Wobble base pairs for improving specificity of RNA editing

Abstract

Disclosed herein are engineered guide RNAs and compositions comprising the same for treatment of diseases or conditions in a subject. Also disclosed herein are methods of treating diseases or conditions in a subject by administering engineered guide RNAs or pharmaceutical compositions described herein.

Description

WOBBLE BASE PAIRS FOR IMPROVING SPECIFICITY OF RNA EDITINGCROSS REFERENCE

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No. 63 / 613,139. filed December 21. 2023, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Payloads that mediate RNA editing can be viable therapies for genetic diseases. However, highly efficacious payloads that can maximize on-target RNA editing while minimizing or eliminating off-target RNA editing and / or splicing are needed.SUMMARY

[0003] Provided herein is a polynucleotide encoding an engineered guide RNA, wherein: hybridization of a target RNA sequence to the engineered guide RNA forms a guide-target RNA scaffold comprising a latent structure; the latent structure compnses one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the engineered guide RNA comprises 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence; the engineered guide RNA facilitates editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and (i) the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by an otherwise comparable reference guide RNA in which the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of a target RNA sequence are each individually replaced with a nucleotide that Watson-Crick base pairs to a corresponding nucleotide in the target RNA sequence when measured in an in vitro assay; (ii) the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on- target editing facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay; (iii) the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay; (iv) the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay; (v) the free energy of the guide-target RNA scaffold is decreased relative to the free energy' of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to the otherwise comparable reference guide RNA when measured in an in vitro assay; or any combination of (i)-(v). In some embodiments, the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence form 3 to 30 wobble base pairs that each individually and independently comprise a G-U wobble base pair, a U-G wobble base pair, I-U wobble base pair, a U-I wobble base pair, a I-A wobble base pair, a A-I wobble base pair, a I-C wobble base pair, or C-I wobble base pair. In some embodiments, the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence are located in a hybridization domain sequence that is upstream of a 5’ symmetric internal loop in the target RNA sequence. In some embodiments, the 3 to 30 noncontiguous nucleotides that w obble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence are located in a hybridization domain sequence that is downstream of a 5’ symmetric internal loop in the target RNA sequence.

[0004] Also provided herein is a polynucleotide encoding an engineered guide RNA, wherein: hybridization of a target RNA sequence to the engineered guide RNA forms a guide-target RNA scaffold comprising a latent structure; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the engineered guide RNA comprises 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence; the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides from the target adenosine in the target RNA sequence; the engineered guide RNA facilitates editing of a target adenosine in the target RNA sequence by an ADAR1 or an ADAR2; and (i) the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by an otherwise comparable reference guide RNA in which the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of a target RNA sequence are each individually replaced with a nucleotide that Watson-Crick base pairs to a corresponding nucleotide in the target RNA sequence when measured in an in vitro assay; (ii) the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay; (iii) the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the otherwise comparablereference guide RNA when measured in an in vitro assay; (iv) the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay; (v) the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to the otherwise comparable reference guide RNA when measured in an in vitro assay; or any combination of (i)-(v). In some embodiments, the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence form 1 or 2 wobble base pairs that each individually and independently comprise a G-U wobble base pair, a U-G wobble base pair, I-U wobble base pair, a U-I wobble base pair, a I-A wobble base pair, a A-I wobble base pair, a I-C wobble base pair, or a C-I wobble base pair. In some embodiments, the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence are each individually located 15 to 30 nucleotides from the target adenosine in the target RNA sequence. In some embodiments, the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence are each individually located 20 to 25 nucleotides from the target adenosine in the target RNA sequence. In some embodiments, the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides 3’ downstream from the target adenosine in the target RNA sequence.

[0005] In some embodiments, the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the reference guidetarget RNA. In some embodiments, the engineered guide RNA facilitates at least about 5% reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates at least about 5% increased on-target RNA editing of the target RNA sequence relative to on-target editing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates aglobal editing specificity of the target RNA sequence that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more higher than the global editing specificity facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates at least about 5% reduced off-target splicing of the target RNA sequence relative to off-target splicing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates at least about 5% increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing speci fici ty facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more higher than the local editing specificity facilitated by a reference guide RNA when measured in an in vitro assay. In some embodiments, the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to a reference guide RNA when measured in an in vitro assay. In some embodiments, the free energy of the guide-target RNA scaffold is decreased 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less relative to the free energy of the reference guide-target RNA scaffold. In some embodiments, the guide-target RNA scaffold binds or recruits an ADAR1 or an ADAR2, resulting in editing of a target adenosine in the target RNA sequence by the AD ARI or the ADAR2. In some embodiments, the referenceguide RNA is a parental engineered guide RNA of the engineered guide RNA. In some embodiments, the reference guide RNA is a parental engineered guide RNA of the engineered guide RNA, that upon hybridization with a target sequence forms a parental guide-target RNA scaffold that comprises the latent structure of the guide-target RNA scaffold. In some embodiments, the latent structure comprises one or more, 2 or more. 3 or more, or 4 or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof. In some embodiments, the latent structure of the guide-target RNA scaffold comprises two internal loops. In some embodiments, the latent structure of the guide-target RNA scaffold comprises an internal loop that is a 5‘ symmetric internal loop positioned upstream of the target adenosine in the target RNA sequence or an internal loop that is a 3’ symmetric internal loop positioned downstream of the target adenosine in the target RNA sequence. In some embodiments, the latent structure of the guide-target RNA scaffold comprises a first internal loop and a second internal loop, wherein the first internal loop is a 5' symmetric internal loop positioned upstream of the target adenosine in the target RNA sequence and the second internal loop is a 3’ symmetric internal loop positioned downstream of the target adenosine in the target RNA sequence. In some embodiments, the guide-target RNA scaffold comprises about: 3 to about 21 wobble base pairs, 3 to about 20 wobble base pairs. 3 to about 19 wobble base pairs, or 3 to about 15 wobble base pairs.

[0006] In some embodiments, the target RNA sequence comprises a sequence from a ABCA4, APP, CFTR, DMPK, DUX4, GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22, SERPINA1, SNCA. or SOD1 RNA. In some embodiments, the target RNA sequence comprises a 3’ untranslated region (UTR) of SNCA. In some embodiments, the target RNA sequence comprises a translation initiation site (TIS) of SNCA. In some embodiments, the target RNA sequence comprises a 3’ UTR of SERPINA1. In some embodiments, the target RNA sequence comprises ABCA4. In some embodiments, the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 16. In some embodiments, the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity , or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 37. In some embodiments, the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 39 - SEQ ID NO: 52. In some embodiments,the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 184 - SEQ ID NO: 188. In some embodiments, the engineered guide RNA comprises any one of SEQ ID NO: 2 - SEQ ID NO: 16. In some embodiments, the engineered guide RNA comprises any one of SEQ ID NO: 18 - SEQ ID NO: 37. In some embodiments, the engineered guide RNA comprises any one of SEQ ID NO: 39 - SEQ ID NO: 52. In some embodiments, the engineered guide RNA comprises any one of SEQ ID NO: 184 - SEQ ID NO: 188.

[0007] Also provided herein is an AAV vector cassette comprising any one or more of the polynucleotide sequences described herein.

[0008] Also provided herein is an AAV vector cassette comprising a polynucleotide sequence encoding a first engineered guide RNA and a second engineered guide RNA, wherein: wherein the first engineered guide RNA and the second engineered guide RNA are independently capable of hybridizing to a target RNA sequence forming a first guide target RNA scaffold and a second guide target RNA scaffold, respectively; the first guide target RNA scaffold and second guide target RNA scaffold comprise a latent structure; the first engineered guide RNA and the second engineered guide RNA share at least 64% sequence identity to each other; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the second engineered guide RNA comprises 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence; the first engineered guide RNA and the second engineered guide RNA facilitate editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and (i) the second engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay; (ii) the second engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay; (iii) the second engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay; (iv) the second engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay; (v) the free energy of the second guide-target RNAscaffold is decreased relative to the free energy of the first guide-target RNA scaffold when measured in an in vitro assay; or any combination of (i)-(v). In some embodiments, the first engineered guide RNA does not comprise the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence.

[0009] Also provided herein is an AAV vector cassette comprising a polynucleotide sequence encoding a first engineered guide RNA and a second engineered guide RNA, wherein: wherein the first engineered guide RNA and the second engineered guide RNA are independently capable of hybridizing to a target RNA sequence forming a first guide target RNA scaffold and a second guide target RNA scaffold, respectively; the first guide target RNA scaffold and second guide target RNA scaffold comprise a latent structure; the first engineered guide RNA and the second engineered guide RNA share at least 64% sequence identity to each other; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the second engineered guide RNA comprises 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence; the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides from the target adenosine in the target RNA sequence; the first engineered guide RNA and the second engineered guide RNA facilitate editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and (i) the second engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay; (ii) the second engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay; (iii) the second engineered guide RNA facilitates a global editing specificity' of the target RNA sequence that is higher than the global editing specificity facilitated by the first engineered guide RNA yvhen measured in an in vitro assay; (iv) the second engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay; (v) the free energy of the second guide-target RNA scaffold is decreased relative to the free energy of the first guide-target RNA scaffold when measured in an in vitro assay; or any combination of (i)- (v). In some embodiments, the first engineered guide RNA does not comprise the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence.

[0010] Also provided herein is a pharmaceutical composition in unit dose form comprising: any one of the polynucleotides encoding the engineered guide RNA described herein, or any one of the AAV vector cassettes described herein; and a pharmaceutically acceptable: excipient, carrier, and / or diluent.

[0011] Also provided herein is a method of increasing ADAR-mediated editing efficiency of a parental engineered guide RNA, the method comprising: providing the parental engineered guide RNA or a polynucleotide encoding the parental engineered guide RNA, wherein: the parental engineered guide RNA comprises a targeting sequence having complementarity to a target RNA sequence that is sufficient for the parental engineered guide RNA to hybridize to the sequence of the target RNA, thereby forming a guide-target RNA scaffold; the guide-target RNA scaffold comprises a latent structure comprising one or more structural features that form upon formation of the guide-target RNA scaffold, wherein the one or more structural features comprise a first internal loop, a second internal loop, and a mismatch; and the guide-target RNA scaffold binds AD ARI or ADAR2, resulting in editing of an adenosine in the sequence of the target RNA by the ADAR1 or ADAR2; and generating a progeny engineered guide RNA by substituting in the parental engineered guide RNA: (i) a first modification comprising substitution of three or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms the first internal loop, the second internal loop, or both in the guide-target RNA scaffold, wherein the first modification maintains the first internal loop and the second internal loop in the guide-target RNA scaffold; and / or (ii) a second modification comprising substitution of three or more nucleotides in a hybridization domain sequence of the parental engineered guide RNA that canonically base pair with corresponding nucleotides in the sequence of the target RNA with three or more nucleotides that wobble base pair with corresponding nucleotides in the sequence of the target RNA- sequence; wherein the progeny engineered guide RNA facilitates increased editing efficiency of the adenosine in the sequence of the target RNA by the AD ARI or the ADAR2, relative to editing efficiency facilitated by the parental engineered guide RNA. In some embodiments, the first internal loop is a 5’ symmetric internal loop that is positioned upstream of the adenosine in the target RNA sequence and the second internal loop is a 3’ symmetric internal loop positioned downstream of the adenosine in the target RNA sequence. In some embodiments, the hybridization domain sequence is upstream of the 5’ symmetric internal loop in the target RNA sequence. In someembodiments, the first modification comprises substituting three or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that produces the 5’ symmetric internal loop, and in a portion of the targeting sequence of the parental engineered guide RNA that produces the 3' symmetric internal loop. In some embodiments, the second modification comprises substituting from 3 to about 21 nucleotides, resulting in from 3 to about 21 wobble base pairs in the progeny guide-target RNA scaffold formed by the progeny engineered guide RNA. In some embodiments, the first modification comprises substituting from 3 to about 6 nucleotides in the first internal loop, the second internal loop, or both.

[0012] Also provided herein is method of increasing ADAR-mediated editing efficiency of a parental engineered guide RNA, the method comprising: providing the parental engineered guide RNA or a polynucleotide encoding the parental engineered guide RNA, wherein: the parental engineered guide RNA comprises a targeting sequence having complementarity to a target RNA sequence that is sufficient for the parental engineered guide RNA to hybridize to the sequence of the target RNA, thereby forming a guide-target RNA scaffold; the guide-target RNA scaffold comprises a latent structure comprising one or more structural features that form upon formation of the guide-target RNA scaffold, wherein the one or more structural features comprise a first internal loop, a second internal loop, and a mismatch; and the guide-target RNA scaffold binds AD ARI or ADAR2, resulting in editing of an adenosine in the sequence of the target RNA by the AD ARI or ADAR2; and generating a progeny engineered guide RNA by substituting in the parental engineered guide RNA: (i) a first modification comprising substitution of one or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms a bulge in the guide-target RNA scaffold, wherein the first modification maintains the first internal loop and the second internal loop in the guide-target RNA scaffold; and / or (ii) a second modification comprising substitution of 1 or 2 nucleotides that are each individually located 10 to 40 nucleotides from the adenosine in the target RNA sequence and that canonically base pair with corresponding nucleotides in the sequence of the target RNA with 1 or 2 nucleotides that wobble base pair with corresponding nucleotides in the sequence of the target RNA; wherein the progeny engineered guide RNA facilitates increased editing efficiency of the adenosine in the sequence of the target RNA by the AD ARI or the ADAR2. relative to editing efficiency facilitated by the parental engineered guide RNA. In some embodiments, the first internal loop is a 5’ symmetric internal loop that is positioned upstream of the adenosine in the target RNA sequence and the second internal loop is a 3’ symmetric internal loop positioned downstream of the adenosine in the target RNA. In someembodiments, the 1 or 2 substituted nucleotides are each individually located 15 to 30 nucleotides from the adenosine in the target RNA sequence. In some embodiments, the 1 or 2 substituted nucleotides are each individually located 20 to 25 nucleotides from the adenosine in the target RNA sequence. In some embodiments, the first modification comprises substituting from 1 to about 4 nucleotides in the bulge.

[0013] In some embodiments, the increased editing efficiency of the progeny engineered guide RNA comprises: (i) a reduced off-target editing of the target RNA sequence relative to off- target AD ARI -mediated or AD AR2 -mediated editing of the target RNA using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2- mediated editing of the target RNA using the parental engineered guide RNA when measured in an in vitro assay; (ii) an increased on-target AD ARI -mediated or ADAR2-mediated editing of the target RNA sequence using the progeny engineered guide RNA as compared to on-target AD ARI -mediated or AD AR2 -mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay; (lii) a higher AD ARI -mediated or ADAR2-mediated global editing specificity of the target RNA sequence using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2- mediated global editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay; (iv) a higher AD ARI -mediated or ADAR2- mediated local editing specificity of the target RNA sequence using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2-mediated local editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay; any combination of (i)-(iv). In some embodiments, the increased editing efficiency of the progeny engineered guide RNA comprises a reduction in off target AD ARI -mediated or ADAR2-mediated editing of the target RNA sequence of least about 5% using the progeny engineered guide RNA, as compared to an amount of off target AD ARI -mediated or AD AR2 -mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay. In some embodiments, the increased editing efficiency of the progeny engineered guide RNA comprises an increase in on target AD ARI -mediated or ADAR2-mediated editing of the target RNA sequence of least about 5% using the progeny engineered guide RNA, as compared to an amount of on target ADAR 1 -mediated or ADAR2-mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay. In some embodiments, the increased editing efficiency of the progeny engineered guide RNA comprises a higher AD ARI -mediated or ADAR2-mediated global editing speci ficity of the target RNA sequence of that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2- mediated global editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay. In some embodiments, the progeny engineered guide RNA facilitates a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by the parental engineered guide RNA when measured in an in vitro assay and / or wherein the progeny engineered guide RNA facilitates an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a parental engineered guide RNA when measured in an in vitro assay. In some embodiments, the increased editing efficiency of the progeny engineered guide RNA comprises a higher ADAR 1 -mediated or AD AR2 -mediated local editing specificity of the target RNA sequence of that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more. 80% or more, 85% or more using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2- mediated local editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay.INCORPORATION BY REFERENCE

[0014] 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

[0015] Novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which exemplary principles of the present disclosure are utilized, and the accompanying drawings of which:

[0016] FIG. 1 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).

[0017] FIG. 2 shows the 5’ target hybridization domain, the central hybridization domain, and the 3 ’ target hybridization domain in a macro-footprint of an engineered guide RNA bound to a target RNA. The 5’ target hybridization domain is the portion of the macro-footprint that is 5‘ of the 5’ symmetric internal loop (5’ in reference to the target adenosine of the target sequence and 3’ in reference to the guide sequence) and the 3’ target hybridization domain is the portion of the macro-footprint 3 ’ of the 3 ' symmetric internal loop (3 ’ in reference to the target adenosine of the target sequence and 5’ in reference to the guide sequence). The target sequence is shown as the strand on the top. the guide sequence is shown as the strand on the bottom.

[0018] FIG. 3 shows a sequence alignment of engineered guide RNAs (SEQ ID NOS: 1-16) that target the 3’ UTR region of SNCA that have 12 to 30 nucleotide substitutions.

[0019] FIGS. 4A-4B show the percent RNA editing in engineered guide RNAs (SEQ ID NOS: 1-16) targeting SNCA 3’UTR. The parental engineered guide RNA (P0), the progeny engineered guide RNAs (P12 to P30), and a structural “Diverse” gRNA, and a negative control “LCOR” gRNA are shown on the X-axis. The Y-axis shows percent RNA editing. FIG. 4A shows total RNA editing for the SNCA 3’ UTR target. FIG. 4B shows on target editing as compared to +3 off-target editing in guide RNAs.

[0020] FIG. 5 shows a sequence alignment of engineered guide RNAs (SEQ ID NOS: 17-26) that target the TIS region of SNCA that have 15 to 30 nucleotide substitutions.

[0021] FIGS. 6A-6B show the percent RNA editing in engineered guide RNAs (SEQ ID NOS: 17-26) targeting SNCA TIS. FIG. 6A shows total RNA editing for the SNCA TIS target. The parental engineered guide RNA (P0), the progeny engineered guide RNAs (P15 to P30), and control (structurally diverse) are show n on the X-axis. The Y-axis shows percent RNA editing.FIG. 6B shows on target editing (position 0) as compared to local off-target editing in different progeny engineered guide RNAs (Pl 5 to P30) as compared to the parent engineered guide RNA (P0).

[0022] FIG. 7 shows a sequence alignment of engineered guide RNAs (SEQ ID NOS: 27-37) that target the T1S region of SNCA that have 12 to 30 nucleotide substitutions.

[0023] FIGS. 8A-8B show the percent RNA editing in engineered guide RNAs (SEQ ID NOS: 27-37) targeting SNCA TIS. FIG. 8A shows total RNA editing for the SNCA TIS target. The parental engineered guide RNA (P0), the progeny engineered guide RNAs (Pl 2 to P30), and control (structurally diverse) are shown on the X-axis. The Y-axis shows percent RNA editing. FIG. 8B shows on target editing (position 0) as compared to local off-target editing in different progeny engineered guide RNAs (Pl 2 to P30) as compared to the parent engineered guide RNA (P0).

[0024] FIG. 9 shows a sequence alignment of engineered guide RNAs (SEQ ID NO: 38-52) that target the SERPINA1 3’ UTR that have 12 to 30 nucleotide substitutions.

[0025] FIGS. 10A-10C show the percent RNA editing in engineered guide RNAs (SEQ ID NOS: 38-52) targeting SERPINA1 3‘ UTR. FIG. 10A shows total RNA editing for the SERPINA1 3’ UTR. The parental engineered guide RNA (P0). the progeny engineered guide RNAs (P12 to P30). and control (structurally diverse, GFP plasmid, and no transfection) are shown on the X-axis. The Y-axis shows percent RNA editing. FIG. 10B shows on target editing (target position 0) as compared to local off-target editing in guide RNAs. FIG. 10C shows a shows a heat map of the editing across the SERPINA1 3’ UTR target RNA. The Y- axis shows the guide RNAs. The X-axis shows the target SERPINA1 3’ UTR sequence relative to the target A at position 0. A darker shade indicates a high editing fraction at aposition, while a lighter shade indicates a low editing fraction.

[0026] FIG. 11 shows a schematic of the effect of GU wobble base pairing on the structure of the double stranded RNA (dsRNA) helix. As shown. GU wobble base pairs have a different base pairing structure than the canonical GC base pair introducing a different angle in the dsRNA helix that is offset by 14.0° and a decrease in the radius of the dsRNA helix by -0.05 A. This may cause the dsRNA with GU wobble base pairs to over-twist as compared to the canonical dsRNA helix without GU wobble base pairs.

[0027] FIG. 12 shows percent RNA editing in engineered guide RNAs (SEQ ID NOS: 38-52) targeting the SERPINA1 3’ UTR is associated with free energy of the binding between target and guide RNAs. FIG. 12 shows total RNA editing for the SERPINA1 3’ UTR target RNA.The parental engineered guide RNA (PO), the progeny engineered guide RNAs (P12 to P30), and control (structurally diverse) are shown on the X-axis. The Y-axis shows percent RNA editing and free energy of ensemble in kcal / mol.

[0028] FIG. 13 shows a sequence alignment of engineered gRNAs (SEQ ID NO: 141 and SEQ ID NO: 143 - SEQ ID NO: 154) that target LCOR that have 12 to 30 nucleotide substitutions. The “P#” indicates the number of substitutions in the engineered guide RNA.

[0029] FIG. 14 shows the percent sequence divergence (“% distance) between the parental and sequence divergent guide RNAs of SEQ ID NO: 141 and SEQ ID NO: 143 - SEQ ID NO: 154, as well as the structural diverse control sequence of (SEQ ID NO: 155) on the bottom panel. As shown on the top panel of FIG. 14, all sequences have the same latent structure (except for the structural diverse design control).

[0030] FIG. 15A shows a bar graph of total LCOR editing (% RNA editing) for each of the engineered parental guide RNA (P0 - SEQ ID NO: 141), the progeny engineered guide RNAs (P12 to P30, SEQ ID NO: 143 - SEQ ID NO: 154, respectively), and controls (structural diverse control - SEQ ID NO: 155, GFP plasmid, and no transfection) guide RNA designs.

[0031] FIG. 15B shows on target editing (target position 0) as compared to local off-target editing in the LCOR target of the parental guide RNA (SEQ ID NO: 141), the P20 guide RNA (SEQ ID NO: 144), and the P21 guide RNA (SEQ ID NO: 145).

[0032] FIG. 16 shows the latent structures of the ABCA4 engineered guide RNAs and the ABCA4 target. Specifically the engineered guide RNA designs of the parental guide RNA sequence (SEQ ID NO: 183), the progeny guide RNA with a removal of the -4 / 4 bulge(SEQ ID NO: 184), the progeny guide RNA with removal of the -4 / 4 bulge and incorporation of 1 wobble base pair between the guide RNA and the target (SEQ ID NO: 185), and the progeny guide RNA with removal of the -4 / 4 bulge and incorporation of 2 wobble base pairs between the guide RNA and the target (SEQ ID NO: 186) are shown.

[0033] FIG. 17A shows a bar graph of on-target ABCA4 RNA editing (% RNA editing) for each of the engineered RNA designs of the parental guide RNA (P0 - SEQ ID NO: 183). the progeny engineered guide RNAs (SEQ ID NO: 184 - SEQ ID NO: 186), and controls (GFP plasmid, and no transfection).

[0034] FIG. 17B shows the local RNA editing specificity of the ABCA4 target of the parental guide RNA (P0 - SEQ ID NO: 183) and the progeny engineered guide RNAs (SEQ ID NO: 184 - SEQ ID NO: 186).

[0035] FIG. 18A shows a bar graph of on-target ABCA4 RNA editing (% RNA editing) for each of the engineered RNA designs of the parental guide RNA (P0 - SEQ ID NO: 183), the progeny engineered guide RNAs (SEQ ID NO: 187 and SEQ ID NO: 188), and controls (structurally diverse. GFP plasmid, and no transfection) for editing of both the pre-mRNA ABCA4 minigene and the mRNA minigene of ABCA4.

[0036] FIG. 18B shows the local RNA editing specificity of the ABCA4 target of the parental guide RNA (P0 - SEQ ID NO: 183) and the progeny engineered guide RNAs (SEQ ID NO: 187 and SEQ ID NO: 188).DETAILED DESCRIPTIONOverview

[0037] Disclosed herein are engineered guide RNAs that hybridize to (target) a sequence of a target RNA. Hybridization of an engineered guide RNA described herein to a target RNA results in formation of a double stranded guide-target RNA scaffold. The guide-target RNA scaffold formed by an engineered guide described herein contains one or more structural features that are formed upon hybridization of the engineered guide RNA to the target RNA. The one or more structural features are formed by one or more bases in the target RNA and / or the engineered guide RNA that are canonically unpaired in the guide-target RNA scaffold. The identity and position of one or more structural features is engineered through manipulation of the guide RNA sequence to result in one or more bases in the guide-target RNA scaffold that are canonically unpaired in the engineered guide RNA and / or the target RNA. Thus, while the one or more structural features are not present in the engineered guide RNA prior to formation of the guide-target RNA scaffold, the sequence necessary to produce the one or more structural features is encoded in the engineered guide RNA, and thus the resulting structural features are “latent” within the engineered guide RNA. In some cases, the guide-target RNA scaffold having one or more structural features is a substrate for an RNA editing enzy me, resulting in editing of one or more bases in the target RNA by the RNA editing enzyme.

[0038] Engineered guide RNAs described herein form one or more latent structural features in the double stranded RNA that is formed upon hybridization of the engineered guide RNA to a target RNA. Described herein are progeny engineered guide RNAs that have one or more substitutions as compared to an engineered parental guide RNA, but maintain the structure of the parental guide-target RNA scaffold. As disclosed herein engineered guide RNAs refer toboth parental engineered guide RNAs and progeny engineered guide RNAs. Progeny engineered guide RNAs disclosed herein can be modified from an engineered parental guide RNA to contain a plurality of substitutions at polynucleotide sequences that produce structural features (e.g., internal loops) in the guide-target RNA scaffold, and / or to contain a plurality’ of substitutions that result in wobble base pairs in the guide-target RNA scaffold. Without wishing to be bound by theory, the addition of wobble base pairs in the guide-target RNA scaffold can result in increased specificity of RNA editing by an RNA editing enzy me and / or increase RNA editing levels by the RNA editing enzy me, relative to a guide-target RNA scaffold lacking the wobble base pairs.RNA Editing

[0039] RNA editing refers to a process by which RNA is enzymatically modified post synthesis at specific nucleosides. RNA editing can comprise any one of an insertion, deletion, or substitution of anucleotide(s). Examples of RNA editing include chemical modifications, such as 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 or from adenosine to inosine, or A-to-I editing). RNA editing can be used to introduce mutations, correct missense mutations, or edit coding or non-coding regions of RNA to inhibit RNA translation and effect protein knockdown.

[0040] Described herein are engineered guide RNAs that facilitate RNA editing by an RNA editing entity (e.g. an adenosine Deaminase Acting on RNA (ADAR)) or biologically active fragments thereof. In some instances, ADARs can be enzymes that catalyze the chemical conversion of adenosines to inosines in RNA. Because the properties of inosine mimic those of guanosine (inosine will form two hydrogen bonds with cytosine, for example), inosine can be recognized as guanosine by the translational cellular machinery'. “Adenosine-to-inosine (A- to-I) RNA editing"’, therefore, effectively changes the primary sequence of RNA targets. In general, ADAR enzymes share a common domain architecture comprising a variable number of amino-terminal dsRNA binding domains (dsRBDs) and a single carboxy-terminal catalytic deaminase domain. Human ADARs possess two or three dsRBDs. Evidence suggests that ADARs can form homodimer as well as heterodimer with other ADARs when bound to doublestranded RNA. however it can be currently inconclusive if dimerization is needed for editing to occur. The engineered guide RNAs disclosed herein can facilitate RNA editing by any of or any combination of the three human ADAR genes that have been identified (ADARs 1-3). ADARs have a typical modular domain organization that includes at least two copies of adsRNA binding domain (dsRBD; AD ARI with three dsRBDs; ADAR2 and ADAR3 each with two dsRBDs) in their N-terminal region followed by a C-terminal deaminase domain. The engineered guide RNAs of the present disclosure facilitate RNA editing by endogenous ADAR enzymes. In some embodiments, exogenous ADAR can be delivered alongside the engineered guide RNAs disclosed herein.

[0041] The present disclosure, in some embodiments, provides engineered guide RNAs that facilitate edits at particular regions in atarget RNA (e.g. , mRNA or pre-mRNA). In some cases, a target RNA can comprise an ABCA4, APP, CFTR. DMPK, DUX4, GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22. SERPINA1, SNCA, or SOD1 target RNA. For example, the engineered guide RNAs of the present disclosure can target an adenosine in a 3’ UTR, a splice site, a translation initiation site (TIS) site, a coding region, or a noncoding region.

[0042] 3’ Untranslated Region (UTR). In some embodiments, the engineered guide RNAs of the present disclosure target a target sequence of a target SNCA RNA that comprises a 3' untranslated region (3' UTR), wherein the adenosine of the 3' UTR is edited. In some embodiments, an engineered guide RNA of the present disclosure can target the 3' UTR of the SNCA transcript. In some embodiments, an engineered guide disclosed herein is at least partially complementary to a target SNCA RNA. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA (e.g., SNCA). In some embodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence. In some embodiments, an engineered guide RNA hybridizes a target RNA and facilitates a protein knockdown. Protein knockdown can also be referred to as reduced expression of wild-type protein. In some instances, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence, resulting in a protein knockdown.

[0043] TIS. In some embodiments, the engineered guide RNAs of the present disclosure target a translation initiation site (TIS). In some embodiments, the engineered guide RNAs of the present disclosure target a target sequence of a target SNCA RNA that comprises a translation initiation site (TIS), wherein the adenosine of the TIS is edited. In some embodiments, an engineered guide RNA of the present disclosure can target the Codon 1 TIS of Exon 2 corresponding to the canonical TIS at nucleotide position 226 of SNCA transcript variant 1 (NCBI Reference Sequence: NM_000345.4). An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA (e.g., SNCA). In someembodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence. In some embodiments, an engineered guide RNA can facilitate a protein knockdown. In some embodiments, the engineered guide RNAs facilitate ADAR-mediated RNA editing of the TIS (AUG) to GUG. In some instances, this results in protein knockdown. Protein knockdown can also be referred to as reduced expression of wild-type protein. In some instances, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in a target RNA sequence and resulting in exon skipping.

[0044] Splice site. In some embodiments, the engineered guide RNAs of the present disclosure target an adenosine at a splice site, thereby facilitating ADAR-mediated RNA editing of an adenosine at the splice site. This can result in truncation of a protein encoded by the pre-mRNA molecule by facilitating exon skipping. In some instances, this results in protein knockdown.

[0045] 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.

[0046] 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.

[0047] As used herein, the term "‘about” a number can refer to that number plus or minus 10% of that number.

[0048] As used herein, the term “engineered guide RNA” can be used interchangeable with “guide RNA” and refers to a designed polynucleotide that is at least partially complementary to a target RNA. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA. Modification of the target RNA includes alteration of RNAsplicing, reduction or enhancement of protein translation, target RNA knockdown, target RNA degradation, and / or ADAR mediated RNA editing of the target RNA. In some cases, guide RNAs facilitate ADAR mediated RNA editing for the purpose of target mRNA knockdown, downstream protein translation reduction or inhibition, downstream protein translation enhancement, correction of mutations (including correction of any G to A mutation, such as missense or nonsense mutations), introduction of mutations (e.g., introduction of an A to I (read as a G by cellular machinery) substitution), or alter the function of any adenosine containing a regulatory motif (e.g., polyadenylation signal, miRNA binding site, etc.). In some cases, a guide RNA can effect a functional outcome (e.g, target RNA modulation, downstream protein translation) via a combination of mechanisms, for example, ADAR-mediated RNA editing and binding and / or degrading target RNA. In some cases, a guide RNA can facilitate introduction of mutations at sites targeted by enzymes in order to modify the affinity of such enzymes for targeting and cleaving such sites. The guide RNAs of this disclosure can contain one or more structural features. A structural feature can be formed from latent structure in latent (unbound) guide RNA upon hybridization of the engineered latent guide RNA to a target RNA. Latent structure refers to a structural feature that forms or substantially forms only upon hybridization of a guide RNA to a target RNA. For example, upon hybridization of the guide RNA to the target RNA, the latent structural feature is formed in the resulting double stranded RNA (also referred herein as guide-target RNA scaffold). In such cases, a structural feature can include, but is not limited to, a mismatch, a wobble base pair, a symmetric internal loop, an asymmetric internal loop, a symmetric bulge, or an asymmetric bulge. In other instances, a structural feature can be a pre-formed structure (e.g.. a GluR2 recruitment hairpin, or a hairpin from U7 snRNA).

[0049] As used herein, the term “targeting sequence” can be used interchangeable with “targeting domain” or “targeting region” and refers to a polynucleotide sequence within an engineered guide RNA sequence that is at least partially complementary to a target polynucleotide. The target polynucleotide (e.g. , a target RNA or a target DNA) may be a region of a polynucleotide of interest, such as a gene or a messenger RNA. As used herein, a “complementary” sequence refers to a sequence that is a reverse complement relative to a second sequence.

[0050] 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 contiguousnucleotides 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.

[0051] 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 ( / .<?., 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 recmit 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.

[0056] In some embodiments, the term “local editing specificity ” when used in reference to editing facilitated by an engineered guide RNA or a guide-target, refers to the precision and accuracy with which a target nucleotide is edited within a target sequence (e.g, a target adenosine within a target RNA sequence) by an ADAR. In some embodiments, local editing specificity can also be characterized by discrimination between the target nucleotide and the off-target nucleotides (e.g., non-target nucleotides) within the target sequence (e.g., non-target adenosines within a target RNA sequence). Local editing specificity is calculated by the following formula: (percent on target editing + 100) / (sum of off target editing percentage at selected off-targets sites + 100).

[0057] In some embodiments, the term ‘'global editing specificity”, when used in reference to editing facilitated by an engineered guide RNA or a guide-target RNA scaffold, refers to the precision and accuracy with which a target sequence (e.g., a target RNA sequence) within a nucleic acid entity (e.g., a genome or a transcriptome) is edited by an ADAR. In some embodiments, global editing specificity can also be characterized by discrimination between the target sequence (e.g., a target RNA sequence) and a non-target sequence (e.g., a non-target RNA sequence) within a nucleic acid entity' (e.g., a genome or a transcriptome). In some embodiments, global editing specificity can be calculated by the following formula: (percent total on target editing + 100) / (sum of off target editing or splicing percentage across all detected sites + 100).

[0058] 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.

[0059] 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.

[0060] The term percent “identity.” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0061] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50%to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows: [(Number of Identical Positions) / (Total Number of Positions in the Test Sequence)] x 100%

[0062] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al.. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed ‘'words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match / mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows: Percent Sequence Identity = ('‘Percent Identity” output value) x (“Query Coverage” output value)

[0063] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions) / (total number of nucleotides in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 1 : AAAAAGGGGG (SEQ ID NO: 113) (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (SEQ ID NO: 114) (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5) / (l 0)] * 100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 115) (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (SEQ ID NO: 116) (length = 10 nucleotides). The percent identity7between test sequence 3 and reference sequence 4 would be [(10) / (20)J * 100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (SEQ ID NO: 116) (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 115) (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10) / (l 0)] *100% = 100%. Test sequence 5 has 100% sequence identity7to reference sequence 6.

[0064] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions) / (total number of amino acids in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (SEQ ID NO: 117) (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (SEQ ID NO: 118) (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5) / (10)] xioo% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 119) (length = 20 ammo acids) to reference sequence 10: FFFFFYYYYY (SEQ ID NO: 1 17) (length = 10 amino acids). The percent identity7between test sequence 9 and reference sequence 10 would be [(10) / (20)] xl00% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11 : FFFFFYYYYY (SEQ ID NO: 117) (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 119) (length = 20 amino acids). The percent identity between test sequence 11 andreference sequence 12 would be [(1O) / (1O)] * 100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12.

[0065] 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. The formation and structure of a latent structural feature upon binding to the target RNA depends on the guide RNA sequence. For example, formation and structure of the latent structural feature may depend on a pattern of complementary and mismatched residues in the guide RNA sequence relative to the target RNA. The guide RNA sequence may be engineered to have a latent structural feature that forms upon binding to the target RNA.

[0066] As disclosed herein, a ‘'macro-footprint” sequence can be positioned such that it flanks a micro-footprint sequence. Further, while a macro-footprint sequence can flank a microfootprint sequence, additional latent structures can be incorporated that flank either end of the macro-footprint as well. In some embodiments, such additional latent structures are included as part of the macro-footprint. In some embodiments, such additional latent structures are separate, distinct, or both separate and distinct from the macro-footprint. In some embodiments, a macro-footprint sequence can comprise a barbell macro-footprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop.

[0067] 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.

[0068] '‘Messenger RNA” or “mRNA” are 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-m RNA” can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions.

[0069] 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.

[0070] As used herein, the term “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5’ to the 3’ end. The term “RNA” is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), IncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA) RNAi (inhibitory RNA). siRNA (small interfering RNA), shRNA (short hairpin RNA). tRNA (transfer RNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA). The term DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. A sequence of a polynucleotide may be provided interchangeably as an RNA sequence (containing U) or a DNA sequence (containing T). A sequence provided as an RNA sequence is intended to also cover the corresponding DNA sequence and the reverse complement RNA sequence or DNA sequence. A sequence provided as a DNA sequence is intended to also cover the corresponding RNA sequence and the reverse complement RNA sequence or DNA sequence.

[0071] 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, orsynthetic 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.

[0072] The term “structured motif’ refers to a combination of two or more structural features in a guide-target RNA scaffold.

[0073] The terms “subject,” “individual,” or “patient” can be used interchangeably herein. A “subject” refers to 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 not necessarily diagnosed or suspected of being at high risk for the disease.

[0074] The term “in vivo” refers to an event that takes place in a subject’s body.

[0075] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay may not be 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 “P? vitro” assay.

[0076] The term “zw 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.

[0077] 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 (or T as shown in a DNA sequence).

[0078] The term “substantially forms” as described herein, when referring to a particular secondary structure, refers to formation of at least 80% of the structure under physiological conditions (e.g. physiological pH, physiological temperature, physiological salt concentration, etc.).

[0079] As disclosed herein, a structured motif comprises two or more structural features in a guide-target RNA scaffold.

[0080] 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.Engineered Guide RNAs

[0081] Disclosed herein are engineered guide RNAs and engineered polynucleotides encoding the same for, selective editing of target RNAs via an RNA editing entity7or a biologically active fragment thereof. An engineered guide RNA of the present disclosure can comprise latent structures, such that when the engineered guide RNA is hybridized to the target RNA to form a guide-target RNA scaffold, at least a portion of the latent structure manifests as at least a portion of a structural feature as described herein. Also disclosed herein are engineered guide RNAs that are engineered to have a plurality7of substitutions (e g., wobble base substitutions) but maintain a latent structure of a guide-target RNA scaffold.

[0082] An engineered guide RNA as described herein comprises a targeting domain with complementarity to a ABCA4, APP, CFTR, DMPK, DUX4, GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22, SERPINA1, SNCA, or SOD1 target RNA described herein. As such, a guide RNA can be engineered to site-specifically / selectively target and hybridize to the ABCA4, APP, CFTR, DMPK, DUX4. GAPDH. GBA, GRN. HEXA, LIPA, LRRK2, MAPT, PINK1 , PMP22, SERPINA1, SNCA, or SOD1 target RNA, thus facilitating editing of specific nucleotide in the target RNA via an RNA editing entity7or a biologically active fragment thereof. The targeting domain can include a nucleotide that is positioned suchthat, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or biologically active 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. However, in some instances there can be some, and in some cases significant, off target editing in addition to the desired edit.

[0083] Hybridization of the target RNA and the targeting domain of the guide RNA produces specific secondary' structures in the guide-target RNA scaffold that manifest upon hybridization, which are referred to herein as "‘latent structures.” 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 guide RNA to facilitate a specific, or selective, 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 facilitate an increased amount of editing of a target adenosine, fewer off target edits, or both, as compared to a construct comprising the mismatch alone or a construct having perfect complementarity to a target RNA. Accordingly, rational design of latent structures in engineered guide RNAs of the present disclosure to produce specific structural features in a guide-target RNA scaffold can be a powerful tool to promote editing of the target RNA with high specificity, selectivity', and robust activity. FIG. 1 illustrates a target RNA scaffold with exemplary' structural features.

[0084] Provided herein are engineered guides and polynucleotides encoding the same; as well as compositions comprising said engineered guide RNAs or said polynucleotides. As used herein, the term “engineered” in reference to a guide RNA or polynucleotide encoding the same refers to a non-naturally occurring guide RNA or polynucleotide encoding the same. For example, the present disclosure provides for engineered polynucleotides encoding engineered guide RNAs. In some embodiments, the engineered guide comprises RNA. In some embodiments, the engineered guide comprises DNA. In some examples, the engineered guide comprises modified RNA bases or unmodified RNA bases. In some embodiments, the engineered guide comprises modified DNA bases or unmodified DNA bases. In some examples, the engineered guide comprises both DNA and RNA bases.

[0085] In some examples, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target a ABCA4, APP, CFTR, DMPK, DUX4,GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22, SERPINA1, SNCA, or SOD1 RNA molecule, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA molecule, 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 molecule by the RNA editing entity. In some cases, engineered guide RNAs can be developed by machine learning and / or a high throughput screen, for example in a cell line described herein or in an in vitro assay. In some cases, an engineered guide can be configured to target the TIS region of SNCA. the 3’ UTR region of SNCA, and / or the 3’ UTR region of SERPINA1.

[0086] A target RNA of an engineered guide RNA of the present disclosure can be a pre- mRNA or mRNA. In some embodiments, the target RNA is ABCA4, APP, CFTR, DMPK, DUX4, GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22, SERPINA1, SNCA. or SOD1 pre-mRNA.

[0087] In some embodiments, the engineered guide RNA of the present disclosure hybridizes to a sequence of the target RNA. In some embodiments, part of the engineered guide RNA (e.g., a targeting domain) hybridizes to the sequence of the target RNA. The part of the engineered guide RNA that hybridizes to the target RNA is of sufficient complementary to the sequence of the target RNA for hybridization to occur.A. Targeting Domain

[0088] Engineered guide RNAs disclosed herein can be engineered in any way suitable for RNA editing. In some examples, an engineered guide RNA generally comprises at least a targeting sequence that allows it to hybridize to a region of a target RNA molecule. A targeting sequence can also be referred to as a “targeting domain” or a “targeting region”.

[0089] The targeting sequence of an engineered guide RNA allows the engineered guide RNA to hybridize to a target polynucleotide (e.g. a target RNA) through base pairing, such as Watson Crick base pairing. A targeting sequence can be located at either the N-terminus or C- terminus of the engineered guide RNA, or both, or the targeting sequence can be within the engineered guide RNA. The targeting sequence can be of any length sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence is 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, 11 1, 1 12, 113, 114, 115, 116, 117, 118, 1 19, 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. or up to about 200 nucleotides in length. In an embodiment, an engineered polynucleotide comprises a targeting sequence that is about 25 to 200, 50 to 150, 75 to 100, 80 to 110, 90 to 120, 95 to 115. 60 to 200, 60 to 180, 60 to 160, 60 to 140, 70 to 200, 70 to 180, 70 to 160, 70 to 140, 80 to 200, 80 to 190. 80 to 170, 80 to 160, 80 to 150. 80 to 140, 80 to 130, 80 to 120, 90 to 200, 90 to 190, 90 to 180, 90 to 170, 90 to 160, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 100 to 200, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130,100 to 120, HO to 200, HO to 190, HO to 180, HO to 170, HO to 160, HO to 150, HO to 140,110 to 120. 120 to 200, 120 to 190, 120 to 180, 120 to 170, 120 to 160, 120 to 150, 120 to 140,130 to 200, 130 to 190, 130 to 180, 130 to 170, 130 to 160, 130 to 150, 140 to 200, 140 to 190,140 to 180, 140 to 170, 140 to 160, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 160 to 200,160 to 190 or 160 to 180 nucleotides in length.

[0090] A targeting sequence comprises at least partial sequence complementarity to a target polynucleotide. The targeting sequence may have a degree of sequence complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to the target polynucleotide. In some cases, the targeting sequence comprises less than 100% complementarity to the target polynucleotide sequence. For example, the targeting sequence may have a single base mismatch relative to the target polynucleotide when bound to the target polynucleotide. In other cases, the targeting sequence 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 relative to the target polynucleotide when bound to the target polynucleotide. In some aspects, nucleotide mismatches can be associated with structural features provided herein. In some aspects, 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 wildt pe polynucleotide of a subject target polynucleotide.

[0091] A targeting sequence comprises nucleotide residues having complementarity to a target polynucleotide. The targeting sequence may have a number of residues with complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. Thecomplementary residues may be contiguous or non-contiguous. In some cases, the targeting sequence comprises at least 50 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 250 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases, the 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 the target polynucleotide. In some cases, the targeting sequence comprises more than 50 nucleotides total and has at least 50 nucleotides having complementarity’ to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 250 nucleotides having complementarity' to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases, the at least 50 nucleotides having complementarity to the target polynucleotide 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 nucleotideshaving complementarity to the target polynucleotide 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 the target polynucleotide 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 the target polynucleotide 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 the target polynucleotide 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 the target polynucleotide, 4 nucleotides form a bulge, and 25 nucleotides are complementarity to the target polynucleotide. As another example, a targeting sequence comprises a total of 118 nucleotides wherein, sequentially, 25 nucleotides are complementarity to the target polynucleotide, 4 nucleotides form a bulge, 25 nucleotides are complementarity to the target polynucleotide, 14 nucleotides form a loop, and 50 nucleotides are complementary to the target polynucleotide.

[0092] In some embodiments, a guide RNA or a polynucleotide encoding a guide RNA disclosed herein can comprise a targeting sequence disclosed in Tables 2-5. In some embodiments, a composition can comprise an engineered guide RNA comprising any one of SEQ ID NO: 1 - SEQ ID NO: 52, SEQ ID NO: 141, SEQ ID NO: 143- SEQ ID NO: 155, SEQ ID NO: 183 - SEQ ID NO: 188. In some embodiments, a composition can comprise an engineered guide RNA with at least about: 80%. 81%. 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 52, SEQ ID NO: 141, SEQ ID NO: 143- SEQ ID NO: 155, SEQ ID NO: 183 - SEQ ID NO: 188. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising any one of SEQ ID NO: 53 - SEQ ID NO: 104, SEQ ID NO: 142, SEQ ID NO: 163- SEQ ID NO: 175. SEQ ID NO: 189- SEQ ID NO: 194. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of any one of SEQ ID NO: 53 - SEQ ID NO: 104. SEQ ID NO: 189 - SEQ ID NO: 194.B. Engineered Guide RNAs Having a Recruiting Domain

[0093] In some examples, a subject engineered guide RNA comprises a recruiting domain that recruits an RNA editing entity (e.g., ADAR), where in some instances, the recruiting domain is formed and present in the absence of binding to the target RNA. A “recruiting domain” can be referred to herein as a “recruiting sequence” or a “recruiting region”. In some examples, 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. Various RNA editing entity recruiting domains can be utilized. In some examples, a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), APOBEC, or Alu.

[0094] In some examples, more than one recruiting domain can be included in an engineered guide of the disclosure. In examples where a recruiting domain can be present, the recruiting domain 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 domain can allow for transient binding of the RNA editing entity to the engineered guide. In some examples, the recruiting domain allows for permanent binding of the RNA editing entity to the engineered guide. A recruiting domain can be of any length. In some cases, a recruiting domain 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 domain can be no more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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 domain can be about 45 nucleotides in length. In some cases, at least a portion of a recruiting domain comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting domain comprises about 45 nucleotides to about 60 nucleotides.

[0095] In an embodiments, a recruiting domain comprises a GluR2 sequence or functional fragment thereof. In some cases, a GluR2 sequence can be recognized by an RNA editingentity, 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.

[0096] In some examples, a recruiting domain comprises a GluR2 sequence, or a sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity and / or length to: GUGGAAUAGUAUAACAAUAUGCUAAAUGUUGUUAUAGUAUCCCAC (SEQ ID NO: 120). 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: 120. In some examples, a recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence homology and / or length to SEQ ID NO: 120.

[0097] Additional, RNA editing entity recruiting domains are also contemplated. In an embodiment, 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 another embodiment, a recruiting domain can be from an Alu domain.

[0098] Any number of recruiting domains can be found in an engineered guide of the present disclosure. In some examples, at least about 1. 2, 3, 4. 5, 6, 7, 8. 9, or up to about 10 recruiting domains can be included in an engineered guide. Recruiting domains can be located at any position of subject guides. In some cases, a recruiting domain can be on an N-terminus, middle, or C-terminus of a polynucleotide. A recruiting domain can be upstream or downstream of a targeting sequence. In some cases, a recruiting domain flanks a targeting sequence of a subject guide. A recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting domain comprising both ribo- and deoxyribonucleotides can in some cases not be excluded.C. Engineered Guide RNAs with Latent Structure

[0099] In some examples, an engineered guide disclosed herein useful for facilitating editing of a target RNA by an RNA editing entity can be an engineered latent guide RNA. An “engineered latent guide RNA"’ refers to an engineered guide RNA that comprises latentstructure. ‘'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.

[0100] 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.” Described herein are structural features that 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).

[0101] FIG. 1 shows a legend of various exemplar}' 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 3nucleotides 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.

[0102] In some examples, the engineered guides disclosed herein lack a recruiting region and recruitment of the RNA editing entity can be effectuated by structural features of the guidetarget RNA scaffold formed by hybridization of the engineered guide RNA and the target RNA. In some examples, the engineered guide, when present in an aqueous solution and not bound to the target RNA molecule, does not comprise structural features that recruit the RNA editing entity (e.g., ADAR). The engineered guide RNA, upon hybridization to a target RNA, form with the target RNA molecule, one or more structural features that recruits an RNA editing entity (e.g. ADAR).

[0103] In cases where a recruiting sequence can be absent, an engineered guide RNA can be still 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 formed in the guide-target RNA scaffold formed upon hybridization of the engineered guide RNA and the target RNA. Structural features can comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge,symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, or any combination thereof.

[0104] 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).

[0105] A double stranded RNA (dsRNA) substrate (i.e.. a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure 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 in a guide RNA is to a G, a C, or a U in the target RNA. For example, a G in the target RNA can mismatch with a G, an A or a U in the guide RNA. In another example, a C in the target RNA can mismatch with a C. an A. or a U in the guide RNA. In another example, a U in the target RNA can mismatch with a U, a G, or a C in the guide RNA. In some embodiments, a mismatch in a guide RNA is to an A in the target RNA. For example, an A in the target RNA can mismatch with an A, a G, or a C in the guide RNA. 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. AG / G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA. In some embodiments, a guide RNA of the present disclosure may not have an A / C mismatch and each A of the target RNA is base paired to a U in the engineered guide RNA.

[0106] In some embodiments, a mismatch positioned 5?of the edit site can facilitate baseflipping 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.

[0107] In another aspect, a structural feature comprises a wobble base. A wobble base pair refers to two bases that weakly base pair. For example, a wobble base pair of the present disclosure can refer to a G paired with a U. Thus, a wobble base pair can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0108] 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.

[0109] A recruitment hairpin, as disclosed herein, can recruit at least in part an RNA editing entity, such as ADAR. In some cases, a recruitment hairpin can be formed and present in the absence of binding to a target RNA. In some embodiments, a recruitment hairpin is a GluR2 domain or portion thereof. In some embodiments, a recruitment hairpin is an Alu domain orportion thereof. A recruitment hairpin, as defined herein, can include a naturally occurring ADAR substrate or truncations thereof. Thus, a recruitment hairpin such as GluR2 is a preformed structural feature that may be present in constructs comprising an engineered guide RNA, not a structural feature formed by latent structure provided in an engineered latent guide RNA.

[0110] 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 mM, 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.

[0111] 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. I l l, 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.

[0112] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. 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. The nucleotides in a bulge of the guide RNA can comprise any nucleotide, in any order so long as they are not complementary’ to their positional counterparts on the target RNA. 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 ofthe 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.

[0113] 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.

[0114] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. 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 guidetarget RNA scaffold target and 2 nucleotides on the target RNA side of the guide-target 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 guidetarget RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNAscaffold. Thus, a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0115] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure 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 guide-target 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 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 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 guidetarget 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 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 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 ofthe 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 guidetarget 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 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.

[0116] A double stranded RNA (dsRNA) substrate (i.e.. a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. 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. Thenucleotides in an internal loop of the guide RNA can comprise any nucleotide, in any order so long as they are not complementary to their positional counterparts on the target RNA. 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.

[0117] One side of the internal loop, either on the target RNA side or the engineered guide RNA 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.

[0118] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. 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 guidetarget 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 11 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 11 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 12 nucleotides on the engineered guide RNAside of the guide-target RNA scaffold target and 12 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 13 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 13 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 14 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 14 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 guide-target 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 guidetarget 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 guide-target 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 guide-target 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 guide-target 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 guide-target 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 theengineered guide RNA side of the guide-target RNA scaffold target and 1 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 120 nucleotides on the engineered guide RNA side of the guidetarget 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 disclosurecan 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 guidetarget 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.

[0119] In some embodiments, a symmetrical internal loop can be positioned upstream (5') of the target A (0 position), downstream (3’) of the target A, or both. In some embodiments, when referring to a location of a structural feature a or negative integer indicates a nucleotide upstream (5’) of the target A or of a specified position, while a positive integer indicates a nucleotide downstream (3 ') of the target A, or of a specified position. In some instances, a first symmetrical internal loop can be downstream of the target A and a second symmetrical internal loop can be upstream of the target A. In some cases, a symmetric internal loop can be from position: -1 to -25, -2 to -10, -4 to -8, -5 to -7, -2 to -15, -4 to -20, -8 to -15, or -10 to -22 relative to the target A. In some cases, a symmetric internal loop can be located at position: -25, -24, - 23, -22, -21, -20. -19, -18, -17. -16, -15, -14, -13, -12. -11, -10, -9, -8, -7. -6, -5, -4, -3, -2, or - 1 relative to the target A. In some cases, a symmetric internal loop can be from position: +1 to +60, +10 to +50, +10 to +40, +20 to +50, +20 to +40, +25 to +45, +31 to +35, +10 to +20, +15 to +30, +25 to +45, or +45 to +60 relative to the target A. In some cases, a symmetric internal loop can be located at position: 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, or +60 relative to the target A. In some cases, a first symmetric internal loop within about: 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of the 5?end of the guide RNA, and a second symmetric internal loop within about: 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of the 3’ end of the guide RNA.

[0120] A double stranded RNA (dsRNA) substrate (z.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. 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 guide-target 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.

[0121] 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 5nucleotides 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 guidetarget 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 guidetarget 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 guidetarget RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-targetRNA 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 guidetarget 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 asymmetncal 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 guidetarget 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 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 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides onthe 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 guidetarget 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 guidetarget 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 guidetarget 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 guidetarget 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 guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guidetarget 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 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 guidetarget 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 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 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 guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guidetarget 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 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 guidetarget 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 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 guidetarget 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 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 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 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 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 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-targetRNA 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 guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guidetarget 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 guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guidetarget 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 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 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 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 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 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 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 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 guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guidetarget 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 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 guidetarget 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 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 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 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 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 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 guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guidetarget 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 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 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 guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guidetarget 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 guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guidetarget 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 guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guidetarget 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 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 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 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 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of theguide-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 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 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 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.

[0122] 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 (e.g, the bases in the targeting sequence of the guide RNA) are paired with opposing bases in the target polynucleotide. 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 to 50, 1 to 75, 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 225, 1 to 250, 1 to 275, 1 to 300, 50 to 75, 50 to 100, 50 to 125, 50 to 150, 50 to 175, 50 to 200, 50 to 225, 50 to 250, 50 to 275, 50 to 300, 60 to 75, 60 to 100, 60 to 125, 60 to 150, 60 to 175, 60 to 200, 60 to 225, 60 to 250, 60 to 275, 60 to 300, 70 to 100, 70 to 125, 70 to 150, 70 to 175, 70 to 200, 70 to 225. 70 to 250, 70 to 275, 70 to 300, 80 to 100, 80 to 125, 80 to 150. 80 to 175, 80 to 200, 80 to 225. 80 to 250, 80 to 275. 80 to 300, 90 to 125. 90 to 150, 90 to 175, 90 to 200, 90 to 225, 90 to 250, 90 to 275, 90 to 300, 100 to 125, 100 to 150, 100 to 175, 100 to 200, 100 to 225, 100 to 250, 100 to 275, 100 to 300, 150 to 200, 150 to 225, 150 to 250. 150 to 275, or 150 to 300 base pairs. In some embodiments, a base paired region has at least 1. 2, 3, 4, 5, 6, 7, 8. 9, 10, 12, 14, 16, 18, 20. 25. 30. 35. 40. 45. 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, 1 16, 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 base pairs.D. Guides with Macro-Footprints

[0123] Guide RNAs of the present disclosure can further comprise a macro-footprint. In some embodiments, the macro-footprint comprises a barbell macro-footprint. A micro-footprint can serve to guide an RNA editing enzy me and direct its activity7towards the target adenosine to be edited. A “barbell” as described herein refers to a pair of internal loop latent structures that manifest upon hybridization of the guide RNA to the target RNA. In some embodiments, each internal loop is positioned towards the 5' end or the 3' end of the guide-target RNA scaffold formed upon hybridization of the guide RNA and the target RNA. In some embodiments, each internal loop flanks opposing sides of the micro-footprint sequence. Insertion of a barbell macro-footprint sequence flanking opposing sides of the micro-footprint sequence, upon hybridization of the guide RNA to the target RNA. results in formation of barbell internal loops on opposing sides of the micro-footprint. In some cases, barbell internal loops can comprise at least one structural feature that facilitates editing of a specific target RNA.

[0124] As described herein, a “micro-footprint” sequence refers to a sequence with latent structures that, when manifested, facilitate editing of the adenosine of a target RNA via an adenosine deaminase enzyme. A macro-footprint can serve to guide an or focus RNA editing entity7(e.g., ADAR) and direct its activity7towards a micro-footprint. In some embodiments, included within the micro-footprint sequence is a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, said nucleotide is opposite the adenosine to be edited by the ADAR enzyme and does not base pair with the adenosine to be edited. This nucleotide is referred to herein as the “mismatched position” or “mismatch” and can be a cytosine. Micro-footprint sequences as described herein have upon hybridization of the engineered guide RNA and target RNA, at least one structural feature selected from the groupconsisting of: a bulge, an internal loop, a mismatch, a hairpin, and any combination thereof. Engineered guide RNAs with superior micro-footprint sequences can be selected based on their ability to facilitate editing of a specific target RNA. Engineered guide RNAs selected for their ability to facilitate editing of a specific target are capable of adopting various micro-footprint latent structures, which can vary on a target-by -target basis.

[0125] In some embodiments, the presence of barbells flanking the micro-footprint can improve one or more aspects of editing. For example, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a higher amount of on target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Additionally, and or alternatively, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Further, while the effect of various microfootprint structural features can vary on a target-by -target basis based on selection in a high throughput screen, the increase in the one or more aspects of editing provided by the barbell macro-footprint structures can be independent of the particular target RNA. For example, macro-footprints (e.g, barbell macro-footprints) and micro-footprints can provide an increased amount of on target adenosine editing relative to an otherwise comparable guide RNA lacking the barbells. In other embodiments, the presence of the barbell macro-footprint in addition to the micro-footprint described here can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA, upon hybridization of the guide RNA and target RNA to form a guide-target RNA scaffold lacking the barbells.

[0126] A dumbbell design in an engineered guide RNA comprises two symmetrical internal loops, wherein the target A to be edited is positioned between the two symmetrical loops for selective editing of the target A. The two symmetrical internal loops are each formed by 6 nucleotides on the guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a dumbbell can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0127] In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell is positioned at least about 5 bases (e.g., 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, or 50 bases) away from the A / C mismatch with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A / C mismatch. In some embodiments, the first internal loop of the barbell or the second internalloop of the barbell is positioned at most about 50 bases away from the A / C mismatch (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A / C mismatch.

[0128] In some embodiments, a first internal loop or a second internal loop independently comprises a number of bases of at least about 5 bases or greater (e.g., 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150); about 150 bases or fewer (e.g., 145, 135, 125, 115, 95, 85, 75. 65. 55. 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9. 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g., 5-150, 6- 145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-1 15, 13-110, 14-105, 15-100, 16-95, 17-90, 18- 85, 19-80, 20-75, 21-70, 22-65, 23-60, 24-55, 25-50) of the engineered guide RNA and a number of bases of at least about 5 bases or greater (e.g., 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40. 50. 60. 70. 80. 90. 100, 110, 120. 130, 140, 150); about 150 bases or fewer (e.g, 145, 135, 125, 115, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g, 5-150, 6-145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-115, 13-110, 14-105, 15-100, 16-95, 17-90, 18-85, 19-80, 20-75, 21-70, 22-65, 23-60, 24-55, 25-50) of the target RNA.

[0129] In some embodiments, provided herein are engineered guide RNAs comprising a barbell macro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a micro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a macro-footprint and a micro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a micro-footprint in the absence of a macro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a macro-footprint in the absence of a micro-footprint.

[0130] In some embodiments, a macro-footprint sequence can comprise a barbell macrofootprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop.

[0131] In some examples, a first internal loop is positioned near the 5' end of the guide-target RNA scaffold and a second internal loop is positioned near the 3' end of the guide-target RNA scaffold. The length of the dsRNA comprises a 5' end and a 3' end, where up to half of the length of the guide-target RNA scaffold at the 5’ end can be considered to be “near the 5' end” while up to half of the length of the guide-target RNA scaffold at the 3' end can be considered “near the 3' end.” Non-limiting examples of the 5' end can include about 50% or less of thetotal length of the dsRNA at the 5' end, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. Non-limiting examples of the 3' end can include about 50% or less of the total length of the dsRNA at the 3' end about 45%, about 40%, about 35%. about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%.

[0132] In some embodiments, the engineered guide RNAs of the disclosure comprising a barbell macro-footprint sequence (that manifests as a first internal loop and a second internal loop) can improve RNA editing efficiency, increase the amount or percentage of RNA editing generally, as well as for on-target nucleotide editing, such as on-target adenosine. In some embodiments, the engineered guide RNAs of the disclosure comprising a first internal loop and a second internal loop can also facilitate a decrease in the amount of or reduce off-target nucleotide editing, such as off-target adenosine or unintended adenosine editing. The decrease or reduction in some examples can be of the number of off-target edits or the percentage of off- target edits.

[0133] Each of the first and second internal loops of the barbell macro-footprint can independently be symmetrical or asymmetrical, where symmetry is determined by the number of bases or nucleotides of the engineered guide RNA and the number of bases or nucleotides of the target RNA, that together form each of the first and second internal loops.E. Nucleotide Substitutions in Guide-Target RNA scaffolds for Improving RNA Editing

[0134] Described herein are progeny engineered guides that are designed to comprise a plurality of substitutions with respect to a parental engineered guide RNA while maintaining the structure of the guide-target RNA scaffold (including any structural features) formed by the parental engineered guide RNA. A engineered parental guide RNA comprises an original targeting sequence that hybridizes to a target RNA, which results in formation of a guide-target RNA scaffold. As described herein the original targeting sequence comprises a sequence that result in one or more canonically unpaired bases in the engineered guide RNA and / or target RNA when both are present in the guide-target RNA scaffold. These one or more canonically unpaired bases produce one or more structural features in the guide-target RNA scaffold. While the one or more structural features are not substantially present in the engineered guide RNA prior to formation of the guide-target RNA scaffold, these structures are encoded by the targeting sequence of the engineered guide RNA and are thus ‘‘latent” within the engineered guide RNA.

[0135] Progeny engineered guide RNAs as described herein are designed to maintain the latent stmcture(s) of a engineered parental guide RNA (and thus the resulting structural feature(s) in the guide-target scaffold), while having a plurality7of nucleic acid substitutions with respect to the parental engineered guide RNA. In some cases, the substitutions can occur at regions that do not base pair in the guide-target RNA scaffold, for example in a mismatched region, a bulge region, or a loop region. In some cases, the substitutions can occur in the symmetric internal loops (e.g., barbell internal loops) of a macro-footprint guide.

[0136] In some cases, progeny engineered guide RNAs as described herein have substitutions in regions that canonically base pair with a corresponding nucleotide of the target RNA in the guide-target RNA scaffold, resulting in additional structural features in the guide-target RNA scaffold. For example, the progeny engineered guide RNA can have one or more substitutions with respect to the parental engineered guide RNA that result in wobble base pairs in the guidetarget RNA scaffold that were not present in the guide-target RNA scaffold formed by the parental engineered guide RNA (referred to herein as “Wobble base substitutions7’). Wobble base substitutions allow for non-canonical base pairing between two nucleotides in polynucleotides that do not follow Watson-Crick base pair rules. For example, wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). In some cases, modifications to the parental engineered guide RNA that produce a plurality of wobble base substitutions can increase specificity of RNA editing and / or increase RNA editing levels of the target RNA by an RNA editing entity, relative to the specificity and level of editing by the RNA editing entity facilitated by the parental engineered guide RNA. In some cases, modifications to the parental engineered guide RNA that produce a plurality of wobble base substitutions can increase local specificity of RNA editing. In some cases, modifications to the parental engineered guide RNA that produce a plurality7of wobble base substitutions can increase global specificity7of RNA editing.

[0137] In some embodiments, progeny engineered guide RNAs herein can have 1 to 50 nucleotide substitutions. 1 to 40 nucleotide substitutions, 1 to 30 nucleotide substitutions, or 1 to 20 nucleotide substitutions as compared to an engineered parental guide RNA. In some cases, progeny engineered guide RNAs herein can have 10 to 30 nucleotide substitutions, 12 to 27 nucleotide substitutions, 15 to 25 nucleotide substitutions, or 18 to 24 nucleotide substitutions as compared to an engineered parental guide RNA. In some embodiments, progeny engineered guide RNAs herein can have at least 1 nucleotide substitution, 2 nucleotide substitutions, 3 nucleotide substitutions, 4 nucleotide substitutions, 5 nucleotide substitutions,6 nucleotide substitutions, 7 nucleotide substitutions, 8 nucleotide substitutions, 9 nucleotide substitutions, 10 nucleotide substitutions, 11 nucleotide substitutions, 12 nucleotide substitutions, 13 nucleotide substitutions, 14 nucleotide substitutions, 15 nucleotide substitutions, 16 nucleotide substitutions, 17 nucleotide substitutions, 18 nucleotide substitutions. 19 nucleotide substitutions, 20 nucleotide substitutions. 21 nucleotide substitutions, 22 nucleotide substitutions, 23 nucleotide substitutions, 24 nucleotide substitutions, 25 nucleotide substitutions, 26 nucleotide substitutions, 27 nucleotide substitutions, 28 nucleotide substitutions, 29 nucleotide substitutions, 30 nucleotide substitutions. 31 nucleotide substitutions, 32 nucleotide substitutions. 33 nucleotide substitutions, 34 nucleotide substitutions, 35 nucleotide substitutions, 36 nucleotide substitutions, 37 nucleotide substitutions, 38 nucleotide substitutions, 39 nucleotide substitutions, or 40 nucleotide substitutions as compared to an engineered parental guide RNA.

[0138] In some embodiments, progeny engineered guide RNAs herein can have 1 to 30 nucleotide substitutions, 1 to 20 nucleotide substitutions, or 1 to 10 nucleotide substitutions in regions (e.g. , loops, bulges and / or mismatched regions) that do not base pair in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some cases, progeny engineered guide RNAs herein can have 5 to 15 nucleotide substitutions, 6 to 14 nucleotide substitutions. 7 to 13 nucleotide substitutions, or 9 to 12 nucleotide substitutions in regions that do not base pair in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some embodiments, progeny engineered guide RNAs herein can have at least 1 nucleotide substitution, 2 nucleotide substitutions, 3 nucleotide substitutions, 4 nucleotide substitutions. 5 nucleotide substitutions. 6 nucleotide substitutions, 7 nucleotide substitutions, 8 nucleotide substitutions, 9 nucleotide substitutions, 10 nucleotide substitutions, 11 nucleotide substitutions, 12 nucleotide substitutions, 13 nucleotide substitutions, 14 nucleotide substitutions, 15 nucleotide substitutions, 16 nucleotide substitutions, 17 nucleotide substitutions. 18 nucleotide substitutions, 19 nucleotide substitutions. 20 nucleotide substitutions. 21 nucleotide substitutions, 22 nucleotide substitutions. 23 nucleotide substitutions, 24 nucleotide substitutions, 25 nucleotide substitutions, 26 nucleotide substitutions, 27 nucleotide substitutions, 28 nucleotide substitutions, 29 nucleotide substitutions, or 30 nucleotide substitutions in regions that do not base pair in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some embodiments, progeny engineered guide RNAs herein can have at least 1 nucleotide substitution, 2 nucleotide substitutions, 3 nucleotide substitutions, 4 nucleotide substitutions, 5 nucleotide substitutions,6 nucleotide substitutions, 7 nucleotide substitutions, 8 nucleotide substitutions, 9 nucleotide substitutions, 10 nucleotide substitutions, 11 nucleotide substitutions, 12 nucleotide substitutions, or more than 12 nucleotide substitutions in a loop of a guide-target RNA scaffold as compared to an engineered parental guide RNA.

[0139] In some embodiments, progeny engineered guide RNAs herein can have 1 to 40 wobble base nucleotide substitutions, 1 to 30 wobble base nucleotide substitutions, 1 to 20 wobble base nucleotide substitutions, or 1 to 10 wobble base nucleotide substitutions in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some cases, progeny engineered guide RNAs herein can have 1 to 15 wobble base nucleotide substitutions, 2 to 10 wobble base nucleotide substitutions, 3 to about 21 wobble base substitutions, 3 to about 15 wobble base substitutions, 3 to about 20 wobble base substitutions, 3 to about 19 wobble base substitutions, 4 to 13 wobble base nucleotide substitutions, or 7 to 10 wobble base nucleotide substitutions in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some embodiments, progeny engineered guide RNAs herein can have at least 1 wobble base nucleotide substitution, 2 wobble base nucleotide substitutions, 3 wobble base nucleotide substitutions, 4 wobble base nucleotide substitutions, 5 wobble base nucleotide substitutions, 6 wobble base nucleotide substitutions, 7 wobble base nucleotide substitutions. 8 wobble base nucleotide substitutions. 9 wobble base nucleotide substitutions, 10 wobble base nucleotide substitutions, 11 wobble base nucleotide substitutions, 12 wobble base nucleotide substitutions, 13 wobble base nucleotide substitutions, 14 wobble base nucleotide substitutions, 15 wobble base nucleotide substitutions, 16 wobble base nucleotide substitutions. 17 wobble base nucleotide substitutions. 18 wobble base nucleotide substitutions, 19 wobble base nucleotide substitutions, 20 wobble base nucleotide substitutions, 21 wobble base nucleotide substitutions, 22 wobble base nucleotide substitutions, 23 wobble base nucleotide substitutions, 24 wobble base nucleotide substitutions, 25 wobble base nucleotide substitutions. 26 wobble base nucleotide substitutions, 27 wobble base nucleotide substitutions, 28 wobble base nucleotide substitutions, 29 wobble base nucleotide substitutions. 30 wobble base nucleotide substitutions, 31 wobble base nucleotide substitutions, 32 wobble base nucleotide substitutions, 33 wobble base nucleotide substitutions, 34 wobble base nucleotide substitutions. 35 wobble base nucleotide substitutions, 36 wobble base nucleotide substitutions, 37 wobble base nucleotide substitutions, 38 wobble base nucleotide substitutions. 39 wobble base nucleotide substitutions, or 40 wobble base nucleotide substitutions in the guide-target RNA scaffold as compared to an engineered parental guide RNA. In some embodiments, theintroduction of GU wobble base pairs to a guide-target RNA scaffold may over twist the double stranded RNA (dsRNA) helix as compared to the dsRNA helix in a guide-target RNA scaffold without GU wobble base pairs.

[0140] In some embodiments, progeny engineered guide RNAs described herein: facilitate a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by a comparable reference guide RNA when measured in an in vitro assay; facilitate an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by a comparable reference guide RNA when measured in an in vitro assay; facilitate a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assay; facilitate local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assay; when hybridized to a target RNA sequence and forms a guide-target RNA scaffold, the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to a comparable reference guide RNA when measured in an in vitro assay; or any combination thereof.

[0141] Accordingly, in some embodiments, engineered guide RNAs provided herein facilitate a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the reference guide-target RNA. In some cases, engineered guide RNAs facilitate at least about 5% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 10% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 20% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 30% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 40% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 50% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 60% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 70% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 80% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 90% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAsfacilitate about 100% reduced off-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%. at least about 60%, at least about 65%. at least about 70%, at least about 75%. at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% reduced off-target editing of the target RNA sequence.

[0142] In some embodiments, progeny engineered guide RNAs herein can have reduced off target editing as compared to an engineered parental guide RNA. In some cases, progeny engineered guide RNAs can have less than 5% editing of an off-target adenosine. In some cases, progeny engineered guide RNAs can have less than 10% editing of an off-target adenosine. In some cases, progeny engineered guide RNAs can have less than 15% editing of an off-target adenosine. In some cases, progeny engineered guide RNAs can have less than 20% editing of an off-target adenosine. In some cases, progeny engineered guide RNAs can have less than 25% editing of an off-target adenosine. In some cases, progeny engineered guide RNAs can have less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%. less than 5%. less than 4%. less than 3%, less than 2%, less than 1%, or 0% editing of an off-target adenosine.

[0143] In some embodiments, engineered guide RNAs provided herein facilitate an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by a comparable reference guide RNA when measured in an in vitro assay. In some cases, engineered guide RNAs facilitate at least about 5% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 10% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 20% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 30% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 40% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 50% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 60% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 70% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitateat least about 80% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 90% increased on-target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate about 100% increased on- target editing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%. at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% increased on-target editing of the target RNA sequence.

[0144] In some cases, progeny engineered guide RNAs can have at least 5% increased RNA editing of the target adenosine as compared to the parental guide RNA. In some cases, progeny engineered guide RNAs can have at least 10% increased RNA editing of the target adenosine as compared to the parental guide RNA. In some cases, progeny engineered guide RNAs can have at least 20% increased RNA editing of the target adenosine as compared to the parental guide RNA. In some cases, progeny engineered guide RNAs can have at least 30% increased RNA editing of the target adenosine as compared to the parental guide RNA. In some cases, progeny engineered guide RNAs can have at least 30%, at least 25%, at least 20%, at least 15%. at least 14%. at least 13%. at least 12%, at least 11%, at least 10%, at least 9%, at least 8%, at least 7%, at least 6%, at least 5%, at least 4%, at least 3%, at least 2%, or at least 1% increased RNA editing of the target adenosine as compared to the parental guide RNA.

[0145] In some embodiments, engineered guide RNAs provided herein facilitate a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assay. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 1% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 5% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 10% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 15% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 20% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 25% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 30% or more. In some cases, engineered guide RNAs facilitate a global editingspecificity of the target RNA sequence that is 35% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 40% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 45% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 50% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 55% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 60% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 65% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 70% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 75% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 80% or more. In some cases, engineered guide RNAs facilitate a global editing specificity of the target RNA sequence that is 85% or more. In some embodiments, engineered guide RNAs provided herein facilitate a global editing specificity of the target RNA sequence that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more higher than the global editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assay.

[0146] In some embodiments, engineered guide RNAs provided herein that increase global editing specificity also facilitate a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by a comparable reference guide RNA when measured in an in vitro assay, an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a comparable reference guide RNA when measured in an in vitro assay, or both.

[0147] In some embodiments, engineered guide RNAs provided herein facilitate a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by a comparable reference guide RNA when measured in an in vitro assay. In some cases, engineered guide RNAs facilitate at least about 5% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 10% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 20% reduced off-targetalternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 30% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 40% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 50% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 60% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 70% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 80% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 90% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate about 100% reduced off-target alternative splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% reduced off-target alternative splicing of the target RNA sequence.

[0148] In some embodiments, engineered guide RNAs provided herein facilitate an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a comparable reference guide RNA when measured in an in vitro assay. In some cases, engineered guide RNAs facilitate at least about 5% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 10% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 20% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 30% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 40% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 50% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 60% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 70% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 80% increased on-target splicing of the target RNA sequence. In some cases,engineered guide RNAs facilitate at least about 90% increased on-target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate about 100% increased on- target splicing of the target RNA sequence. In some cases, engineered guide RNAs facilitate at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% increased on-target splicing of the target RNA sequence.

[0149] In some embodiments, engineered guide RNAs provided herein facilitate local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assay. In some cases, engineered guide RNAs facilitate a local editing specificity' of the target adenosine in the target RNA sequence that is 1% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 5% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 10% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 15% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 20% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 25% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 30% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 35% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 40% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 45% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 50% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 55% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 60% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNAsequence that is 65% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 70% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 75% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 80% or more. In some cases, engineered guide RNAs facilitate a local editing specificity of the target adenosine in the target RNA sequence that is 85% or more. In some embodiments, engineered guide RNAs provided herein facilitate a local editing speci ficity of the target adenosine in the target RNA sequence that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more higher than the local editing specificity facilitated by a comparable reference guide RNA when measured in an in vitro assav.

[0150] In some embodiments, the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to a comparable reference guide RNA when measured in an in vitro assay. In some cases, the free energy of the guide-target RNA scaffold is decreased 20% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 19% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 18% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 17% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 16% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 15% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 14% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 13% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 12% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 11% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 10% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 9% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 8% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 7% or less. In some cases, the free energy of the guidetarget RNA scaffold is decreased 6% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 5% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 4% or less. In some cases, the free energy of the guide-target RNAscaffold is decreased 3% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 2% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 1% or less. In some cases, the free energy of the guide-target RNA scaffold is decreased 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less. 12% or less. 11% or less. 10% or less, 9% or less, 8% or less. 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less relative to the free energy' of the reference guide-target RNA scaffold.

[0151] In some embodiments, a comparable reference guide RNA is a guide RNA that do not comprise the nucleotide(s) that wobble base pair with the corresponding nucleotide of a target RNA sequence. In some embodiments, a comparable reference guide RNA is a guide RNA in which the nucleotide(s) that wobble base pair with the corresponding nucleotide of a target RNA sequence are each individually replaced with a nucleotide that canonically or Watson- Crick base pairs to the corresponding nucleotide in the target RNA sequence. In some embodiments, a comparable reference guide RNA is a parental engineered guide RNA of the engineered guide RNA (e.g., a progeny guide RNA). In some embodiments, activity7of a comparable reference guide RNA can be measured in an appropriate in vitro assay, for example, having the same or similar conditions and parameters as described in the Examples herein.

[0152] As shown in FIG. 2, the 5’ hybridization domain is the portion of the macro-footprint that is 5’ (in reference to the target sequence) of the 5’ symmetric internal loop in a guide-target RNA scaffold and the 3‘ hybridization domain is the portion of the macro-footprint that is 3’ (in reference to the target sequence) of the 3?symmetric internal loop in the guide-target RNA scaffold. The central hybridization domain is the portion of the macro-footprint that is in between the 5’ and 3’ symmetric internal loops in the guide-target RNA scaffold. As referred to herein, the 5’ hybridization domain sequence, the 3‘ hybridization domain sequence, and the central hybridization domain sequence of the engineered guide RNA is the portion of the engineered guide RNA that forms the 5’ hybridization domain, the 3’ hybridization domain, and the central hybridization domain respectively, of the macro-footprint of the guide-target RNA scaffold.

[0153] In some cases, a progeny engineered guide RNA can have different polynucleotide sequences for the 5’ hybridization domain sequence, the central hybridization domain sequence, and / or the 3:hybridization domain sequence as compared to an engineered parental guide RNA and maintain the latent structure of a guide-target scaffold of an engineered parentalguide RNA. In some cases, non-canonical Watson-Crick interactions (e.g., wobble base pairs) can be incorporated into the 5’, central, and / or 3’ hybridization domain of the guide-target RNA scaffold with substantially no effect to the macro-footprint or guide-target RNA scaffold of an engineered parental guide RNA. In some cases, one or more nucleotide substitutions can be incorporated in a portion of the targeting sequence of the parental engineered guide RNA that from the loop(s), bulge(s), or other structural features in the guide-target RNA scaffold with substantially no effect to the macro-footprint or guide-target RNA scaffold of an engineered parental guide RNA. In some embodiments, such modifications can be made in addition to modifications made in the 5’ hybridization domain sequence, the central hybridization domain sequence, the 3’ hybridization domain sequence, or all hybridization domain sequences described herein. For example, in some embodiments, an engineered guide RNA comprises a first modification comprising substitution of one or more, two or more, three or more nucleotides, four or more, five or more, or six or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms a first internal loop, a second internal loop, or both in the guide-target RNA scaffold, wherein the first modification maintains the first internal loop and / or the second internal loop in the guide-target RNA scaffold. For another example, in some embodiments, an engineered guide RNA comprises a first modification comprising substitution of one or more, two or more, three or more nucleotides, or four or more, nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms a bulge in the guide-target RNA scaffold, wherein the first modification maintains a first internal loop and / or a second internal loop in the guide-target RNA scaffold.

[0154] In some cases, the 5’ hybridization domain sequence, the central hybridization domain sequence, the 3’ hybridization domain sequence, or all hybridization domain sequences of a progeny engineered guide RNA can comprise 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 or more than 25 wobble base substitutions as compared to a corresponding 5‘ hybridization domain sequence, central hybridization domain sequence, 3' hybridization domain sequence, or any combination of hybridization domain sequences of an engineered parental guide RNA.

[0155] In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise 3 or more wobble base pairs in a guide-target scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise 4 or more wobble base pairs in a guide-target scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise 5 or more wobble base pairs in aguide-target scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise 6 or more wobble base pairs in a guide-target scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 1 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the 5‘ hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 10 wobble base pairs in a guide- target scaffold. In some cases, the 5‘ hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 5 wobble base pairs in a guidetarget scaffold. In some cases, the 5’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 5 to about 8 wobble base pairs in a guide-target scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise 3 or more wobble base pairs in a guide-target scaffold. In some cases, the 3' hybridization domain sequence of a progeny engineered guide RNA can comprise 4 or more wobble base pairs in a guide-target scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise 5 or more wobble base pairs in a guide-target scaffold. In some cases, the 3‘ hybridization domain sequence of a progeny engineered guide RNA can comprise 6 or more wobble base pairs in a guide-target scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 1 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the 3' hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 10 wobble base pairs in a guide-target scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 5 wobble base pairs in a guidetarget scaffold. In some cases, the 3’ hybridization domain sequence of a progeny engineered guide RNA can comprise about 5 to about 8 wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise 3 or more wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise 4 or more wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise 5 or more wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progenyengineered guide RNA can comprise 6 or more wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise about 1 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 15 wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 10 wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise about 3 to about 5 wobble base pairs in a guide-target scaffold. In some cases, the central hybridization domain sequence of a progeny engineered guide RNA can comprise about 5 to about 8 wobble base pairs in a guide-target scaffold.

[0156] In some embodiments, one or more wobble base pairs are each individually located 10 to 40 nucleotides from the target adenosine in the target RNA sequence. In some embodiments, one or more wobble base pairs are each individually located 15 to 30 nucleotides from the target adenosine in the target RNA sequence. In some embodiments, one or more wobble base pairs are each individually located 20 to 25 nucleotides from the target adenosine in the target RNA sequence. In some embodiments, one or more wobble base pairs can be located 10 to 40 nucleotides 3’ downstream from the target adenosine in the target RNA sequence.

[0157] In some cases, the 5’ hybridization domain sequence, the central hybridization domain sequence, the 3’ hybridization domain sequence, or any combination of hybridization domain sequences of a progeny engineered guide sequence can have a sequence identity of more than, or equal to about: 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% or 99% with respect to a corresponding 5‘ hybridization domain sequence, a central hybridization domain sequence, a 3’ hybridization domain sequence, or any combination of hybridization domain sequences of an engineered parental guide RNA. In some cases, the 5’ hybridization domain sequence, the central hybridization domain sequence, the 3’ hybridization domain sequence, or any combination of hybridization domain sequences of a progeny engineered guide RNA sequence can have a sequence identity of about 60% to about 100%, 70% to about 100%, about 60% to about 70%, about 70% to about 80%. about 75% to about 85%. 80% to about 90%, 85% to about 95%, or about 90% to about 98%, with respect to a corresponding 5’ hybridization domain sequence, a central hybridization domain sequence, a 3’ hybridization domainsequence, or any combination of hybridization domain sequences of an engineered parental guide RNA.

[0158] An engineered polynucleotide as described herein can comprise one or more polynucleotide sequence(s) that encode one or more engineered guide RNA(s). For example, an engineered polynucleotide can comprise 1. 2, 3, 4. or more than 4 polynucleotide sequence(s) that encode 1, 2, 3, 4, or more than 4 engineered guide RNAs.

[0159] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target): (1) different target sequences of the same target RNA, or (2) different target sequences of different target RNAs. For example, a first engineered guide RNA encoded by a first polynucleotide sequence can hybridize to a target sequence of a first target RNA while a second engineered guide RNA encoded by a second polynucleotide sequence can hybridize to a target sequence of a second target RNA, in some instances resulting in ADAR- mediated editing of an adenosine in the target sequence of the first target RNA and an adenosine in the target sequence of the second target RNA.

[0160] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target) the same target sequence of a target RNA. For example, the one or more engineered guide RNA(s) encoded by the one or more polynucleotide sequence(s) can each independently hybridize to a target sequence of a target RNA and / or facilitate editing of the same adenosine in the target sequence of the target RNA via ADAR. In some cases, the one or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA have identical sequences (z.e., the one or more engineered guide RNAs are copies of each other).

[0161] Alternatively, two or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA can comprise different sequences. For example, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to: 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%, or 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some instances, a first engineered guide RNA encoded by anengineered polynucleotide can have at least about 70% to about 99% sequence identity, at least about 60% to about 99% sequence identity, at least about 80% to about 99% sequence identity, at least about 60% to about 70% sequence identity, at least about 70% to about 80% sequence identity, at least about 75% to about 85% sequence identity, at least about 85% to about 99% sequence identity, at least about 85% to about 90% sequence identity, at least about 88% to about 93% sequence identity, at least about 90% to about 95% sequence identity, at least about 92% to about 99% sequence identity’, or at least about 95% to about 99% sequence identity to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 60% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 61% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 62% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 63% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity’ of less than, greater than, or equal to about 64% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 65% to a second engineered guide RNA encoded by the engineered polynucleotide, where the secondengineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 66% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity7of less than, greater than, or equal to about 67% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 68% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 69% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity7of less than, greater than, or equal to about 70% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 71% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 72% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 73% to a second engineered guide RNA encoded by the engineeredpolynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greater than, or equal to about 74% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 75% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 76% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 77% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 78% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 79% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 80% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 81% to a second engineered guide RNA encodedby the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 82% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 83% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 84% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity7of less than, greater than, or equal to about 85% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 86% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 87%, to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity7of less than, greater than, or equal to about 88% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 89% to a secondengineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 90% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 91% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greater than, or equal to about 92% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greater than, or equal to about 93% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 94% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greater than, or equal to about 95% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greater than, or equal to about 96% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity' of less than, greaterthan, or equal to about 97% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 98% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some embodiments, polynucleotides encoding a first engineered guide RNA, a second engineered guide RNA, or both can be delivered via an AAV. In some instances, the AAV can be formulated in a composition, such as any of the pharmaceutical compositions disclosed herein.F. Additional Engineered Guide RNA Components

[0162] The present disclosure provides for engineered guide RNAs with additional structural features and components. For example, an engineered guide RNA described herein can be circular. In another example, an engineered guide RNA described herein can comprise a U7, an SmOPT sequence, or a combination of both.

[0163] In some cases, an engineered guide RNA can be circularized. In some cases, an engineered guide RNA provided herein can be circularized or in a circular configuration. In some aspects, an at least partially circular guide RNA lacks a 5’ hydroxyl or a 3’ hydroxyl.

[0164] In some examples, an engineered guide RNA can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some examples, 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.

[0165] 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 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. In some instances, a backbone of anengineered guide 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 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.

[0166] As described herein, an engineered guide can comprise a circular structure. An engineered polynucleotide can be circularized from a precursor engineered polynucleotide. Such a precursor engineered polynucleotide can be a precursor engineered linear polynucleotide. In some cases, a precursor engineered linear polynucleotide can be a precursor for a circular engineered guide RNA. For example, a precursor engineered linear polynucleotide can be a linear mRNA transcribed from a plasmid, which can be configured to circularize within a cell using the techniques described herein. A precursor engineered linear polynucleotide can be constructed with domains such as a ribozyme domain and a ligation domain that allow for circularization when inserted into a cell. A ribozyme domain can include a domain that is capable of cleaving the linear precursor RNA at specific sites (e.g., adjacent to the ligation domain). A precursor engineered linear polynucleotide can comprise, from 5’ to 3’: a 5’ ribozyme domain, a 5’ ligation domain, a circularized region, a 3’ ligation domain, and a 3’ ribozyme domain. In some cases, a circularized region can comprise a guide RNA described herein. In some cases, the precursor polynucleotide can be specifically processed at both sites by the 5’ and the 3’ ribozymes, respectively, to free exposed ends on the 5‘ and 3’ ligation domains. The free exposed ends can be ligation competent, such that the ends can be ligated to form a mature circularized structure. For instance, the free ends can include a 5’-OH and a 2’, 3 ’-cyclic phosphate that are ligated via RNA ligation in the cell. The linear polynucleotide with the ligation and ribozy me domains can be transfected into a cell where it can circularize via endogenous cellular enzymes. In some cases, a polynucleotide can encode an engineered guide RNA comprising the ribozyme and ligation domains described herein, which can circularize within a cell. For example, PCT / US2021 / 034301 provides a description of circular guide RNAs and their structures, sequences of circular guide RNAs, and methods of engineering circularized polynucleotide domains, and each of these descriptions in PCT / US2021 / 034301 is herein incorporated by reference.

[0167] An engineered polynucleotide as described herein (e.g., a circularized guide RNA) can include spacer domains. As described herein, a spacer domain can refer to a domain thatprovides space between other domains. A spacer domain can be used to between a region to be circularized and flanking ligation sequences to increase the overall size of the mature circularized guide RNA. Where the region to be circularized includes a targeting domain as described herein that is configured to associate to a target sequence, the addition of spacers can provide improvements (e.g. increased specificity, enhanced editing efficiency, etc.) for the engineered polynucleotide to the target polynucleotide, relative to a comparable engineered polynucleotide that lacks a spacer domain. In some instances, the spacer domain is configured to not hybridize with the target RNA. In some embodiments, a precursor engineered polynucleotide or a circular engineered guide, can comprise, in order of 5?to 3’: a first ribozyme domain; a first ligation domain; a first spacer domain; a targeting domain that can be at least partially complementary to a target RNA, a second spacer domain, a second ligation domain, and a second ribozy me domain. In some cases, the first spacer domain, the second spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA.

[0168] A circular or looped RNA can be formed by employing a self-cleaving entity, 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 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 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, a peptidyl transferase 23 S rRNA, a GIRI branching ribozyme, a leadzyme, a group II intron, a hairpin ribozyme, a V S ribozyme, a CPEB3 ribozy me, a CoTC ribozyme, or a group I intron. In some cases, the self-cleaving ribozyme can be a trans-acting ribozyme that joins one RNA end on which it is present to a separate RNA end. In some embodiments, an aptamer can be added to each end of the engineered guide RNA. A ligase can be contacted with the aptamers at each end of the engineered guide RNA to form a covalent linkage between the aptamers thereby forming a circular engineered guide RNA. In some cases, a self-cleaving element or an aptamer can be configured to facilitate self-circularization of an engineered polynucleotide or a propolynucleotide (e.g, from a precursor engineered polypeptide) after transcription in a cell. In some instances, circularization of a guide RNA can be shown by PCR. For example, primerscan by developed that bind to the end of a guide RNA and are directed outward such that a product is only formed when guides are circularized.

[0169] In some cases, circularization can occur by back-slicing and ligation of an exon. For example, an RNA can be engineered from 5’ to 3' to comprise a forward complementary sequence intron, an exon (which can comprise the guide sequence), followed by a reverse complementary sequence intron. Once transcribed, the complementary sequence introns can hybridize and form dsRNA. The internal exon containing the guide sequence can be removed by splicing and ligated by an endogenous ligase to form a circular guide. In one example, an engineered guide RNA can initiate circularization in a cell by autocatalytic reactions of encoded ribozymes. After cleavage by one or more ribozymes, the linear polynucleotide will undergo intracellular RNA ligation of the 5’ and the 3’ end of ligation sequences by an endogenous ligase to circularize the guide RNA.

[0170] A suitable self-cleaving molecule can include a ribozyme. For example, a ribozyme domain can create an autocatalytic RNA. A ribozyme can comprise an RNase P, an rRNA (such as a Peptidyl transferase 23S rRNA), Leadzyme, Group I intron ribozyme, Group II intron ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a hairpin ribozyme, a Hammerhead ribozyme, an HDV ribozyme, a Twister ribozyme, a Twister sister ribozyme, a VS ribozyme, a Pistol ribozyme, a Hatchet ribozyme, a viroid, or any combination thereof. A ribozyme can include a P3 twister U2A ribozyme. A ribozyme can comprise 5’ GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGC CT 3’ (SEQ ID NO: 121). A ribozyme can comprise 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3’ (SEQ ID NO: 122). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology7to 5’GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGC CT 3’ (SEQ ID NO: 121). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%. 95%, or 100% sequence homology to 5’GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3’ (SEQ ID NO: 122). A ribozyme can include a Pl Twister Ribozyme. A ribozy me can include 5 ’AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 123). A ribozyme can include 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC 3’ (SEQ ID NO: 124). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 123). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%. or 100% sequence homology to 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 3’ (SEQ ID NO: 124).

[0171] A ligation domain can facilitate a linkage, covalent or non-covalent, of a first nucleotide to a second nucleotide. In some embodiments, a ligation domain can recruit a ligating entity to facilitate a ligation reaction. In some cases, a ligation domain can recruit a recombining entity to facilitate a homologous recombination. In some instances, a first ligation domain can facilitate a linkage, covalent or non-covalent, to a second ligation domain. In some embodiments, a first ligation domain can facilitate the complementary pairing of a second ligation domain. In some cases, a ligation domain can comprise 5’ AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 125). In some embodiments, a ligation domain can comprise 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 126). In some cases, a ligation domain can comprise 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 127). In some cases, a ligation domain can comprise 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 128). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 125). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%. 85%, 90%, 95%, or 100% sequence homology to 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 126). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 127). In some cases, a ligation domain can comprise at least about: 70%. 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5' GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 128).

[0172] The compositions and methods of the present disclosure provide engineered polynucleotides encoding for guide RNAs that are operably linked to a portion of a small nuclear ribonucleic acid (snRNA) sequence. The engineered polynucleotide can include at least a portion of a small nuclear ribonucleic acid (snRNA) sequence. The U7 and U1 small nuclear RNAs, whose natural role is in spliceosomal processing of pre-mRNA, have for decades beenre-engineered to alter splicing at desired disease targets. Replacing the first 18 nt of the U7 snRNA (which naturally hybridizes to the spacer element of histone pre-mRNA) with a short targeting (or antisense) sequence of a disease gene, redirects the splicing machinery to alter splicing around that target site. Furthermore, converting the wild type U7 Sm-domain binding site to an optimized consensus Sm-binding sequence (SmOPT) can increase the expression level, activity, and subcellular localization of the artificial antisense-engineered U7 snRNA. Many subsequent groups have adapted this modified U7 SmOPT snRNA chassis with antisense sequences of other genes to recruit spliceosomal elements and modify RNA splicing for additional disease targets.

[0173] An snRNA is a class of small RNA molecules found within the nucleus of eukaryotic cells. They are involved in a variety of important processes such as RNA splicing (removal of introns from pre-mRNA), regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA). and maintaining the telomeres. They are always associated with specific proteins, and the resulting RNA-protein complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. There are many snRNAs, which are denominated Ul, U2, U3, U4, U5, U6, U7, U8, U9, and U10.

[0174] The snRNA of the U7 type is normally involved in the maturation of histone mRNA. This snRNA has been identified in a great number of eukaryotic species (56 so far) and the U7 snRNA of each of these species should be regarded as equally convenient for this disclosure.

[0175] Wild-type U7 snRNA includes a stem-loop structure, the U7-specific Sm sequence, and a sequence antisense to the 3' end of histone pre-mRNA.

[0176] In addition to the SmOPT domain, U7 comprises a sequence antisense to the 3' end of histone pre-mRNA. When this sequence is replaced by a targeting sequence that is antisense to another target pre-mRNA, U7 is redirected to the new target pre-mRNA. Accordingly, the stable expression of modified U7 snRNAs containing the SmOPT domain and a targeting antisense sequence has resulted in specific alteration of mRNA splicing.

[0177] The engineered polynucleotide can comprise at least in part an snRNA sequence. The snRNA sequence can be Ul, U2, U3, U4, U5, U6, U7, U8, U9, or a U10 snRNA sequence.

[0178] In some instances, an engineered polynucleotide that comprises at least a portion of an snRNA sequence (e.g. an snRNA promoter, an snRNA hairpin, and the like) can have superior properties for treating or preventing a disease or condition, relative to a comparable polynucleotide lacking such features. For example, as described herein an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate exonskipping of an exon at a greater efficiency than a comparable polynucleotide lacking such features. Further, as described herein an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate an editing of a base of a nucleotide in a target RNA (e.g. a pre-mRNA or a mature RNA) at a greater efficiency than a comparable polynucleotide lacking such features. Promoters and snRNA components are described in PCT / US2021 / 028618 and PCT / US2022 / 078801, and each of these descriptions in PCT / US2021 / 028618 and PCT / US2022 / 078801 are herein incorporated by reference.

[0179] Disclosed herein are engineered RNAs comprising (a) an engineered guide RNA as described herein, and (b) a U7 snRNA hairpin sequence, a SmOPT sequence, or a combination thereof. In some embodiments, the U7 hairpin comprises a human U7 Hairpin sequence, or a mouse U7 hairpin sequence. In some cases, a human U7 hairpin sequence comprises TAGGCTTTCTGGCTTTTTACCGGAAAGCCCCT (SEQ ID NO: 129 or RNA: UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU (SEQ ID NO: 130). In some cases, a mouse U7 hairpin sequence compnses CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 131 or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU SEQ ID NO: 132). In some embodiments, the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 133 or RNA: AAUUUUUGGAG SEQ ID NO: 134). In some embodiments, a guide RNA can comprise a guide RNA comprising a U7 hairpin sequence (e.g, a human or a mouse U7 hairpin sequence), an SmOPT sequence, or a combination thereof. In some cases, a combination of a U7 hairpin sequence and a SmOPT sequence can comprise a SmOPT U7 hairpin sequence, wherein the SmOPT sequence is linked to the U7 sequence. In some cases, a U7 hairpin sequence, an SmOPT sequence, or a combination thereof is downstream (e.g.. 3’) of the engineered guide RNA disclosed herein.

[0180] Also disclosed herein are promoters for driving the expression of a guide RNA disclosed herein. In some cases, the promoters for driving expression can be 5 ' to the guide RNA sequence disclosed herein. In some cases, a promoter can comprise a U1 promoter, a U7 promoter, a U6 promoter or any combination thereof. In some cases, a promoter can comprise a CMV promoter. In some cases, a U7 promoter, or a U6 promoter can be a mouse U7 promoter, or a mouse U6 promoter. In some cases, a U1 promoter, a U7 promoter, or a U6 promoter can be a human U1 promoter, a human U7 promoter, or a human U6 promoter. In some cases, a human U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:GAGGGC CTATTTCC C ATGATTC CTTC ATATTTGC AT ATAC GATAC AAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA CGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTT AAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTT ATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 135). In some cases, a mouse U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%. or 99% sequence identity to:GTACTGAGTCGCCCAGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGC CGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTA ATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACA GATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTA TAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTC ACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAA AGCCTTGTTTG (SEQ ID NO: 136). In some cases, a human U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TTAACAACAACGAAGGGGCTGTGACTGGCTGCTTTCTCAACCAATCAGCACCGA ACTCATTTGCATGGGCTGAGAACAAATGTTCGCGAACTCTAGAAATGAATGACTT AAGTAAGTTCCTTAGAATATTATTTTTCCTACTGAAAGTTACCACATGCGTCGTTG TTTATACAGTAATAGGAACAAGAAAAAAGTCACCTAAGCTCACCCTCATCAATT GTGGAGTTCCTTTATATCCCATCTTCTCTCCAAACACATACGCA (SEQ ID NO: 137). In some cases, a mouse U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity' to:TTAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACTGACTCATT TGCATAGCCTTTACAAGCGGTCACAAACTCAAGAAACGAGCGGTTTTAATAGTCT TTTAGAATATTGTTTATCGAACCGAATAAGGAACTGTGCTTTGTGATTCACATAT CAGTGGAGGGGTGTGGAAATGGCACCTTGATCTCACCCTCATCGAAAGTGGAGT TGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCCGC (SEQ ID NO: 138). In some cases, a human U1 promoter can comprise a sequence with at least about: 70%, 75%, 80%. 85%, 90%, 95%, or 99% sequence identity to:TAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGG GAGAGGCAGACGTCACTTCCTCTTGGCGACTCTGGCAGCAGATTGGTCGGTTGAG TGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGAC AGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCT GCTTCGCCACGAAGGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGC GGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCA GAGCCCGAAGATCTC (SEQ ID NO: 139). In some cases, a CMV promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: ATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAA CCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAC CGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACC (SEQ ID NO: 140).G. Chemically modified guide RNAs

[0181] An engineered guide RNA as described herein for use in treating a disease or condition in a subject can comprise at least one chemical modification. In some embodiments, the engineered guide RNA can comprise at least one, two, three, four, five, six, seven, eight, nine, ten, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 50, 100, or more chemical modifications. In some embodiments, the engineered guide RNA described herein may not comprise a chemical modification. In some cases, the engineered guide RNAs disclosed herein with barbell macrofootprints can be manufactured, chemically modified, and delivered directly to a subject in need thereof as RNA (without a vector, such as an AAV).

[0182] Exemplary chemical modifications comprise any one of: 5' adenylate, 5' guanosinetriphosphate 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.

[0183] A chemical modification can be made at any location of the engineered guide RNA. In some cases, a modification may be located in a 5’ or 3’ end, or both. In some cases, a polynucleotide can comprise 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, 1 15, 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. In some cases, more than one modification can be made to the engineered guide RNA. In some cases, a modification can be permanent. In other cases, a modification can be transient. In some cases, multiple modifications may be made to the engineered guide RNA. The engineered guide RNA modification can alter physio-chemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.

[0184] In some embodiments, a chemical modification can also be a phosphorothioate substitute. In some cases, a natural phosphodi ester 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 may 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 caninhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases.

[0185] In some embodiments, a chemical modification can occur at 3 ’OH, group, 5 ’OH group, at the backbone, at the sugar component, or at the nucleotide base. Chemical modification can include non-naturally occurring linker molecules of interstrand or intrastrand cross links. In one aspect, the chemically modified nucleic acid comprises modification of one or more of the 3 ’OH or 5 ’OH group, the backbone, the sugar component, or the nucleotide base, or addition of non-naturally occurring linker molecules. In some embodiments, a chemically modified backbone comprises a backbone other than a phosphodiester backbone. In some embodiments, a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In some embodiments, a modified base comprises a base other than adenine, guanine, cytosine, thymine or uracil. In some embodiments, the engineered guide RNA comprises at least one chemically modified base. In some instances, an engineered guide RNA can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more modified bases. In some cases, chemical modifications to the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, and purine or pyrimidine bases.

[0186] In some embodiments, a chemical modification of the engineered guide RNA can comprise 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. Chemical modifications to the engineered guide RNA include any modification contained herein, while some exemplary modifications are recited in Table 1.Table 1. Exemplary Chemical ModificationModification of phosphate backbone

[0187] In some embodiments, the chemical modification can comprise modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage or modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage. As used herein, “alkyl” may be meant to refer to a saturated hydrocarbon group which may be straight-chained or branched. Example alk l 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” may 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” may refer to an aliphatic group containing at least one double bond. As used herein, “alkynyl” may refer to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alky nyl groups can include ethynyl, propargyl, or 3-hexynyl. “Arylalkyl” or “aralkyl” may refer to an alkyl moiety in which an alkyl hydrogen atom may 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” may 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” may 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” may 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.

[0188] 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 may be a stereogenic center. The stereogemc phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some cases, the engineered guide RNA can comprise stereopure nucleotides comprising S conformation of phosphorothioate or R conformation of phosphorothioate. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 95%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 96%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 97%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 98%. In some embodiments, the chiral phosphate product may 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 nonbridging 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., alkyd or aryl). In some embodiments, the phosphate linker can also be modified by replacement of a bridging oxygen, (z.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). In some cases, the replacement can occur at either or both of the linking oxygens.

[0189] In certain 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 are 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-Si(H)2-O-); andN,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH?)). 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. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0190] In some cases, 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 may also be used.

[0191] 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.

[0192] In some instances, a phosphorous derivative (or modified phosphate group) may be attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like.

[0193] 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 may 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.

[0194] In some cases, substitutes for the phosphate include, for example, short chain alkyd or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalky 1 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; alkenecontaining 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 may be also 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 may be also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. In some cases, 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-oxycholesterol moiety.

[0195] In some embodiments, a chemical modification described herein can comprise modification of a phosphate backbone. In some embodiments, the engineered guide RNA described herein can comprise 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 RNA 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). 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 may 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 may be to say that a phosphorous atom in a phosphate group modified in this way may 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 RNA can be stereopure (e.g., S or R confirmation). In some cases, a chemically modified engineered guide RNA comprises stereopure phosphate modification. For example, the chemically modified engineered guide RNA can comprise S conformation of phosphorothioate or R conformation of phosphorothioate.

[0196] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates may be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both nonbridging 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, alky l or aryl).

[0197] In some cases, the phosphate linker can also be modified by replacement of a bridging oxygen, (z. 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.Replacement of phosphate moiety

[0198] In some embodiments, at least one phosphate group of the engineered guide RNA can be chemically modified. In some embodiments, the phosphate group can be replaced by nonphosphorus 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 alkyd phosphonates including 3 ’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g, 3’-amino phosphoramidate and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotri esters, and boranophosphates. In some cases, the phosphate or modified phosphate linkage between twonucleotides 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’.Substitution of phosphate group

[0199] In some embodiments, a chemical modification described herein can comprise modification by replacement of a phosphate group. In some embodiments, the engineered guide RNA described herein can comprise 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.Modification of the Ribophosphate Backbone

[0200] In some embodiments, the chemical modification described herein can comprise modifying ribophosphate backbone of the engineered guide RNA. In some embodiments, the engineered guide RNA described herein can comprise 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 may 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.Modification of sugar

[0201] In some embodiments, the chemical modification described herein can comprise modifying of sugar. In some embodiments, the engineered guide RNA described herein can comprise 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 intramolecularnucleophilic atack 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, ar l, 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 methoxyethyl 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. heteroarylamino, diheteroarylamino, or amino acid); NH(CEl2CH2NH)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 a nucleobase 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 may be in the L form, e.g.. L-nucleosides. In some aspects, the engineered guide RNA described herein includes the sugar group ribose, which may be a 5-membered ring having an oxygen. Exemplary modified nucleosides andmodified 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 may be replaced by glycol units attached to phosphodiester bonds), threose nucleic acid. In some embodiments, the modifications to the sugar of the engineered guide RNA comprises modifying the engineered guide RNA to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), or bridged nucleic acid (BNA).Modification of a constituent of the ribose sugar

[0202] In some embodiments, the engineered guide RNA described herein can comprise 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 -0- methoxy-ethyl (2’-M0E). 2’-fluoro, 2’ -aminoethyl, 2’-deoxy-2’-fuloarabinou-cleic acid. 2'- deoxy, 2'-O-methyl, 3 '-phosph orothioate, 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 ’-CEE-substituted 2’-O-protected nucleosides. In some cases, unnatural nucleic acids include amide linked nucleoside dimers that can be prepared for incorporation into oligonucleotides. In some cases, the 3‘ linked nucleoside in the dimer (5’ to 3’) comprises a 2’- OCHs and a 5’-(S)-CH?. Unnatural nucleic acids can include 2 ’-substituted 5’-CH2 (or O) modified nucleosides. Unnatural nucleic acids can include 5 ’-methylenephosph onate 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 may beunsubstituted or substituted with a thio-tert-butyl group (SCtCHs)?) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof).

[0203] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids can contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property'. In certain embodiments, nucleic acids can 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, Ci-C 12 alkyl or a protecting group); and combinations thereof.

[0204] In some instances, the engineered guide RNA described herein can comprise 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 may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-0-alkyl-substituted deoxyribonucleosides and 2’-O- ethy 1 enegly col -li ke ribonucl eosi des .

[0205] In some cases, 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-, or N-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]mCH3,-O(CH2)nOCHs,- O(CH2)nNH2,-O(CH2)nCH3,-O(CH2)nONH2. and-O(CH2)nON[(CH2)n CH3)]2, where n and m may 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, SCHs, OCN, Cl, Br, CN, CFs, OCFs, SOCHs, SO2 CHs, ONO2, NO2, Ns, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkyl amino, 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 may 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(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rmand Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.

[0206] In certain embodiments, nucleic acids described herein can 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 can 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 '-(042)2-0-2’ (ENA); 4 -CH(CH3)-O-2’ and 4’-CH(CH2OCH3)-O-2', and analogs thereof; 4’-C(CH3)(CH3)- 0-2 ’and analogs thereof.Modifications on the base of nucleotide

[0207] In some embodiments, the chemical modification described herein can comprise 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 RNA 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.

[0208] In some embodiments, the chemical modification described herein can comprise modifying an uracil. In some embodiments, the engineered guide RNA described herein cancomprise at least one chemically modified uracil. Exemplary7chemically 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-carboxymethyl-pseudouridine, 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-taurinomethyl-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- 1 -methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -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'-0-methyl-pseudouridine. 2-thio-2’-O- methyl-uridine, 5-methoxycarbonylmethyl-2’-O-methyl -uridine, 5-carbamoylmethyl-2’-O- methyl-uridine, 5-carboxymethylaminomethyl-2’-O-methyl-uridine, 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.

[0209] In some embodiments, the chemical modification described herein can comprise modifying a cytosine. In some embodiments, the engineered guide RNA described herein can comprise 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-1-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 -0- methyl-cytidine, 5,2’ -O-dimethyl-cyti dine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O- dimethyl-cytidine, 5-formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1-thio- cytidine, 2'-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0210] In some embodiments, the chemical modification described herein can comprise modifying an adenine. In some embodiments, the engineered guide RNA described herein can comprise 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-diamin opurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, 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 ’-deoxy adenosine, N6, N6, 2’-O-trimethyl-adenosine, 1 ,2’-O-dimethyl-adenosine, 2’-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-aden...

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A polynucleotide encoding an engineered guide RNA, wherein: hybridization of a target RNA sequence to the engineered guide RNA forms a guide-target RNA scaffold comprising a latent structure; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the engineered guide RNA comprises 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence; the engineered guide RNA facilitates editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and(i) the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by an otherwise comparable reference guide RNA in which the 3 to 30 noncontiguous nucleotides that wobble base pair with 3 to 30 noncontiguous nucleotides of a target RNA sequence are each individually replaced with a nucleotide that Watson-Crick base pairs to a corresponding nucleotide in the target RNA sequence when measured in an in vitro assay;(ii) the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(iii) the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(iv) the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(v) the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to the otherwise comparable reference guide RNA when measured in an in vitro assay; or(vi) any combination of (i)-(v).

2. A polynucleotide encoding an engineered guide RNA, wherein: hybridization of a target RNA sequence to the engineered guide RNA forms a guide-target RNA scaffold comprising a latent structure; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the engineered guide RNA comprises 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence; the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides from the target adenosine in the target RNA sequence; the engineered guide RNA facilitates editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and(i) the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by an otherwise comparable reference guide RNA in which the 1 or 2 noncontiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of a target RNA sequence are each individually replaced with a nucleotide that Watson-Crick base pairs to a corresponding nucleotide in the target RNA sequence when measured in an in vitro assay;(ii) the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(iii) the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(iv) the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the otherwise comparable reference guide RNA when measured in an in vitro assay;(v) the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNA scaffold formed upon hybridization of a target RNA sequence to the otherwise comparable reference guide RNA when measured in an in vitro assay; or(vi) any combination of (i)-(v).

3. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the reference guide-target RNA.

4. The polynucleotide of claim 3, wherein the engineered guide RNA facilitates at least about 5% reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by a reference guide RNA when measured in an in vitro assay.

5. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by a reference guide RNA when measured in an in vitro assay.

6. The polynucleotide of claim 5, wherein the engineered guide RNA facilitates at least about 5% increased on-target RNA editing of the target RNA sequence relative to on- target editing facilitated by a reference guide RNA when measured in an in vitro assay.

7. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by a reference guide RNA when measured in an in vitro assay.

8. The polynucleotide of claim 7, wherein the engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is 1% or more. 5% or more, 10% or more. 15% or more, 20% or more. 25% or more, 30% or more. 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more higher than the global editing specificity facilitated by a reference guide RNA when measured in an in vitro assay.

9. The polynucleotide of claim 7 or 8. wherein the engineered guide RNA facilitates a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by a reference guide RNA when measured in an in vitro assay.

10. The polynucleotide of claim 9, wherein the engineered guide RNA facilitates at least about 5% reduced off-target splicing of the target RNA sequence relative to off-target splicing facilitated by a reference guide RNA when measured in an in vitro assay.

11. The polynucleotide of claim 7 or 8. wherein the engineered guide RNA facilitates an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a reference guide RNA when measured in an in vitro assay.

12. The polynucleotide of claim 11, wherein the engineered guide RNA facilitates at least about 5% increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a reference guide RNA when measured in an in vitro assay.

13. The polynucleotide of claim 1 or 2. wherein the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by a reference guide RNA when measured in an in vitro assay.

14. The polynucleotide of claim 13, wherein the engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more. 35% or more, 40% or more. 45% or more, 50% or more. 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more higher than the local editing specificity facilitated by a reference guide RNA when measured in an in vitro assay.

15. The polynucleotide of claim 1 or 2, wherein the free energy of the guide-target RNA scaffold is decreased relative to the free energy of a reference guide-target RNAscaffold formed upon hybridization of a target RNA sequence to a reference guide RNA when measured in an in vitro assay.

16. The polynucleotide of claim 15, wherein the free energy of the guide-target RNA scaffold is decreased 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less relative to the free energy of the reference guide-target RNA scaffold.

17. The polynucleotide of claim 1 or 2, wherein the guide-target RNA scaffold binds or recruits an AD ARI or an ADAR2, resulting in editing of a target adenosine in the target RNA sequence by the AD ARI or the ADAR2.

18. The polynucleotide of claim 1, wherein the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence form 3 to 30 wobble base pairs that each individually and independently comprise a G- U wobble base pair, a U-G wobble base pair, I-U wobble base pair, a U-I wobble base pair, a I- A wobble base pair, a A-I wobble base pair, a I-C wobble base pair, or C-I wobble base pair.

19. The polynucleotide of claim 2, wherein the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence form 1 or 2 wobble base pairs that each individually and independently comprise a G- U wobble base pair, a U-G wobble base pair, I-U wobble base pair, a U-I wobble base pair, a I-A wobble base pair, a A-I wobble base pair, a I-C wobble base pair, or a C-I wobble base pair.

20. The polynucleotide of claim 1 or 2, wherein the reference guide RNA is a parental engineered guide RNA of the engineered guide RNA.

21. The polynucleotide of claim 1 or 2, wherein the reference guide RNA is a parental engineered guide RNA of the engineered guide RNA, that upon hybridization with a target sequence forms a parental guide-target RNA scaffold that comprises the latent structure of the guide-target RNA scaffold.

22. The polynucleotide of claim 1 or 2, the latent structure comprises one or more, 2 or more, 3 or more, or 4 or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof.

23. The polynucleotide of claim 22, wherein the latent structure of the guide-target RNA scaffold comprises two internal loops.

24. The polynucleotide of claim 22, wherein the latent structure of the guide-target RNA scaffold comprises an internal loop that is a 5’ symmetric internal loop positioned upstream of the target adenosine in the target RNA sequence or an internal loop that is a 3 ’ symmetric internal loop positioned downstream of the target adenosine in the target RNA sequence.

25. The polynucleotide of claim 22, wherein the latent structure of the guide-target RNA scaffold comprises a first internal loop and a second internal loop, wherein the first internal loop is a 5 ' symmetric internal loop positioned upstream of the target adenosine in the target RNA sequence and the second internal loop is a 3?symmetric internal loop positioned downstream of the target adenosine in the target RNA sequence.

26. The polynucleotide of claim 1, wherein the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence are located in a hybridization domain sequence that is upstream of a 5’ symmetric internal loop in the target RNA sequence.

27. The polynucleotide of claim 1, wherein the 3 to 30 non-contiguous nucleotides that w obble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence are located in a hybridization domain sequence that is downstream of a 5’ symmetric internal loop in the target RNA sequence.

28. The polynucleotide of claim 1, wherein the guide-target RNA scaffold compnses about: 3 to about 21 w obble base pairs, 3 to about 20 wobble base pairs, 3 to about 19 w obble base pairs, or 3 to about 15 w obble base pairs.

29. The polynucleotide of claim 2, wherein the 1 or 2 non-contiguous nucleotides that w-obble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequenceare each individually located 15 to 30 nucleotides from the target adenosine in the target RNA sequence.

30. The polynucleotide of claim 2, wherein the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence are each individually located 20 to 25 nucleotides from the target adenosine in the target RNA sequence.

31. The polynucleotide of claim 1, wherein the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides 3‘ downstream from the target adenosine in the target RNA sequence.

32. The polynucleotide of claim 1 or 2, wherein the target RNA sequence comprises a sequence from a ABCA4, APP, CFTR, DMPK, DUX4, GAPDH, GBA, GRN, HEXA, LIPA, LRRK2, MAPT, PINK1, PMP22. SERPINA1, SNCA, or SOD1 RNA.

33. The polynucleotide of claim 32, wherein the target RNA sequence comprises a 3’ untranslated region (UTR) of SNCA.

34. The polynucleotide of claim 32. wherein the target RNA sequence comprises a translation initiation site (TIS) of SNCA.

35. The polynucleotide of claim 32, wherein the target RNA sequence comprises a 3‘ UTR of SERPINA1.

36. The polynucleotide of claim 32, wherein the target RNA sequence comprises ABCA4.

37. The polynucleotide of claim 1 or 2. wherein the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 1638. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA has at least 95% sequence identity’, at least 96% sequence identity', at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 37.

39. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity', at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 39 - SEQ ID NO: 52.

40. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA has at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NO: 184 - SEQ ID NO: 188.

41. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA comprises any one of SEQ ID NO: 2 - SEQ ID NO: 1642. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA comprises any one of SEQ ID NO: 18 SEQ ID NO: 37.

43. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA comprises any one of SEQ ID NO: 39 - SEQ ID NO: 52.

44. The polynucleotide of claim 1 or 2, wherein the engineered guide RNA comprises any one of SEQ ID NO: 184 - SEQ ID NO: 188.

45. An AAV vector cassette comprising the polynucleotide sequence of any one of claims 1 to 44.

46. An AAV vector cassette comprising a polynucleotide sequence encoding a first engineered guide RNA and a second engineered guide RNA, wherein: wherein the first engineered guide RNA and the second engineered guide RNA are independently capable of hybridizing to a target RNA sequence forming a first guide target RNA scaffold and a second guide target RNA scaffold, respectively; the first guide target RNA scaffold and second guide target RNA scaffold comprise a latent structure; the first engineered guide RNA and the second engineered guide RNA share at least 64% sequence identity to each other; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof;the second engineered guide RNA comprises 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence; the first engineered guide RNA and the second engineered guide RNA facilitate editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and(i) the second engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay;(ii) the second engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay;(iii) the second engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay;(iv) the second engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay;(v) the free energy of the second guide-target RNA scaffold is decreased relative to the free energy of the first guide-target RNA scaffold when measured in an in vitro assay; or(vi) any combination of (i)-(v).

47. The AAV vector of claim 46, wherein the first engineered guide RNA does not comprise the 3 to 30 non-contiguous nucleotides that wobble base pair with 3 to 30 non-contiguous nucleotides of the target RNA sequence.

48. An AAV vector cassette comprising a polynucleotide sequence encoding a first engineered guide RNA and a second engineered guide RNA, wherein: wherein the first engineered guide RNA and the second engineered guide RNA are independently capable of hybridizing to a target RNA sequence forming a first guide target RNA scaffold and a second guide target RNA scaffold, respectively;the first guide target RNA scaffold and second guide target RNA scaffold comprise a latent structure; the first engineered guide RNA and the second engineered guide RNA share at least 64% sequence identity to each other; the latent structure comprises one or more structural features selected from the group consisting of: a bulge, an internal loop, a hairpin, and any combination thereof; the second engineered guide RNA comprises 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 non-contiguous nucleotides of the target RNA sequence; the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence are each individually located 10 to 40 nucleotides from the target adenosine in the target RNA sequence; the first engineered guide RNA and the second engineered guide RNA facilitate editing of a target adenosine in the target RNA sequence by an AD ARI or an ADAR2; and(i) the second engineered guide RNA facilitates a reduced off-target editing of the target RNA sequence relative to off-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay;(ii) the second engineered guide RNA facilitates an increased on-target editing of the target RNA sequence relative to on-target editing facilitated by the first engineered guide RNA when measured in an in vitro assay;(iii) the second engineered guide RNA facilitates a global editing specificity of the target RNA sequence that is higher than the global editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay;(iv) the second engineered guide RNA facilitates a local editing specificity of the target adenosine of the target RNA sequence that is higher than the local editing specificity facilitated by the first engineered guide RNA when measured in an in vitro assay;(v) the free energy of the second guide-target RNA scaffold is decreased relative to the free energy of the first guide-target RNA scaffold when measured in an in vitro assay; or(vi) any combination of (i)-(v).

49. The AAV vector of claim 48, wherein the first engineered guide RNA does not comprise the 1 or 2 non-contiguous nucleotides that wobble base pair with 1 or 2 noncontiguous nucleotides of the target RNA sequence.

50. A pharmaceutical composition in unit dose form comprising: the polynucleotide encoding the engineered guide RNA of any one of claims 1 to 44, or the AAV vector cassette of any one of claims 45 to 49; and a pharmaceutically acceptable: excipient, carrier, and / or diluent.

51. A method of increasing ADAR-mediated editing efficiency of a parental engineered guide RNA, the method comprising:(a) providing the parental engineered guide RNA or a polynucleotide encoding the parental engineered guide RNA, wherein:(i) the parental engineered guide RNA comprises a targeting sequence having complementarity to a target RNA sequence that is sufficient for the parental engineered guide RNA to hybridize to the sequence of the target RNA, thereby forming a guide-target RNA scaffold;(ii) the guide-target RNA scaffold comprises a latent structure comprising one or more structural features that form upon formation of the guidetarget RNA scaffold, wherein the one or more structural features comprise a first internal loop, a second internal loop, and a mismatch; and(iii) the guide-target RNA scaffold binds ADAR1 or ADAR2, resulting in editing of an adenosine in the sequence of the target RNA by the AD ARI or ADAR2; and(b) generating a progeny engineered guide RNA by substituting in the parental engineered guide RNA: (i) a first modification comprising substitution of three or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms the first internal loop, the second internal loop, or both in the guide-target RNA scaffold, wherein the first modificationmaintains the first internal loop and the second internal loop in the guide-target RNA scaffold; and / or (ii) a second modification comprising substitution of three or more nucleotides in a hybridization domain sequence of the parental engineered guide RNA that canonically base pair with corresponding nucleotides in the sequence of the target RNA with three or more nucleotides that wobble base pair with corresponding nucleotides in the sequence of the target RNA- sequence; wherein the progeny engineered guide RNA facilitates increased editing efficiency of the adenosine in the sequence of the target RNA by the AD ARI or the ADAR2, relative to editing efficiency facilitated by the parental engineered guide RNA.

52. The method of claim 51 , wherein the first internal loop is a 5’ symmetric internal loop that is positioned upstream of the adenosine in the target RNA sequence and the second internal loop is a 3‘ symmetric internal loop positioned downstream of the adenosine in the target RNA sequence.

53. The method of claim 52, wherein the hybridization domain sequence is upstream of the 5’ symmetric internal loop in the target RNA sequence.

54. The method of claim 52, wherein the first modification comprises substituting three or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that produces the 5’ symmetric internal loop, and in a portion of the targeting sequence of the parental engineered guide RNA that produces the 3' symmetric internal loop.

55. The method of claim 51, wherein the second modification comprises substituting from 3 to about 21 nucleotides, resulting in from 3 to about 21 wobble base pairs in the progeny guide-target RNA scaffold formed by the progeny engineered guide RNA.

56. The method of claim 51, wherein the first modification comprises substituting from 3 to about 6 nucleotides in the first internal loop, the second internal loop, or both.

57. A method of increasing ADAR-mediated editing efficiency of a parental engineered guide RNA, the method comprising:(a) providing the parental engineered guide RNA or a polynucleotide encoding the parental engineered guide RNA, wherein:(i) the parental engineered guide RNA comprises a targeting sequence having complementarity to a target RNA sequence that is sufficient for the parental engineered guide RNA to hybridize to the sequence of the target RNA, thereby forming a guide-target RNA scaffold;(ii) the guide-target RNA scaffold comprises a latent structure comprising one or more structural features that form upon formation of the guidetarget RNA scaffold, wherein the one or more structural features comprise a first internal loop, a second internal loop, and a mismatch; and(iii) the guide-target RNA scaffold binds ADAR1 or ADAR2, resulting in editing of an adenosine in the sequence of the target RNA by the AD ARI or ADAR2; and(b) generating a progeny engineered guide RNA by substituting in the parental engineered guide RNA: (i) a first modification comprising substitution of one or more nucleotides in a portion of the targeting sequence of the parental engineered guide RNA that forms a bulge in the guide-target RNA scaffold, wherein the first modification maintains the first internal loop and the second internal loop in the guide-target RNA scaffold; and / or (ii) a second modification comprising substitution of 1 or 2 nucleotides that are each individually located 10 to 40 nucleotides from the adenosine in the target RNA sequence and that canonically base pair with corresponding nucleotides in the sequence of the target RNA with 1 or 2 nucleotides that wobble base pair with corresponding nucleotides in the sequence of the target RNA; wherein the progeny engineered guide RNA facilitates increased editing efficiency of the adenosine in the sequence of the target RNA by the AD ARI or the ADAR2, relative to editing efficiency facilitated by the parental engineered guide RNA.

58. The method of claim 51, wherein the first internal loop is a 5’ symmetric internal loop that is positioned upstream of the adenosine in the target RNA sequence and the second internal loop is a 3’ symmetric internal loop positioned downstream of the adenosine in the target RNA.

59. The method of claim 58, wherein the 1 or 2 substituted nucleotides are each individually located 15 to 30 nucleotides from the adenosine in the target RNA sequence.

60. The method of claim 58, wherein the 1 or 2 substituted nucleotides are each individually located 20 to 25 nucleotides from the adenosine in the target RNA sequence.

61. The method of claim 56. wherein the first modification comprises substituting from 1 to about 4 nucleotides in the bulge.

62. The method of claim 51 or 56, wherein the increased editing efficiency of the progeny engineered guide RNA comprises:(i) a reduced off-target editing of the target RNA sequence relative to off- target AD ARI -mediated or ADAR2-mediated editing of the target RNA using the progeny engineered guide RNA as compared to an amount of on target ADAR 1 -mediated or ADAR2-mediated editing of the target RNA using the parental engineered guide RNA when measured in an in vitro assay;(ii) an increased on-target AD ARI -mediated or ADAR2-mediated editing of the target RNA sequence using the progeny engineered guide RNA as compared to on-target ADAR 1 -mediated or AD AR2 -mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay;(iii) a higher AD ARI -mediated or AD AR2 -mediated global editing specificity of the target RNA sequence using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2-mediated global editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay;(iv) a higher AD ARI -mediated or ADAR2-mediated local editing specificity of the target RNA sequence using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2-mediated local editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay;(v) any combination of (i)-(iv).

63. The method of claim 62. wherein the increased editing efficiency of the progeny engineered guide RNA comprises a reduction in off target AD ARI -mediated or ADAR2 -mediated editing of the target RNA sequence of least about 5% using the progeny engineered guide RNA, as compared to an amount of off target AD ARI - mediated or ADAR2-mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay.

64. The method of claim 62, wherein the increased editing efficiency of the progeny engineered guide RNA comprises an increase in on target AD ARI -mediated or ADAR2-mediated editing of the target RNA sequence of least about 5% using the progeny engineered guide RNA, as compared to an amount of on target AD ARI - mediated or ADAR2-mediated editing of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay.

65. The method of claim 62, wherein the increased editing efficiency of the progeny engineered guide RNA comprises a higher ADARl-mediated or ADAR2-mediated global editing specificity of the target RNA sequence of that is 1 % or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more using the progeny engineered guide RNA as compared to an amount of on target ADARl-mediated or ADAR2- mediated global editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay.

66. The method of claim 65, wherein the progeny engineered guide RNA facilitates a reduced off-target alternative splicing of the target RNA sequence relative to off-target alternative splicing facilitated by the parental engineered guide RNA when measured in an in vitroassay and / or wherein the progeny engineered guide RNA facilitates an increased on-target splicing of the target RNA sequence relative to on-target splicing facilitated by a parental engineered guide RNA when measured in an in vitro assay.

67. The method of claim 62, wherein the increased editing efficiency of the progeny engineered guide RNA comprises a higher ADAR 1 -mediated or ADAR2-mediated local editing specificity of the target RNA sequence of that is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more. 45% or more, 50% or more, 55% or more, 60% or more, 65% or more. 70% or more, 75% or more, 80% or more, 85% or more using the progeny engineered guide RNA as compared to an amount of on target AD ARI -mediated or ADAR2-mediated local editing specificity of the target RNA sequence using the parental engineered guide RNA when measured in an in vitro assay.

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