herein, is the resulting double stranded RNA formed upon hybridization of a guide RNA, with latent structure, to a target RNA. A guide-target RNA scaffold has one or more structural features formed within the double stranded RNA duplex upon hybridization. For example, the guide-target RNA scaffold can have one or more structural features selected from a bulge, mismatch, internal loop, hairpin, or wobble base pair.

[032] As disclosed herein, a “hairpin” includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion. [033] “Homology” or “identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure. Sequence homology can refer to a % identity of a sequence to a reference sequence. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be allowed.

[034] In some cases, the identity between a reference sequence (query sequence, i.e., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program-based son the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB amino acid alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be shorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction can be made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the N- and C-terminal of the subject sequence, which can be not matched/ aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which can be not matched/aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest bland C-terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching/ alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the N- or C-termini of the subject sequence which can be not matched/aligned with the query. In this case, the percent identity calculated by FASTDB can be not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched/aligned with the query sequence can be manually corrected for.

[035] In some cases, the identity between a reference sequence (query sequence, i.e., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB amino acid alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be shorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction can be made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the N- and C-terminal of the subject sequence, which can be not matched/ aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which can be not matched/aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest bland C-terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching/ alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the N- or C-termini of the subject sequence which can be not matched/aligned with the query. In this case, the percent identity calculated by FASTDB can be not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched/aligned with the query sequence can be manually corrected for.

[036] As disclosed herein, an “internal loop” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a “bulge” or a “mismatch,” depending on the size of the structural feature. A “symmetrical internal loop” is formed when the same number of nucleotides is present on each side of the internal loop. An “asymmetrical internal loop” is formed when a different number of nucleotides is present on each side of the internal loop.

[037] “Latent structure” refers to a structural feature that substantially forms only upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked.

[038] “Messenger RNA” or “mRNA” can be RNA molecules comprising a sequence that encodes a polypeptide or protein. In general, RNA can be transcribed from DNA. In some cases, precursor mRNA containing non-protein coding regions in the sequence can be transcribed from DNA and then processed to remove all or a portion of the non-coding regions (introns) to produce mature mRNA. As used herein, the term “pre-mRNA” can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions. [039] As disclosed herein, a “mismatch” refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a “bulge” or an “internal loop,” depending on the size of the structural feature.

[040] The term “mutation” as used herein, can refer to an alteration to a nucleic acid sequence or a polypeptide sequence that can be relative to a reference sequence. A mutation can occur in a DNA molecule, a RNA molecule (e.g., tRNA, mRNA), or in a polypeptide or protein, or any combination thereof. The reference sequence can be obtained from a database such as the NCBI Reference Sequence Database (RefSeq) database. Specific changes that can constitute a mutation can include a substitution, a deletion, an insertion, an inversion, or a conversion in one or more nucleotides or one or more amino acids. Non-limiting examples of mutations in a nucleic acid sequence that, without the mutation, encodes for a polypeptide sequence, include: “missense” mutations that can result in the substitution of one codon for another, a “nonsense” mutations that can change a codon from one encoding a particular amino acid to a stop codon (which can result in truncated translation of proteins), or a “silent” mutations that can be those which have no effect on the resulting protein. The mutation can be a “point mutation,” which can refer to a mutation affecting only one nucleotide in a DNA or RNA sequence. The mutation can be a “splice site mutations,” which can be present pre-mRNA (prior to processing to remove introns) resulting in mistranslation and often truncation of proteins from incorrect delineation of the splice site. The mutation can be a fusion gene. A fusion pair or a fusion gene can result from a mutation, such as a translocation, an interstitial deletion, a chromosomal inversion, or any combination thereof. A mutation can constitute variability in the number of repeated sequences, such as triplications, quadruplications, or others. For example, a mutation can be an increase or a decrease in a copy number associated with a given sequence (i.e., copy number variation, or CNV). A mutation can include two or more sequence changes in different alleles or two or more sequence changes in one allele. A mutation can include two different nucleotides at one position in one allele, such as a mosaic. A mutation can include two different nucleotides at one position in one allele, such as a chimeric. A mutation can be present in a malignant tissue. A mutation can comprise a single nucleotide variation (SNV). A mutation can comprise a sequence variant, a sequence variation, a sequence alteration, or an allelic variant.

[041] A presence or an absence of a mutation can indicate an increased risk to develop a disease or condition. A presence or an absence of a mutation can indicate a presence of a disease or condition. A mutation can be present in a benign tissue. Absence of a mutation can indicate that a tissue or sample can be benign. As an alternative, absence of a mutation may not indicate that a tissue or sample can be benign. Methods as described herein can comprise identifying a presence of a mutation in a sample.

[042] The terms “polynucleotide” and “oligonucleotide” can be used interchangeably and can refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following can be non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by nonnucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term can also refer to both double- and singlestranded molecules. Unless otherwise specified or required, any embodiment of this invention that can be a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. [043] A polynucleotide can be composed of a specific sequence of nucleotides. A nucleotide comprises a nucleoside and a phosphate group. A nucleotide comprises a sugar (e.g., ribose or 2 ’deoxyribose) and a nucleobase, such as a nitrogenous base. Non-limiting examples of nucleobases include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), and inosine (I). In some embodiments, I can be formed when hypoxanthine can be attached to ribofuranose via a P-N9-glycosidic bond, resulting in the chemical structure:

[044] Some polynucleotide embodiments refer to a DNA sequence. In some embodiments, the DNA sequence can be interchangeable with a similar RNA sequence. Some embodiments refer to an RNA sequence. In some embodiments, the RNA sequence can be interchangeable with a similar DNA sequence. In some embodiments, any of the Us and Ts can be interchanged in a sequence provided herein.

[045] The term “protein”, “peptide” and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence. As used herein the term “amino acid” can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term “fusion protein” can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function. In this regard, the term “linker” can refer to a protein fragment that can be used to link these domains together - optionally to preserve the conformation of the fused protein domains, prevent unfavorable interactions between the fused protein domains which can compromise their respective functions, or both.

[046] The term “stop codon” can refer to a three nucleotide contiguous sequence within messenger RNA that signals a termination of translation. Non-limiting examples include in RNA, UAG (amber), UAA (ochre), UGA (umber, also known as opal) and in DNA TAG, TAA or TGA. Unless otherwise noted, the term can also include nonsense mutations within DNA or RNA that introduce a premature stop codon, causing any resulting protein to be abnormally shortened.

[047] The term “structured motif’ refers to a combination of two or more features in a dsRNA substrate.

[048] The terms “subject,” “individual,” or “patient” can be used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is in some cases not necessarily diagnosed or suspected of being at high risk for the disease

[049] The term “/« vivo” refers to an event that takes place in a subject’s body.

[050] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay is in some cases not performed on a subject. Rather, it can be performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample can be an “/« vitro” assay.

[051] The term “/« vitro” refers to an event that takes places contained in a container for holding laboratory reagent such that it can be separated from the biological source from which the material can be obtained. In vitro assays can encompass cell-based assays in which living or dead cells can be employed. In vitro assays can also encompass a cell-free assay in which no intact cells can be employed.

[052] The term “wobble base pair” refers to two bases that weakly pair. For example, a wobble base pair can refer to a G paired with a U.

[053] As used herein, the terms “treatment” or “treating” can be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit can refer to eradication or amelioration of one or more symptoms of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement can be observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made.

[054] The section headings used herein can be for organizational purposes only and are not to be construed as limiting the subject matter described.

TARGETING OF RIBONUCLEIC ACID

[055] In some embodiments, an engineered guide is used herein to target a nucleotide of an RNA. In some embodiments, the engineered guide is an SLS guide, and the SLS guide is used herein to target a nucleotide of an RNA. In some embodiments, an SLS guide is used herein to target a nucleotide of an RNA with a mutation. In some embodiments, an SLS guide is used herein to target a nucleotide of an RNA 5’ UTR, 3’UTR, splice site, translation initiation site, or stop codon. Targeting an RNA can be a process by which RNA can be enzymatically modified post synthesis on specific nucleosides or bases. Targeting of RNA can be a way to modulate expression of a polypeptide. For example, through modulation of polypeptide-encoding dsRNA substrates that enter the RNA interference (RNAi) pathway. This modulation can then act at the chromatin level to modulate expression of the polypeptide. Targeting of RNA can also be a way to regulate gene translation. RNA editing can be a mechanism in which to regulate transcript recoding by altering a nucleotide base of the triplet codon to introduce silent mutations and/or non-synonymous mutations.

[056] Specific RNA editing can lead to transcript recoding. Because inosine shares the base pairing properties of guanosine, the translational machinery interprets edited adenosines as guanosine, altering the triplet codon, which can result in amino acid substitutions in protein products. More than half the triplet codons in the genetic code could in theory be reassigned through RNA editing. Due to the degeneracy of the genetic code, RNA editing can cause both silent and non-synonymous amino acid substitutions.

[057] Targeting an RNA can chemically transform a base of a nucleotide in a targeted RNA. In some cases, targeting an RNA can affect splicing. Adenosines targeted for editing can be disproportionately localized near splice junctions in pre-mRNA. For example, during formation of a dsRNA ADAR substrate (e.g., a guide-target RNA scaffold), intronic cis-acting sequences can form RNA duplexes encompassing splicing sites and potentially obscuring them from the splicing machinery. As another example, after the formation of a dsRNA ADAR substrate (e.g., a guide-target RNA scaffold), modification of select adenosines by ADARs can create or eliminate splicing sites, broadly affecting later splicing of the transcript. Similar to the translational machinery, the spliceosome interprets inosine as guanosine, and therefore, a canonical GU 5' splice site and AG 3' acceptor site can be created via the deamination of AU (IU = GU) and AA (Al = AG), respectively. Correspondingly, RNA editing can destroy a canonical AG 3' splice site (IG = GG).

[058] A chemical transformation of a targeted RNA can result in an increased level of a protein or fragment thereof after translation of the targeted RNA with the chemical transformation (e.g., after chemical transformation of an adenosine to an inosine by ADAR), relative to an otherwise comparable RNA lacking the chemical transformation. In some embodiments, a chemical transformation of a targeted RNA can result in a decreased level of a protein or fragment thereof after translation of the targeted RNA with the chemical transformation (e.g., after chemical transformation of an adenosine to an inosine by ADAR), relative to an otherwise comparable RNA lacking the chemical transformation. In some embodiments, a chemical transformation can convert a sense codon into a stop codon. In some embodiments, a chemical transformation can convert a stop codon into a sense codon. In some embodiments, a chemical transformation can convert a first sense codon into a second sense codon. In some embodiments, a chemical transformation can convert a first stop codon into a second stop codon. In some embodiments, a chemical transformation can alter a translation initiation site. In some embodiments, a chemical transformation can alter the localization, folding, stability, or synthesis of a protein or fragment thereof after translation of a targeted RNA with the chemical transformation (e.g., after chemical transformation of an adenosine to an inosine by ADAR), relative to an otherwise comparable RNA lacking the chemical transformation. In some cases, a chemical transformation can alter the localization, folding, stability, or synthesis of a targeted RNA with the chemical transformation, relative to an otherwise comparable target RNA lacking the chemical transformation. In some cases, a target RNA can comprise a coding or a non-coding RNA.

[059] Targeting of RNA can comprise any one of an insertion, deletion, or substitution of a base. Examples of RNA targeting include pseudouridylation (the isomerization of uridine residues) and deamination (removal of an amine group from cytidine to give rise to uridine, or C- to-U editing). Examples of RNA targeting also include deamination of adenosine, to give rise to A-to-I editing.

[060] The engineered guide RNAs disclosed herein, such as SLS guides, can facilitate editing of a target RNA sequence(s). The target RNA sequence can at least partially encode a protein implicated in a disease. For example, the target RNA sequence at least partially encodes a protein implicated in a neurodegenerative disease (such as an amyloid precursor protein, a Tau protein, or alpha-synuclein). For example, the target RNA sequences at least partially encodes a protein implicated in a liver/lung disease (e.g., AAT-1 protein). For example, the target RNA sequence at least partially encodes a protein implicated in macular degeneration (e.g., ABCA4 or EVOLO4 protein).

[061] Furthermore, the RNA editing entity can be ADAR and upon association with a SLS guide can edit an adenosine in a target RNA polynucleotide sequence to an inosine. More than one composition can be developed and administered, such as a first composition for editing of an RNA sequence that encodes a first protein and a second composition for editing an RNA sequence that encodes a second protein. A composition for editing an RNA sequence can result in knockdown of expression of a target. A composition for editing an RNA sequence can result in prevention or reduction in cleavage of a protein. A composition for editing an RNA sequence can result in expression of the wild-type protein.

[062] Compositions and methods provided herein can be utilized to modulate expression of a target RNA. Modulation can refer to altering the expression of a gene or portion thereof at one of various stages, with a view to alleviate a disease or condition associated with the gene or a mutation in the gene. Modulation can be mediated at the level of transcription or post- transcriptionally. Modulating transcription can correct aberrant expression of splice variants generated by a mutation in a gene. In some cases, compositions and methods provided herein can be utilized to regulate RNA translation of a target.

[063] In some cases, a target RNA sequence can be associated with a disease or condition. In some cases, a disease or condition can be associated with a mutation in a DNA molecule or RNA molecule encoding ABCA4, AAT, SERPINA1, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, Tau, GBA, PINK1, RAB7A, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, a fragment any of these, or any combination thereof. In some examples, a protein encoded for by a mutated DNA molecule or mutated RNA molecule selected from the group consisting of: ABCA4, AAT, SERPINA1, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, Tau, GBA, PINK1, RAB7A, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, PCSK9 start site, SCNN1A start site, or any combination thereof contributes to, the pathogenesis or progression of a disease. In some examples, the mutation in the DNA or RNA molecule can be relative to an otherwise identical reference DNA or RNA molecule. In some cases, a disease or condition can be associated with expression of a protein from a target RNA, wherein a translation initiation site, 5’UTR, 3’UTR, splice site, or stop codon of the target RNA is to be edited to treat the disease or condition.

COMPOSITIONS

1. Engineered guide RNAs

[064] Provided herein are engineered guides (e.g., SLS guides), engineered polynucleotides encoding engineered guide RNAs(e.g., SLS guides), and compositions comprising said engineered guide RNAs (e.g., SLS guides) and said engineered polynucleotides (e.g., polynucleotides comprising SLS guides). Herein, the term “engineered guide” can be used interchangeably with the term “engineered guide RNA.” In some examples, the engineered guide can be an SLS guide. In some examples, the engineered guide can be encoded by an engineered polynucleotide. In some embodiments, the engineered guide RNA comprises a targeting domain that at least partially binds to the target RNA. In some embodiments, the engineered polynucleotide codes for an engineered guide RNA (e.g., an SLS guide.

[065] In some embodiments, an engineered guide (e.g., an SLS guide) of the disclosure can be utilized for RNA editing, for example to prevent or treat a disease or condition. In some cases, an engineered guide can be used in association with a subject RNA editing entity to edit a target RNA or modulate expression of a polypeptide encoded by the target RNA. In some embodiments, compositions disclosed herein can include engineered guides capable of facilitating editing by subject RNA editing entities such as ADAR polypeptides or biologically active fragments thereof.

[066] In some embodiments, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target RNA, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA (e.g., guide-target RNA scaffold), wherein the guide-target RNA scaffold comprises at least one structural feature, and wherein the guide-target RNA scaffold recruits an RNA editing entity and facilitates a chemical modification of a base of a nucleotide in the target RNA by the RNA editing entity.

[067] In some embodiments, the engineered guides disclosed herein comprise at least one nucleic acid structural feature, wherein the engineered guide can be configured to facilitate editing of a nucleotide base of a nucleotide of a target RNA.

[068] In some embodiments, the targeting domains disclosed herein comprise at least one nucleic acid structural feature, wherein the targeting domain can be configured to facilitate editing of a nucleotide base of a nucleotide of a target RNA.

[069] In some embodiments, the engineered guides (e.g., an SLS guide) disclosed herein comprise: (a) at least one RNA-editing enzyme recruiting domain; (b) at least one nucleic acid structural feature; and wherein the engineered guide can be configured to facilitate editing of a nucleotide base of a nucleotide of a target RNA.

[070] In some embodiments, chemical modification of the base of the nucleotide in the target RNAs can be confirmed by an in vitro assay. In some examples, the in vitro assay is a sequencing assay. In some cases, chemical modification of the base can be confirmed by delivering the engineered guide to cells in vitro or in vivo and measuring the amount of mutated target bases in the target RNA.

[071] In some embodiments, the engineered guide RNA (e.g., an SLS guide) is singlestranded. In some cases, the engineered guide RNA is at least partially single-stranded. In some cases, the engineered guide RNA is partially single-stranded.

[072] In some embodiments, an engineered guide RNA (e.g., an SLS guide) can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some cases, a backbone of an engineered guide RNA can comprise a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA.

[073] In some embodiments, a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to a solvent. In some embodiments, a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to nucleases. In some embodiments, a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes. In some instances, a backbone of an engineered guide RNA can be represented as a polynucleotide sequence in a circular 2- dimensional format with one nucleotide after the other. In some instances, a backbone of an engineered guide RNA can be represented as a polynucleotide sequence in a looped 2- dimensional format with one nucleotide after the other. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be joined through a phosphorus-oxygen bond. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be modified into a phosphoester with a phosphorus- containing moiety.

2. Structural loop stabilized (SLS) guide RNAs

A. SLS Guide Overview

[074] Engineered guide RNAs disclosed herein further comprise structural loop stabilized (SLS) scaffold. These can be referred to as structural loop stabilized (SLS) guide RNAs. [075] In some embodiments, an SLS scaffold can comprise a 5’ end and 3’ end that together form a secondary or tertiary structure, which is present in the absence of binding to a target RNA. Therefore, the SLS scaffold is a preformed structural feature that can be present in the SLS gRNA, and not a structural feature formed by latent structure provided in a targeting domain. For example, a SLS guide RNA can comprise a stem-loop structure in which the 5’ end and the 3’ end together form part of the stem loop structure. As another example, an SLS guide RNA utilizes a structured loop stabilized scaffold, such as a tRNA scaffold, to impart beneficial properties onto the guide RNA. In some embodiments, an engineered guide comprising a SLS scaffold can provide greater stability, improved recruitment of RNA-editing entities (such as endogenous RNA editing enzymes), longer half-lives, and/or improved RNA-editing efficiency as compared to engineered guides lacking the SLS scaffold.

[076] In some embodiments, the structural loop stabilized scaffold comprises nucleic acid structures such as RNA structures. In one aspect, the SLS scaffold comprises a secondary structure. In some embodiments, the 5’ end and the 3’ end of the SLS scaffold together form the secondary structure. In some cases, the SLS scaffold blocks or partially blocks a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, from exposure to a solvent. In some cases, the SLS guide blocks or paritally blocks a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, from exposure to a solvent. In some cases, the secondary structure or the RNA structures can take the form of a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof. In one aspect, the secondary structure comprises an RNA editing entity recruiting domain. In some cases, the SLS scaffold comprises a recruiting sequence for an RNA editing entity. In some cases, the SLS scaffold comprises a targeting domain that can form latent structural features for an RNA editing entity. In some cases, the structural loop stabilized scaffold comprises secondary RNA structure, tertiary RNA structure, quaternary structure, or a combination thereof. In some cases, the structural loop stabilized scaffold comprises structures of an RNA aptamer. In some instances, the structural loop stabilized scaffold comprises structures of any RNA species (e.g., ribosomal RNA, regulatory RNA, or tRNA). In some instances, the structural loop stabilized scaffold comprises a secondary structure comprising: an acceptor stem composed of a plurality of ribonucleotides of the 5' end of the ribonucleotide chain and the plurality of ribonucleotides preceding the last 4 ribonucleotides of the 3' end of the ribonucleotide chain, thus forming a double-stranded structure comprising a plurality of pairs of ribonucleotides. In some cases, it the ribonucleotides constituted by the ribonucleotide of the 5' end of the ribonucleotide chain and the ribonucleotide that precedes the last 4 ribonucleotides of the 3' end of the ribonucleotide chain can be unpaired. The structural loop stabilized scaffold can further comprise a secondary structure comprising a D arm comprising a plurality of pairs of ribonucleotides and a D loop comprising 1 to 100 ribonucleotides, formed by folding of a part of the ribonucleotide chain that follows the plurality of ribonucleotides of the 5' end of the ribonucleotide chain. In some embodiments, the structural loop stabilized scaffold comprises a secondary structure comprising a stem that can be an equivalent of an anticodon region of a tRNA and a loop of the anticodon region of the tRNA (stem-loop of the anticodon), formed by the folding of a part of the ribonucleotide chain that follows the D arm and the D loop. In some embodiments, the structural loop stabilized scaffold comprises a secondary structure comprising a variable loop constituted by from 1 to 100 ribonucleotides and formed by a part of the ribonucleotide chain that follows the stem of the anticodon and the loop of the anticodon. In some embodiments, the structural loop stabilized scaffold comprises a secondary structure comprising a T arm comprising a plurality of pairs of ribonucleotides, and a T loop comprising 1 to 100 ribonucleotides, formed by the folding of a part of the ribonucleotide chain that follows the variable loop and precedes the ribonucleotides of the 3' end of the ribonucleotide chain of the acceptor stem. In some embodiments, the scaffold described herein comprises a tRNA scaffold, where the structures of the tRNA can be incorporated into the engineered guide RNA described herein.

[077] In some embodiments, the structural loop stabilized scaffold comprises a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof. In one aspect, the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 stem loop structures. In one aspect, the structural loop stabilized scaffold comprises no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 stem loop structures. In one aspect, the structural loop stabilized scaffold comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 stem loop structures. In one aspect, the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 junction structures. In one aspect, the structural loop stabilized scaffold comprises no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 junction structures. In one aspect, the structural loop stabilized scaffold comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 junction structures. In one aspect, the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 T junction structures. In one aspect, the structural loop stabilized scaffold comprises no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 T junction structures. In one aspect, the structural loop stabilized scaffold comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 T junction structures. In one aspect, the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cloverleaf structures. In one aspect, the structural loop stabilized scaffold comprises no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 clover leaf structures. In one aspect, the structural loop stabilized scaffold comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cloverleaf structures. In one aspect, the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 pseudoknot structures. In one aspect, the structural loop stabilized scaffold comprises no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 psueudoknot structures. In one aspect, the structural loop stabilized scaffold comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pseudoknot structures.

[078] In some embodiments, the structural loop stabilized guide RNAs comprise a series of stem-loop structures. For example, a structural loop stabilized guide RNA of the present disclosure can have at least 2 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have 2 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have at least 3 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have at 3 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have at least 4 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have 4 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have at least 5 stem loop structures. A structural loop stabilized guide RNA of the present disclosure can have 5 stem loop structures. [079] In some embodiments, the SLS scaffold comprises atRNA scaffold. In some cases, the SLS scaffold comprises a tRNA comprising a targeting sequence inserted into the anticodon region, wherein the targeting sequence binds to at least a part of the target RNA. In some cases, the tRNA can be an endogenous tRNA with a modified anticodon stem region recognizing the codon in the target RNA comprising a mutation. In some cases, the SLS scaffold can be a tRNA scaffold that cannot be charged with an amino acid. In some cases, the tRNA scaffold is an orthogonal tRNA charged with a non-canonical amino acid. In some cases, the SLS guide RNA can be administered along with a corresponding tRNA synthetase. In some instances, the corresponding synthetase can be E. coli Glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-canonical amino acid can be pyrrolysine.

[080] In some embodiments, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a T loop secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a T loop secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a D loop secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a D loop secondary structure. [081] In some embodiments, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms an anticodon loop secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms an anticodon loop secondary structure.

[082] In some embodiments, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA variable arm secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA variable arm secondary structure.

[083] In some embodiments, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA acceptor stem secondary structure. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA acceptor stem secondary structure.

[084] In some embodiments, the SLS scaffold comprises at least one sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA pseudoknot secondary structure in the acceptor arm. In one aspect, the SLS scaffold comprises at least one sequence that has no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or 99% similarity to a tRNA sequence that forms a tRNA pseudoknot secondary structure in the acceptor arm.

[085] An engineered guide RNA comprising a SLS scaffold can be formed directly or indirectly by forming a linkage (such as a covalent linkage) between more than one end of a RNA sequence, such as a 5’ end and a 3’ end. An RNA sequence can comprise an engineered guide RNA (such as a recruiting domain, targeting domain, or both). A linkage can be formed by employing an enzyme, such as a ligase. A suitable ligase (or synthetase) can include a ligase that forms a covalent bond. A covalent bond can include a carbon-oxy gen bond, a carbon-sulfur bond, a carbon-nitrogen bond, a carbon-carbon bond, a phosphoric ester bond, or any combination thereof. A linkage can also be formed by employing a recombinase. An enzyme can be recruited to an RNA sequence to form a linkage. A circular or looped RNA can be formed by ligating more than one end of an RNA sequence using a linkage element. In some embodiments, a linkage can be formed by a ligation reaction. In some instances, a linkage can be formed by a homologous recombination reaction. A linkage element can employ click chemistry to form a circular or looped RNA. A linkage element can be an azide-based linkage. A circular or looped RNA can be formed by genetically encoding or chemically synthesizing the circular or looped RNA. A circular or looped RNA can be formed by employing a self-cleaving enzyme, such as a ribozyme, tRNA, aptamer, catalytically active fragment of any of these, or any combination thereof. For example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ end, a 5’ end, or both of a precursor of the engineered RNA. In another example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ terminal end, a 5’ terminal end, or both of a precursor of the engineered RNA. A self-cleaving ribozyme can comprise, for example, an RNase P RNA a Hammerhead ribozyme (e.g. a Schistosoma mansoni ribozyme), a glmS ribozyme, an HDV-like ribozyme, an R2 element, or a group I intron. In some cases, the self-cleaving ribozyme can be a /ra -acting ribozyme that joins one RNA end on which it can be present to a separate RNA end. In some cases, a self-cleaving element or an aptamer can be configured to facilitate self-circularization of an engineered guide RNA described herein. i. Spacer domains

[086] An SLS guide RNA as described herein can include spacer domains. As described herein, a spacer domain can refer to a domain that provides space between other domains. A spacer domain can be used between the SLS scaffold and the targeting domain and/or RNA editing entity recruiting domain. In some embodiments, the spacer sequence is between a portion of the SLS scaffold and the targeting domain and/or RNA editing entity recruiting domain. For example, a targeting domain and/or RNA editing entity recruiting domain located in the anticodon region of the SLS scaffold (e.g., a tRNA scaffold) is flanked by spacer domains. In some embodiments, a spacer domain is 5’ of the targeting domain and/or RNA editing entity recruiting domain located in the anticodon region of the SLS scaffold. In some embodiments, a spacer domain is 3’ of the targeting domain and/or RNA editing entity recruiting domain located in the anticodon region of the SLS scaffold. Where the region of the SLS scaffold includes a targeting domain as described herein that is configured to associate to a target polynucleotide, the addition of spacers can provide improvements (e.g., increased specificity, enhanced editing efficiency, etc.) in the engineered SLS gRNA comprising a spacer domain relative to a comparable engineered SLS gRNA that lacks a spacer domain. In some instances, the spacer domain is configured to not hybridize with the target RNA in a contiguous fashion with the target RNA. In such a configuration, the spacer domain is not simply increasing the amount of overlap between the target domain of the engineered guide and the target RNA. Rather, the spacer domain can be used to elongate the SLS scaffold outside of the overlap region between the targeting domain and the target RNA (e.g. increase the size of an engineered SLS gRNA). By increasing the size of the engineered SLS gRNA outside of the overlap region, overall binding efficiency between the targeting domain and target RNA can be improved. In some cases, this improvement can result from providing a more optimal geometry for the targeting domain of the engineered SLS gRNA to bind to the target RNA.

[087] In some cases, the SLS guide comprising the spacer domain can have a lower Gibbs free energy (AG) of binding of the targeting domain and/or RNA editing entity recruiting domain to the target RNA, relative to a AG of binding of a corresponding SLS guide that lacks the spacer domain, to the target RNA, as determined by KPFM. In some cases, the targeting domain can be configured to at least partially associate with an untranslated region of the target RNA, wherein the association of the targeting domain with the untranslated region of the target RNA facilitates a reduction in an expression level of a polypeptide encoded for by the target RNA and wherein association of the targeting domain with a sequence of the target RNA facilitates an edit of a base of a nucleotide of the target RNA by an RNA editing entity.

[088] In some embodiments, an SLS guide can comprise a targeting domain that can be at least partially complementary to a target RNA, an RNA editing entity recruiting domain, and a spacer domain. In some cases, a spacer domain of an SLS guide can enlarge the SLS guide by the addition of one or more nucleotides. In some instances, the targeting domain can be configured to at least partially associate with a coding region of the target RNA. In some cases, the association of the targeting domain with the coding region of the target RNA can facilitate an edit of a base of a nucleotide of the target RNA by an RNA editing entity

[089] In some cases, a spacer domain can have a sequence length of from about: 1 nucleotide to about 1,000 nucleotides, 2 nucleotides to about 20 nucleotides, 10 nucleotides to about 100 nucleotides, 50 nucleotides to about 500 nucleotides or about 400 nucleotides to about 1000 nucleotides in length. In some cases, a spacer domain can have a sequence length of at least

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,

125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,

144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,

163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,

182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,

201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,

220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,

239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,

258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,

277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,

296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,

315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,

334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,

372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,

391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,

410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,

429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,

448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,

467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,

486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 600, 700, 800, 900,

1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides, or any number of nucleotides therebetween.

[090] In some embodiments, about 80% of the nucleotides of a spacer domain can be non- complementary to the target RNA. In some cases, a spacer domain can have a sequence length of about 5 nucleotides. In some cases, a spacer domain can have a sequence length of about 10 nucleotides. In some cases, a spacer domain can have a sequence length of about 15 nucleotides. In some cases, a spacer domain can have a sequence length of about 20 nucleotides. In some embodiments, a spacer domain can comprise a polynucleotide sequence of 5’ AT ATA 3 ’ (SEQ ID 11), 5’ ATAAT 3’ (SEQ ID 12), or any combination thereof. In some cases, a spacer domain can comprise a sequence of 5’AUAAU 3’ (SEQ ID 13), 5’AUAUA 3’ (SEQ ID 14), 3’AUAUA 5’ (SEQ ID 15), or 3’AUAAU 5’ (SEQ ID 16). In some embodiments, a spacer domain can be at least a single nucleotide, such as A, T, G, C or U.

[091] A spacer domain can be located proximal to a targeting domain, proximal to a ligation domain, proximal to a ribozyme domain, proximal to a RNA editing recruiting domain, proximal to another spacer domain, proximal to an SLS scaffold, or proximal to a portion of an SLS scaffold, where proximal can mean separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,

131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,

150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,

169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,

188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,

207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,

226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,

245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,

264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,

283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,

302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,

321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,

340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,

359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,

378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,

397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,

416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,

435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,

454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,

473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,

492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides.

[092] In some embodiments, a spacer domain can be separated from a targeting domain by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,

82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,

125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,

144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,

163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,

182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,

201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,

220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,

239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,

258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,

277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,

296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,

315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,

334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,

372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,

391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,

410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,

429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,

448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,

467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,

486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides.

[093] In some embodiments, a spacer domain can be separated from a SLS scaffold or portion of an SLS scaffold by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,

100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,

119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,

138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,

157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides.

[094] An SLS guide can comprise a single spacer domain. In some cases, an SLS guide can comprise a second spacer domain. In some cases, the first spacer domain, the second spacer domain or both can be configured to not bind to the target RNA when the targeting domain binds to the target RNA. In some embodiments an SLS guide can comprise multiple spacer domains, for example 2, 3, 4, 5, 6, 7, or 8 spacer domains.

[095] In some cases, a spacer sequence can comprise about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,

57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% the total sequence of an SLS guide. In some cases, a engineered guide can comprise about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,

26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% the total sequence of an SLS guide. In some cases, an SLS guide can comprise 1, 2, 3, 4, or more spacer sequences.

[096] A spacer domain can be configured to facilitate an engineered guide (e.g., SLS guide) adopting a conformation that facilitates at least partial binding to a target RNA. In some cases, a spacer domain can change the geometry of a targeting domain of an engineered guide (e.g., SLS guide) so that the targeting domain of the polynucleotide can be substantially linear.

[097] An engineered guide (e.g., SLS guide), comprising a spacer domain can have an increase in the binding specificity to a target RNA, among a plurality of other RNAs, relative to the binding specificity of a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain. In some embodiments, an increase in the binding specificity to a target RNA can be determined by sequencing of a target RNA and plurality of other RNAs after contacting to an engineered guide (e.g., SLS guide) comprising a spacer domain compared to a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain. In some instances, a spacer domain can be configured to facilitate a lower entropy (AS) of binding of a engineered guide (e.g., SLS guide) to a target RNA. In some embodiments, a spacer domain can be configured to at least maintain an editing efficiency of a engineered guide (e.g., SLS guide) to a target RNA, relative to the editing efficiency of a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain. In some cases, an editing efficiency can be determined by sequencing of a target RNA after contacting to a engineered guide (e.g., SLS guide) comprising a spacer domain or to a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain. In some embodiments, an at least maintain can comprise an increase. In some instances, an editing efficiency can be determined by mass spectroscopy of a target RNA after contacting to an engineered guide (e.g., SLS guide) comprising a spacer domain or a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain.

[098] In some embodiments, the editing efficiency of an engineered guide (e.g., SLS guide) comprising a spacer domain can be from about 2x to about 5x greater than a comparable engineered guide (e.g., SLS guide) that lacks the spacer. In some instances, the editing efficiency of an engineered guide (e.g., SLS guide) comprising a spacer delivered to a cell or to a subject can be from about 3x to about 6x greater than a comparable engineered guide (e.g., SLS guide) that lacks the spacer. In some cases, the percent editing of a target RNA by an engineered guide (e.g., SLS guide) comprising a spacer can be about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% greater than a comparable engineered guide (e.g., SLS guide)that lacks the spacer.

[099] In some cases, Kelvin Probe Force Microscopy (KPFM) can be used to determine the Gibbs free energy (AG) of binding of an engineered guide (e.g., SLS guide) comprising a spacer domain to a target RNA, relative to a AG of binding of a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain: A target RNA can be immobilized on a bare gold nanoparticle. An engineered guide (e.g., SLS guide) comprising or lacking the spacer domain, can be added and allowed to bind to the target RNA immobilized on the gold particle. The topography and surface potential images can then be measured with KPFM and used to calculate the AG of binding between the engineered guide (e.g., SLS guide) comprising or lacking the spacer domain, and the target RNA.

[100] An in vitro half-life of an engineered guide (e.g., SLS guide) comprising a spacer domain can be at least about: lx, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 5x, lOx, 20x longer or more as compared to a substantially comparable engineered guide (e.g., SLS guide) that lacks the spacer domain. An in vivo half-life of an an engineered guide (e.g., SLS guide) comprising a spacer domain can be at least about: lx, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 5x, lOx, 20x longer or more as compared to a substantially comparable engineered guide (e.g., SLS guide) that lacks the spacer domain. A dosage of a composition comprising an engineered guide (e.g., SLS guide) comprising a spacer domain administered to a subject in need thereof can be at least about: lx, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 5x, lOx, or 20x less as compared to a composition comprising a substantially comparable engineered guide (e.g., SLS guide)that lacks the spacer domain administered to a subject in need thereof. A composition comprising an engineered guide (e.g., SLS guide) comprising a spacer domain administered to a subject in need thereof can be given as a single time treatment as compared to a composition comprising a substantially comparable engineered guide (e.g., SLS guide) that lacks the spacer domain given as a two-time treatment or more.

[101] An engineered guide (e.g., SLS guide) comprising a spacer domain can comprise a half-life at least about: 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or lOx greater than a comparable engineered guide (e.g., SLS guide) that lacks the spacer. A half-life of an engineered guide (e.g., SLS guide) comprising a spacer domain can be from about 2x to about 5x greater than a comparable engineered guide (e.g., SLS guide) that lacks the spacer. A half-life of an engineered guide (e.g., SLS guide) comprising a spacer delivered to a cell or to a subject can be from about 3x to about 6x greater than a comparable engineered guide (e.g., SLS guide) that lacks the spacer.

[102] In some embodiments, an engineered guide can comprise: an SLS scaffold; a targeting domain that is at least partially complementary to a target RNA; an RNA editing entity recruiting domain, wherein the RNA editing entity recruiting domain is configured to at least transiently associate with an RNA editing entity; and a spacer domain. In some embodiments, a SLS guide can comprise: an SLS scaffold; an engineered guide comprising at least one nucleic acid structural feature, wherein the engineered guide can be configured to facilitate editing of a nucleotide base of a nucleotide of a target RNA; and a spacer domain. In some cases, the engineered guide (e.g., SLS guide) comprising the spacer domain can have a lower Gibbs free energy (AG) of binding of the engineered guide (e.g., SLS guide) to the target RNA, relative to a AG of binding of a corresponding engineered guide (e.g., SLS guide) that lacks the spacer domain, as determined by Kelvin Probe Force Microscopy. In some cases, the targeting domain can be configured to at least partially associate with a coding region of the target RNA. In some cases, the association of the targeting domain with the coding region of the target RNA can facilitate an edit of a base of a nucleotide of the target RNA by an RNA editing entity.

B. Benefits of SLS guide RNAs

[103] In some cases, an engineered guide comprising a SLS scaffold can provide various benefits as compared to an engineered guide lacking the SLS scaffold. An engineered guide comprising a SLS scaffold can provide greater stability, improved recruitment of RNA-editing entities (such as endogenous RNA editing enzymes), longer half-lives, improved RNA-editing efficiency, or any combination thereof, as compared to an engineered guide lacking the SLS scaffold. An engineered guide comprising a SLS scaffold an provide one or more of these improved qualities and can retain genetic encodability as compared guide polynucleotides comprising other types of modifications designed to improve guide stability — such as chemical modifications or sugar additions. An engineered guide comprising a SLS scaffold can be capable of being genetically encoded, capable of being delivered by a vector, and retain improved stability.

[104] In some instances, an engineered guide comprising a SLS scaffold (e.g., SLS guide) can have significantly increased half-life as compared to a comparable engineered guide lacking a SLS scaffold. In some embodiments, forming a SLS scaffold can significantly increase a half- life of an engineered guide RNA when delivered in vivo, as compared to a comparable engineered guide RNA lacking the SLS scaffold. In some cases, forming an SLS scaffold can significantly reduce an amount (such as a therapeutically effective amount) of the engineered guide RNA dosed to a subject, as compared to a comparable engineered guide RNA lacking a SLS scaffold. In some embodiments, an engineered guide RNA comprising an SLS scaffold can significantly enhance efficiency of editing, can significantly reduce off-target editing, enhance efficiency of recruiting an RNA editing enzyme, or a combination thereof, as compared to a comparable engineered guide RNA lacking an SLS scaffold. In some embodiments, an engineered guide RNA comprising a SLS scaffold can significantly increase the transport of the engineered guide RNA into a cell, as compared to a comparable engineered guide RNA lacking the SLS scaffold. In some cases, an engineered guide RNA comprising an SLS scaffold can significantly increase the intracellular retention of the engineered guide RNA, as compared to a comparable engineered guide RNA that lacks an SLS scaffold.

[105] In some embodiments, the SLS guide, upon binding to the target RNA, can be more efficient in recruiting the RNA editing enzyme for editing the target RNA relative to an otherwise identical reference polynucleotide (e.g. reference guide lacking a SLS scaffold). In some embodiments, the SLS guide can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more efficient in recruiting the RNA editing enzyme for editing the target RNA relative to an otherwise identical reference guide lacking a SLS scaffold. In some embodiments, the efficiency of editing the target RNA can be measured to amplifying and sequencing the edited target RNA by methods such as Sanger sequencing or sequencing of ddPCR product.

[106] In some embodiments, the engineered guide RNA comprising a SLS scaffold (e.g., SLS guide), upon binding to the target RNA, can be more specific in recruiting the RNA editing enzyme for editing the target RNA relative to an otherwise identical reference engineered guide RNA lacking a SLS scaffold. In some embodiments, the engineered guide RNA comprising an SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more specific in recruiting the RNA editing enzyme for editing the target RNA relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the specificity of editing of the target RNA can be determined by amplifying and sequencing the edited target RNA by methods such as Sanger sequencing or sequencing of ddPCR product. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) comprises an increased resistance towards degradation by hydrolysis compared to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by hydrolysis relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by hydrolysis relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be contacted with a cell. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by hydrolysis relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be administered to a subject in need thereof. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by hydrolysis relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be in circulation in the subject. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by hydrolysis relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be contacted with the target RNA.

[107] In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) comprises an increased resistance towards degradation by nuclease digestion compared to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by nuclease digestion relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by nuclease digestion relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be contacted with a cell. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by nuclease digestion relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be administered to a subject in need thereof. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by nuclease digestion relative to an otherwise identical reference engineered lacking a SLS scaffold, when the engineered guide can be in circulation in the subject. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more resistant towards degradation by nuclease digestion relative to an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be contacted with the target RNA.

[108] In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less immunogenicity relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, fivefold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce immunogenicity relative to immunogenicity induced by an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less immunogenicity relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce immunogenicity relative to immunogenicity induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be in a cell. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less immunogenicity relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce immunogenicity relative to immunogenicity induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be administered to a subject in need thereof. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less immunogenicity relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce immunogenicity relative to immunogenicity induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be in circulation in the subject. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less immunogenicity relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce immunogenicity relative to immunogenicity induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide can be contacted with the target RNA. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less innate immune response relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce innate immune response relative to innate immune response induced by an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less innate immune response relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce innate immune response relative to innate immune response induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide RNA can be in a cell. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less innate immune response relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce innate immune response relative to innate immune response induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide RNA can be administered to a subject in need thereof. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less innate immune response relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce innate immune response relative to innate immune response induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide RNA can be in circulation in the subject. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) induces less innate immune response relative to an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide)may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce innate immune response relative to innate immune response induced by an otherwise identical reference engineered guide lacking a SLS scaffold, when the engineered guide RNA can be contacted with the target RNA.

[109] In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide), when contacted with the target RNA, can be less likely to induce off-target editing of the target RNA by the RNA editing enzyme relative to the off-target editing of the target RNA by the same RNA editing enzyme induced by an otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the engineered guide comprising a SLS scaffold (e.g., SLS guide) can be at least at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 500 fold, 1000 fold, or more less likely to induce off-target editing relative to the otherwise identical reference engineered guide lacking a SLS scaffold. In some embodiments, the prevalence of the off-targeting can be measured to amplifying and sequencing the edited target RNA by methods such as Sanger sequencing or sequencing of ddPCR product.

3. Targeting Domain

[110] An engineered guide RNA of the present disclosure can comprise a targeting domain. For example, an SLS guide RNA can comprise a targeting domain. Engineered guides (e.g., an SLS guide) as disclosed herein can be engineered in any way suitable for RNA editing. In some examples, an engineered guide generally comprises at least a targeting sequence that allows it to hybridize to a region of a target RNA. A targeting sequence can also be referred to as a “targeting domain” or a “targeting region”.

[111] In some embodiments, a targeting sequence of an engineered guide allows the engineered guide to target an RNA sequence through base pairing, such as Watson Crick base pairing. In some examples, the targeting sequence can be located at either the N-terminus or C- terminus of the engineered guide. In some cases, the targeting sequence can be located at both termini. The targeting sequence can be of any length. In some cases, the targeting sequence can be at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,

121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,

140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or up to about 200 nucleotides in length.

In some cases, the targeting sequence can be no greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,

131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,

150, or 200 nucleotides in length. In some examples, an engineered guide comprises a targeting sequence that can be about 75-100, 80-110, 90-120, or 95-115 nucleotides in length. In some examples, an engineered guide comprises a targeting sequence that can be about 100 nucleotides in length.

[112] In some embodiments, a targeting sequence comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to a target RNA. In some cases, a targeting sequence comprises less than 100% complementarity to a target RNA sequence. For example, a targeting sequence and a region of a target RNA that can be bound by the targeting sequence can have a single base mismatch. In other cases, the targeting sequence of a subject engineered guide RNA comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 30, 40 or up to about 50 base mismatches. In other cases, the targeting sequence of a subject engineered guide RNA comprises nor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 30, 40 or 50 base mismatches. In some examples, nucleotide mismatches can be associated with structural features provided herein. In some examples, a targeting sequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or up to about 15 nucleotides that differ in complementarity from a wildtype RNA of a subject target RNA. In some examples, a targeting sequence comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides that differ in complementarity from a wildtype RNA of a subject target RNA. In some cases, a targeting sequence comprises at least 50 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 150 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 200 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 250 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 300 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 191, 192, 193,

194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,

213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253,

254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,

273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,

292, 293, 294, 295, 296, 297, 298, 299, or 300 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises more than 50 nucleotides total and has at least 50 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 150 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 200 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 250 nucleotides having complementarity to a target RNA. In some cases, a targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 300 nucleotides having complementarity to a target RNA. In some cases, the at least 50 nucleotides having complementarity to a target RNA are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 150 nucleotides having complementarity to a target RNA are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 200 nucleotides having complementarity to a target RNA are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 250 nucleotides having complementarity to a target RNA are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 300 nucleotides having complementarity to a target RNA are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. For example, a targeting sequence comprises a total of 54 nucleotides wherein, sequentially, 25 nucleotides are complementarity to a target RNA, 4 nucleotides form a bulge, and 25 nucleotides are complementarity to a target RNA. As another example, a targeting sequence comprises a total of 118 nucleotides wherein, sequentially, 25 nucleotides are complementarity to a target RNA, 4 nucleotides form a bulge, 25 nucleotides are complementarity to a target RNA, 14 nucleotides form a loop, and 50 nucleotides are complementary to a target RNA.

[113] The engineered guide RNAs (e.g., SLS guides) disclosed herein can have a targeting domain (e.g., targeting sequence) that can be at least partially complementary to a sequence in a target polynucleotide (e.g., SERPINA1). Said sequence can be within a targeting domain of the engineered guide RNA. For example, the engineered guide RNA can be sufficiently complementary to a sequence of the target polynucleotide to ensure hybridization of the engineered guide RNA to said sequence. In some embodiments, the engineered guide RNA comprises a targeting domain that can be at least 75% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a targeting sequence that can be at least 80% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be at least 90% complementary to the sequence in the target polynucleotide (e.g., SERPINAE) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be at least 92% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be at least 95% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be at least 97% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be at least 99% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. In some embodiments, the engineered guide RNA comprises a sequence that can be 100% complementary to the sequence in the target polynucleotide (e.g., SERPINA1) and hybridizes to the sequence. 4. Recruiting Domain

[114] An engineered guide RNA of the present disclosure can comprise a recruiting domain. For example, an SLS guide RNA can comprise a recruiting domain. In some cases, a subject engineered guide comprises an RNA editing entity recruiting domain. An RNA editing entity can be recruited by an RNA editing entity recruiting domain on an engineered guide. In some cases, a subject engineered guide can be configured to facilitate editing of a base of a nucleotide of a polynucleotide of a region of a subject target RNA, modulation expression of a polypeptide encoded by the subject target RNA, or both. In some cases, an engineered guide can be configured to facilitate an editing of a base of a nucleotide or polynucleotide of a region of an RNA by a subject RNA editing entity. In order to facilitate editing, an engineered guide RNA of the disclosure can recruit an RNA editing entity.

[115] In some embodiments, a recruiting domain can comprise a recruiting sequence for an

RNA editing entity. Various recruiting domains for different RNA editing entities can be utilized. In some examples, a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), APOBEC, MS2-bacteriophage-coat-protein-recruiting domain, Alu, a TALEN recruiting domain, a Zn-finger polypeptide recruiting domain, a mega-TAL recruiting domain, or a Casl3 recruiting domain, combinations thereof, or modified versions thereof. In some examples, more than one recruiting sequence can be included in an engineered guide of the disclosure. In examples where a recruiting sequence can be present, the recruiting sequence can be utilized to position the RNA editing entity to effectively react with a subject target RNA after the targeting sequence, for example an antisense sequence, hybridizes to a target RNA. In some cases, a recruiting sequence can allow for transient binding of the RNA editing entity to the engineered guide. In some examples, the recruiting sequence allows for permanent binding of the RNA editing entity to the engineered guide. A recruiting sequence can be of any length. In some cases, a recruiting sequence can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, up to about 80 nucleotides in length. In some cases, a recruiting sequence can be in some cases no more than about 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 80 nucleotides in length. In some cases, a recruiting sequence can be about 45 nucleotides in length. In some cases, at least a portion of a recruiting sequence comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting sequence comprises about 45 nucleotides to about 60 nucleotides.

[116] Any number of recruiting sequences can be found in a polynucleotide. In some cases, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting sequences can be included in a polynucleotide. Recruiting sequences can be located at any position of subject polynucleotides. In some cases, a recruiting sequence can be on an N-terminus, middle, or C-terminus of a polynucleotide. A recruiting sequence can be upstream or downstream of a targeting sequence. In some cases, a recruiting sequence flanks a targeting sequence of a subject polynucleotide.

[117] In some embodiments, an RNA editing entity recruiting domain comprises a GluR2 sequence or functional fragment thereof. In some cases, a GluR2 sequence can be recognized by an RNA editing entity, such as an ADAR or biologically active fragment thereof. In some embodiments, a GluR2 sequence can be a non-naturally occurring sequence. In some cases, a GluR2 sequence can be modified, for example for enhanced recruitment. In some embodiments, a GluR2 sequence can comprise a portion of a naturally occurring GluR2 sequence and a synthetic sequence.

[118] In some embodiments, a recruiting domain comprises a GluR2 sequence, or a sequence having at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to: SEQ ID NO: 8. In some cases, a recruiting domain can comprise at least about 80% sequence homology to at least about 10, 15, 20, 25, or 30 nucleotides of SEQ ID NO: 8. In some cases, a recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO: 8.

[119] Any number of recruiting sequences can be found in an engineered guide of the present disclosure. In some cases, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting sequences can be included in an engineered guide. Recruiting sequences can be located at any position of subject guides. In some cases, a recruiting sequence can be on an N- terminus, middle, or C-terminus of a polynucleotide. A recruiting sequence can be upstream or downstream of a targeting sequence. In some cases, a recruiting sequence flanks a targeting sequence of a subject guide. A recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting sequence comprising both ribo- and deoxyribonucleotides can in some cases not be excluded.

[120] In some embodiments, a recruiting domain comprises a CRISPR associated recruiting domain sequence. For example, a CRISPR associated recruiting sequence can comprise a Cas protein sequence. In some cases, a Casl3 recruiting domain can comprise a Casl3a recruiting domain, a Casl3b recruiting domain, a Casl3c recruiting domain, or a Casl3d recruiting domain. In some examples, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 20 nucleic acids of a Cast 3b recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a Cast 3b recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of a Casl3b domain. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a Cast 3b domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to a Cast 3b domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to a Cast 3b domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to a Casl3b domain encoding sequence. In some cases, a Casl3b-domain-encoding sequence can be a non-naturally occurring sequence. In some cases, a Casl3b-domain-encoding sequence can comprise a modified portion. In some cases, a Casl3b- domain-encoding sequence can comprise a portion of a naturally occurring Casl3b-domain- encoding-sequence.

[121] Additional, RNA editing entity recruiting domains can be contemplated. In some embodiments, a recruiting domain comprises an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) domain. In some cases, an APOBEC domain can comprise a non-naturally occurring sequence or naturally occurring sequence. In some embodiments, an APOBEC-domain-encoding sequence can comprise a modified portion. In some cases, an APOBEC-domain-encoding sequence can comprise a portion of a naturally occurring APOBEC- domain-encoding-sequence. In some examples, a recruiting domain can be from an MS2- bacteriophage-coat-protein-recruiting domain. In another embodiment, a recruiting domain can be from an Alu domain. In some examples, a recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleotides of an APOBEC, MS2-bacteriophage-coat-protein-recruiting domain, or Alu domain.

[122] A recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting sequence comprising both ribo- and deoxyribonucleotides is in some cases not excluded. [123] In some cases, the recruiting domain and the targeting domain of the engineered guide RNA of the SLS guide at least partially overlap. In some cases, the recruiting domain and the targeting domain comprise at least one nucleotide shared by both domains. In some cases, the recruiting domain and the targeting domain comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more nucleotides shared by both domains. In some cases, the recruiting domain and the targeting domain completely overlap. In some cases, the editing entity recruiting domain and the targeting domain can be the same region of the engineered guide RNA.

[124] In cases where a recruiting sequence is present, the sequence is utilized to position the RNA editing entity to effectively react with the RNA after the targeting sequence, for example the antisense sequence, hybridizes to a target RNA. The recruiting sequence can allow for transient binding of the RNA editing entity to the polynucleotide. In other cases, the recruiting sequence allows for permanent binding of the RNA editing entity to the polynucleotide. A recruiting sequence can be of any length. In some cases, a recruiting sequence is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, up to about 80 bases in length. In some cases, a recruiting sequence is about 45 nucleotides in length. In some cases, at least a portion of a recruiting sequence comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting sequence comprises about 45 nucleotides to about 60 nucleotides.

[125] In some embodiments, an RNA editing entity recruiting domain sequence can comprise at least about 80% sequence homology to at least about 10, 15, 20, 30, 40, or 50 nucleotides of: an Alu domain, an APOBEC recruiting domain, a GluR2 domain, a TALEN recruiting domain, a Zn-finger polypeptide recruiting domain, a mega-TAL recruiting domain, or a Cast 3 recruiting domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 20 nucleotides of an Alu domain. In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an Alu-recruiting domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of an Alu domain. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an Alu domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to an Alu domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to an Alu domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to an Alu domain encoding sequence. In some cases, an Alu-domain-encoding sequence can be a non-naturally occurring sequence. In some embodiments, an Alu-domain-encoding sequence can comprise a modified portion. In some cases, an Alu-domain-encoding sequence can comprise a portion of a naturally occurring Alu- domain-encoding-sequence.

[126] In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 10, 15, 20, 25, or 30 nucleotides of a GluR2 domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a GluR2-recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of a GluR2 domain. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a GluR2 domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to a GluR2 domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to a GluR2 domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to a GluR2 domain encoding sequence. In some embodiments, a GluR2-domain-encoding sequence can be a non-naturally occurring sequence. In some cases, a GluR2-domain-encoding sequence can comprise a modified portion. In some embodiments, a GluR2-domain-encoding sequence can comprise a portion of a naturally occurring GluR2-domain-encoding sequence. In some cases, at least a portion of a recruiting domain can comprise at least about 80% sequence identity to an encoding sequence that recruits an ADAR or biologically active fragment thereof.

[127] In some cases, a recruiting sequence can comprise a GluR2 sequence or a sequence having at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 8. In some cases, the GluR2 sequence is recognized by the dsRBD of an RNA editing entity, such as an ADAR or biologically active fragment thereof. “Glur2 mRNA” as used herein can refer to the mRNA encoding ionotropic AMPA glutamate receptor 2 (“Glur2”) which undergoes adenosine to inosine (A -> I) editing. This mRNA can recruit ADARs in a site-specific manner. [128] In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 20 nucleotides of an MS2-bacteriophage-coat-protein- recruiting domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an MS2-bacteriophage-coat-protein-recruiting- recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of an MS2-bacteriophage-coat-protein-recruiting domain. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an MS2-bacteriophage-coat-protein-recruiting domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to an MS2-bacteriophage-coat-protein- recruiting domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to an MS2- bacteriophage-coat-protein-recruiting domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to an MS2-bacteriophage-coat-protein-recruiting domain encoding sequence. In some embodiments, an MS2-bacteriophage-coat-protein-recruiting-domain-encoding sequence can be a non-naturally occurring sequence. In some cases, an MS2-bacteriophage-coat-protein- recruiting-domain-encoding sequence can comprise a modified portion.

[129] In some cases, a recruiting sequence is an Alu domain. In some cases, a recruiting sequence can comprise an Alu sequence as is known to one skilled in the art.

[130] In some embodiments, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 20 nucleotides of an APOBEC domain. In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an APOBEC-recruiting domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of an APOBEC domain. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to an APOBEC domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to an APOBEC domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to an APOBEC domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to an APOBEC domain encoding sequence. In some cases, an APOBEC-domain-encoding sequence can be a non- naturally occurring sequence. In some embodiments, an APOBEC-domain-encoding sequence can comprise a modified portion. In some cases, an APOBEC-domain-encoding sequence can comprise a portion of a naturally occurring APOBEC-domain-encoding-sequence.

[131] In some cases, a recruiting sequence is an apolipoprotein B mRNA catalytic polypeptide like (APOBEC). In some cases, a recruiting sequence can comprise an APOBEC sequence or a sequence as is known to one skilled in the art.

[132] In some embodiments, a polynucleotide comprises a CRISPR associated recruiting domain sequence. For example, a CRISPR associated recruiting sequence can comprise a Cas protein sequence. In some cases, a Casl3 recruiting domain can comprise a Casl3a recruiting domain, a Cas 13b recruiting domain, a Cas 13c recruiting domain, or a Cas 13d recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to at least about 20 nucleic acids of a Cas 13b recruiting domain. In some embodiments, an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a Cas 13b recruiting domain. In some cases, an RNA editing entity recruiting domain can comprise at least about: 80%, 85%, 90%, or 95% sequence homology to at least about: 15, 20, 25, 30, or 35 nucleic acids of a Casl3b domain. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 80% sequence homology to a Cas 13b domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 85% sequence homology to a Cas 13b domain encoding sequence. In some embodiments, at least a portion of an RNA editing entity recruiting domain can comprise at least about 90% sequence homology to a Cas 13b domain encoding sequence. In some cases, at least a portion of an RNA editing entity recruiting domain can comprise at least about 95% sequence homology to a Cas 13b domain encoding sequence. In some embodiments, a Casl3b-domain-encoding sequence can be a non-naturally occurring sequence. In some cases, a Casl3b-domain-encoding sequence can comprise a modified portion. In some embodiments, a Casl3b-domain-encoding sequence can comprise a portion of a naturally occurring Casl3b-domain-encoding-sequence.

[133] Any number of recruiting sequences can be found in a polynucleotide. In some cases, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting sequences can be included in a polynucleotide. Recruiting sequences can be located at any position of subject polynucleotides. In some cases, a recruiting sequence can be on an N-terminus, middle, or C-terminus of a polynucleotide. A recruiting sequence can be upstream or downstream of a targeting sequence. In some cases, a recruiting sequence flanks a targeting sequence of a subject polynucleotide.

5. Engineered Guides having Latent Structure

[134] In some embodiments, the present disclosure provides for engineered guide RNAs having latent structure. An “engineered latent guide RNA” refers to an engineered guide RNA that comprises latent structure. “Latent structure” refers to a structural feature that substantially forms upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked.

[135] A double stranded RNA (dsRNA) substrate is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. The resulting dsRNA substrate is also referred to herein as a “guide-target RNA scaffold.”

[136] FIG. 6 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side). Unless otherwise noted, the number of participating nucleotides in a given structural feature is indicated as the nucleotides on the target RNA side over nucleotides on the guide RNA side. Also shown in this legend is a key to the positional annotation of each figure. For example, the target nucleotide to be edited is designated as the 0 position.

Downstream (3’) of the target nucleotide to be edited, each nucleotide is counted in increments of +1. Upstream (5’) of the target nucleotide to be edited, each nucleotide is counted in increments of -1. Thus, the example 2/2 symmetric bulge in this legend is at the +12 to +13 position in the guide-target RNA scaffold. Similarly, the 2/3 asymmetric bulge in this legend is at the -36 to-37 position in the guide-target RNA scaffold. As used herein, positional annotation is provided with respect to the target nucleotide to be edited and on the target RNA side of the guide-target RNA scaffold. As used herein, if a single position is annotated, the structural feature extends from that position away from position 0 (target nucleotide to be edited). For example, if a latent guide RNA is annotated herein as forming a 2/3 asymmetric bulge at position -36, then the 2/3 asymmetric bulge forms from -36 position to the -37 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold. As another example, if a latent guide RNA is annotated herein as forming a 2/2 symmetric bulge at position +12, then the 2/2 symmetric bulge forms from the +12 to the +13 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold.

[137] A latent guide RNA can comprise a portion of sequence that, upon hybridization to a target RNA, forms at least a portion of a structural feature, other than a single A/C mismatch feature at the target adenosine to be edited. In some embodiments, the SLS guide comprises a latent guide. In some embodiments, the SLS guide is a latent guide. In some embodiments, a targeting domain is a latent guide. In some embodiments, a latent structural feature formed upon hybridization to a target RNA includes at least two contiguous nucleotides of the engineered guide RNA. In some instances, a latent structural feature can include a mismatch that is in addition to the A/C mismatch feature at the target adenosine to be edited, with this additional mismatch providing an increase in an amount of editing of the target RNA in the presence of the RNA editing entity, relative to an otherwise comparable engineered guide RNA lacking the additional mismatch. In some embodiments, the engineered guide RNAs disclosed herein lack an RNA editing entity recruiting domain that is formed and present in the absence of binding to the target RNA. A double stranded substrate can also be referred to herein as a guide-target RNA scaffold. A guide-target RNA scaffold, as disclosed herein, can be a resulting double stranded RNA duplex formed upon hybridization of an engineered guide RNA to a target RNA, where the engineered guide RNA prior to hybridizing to the target RNA comprise a portion of sequence that, upon hybridization to a target RNA, forms at least a portion of a structural feature, other than a single A/C mismatch feature at the target adenosine to be edited. Accordingly, a guide-target RNA scaffold has structural features formed within the double stranded RNA duplex. For example, the guide-target RNA scaffold can have two or more features selected from a bulge, mismatch, internal loop, hairpin, or wobble base pair. In some embodiments, engineered guide RNAs with latent structure lack an RNA editing entity recruiting domain that is formed and present in the absence of binding to the target RNA. In some embodiments, engineered guide RNAs with latent structure further comprise a recruiting domain that is formed and present in the absence of binding to the target RNA.

[138] In some examples, the engineered guides (e.g., SLS guides) disclosed herein lack a recruiting region and recruitment of the RNA editing entity can be effectuated by the guidetarget RNA scaffold formed by the engineered guide and the target RNA (e.g., by the guidetarget RNA scaffold). In some examples, an engineered guide disclosed herein, when present in an aqueous solution and not bound to the target RNA, does not recruit an RNA editing entity. In some examples, when present in an aqueous solution and not bound to the target RNA, if it binds to the RNA editing entity, an engineered guide disclosed herein does so with a dissociation constant of about greater than or equal to 500 nM. In some examples, the dissociation constant can be about 22 nM. In some examples, the engineered guides disclosed herein, when present in an aqueous solution and not bound to the target RNA, lack a structural feature. In some examples, the engineered guides disclosed herein, when present in an aqueous solution and not bound to the target RNA, lack a bulge, an internal loop, a hairpin, or any combination thereof. In some examples, the engineered guides disclosed herein, when present in an aqueous solution and not bound to the target RNA, can be linear and do not comprise any structural features.

[139] In some examples, an engineered guide (e.g., SLS guide) can be configured to facilitate an editing of a base of a nucleotide or polynucleotide of a region of an RNA by a subject RNA editing entity. In order to facilitate editing, an engineered guide of the disclosure can recruit an RNA editing entity.

[140] In cases where a recruiting sequence can be absent, an engineered guide (e.g., SLS guide) can still be capable of associating with a subject RNA editing entity (e.g., ADAR) to facilitate editing of a target RNA and/or modulate expression of a polypeptide encoded by a subject target RNA. This can be achieved through structural features. Structural features can comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge, symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, a structured motif, circularized RNA, chemical modification, or any combination thereof. In an aspect, a guide-target RNA scaffold, for example hybridized polynucleotide strands, can be formed upon hybridization of an engineered guide (e.g., SLS guide) to a target RNA.

[141] Described herein are structural features which can be present in a guide-target RNA scaffold of the present disclosure. Examples of features include a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a non-recruiting hairpin). Engineered guide RNAs of the present disclosure can have from 1 to 50 features. Engineered guide RNAs of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features. In some embodiments, structural features (e.g., mismatches, bulges, internal loops) can be formed from latent structure in an engineered latent guide RNA upon hybridization of the engineered latent guide RNA to a target RNA and, thus, formation of a guide-target RNA scaffold. In some embodiments, structural features are not formed from latent structures and are, instead, pre-formed structures (e.g., a GluR2 recruitment hairpin or a hairpin from U7 snRNA).

[142] As disclosed herein, a structured motif comprises two or more features in a dsRNA substrate.

[143] A double stranded RNA (dsRNA) substrate (a guide-target RNA scaffold) is formed upon hybridization of an engineered guide (e.g., SLS guide) to a target RNA. As disclosed herein, a mismatch refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a bulge or an internal loop, depending on the size of the structural feature. In some embodiments, a mismatch is an A/C mismatch. An A/C mismatch can comprise a C in an engineered guide RNA of the present disclosure opposite an A in a target RNA. An A/C mismatch can comprise an A in an engineered guide RNA of the present disclosure opposite a C in a target RNA. A G/G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA. [0044] In some embodiments, a mismatch positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited. A mismatch can also help confer sequence specificity. Thus, a mismatch can be a structural feature formed from latent structure provided by an engineered latent guide RNA. In an embodiment, a mismatch comprises a G/G mismatch. In an embodiment, a mismatch comprises an A/C mismatch, wherein the A can be in the target RNA and the C can be in the targeting sequence of the engineered guide RNA. In another embodiment, the A in the A/C mismatch can be the base of the nucleotide in the target RNA edited by a subject RNA editing entity. [144] In an aspect, a structural feature can form in engineered guide (e.g., SLS guide) independently. In some cases, a structural feature can form when an engineered guide RNA binds to a target RNA. A structural feature can also form when an engineered guide RNA associates with other molecules such as a peptide, a nucleotide, or a small molecule. In certain embodiments, a structural feature of an engineered guide RNA can be formed independent of a target RNA, and its structure can change as a result of the engineered polypeptide hybridization with a target RNA region (i.e., the resulting structure that forms upon hybridization is latent, while the structure that is present prior to hybridization is not latent). In certain embodiments, a structural feature is present when an engineered guide RNA is in association with a target RNA.

[145] In some examples, a structural feature is present when an engineered guide (e.g., SLS guide) is in association with a target RNA. A structural feature of an engineered guide RNA can form a substantially linear two-dimensional structure. A structural feature of an engineered guide RNA can comprise a linear region, a stem-loop, a cruciform, a toe hold, a mismatch bulge, or any combination thereof. In some instances, a structural feature can comprise a stem, a hairpin loop, a pseudoknot, a bulge, an internal loop, a multiloop, a G-quadruplex, or any combination thereof. In some examples, an engineered guide RNA can adopt an A-form, a B-form, a Z-form, or any combination thereof.

[146] In some cases, a structural feature can be a hairpin. As disclosed herein, a hairpin includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion. A hairpin can have from 10 to 500 nucleotides in length of the entire duplex structure. The loop portion of a hairpin can be from 3 to 15 nucleotides long. A hairpin can be present in any of the engineered guide RNAs disclosed herein. The engineered guide RNAs disclosed herein can have from 1 to 10 hairpins. In some embodiments, the engineered guide RNAs disclosed herein have 1 hairpin. In some embodiments, the engineered guide RNAs disclosed herein have 2 hairpins. As disclosed herein, a hairpin can include a recruitment hairpin or a non-recruitment hairpin. A hairpin can be located anywhere within the engineered guide RNAs of the present disclosure. In some embodiments, one or more hairpins is proximal to or present at the 3’ end of an engineered guide RNA of the present disclosure, proximal to or at the 5’ end of an engineered guide RNA of the present disclosure, proximal to or within the targeting domain of the engineered guide RNAs of the present disclosure, or any combination thereof.

[147] In some aspects, a structural feature comprises a non-recruitment hairpin. A nonrecruitment hairpin, as disclosed herein, does not have a primary function of recruiting an RNA editing entity. A non-recruitment hairpin, in some instances, does not recruit an RNA editing entity. In some instances, a non-recruitment hairpin has a dissociation constant for binding to an RNA editing entity under physiological conditions that is insufficient for binding. For example, a non-recruitment hairpin has a dissociation constant for binding an RNA editing entity at 25 °C that is greater than about 1 mM, 10 M, 100 mM, or 1 M, as determined in an in vitro assay. A non-recruitment hairpin can exhibit functionality that improves localization of the engineered guide RNA to the target RNA. In some embodiments, the non-recruitment hairpin improves nuclear retention. In some embodiments, the non-recruitment hairpin comprises a hairpin from U7 snRNA. Thus, a non-recruitment hairpin such as a hairpin from U7 snRNA is a pre-formed structural feature that can be present in constructs comprising engineered guide RNA constructs, not a structural feature formed by latent structure provided in an engineered latent guide RNA.

[148] In another aspect, a structural feature of an engineered guide (e.g., SLS guide) comprises a wobble base. A “wobble base pair” refers to two bases that weakly pair. For example, a wobble base pair of the present disclosure can refer to a G paired with a U.

[149] A hairpin of the present disclosure can be of any length. In an aspect, a hairpin can be from about 10-500 or more nucleotides. In some cases, a hairpin can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,

131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,

150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,

169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,

188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,

207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,

226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,

245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,

264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,

302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,

321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,

340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,

359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,

378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,

397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,

416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,

435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,

454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,

473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,

492, 493, 494, 495, 496, 497, 498, 499, 500 or more nucleotides. In other cases, a hairpin can also comprise 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to

190, 10 to 200, 10 to 210, 10 to 220, 10 to 230, 10 to 240, 10 to 250, 10 to 260, 10 to 270, 10 to

280, 10 to 290, 10 to 300, 10 to 310, 10 to 320, 10 to 330, 10 to 340, 10 to 350, 10 to 360, 10 to

370, 10 to 380, 10 to 390, 10 to 400, 10 to 410, 10 to 420, 10 to 430, 10 to 440, 10 to 450, 10 to

460, 10 to 470, 10 to 480, 10 to 490, or 10 to 500 nucleotides.

[150] As disclosed herein, a bulge refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA (SLS guide) or the target RNA are not complementary to their positional counterparts on the opposite strand. A bulge can change the secondary or tertiary structure of the guide-target RNA scaffold. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair - a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a mismatch. Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an internal loop. In some embodiments, the guide-target RNA scaffold of the present disclosure has 2 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 4 bulges. Thus, a bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[151] In some embodiments, the presence of a bulge in a guide-target RNA scaffold can position or can help to position ADAR to selectively edit the target A in the target RNA and reduce off-target editing of non-target A(s) in the target RNA. In some embodiments, the presence of a bulge in a guide-target RNA scaffold can recruit or help recruit additional amounts of ADAR. Bulges in guide-target RNA scaffolds disclosed herein can recruit other proteins, such as other RNA editing entities. In some embodiments, a bulge positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited. A bulge can also help confer sequence specificity for the A of the target RNA to be edited, relative to other A(s) present in the target RNA. For example, a bulge can help direct ADAR editing by constraining it in an orientation that yields selective editing of the target A.

[152] A guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure (SLS guide) to a target RNA. A bulge can be a symmetrical bulge or an asymmetrical bulge. A symmetrical bulge is formed when the same number of nucleotides is present on each side of the bulge. For example, a symmetrical bulge in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 2 nucleotides on the target RNA side of the guidetarget RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[153] A guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure (SLS guide) to a target RNA. A bulge can be a symmetrical bulge or an asymmetrical bulge. An asymmetrical bulge is formed when a different number of nucleotides is present on each side of the bulge. For example, an asymmetrical bulge in a guidetarget RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 1 nucleotide on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[154] In some cases, a structural feature can be an internal loop. As disclosed herein, an internal loop refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a bulge or a mismatch, depending on the size of the structural feature. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. Internal loops present in the vicinity of the edit site can help with base flipping of the target A in the target RNA to be edited.

[155] One side of the internal loop, either on the target RNA side or the engineered guide RNA (SLS guide) side of the guide-target RNA scaffold, can be formed by from 5 to 150 nucleotides. One side of the internal loop can be formed by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 120, 135, 140, 145, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides, or any number of nucleotides therebetween. One side of the internal loop can be formed by 5 nucleotides. One side of the internal loop can be formed by 10 nucleotides. One side of the internal loop can be formed by 15 nucleotides. One side of the internal loop can be formed by 20 nucleotides. One side of the internal loop can be formed by 25 nucleotides. One side of the internal loop can be formed by 30 nucleotides. One side of the internal loop can be formed by 35 nucleotides. One side of the internal loop can be formed by 40 nucleotides. One side of the internal loop can be formed by 45 nucleotides. One side of the internal loop can be formed by 50 nucleotides. One side of the internal loop can be formed by 55 nucleotides. One side of the internal loop can be formed by 60 nucleotides. One side of the internal loop can be formed by 65 nucleotides. One side of the internal loop can be formed by 70 nucleotides. One side of the internal loop can be formed by 75 nucleotides. One side of the internal loop can be formed by 80 nucleotides. One side of the internal loop can be formed by 85 nucleotides. One side of the internal loop can be formed by 90 nucleotides. One side of the internal loop can be formed by 95 nucleotides. One side of the internal loop can be formed by 100 nucleotides. One side of the internal loop can be formed by 110 nucleotides. One side of the internal loop can be formed by 120 nucleotides. One side of the internal loop can be formed by 130 nucleotides. One side of the internal loop can be formed by 140 nucleotides. One side of the internal loop can be formed by 150 nucleotides. One side of the internal loop can be formed by 200 nucleotides. One side of the internal loop can be formed by 250 nucleotides. One side of the internal loop can be formed by 300 nucleotides. One side of the internal loop can be formed by 350 nucleotides. One side of the internal loop can be formed by 400 nucleotides. One side of the internal loop can be formed by 450 nucleotides. One side of the internal loop can be formed by 500 nucleotides. One side of the internal loop can be formed by 600 nucleotides. One side of the internal loop can be formed by 700 nucleotides. One side of the internal loop can be formed by 800 nucleotides. One side of the internal loop can be formed by 900 nucleotides. One side of the internal loop can be formed by 1000 nucleotides. Thus, an internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[156] An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. A symmetrical internal loop is formed when the same number of nucleotides is present on each side of the internal loop. For example, a symmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 8 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 9 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 15 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 15 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 20 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 20 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 30 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 30 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 40 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 40 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 50 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 60 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 60 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 70 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 70 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 80 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 80 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 90 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 90 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 100 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 110 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 110 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 120 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 120 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 130 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 130 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 140 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 140 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 150 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 200 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 250 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 250 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 300 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 350 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 350 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 400 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 450 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 450 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 500 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 600 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 600 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 700 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 800 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 800 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 900 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 900 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 1000 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[157] An asymmetrical internal loop is formed when a different number of nucleotides is present on each side of the internal loop. For example, an asymmetrical internal loop in a guidetarget RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.

[158] An asymmetrical internal loop of the present disclosure can be formed by from 5 to 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 150 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by from 5 to 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 1000 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guidetarget RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guidetarget RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide- target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guidetarget RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[159] Structural features that comprise a loop can be of any size greater than 5 bases. In some cases, a loop comprise at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bases. In some cases, a loop comprise at least about 5-10, 5-15, 10-20, 15-25, 20-30, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 5-110, 5-120, 5-130, 5-140, 5-150, 5-200, 5-250, 5-300, 5-350, 5-400, 5-450, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 20- 50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-110, 20-120, 20-130, 20-140, 20-150, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-110, 30-120, 30-130, 30-140, 30-150, 30-200, 30-250, 30-300, 30-350, 30-400, 30-450, 30-500, 30-600, 30-700, 30-800, 30-900, 30-1000, 40-50, 40- 60, 40-70, 40-80, 40-90, 40-100, 40-110, 40-120, 40-130, 40-140, 40-150, 40-200, 40-250, 40- 300, 40-350, 40-400, 40-450, 40-500, 40-600, 40-700, 40-800, 40-900, 40-1000, 50-60, 50-70, 50-80, 50-90, 50-100, 50-110, 50-120, 50-130, 50-140, 50-150, 50-200, 50-250, 50-300, 50-350, 50-400, 50-450, 50-500, 50-600, 50-700, 50-800, 50-900, 50-1000, 60-70, 60-80, 60-90, 60-100, 60-110, 60-120, 60-130, 60-140, 60-150, 60-200, 60-250, 60-300, 60-350, 60-400, 60-450, 60- 500, 60-600, 60-700, 60-800, 60-900, 60-1000, 70-80, 70-90, 70-100, 70-110, 70-120, 70-130, 70-140, 70-150, 70-200, 70-250, 70-300, 70-350, 70-400, 70-450, 70-500, 70-600, 70-700, 70- 800, 70-900, 70-1000, 80-90, 80-100, 80-110, 80-120, 80-130, 80-140, 80-150, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 80-600, 80-700, 80-800, 80-900, 80-1000, 90-100, 90- 110, 90-120, 90-130, 90-140, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-450, 90-500, 90-600, 90-700, 90-800, 90-900, 90-1000, 100-110, 100-120, 100-130, 100-140, 100-150, 100- 200, 100-250, 100-300, 100-350, 100-400, 100-450, 100-500, 100-600, 100-700, 100-800, 100- 900, 100-1000, 110-120, 110-130, 110-140, 110-150, 110-200, 110-250, 110-300, 110-350, 110- 400, 110-450, 110-500, 110-600, 110-700, 110-800, 110-900, 110-1000, 120-130, 120-140, 120- 150, 120-200, 120-250, 120-300, 120-350, 120-400, 120-450, 120-500, 120-600, 120-700, 120- 800, 120-900, 120-1000, 130-140, 130-150, 130-200, 130-250, 130-300, 130-350, 130-400, 130- 450, 130-500, 130-600, 130-700, 130-800, 130-900, 130-1000, 140-150, 140-200, 140-250, 140- 300, 140-350, 140-400, 140-450, 140-500, 140-600, 140-700, 140-800, 140-900, 140-1000, 150- 200, 150-250, 150-300, 150-350, 150-400, 150-450, 150-500, 150-600, 150-700, 150-800, 150- 900, 150-1000, 200-250, 200-300, 200-350, 200-400, 200-450, 200-500, 200-600, 200-700, 200- 800, 200-900, 200-1000, 250-300, 250-350, 250-400, 250-450, 250-500, 250-600, 250-700, 250-800, 250-900, 250-1000, 300-350, 300-400, 300-450, 300-500, 300-600, 300-700, 300-800, 300-900, 300-1000, 350-400, 350-450, 350-500, 350-600, 350-700, 350-800, 350-900, 350- 1000, 400-450, 400-500, 400-600, 400-700, 400-800, 400-900, 400-1000, 500-600, 500-700, 500-800, 500-900, 500-1000, 600-700, 600-800, 600-900, 600-1000, 700-800, 700-900, 700- 1000, 800-900, 800-1000, or 900-1000 bases in total. [160] As disclosed herein, a “base paired (bp) region” refers to a region of the guide-target RNA scaffold in which bases in the guide RNA are paired with opposing bases in the target RNA. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to the other end of the guide-target RNA scaffold. Base paired regions can extend between two structural features. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to a structural feature. Base paired regions can extend from a structural feature to the other end of the guide-target RNA scaffold. In some embodiments, a base paired region has from 1 bp to 100 bp, from 1 bp to 90 bp, from 1 bp to 80 bp, from 1 bp to 70 bp, from 1 bp to 60 bp, from 1 bp to 50 bp, from 1 bp to 45 bp, from 1 bp to 40 bp, from 1 bp to 35 bp, from 1 bp to 30 bp, from 1 bp to 25 bp, from 1 bp to 20 bp, from 1 bp to 15 bp, from 1 bp to 10 bp, from 1 bp to 5 bp, from 5 bp to 10 bp, from 5 bp to 20 bp, from 10 bp to 20 bp, from 10 bp to 50 bp, from 5 bp to 50 bp, at least 1 bp, at least 2 bp, at least 3 bp, at least 4 bp, at least 5 bp, at least 6 bp, at least 7 bp, at least 8 bp, at least 9 bp, at least 10 bp, at least 12 bp, at least 14 bp, at least 16 bp, at least 18 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40 bp, at least 45 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp.

[161] In some cases, the engineered guide (e.g., SLS guide), when present in an aqueous solution and not bound to the target RNA molecule, if it binds to the RNA editing entity, does so with a dissociation constant of less than about 500 nM. In some cases, the engineered guide, when present in an aqueous solution and not bound to the target RNA molecule, if it binds to the RNA editing entity, does so with a dissociation constant of about 22 nM. In some cases, the engineered guide, when present in an aqueous solution and not bound to the target RNA molecule, lacks a bulge, an internal loop, a hairpin, or any combination thereof.

[162] In some embodiments, a double stranded RNA (dsRNA) substrate (a guide-target RNA scaffold) is formed upon hybridization of an engineered guide (e.g., SLS guide) as disclosed herein to a target RNA. In some examples, the target RNA forming the guide-target RNA scaffold comprises a portion of an mRNA or pre-mRNA molecule encoded by the SERPINA1 gene. In some examples, the targeting region of the engineered guide forming the guide-target RNA scaffold is, at least in part, complementary to a portion of an mRNA or pre- mRNA molecule encoded by the SERPINA1 gene. In some examples, the guide-target RNA scaffold comprises a single A/C mismatch. In some examples, the engineered substrate additionally comprises one or two bulges. In some examples, the guide-target RNA scaffold is formed by a target RNA comprising an mRNA or pre-mRNA encoded by the SERPENA1 gene and an engineered guide complementary to a portion of the mRNA encoded by the SERPINA1 gene, wherein the engineered substrate comprises a single A/C mismatch. In some examples, the guide-target RNA scaffold is formed by a target RNA comprising an mRNA or pre-mRNA encoded by the SERPINA1 gene and an engineered guide complementary to a portion of the mRNA or pre-mRNA encoded by the SERPENA1 gene, wherein the engineered substrate comprise a single A/C mismatch, wherein the mismatch comprises a C in the engineered guide and an A in the target RNA, and wherein the engineered substrate comprises additional structural features.

[163] In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of any of SEQ ID NOS: 3-10. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 3. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 4. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 5. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 6. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 7. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 8. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 9. In certain examples, the engineered guide (e.g., SLS guide) comprises the sequence of SEQ ID NO: 10.

[164] In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to any of SEQ ID NOS: 3-10. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 3. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 4. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 5. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 6. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 7. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 8. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 9. In some examples, the engineered guide (e.g., SLS guide) comprises a polynucleotide stretch having at least 99% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity to SEQ ID NO: 10.

[165] In some embodiments, a guide-target RNA scaffold is formed upon hybridization of an engineered guide (e.g., SLS guide) to a target RNA. In some examples, the guide-target RNA scaffold comprises structural features that mimic a naturally occurring ADAR substrate. In some examples, the naturally occurring substrate can be a drosophila ADAR substrate. In some examples, the guide-target RNA scaffold mimics one or more structural features of the naturally occurring ADAR substrate and comprises a target mRNA encoded by the ABCA4 gene and an engineered guide that can be complementary, at least in part, to a portion of the target mRNA. In some examples, guide-target RNA scaffold contains one or more structural features which mimic structural features of a naturally occurring drosophila substrate. In some examples, the guidetarget RNA scaffold comprises 1, 2, 3, 4, 5, 6 or 7 bulges. In some examples, the guide-target RNA scaffold mimics one or more structural features of the naturally occurring ADAR substrate and comprises a target mRNA encoded by the ABCA4 gene and an engineered guide that can be complementary, at least in part, to a portion of the target mRNA, wherein the guide-target RNA scaffold contains one or more structural features which mimic structural features of a naturally occurring drosophila substrate, for example, 1, 2, 4, 5, 6 or 7 bulges.

[166] In some cases, a structural feature can be a structured motif. As disclosed herein, a structured motif comprises two or more structural features in a guide-target RNA scaffold. A structured motif can comprise of any combination of structural features, such as in the above claims, to generate an ideal substrate for ADAR editing at a precise location(s). These structural motifs could be artificially engineered to maximized ADAR editing, and/or these structural motifs can be modeled to recapitulate known ADAR substrates.

[167] In some cases, a structural feature comprises an at least partial circularization of a polynucleotide. In some cases, a polynucleotide provided herein can be circularized or in a circular configuration. In some aspects, an at least partially circular polynucleotide lacks a 5’ hydroxyl or a 3’ hydroxyl.

[168] In some cases, an engineered guide (e.g., SLS guide) can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some cases, a SLS guide can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some examples, a backbone of an engineered guide can comprise a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA. In some examples, a backbone of an SLS guide can comprise a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA.

[169] In some cases, a backbone of engineered guide (e.g., SLS guide) as disclosed herein can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to a solvent. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to nucleases. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes.

[1] In some cases, the structural a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a hairpin comprising a non-targeting domain). Engineered guide RNAs (e.g., SLS guides) of the present disclosure can have from 1 to 50 features. Engineered guide RNAs (e.g., SLS guides) of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features.

6. Target RNA sequence

[170] In some embodiments, an engineered guide (e.g., SLS guide) can be used to facilitate a mutation in a target RNA sequence. In some examples, the target RNA sequence can be an mRNA molecule or a pre-mRNA molecule. FIGS. 1A and IB illustrate using engineered guides disclosed herein to target both pre-mRNA molecules (FIG. 1A) and mRNA molecules (FIG. IB). As illustrated in FIG. 1A, the engineered guide can be complementary, at least in part, to both an intron and an exon of a pre-mRNA molecule. In some examples, the engineered guide can be complementary only to an exon region of a pre-mRNA molecule.

[171] In some embodiments, the target RNA sequence can be an mRNA molecule. In some examples, the mRNA molecule comprises a premature stop codon. In some examples, the mRNA comprises 1, 2, 3, 4 or 5 premature stop codons. In some examples, the stop codon can be an amber stop codon (UAG), an ochre stop codon (UAA), or an opal stop codon (UGA), or a combination thereof. In some examples, the premature stop codon can be created by a point mutation. In some examples, the premature stop codon causes translation termination of an expression product expressed by the mRNA molecule. In some examples, the premature stop codon can be produced by a point mutation on an mRNA molecule in combination with two additional nucleotides. In some examples, the two additional nucleotides can be (i) a U and (ii) an A or a G, on a 5’ and a 3’ end of the point mutation.

[172] In some embodiments, the target RNA sequence can be a pre-mRNA molecule. In some examples, the pre-mRNA molecule comprises a splice site mutation. In some examples, the splice site mutation facilitates unintended splicing of a pre-mRNA molecule. In some examples, the splice site mutation results in mistranslation and/or truncation of a protein encoded for by the pre-mRNA molecule.

[173] Compositions and methods provided herein (e.g., an engineered guide, such as a SLS guide) can be utilized to target suitable RNA polypeptides and portions thereof. A suitable RNA can comprise a non-protein coding region or a protein coding region. Exemplary non-protein coding regions include but are not always limited to a three prime untranslated region (3’UTR), five prime untranslated region (5’UTR), a translation initiation site, poly(A) tail, a microRNA response element (MRE), AU-rich element (ARE), or any combination thereof. A suitable RNA can also comprise an intron, exon, or any combination thereof.

[174] In some cases, a suitable RNA to target includes but is not necessarily limited to: a precursor-mRNA, a pre-messenger RNA, a messenger RNA, a ribosomal RNA, a transfer RNA, a long non-coding RNA, a small RNA, a nuclear RNA, a cytoplasmic RNA, a prokaryotic RNA, a synthesized RNA, a purified RNA, a single-stranded RNA, a double-stranded RNA, a mitochondrial RNA, and any combination thereof. In some embodiments, a suitable RNA to target can comprise a ribozyme, isolated RNA of a sequence, sgRNA, guide RNA, snRNA, long non-coding RNA, long intergenic non-coding RNA, enhancer RNA, extracellular RNA, Y RNA, hnRNA, scaRNA, circRNA, snoRNA, siRNA, miRNA, tRNA-derived small RNA (tsRNA), antisense RNA, shRNA, small rDNA-derived RNA (srRNA), and any combination thereof. [175] A messenger RNA or mRNA can comprise a nucleic acid molecule that can be transcribed from DNA and then processed to remove non-coding sections known as introns. The resulting mRNA can be exported from the nucleus (or another locus where the DNA can be present) and translated into a protein. A pre-mRNA can comprise the nucleic acid strand prior to processing to remove non-coding sections.

[176] In one aspect, the target RNA encodes for a protein selected from ABCA4, AAT, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, GBA, PINK1, Tau, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, PCSK9 start site, or SCNN1A start site, a fragment any of these, or any combination thereof.

7. Chemical modifications

[177] In some embodiments, engineered guide RNAs, such as an engineered SLS guide RNA, can comprise a modification. A modification can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof. In some cases, a modification can be a chemical modification. Suitable chemical modifications comprise any one of: 5'adenylate, 5' guanosine-triphosphate cap, 5'N7- Methylguanosine-triphosphate cap, 5 'triphosphate cap, 3 'phosphate, 3 'thiophosphate, 5'phosphate, 5 'thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3'-3' modifications, 5'-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3 'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2'deoxyribonucleoside analog purine, 2'deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2'fluoro RNA, 2'0-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5- methylcytidine-5 '-triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof.

[178] In some embodiments, the at least one chemical modification of the engineered guide (e.g., SLS guide) comprises a modification of any one of or any combination of: modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage; modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; modification of a constituent of the ribose sugar; Replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring nucleobase; modification of the ribose-phosphate backbone; modification of 5’ end of polynucleotide; modification of 3’ end of polynucleotide; modification of the deoxyribose phosphate backbone; substitution of the phosphate group; modification of the ribophosphate backbone; modifications to the sugar of a nucleotide; modifications to the base of a nucleotide; or stereopure of nucleotide. Exemplary chemical modification to the engineered guide RNA can be seen in Table 2

Table 2. Exemplary Chemical Modifications

[179] In some embodiments, the chemical modification comprises modification of one or both of the non-linking phosphate oxygens in the phosphodi ester backbone linkage or modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage. As used herein, “alkyl” can be meant to refer to a saturated hydrocarbon group which can be straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n- pentyl, isopentyl, or neopentyl). An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. As used herein, “aryl” can refer to monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, aryl groups have from 6 to about 20 carbon atoms. As used herein, “alkenyl” can refer to an aliphatic group containing at least one double bond. As used herein, “alkynyl” can refer to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups can include ethynyl, propargyl, or 3-hexynyl. “Arylalkyl” or “aralkyl” can refer to an alkyl moiety in which an alkyl hydrogen atom can be replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3- phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups. “Cycloalkyl” can refer to a cyclic, bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. “Heterocyclyl” can refer to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. “Heteroaryl” can refer to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties can include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

[180] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. In some embodiments, the chemically modified nucleotide can include replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Examples of modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group can be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. A phosphorous atom in a phosphate group modified in this way can be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some cases, the engineered guide (e.g., SLS guide) can comprise stereopure nucleotides comprising S conformation of phosphorothioate or R conformation of phosphorothioate. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 95%. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 96%. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 97%. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 98%. In some embodiments, the chiral phosphate product can be present in a diastereomeric excess of 99%. In some embodiments, both non-bridging oxygens of phosphorodithioates can be replaced by sulfur. The phosphorus center in the phosphorodithioates can be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the nonbridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). In some embodiments, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either or both of the linking oxygens.

[181] In some embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any inter nucleic acid linkage. The two main classes of inter nucleic acid linking groups can be defined by the presence or absence of a phosphorus atom. Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-SiQ fi-O-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification.

[182] Unnatural nucleic acids can contain multiple modifications within one of the moi eties or between different moieties. Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or nonbridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and can be used in any combination. Other non-phosphate linkages can also be used. In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate intemucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo.

[183] In some embodiments, a phosphorous derivative (or modified phosphate group) can be attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. In some cases, backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Examples of such modifications include: anionic intemucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral intemucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos. A modified nucleic acid can comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.

[184] Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. It can also be understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). It can be possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.

[185] In some embodiments, the chemical modification described herein comprises modification of a phosphate backbone. In some embodiments, engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified phosphate backbone. Exemplary chemically modification of the phosphate group or backbone can include replacing one or more of the oxygens with a different substituent. Furthermore, the modified nucleotide present in the engineered guide (e.g., SLS guide) can include the replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations resulting in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Exemplary modified phosphate groups can include, phosphorothioate, phosphonothioacetate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BRs (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group can be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that can be to say that a phosphorous atom in a phosphate group modified in this way can be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In such case, the chemically modified engineered guide (e.g., SLS guide) can be stereopure (e.g. S or R confirmation). In some cases, the chemically modified engineered guide (e.g., SLS guide) comprises stereopure phosphate modification. For example, the chemically modified engineered guide (e.g., SLS guide) can comprise S conformation of phosphorothioate or R conformation of phosphorothioate.

[186] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates can be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non- bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

A. Replacement of phosphate moiety

[187] In some embodiments, at least one phosphate group of the engineered guide (e.g., SLS guide) can be chemically modified. In some embodiments, the phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the phosphate moiety can be replaced by dephospho linker. In some embodiments, the charge phosphate group can be replaced by a neutral group. In some cases, the phosphate group can be replaced by methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In some embodiments, nucleotide analogs described herein can also be modified at the phosphate group. Modified phosphate group can include modification at the linkage between two nucleotides with phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3 ’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g. 3’-amino phosphorami date and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. The phosphate or modified phosphate linkage between two nucleotides can be through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’.

B. Substitution of phosphate group

[188] In some embodiments, the chemical modification described herein comprises modification by replacement of a phosphate group. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modification comprising a phosphate group substitution or replacement. Exemplary phosphate group replacement can include non-phosphorus containing connectors. In some embodiments, the phosphate group substitution or replacement can include replacing charged phosphate group can by a neutral moiety. Exemplary moieties which can replace the phosphate group can include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

C. Mollification of the Riboyhosyhate Backbone

[189] In some embodiments, the chemical modification described herein comprises modifying ribophosphate backbone of the engineered guide (e.g., SLS guide). In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified ribophosphate backbone. Exemplary chemically modified ribophosphate backbone can include scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar can be replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

D. Mollification of sugar

[190] In some embodiments, the chemical modification described herein comprises modifying of sugar. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified sugar. Exemplary chemically modified sugar can include 2’ hydroxyl group (OH) modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2’ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2’- alkoxide ion. The 2’-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxy"-2’ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2’ hydroxyl group modification can include (LNA, in which the 2’ hydroxyl can be connected, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2’ hydroxyl group modification can include the methoxy ethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative). In some cases, the deoxy modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH2CH2NH)_nCH2CH2-amino (wherein amino can be, e.g., as described herein), NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which can be optionally substituted with e.g., an amino as described herein. In some instances, the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the T position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack anucleobase at C-. The abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that can be in the L form, e.g. L-nucleosides. In some aspects, the engineered guide (e.g., SLS guide) described herein includes the sugar group ribose, which can be a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-or 7- membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose can be replaced by glycol units attached to phosphodi ester bonds), threose nucleic acid. In some embodiments, the modifications to the sugar of the engineered guide (e.g., SLS guide) comprises modifying the engineered guide (e.g., SLS guide) to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), or bridged nucleic acid (BNA).

E. Mollification of a constituent of the ribose sugar

[191] In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemical modification of a constituent of the ribose sugar. In some embodiments, the chemical modification of the constituent of the ribose sugar can include 2’-O- methyl, 2’-O-methoxy-ethyl (2’-M0E), 2’-fluoro, 2 ’-aminoethyl, 2’-deoxy-2’-fuloarabinou-cleic acid, 2'-deoxy, 2'-O-methyl, 3'-phosphorothioate, 3'-phosphonoacetate (PACE), or 3'- phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the constituent of the ribose sugar comprises unnatural nucleic acid. In some instances, the unnatural nucleic acids include modifications at the 5 ’-position and the 2’-position of the sugar ring, such as 5’-CH2-substituted 2’-O-protected nucleosides. In some cases, unnatural nucleic acids include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH3 and a 5’-(S)-CH3. Unnatural nucleic acids can include 2 ’-substituted 5’-CH2 (or O) modified nucleosides.

Unnatural nucleic acids can include 5’-methylenephosphonate DNA and RNA monomers, and dimers. Unnatural nucleic acids can include 5 ’-phosphonate monomers having a 2’ -substitution and other modified 5 ’-phosphonate monomers. Unnatural nucleic acids can include 5 ’-modified methylenephosphonate monomers. Unnatural nucleic acids can include analogs of 5’ or 6’- phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and/or 6’-position. Unnatural nucleic acids can include 5 ’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group. Unnatural nucleic acids can include nucleosides having a 6’ -phosphonate group wherein the 5’ or/and 6’-position can be unsubstituted or substituted with a thio-tert-butyl group (SC(CH₃)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof).

[192] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides can impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5’ and/or 2’ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids; replacement of the ribosyl ring oxygen atom with S, N(R), or C(RI)(R2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof.

[193] In some instances, the engineered guide (e.g., SLS guide) described herein comprises modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. The sugar can be in a pyranosyl or furanosyl form. The sugar moiety can be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’- amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy-or amino-RNA/DNA chimeras. For example, a sugar modification can include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’ -O-ethylenegly col-like ribonucleosides.

[194] Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to C10, alkyl or C2 to C10 alkenyl and alkynyl. 2’ sugar modifications also include but are not limited to-O[(CH2)nO]m CH3,-O(CH2)nOCH3,-O(CH2)nNH2,-O(CH2)nCH3,- O(CH₂)nONH₂, and-O(CH2)nON[(CH2)n CH3)]2, where n and m can be from 1 to about 10.

Other chemical modifications at the 2’ position include but are not limited to: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of the 5’ terminal nucleotide. Chemically modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Examples of nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’-O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-(Ci-Cio alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O- N(R_m)(Rn), and O-CH2-C(=O)-N(R_m)(Rn), where each R_m and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.

[195] In certain embodiments, nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms. In certain embodiments, nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid. Examples of such 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4’-(CH₂)-O-2’ (LNA); 4’-(CH₂)-S-2’; 4’-(CH₂)2-O-2’ (ENA); 4’-CH(CH₃)-O-2’ and 4’-CH(CH2OCH3)-O-2’, and analogs thereof; 4’-C(CH3)(CH3)-O-2’and analogs thereof.

F. Modifications on the base of nucleotide

[196] In some embodiments, the chemical modification described herein comprises modification of the base of nucleotide (e.g. the nucleobase). Exemplary nucleobases can include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or replaced to in the engineered guide (e.g., SLS guide) described herein. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can be naturally-occurring or synthetic derivatives of a base.

[197] In some embodiments, the chemical modification described herein comprises modifying an uracil. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified uracil. Exemplary chemically modified uracil can include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5- aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5 -bromo-uridine), 3 -methyl -uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1 -carboxy methyl-pseudouri dine, 5-carboxyhydroxymethyl- uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl- uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5- carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl- 2 -thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1- taurinomethyl-pseudouridine, 5 -taurinomethy 1-2 -thio-uridine, l-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine, 1 methyl-pseudouridine, 5-methyl-2 -thio-uridine, l-methyl-4- thio-pseudouridine, 4-thio-l -methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-l -methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio-l -methyl- 1 -deaza-pseudouridine, dihydroundine, dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl-pseudouridine, 3-(3- amino-3-carboxypropyl) uridine, l-methyl-3-(3-amino-3-carboxypropy pseudouridine, 5- (isopentenylaminomethyl) uridine, 5-(isopentenylaminomethy])-2 -thio-uridine, a-thio-uridine, 2’-O-methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl-pseudouridine, 2-thio-2’-O-methyl- uridine, 5-methoxycarbonylmethyl-2’-O-methyl-uridine, 5-carbamoylmethyl-2’-O-methyl- uridine, 5 -carboxy methylaminomethy 1-2 ’-O-methyl-uri dine, 3,2’-O-dimethyl-uridine, 5- (isopentenylaminomethyl)-2’-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2’-F-ara- uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-( 1-E- propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

[198] In some embodiments, the chemical modification described herein comprises modifying a cytosine. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified cytosine. Exemplary chemically modified cytosine can include 5 -aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3 -methyl -cytidine, N4- acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5- hydroxymethyl-cytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza-pseudoisocytidine, 1 -methyl-l-deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O- methyl-cytidine, 5,2’ -O-dimethyl-cyti dine, N4-acety 1-2 ’-O-methyl-cyti dine, N4,2’-O-dimethyl- cytidine, 5 -formyl-2’ -O-methyl-cyti dine, N4,N4,2’-O-trimethyl-cytidine, 1 -thio-cytidine, 2’-F- ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[199] In some embodiments, the chemical modification described herein comprises modifying an adenine. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified adenine. Exemplary chemically modified adenine can include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6- chloro-purine), 6-halo-purine (e.g., 6-chloi-purine), 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl- adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyl- adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2- methylthio-N6-threonylcarbamoyl-adenosine, N6, N6-dimethyl-adenosine, N6- hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2’-O-methyl-adenosine, N6, 2’-O-dimethyl-adenosine, N6-Methyl-2’- deoxyadenosine, N6, N6, 2’-O-trimethyl-adenosine, l,2’-O-dimethyl-adenosine, 2’-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[200] In some embodiments, the chemical modification described herein comprises modifying a guanine. In some embodiments, the engineered guide (e.g., SLS guide) described herein comprises at least one chemically modified guanine. Exemplary chemically modified guanine can include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undemriodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2, N2-dimethyl-guanosine, N2, 7-dimethyl-guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1-meththio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio- guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2, 7 -dimethyl -2’- O-methyl-guanosine, 2’-O-methyl-inosine, l,2’-O-dimethyl-inosine, 6-O-phenyl-2’- deoxyinosine, 2’-O-ribosylguanosine, 1 -thio-guanosine, 6-O-methyguanosine, O⁶-Methyl-2’- deoxyguanosine, 2’-F-ara-guanosine, and 2’-F-guanosine.

[201] In some cases, the chemical modification of the engineered guide (e.g., SLS guide) can include introducing or substituting a nucleic acid analog or an unnatural nucleic acid into the engineered guide RNA. In some embodiments, nucleic acid analog can be any one of the chemically modified nucleic acid described herein. Exemplary nucleic acid analog can be found in PCT/US2015/025175, PCT/US2014/050423, PCT/US2016/067353, PCT/US2018/041503, PCT/US 18/041509, PCT/US2004/011786, or PCT/US2004/011833, all of which are expressly incorporated by reference in their entireties. The chemically modified nucleotide described herein can include a variant of guanosine, uridine, adenosine, thymidine, and cytosine, including any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytidine that has been altered chemically, for example by acetylation, methylation, hydroxylation. Exemplary chemically modified nucleotide can include 1-methyl-adenosine, 1- methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2’-amino-2’- deoxyadenosine, 2’ -amino-2’ -deoxy cytidine, 2 ’-amino-2’ -deoxy guanosine, 2’-amino-2’- deoxyuridine, 2-amino-6-chloropurineriboside, 2-aminopurine-riboside, 2’-araadenosine, 2’- aracytidine, 2’-arauridine, 2’-azido-2’-deoxyadenosine, 2’ -azi do-2’ -deoxy cytidine, 2’-azido-2’- deoxyguanosine, 2’-azido-2’-deoxyuridine, 2-chloroadenosine, 2’-fluoro-2’-deoxyadenosine, 2’- fluoro-2’ -deoxy cytidine, 2’-fluoro-2’-deoxyguanosine, 2’-fluoro-2’-deoxyuridine, 2’- fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl- adenosine, 2’-O-methyl-2-aminoadenosine, 2’-O-methyl-2’-deoxyadenosine, 2’ -O-methyl-2’ - deoxycytidine, 2 ‘-O-methyl-2’ -deoxy guanosine, 2, -O-methyl-2’ -deoxyuridine, 2’-O-methyl-5- methyluridine, 2’-O-methylinosine, 2’-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6- dihydrouridine, 5-aminoallylcytidine, 5 -aminoally 1-deoxy uridine, 5-bromouridine, 5- carboxy methy laminomethyl-2-thio-uracil, 5 -carboxy methylamonomethyl-uracil, 5 -chloro-ara- cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5 -methoxy -uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6- chloropurineriboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2’- deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D- mannosyl-queosine, dihydro-uridine, inosine, N1 -methyladenosine, N6-([6-ami nohexyl] carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl- xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 2-amino-6-chloropurineriboside-5’- triphosphate, 2-aminopurine-riboside-5 ’ -triphosphate, 2-aminoadenosine-5’ -triphosphate, 2’- amino-2’ -deoxy cytidine-triphosphate, 2-thiocytidine-5 ’ -triphosphate, 2-thiouridine-5 ’ - triphosphate, 2 ’-fluorothymidine-5’ -triphosphate, 2’-O-methyl-inosine-5’-triphosphate, 4- thiouridine-5 ’ -triphosphate, 5 -aminoally lcytidine-5 ’ -triphosphate, 5 -aminoallyluridine-5 ’ - triphosphate, 5 -bromocytidine-5 ’ -triphosphate, 5-bromouridine-5’ -triphosphate, 5-bromo-2’- deoxycytidine-5 ’-triphosphate, 5-bromo-2’-deoxyuridine-5’ -triphosphate, 5-iodocytidine-5’- triphosphate, 5-iodo-2’-deoxycytidine-5’-triphosphate, 5-iodouridine-5’ -triphosphate, 5-iodo-2’- deoxyuridine-5 ’ -triphosphate, 5 -methy lcytidine-5 ’ -triphosphate, 5 -methyluridine-5 ’ - triphosphate, 5-propynyl-2’-deoxycytidine-5 ’-triphosphate, 5-propynyl-2’-deoxyuridine-5’- triphosphate, 6-azacytidine-5 ’ -triphosphate, 6-azauridine-5’ -triphosphate, 6- chloropurineriboside-5 ’-triphosphate, 7-deazaadenosine-5’ -triphosphate, 7-deazaguanosine-5’- triphosphate, 8-azaadenosine-5’ -triphosphate, 8-azidoadenosine-5’ -triphosphate, benzimidazole- riboside-5 ’ -triphosphate, N 1 -methyladenosine-5 ’ -triphosphate, N 1 -methy lguanosine-5 ’ - triphosphate, N6-methyladenosine-5 ’ -triphosphate, 6-methy lguanosine-5 ’ -triphosphate, pseudouridine-5’ -triphosphate, puromycin-5’ -triphosphate, or xanthosine-5 ’ -triphosphate. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2 -thio-5 -aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1 -tauri nomethylpseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1- methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the artificial nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-th io-l-methyl-

1-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 2-aminopurine, 2, 6-diaminopurine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza- 2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7 -methyl adenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, wyosine, wybutosine,

7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine. In certain embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5- aminoallyl-uridine, 5 -iodo-uridine, Nl-methyl-pseudouri dine, 5,6-dihydrouridine, alpha-thio- uridine, 4-thio-uridine, 6-aza-uridine, 5 -hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2- amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine,

8-azido-adenosine, 7-deaza-adenosine.

[202] A modified base of a unnatural nucleic acid includes, but is in some cases not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5 -methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine,

2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3 -deazaguanine and 3 -deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, 0-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine,

5 -methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,

6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5- halocytosine, 5-propynyl (-C=C-CH3) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine( [5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9-(2- aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin- 2-one), those in which the purine or pyrimidine base can be replaced with other heterocycles, 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5- fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5 -nitrocytosine, 5 -bromouracil, 5-chlorouracil, 5-fluorouracil, and 5- iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2’- deoxyuridine, or 2-amino-2’ -deoxy adenosine.

[203] In some cases, the at least one chemical modification can comprise chemically modifying the 5’ or 3’ end such as 5’ cap or 3’ tail of the engineered guide (e.g., SLS guide). In some embodiments, the engineered guide (e.g., SLS guide) can comprise a chemical modification comprising 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridines can be replaced with modified uridines, e.g., 5-(2-amino) propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O-and N-alkylated nucleotides, e.g., N6-methyladenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2’ OH-group can be replaced by a group selected from H,-OR,-R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2 ’-sugar modified, such as, 2-F 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’-O- methoxyethyladenosine (Aeo ), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo ), or any combinations thereof.

[204] A modification can be made at any location of an engineered guide (e.g., SLS guide). In some cases, a modification can be located in a 5’ or 3’ end. In some cases, an engineered guide (e.g., SLS guide) comprises a modification at a base selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,

110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, or more. More than one modification can be made to an engineered guide (e.g., SLS guide). In some cases, a modification can be permanent. In other cases, a modification can be transient. In some cases, multiple modifications can be made to a polynucleic acid. A polynucleic acid modification can alter physio-chemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.

[205] A modification can also be a phosphorothioate substitute. In some cases, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and; a modification of intemucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a polynucleic acid. A modification can also enhance biological activity. In some cases, a phosphorothioate enhanced RNA polynucleic acid can inhibit RNase A, RNase Tl, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA polynucleic acids to be used in applications where exposure to nucleases can be of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of a polynucleic acid which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases.

[206] An engineered guide (e.g., SLS guide) can have any frequency of bases. For example, an engineered guide (e.g., SLS guide) can have a percent adenine of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%, 8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. An engineered guide (e.g., SLS guide) can have a percent cytosine of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%, 8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. An engineered guide (e.g., SLS guide) can have a percent thymine of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%, 8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. An engineered guide (e.g., SLS guide) can have a percent guanine of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%, 8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. An engineered guide (e.g., SLS guide) can have a percent uracil of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%, 8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%.

8. Quality control of compositions

[207] In some cases, an engineered guide (e.g., SLS guide) can undergo quality control after a modification. In some cases, quality control can include PAGE, HPLC, MS, or any combination thereof. In some cases, a mass of an engineered guide (e.g., SLS guide) can be determined. A mass can be determined by LC-MS assay. A mass can be 30,000 amu, 50,000amu, 70,000 amu, 90,000 amu, 100,000 amu, 120,000 amu, 150,000 amu, 175,000 amu, 200,000 amu, 250,000 amu, 300,000 amu, 350,000 amu, 400,000 amu, to about 500,000 amu. A mass can be of a sodium salt of an engineered guide (e.g., SLS guide).

[208] In some cases, an endotoxin level of an engineered guide (e.g., SLS guide) or a polynucleotide encoding the engineered guide (e.g., SLS guide) can be determined. A clinically/therapeutically acceptable level of an endotoxin can be less than 3 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 10 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 8 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 5 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 4 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 3 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 2 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 1 EU/mL. A clinically/therapeutically acceptable level of an endotoxin can be less than 0.5 EU/mL

[209] In some cases, an engineered guide (e.g., SLS guide) or a polynucleotide encoding the engineered guide (e.g., SLS guide) can undergo sterility testing. A clinically/therapeutically acceptable level of a sterility testing can be 0 or denoted by no growth on a culture. A clinically/therapeutically acceptable level of a sterility testing can be less than 0.5% growth. A clinically/therapeutically acceptable level of a sterility testing can be less than 1% growth.

9. Modulation and measurement of target expression

[210] Compositions and methods provided herein can be utilized to modulate expression of a target. Modulation can refer to altering the expression of a gene or portion thereof at one of various stages, with a view to alleviate a disease or condition associated with the gene or a mutation in the gene. Modulation can be mediated at the level of transcription or post- transcriptionally. Modulating transcription can correct aberrant expression of splice variants generated by a mutation in a gene. In some cases, compositions and methods provided herein can be utilized to regulate gene translation of a target. Modulation can refer to decreasing or knocking down the expression of a gene or portion thereof by decreasing the abundance of a transcript. The decreasing the abundance of a transcript can be mediated by decreasing the processing, splicing, turnover or stability of the transcript; or by decreasing the accessibility of the transcript by translational machinery such as ribosome. In some cases, an engineered guide RNA (e.g., SLS guide) described herein can facilitate a knockdown. A knockdown can reduce the expression of a target RNA. In some cases, a knockdown can be accompanied by editing of an mRNA. In some cases, a knockdown can occur with substantially little to no editing of an mRNA. In some instances, a knockdown can occur by targeting an untranslated region of the target RNA, such as a 3’ UTR, a 5’ UTR or both. In some cases, a knockdown can occur by targeting a translation initiation site of the target RNA. In some cases, a knockdown can occur by targeting a coding region of the target RNA. In some instances, a knockdown can be mediated by an RNA editing enzyme (e.g. ADAR). In some instances, an RNA editing enzyme can cause a knockdown by hydrolytic deamination of multiple adenosines in an RNA. Hydrolytic deamination of multiple adenosines in an RNA can be referred to as hyper-editing. In some cases, hyper-editing can occur in cis (e.g. in an Alu element) or in trans (e.g. in a target RNA by an engineered guide (e.g., SLS guide)).

[211] In some embodiments, RNA editing can be evaluated by determining by the percent RNA editing of a target RNA. In some cases, RNA editing can be determined by changes in a level of protein. In some cases, a level of a protein can be measured by a Western blot. In some cases, a level of a protein can be measured by densitometry with a quantitative protein gel. In some cases, the percent RNA editing of a target RNA can be determined at different time points (e.g. 24 hours, 48 hours, 96 hours) after transfection with an engineered guide (e.g., SLS guide) by reverse transcribing the target RNA to cDNA then using Sanger sequencing to determine the percent RNA editing of a target RNA. In some cases, the cDNA can be amplified prior to sequencing by polymerase chain reaction. Sanger traces from Sanger sequencing can be analyzed to assess the editing efficiency of guide RNAs. In some cases, next generation sequencing technologies (e.g. sequencing by synthesis) can be used to determine percent RNA editing of a target RNA. For example, RNA sequencing can be used to determine the percent RNA editing of a target RNA after transfection with a guide RNA or guide polynucleotide. In some instances, the individual sequencing reads can be analyzed to determine the percent RNA editing. In some cases, chemical modification of the base of the nucleotide in a target RNA can be confirmed by an in vitro assay. In some examples, the in vitro assay can be a sequencing or ELISA assay. In some examples, chemical modification of the base can be confirmed by delivering the engineered guide to cells in vitro or in vivo and measuring cell cytokine responses (e.g., IFN production) using an ELISA assay.

METHODS OF TREATMENT

[212] Disclosed herein are methods of delivering any engineered guide disclosed herein, such as an SLS guide RNA to a cell or a subject. In some examples, methods of delivering an engineered guide (e.g., SLS guide) to a cell comprises delivering directly or indirectly to the cell an engineered guide that at least partially hybridizes to and forms, at least in part, a guide-target RNA scaffold with at least a portion of a target RNA. In some cases, the guide-target RNA scaffold comprises at least one structural feature, and the guide-target RNA scaffold can recruit an RNA editing entity. In some cases, the RNA editing entity facilitates a chemical modification of a base of a nucleotide in the target RNA by the RNA editing entity. The engineered guide (e.g., SLS guide) as described herein can be used to treat Alpha-1 antitrypsin deficiency (AATD), liver cirrhosis, Stargardt disease and neurodegenerative diseases. In some cases, the engineered guide (e.g., SLS guide) is encoded by a polynucleotide. In some instances, a vector can comprise an engineered guide (e.g., SLS guide). In some instances, an engineered guide (e.g., SLS guide) disclosed herein can be comprised in a composition, pharmaceutical composition, isolated cell, or plurality of cells disclosed herein.

[213] Also disclosed herein are methods of treating a disease or condition in a subject in need thereof comprising administering to the subject any engineered guide, such as a SLS guide RNA disclosed herein. In some examples, the methods of treating or preventing a disease or a condition in a subject in need thereof comprise administering to the subject having the disease or the condition an engineered guide, thereby treating or preventing the disease or the condition in the subject, wherein the engineered guide: (a) at least in part associates with at least a portion of a target RNA; (b) in association with the target RNA, forms a guide-target RNA scaffold comprising at least one structural feature, and wherein the guide-target RNA scaffold recruits an RNA editing entity; and (c) facilitates a chemical modification of a base of a nucleotide in the target RNA by the RNA editing entity. In some examples, the methods of treating or preventing a disease or a condition in a subject in need thereof comprises administering to the subject having the disease or the condition an engineered guide, thereby treating or preventing the disease or the condition in the subject, wherein the engineered guide: (a) at least in part associates with at least a portion of a target RNA; (b) in association with the target RNA, forms a guide-target RNA scaffold comprising at least one structural feature, and wherein the guidetarget RNA scaffold recruits an RNA editing entity; and (c) facilitates a chemical modification of a base of a nucleotide in the target RNA by the RNA editing entity, and wherein the engineered RNA comprises an SLS scaffold. In some examples, chemical modification of the base can be confirmed by delivering the engineered guide (e.g., SLS guide) to cells in vitro or in vivo and measuring cell cytokine responses (e.g., IFN production) using an ELISA assay or by sequencing the target RNA.

1. Indications

[214] Disclosed herein are methods of treating a disease or condition in a subject in need thereof comprising administering to the subject any engineered guide (e.g., an engineered guide (e.g., SLS guide), a vector encoding or comprising an engineered guide (e.g., SLS guide)) disclosed herein.

[215] In some embodiments, the disease or condition can be associated with a mutation in a DNA molecule or RNA molecule encoding, for example, ABCA4, AAT, SERPINA1 E342K, HEXA, GBA, PINK1, Tau, LRRK2, SNCA, APP, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, a fragment any of these, or any combination thereof. In some examples, a protein encoded for by a mutated DNA molecule or mutated RNA molecule selected from the group consisting of: ABCA4, AAT, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, GBA, PINK1, Tau, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, aPCSK9 start site, or a SCNN1A start site, or any combination thereof that contributes to, the pathogenesis or progression of a disease. In some examples, the mutation in the DNA or RNA molecule can be relative to an otherwise identical reference DNA or RNA molecule. In some examples, a translation initiation site of an RNA that expresses a protein associated with a disease or condition, can be targeted by the engineered guide (e.g., SLS guide) to treat the disease or condition in the subject, for example, the PCSK9 start site or the SCNN1 A start site. In some examples, other regions of an RNA that expresses a protein associated with a disease or condition, can be targeted by the engineered guide (e.g., SLS guide) to treat the disease or condition in the subject, such as a 5’ UTR, 3’UTR, splice site, translation initiation site, or stop codon. [216] In some embodiments, the disease or condition can be associated with a mutation in a SERPINA1 gene. In some examples, the disease or condition can be Alpha- 1 antitrypsin deficiency (AATD). In some cases, the disease or condition can be a lung disease, a liver disease or both. In some cases, the disease or condition can comprise chronic obstructive pulmonary disease (COPD) or liver cirrhosis. In some cases, the disease or condition can comprise emphysema or chronic bronchitis. In some examples, the AAT deficiency can be caused, at least in part, by a mutation in a SERPINA1 gene. In some examples, the mutation is a substitution of a G with an A at nucleotide position 9989 within the SERPINA1 gene, SEQ ID NO: [1].

[217] In some embodiments, the disease or condition can be associated with a mutation in an ABCA4 gene. In some examples, the disease or condition can be Stargardt macular degeneration. In some examples, the Stargardt macular degeneration can be caused, at least in part, by a mutation in an ABCA4 gene. In some examples, the mutation comprises a substitution of a G with an A at nucleotide position 5882 in an ABCA4 gene, SEQ ID NO: [2], In some examples, the mutation comprises a G with an A at nucleotide position 5714 in a ABAC4 gene, SEQ ID NO: [2], In some examples, the mutation comprises a substitution of a G with an A at nucleotide position 6320 in an ABAC4 gene, SEQ ID NO: [2],

[218] In one aspect, the disease or condition can be associated with, at least in part, with mutations in RAB7A, ABCA4, AAT, SERPINA1 E342K, HEXA, LRRK2, GBA, PINK1, Tau, SNCA, APP, CFTR, ALAS1, ATP7B, ATP7B G1226R, HEE C282Y, LIPA c.894 G>A, aPCSK9 start site, or SCNN1A start site, or any combination thereof. In one aspect, the disease or condition can be associated with, at least in part, with mutations in ABCA4, AAT, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, GBA, PINK1, Tau, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, a fragment any of these, or any combination thereof.

[219] In some embodiments, the disease or condition can be associated with expression of or cleavage products of an amyloid precursor protein (APP). In some examples, the disease or condition associated with Amyloid beta (A[3 or Abeta) peptide deposition in the brain or blood vessels. In some examples, the Abeta deposition can be produced by the cleavage of APP by beta secretase (BACE) or gamma secretase. In some examples, the disease can be a neurodegenerative disease. In some examples, the disease comprises Alzheimer’s disease, Parkinson’s disease, corticobasal degeneration, dementia with Lewy bodies, Lewy body variant of Alzheimer’s disease, Parkinson’s disease with dementia, Pick’s disease, progressive supranuclear palsy, dementia, fronto-temporal dementia with Parkinsonism linked to tau mutations on chromosome 17, or any combination thereof. In some examples, the engineered guides can be administered to knockdown expression of APP or to edit a cut site to prevent Abeta fragment formation from APP.

[220] In some embodiments, a disease or condition comprises a neurodegenerative disease, a muscular disorder, a metabolic disorder, an ocular disorder (e.g. an ocular disease), a cancer, a liver disease (e.g., Alpha- 1 antitrypsin deficiency (AATD)), or any combination thereof. In some examples, the disease comprises cystic fibrosis, albinism, alpha- 1 -antitrypsin deficiency, Alzheimer disease, Amyotrophic lateral sclerosis, Asthma, P-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, Chronic Obstructive Pulmonary Disease (COPD), dementia, Distal Spinal Muscular Atrophy (DSMA), Duchenne/Becker muscular dystrophy, Dystrophic Epidermolysis bullosa, Epidermylosis bullosa, Fabry disease, Factor V Leiden associated disorders, Familial Adenomatous, Polyposis, Galactosemia, Gaucher's Disease, Glucose-6- phosphate dehydrogenase, Haemophilia, Hereditary Hematochromatosis, Hunter Syndrome, Huntington's disease, Hurler Syndrome, Inflammatory Bowel Disease (IBD), Inherited polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, Mucopolysaccharidosis, Muscular Dystrophy, Myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-esol related cancer, Parkinson's disease, Peutz-Jeghers Syndrome, Phenylketonuria, Pompe's disease, Primary Ciliary Disease, Prothrombin mutation related disorders, such as the Prothrombin G20210A mutation, Pulmonary Hypertension, Retinitis Pigmentosa, Sandhoff Disease, Severe Combined Immune Deficiency Syndrome (SCID), Sickle Cell Anemia, Spinal Muscular Atrophy, Stargardt's Disease, Tay-Sachs Disease, Usher syndrome, Wolman disease, X-linked immunodeficiency, various forms of cancer (e.g., BRCA1 and 2 linked breast cancer and ovarian cancer). In some cases, a treatment of a disease or condition such as a neurodegenerative disease (e.g. Alzheimer’s, Parkinson’s) can comprise producing an edit, a knockdown or both of amyloid precursor protein (APP), tau, alpha-synuclein, or any combination thereof. In some cases, APP, tau, and alpha-synuclein can comprise a pathogenic variant. In some instances, APP can comprise a pathogenic variant such as A673V mutation or A673T mutation. In some cases, a treatment of a disease or condition such as a neurodegenerative disease (Parkinson’s) can comprise producing an edit, a knockdown or both of a pathogenic variant of LRRK2. In some cases, a pathogenic variant of LRRK can comprise a G2019S mutation. The disease or condition can comprise a muscular dystrophy, an ornithine transcarbamylase deficiency, a retinitis pigmentosa, a breast cancer, an ovarian cancer, Alzheimer’s disease, pain, Stargardt macular dystrophy, Charcot-Marie-Tooth disease, Rett syndrome, or any combination thereof. [221] In some embodiments, the disease or condition can be caused or contributed to, at least in part, by a protein encoded by an mRNA comprising a premature stop codon. In some cases, the premature stop codon results in a truncated version of the polypeptide or protein. In some cases, the disease, disorder, or condition can be caused by an increased level of a truncated version of the polypeptide, or a decreased level of substantially full-length polypeptide. In some examples, the premature stop codon can be created by a point mutation. In some examples, the premature stop codon can be produced by a point mutation on an mRNA molecule in combination with two additional nucleotides. In some examples, the mRNA molecule comprises one, two, three, or for premature stop codons. In some examples, the disease or condition is caused or contributed to, at least in part, by a splice site mutation on a pre-mRNA molecule. In some examples, the splice site mutation facilitates unintended splicing of a pre-mRNA molecule. In some examples, the splice site mutation results in mistranslation and/or truncation of a protein caused by incorrect delineation of a pre-mRNA splice site.

[222] In some embodiments, in methods disclosed herein, the subject can be diagnosed with the disease or condition. In some examples, the subject can be diagnosed with the disease or condition by an in vitro assay.

[223] In some embodiments, administration of a composition or engineered guide disclosed herein: (a) decreases expression of a gene relative to an expression of the gene prior to administration; (b) edits at least one point mutation in a subject, such as a subject in need thereof; (c) edits at least one stop codon in the subject to produce a readthrough of a stop codon; (d) produces an exon skip in the subject, or (e) any combination thereof.

[224] In some embodiments, administration of a composition or engineered guide (e.g., SLS guide) disclosed herein: (a) decreases expression of a gene relative to an expression of the gene prior to administration; (b) edits at least one point mutation in a subject, such as a subject in need thereof; (c) edits at least one stop codon in the subject to produce a readthrough of a stop codon; (d) produces an exon skip in the subject, or (e) any combination thereof.

2. Administration, Additional Therapies, and Prodrugs

[225] Methods described herein can comprise administration to a subject one or more engineered guides (e.g., SLS guides), polynucleotides, compositions, pharmaceutical compositions, vectors, cells and isolated cells as described herein. For example, methods described herein can comprise administration to a subject one or more SLS guides or vectors encoding a SLS guide. Methods of determining the most effective means and dosage of administration can be known to those of skill in the art and can vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.

[226] The appropriate dosage and treatment regimen for the methods of treatment described herein vary with respect to the particular disease being treated, the engineered guide (e.g., SLS guide) and/or ADAR (or a vector encoding engineered guide (e.g., SLS guide) and/or ADAR) being delivered, and the specific condition of the subject. In some examples, the administration can be over a period of time until the desired effect (e.g., reduction in symptoms can be achieved). In some examples, administration can be 1, 2, 3, 4, 5, 6, or 7 times per week. In some examples, administration or application of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In some examples, administration can be over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some examples, administration can be over a period of 2, 3, 4, 5, 6 or more months. In some examples, administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject. In some examples, administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more. In some examples, treatment can be resumed following a period of remission.

[227] In some examples, administration of the engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be performed for a treatment duration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99, or 100 days consecutive or nonconsecutive days. In some examples, administration of the engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be performed for a treatment duration of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 days consecutive or nonconsecutive days.

[228] In some cases, a treatment duration can be from about 1 to about 30 days, from about

2 to about 30 days, from about 3 to about 30 days, from about 4 to about 30 days, from about 5 to about 30 days, from about 6 to about 30 days, from about 7 to about 30 days, from about 8 to about 30 days, from about 9 to about 30 days, from about 10 to about 30 days, from about 11 to about 30 days, from about 12 to about 30 days, from about 13 to about 30 days, from about 14 to about 30 days, from about 15 to about 30 days, from about 16 to about 30 days, from about 17 to about 30 days, from about 18 to about 30 days, from about 19 to about 30 days, from about 20 to about 30 days, from about 21 to about 30 days, from about 22 to about 30 days, from about 23 to about 30 days, from about 24 to about 30 days, from about 25 to about 30 days, from about 26 to about 30 days, from about 27 to about 30 days, from about 28 to about 30 days, or from about 29 to about 30 days.

[229] In some examples, administration of the engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In some examples, administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject. In some examples, administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more.

[230] In some examples, administration of the engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times a day. In some examples, administration or application of composition disclosed herein is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a week. In some examples, administration of the engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times a month.

[231] In some examples, an engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be administered/ applied as a single dose or as divided doses. In some examples, engineered guides (e.g., SLS guides), polynucleotides, compositions, pharmaceutical compositions, vectors, or cells disclosed herein can be administered at a first time point and a second time point. In some examples, an engineered guide (e.g., SLS guide), polynucleotide, composition, pharmaceutical composition, vector, or cell disclosed herein can be administered such that a first administration can be administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more.

[232] In some embodiments, the pharmaceutical composition can be administered to a subject by any means which will contact the engineered guide (e.g., SLS guide) and/or ADAR (or a vector encoding the engineered guide (e.g., SLS guide) and/or ADAR) with a target cell. In some examples, the specific route will depend upon certain variables such as the target cell and can be determined by the skilled practitioner. In some examples, the pharmaceutical composition can be administered by intravenous administration, intraperitoneal administration, intravascular administration, an infusion, intramuscular administration, parenteral administration, intracoronary administration, intravitreal administration, retinal administration, intracerebroventricular administration, intraparenchymal administration, intraduodenal administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracerebral administration, nasal administration, intraocular administration, oral administration, rectal administration, pulmonary administration, impregnation of a catheter, or direct injection into a tissue, or any combination thereof. In some examples, the target cells can be in or near a tumor and administration can be by direct injection into the tumor or tissue surrounding the tumor. In some examples, the tumor can be a breast tumor and administration comprises impregnation of a catheter and direct injection into the tumor. In some examples, aerosol (inhalation) delivery can be performed using methods known in the art, such as methods described in, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992, which can be incorporated by reference herein. In some examples, oral delivery can be performed by complexing a ttRNA (or a vector encoding a ttRNA) to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art.

[233] In some examples, direct injection techniques can be used for administering the engineered guide (e.g., SLS guide) and/or ADAR (or a vector encoding engineered guide (e.g., SLS guide) and/or ADAR) to a cell or tissue that can be accessible by surgery, and on or near the surface of the body. In some examples, administration of a composition locally within the area of a target cell comprises injecting the composition centimeters and preferably, millimeters from the target cell or tissue.

[234] In some examples, a pharmaceutical composition disclosed herein can be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect.

[235] In some examples, methods described herein can comprise administering a cotherapy. In some examples, a co-therapy can comprise a cancer treatment (e.g. radiotherapy, chemotherapy, CAR-T therapy, immunotherapy, hormone therapy, cryoablation). In some examples, a co-therapy can comprise surgery. In some example, a co-therapy can comprise a laser therapy.

[236] In some examples, the pharmaceutical composition comprises a first active ingredient (e.g., an engineered guide (e.g., SLS guide) disclosed herein, a composition disclosed herein, an isolated cell disclosed herein, or an isolated plurality of cells disclosed herein). In some examples, the pharmaceutical can comprise a second, third or fourth active ingredient. In some examples, the pharmaceutical composition comprises an additional therapeutic agent. In some examples, the second, third, or fourth active ingredient can be the additional therapeutic agent. In some examples, the additional therapeutic agent treats macular degeneration. In some examples, the additional therapeutic agent can be for treating a neurological disease or disorder (e.g., Parkinson’s disease, Alzheimer’s disease, or dementia). In some examples, the additional therapeutic agent can be for treating a liver disease or disorder (e.g., liver cirrhosis or alpha- 1 antitrypsin deficiency). In some instances, amyloid-beta aggregation (Abeta plauqes) contribute to the pathology of Alzheimer’s disease. Abeta can be derived from sequential proteolysis of amyloid precursor protein (APP) as variable-length fragments. In some examples, the additional therapeutic agent can be for preventing beta-amyloid from clumping into plaques or remove beta-amyloid plaques that have formed.

[237] In some examples, the additional therapeutic agent can be a 5-HT 6 antagonist, a 5- HT2A inverse agonist, an AB42 lowering agent, an acetylcholinesterase inhibitor, an alpha secretase enhancer, an alpha- 1 adrenoreceptor antagonist, an ammonia reducer, an angiotensin II receptor blocker, an alpha-2 adrenergic agonist, an anti-amyloid antibody, an anti-aggregation agent, an anti-amyloid immunotherapy, an anti-inflammatory agent, a glial cell modulator, an antioxidant, anti-tau antibody, an anti-tau immunotherapy, an anti-VEGF agent, an antiviral drug, a BACE inhibitory beta-adrenergic blocking agents, a beta-2 andrenergic receptor agonist, an arginase inhibitor, a beta blocker, a beta-HSDl inhibitor, a calcium channel blocker, a cannabinoid, a CB1 or CB2 endocannabinoid receptor agonist, a cholesterol lowering agent, a D2 receptor agonist, a dopamine-norepinephrine reuptake inhibitor, a FLNA inhibitor, a gamma secretase inhibitor, a GABA receptor modulator, a glucagon-like peptide 1 receptor agonist, a glutamate modulator, a glutamate receptor antagonist, a glycine transporter 1 inhibitor, a gonadotropin-releasing hormone receptor agonist, a GSK-3B inhibitor, a hepatocyte growth factor, ahistone deacetylase inhibitor, a IgGl-Fc-GAIM fusion protein, an ion channel modulator, an iron chelating agent, a meukotriene receptor antagonist, a MAPT RNA inhibitor, a mast cell stabilizer, a melatonin receptor agonist, a microtubule protein modulator, a mitochondrial ATP synthase inhibitor, a monoamine oxidase B inhibitor, a muscarinic agonist, a nicotinic acetylcholine receptor agonist, an NMDA antagonist, an NMDA receptor modulator, a nonhormonal estrogen receptor B agonist, a nonnucleoside reverse transcriptase inhibitor, a nonsteroidal anti-inflammatory agent, an omega-3 fatty acid, a P38 MAPK inhibitor, a P75 neurotrophin receptor ligand, a PDE 5 inhibitor, a PDE-3 inhibitor, a PDE4D inhibitor, a positive allosteric modulator of GABA-A receptors, a PPAR-gamma agonist, a protein kinase C modulator, a RIPK1 inhibitor, a secretase inhibitor, a selective inhibitor of APP production, a selective norepinephrine reuptake inhibitor, a selective serotonin reuptake inhibitor, a selective tyrosine kinase inhibitor, a SGLT2 inhibitor, a SIGLEC-3 inhibitor, a sigma-1 receptor agonist, a sigma-2 receptor antagonist, a stem cell therapy, an SV2A modulator, a synthetic hormone, a synthetic granulocyte colony stimulator, synthetic thiamine, a tau protein aggregation inhibitor, a telomerase reverse transcriptase vaccine, a thrombin inhibitor, a transport protein ABCC1 activator, a TREM2 inhibitor, a vascular endothelial growth factor (VEGF) inhibitor, a vitamin or any combination thereof.

[238] In some examples, the additional therapeutic agent can be an ammonia reducer, a beta blocker, a synthetic hormone, an antibiotic, or an antiviral drug, a vascular endothelial growth factor (VEGF) inhibitor, a stem cell treatment, a vitamin or modified form thereof, or any combination thereof.

[239] In some examples, the additional therapeutic agent can be AADvacl, AAVrh.lOhAPOE2, ABBV-8E12, ABvac40, AD-35, aducanumab, aflibercept, AGB101, AL002, AL003, allopregnanolone, amlopidine, AMX0035, ANAVEX 2-73, APH-1105, AR1001, AstroStem, atorvastatin, AVP-786, AXS-05, BAC, benfotiamine, BHV4157, BI425809, BIIB092, BIIP06, bioactive dietary polyphenol preparation, BPN14770, brexpiprazole, brolucizumab, byrostatin, CAD 106, candesartan, CERE- 110, cilostazol, CKD- 355, CNP520, COR388, crenezumab, cromolyn, CT1812, curcumin, dabigatran, DAOI, dapagliflozin, deferiprone, DHA, DHP1401, DNL747, dronabinol, efavirenz, elderberry juice, elenbecestat, escitalopram, formoterol, gantenerumab, ginkgo biloba, grapeseed extract, GRF6019, guanfacine, GV1001, hUCB-MSCs, ibuprofen, icosapent ethyl, ID1201, insulin aspart, insulin glulisine, IGNIS MAPTRx, J147, JNJ-63733657, lactulose, lactitol, lemborexant, leuprolide acetate depot, levetiracetam, liraglutide, lithium, LM11A-31-BHS, losartan, L-serine, L-omithine phenylacetate, Lu AF20513, LY3002813, LY3303560, LY3372993, masitinib, methylene blue, methylphenidate, metronidazole, mirtazapine, ML-4334, MLC901, montelukast, MP-101, nabilone, NDX-1017, neflamapimod, neomycin, nicotinamide, nicotine, nilotinib, nitazoxanide, NPT08, octagam 10%, octohydroaminoacridine succinate, omega-3 PUFA, perindopril, pimavanserin, piromelatine, posiphen, prazosin, PTI-125, ranibizumab rasagiline, rifaximin, riluzole, RG7105705, RPh201, sagramostim, salsalate, S-equol, sodium benzoate, sodium phenylacetate, solanezumab, SUVN-502, telmisartan, TEP, THN201, TPI-287, traneurocin, TRxO237, UB-311, valacyclovir, venlafaxine hMSCs (human mesenchymal stem cells), vorinostat, xanamem, zolpidem, or any combination thereof.

[240] In some examples, the pharmaceutical composition can be formulated in unit dose forms or multiple-dose forms. In some examples, the unit dose forms can be physically discrete units suitable for administration to human or non-human subjects (e.g., animals). In some examples, the unit dose forms can be packaged individually. In some examples, each unit dose contains a predetermined quantity of an active ingredient(s) that can be sufficient to produce the desired therapeutic effect in association with pharmaceutical carriers, diluents, excipients, or any combination thereof. In some examples, the unit dose forms comprise ampules, syringes, or individually packaged tablets and capsules, or any combination thereof. In some instances, a unit dose form can be comprised in a disposable syringe. In some instances, unit-dosage forms can be administered in fractions or multiples thereof. In some examples, a multiple-dose form comprises a plurality of identical unit dose forms packaged in a single container, which can be administered in segregated a unit dose form. In some examples, multiple dose forms comprise vials, bottles of tablets or capsules, or bottles of pints or gallons. In some instances, a multipledose forms comprise the same pharmaceutically active agents. In some instances, a multipledose forms comprise different pharmaceutically active agents.

[241] In some embodiments, pharmaceutical compositions described herein can be prepared as prodrugs. A "prodrug" can refer to an agent that can be converted into the parent drug in vivo. Prodrugs can be often useful because, in some situations, they can be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent can be not. The prodrug can also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a pharmaceutical composition described herein, which can be administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility can be detrimental to mobility but which then can be metabolically hydrolyzed to the carboxylic acid, the active enzyme, once inside the cell where water-solubility can be beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide can be metabolized to reveal the active moiety. In certain embodiments, upon in vivo administration, a prodrug can be chemically converted to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition. In certain embodiments, a prodrug can be enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition.

[242] Prodrug forms of the pharmaceutical compositions, wherein the prodrug can be metabolized in vivo to produce an agent as set forth herein can be included within the scope of the claims. Prodrug forms of the herein described pharmaceutical compositions, wherein the prodrug can be metabolized in vivo to produce an agent as set forth herein can be included within the scope of the claims. In some cases, some of the pharmaceutical compositions described herein can be a prodrug for another derivative or active compound. In some embodiments described herein, hydrazones can be metabolized in vivo to produce a pharmaceutical composition. 3. Excipients, Carriers, and Diluents

[243] In some examples, the pharmaceutical composition comprises an engineered guide (e.g., SLS guide) and an excipient (e.g., a pharmaceutically acceptable excipient). In some examples, the excipient comprises a buffering agent, a cryopreservative, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, or a coloring agent, or any combination thereof.

[244] In some examples, an excipient comprises a buffering agent. In some examples, the buffering agent comprises sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, calcium bicarbonate, or any combination thereof. In some examples, the buffering agent comprises sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, or calcium hydroxide and other calcium salts, or any combination thereof.

[245] In some examples, an excipient comprises a cryopreservative. In some examples, the cryopreservative comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. In some examples, a cryopreservative comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof. In some examples, an excipient comprises a pH agent (to minimize oxidation or degradation of a component of the composition), a stabilizing agent (to prevent modification or degradation of a component of the composition), a buffering agent (to enhance temperature stability), a solubilizing agent (to increase protein solubility), or any combination thereof. In some examples, an excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof. In some examples, an excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerin, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. In some examples, the excipient can be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

[246] In some examples, the excipient comprises a preservative. In some examples, the preservative comprises an antioxidant, such as alpha-tocopherol and ascorbate, an antimicrobial, such as parabens, chlorobutanol, and phenol, or any combination thereof. In some examples, the antioxidant comprises EDTA, citric acid, ascorbic acid, butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol or N- acetyl cysteine, or any combination thereof. In some examples, the preservative comprises validamycin A, TL-3, sodium ortho vanadate, sodium fluoride, N-a-tosyl-Phe- chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitor, phosphatase inhibitor, caspase inhibitor, granzyme inhibitor, cell adhesion inhibitor, cell division inhibitor, cell cycle inhibitor, lipid signaling inhibitor, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitors, or any combination thereof.

[247] In some examples, the excipient comprises a binder. In some examples, the binder comprises starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, or any combination thereof.

[248] In some examples, the binder can be a starch, for example a potato starch, com starch, or wheat starch; a sugar such as sucrose, glucose, dextrose, lactose, or maltodextrin; a natural and/or synthetic gum; a gelatin; a cellulose derivative such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, or ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); a wax; calcium carbonate; calcium phosphate; an alcohol such as sorbitol, xylitol, mannitol, or water, or any combination thereof.

[249] In some examples, the excipient comprises a lubricant. In some examples, the lubricant comprises magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, or light mineral oil, or any combination thereof. In some examples, the lubricant comprises metallic stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffins, hydrogenated vegetable oils, leucine, polyethylene glycols (PEG), metallic lauryl sulphates (such as sodium lauryl sulphate, magnesium lauryl sulphate), sodium chloride, sodium benzoate, sodium acetate or talc or a combination thereof.

[250] In some examples, the excipient comprises a dispersion enhancer. In some examples, the dispersion enhancer comprises starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate, or microcrystalline cellulose, or any combination thereof as high HLB emulsifier surfactants.

[251] In some examples, the excipient comprises a disintegrant. In some examples, a disintegrant comprises a non-effervescent disintegrant. In some examples, a non-effervescent disintegrants comprises starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, or gums such as agar, guar, locust bean, karaya, pectin, and tragacanth, or any combination thereof. In some examples, a disintegrant comprises an effervescent disintegrant. In some examples, a suitable effervescent disintegrant comprises bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

[252] In some examples, the excipient comprises a sweetener, a flavoring agent or both. In some examples, a sweetener comprises glucose (com syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like, or any combination thereof. In some cases, flavoring agents incorporated into a composition comprise synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; or any combination thereof. In some embodiments, a flavoring agent comprises a cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citms oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot, or any combination thereof.

[253] In some examples, the excipient comprises a pH agent (e.g., to minimize oxidation or degradation of a component of the composition), a stabilizing agent (e.g., to prevent modification or degradation of a component of the composition), a buffering agent (e.g., to enhance temperature stability), a solubilizing agent (e.g., to increase protein solubility), or any combination thereof. In some examples, the excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a trigylceride, an alcohol, or any combination thereof. In some examples, the excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerine, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. In some examples, the excipient comprises a cryo-preservative. In some examples, the excipient comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. In some examples, the excipient comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof.

[254] In some examples, the pharmaceutical composition comprises an engineered guide (e.g., SLS guide) and a diluent. In some examples, the diluent comprises water, glycerol, methanol, ethanol, or other similar biocompatible diluents, or any combination thereof. In some examples, a diluent comprises an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or any combination thereof. In some examples, a diluent comprises an alkaline metal carbonates such as calcium carbonate; alkaline metal phosphates such as calcium phosphate; alkaline metal sulphates such as calcium sulphate; cellulose derivatives such as cellulose, microcrystalline cellulose, cellulose acetate; magnesium oxide, dextrin, fructose, dextrose, glyceryl palmitostearate, lactitol, choline, lactose, maltose, mannitol, simethicone, sorbitol, starch, pregelatinized starch, talc, xylitol and/or anhydrates, hydrates and/or pharmaceutically acceptable derivatives thereof or combinations thereof.

[255] In some examples, the pharmaceutical composition comprises an engineered guide (e.g., SLS guide) and a carrier. In some examples, the carrier comprises a liquid or solid filler, solvent, or encapsulating material. In some examples, the carrier comprises additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldolic acids, esterified sugars and the like; and polysaccharides or sugar polymers), alone or in combination.

[256] In some embodiments, a pharmaceutical composition exists as an enantiomer, diastereomer, or other steroisomeric form. The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms as well as mixtures thereof. 4. Methods and Systems for Delivery

[257] The present disclosure also provides for vectors that comprise or encode an engineered guide (e.g., SLS guide RNA). The compositions provided herein can be delivered by any suitable means. In some cases, a polynucleotide can encode engineered guide (e.g., SLS guide RNA). For example, a plasmid can encode engineered guide (e.g., SLS guide RNA). Any vector system can be used utilized, including but not limited to: plasmid vectors, minicircle vectors, linear DNA vectors, doggy bone vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, a liposome, a nanoparticle, an exosome, an extracellular vesicle, a nanomesh, modified versions thereof, chimeras thereof, and any combination thereof. In some cases, a vector can be used to introduce a polynucleotide provided herein. In some cases, the polynucleotide comprises a targeting sequence that hybridizes to a region of an RNA provided herein.

[258] Vectors provided herein can be used to deliver polynucleotide compositions provided herein. In some cases, at least about 2, 3, 4, or up to 5 different polynucleotides are delivered using a single vector. In some cases, multiple vectors are delivered. In some cases, at least two engineered guide RNAs are delivered in a single vector. In other cases, at least two engineered guide RNAs are delivered on separate vectors. Engineered guide RNAs can also be delivered as naked polynucleotides. In some cases, multiple vector delivery can be co-current or sequential. In some cases, at least two SLS guide RNAs are delivered in a single vector. In other cases, at least two SLS guide RNAs are delivered on separate vectors. SLS guide RNAs can also be delivered as naked polynucleotides. Any combination of vector and/or anon-vector approach can be taken.

[259] A vector can be employed to deliver a nucleic acid. A vector can comprise DNA, such as double stranded DNA or single stranded DNA. A vector can comprise RNA. In some cases, the RNA can comprise a base modification. The vector can comprise a recombinant vector. The vector can be a vector that is modified from a naturally occurring vector. The vector can comprise at least a portion of a non-naturally occurring vector. Any vector can be utilized.

A. Viral methods of delivery

[260] In some embodiments, an engineered guide, such as an SLS guide RNA, can be delivered by a viral vector. A viral vector can comprise an adenoviral vector, an adeno- associated viral vector (AAV), a lentiviral vector, a retroviral vector, a portion of any of these, or any combination thereof. In some cases, a vector can comprise an AAV vector. A vector can be modified to include a modified VP1 protein (such as an AAV vector modified to include a VP1 protein). In an aspect an AAV vector is a recombinant AAV (rAAV) vector. rAAVs can be composed of substantially similar capsid sequence and structure as found in wild-type AAVs (wtAAVs). However, rAAVs encapsidate genomes that are substantially devoid of AAV proteincoding sequences and have therapeutic gene expression cassettes, such as subject polynucleotides, designed in their place. In some cases, sequences of viral origin can be the ITRs, which can be needed to guide genome replication and packaging during vector production. Suitable AAV vectors can be selected from any AAV serotype or combination of serotypes. For example, an AAV vector can be any one of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV-rh74, AAV-rhlO, AAV-2i8, or any combination thereof. In some cases, a vector is selected based on its natural tropism. In some cases, a vector serotype is selected based on its ability to cross the blood brain barrier. AAV9 and AAV 10 have been shown to cross the blood brain barrier to transduce neurons and glia. In an aspect, an AAV vector is AAV2, AAV5, AAV6, AAV8, or AAV9. In some cases, an AAV vector is a chimera of at least two serotypes. In some cases, a AAV vector can comprise a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self- complementary AAV (scAAV) vector, or a single-stranded AAV. In some cases, the AAV vector can comprise a genome comprising a replication gene and inverted terminal repeats from a first AAV serotype and a capsid protein from a second AAV serotype. In some cases, an AAV vector can be a chimera of one or more serotypes (e.g., an AAV2/5 virus having Rep and ITRs from AAV2 and capsid polypeptides from AAV5). In some cases, an AAV vector comprises an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector. In some cases, the inverted terminal repeats can comprise a 5’ inverted terminal repeat, a 3’ inverted terminal repeat, and a mutated inverted terminal repeat. In one aspect, the mutated inverted terminal repeat lacks a terminal resolution site. In some cases, rep, cap, and ITR sequences can be mixed and matched from all the of the different AAV serotypes provided herein.

B. Non-viral methods of delivery

[261] In some embodiments, an engineered guide, such as an SLS guide RNA can be delivered by a non-viral delivery method. Chemical means for introducing the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme into the cell can include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo can be a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids can be available, such as delivery of engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme with targeted nanoparticles or other suitable sub-micron sized delivery system.

[262] In the case where a non-viral delivery system can be utilized, an exemplary delivery vehicle can be a liposome. The use of lipid formulations can be contemplated for the introduction of the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme into a cell (in vitro, ex vivo or in vivo). In another aspect, the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme can be associated with a lipid. The engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme associated with a lipid, in some embodiments, can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that can be associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions can be not limited to any particular structure in solution. For example, in some embodiments, they can be present in a bilayer structure, as micelles, or with a “collapsed” structure. Alternately, they can be simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids can be fatty substances which are, in some embodiments, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[263] Lipids suitable for use can be obtained from commercial sources. For example, in some embodiments, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; in some embodiments, dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”), in some embodiments, can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”). Stock solutions of lipids in chloroform or chloroform/methanol can be often stored at about-20 °C. Chloroform can be used as the only solvent since it can be more readily evaporated than methanol. “Liposome” can be a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids can be suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure can be encompassed. For example, the lipids, in some embodiments, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated can be lipofectamine-nucleic acid complexes.

[264] In some cases, the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme described herein can be packaged and delivered to the cell via extracellular vesicles. The extracellular vesicles can be any membrane-bound particles. In some embodiments, the extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized without a cell. In some cases, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles. In some cases, the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme described herein can be administered to the subject in need thereof via the use of the transgenic cells generated by introduction of the engineered guide (e.g., SLS guide RNA) or vector encoding the engineered guide (e.g., SLS guide RNA) or the RNA editing enzyme first into allogeneic or autologous cells. In some cases, the cell can be isolated. In some embodiments, the cell can be isolated from the subject. In some embodiments, the cell can be an immune cell such as T cell.

5. Methods and Systems for Diagnosing a Disease or Monitoring a Disease Progression

[265] Doctors will use medical history, physical examination of the eye and use of opthalmoscopic tests such as the use of autofluorescence test to measure the retinal layer, fundoscopy or fluorescein angiography to measure leakage of blood vessels in the retina, Optiocal Coherence Tomography 9OCT), Tonometry etc, as a measure of various parameters relevant to eye health and that can be used for diagnosis of macular degeneration. In other cases, a home monitoring device such as an Amsler Grid can be used for detection of the retinal health. Genetic tests can include testing for mutations in ABCA4, ELOVL4, RPE65 gene in the subject. [266] In another aspect, the diagnostic test can include a genetic test for mutations in RAB7A, ABCA4, AAT, SERPINA1 E342K, HEXA, LRRK2, SNCA, GBA, PINK1, Tau, APP, CFTR, ALAS1, ATP7B, ATP7B G1226R, HEE C282Y, LIPA c.894 G>A, or any combination thereof. In one aspect, the diagnostic test can include a functional test of liver enzymes or hepatic function. In one aspect, the diagnostic test can include a functional test of lung function. In one aspect, the diagnostic test can include imaging of lung tissue. In one aspect, the diagnostic test can include ultrasound imaging of liver tissue. Doctors will use medical history, physical exam, neurological exam, mental status test, genetic test, and brain imaging to diagnose neurological diseases such as Alzheimer’s disease, Parkinson’s disease etc. Medical history consultation can comprise examining whether there can be current or past illness or if family members can have Alzheimer’s disease. Physical exam can help identify medical issues causing dementia-like symptoms. Physical exam can comprise examining diet, nutrition, alcohol use, medications, blood pressure, temperature, pulse, heart and lung functions, or other health conditions. Physical exam can also comprise blood and urine test. Neurological exam can evaluate if a patient has other brain disorders other than Alzheimer’s disease. Neurological exam can comprise testing reflexes, coordination, muscle tone/strength, eye movement, speech, or sensation. Neurological exam can also comprise brain imaging study including but not limited to Magnetic resonance imaging (MRI), computerized tomography (CT), or Positron emission tomography (PET). Mental status test can evaluate memory, problem-solving ability, or other cognitive abilities. Mental status test can comprise examining self-awareness, temporal or spatial awareness, memory, calculation ability, or others cognitive abilities. Mental status test can also comprise Mini-Mental State Exam (MMSE), the Mini-Cog test, FDA-approved computerized tests, mood assessment, or others. FDA-approved computerized tests can comprise the Cantab Mobile, Cognigram, Cognivue, Cognision and Automated Neuropsychological Assessment Metrics (ANAM) devices. Genetic testing can comprise testing APP, PSEN-1, PSEN-2, or apoE4. Other risk genes of Alzheimer’s disease include ABCA7, CLU, CR1, PICALM, PLD3, TREM2, or SORL1. With all the information listed above, a doctor can determine if a patient has “possible Alzheimer’s dementia” (dementia can be due to another cause), “probable Alzheimer’s dementia” (no other cause for dementia can be found), or some other problems.

KITS

[267] Disclosed herein are kits comprising compositions that comprise an engineered guide (e.g., SLS guide RNA), pharmaceutical compositions that comprise an engineered guide (e.g., SLS guide RNA), and isolated cells that comprise an engineered guide (e.g., SLS guide RNA) as disclosed herein. In some examples, a kit comprises one or more compositions, pharmaceutical compositions, or isolated cells disclosed herein and a container. In some examples, the kit comprises a pharmaceutical composition disclosed herein, which comprises an engineered guide disclosed herein or a polynucleotide encoding the engineered guide disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent. In some examples, the kit comprises one or more delivery vectors disclosed herein which comprise the polynucleotide encoding the engineered guide. In some examples, the kit comprises one or more isolated cells described herein. In some instances, the container can be plastic, glass, metal, or any combination thereof. In some examples, the container can be compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. In some example, the container can be a bottle, a vial, a syringe, or a test tube.

[268] In some embodiments, in addition to the composition, pharmaceutical composition, or isolated cell disclosed herein, a kit disclosed herein further comprises an additional therapeutic agent disclosed herein. In some examples, the additional therapeutic agent comprises a vascular endothelial growth factor (VEGF) inhibitor, a stem cell treatment, or a vitamin or modified form thereof, or any combination thereof.

[269] In some embodiments, a kit comprises instructions for use, such as instructions for administration to a subject in need thereof.

[270] In some cases, the kit comprises packaging for a composition or pharmaceutical composition described herein. In some examples, the packaging can be properly labeled. In some instances, the pharmaceutical composition described herein can be manufactured according to good manufacturing practice (cGMP) and labeling regulations.

[271] Also disclosed herein are methods of making a kit disclosed herein. In some examples methods of making the kits herein comprises contacting any of the engineered guides, compositions, pharmaceutical compositions, isolated cells, or isolated plurality of cells disclosed herein with a container. In some examples, methods of making a kit disclosed herein comprising placing an engineered guide, composition, pharmaceutical composition or isolated cell or plurality of cells disclosed herein in a container disclosed herein. In some examples, such methods further comprise placing instructions for use in the container.

[272] Kits can comprise a suitably aliquoted composition comprising an engineered guide (e.g., SLS guide RNA). The components of the kits can be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component can be placed, and preferably, suitably aliquoted. Where there can be more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components can be separately placed. However, various combinations of components can be comprised in a vial. The kits also will typically include a means for containing the components in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which the desired vials can be retained.

[273] However, the components of the kit can be provided as dried powder(s). When reagents and/or components can be provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It can be envisioned that the solvent can also be provided in another container means.

[274] In some embodiments, a kit can comprise the engineered guide RNAs (e.g., SLS guides), vectors, cells, or pharmaceutical compositions placed in a container. In some instances, a container can be plastic, glass, metal, or any combination thereof.

[275] In some cases, a packaged product comprising a composition described herein can be properly labeled. In some instances, the pharmaceutical composition described herein can be manufactured according to good manufacturing practice (cGMP) and labeling regulations. In some cases, a pharmaceutical composition disclosed herein can be aseptic.

NUMBERED EMBODIMENTS

[276] A number of compositions, and methods are disclosed herein. Specific exemplary embodiments of these compositions and methods are disclosed below. The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. [277] Embodiment 1. An engineered guide RNA comprising: (a) a targeting domain that binds to a target RNA, and (b) a structural loop stabilized scaffold; wherein the engineered guide RNA is configured, upon association with the target RNA, to facilitate a chemical modification of a base of a nucleotide in the target RNA by an RNA editing entity.

[278] Embodiment 2. The engineered guide RNA of embodiment 1, wherein the structural loop stabilized scaffold comprises a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof.

[279] Embodiment 3. The engineered guide RNA of embodiment 1 or 2, wherein the structural loop stabilized scaffold comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 stem loop structures.

[280] Embodiment 4. The engineered guide RNA of any one of embodiments 1-2, wherein the structural loop stabilized scaffold comprises a tRNA scaffold.

[281] Embodiment 5. The engineered guide RNA of any one of embodiments 1-4, wherein the engineered guide RNA is configured upon binding to the target RNA to form a guide-target RNA scaffold in conjunction with the target RNA, wherein the guide-target RNA scaffold comprises a structural feature that recruits the RNA editing entity.

[282] Embodiment 6. The engineered guide RNA of any one of embodiments 1-5, wherein the target RNA encodes: ABCA4, AAT, HEXA, LRRK2, APP, CFTR, ALAS1, ATP7B, HFE, PCSK9, SCNN1A, SNCA, GBA, PINK1, Tau, or LIPA, a biological active fragment of any of these, or any combination thereof.

[283] Embodiment 7. The engineered guide RNA of any one of embodiments 1-5, wherein the target RNA is a mRNA, a pre-mRNA, a tRNA, a IncRNA, a lincRNA, a miRNA, a rRNA, a snRNA, a siRNA, a piRNA, a snoRNA, an exRNA, a scaRNA, a YRNA, an eRNA, or a hnRNA molecule.

[284] Embodiment 8. The engineered guide RNA of any one of embodiments 1-7, wherein the engineered guide RNA comprises a modified DNA base, a modified DNA nucleotide, an unmodified DNA nucleotide, an unmodified DNA base, or a combination thereof.

[285] Embodiment 9. The engineered guide RNA of any one of embodiments 1-7, wherein the engineered guide RNA comprises a modified RNA nucleotide, a modified RNA base, an unmodified RNA base, an unmodified RNA nucleotide, or a combination thereof.

[286] Embodiment 10. The engineered guide RNA of any one of embodiments 1-9, wherein the engineered guide RNA is at least partially single-stranded. [287] Embodiment 11. The engineered guide RNA of any one of embodiments 1-10, wherein the targeting domain comprises from 4 contiguous nucleotides to about 1000 nucleotides.

[288] Embodiment 12. The engineered guide RNA of embodiment 11, wherein the targeting domain comprises from about 20 nucleotides to about 100 nucleotides.

[289] Embodiment 13. The engineered guide RNA of embodiment 5, wherein the guidetarget RNA scaffold formed upon association of the targeting domain with the target RNA comprises a nucleotide mismatch.

[290] Embodiment 14. The engineered guide RNA of embodiment 13, wherein the mismatch is: (a) an A to C mismatch; (b) a G to 5’ G mismatch; (c) a wobble base pair; or (d) or any combination of (a) to (c).

[291] Embodiment 15. The engineered guide RNA of any one of embodiments 13-14, wherein the mismatch comprises a base in the engineered guide RNA opposite to and unpaired with a base in the target RNA.

[292] Embodiment 16. The engineered guide RNA of any one of embodiments 13-15, wherein the mismatched nucleotide comprises a cytosine.

[293] Embodiment 17. The engineered guide RNA of any one of embodiments 13-15, wherein the mismatched nucleotide comprises an adenine.

[294] Embodiment 18. The engineered guide RNA of embodiment 13 or 14, wherein the mismatch is an Adenosine (A)ZCytosine (C) mismatch and wherein the Adenosine (A) is in the target RNA and the Cytosine (C) is in the engineered guide RNA.

[295] Embodiment 19. The engineered guide RNA of embodiment 13 or 14, wherein the mismatch is a Guanine (G)/Guanine (G) mismatch and wherein the Guanine (G) is in the target RNA and the Guanine (G) is in the engineered guide RNA.

[296] Embodiment 20. The engineered guide RNA of embodiment 13 or 14, wherein the Adenosine in the Adenosine (A)/ Cytosine (C) mismatch in the base of the nucleotide in the target RNA is chemically modified by the RNA editing entity to an Inosine (I).

[297] Embodiment 21. The engineered guide RNA of embodiment 13 or 14, wherein the engineered guide RNA comprises a C opposite the base of the nucleotide in the target RNA chemically modified by the RNA editing entity.

[298] Embodiment 22. The engineered guide RNA of any one of embodiments 13-21, wherein the target RNA comprises a 5’ G adjacent to the base of the nucleotide in the target RNA chemically modified by the RNA editing entity. [299] Embodiment 23. The engineered guide RNA of any one of embodiments 13-21, wherein the engineered guide RNA comprises a 5’ G adjacent to the C opposite to and unpaired with the A in the target RNA chemically modified by the RNA editing entity.

[300] Embodiment 24. The engineered guide RNA of any one of embodiments 1-23, wherein the target RNA comprises a point mutation that is associated with a disease or a condition.

[301] Embodiment 25. The engineered guide RNA of embodiment 24, wherein the point mutation comprises a missense mutation.

[302] Embodiment 26. The engineered guide RNA of embodiment 25, wherein the missense mutation results in an A at the mutated nucleotide.

[303] Embodiment 27. The engineered guide RNA of embodiment 25, wherein the point mutation facilitates unintended splicing of the target RNA.

[304] Embodiment 28. The engineered guide RNA of any one of embodiments 24-27, wherein the point mutation is a splice site mutation positioned adjacent to a C and a G on a 5’ and a 3’ end of the point mutation, respectively.

[305] Embodiment 29. The engineered guide RNA of any one of embodiments 1-28, wherein the RNA editing entity is: a) Adenosine deaminase acting on RNA (ADAR) or Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) enzyme; b) a catalytically active fragment of ADAR or APOBEC; c) fusion polypeptide comprising (a) or (b); or d) any combination of the above.

[306] Embodiment 30. The engineered guide RNA of embodiment 29, wherein the RNA editing entity is the ADAR, wherein the ADAR comprises human ADAR (hADAR).

[307] Embodiment 31. The engineered guide RNA of any one of embodiments 29-30, wherein the RNA editing entity is the ADAR, wherein the ADAR comprises AD ARI, ADAR2, or a combination thereof.

[308] Embodiment 32. The engineered guide RNA of embodiment 5, wherein the structural feature comprises: (a) a hairpin loop; (b) an internal loop; (c) a polynucleotide loop; (d) a wobble base pair; (e) a bulge; (1) a structured motif; or (g) or any combination of (a) to (1).

[309] Embodiment 33. The engineered guide RNA of embodiment 5, wherein the engineered guide RNA comprises from 1 to about 50 structural features.

[310] Embodiment 34. The engineered guide RNA of embodiment 33, wherein the engineered guide RNA comprises from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features.

[311] Embodiment 35. The engineered guide RNA of embodiment 32, wherein the structural feature comprises a bulge.

[312] Embodiment 36. The engineered guide RNA of embodiment 35, wherein the bulge comprises from 2 to about 100 nucleotides that are mismatched between the engineered guide RNA side and the target RNA in the guide-target RNA scaffold.

[313] Embodiment 37. The engineered guide RNA of embodiment 35, wherein the engineered guide RNA comprises 2 bulges, 3 bulges, or 4 bulges, upon associating with the target RNA.

[314] Embodiment 38. The engineered guide RNA of embodiment 35, wherein the bulge comprises an asymmetric bulge.

[315] Embodiment 39. The engineered guide RNA of embodiment 38, wherein the asymmetric bulge comprises 2 nucleotides, 3 nucleotides, or 4 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold.

[316] Embodiment 40. The engineered guide RNA of embodiment 39, wherein the asymmetric bulge comprises 2 nucleotides, 3 nucleotides, or 4 nucleotides on the target RNA side of the guide-target RNA scaffold.

[317] Embodiment 41. The engineered guide RNA of embodiment 35, wherein the bulge comprises a symmetric bulge.

[318] Embodiment 42. The engineered guide RNA of embodiment 41, wherein the symmetrical bulge comprises 2 nucleotides on the engineered guide RNA side of a dsRNA target and 2 nucleotides on the target RNA side of the guide-target RNA scaffold.

[319] Embodiment 43. The engineered guide RNA of embodiment 41, wherein the symmetrical bulge comprises 3 nucleotides on the engineered guide RNA side of a dsRNA target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold.

[320] Embodiment 44. The engineered guide RNA of embodiment 41, wherein the symmetrical bulge comprises 4 nucleotides on the engineered guide RNA side of a dsRNA target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.

[321] Embodiment 45. The engineered guide RNA of embodiment 32, wherein the structural feature comprises an internal loop.

[322] Embodiment 46. The engineered guide RNA of embodiment 45, wherein the internal loop comprises an asymmetric loop. [323] Embodiment 47. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 5 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold and 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[324] Embodiment 48. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 6 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold and 5 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[325] Embodiment 49. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 7 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[326] Embodiment 50. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 8 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 9 nucleotides, or 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[327] Embodiment 51. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 9 nucleotides on the engineered guide RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, or 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[328] Embodiment 52. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 5 nucleotides on target RNA side of the guide-target RNA scaffold a 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.

[329] Embodiment 53. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 6 nucleotides on the target RNA side of a guide-target RNA scaffold and 5 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.

[330] Embodiment 54. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 7 nucleotides on the target RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. [331] Embodiment 55. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 8 nucleotides on the target RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 9 nucleotides, or 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.

[332] Embodiment 56. The engineered guide RNA of embodiment 46, wherein the asymmetrical internal loop comprises 9 nucleotides on the target RNA side of a guide-target RNA scaffold and comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, or 10 nucleotides on the engineered guide RNA of the guide-target RNA scaffold.

[333] Embodiment 57. The engineered guide RNA of embodiment 45, wherein the internal loop is a symmetric loop.

[334] Embodiment 58. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 5 nucleotides on the engineered guide RNA side of a dsRNA target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold.

[335] Embodiment 59. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 6 nucleotides on the engineered guide RNA side of a dsRNA target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold.

[336] Embodiment 60. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 7 nucleotides on the engineered guide RNA side of a dsRNA target and 7 nucleotides on the target RNA side of the guide-target RNA scaffold.

[337] Embodiment 61. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 8 nucleotides on the engineered guide RNA side of a dsRNA target and 8 nucleotides on the target RNA side of the guide-target RNA scaffold.

[338] Embodiment 62. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 9 nucleotides on the engineered guide RNA side of a dsRNA target and 9 nucleotides on the target RNA side of the guide-target RNA scaffold.

[339] Embodiment 63. The engineered guide RNA of embodiment 57, wherein the symmetric loop comprises 10 nucleotides on the engineered guide RNA side of a dsRNA target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold.

[340] Embodiment 64. The engineered guide RNA of embodiment 32, wherein the structural feature comprises a hairpin loop.

[341] Embodiment 65. The engineered guide RNA of embodiment 64, wherein the engineered guide RNA comprises from 1 to 10 hairpin loops. [342] Embodiment 66. The engineered guide RNA of any one of embodiments 64-65, wherein the hairpin loop is present at (i) 3’ of the engineered guide RNA, (ii) 5’ end of the engineered guide RNA, or (iii) within the engineered guide RNA.

[343] Embodiment 67. The engineered guide RNA of embodiment 64, wherein the hairpin loop comprises a recruitment hairpin loop.

[344] Embodiment 68. The engineered guide RNA of embodiment 67, wherein the recruitment hairpin comprises at least a part of a GluR2 domain.

[345] Embodiment 69. The engineered guide RNA of embodiment 67, wherein the recruitment hairpin comprises at least a part of an Alu domain.

[346] Embodiment 70. The engineered guide RNA of embodiment 64, wherein the hairpin loop comprises a non-recruitment hairpin loop.

[347] Embodiment 71. The engineered guide RNA of embodiment 70, wherein the nonrecruitment hairpin comprises a hairpin loop from a U7 snRNA.

[348] Embodiment 72. The engineered guide RNA of any one of embodiments 64-71, wherein the hairpin loop comprises from about 10 to 500 nucleotides.

[349] Embodiment 73. The engineered guide RNA of any one of embodiments 64-71, wherein the hairpin loop comprises a stem loop structure.

[350] Embodiment 74. The engineered guide RNA of embodiment 73, wherein the stem loop comprises from 3 to 15 nucleotides.

[351] Embodiment 75. The engineered guide RNA of embodiment 32, wherein the structural feature comprises a wobble base pair.

[352] Embodiment 76. The engineered guide RNA of embodiment 75, wherein the wobble base pair comprises a G paired with a U.

[353] Embodiment 77. The engineered guide RNA of any one of embodiments 1-76, wherein the engineered guide RNA comprises a 5’ hydroxyl, a 3’ hydroxyl, or both, capable of being exposed to solvent.

[354] Embodiment 78. The engineered guide RNA of any one of embodiments 1-77, wherein the engineered guide RNA does not comprise a 5’ hydroxyl, a 3’ hydroxyl, or both, capable of being exposed to solvent.

[355] Embodiment 79. The engineered guide RNA of any one of embodiments 77-78, wherein the 3’ hydroxyl or the 5’ hydroxyl comprises a chiral center and wherein the chiral center is independently in the (R)- or (S)- configuration. [356] Embodiment 80. The engineered guide RNA of any one of embodiments 35, or 77- 79, wherein the structural feature is proximal to the 5’ hydroxyl or the 3’ hydroxyl.

[357] Embodiment 81. The engineered guide RNA of embodiment 32, wherein the structured motif comprises at least two structural features selected from (a)- (1).

[358] Embodiment 82. The engineered guide RNA of any one of embodiments 1-81, wherein the structural loop stabilized scaffold comprises at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with at least a portion of any one of SEQ ID NOs: 3-10, as determined by the BLAST.

[359] Embodiment 83. The engineered guide RNA of any one of embodiments 1-82, wherein the targeting domain, in association with the target RNA, comprises a structural feature that mimics a structure of a naturally occurring substrate for the RNA editing entity.

[360] Embodiment 84. The engineered guide RNA of any one of embodiments 1-83, wherein the engineered guide RNA is isolated, or purified, or both.

[361] Embodiment 85. A polynucleotide encoding the engineered guide RNA of any one of embodiments 1-84.

[362] Embodiment 86. A delivery vector comprising the engineered guide RNA of any one of embodiments 1-84 or the polynucleotide of embodiment 85.

[363] Embodiment 87. The delivery vector of embodiment 86, wherein the vector comprises an isolated cell.

[364] Embodiment 88. The delivery vector of embodiment 87, wherein the delivery vector comprises a viral vector.

[365] Embodiment 89. The delivery vector of embodiment 88, wherein the viral vector comprises a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno-associated viral (AAV) vector, or a poxvirus vector.

[366] Embodiment 90. The delivery vector of embodiment 89, wherein the viral vector comprises the AAV vector.

[367] Embodiment 91. The delivery vector of embodiment 90, wherein the AAV vector is of a serotype selected from the group consisting of: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-rh74, AAV-rhlO, and AAV-2i8.

[368] Embodiment 92. The delivery vector of any one of embodiments 90-91, wherein the AAV vector comprises a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. [369] Embodiment 93. The vector of any one of embodiments 90-92, wherein the AAV vector comprises a genome comprising a replication gene and inverted terminal repeats from a first AAV serotype and a capsid protein from a second AAV serotype.

[370] Embodiment 94. The delivery vector of any one of embodiments 90-93, wherein the AAV vector comprises an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector.

[371] Embodiment 95. The delivery vector of embodiment 93, wherein the inverted terminal repeats comprise a 5’ inverted terminal repeat, a 3’ inverted terminal repeat, and a mutated inverted terminal repeat.

[372] Embodiment 96. The delivery vector of embodiment 95, wherein the mutated inverted terminal repeat lacks a terminal resolution site.

[373] Embodiment 97. The delivery vector of embodiment 87, wherein the delivery vector is a non-viral delivery vector.

[374] Embodiment 98. The delivery vector of embodiment 97, wherein the non-viral delivery vector comprises a microvesicle, a nanovesicle, a microparticle, or a nanoparticle.

[375] Embodiment 99. The isolated cell of embodiment 98, wherein the isolated cell comprises the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, or the vector of any one of embodiments 86-98.

[376] Embodiment 100. The isolated cell of embodiment 99, wherein the isolated cell comprises an immune cell.

[377] Embodiment 101. The isolated cell of embodiment 100, wherein the immune cell comprises a T cell.

[378] Embodiment 102.A pharmaceutical composition comprising: (a) the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98 or the isolated cell of any one of embodiments 99-101, and;

(b) a pharmaceutically acceptable: excipient, carrier, or diluent.

[379] Embodiment 103. The pharmaceutical composition of embodiment 102 that is in unit dose form.

[380] Embodiment 104. A kit comprising: (a) the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, the isolated cell of any one of embodiments 99-101, or the pharmaceutical composition of any one of embodiments 102-103, and; (b) a container. [381] Embodiment 105. A method of making the kit of embodiment 104, the method comprising: contacting the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, the isolated cell of any one of embodiments 99-101, or the pharmaceutical composition of any one of embodiments 102-103, and, with the container.

[382] Embodiment 106. A method comprising delivering to a cell a composition comprising the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, or the pharmaceutical composition of any one of embodiments 102-103.

[383] Embodiment 107. A method of treating a disease or a condition in a subject in need thereof, the method comprising: administering to a subject, a first therapeutic comprising the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, or the isolated cell of any one of embodiments 99-101, in a therapeutically effective amount to treat the disease or condition.

[384] Embodiment 108. A method of preventing a disease or a condition in a subject in need thereof, the method comprising: administering to a subject, a first therapeutic comprising the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, the isolated cell of any one of embodiments 99-101, or the pharmaceutical composition of any one of embodiments 102-103, in a therapeutically effective amount to prevent the disease or condition.

[385] Embodiment 109. The method of any one of embodiments 107-108, wherein the administering of the therapeutic results in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or condition.

[386] Embodiment 110. The method of embodiment 109, wherein the RNA editing entity chemically modifies a cytosine to an uracil, thereby generating the modified target RNA.

[387] Embodiment 111. The method of embodiment 109, wherein the RNA editing entity chemically modifies an adenosine to an inosine, thereby generating the modified target RNA.

[388] Embodiment 112. The method of any one of embodiments 109-111, wherein the RNA editing enzyme is exogenously provided.

[389] Embodiment 113. The method of any one of embodiments 109-111, wherein the RNA editing enzyme is endogenous. [390] Embodiment 114. The method of embodiment 109, further comprising generating a modified expression product from the modified target RNA, wherein the modified expression product comprises a modified amino acid that exhibits an altered physicochemical property compared to the expression product from an otherwise comparable target RNA that does not contain the chemical modification.

[391] Embodiment 115. The method of embodiment 114, wherein the physicochemical property is charge, hydrophobicity, polarity, or any combination thereof.

[392] Embodiment 116. The method of any one of embodiments 109-115, wherein the modified amino acid is a glutamine, arginine, glycine, or valine.

[393] Embodiment 117. The method of embodiment 109, further comprising generating a modified expression product from the modified target RNA, wherein the modified expression product is expressed at an altered level compared to the expression product from an otherwise comparable target RNA that does not contain the chemical modification. Embodiment 118. The method of embodiment 109, further comprising generating a modified expression product from the modified target RNA, wherein the modified expression product either lacks a cleavage site for a cleavage enzyme or is more resistant to cleavage by a cleavage enzyme than the expression product from an unmodified target RNA.

[394] Embodiment 119. The method of embodiment 118, where in the cleavage enzyme is BACE.

[395] Embodiment 120. The method of embodiment 118, wherein the target RNA is APP.

[396] Embodiment 121. The method of any one of embodiments 109-118, wherein the target RNA is encoded by the serpin family A member 1 (SERPINA1) gene.

[397] Embodiment 122. The method of embodiment 121, wherein the SERPINA1 gene comprises a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1.

[398] Embodiment 123. The method of any one of embodiments 109-118, wherein the target RNA is encoded by an ABCA4 gene, or a portion thereof.

[399] Embodiment 124. The method of embodiment 123, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2.

[400] Embodiment 125. The method of embodiment 123, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2.

[401] Embodiment 126. The method of embodiment 123, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2. [402] Embodiment 127. The method of any one of embodiments 107-126, wherein the administering is intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above.

[403] Embodiment 128. The method of any one of embodiments 107-126, further comprising administering to the subject a dose of the first therapeutic via lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations, or lipid: nucleic acid conjugates.

[404] Embodiment 129. The method of any one of embodiments 107-128, where the first therapeutic comprises a tissue targeting moiety.

[405] Embodiment 130. The method of embodiment 129, wherein the tissue targeting moiety targets liver, eye, lung or brain.

[406] Embodiment 131. The method of any one of embodiments 107-130, comprising administering to the subject an additional dose of the first therapeutic.

[407] Embodiment 132. The method of embodiment 131, wherein the subject is administered the additional dose of the first therapeutic at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 70, 100, 120, 180, 240, 300, 400, or 1000 times.

[408] Embodiment 133. The method of embodiment 131, wherein the subject is administered the additional dose of the first therapeutic 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 times a day, or chronically.

[409] Embodiment 134. The method of embodiment 131, wherein the subject is administered the additional dose of the first therapeutic once every 1, 2, 3, or 4 weeks, 2 months, 3 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 5 years, or 10 years.

[410] Embodiment 135. The method of any one of embodiments 107-134, further comprising administering a second therapeutic to the subject.

[411] Embodiment 136. The method of embodiment 135, wherein the second therapeutic comprises the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, the isolated cell of any one of embodiments 99-101, or the pharmaceutical composition of any one of embodiments 102- 103.

[412] Embodiment 137. The method of embodiment 135, wherein the second therapeutic does not comprise the engineered guide RNA of any one of embodiments 1-84, the polynucleotide of embodiment 85, the delivery vector of any one of embodiments 86-98, the isolated cell of any one of embodiments 99-101, or the pharmaceutical composition of any one of embodiments 102- 103.

[413] Embodiment 138. The method of embodiment 135, wherein the second therapeutic comprises an ammonia reducer, a beta blocker, a synthetic hormone, an antibiotic, or an antiviral drug, or a combination thereof, for the treatment of a liver disease or disorder.

[414] Embodiment 139. The method of embodiment 135, wherein the second therapeutic comprises a vascular endothelial growth factor (VEGF) inhibitor, a stem cell treatment, a vitamin or modified form thereof, for the treatment of macular degeneration.

[415] Embodiment 140. The method of any one of embodiments 135-139, wherein the second therapeutic is administered concurrently with the first therapeutic.

[416] Embodiment 141. The method of any one of embodiments 135-139, wherein the second therapeutic is administered consecutively with the first therapeutic.

[417] Embodiment 142. The method of any one of the embodiments 107-141, wherein the subject is diagnosed with the disease or the condition.

[418] Embodiment 143. The method of embodiment 142, wherein the diagnosing comprises an in-vitro diagnostic test.

[419] Embodiment 144. The method of embodiment 143, wherein the in vitro diagnostic test comprises a companion test.

[420] Embodiment 145. The method of any one of embodiments 107-144, wherein the disease or the condition comprises a neurological disease or disorder.

[421] Embodiment 146. The method of embodiment 145, wherein the neurological disease or disorder comprises Parkinson’s disease, Alzheimer’s disease, or dementia.

[422] Embodiment 147. The method of any one of embodiments 107-144, wherein the disease or the condition comprises a liver disease or disorder.

[423] Embodiment 148. The method of embodiment 147, wherein the liver disease or disorder comprises liver cirrhosis.

[424] Embodiment 149. The method of embodiment 148, wherein the liver disease or disorder comprises alpha-1 antitrypsin deficiency (AAT deficiency).

[425] Embodiment 150. The method of any one of embodiments 107-144 wherein the disease or condition comprises macular degeneration.

[426] Embodiment 151. The method of embodiment 150, wherein the macular degeneration comprises Stargardt disease. [427] Embodiment 152. The method of any one of embodiments 107-144, wherein the disease or the condition comprises a lung disease or disorder.

[428] Embodiment 153. The method of embodiment 152, wherein the lung disease or disorder comprises chronic obstructive pulmonary disease (COPD).

[429] Embodiment 154. The method of any one of embodiments 107-153, wherein the chemical modification is confirmed by an in vitro assay.

[430] Embodiment 155. The method of embodiment 119, wherein the in vitro assay comprises: (i) contacting the target RNA with the engineered guide RNA; (a) in a presence of the RNA editing entity; and (b) in an absence of the RNA editing entity; and (ii) comparing the sequence of the target RNA in (a) and (b) to detect the chemical modification.

[431] Embodiment 156. The method of embodiment 155, wherein in vitro assay comprises sanger sequencing, next-generation sequencing, or a combination thereof.

[432] Embodiment 157. The engineered guide RNA of any one of embodiments 1-4, wherein the engineered guide RNA comprises an RNA editing entity recruiting domain.

EXAMPLES

[433] The following examples can be included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Treatment of Alpha- 1 antitrypsin deficiency (AATD)

[434] This example describes treatment of AATD disease using the engineered guide RNAs of the present disclosure, such as an SLS guide disclosed herein. A subject will be diagnosed with AATD disease. The subject will be prescribed a dosing regimen of a pharmaceutical composition. The pharmaceutical composition will comprise an engineered guide RNA that edits a mutation in the SERPINA1 gene that causes misfolding of AAT. The pharmaceutical composition will be administered to the subject by intravenous injection or intramuscular injection. An amount of the AAT circulation in the serum following treatment with the engineered guide RNA will be at least 4-fold more than an amount of AAT circulating in the serum without the treatment.

Example 2: Multiplexed compositions for treatment of AATD

[435] This example describes multiplexed compositions (e.g., two engineered guide RNA (e.g., two SLS guides) targeting different RNA polynucleotides) for treatment of AATD. A subject will be diagnosed with AATD. The subject will be prescribed a dosing regimen of a pharmaceutical composition. The pharmaceutical composition will comprise a multiplexed composition comprising an engineered guide RNA for editing of an Arg³⁹ to Cyst³⁹ (CGC to TGC) in the SERPINA1 gene and an engineered guide RNA for editing of the Glu³⁷⁶-Asp³⁷⁶ (GAA to GAC) mutation in the SERPINA1 gene. The pharmaceutical composition will be administered to the subject by parenchymal injection, ICM injection, or ICV injection in an effective amount to treat the resulting AATD in the patient.

Example 3: Multiplexed Vectors for Treatment of AATD and Stargardt disease

[436] This example describes multiplexed vectors for treatment of AATD and Stargradt disease. A subject will be diagnosed with an AATD and Stargardt disease caused by mutations in SERPINA1 W ABCA4 genes respectively. The subject will be prescribed a dosing regimen of a pharmaceutical composition. The pharmaceutical composition will comprise a first vector comprising a first engineered guide RNA (e.g., a first SLS guide) that will be directed to correct the mutation in ABCA4, and a second vector comprising a second engineered guide RNA (e.g., a second SLS guide) that will be directed to correct the mutation in SERPINA1. The pharmaceutical composition will be administered to the subject by parenchymal injection in an effective amount to treat the AATD and Stargardt disease.

Example 4: Delivery of multiplexed engineered guide RNAs with a single vector.

[437] The modularity of the RNA editing entity and the RNA targeting polynucleotide allows the multiplexed targeting to be carried out in various ways. Multiplexed targeting will be used to treat Alzheimer’s disease patients with contributing polymorphisms in APP and Tau or SNCA. Two sequences encoding two engineered guide RNAs (e.g., two SLS guides) are generated. The first coding sequence codes for an engineered guide RNA that affects the proteolytic cleavage and processing of APP and thereby alters the ratio of the Abeta 40/Abeta 42 in the subject. The second coding sequence codes for an engineered guide RNA that targets the start ATG of any one of the Tau or SNCA mRNA and converts a nucleotide of the start ATG into a different nucleotide. Since the start ATG is removed, the expression of Tau or SNCA will decrease. These two coding sequences can be each paired with a Polymerase III promoter and each cloned into a single viral vector — such as an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, or a retroviral vector — to express each coding sequence individually. The vectors will be injected into the brain of the patient by intracerebroventricular injection. The patient will show improved memory as compared to their memory before treatment. Example 5: Example Workflow

[438] In an example of a workflow of the methods described herein, first a patient with m- RNA or pre-mRNA comprising one or more disease relevant mutations can be isolated and immortalized. Second, mRNA expression of the mutation or mutations can be verified using DNA or RNA sequencing (e.g., Sanger sequencing). Third an engineered RNA comprising a SLS scaffold and a targeting region complementary to the region of pre-mRNA or mRNA comprising the mutation can be recombinantly produced. Fourth, the engineered RNA can be administered to the patient cells (e.g., via a viral vector). After treatment, to verify editing has occurred, the patient RNA can be isolated and converted to cDNA (and then sequenced by Sanger sequencing).

Example 6: Editing of SERPINA1 in patient cells using SLS guide RNA.

[439] This example describes editing of SERPINA1 in patient cells using engineered SLS guide RNAs. Engineered SLS guide RNAs were designed to target SERPINA1 pre-mRNA and mRNA, as shown in FIGs. 1A-B. Engineered SLS guide RNAs were designed to span intron/ exon junctions, and in mature mRNA, exon/exon junctions. The structure of an exemplary SLS guide RNA of the present disclosure, a non-limiting example of a tRNA scaffold containing the targeting domain (noted as gRNA) targeting SERPINA1, is shown in FIG. 2. The gRNA targeting SERPINA1 comprises a C at a position opposite the target A in SERPINA1 to be edited, thus yielding a mismatch upon hybridization of the gRNA to the target sequence and formation of a guide-target RNA scaffold. In this exemplary SLS guide RNA, the gRNA targeting SERPENA1 is positioned in the anticodon region of the tRNA scaffold.

[440] FIGs. 3A-3B show data verifying that the immortalized fibroblasts from patients with the SERPINA1 mutation, did indeed contain SERPINA1 with the E342K mutation. FIG. 3A, at top, shows the target A (under the arrow) at the expected position for SERPINA1 carrying the E342K mutation. FIG. 3A, at bottom, shows that SERPINA1 was expressed in the fibroblasts. FIG. 3B shows a Western blot of protein expression in the fibroblasts, including AD ARI, ADAR2, and GAPDH (a control). In the fibroblasts carrying the SERPINA1 mutation (column 3), AD ARI was expressed and ADAR2 was not expressed.

[441] Immortalized cells (fibroblasts) from patients carrying the E342K mutation were grown in culture. The mRNA expression of the mutant isoform of AAT from the mutated SERPINA1 gene in the patient was verified using RT-PCR or a suitable antibody that can recognize the mutated isoform. An engineered linear gRNA against Rab7a (“control gRNA”; negative control), an engineered linear gRNA against SERPINA1 (“WT gRNA”; positive control), and an engineered SLS guide RNA (“WT_tRNA”) were nucleofected in the fibroblasts. 2x10^A5 cells were used per transfection and cells were transfected with 60 pmoles of the engineered guide RNA. cDNA synthesized from the isolated RNA was PCR amplified, followed by Sanger sequencing. Percent editing was quantified using the EditR software. As shown in FIG. 4 (at left), the engineered SLS guide RNAs displayed the highest percent editing of SERPINA1. Sanger sequencing traces (FIG. 4 at right) show the same.

[442] In order to explore off-target editing of adenosines in SERPINA1, the engineered guide RNAs were modified to incorporate Gs opposite off-target. As in the target SERPINA1 sequence, as shown in FIG. 5 (schematic at left). Transfection and measurement of percent editing was carried out as described above. The engineered SLS guide RNA containing the single A/C mismatch upon hybridization to the target (WT_tRNA) displayed the highest levels of SERP NA1 editing.

Example 7: Compositions for treatment of Alzheimer’s disease

[443] A subject will be diagnosed with an Alzheimer’s disease. The subject is prescribed a dosing regimen of a pharmaceutical composition. The pharmaceutical composition will comprise a multiplex targeting scheme of a first vector comprising a first polynucleotide encoding a first engineered guide RNA (e.g., a first SLS guide) that targets the ratio of processed Abeta 40/42 and a second vector comprising a second polynucleotide encoding a second engineered guide RNA (e.g., a second SLS guide) that targets Tau/SNCA. The pharmaceutical composition is administered to the subject by direct injection to the central nervous system (CNS) in an effective amount to treatment the Alzheimer’s disease.

[444] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments can be provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following enumerated embodiments define the scope of the invention and that methods and structures within the scope of these enumerated embodiments and their equivalents be covered thereby. Table 3. SEQUENCES

Claims

CLAIMS What is claimed is:

1. An engineered guide RNA comprising:

(a) a targeting domain that binds to a target RNA, and

(b) a structural loop stabilized scaffold; wherein the engineered guide RNA is configured, upon association with the target RNA, to facilitate a chemical transformation of a base of a nucleotide in the target RNA by an RNA editing entity.

2. The engineered guide RNA of claim 1, wherein the structural loop stabilized scaffold comprises a 5’ end and a 3’ end that together form a secondary structure or a tertiary structure.

3. The engineered guide RNA of any one of claims 1-2, wherein the structural loop stabilized scaffold comprises a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof.

4. The engineered guide RNA of any one of claims 1-3, wherein the structural loop stabilized scaffold comprises at least 2 stem loop structures.

5. The engineered guide RNA of any one of claims 1-4, wherein the structural loop stabilized scaffold comprises a tRNA scaffold.

6. The engineered guide RNA of any one of claims 1-5, wherein engineered guide RNA comprises an RNA editing entity recruiting domain.

7. The engineered guide RNA of any one of claims 1-6, wherein the engineered guide RNA is configured upon binding to the target RNA to form a guide-target RNA scaffold in conjunction with the target RNA, and wherein the guide-target RNA scaffold comprises a structural feature that recruits the RNA editing entity.

8. The engineered guide RNA of any one of claims 1-7, wherein the target RNA encodes: ABCA4, AAT, HEXA, LRRK2, APP, CFTR, ALAS1, ATP7B, HFE, PCSK9, SCNN1A, SNCA, GBA, PINK1, Tau, or LIPA, a biological active fragment of any of these, or any combination thereof.

9. The engineered guide RNA of any one of claims 1-8, wherein the engineered guide RNA comprises a modified RNA nucleotide, a modified RNA base, an unmodified RNA base, an unmodified RNA nucleotide, or a combination thereof.

10. The engineered guide RNA of any one of claims 1-9, wherein the targeting domain comprises from about 20 nucleotides to about 200 nucleotides.

11. The engineered guide RNA of any one of claims 7-10, wherein the guide-target RNA scaffold formed upon association of the targeting domain with the target RNA comprises a nucleotide mismatch.

12. The engineered guide RNA of claim 11, wherein the mismatch is:

(a) an A to C mismatch;

(b) a G to 5’ G mismatch;

(c) a wobble base pair; or

(d) or any combination of (a) to (c).

13. The engineered guide RNA of any one of claims 1-12, wherein the target RNA comprises a point mutation that is associated with a disease or a condition.

14. The engineered guide RNA of claim 13, wherein the point mutation comprises a missense mutation.

15. The engineered guide RNA of any one of claims 1-14, wherein the RNA editing entity is: a) Adenosine deaminase acting on RNA (ADAR) or Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) enzyme; b) a catalytically active fragment of ADAR or APOBEC; c) fusion polypeptide comprising (a) or (b); or d) any combination of (a) to (c).

16. The engineered guide RNA of claim 15, wherein the RNA editing entity is the ADAR, wherein the ADAR comprises human ADAR (hADAR).

17. The engineered guide RNA of claim 15, wherein the RNA editing entity is the ADAR, wherein the ADAR comprises AD ARI , ADAR2, or a combination thereof.

18. The engineered guide RNA of any one of claims 7-17, wherein the structural feature comprises:

(a) a hairpin loop;

(b) an internal loop;

(c) a polynucleotide loop;

(d) a wobble base pair;

(e) a bulge; or

(g) or any combination of (a) to (e).

19. The engineered guide RNA of claim 18, wherein the engineered guide RNA comprises from 1 to 50 structural features.

20. The engineered guide RNA of any one of claims 7-19, wherein the structural feature comprises a bulge.

21. The engineered guide RNA of claim 20, wherein the bulge comprises from 2 to 4 nucleotides that are mismatched between the engineered guide RNA side and the target RNA in the guide-target RNA scaffold.

22. The engineered guide RNA of claim 20, wherein the bulge comprises an asymmetric bulge.

23. The engineered guide RNA of claim 20, wherein the bulge comprises a symmetric bulge.

24. The engineered guide RNA of any one of claims 7-19, wherein the structural feature comprises an internal loop.

25. The engineered guide RNA of claim 24, wherein the internal loop comprises an asymmetric loop.

26. The engineered guide RNA of claim 24, wherein the internal loop is a symmetric loop.

27. The engineered guide RNA of any one of claims 7-19, wherein the structural feature comprises a hairpin loop.

28. The engineered guide RNA of claim 27, wherein the engineered guide RNA comprises from 1 to 10 hairpin loops.

29. The engineered guide RNA of any one of claims 27-28, wherein the hairpin loop is present at (i) 3’ of the engineered guide RNA, (ii) 5’ of the engineered guide RNA, or (iii) within the engineered guide RNA.

30. The engineered guide RNA of claim 27, wherein the hairpin loop comprises a nonrecruitment hairpin loop and the non-recruitment hairpin loop is from a U7 snRNA.

31. The engineered guide RNA of any one of claims 27-30, wherein the hairpin loop comprises from about 10 to 500 nucleotides.

32. The engineered guide RNA of any one of claims 7-19, wherein the structural feature comprises a wobble base pair and wherein the wobble base pair comprises a G paired with a U.

33. The engineered guide RNA of any one of claims 1-32, wherein the structural loop stabilized scaffold comprises at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with at least a portion of any one of SEQ ID NOs: 3-10, as determined by BLAST.

34. The engineered guide RNA of any one of claims 1-33, further comprising a spacer domain.

35. A polynucleotide encoding the engineered guide RNA of any one of claims 1-34.

175

36. A delivery vector comprising the engineered guide RNA of any one of claims 1-34, or the polynucleotide of claim 35.

37. The delivery vector of claim 36, wherein the vector comprises a viral vector.

38. The delivery vector of claim 37, wherein the viral vector comprises a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno-associated viral (AAV) vector, or a poxvirus vector.

39. The delivery vector of claim 38, wherein the viral vector comprises the AAV vector.

40. The delivery vector of claim 39, wherein the AAV vector is of a serotype selected from the group consisting of: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-rh74, AAV-rhlO, and AAV-2i8.

41. The delivery vector of any one of claims 39-40, wherein the AAV vector comprises a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self- complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof.

42. The delivery vector of claim 36, wherein the delivery vector is a non-viral delivery vector.

43. The vector of claim 42, wherein the non-viral delivery vector comprises a microvesicle, a nanovesicle, a microparticle, or a nanoparticle.

44. A pharmaceutical composition in unit dose form comprising:

(a) the engineered guide RNA of any one of claims 1-34, the polynucleotide of claim 35, or the delivery vector of any one of claims 36-43, and;

(b) a pharmaceutically acceptable: excipient, carrier, or diluent.

45. A method of treating a disease or a condition in a subject in need thereof, the method comprising: administering to a subject, a therapeutic comprising the engineered guide RNA of any one of claims 1-34, the polynucleotide of claim 35, or the delivery vector of any one of claims 36-43 in a therapeutically effective amount to treat the disease or condition.

46. The method of claim 45, wherein the administering of the therapeutic results in recruitment of an RNA editing entity to chemically modify a base of a nucleotide in a target RNA, thereby generating a modified target RNA and treating the disease or the condition.

47. The method of claim 46, wherein the target RNA is encoded by the serpin family A member 1 (SER INA 7) gene.

48. The method of claim 47, wherein the SERPINA1 gene comprises a substitution of a G with an A at nucleotide position 9989 within SEQ ID NO: 1.

176

49. The method of claim 46, wherein the target RNA is encoded by an ABCA4 gene, or a portion thereof.

50. The method of claim 49, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 5882 within SEQ ID NO: 2.

51. The method of claim 49, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 5714 within SEQ ID NO: 2.

52. The method of claim 49, wherein the ABCA4 gene comprises a substitution of a G with an A at nucleotide position 6320 within SEQ ID NO: 2.

53. The method of any one of claims 45-52, wherein the administering is intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a route that is a combination of the above.

54. The method of any one of claims 45-53, wherein the disease or the condition comprises Parkinson’s disease, Alzheimer’s disease, a dementia, liver cirrhosis, alpha-1 antitrypsin deficiency (AAT deficiency), Stargardt disease, chronic obstructive pulmonary disease (COPD), or any combination thereof.

55. The method of any one of claims 46-54, wherein the chemical modification is confirmed by an in vitro assay.

177