Engineered constructs with cassette arrangements for increased transcription of RNA payloads

Abstract

Described herein are viral vectors comprising a plurality of expression cassettes encoding small RNA payloads, such as engineered guide RNAs. The viral vectors comprising a plurality of expression cassettes may be engineered to increase expression of the small RNA payload encoded by one or more expression cassette. The viral vectors comprising a plurality of expression cassettes include various sequence elements that may enhance expression of the small RNA payload, such as transcription factor binding sequences, transcriptional termination sequences, and core promoter sequences. Sequence elements may be combined or interchanged to tune small RNA payload expression levels. The expression cassettes may be arranged in an orientation to enhance small RNA payload expression levels. Also described herein are methods of editing a target gene using a small RNA payload encoded by a viral vector comprising a plurality of expression cassettes.

Description

ENGINEERED CONSTRUCTS WITH CASSETTE ARRANGEMENTS FOR INCREASED TRANSCRIPTION OF RNA PAYLOADSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 466,627, filed May 15, 2023, U.S. Provisional Application No. 63 / 613,541, filed December 21, 2023, U.S. Provisional Application No. 63 / 559,087, filed February 28, 2024, and U.S. Provisional Application No. 63 / 569,019, filed March 22, 2024, which applications are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 6, 2024, is named “421688- 722021_SL. xml” and is 117,230 bytes in size.BACKGROUND

[0003] A wide variety of diseases and disorders are caused by mutations, deletions, altered expression, or altered splicing of genes. RNAs can serve as a mechanism for gene therapy, such as by editing a mutated RNA sequence associated with a disease. There is a need for engineered vectors to increase or modulate expression of RNA payloads.SUMMARY

[0004] In various aspects, the present disclosure provides a polynucleotide comprising a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence, and the first and second expression cassette sequences are different sequences.

[0005] In some aspects, the first expression cassette sequence and the second expression cassette sequence are orientated in a tandem read orientation. In some aspects, the DNA sequence encoding a promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 67. In some aspects, the DNA sequence encoding a transcription termination comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. In some aspects, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92.

[0006] In some aspects, the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, and the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79.

[0007] In some aspects, the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0008] In some aspects, the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, and the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67.

[0009] In some aspects, the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0010] In some aspects, the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprisesat least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79.

[0011] In some aspects, the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0012] In some aspects, the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78, the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79.

[0013] In some aspects, the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79, the DNA sequence encoding a promoter sequence of thesecond expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80.

[0014] In various aspects, the present disclosure provides a polynucleotide comprising a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising a small RNA payload, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5’ to 3’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences.

[0015] In some aspects, the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of reverse. In some aspects, the first expression cassette sequence has a read directionality of reverse and the second expression cassette sequence has a read directionality of forward. In some aspects, the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of forward.

[0016] In some aspects, the plurality of expression cassette sequences comprises two expression cassette sequences, three expression cassette sequences, four expression cassette sequences, five expression cassette sequences, six expression cassette sequences, seven expression cassette sequences, eight expression cassette sequences, nine expression cassette sequences, or ten expression cassette sequences.

[0017] In some aspects, the small RNA payload comprises an engineered guide RNA sequence. In some aspects, the engineered guide RNA sequence is capable of hybridizing to a target sequence. In some aspects, the engineered guide RNA sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% reverse complementary to the target sequence.

[0018] In some aspects, the engineered guide RNA sequence is capable of forming a guidetarget RNA scaffold comprising one or more structural features upon hybridization to a target sequence. In some aspects, the one or more structural features comprise a bulge, a mismatch, anintemal loop, a hairpin, or combinations thereof. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is a symmetric bulge. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is an asymmetric bulge. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is a symmetric internal loop. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is an asymmetric internal loop. In some aspects, the one or more structural features comprises the hairpin, and wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin. In some aspects, the guide-target RNA scaffold comprises one or more wobble base pairs. In some aspects, the one or more of the wobble base pairs are GU wobble base pairs.

[0019] In some aspects, the engineered guide RNA sequence comprises at least one base pair mismatch relative to the target sequence. In some aspects, the target sequence comprises an adenosine residue. In some aspects, the target sequence is an RNA sequence. In some aspects, the RNA sequence is a mRNA or a pre-mRNA. In some aspects, the target sequence comprises a G to A mutation relative to a wild type sequence. In some aspects, the target sequence comprises a missense mutation or a nonsense mutation relative to a wild type sequence.

[0020] In some aspects, the target sequence encodes a-synuclein (SNCA). In some aspects, the target sequence encodes peripheral myelin protein 22 (PMP22). In some aspects, the target sequence encodes double homeobox 4 (DUX4). In some aspects, the target sequence encodes leucine rich repeat kinase 2 (LRRK2). In some aspects, the target sequence encodes Tau (MAPT). In some aspects, the target sequence encodes ATP-binding cassette sub-family A member 4 (ABCA4). In some aspects, the target sequence encodes alpha- 1 antitrypsin (SERPINA1). In some aspects, the target sequence encodes methyl CpG binding protein 2 (MECP2).

[0021] In some aspects, the engineered guide RNA sequence is not less than 20 nucleotide residues and not more than 500 nucleotide residues long. In some aspects, the engineered guide RNA sequence is not less than 60 and not more than 100 residues long. In some aspects, the engineered guide RNA sequence is not less than 80 and not more than 120 residues long. In some aspects, the engineered guide RNA sequence is not less than 100 and not more than 140 residues long. In some aspects, the engineered guide RNA sequence is not less than 130 and not more than 170 residues long.

[0022] In some aspects, the promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91. In some aspects, the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

[0023] In some aspects, the payload sequence further comprises an Sm binding sequence or a hairpin sequence. In some aspects, the hairpin sequence comprises a U7 hairpin.

[0024] In some aspects, the first expression cassette sequence, the second expression cassette sequence, or each expression cassette of the plurality of expression cassette sequences independently has a length of not less than 1300 nucleotide residues and not more than 2160 nucleotide residues. In some aspects, the first expression cassette sequence, the second expression cassette sequence, or each expression cassette of the plurality of expression cassette sequences independently comprises at least 80% sequence identity to a U1 sequence or a U7 sequence. In some aspects, the U1 sequence is a mouse U1 sequence or a human U1 sequence. In some aspects, the U7 sequence is a mouse U7 sequence or a human U7 sequence.

[0025] In various aspects, the present disclosure provides a viral vector encoding the polynucleotide of the present disclosure.

[0026] In some aspects, the viral vector is an adeno-associated viral vector. In some aspects, the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, variants or derivatives thereof, and combinations thereof.

[0027] In various aspects, the present disclosure provides a pharmaceutical composition comprising the polynucleotide of the present disclosure or the viral vector of the present disclosure and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

[0028] In various aspects, the present disclosure provides a method of expressing an engineered guide RNA in a cell, the method comprising: (i) delivering a composition comprising a polynucleotide to a cell, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNAsequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5’ to 3’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequences; and (ii) expressing the engineered guide RNA (gRNA) sequence in the cell.

[0029] In various aspects, the present disclosure provides a method of editing a target sequence, the method comprising: (i) delivering a composition comprising a polynucleotide to a cell encoding a target sequence, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequences; (ii) expressing the engineered guide RNA (gRNA) sequence in the cell; (iii) forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA (gRNA) sequence to the target sequence; (iv) recruiting an editing enzyme to the target sequence; and (v) editing the target sequence with the editing enzyme.

[0030] In various aspects, the present disclosure provides a method of expressing an engineered guide RNA sequence in a cell, the method comprising: (i) delivering a composition comprising a polynucleotide to a cell, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassettesequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences; and (ii) expressing the engineered guide RNA sequence in the cell.

[0031] In various aspects, the present disclosure provides a method of editing a target sequence, the method comprising: (i) delivering a composition comprising a polynucleotide to a cell encoding a target sequence, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences; (ii) expressing the engineered guide RNA sequence in the cell; (iii) forming a guidetarget RNA scaffold upon hybridization of the engineered guide RNA sequence to the target sequence; (iv) recruiting an editing enzyme to the target sequence; and (v) editing the target sequence with the editing enzyme.

[0032] In various aspects, the present disclosure provides a method of administering a polynucleotide to a subject with a disease, the method comprising: (i) administering to the subject a composition comprising the polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and thesecond expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequence; (ii) delivering the polynucleotide to a cell of the subject; and (iii) expressing the engineered guide RNA (gRNA) sequence in the cell.

[0033] In various aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising: (i) administering to the subject a composition comprising a polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5’ to 3’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequence; (ii) delivering the polynucleotide to a cell of the subject; and (iii) expressing the engineered guide RNA (gRNA) sequence in the cell, thereby treating the disease.

[0034] In various aspects, the present disclosure provides a method of administering a polynucleotide to a subject with a disease, the method comprising: (i) administering to the subject a composition comprising the polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences; (ii) delivering the therapeutic polynucleotide to a cell of the subject; and (iii) expressing the engineered guide RNA sequence in the cell.

[0035] In various aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising: (i) administering to the subject a composition comprising a polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences; (ii) delivering the therapeutic polynucleotide to a cell of the subject; and (iii) expressing the engineered guide RNA sequence in the cell, thereby treating the disease.

[0036] In some aspects, the cell is in a central nervous system tissue. In some aspects, the cell is in a liver tissue, muscle tissue, ocular tissue, retinal tissue, heart tissue, skeletal muscle tissue, or kidney tissue. In some aspects, the composition is the pharmaceutical composition of the present disclosure. In some aspects, the composition comprises the polynucleotide of the present disclosure or the viral vector of the present disclosure. In some aspects, the disease is a synucleinopathy, Parkinson’s disease, Lewy body dementia, multiple system atrophy, Charcot- Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, Yuan-Harel- Lupski syndrome, a tauopathy, Alzheimer’s disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, autism, traumatic brain injury, Dravet syndrome, Crohn’s disease, muscular dystrophy, B-cell leukemia, Dejerine-Sottas disease, Stargardt disease, alpha- 1 antitrypsin deficiency, Tay-Sachs disease, cystic fibrosis, liposomal acid lipase deficiency, or Gaucher disease.

[0037] In some aspects, the engineered guide RNA sequence hybridizes to a target sequence, and wherein the cell encodes the target sequence. In some aspects, the method further comprises forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA to the target sequence, recruiting an editing enzyme to the target sequence, and editing the target sequence with the editing enzyme.

[0038] In some aspects, the target sequence encodes a-synuclein (SNCA). In some aspects, the target sequence encodes peripheral myelin protein 22 (PMP22). In some aspects, the target sequence encodes double homeobox 4 (DUX4). In some aspects, the target sequence encodes leucine rich repeat kinase 2 (LRRK2). In some aspects, the target sequence encodes Tau (MAPT). In some aspects, the target sequence encodes ATP-binding cassette sub-family A member 4 (ABCA4). In some aspects, the target sequence encodes alpha- 1 antitrypsin(SERPINA1). In some aspects, the target sequence encodes methyl CpG binding protein 2 (MECP2).

[0039] In some aspects, the target sequence comprises a mutation relative to a wild type sequence. In some aspects, editing the target sequence corrects the mutation in the target sequence. In some aspects, the mutation is a missense mutation. In some aspects, the mutation is a nonsense mutation. In some aspects, the mutation is a G to A mutation. In some aspects, the mutation is associated with the disease.

[0040] In some aspects, editing the target sequence comprises editing an untranslated region of the target sequence. In some aspects, the untranslated region is a 5’ untranslated region or a 3’ untranslated region. In some aspects, the 3’ untranslated region is a polyadenylation sequence. In some aspects, editing the target sequence comprises editing a translation initiation site.

[0041] In some aspects, editing the target sequence alters expression of the target sequence. In some aspects, editing the target sequence increases expression of the target sequence. In some aspects, editing the target sequence decreases expression of the target sequence. In some aspects, the editing enzyme comprises an ADAR, an APOBEC, or a Cas nuclease. In some aspects, the ADAR comprises AD ARI, ADAR2, or a combination thereof. In some aspects, the target sequence comprises RNA or DNA. In some aspects, the target sequence is a mRNA or a pre- mRNA. In some aspects, editing the target sequence comprises deamidating a nucleotide of the target sequence. In some aspects, the target sequence is edited with an efficiency of at least 10%, at least 20%, or at least 25%.

[0042] In various aspects, the present disclosure provides a method of increasing a vector genome integrity of a multi-expression cassette vector comprising a first expression cassette and a second expression cassette, the method comprising: a) generating a sequence of the second expression cassette by altering a sequence of the first expression cassette, wherein the first expression cassette and the second expression cassette each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; b) generating the multi-expression cassette vector by combining the sequence of the first expression cassette and the sequence of the second expression cassette; and c) increasing the vector genome integrity as compared to a vector comprising a first expression cassette and a second expression cassette that each have the sequence of the first expression cassette.

[0043] In some aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a promoter sequence. Insome aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a transcription termination sequence. In some aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence. In some aspects, the DNA sequence encoding a promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91. In some aspects, the DNA sequence encoding a transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

[0044] In various aspects, the present disclosure provides a polynucleotide comprising a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising a small RNA payload, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5’ to 3’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences.

[0045] In some aspects, the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of reverse. In some aspects, the first expression cassette sequence has a read directionality of reverse and the second expression cassette sequence has a read directionality of forward. In some aspects, the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of forward. In some aspects, the plurality of expression cassette sequences comprises two expression cassette sequences, three expression cassette sequences, four expression cassette sequences, five expression cassette sequences, six expression cassette sequences, seven expression cassette sequences, eight expression cassette sequences, nine expression cassette sequences, or ten expression cassette sequences.

[0046] In some aspects, the small RNA payload comprises an engineered guide RNA sequence capable of hybridizing to a target sequence. In some aspects, the first expression cassette sequence comprises a first engineered guide RNA sequence capable of hybridizing to a target sequence and the second expression cassette comprises a second engineered guide RNAsequence capable of hybridizing to the target sequence. In some aspects, the second engineered guide RNA sequence has at least one and no more than 30 nucleotide alterations from the first engineered guide RNA sequence. In some aspects, the nucleotide alterations from the first engineered guide RNA sequence are dispersed at a frequency of at least one and no more than 4 nucleotide alterations per every 10 nucleotides in the second engineered guide RNA sequence. In some aspects, the nucleotide alterations from the first engineered guide RNA sequence are dispersed at a frequency of 3 nucleotide alterations per every 10 nucleotides in the second engineered guide RNA sequence.

[0047] In some aspects, the engineered guide RNA sequence, the first engineered guide RNA sequence, or the second engineered guide RNA sequence are each independently at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% reverse complementary to the target sequence. In some aspects, the engineered guide RNA sequence, the first engineered guide RNA sequence, or the second engineered guide RNA sequence are each independently capable of forming a guide-target RNA scaffold comprising one or more structural features upon hybridization of the small RNA payload to a target sequence.

[0048] In some aspects, the guide-target RNA scaffold of the first engineered guide RNA sequence and the guide -target RNA scaffold of the second engineered guide RNA sequence comprise the same one or more structural features. In some aspects, the one or more structural features comprise a bulge, a mismatch, an internal loop, a hairpin, or combinations thereof. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is a symmetric bulge. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is an asymmetric bulge. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is a symmetric internal loop. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is an asymmetric internal loop. In some aspects, the one or more structural features comprises the hairpin, and wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin.

[0049] In some aspects, the guide-target RNA scaffold comprises one or more wobble base pairs. In some aspects, the one or more of the wobble base pairs are GU wobble base pairs. In some aspects, the guide-target RNA scaffold of the second engineered guide RNA sequence has between at least one and no more than 15 additional wobble base pairs than the guide-target RNA scaffold of the first engineered guide RNA sequence. In some aspects, the engineered guide RNA sequence, the first engineered guide RNA sequence, or the second engineered guide RNA sequence comprise at least one base pair mismatch relative to the target sequence.

[0050] In various aspects, the present disclosure provides a method of editing a target sequence in a cell with an increased specificity, the method comprising: delivering the polynucleotide as described herein, the viral vector as described herein, or the pharmaceutical composition as described herein to a cell encoding the target sequence; expressing the small RNA payload in the cell, wherein, the small RNA payload comprises an engineered guide RNA sequence capable of hybridizing to the target sequence; forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA sequence to the target sequence, wherein the guide-target RNA scaffold comprises at least one and no more than 15 wobble base pairs; recruiting an editing enzyme to the target sequence; and editing the target sequence with the editing enzyme with the increased specificity as compared to a specificity of a guide-target RNA scaffold comprising 0 wobble base pairs.

[0051] In some aspects, the wobble base pairs comprise one or more GU wobble base pairs. In some aspects, the method further comprises over twisting a helical structure of the guide-target RNA scaffold.

[0052] In various aspects, the present disclosure provides a method of increasing a vector genome integrity of a vector with a first expression cassette and a second expression cassette, the method comprising: a) generating a sequence of the second expression cassette by altering a sequence of the first expression cassette, wherein the first expression cassette and the second expression cassette each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising a small RNA payload, and a transcription termination sequence; b) generating the multiple payload vector by combining the sequence of the first expression cassette sequence and the sequence of the second expression cassette; and c) increasing the vector genome integrity as compared to a vector comprising a first expression cassette and a second expression cassette that each have the sequence of the first expression cassette.

[0053] In some aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different promoter sequences. In some aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different transcription termination sequences. In some aspects, the sequence of the first expression cassette and the sequence of the second expression cassette have different payload sequences. In some aspects, the promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91. In some aspects, the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100%sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

[0054] In some aspects, the method further comprises a) altering a sequence of a first payload comprising a first engineered guide RNA sequence to generate a second engineered guide RNA sequence to be comprised by a second payload by: i) hybridizing the first engineered guide RNA sequence to a target sequence; ii) forming a first guide -target RNA scaffold comprising one or more structural features; iv) altering at least 20 and no more than 40 nucleotides in the first engineered guide RNA sequence to a different nucleotide resulting in the second engineered guide RNA sequence, wherein hybridizing the second engineered guide RNA sequence to the target sequence forms a second guide-target RNA scaffold comprising the same one or more features at the first guide-target RNA scaffold; b) encoding the first engineered guide RNA sequence in the first payload and the second engineered guide RNA sequence in the second payload in the multiple payload vector; and c) increasing the vector genome integrity of the multiple payload vector as compared to a multiple payload vector comprising a first and second payload each comprising the first engineered guide RNA sequence.

[0055] In some aspects, the one or more structural features comprise a bulge, a mismatch, an internal loop, a hairpin, or combinations thereof. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is a symmetric bulge. In some aspects, the one or more structural features comprises the bulge, and wherein the bulge is an asymmetric bulge. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is a symmetric internal loop. In some aspects, the one or more structural features comprises the internal loop, and wherein the internal loop is an asymmetric internal loop. In some aspects, the one or more structural features comprises the hairpin, and wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin.

[0056] In some aspects, the second guide-target RNA scaffold comprises at least one and no more than 15 additional wobble base pairs as compared to the first guide -target RNA scaffold. In some aspects, the one or more of the additional wobble base pairs is a GU wobble base pair. In some aspects, the structure of the first guide-target RNA scaffold and the structure of the second guide-target RNA scaffold comprise a helical structure. In some aspects, the helical structure is over twisted in the structure of the second guide-target RNA scaffold compared to the structure of the first guide-target RNA scaffold.

[0057] In various aspects, the present disclosure provides a viral vector comprising: a plurality of expression cassettes, wherein each expression cassette independently comprises: a promoter sequence; a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising a small RNA payload; and a transcription termination sequence,wherein each expression cassete of the plurality of expression cassettes is arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse.

[0058] In some aspects, the plurality of expression cassettes comprises two expression cassettes, three expression cassettes, four expression cassettes, five expression cassettes, six expression cassettes, seven expression cassettes, eight expression cassettes, nine expression cassettes, or ten expression cassettes.

[0059] In some aspects, the plurality of expression cassettes comprises a first expression cassette and a second expression cassette, wherein: a) the first expression cassette has the read directionality of forward and the second expression cassette has the read directionality of reverse; b) the first expression cassette has the read directionality of reverse and the second expression cassette has the read directionality of forward; c) the first expression cassette has the read directionality of forward and the second expression cassette has the read directionality of forward; or d) the first expression cassette has the read directionality of reverse and the second expression cassette has the read directionality of reverse.

[0060] In some aspects, the plurality of expression cassettes comprises a first expression cassette, a second expression cassette and a third expression cassette, wherein: a) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of reverse; b) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of forward; c) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of reverse; d) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of forward; e) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of reverse; f) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of forward; g) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of forward; or h) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of reverse.

[0061] In some aspects, the plurality of expression cassettes comprises a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette, wherein: a) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; b) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; c) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse; d) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; e) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward; f) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse; g) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward; h) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse; i) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; j) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse; k) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality offorward; 1) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse; m) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse; n) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward; the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse; or p) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse.

[0062] In some aspects, the plurality of expression cassettes comprises a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette, wherein: a) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; b) the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; c) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse; d) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward; or e) the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward.

[0063] In some aspects, the first expression cassette and the second expression cassette comprise a different promoter sequence. In some aspects, the first expression cassette and the second expression cassette comprise same promoter sequence. In some aspects, the first expression cassette, the second expression cassette and the third expression cassette each comprise a different promoter sequence. In some aspects, at least two expression cassettes comprise same promoter sequence. In some aspects, the first expression cassette, the second expression cassette, the third expression cassette and fourth expression cassette each comprise a different promoter sequence. In some aspects, at least two of the four expression cassettes comprise same promoter sequence.

[0064] In various aspects, the present disclosure provides a viral vector encoding the polynucleotide as described herein.

[0065] In some aspects, the viral vector is an adeno-associated viral vector. In some aspects, the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, variants or derivatives thereof, and combinations thereof.

[0066] In some aspects, the promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91. In some aspects, the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16. In some aspects, the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

[0067] In some aspects, the small RNA payload comprises an engineered guide RNA capable of hybridizing to a target sequence. In some aspects, the engineered guide RNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% reversecomplementary to the target sequence. In some aspects, the engineered guide RNA comprises at least one base pair mismatch relative to the target sequence. In some aspects, the target sequence comprises an adenosine residue. In some aspects, the target sequence is an RNA sequence. In some aspects, the RNA sequence is a mRNA or a pre-mRNA. In some aspects, the target sequence comprises a G to A mutation relative to a wild type sequence. In some aspects, the target sequence comprises a missense mutation or a nonsense mutation relative to a wild type sequence.

[0068] In some aspects, the target sequence encodes a-synuclein (SNCA), peripheral myelin protein 22 (PMP22), double homeobox 4 (DUX4), leucine rich repeat kinase 2 (LRRK2), Tau (MAPT), progranulin (GRN), a duplication of the PMP22 associated with Charcot-Marie-Tooth disease type 1A (CMT1A), ATP -binding cassette sub-family A member 4 (ABCA4), amyloid precursor protein (APP), alpha- 1 antitrypsin (SERPINA1), hexosaminidase A (HEXA), cystic fibrosis transmembrane conductance regulator (CFTR), lipase A (LIPA), glucosylceramidase beta (GBA), PTEN-induced kinase 1 (PINK1), or methyl CpG binding protein 2 (MECP2).

[0069] In some aspects, the small RNA payload comprises an antisense oligonucleotide, an siRNA, an shRNA, a miRNA, or a tracrRNA. In some aspects, the small RNA payload is not less than 20 nucleotide residues and not more than 500 nucleotide residues long. In some aspects, the small RNA payload is not less than 60 and not more than 100 residues long. In some aspects, the small RNA payload is not less than 80 and not more than 120 residues long. In some aspects, the small RNA payload is not less than 100 and not more than 140 residues long. In some aspects, the small RNA payload is not less than 130 and not more than 170 residues long.

[0070] In some aspects, the payload sequence further comprises an Sm binding sequence or a hairpin sequence. In some aspects, the hairpin sequence comprises a U7 hairpin. In some aspects, each expression cassette of the plurality of expression cassettes independently has a length of not less than 1300 nucleotide residues and not more than 2160 nucleotide residues. In some aspects, each expression cassette of the plurality of expression cassettes independently comprises at least 80% sequence identity to a U1 sequence or a U7 sequence. In some aspects, the U1 sequence is a mouse U1 sequence or a human U1 sequence. In some aspects, the U7 sequence is a mouse U7 sequence or a human U7 sequence.

[0071] In some aspects, the engineered guide RNA is capable of forming a guide-target RNA scaffold comprising a structural feature upon hybridization of the small RNA payload to a target sequence. In some aspects, the structural feature is a bulge, a mismatch, an internal loop, a hairpin, or combinations thereof. In some aspects, the structural feature comprises the bulge, and wherein the bulge is a symmetric bulge. In some aspects, the structural feature comprises the bulge, and wherein the bulge is an asymmetric bulge. In some aspects, the structural featurecomprises the internal loop, and wherein the internal loop is a symmetric internal loop. In some aspects, the structural feature comprises the internal loop, and wherein the internal loop is an asymmetric internal loop. In some aspects, the structural feature comprises the hairpin, and wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin. In some aspects, the guide-target RNA scaffold comprises a wobble base pair.

[0072] In various aspects, the present disclosure provides a pharmaceutical composition comprising the viral vector as described herein and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

[0073] In various aspects, the present disclosure provides a pharmaceutical composition comprising the polynucleotide as described herein or the viral vector as described herein and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

[0074] In various aspects, the present disclosure provides a method of expressing a small RNA payload in a cell, the method comprising delivering the viral vector as described herein or the pharmaceutical composition as described herein to a cell and expressing the small RNA payload encoded by the expression cassette in the cell. In various aspects, the method of expressing a small RNA payload in a cell is conducted in vitro or ex vivo.

[0075] In various aspects, the present disclosure provides a method of expressing a small RNA payload in a cell, the method comprising delivering the polynucleotide as described herein, the viral vector as described herein, or the pharmaceutical composition as described herein to a cell and expressing the small RNA payload encoded by the expression cassette in the cell. In various aspects, the method of expressing a small RNA payload in a cell is conducted in vitro or ex vivo.

[0076] In various aspects, the present disclosure provides a method of editing a target sequence, the method comprising: delivering the viral vector as described herein or the pharmaceutical composition as described herein to a cell encoding the target sequence, expressing the small RNA payload in the cell; forming a guide-target RNA scaffold upon hybridization of the small RNA payload to the target sequence; recruiting an editing enzyme to the target sequence; and editing the target sequence with the editing enzyme. In various aspects, the method of editing a target sequence is conducted in vitro or ex vivo.

[0077] In various aspects, the present disclosure provides a method of editing a target sequence, the method comprising: delivering the polynucleotide as described herein, the viral vector as described herein, or the pharmaceutical composition as described herein to a cell encoding the target sequence, expressing the small RNA payload in the cell; forming a guide-target RNA scaffold upon hybridization of the small RNA pay load to the target sequence; recruiting an editing enzyme to the target sequence; and editing the target sequence with the editing enzyme. In various aspects, the method of editing a target sequence is conducted in vitro or ex vivo.

[0078] In various aspects, the present disclosure provides a method of administering a viral vector to a subject with a disease, the method comprising: administering to the subject a composition comprising the viral vector as described herein or the pharmaceutical composition as described herein; delivering the expression cassette to a cell of the subject; and expressing a small RNA payload in the cell.

[0079] In various aspects, the present disclosure provides a method of administering a therapeutic polynucleotide to a subject with a disease, the method comprising: administering to the subject a composition comprising the polynucleotide as described herein, the viral vector as described herein, or the pharmaceutical composition as described herein; delivering the therapeutic polynucleotide to a cell of the subject; and expressing a small RNA payload in the cell.

[0080] In various aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising: administering to the subject a composition comprising the viral vector as described herein or the pharmaceutical composition of claim 46as described herein; delivering the expression cassette to a cell of the subject; and expressing a small RNA payload in the cell, thereby treating the disease.

[0081] In various aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising: administering to the subject a composition comprising the polynucleotide as described herein, the viral vector as described herein, or the pharmaceutical composition as described herein; delivering a therapeutic polynucleotide to a cell of the subject; and expressing a small RNA payload in the cell, thereby treating the disease.

[0082] In some aspects, the disease is a synucleinopathy, Parkinson’s disease, Lewy body dementia, multiple system atrophy, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, Yuan-Harel-Lupski syndrome, a tauopathy, Alzheimer’s disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, autism, traumatic brain injury, Dravet syndrome, Crohn’s disease, muscular dystrophy, B-cell leukemia, Dejerine-Sottas disease, Stargardt disease, alpha- 1 antitrypsin deficiency, Tay-Sachs disease, cystic fibrosis, liposomal acid lipase deficiency, or Gaucher disease.

[0083] In some aspects, the target sequence encodes a-synuclein (SNCA), peripheral myelin protein 22 (PMP22), double homeobox 4 (DUX4), leucine rich repeat kinase 2 (LRRK2), Tau (MAPT), progranulin (GRN), a duplication of the PMP22 associated with Charcot-Marie-Tooth disease type 1A (CMT1A), ATP -binding cassette sub-family A member 4 (ABCA4), amyloid precursor protein (APP), alpha- 1 antitrypsin (SERPINA1), hexosaminidase A (HEXA), cysticfibrosis transmembrane conductance regulator (CFTR), lipase A (LIPA), glucosylceramidase beta (GBA), PTEN-induced kinase 1 (PINK1), or methyl CpG binding protein 2 (MECP2).

[0084] In some aspects, the small RNA payload comprises an engineered guide RNA that hybridizes to a target sequence, and wherein the cell encodes the target sequence. In some aspects, the method further comprises forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA to the target sequence, recruiting an editing enzyme to the target sequence, and editing the target sequence with the editing enzyme. In some aspects, the target sequence comprises a mutation relative to a wild type sequence. In some aspects, editing the target sequence corrects the mutation in the target sequence. In some aspects, the mutation is a missense mutation. In some aspects, the mutation is a nonsense mutation. In some aspects, the mutation is a G to A mutation. In some aspects, the mutation is associated with the disease. In some aspects, editing the target sequence comprises editing an untranslated region of the target.

[0085] In some aspects, the untranslated region is a 5 ’ untranslated region or a 3’ untranslated region. In some aspects, the 3’ untranslated region is a polyadenylation sequence. In some aspects, editing the target sequence comprises editing a translation initiation site. In some aspects, editing the target sequence alters expression of the target sequence. In some aspects, editing the target sequence increases expression of the target sequence. In some aspects, editing the target sequence decreases expression of the target sequence.

[0086] In some aspects, the editing enzyme comprises an ADAR, an APOBEC, or a Cas nuclease. In some aspects, the ADAR comprises AD ARI, ADAR2, ADAR3, or combinations thereof. In some aspects, the target sequence comprises RNA or DNA. In some aspects, the target sequence is a mRNA or a pre-mRNA. In some aspects, editing the target sequence comprises deamidating a nucleotide of the target sequence. In some aspects, the target sequence is edited with an efficiency of at least 10%, at least 20%, or at least 25%.INCORPORATION BY REFERENCE

[0087] 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

[0088] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0089] FIG. 1A schematically illustrates an example configuration that includes an engineered guide RNA expression cassette based on a human U 1 cassette in the forward read direction.

[0090] FIG. IB schematically illustrates an example configuration that includes a human U1 expression cassette having a forward read directionality and a mouse U7 expression cassette having a forward read directionality.

[0091] FIG. 1C, schematically illustrates an example configuration that includes a mouse U7 expression cassette having a reverse read directionality and a human U1 expression cassette having a forward read directionality.

[0092] FIG. ID schematically illustrates an example configuration that includes a human U 1 cassette having a forward read directionality and a mouse U7 cassette having a reverse read directionality.

[0093] FIG. 2A is a bar graph showing expression of guide RNA luciferase reporter, Reporter 1, when configurations described in FIG. 1A - FIG. ID are tested using plasmid delivery of vectors including: a viral vector with a human U1 cassette in the forward read direction with a PMP22 guide RNA payload (SEQ ID NO: 26); a viral vector with a human U1 cassette having a forward read directionality and a mouse U7 cassette having a forward read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 27); a viral vector with mouse U7 cassette having a reverse read directionality and a human U 1 cassette having a forward read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 28); and a viral vector with human U1 cassette having a forward read directionality and a mouse U7 cassette having a reverse read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 29) compared to the expression of no plasmid delivery (“no plasmid”).

[0094] FIG. 2B is a bar graph showing expression of guide RNA luciferase reporter, Reporter 2, when configurations described in FIG. 1A - FIG. ID are tested using plasmid delivery of vectors including: a viral vector with a human U1 cassette in the forward read direction with an SNCA guide RNA payload (SEQ ID NO: 30); a viral vector with a human U1 cassette having a forward read directionality and a mouse U7 cassette having a forward read directionality each having an SNCA guide RNA payload (SEQ ID NO: 31); a viral vector with mouse U7 cassette having a reverse read directionality and a human U 1 cassette having a forward read directionality each having an SNCA guide RNA payload (SEQ ID NO: 32); and a viral vector with human U1 cassette having a forward read directionality and a mouse U7 cassette having a reverse read directionality each having an SNCA guide RNA payload (SEQ ID NO: 33) compared to the expression of no plasmid delivery (“no plasmid”).

[0095] FIG. 2C is a bar graph showing expression of guide RNA luciferase reporter, Reporter 1 , when configurations described in FIG. 1A - FIG. ID are tested using viral (AAV) delivery ofvectors including: a viral vector with a human U1 cassette in the forward read direction with a PMP22 guide RNA payload (SEQ ID NO: 26); a viral vector with a human U1 cassette having a forward read directionality and a mouse U7 cassette having a forward read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 27); a viral vector with mouse U7 cassette having a reverse read directionality and a human U 1 cassette having a forward read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 28); and a viral vector with human U1 cassette having a forward read directionality and a mouse U7 cassette having a reverse read directionality each having a PMP22 guide RNA payload (SEQ ID NO: 29) compared to the expression of no plasmid delivery (“no plasmid”).

[0096] FIG. 2D is a bar graph showing expression of guide RNA luciferase reporter, Reporter 2, when configurations described in FIG. 1A - FIG. ID are tested using viral (AAV) delivery of vectors including: a viral vector with a human U1 cassette in the forward read direction with an SNCA guide RNA payload (SEQ ID NO: 30); a viral vector with a human U1 cassette having a forward read directionality and a mouse U7 cassette having a forward read directionality each having an SNCA guide RNA payload (SEQ ID NO: 31); a viral vector with mouse U7 cassette having a reverse read directionality and a human U 1 cassette having a forward read directionality each having an SNCA guide RNA payload (SEQ ID NO: 32); and a viral vector with human U1 cassette having a forward read directionality and a mouse U7 cassette having a reverse read directionality each having an SNCA guide RNA payload (SEQ ID NO: 33). compared to the expression of no plasmid delivery (“no plasmid”).

[0097] FIG. 3A is a line graph showing expression of the guide RNA luciferase reporter over the GAPDH control (Guide / GAPDH) over increasing multiplicity of infection (MOI) of the wild type single copy vector, as shown in FIG. 3C, and the engineered two copy vector as shown in FIG. 3C.

[0098] FIG. 3B shows a line graph showing Sanger editing of an ATG sequence to GTG to evaluate expression and editing activity over increasing multiplicity of infection (MOI) of the wild type single copy vector, as shown in FIG. 3C, and the engineered two copy vector as shown in FIG. 3C.

[0099] FIG. 3C shows a schematic of the wild type single copy vector (top) and the engineered two copy vector (bottom).

[0100] FIG. 4A schematically illustrates the 16 permutations of 4 expression cassettes with each cassette having a forward (F) or a reverse (R) read directionality.

[0101] FIG. 4B schematically illustrates the 5 permutations of 4 expression cassettes where the expression cassettes do not read into each other.

[0102] FIG. 5A shows a line graph showing Sanger editing of an ATG sequence to GTG to evaluate editing activity of vector constructs in mouse primary neurons. Editing activity was tested for a control AAV transduction without a vector (“Control”), an AAV transduction of a vector with one expression cassette (“WT single copy”), an AAV transduction of a vector with two expression cassettes with the first cassette having a reverse read orientation and the second cassette having a forward read orientation (“Eng two copy”), and a vector with two expression cassettes with the first cassette having a reverse read orientation and the second cassette having a forward read orientation, and with additional engineered sequence elements, including an hnRNP motif, an OPT sequence, and a hairpin sequence (“Eng two copy + gRNA opt”).

[0103] FIG. 5B shows a line graph showing Sanger editing of an ATG sequence to GTG to evaluate editing activity of vector constructs in SH-SY5Y cells. Editing activity was tested for a control AAV transduction without a vector (“Control”), an AAV transduction of a vector with one expression cassette (“WT single copy”), an AAV transduction of a vector with two expression cassettes with the first cassette having a reverse read orientation and the second cassette having a forward read orientation (“Eng two copy”), and a vector with two expression cassettes with the first cassette having a reverse read orientation and the second cassette having a forward read orientation, and with additional engineered sequence elements, including an hnRNP motif, an OPT sequence, and a hairpin sequence (“Eng two copy + gRNA opt”).

[0104] FIG. 6 shows a schematic of the two-copy vectors tested in two orientations including a tandem orientation wherein the guide RNAs have the same read directionality of forward and a bidirectional orientation wherein the guide RNAs have different read directionalities.Specifically, the bidirectional orientation has a first guide RNA with a read directionality of reverse, and a second guide RNA with a read directionality of forward and the tandem orientation has a first guide RNA with a read directionality of forward, and a second guide RNA with a read directionality of forward.

[0105] FIG. 7A shows a bar graph of a guide RNA (gRNAl) expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (2X-bidirectional) two-copy vector, a tandem orientation (2X-tandem) two-copy vector, and a vector with a single guide RNA (Single) that were delivered via plasmid transfection.

[0106] FIG. 7B shows a bar graph of a guide RNA (gRNAl) expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (2X-bidirectional) two-copy vector, a tandem orientation (2X-tandem) two-copy vector, and a vector with a single guide RNA (Single) that were delivered via AAV transfection.

[0107] FIG. 7C shows a bar graph of a guide RNA (gRNAl) expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (Bidirectional) two-copy vector, a tandemorientation (Tandem) two-copy vector, and a vector with a single guide RNA (Single) that were delivered via AAV transfection at different multiplicities of infection (MOI) of 10k and 100k.

[0108] FIG. 8A shows a bar graph of a guide RNA (gRNA2) expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (2X-bidirectional) two-copy vector, a tandem orientation (2X-tandem) two-copy vector, and a vector with a single guide RNA (Single) that were delivered via plasmid transfection.

[0109] FIG. 8B shows a bar graph of a guide RNA (gRNA2) expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (2X-bidirectional) two-copy vector, a tandem orientation (2X-tandem) two-copy vector, and a vector with a single guide RNA (Single) that were delivered via AAV transfection.

[0110] FIG. 8C shows a bar graph of a guide RNA (gRNA2) expression relative to a GAPDH control (gRNA / GAPDH) of a tandem orientation (Tandem) two-copy vector and a vector with a single guide RNA (Single) that were delivered via AAV transfection at different multiplicities of infection (MOI) of 10k and 100k.

[0111] FIG. 9A shows a bar graph of percent A to G editing of a CNS target in mouse primary neurons of a bidirectional orientation (2X bidirectional v2.0) two-copy vector and a single-copy guide RNA (single WT) vector.

[0112] FIG. 9B shows a plot of guide expression relative to a GAPDH control (gRNA / GAPDH) of a bidirectional orientation (2X bidirectional v2.0) two-copy vector and a single-copy guide RNA (single WT) vector across various multiplicities of infection (MOIs) including 10, 100, 1,000, and 10,000.

[0113] FIG. 10 shows an alkaline gel evaluation of vector genome integrity of two-copy vectors comprising two expression cassettes each with the same guide RNA, hairpin structure, and terminator sequence (about 250 nucleotides of sequence homology) also compared to singlecopy vectors.

[0114] FIG. 11 shows an alkaline gel evaluation of vector genome integrity of two-copy vectors comprising two guide RNAs with different sequences (distinct sequences), vectors with two guide RNAs with similar sequences (diverged guide RNAs), and vectors with two guide RNAs with the same sequence (identical guide RNAs). The two-copy vectors were in a bidirectional orientation, as shown in the schematic in FIG. 11, or were in a tandem orientation.

[0115] FIG. 12 shows an alkaline gel evaluation of vector genome integrity of two-copy vectors comprising two guide RNAs with 1 Obp of divergence with each expression cassette and with distinct hairpin and terminator sequences. The two-copy vectors were tested in both bidirectional and tandem orientations.

[0116] FIG. 13 shows a schematic of sequence diverged guide RNA designs. lOOmer gRNAs were designed and tested that had 10 regions of 1 nucleotide alteration (10X1), 10 regions of 2 nucleotide alterations (10X2), 10 regions of 3 nucleotide alterations (10X3), 2 regions of 5 nucleotide alterations (2X5), 4 regions of 5 nucleotide alterations (4X5), 6 regions of 5 nucleotide alterations (6X5), 1 region of 10 nucleotide alterations (1X10), 2 regions of 10 nucleotide alterations (2X10), 3 regions of 10 nucleotide alterations (3X10), 2 regions of 15 nucleotide alterations (2X15), 1 region of 20 nucleotide alterations (1X20), or 1 region of 30 nucleotide alterations (1X20).

[0117] FIG. 14 shows an alkaline gel evaluation of vector genome integrity of two-copy vectors in a bidirectional orientation comprising sequence divergent guide RNAs designed as provided in FIG. 13.

[0118] FIG. 15 shows a bar graph of the percent intact for each of the vector genomes evaluated in the alkaline gel of FIG. 14. The percent intact values for each of the lOOmer gRNAs were calculated using the intensities of each gel band as measured by ImageJ analysis software and the following equation: Percent Intact = (Intensity(Fuii length vector) / (Intensity(Fuii length vector) + Intensity(vector truncations)))* 100%; wherein the Intensity^ length vector) is the intensity of the highest molecular weight band in the gel and the Intensity(vector truncations) is the intensity of the lower molecular weight band.

[0119] FIG. 16 shows the alkaline gel evaluation of vector genome integrity of FIG. 14 with the contrast increased to show the presence of additional lower bands around 1.3kb, as indicated by the brackets.

[0120] FIG. 17 shows an alkaline gel evaluation of vector genome integrity of two-copy vectors in a tandem orientation comprising sequence divergent guide RNAs designed as provided in FIG. 13.

[0121] FIG. 18 shows a bar graph of the percent intact for each of the vector genomes evaluated in the alkaline gel of FIG. 17. The percent intact values for each of the lOOmer gRNAs were calculated using the intensities of each gel band as measured by ImageJ analysis software and the following equation: Percent Intact = (Intensity^ length vector) / (Intensity^ length vector) + Intensity(vector truncations)))* 100%; wherein the Intensity^ length vector) is the intensity of the highest molecular weight band in the gel and the Intensity(vector truncations) is the intensity of the lower molecular weight band.

[0122] FIG. 19 shows the alkaline gel evaluation of vector genome integrity of FIG. 17 with the contrast increased to show the presence of additional lower bands.

[0123] FIG. 20 shows a schematic for developing guide RNAs with sequence divergence. Nucleotide alterations were introduced at nucleotide positions in the guide RNA sequence thatwould either be an alternative mismatch position in the guide-target RNA scaffold or would introduce a GU wobble base pair in the guide-target RNA scaffold. Alternative mismatches were introduced in regions of internal structure in the guide-target RNA scaffold (e.g., internal loops) and GU wobble base pairs were introduced at A and C nucleotides by replacement with G and T nucleotides, respectively.

[0124] FIG. 21 shows a schematic of sequence divergent guide RNA designs. Sequence divergent guide RNAs were designed with i) only alternative mismatches, ii) only GU wobble base pairs and iii) combinatorial design with both alternative mismatches and GU wobble base pairs.

[0125] FIG. 22A shows a schematic of a sequence divergent guide RNA design. The sequence divergent guide RNA had both GU wobble base pairs introduced into the guide-target RNA scaffold (dark blue circles) and alternative mismatches in the internal loops of the guide-target RNA scaffold (light blue circles).

[0126] FIG. 22B shows a bar graph of RNA editing (% RNA editing) from the sequence divergent guide RNAs with alternative mismatches (gRNA 1-6), GU wobble base pairs, and the combinatorial designs of both alternative mismatches and GU wobble base pairs compared to the RNA editing to the original guide RNA sequence (gRNA, “primary design”).

[0127] FIG. 23A shows a schematic of sequence divergent guide RNAs that were developed by introducing alternative mismatches and GU wobble base pairs. The nucleotide alterations were confirmed to not change the guide-target RNA scaffold as shown for 12 (“P12”) and 30 (“P30”) nucleotide alterations.

[0128] FIG. 23B shows a bar graph of RNA editing (% RNA editing) of sequence divergent guide RNAs that were developed by introducing alternative mismatches and GU wobble base pairs for a total of 12 (“P12”), 16 (“P16”), 18 (“Pl 8”), 20 (“P20”), 21 (“P21”), 22 (“P22”), 23 (“P23”), 24 (“P24”), 25 (“P25”), 26 (“P26”), 27 (“P27”), 28 (“P28”), 29 (“P29”), and 30 (“P30”) total nucleotide alterations from the original guide RNA sequence (“P0”). The RNA editing profiles for the original guide RNA sequence (“P0”) and the sequence divergent guide RNA with 20 nucleotide alterations (“P20”) are also provided.

[0129] FIG. 24 shows a graph of the mean percent editing for each position of a target sequence by sequence divergent guide RNAs that were developed by introducing alternative mismatches and GU wobble base pairs for a total of 12 (“Pl 2”), 16 (“Pl 6”), 18 (“Pl 8”), 20 (“P20”), 21 (“P21”), 22 (“P22”), 23 (“P23”), 24 (“P24”), 25 (“P25”), 26 (“P26”), 27 (“P27”), 28 (“P28”), 29 (“P29”), and 30 (“P30”) total nucleotide alterations from the original guide RNA sequence (“P0”). The RNA editing profiles for the original guide RNA sequence (“P0”) and the sequence divergent guide RNA with 24 nucleotide alterations (“P24”) are also provided.

[0130] FIG. 25 shows a bar graph of RNA editing of a CNS target sequence by a sequence diverged guide RNA with 20 total nucleotide alterations (“P20”) compared to the original guide RNA sequence (“P0”). The total editing by a sequence diverged guide RNA with 20 total nucleotide alterations (“P20”) was 58% and the original guide RNA sequence (“P0”) was 49%. The RNA editing is also shown for each target position of the CNS target sequence and shows that the sequence diverged guide RNA with 20 total nucleotide alterations (“P20”) had less RNA editing at off-target positions and increased editing at the target position (0 target position) compared to the original guide RNA sequence (“P0”).

[0131] FIG. 26 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 -0.05A. 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.

[0132] FIG. 27 shows a schematic of the two-copy vector designs evaluated for transduction marker-free vector designs and includes the bidirectional vector designs (e.g., top of FIG. 27) and tandem vector designs (e.g., middle of FIG. 27) that were compared to single copy vector designs (e.g., bottom of FIG. 27).

[0133] FIG. 28A shows an alkaline gel evaluation of the vector genome integrity for two-copy vector designs evaluated for transduction marker-free vector designs. The two-copy vector designs evaluated for transduction marker-free vector designs included bidirectional vector designs (Bidirectional) and tandem vector designs (Tandem) that were compared to the genome integrity of single copy vector designs (Single).

[0134] FIG. 28B shows a bar graph of the percent intact for each of the vector genomes evaluated in the alkaline gel of FIG. 28A. The percent intact values for each of the transduction marker-free vectors were calculated using the intensities of each gel band as measured by ImageJ analysis software and the following equation: Percent Intact = (Intensity(Fuii length vector) / (Intensity(Fuii length vector) + Intensity! vector truncations)))* 100%, wherein the Intensity i uii length vector) is the intensity of the highest molecular weight band in the gel and the Intensity(vector truncations) is the intensity of the lower molecular weight band.

[0135] FIG. 29A shows a bar graph of the RNA editing of a target sequence (On Target Editing (%)) for each of the transduction marker- free vector designs. The two-copy vector designs evaluated for transduction marker- free vector designs included bidirectional vector designs and tandem vector designs that were compared to the RNA editing of a two-copy vector design witha transduction marker. Each of the vector designs were tested at 5k, 50k, and 500k multiplicities of infection (MOI).

[0136] FIG. 29B shows a bar graph of the fold change RNA editing of the transduction marker- free vector designs including the bidirectional vector designs and the tandem vector designs relative to the RNA editing of a two-copy vector design with a transduction marker. Each of the fold change values are provided at 5k, 50k, and 500k multiplicities of infection (MOI).

[0137] FIG. 30A shows a bar graph of the expression of a guide RNA (“05450”) relative to a GAPDH control (gRNA / GAPDH) for the transduction marker-free vector designs including the bidirectional vector designs and the tandem vector designs and compared to the gRNA expression of a two-copy vector design with a transduction marker. Each of the gRNA expression values are provided at 5k, 50k, and 500k multiplicities of infection (MOI).

[0138] FIG. 30B shows a bar graph of the expression of a guide RNA (“38764”) relative to a GAPDH control (gRNA / GAPDH) for the transduction marker-free vector designs including the bidirectional vector designs and the tandem vector designs and compared to the gRNA expression of a two-copy vector design with a transduction marker. Each of the gRNA expression values are provided at 5k, 50k, and 500k multiplicities of infection (MOI).

[0139] FIG. 31A and FIG. 3 IB provide schematics of vector with synthetic filler additions in three different positions, addition on the 5’ end of the expression cassette (5’), addition on both the 5’ and 3’ end of the expression cassette (Mid), and addition on the 3’ end of the expression cassette (3 ’).

[0140] FIG. 32A, FIG. 32B, FIG. 32C, and FIG. 32D provide bar graphs of SNCA-TIS editing by vectors with synthetic filler additions in three different positions, addition on the 5’ end of the expression cassette (5’), addition on both the 5’ and 3’ end of the expression cassette (Mid), and addition on the 3’ end of the expression cassette (3’) at four different multiplicities of infection (MOIs) of the AAV dosage including 5 MOI (FIG. 32A), 500 MOI (FIG. 32B), 5000 MOI (FIG. 32C), and 50000 MOI (FIG. 32D).

[0141] FIG. 33A provides a bar graph of percent SNCA-TIS editing by vector constructs with extended termination sequences as compared to vector constructs with the non-extended termination sequences equivalent of each termination sequence tested.

[0142] FIG. 33B provides a bar graph of GFP fluorescence by vector constructs with extended termination sequences as compared to vector constructs with the non-extended termination sequences equivalent of each termination sequence tested.

[0143] FIG. 34A provides a bar graph of percent SNCA-TIS editing by vector constructs with extended promoter sequences as compared to vector constructs with the non-extended promoter sequences equivalent of each promoter sequence tested.

[0144] FIG. 34B provides a bar graph of GFP fluorescence by vector constructs with extended promoter sequences as compared to vector constructs with the non-extended promoter sequences equivalent of each promoter sequence tested and a vehicle-only control to show the baseline fluorescence (“Baseline”).

[0145] FIG. 35 provides a graph of percent A->G SNCA TIS editing as a function of AAV dosage MOI for tandem, bidirectional, and single expression cassette vector constructs.

[0146] FIG. 36A provides a bar graph of SNCA TIS editing at an AAV dosage of 5 MOI of the vector constructs tested.

[0147] FIG. 36B provides a bar graph of SNCA TIS editing at an AAV dosage of 50 MOI of the vector constructs tested.

[0148] FIG. 37A provides a schematic of the tandem orientation of the developmental vector constructs tested.

[0149] FIG. 37B shows an alkaline gel evaluation of the tandem developmental vector genome integrity for the vector designs provided in TABLE 9.

[0150] FIG. 37C shows a bar graph of the percent intact for each of the vector genomes evaluated in the alkaline gel of FIG. 37A. The percent intact values for each of the tandem developmental vector genome were calculated using the intensities of each gel band as measured by ImageJ analysis software and the following equation: Percent Intact = (Intensity(Fuii length vector) / (Intensity(Fuii length vector) + Intensity(Vector truncations)))* 100%; wherein the Intensity(Fuii length vector) is the intensity of the highest molecular weight band in the gel and the Intensity(vector truncations) is the intensity of the lower molecular weight band.

[0151] FIG. 38 provides a schematic of moving to the developmental vector design from the research vector design by removing the transduction marker sequence and extending the cassette element sequences to retain vector size.

[0152] FIG. 39 provides a graph of percent SNCA TIS RNA editing in HEK293 cells compared between the developmental vector design (“dual cassette”), a single expression cassette vector (“single cassette”), and the research vector design with a transduction marker sequence (“research vector”).

[0153] FIG. 40 provides a bar graph of percent SNCA TIS RNA editing of vector constructs with elongated expression cassette elements (e.g., promoter or termination sequences) as present in the developmental vector design as compared to the non-elongated expression cassette elements as present in the research vector design.

[0154] FIG. 41A provides a graph of guide RNA expression in mouse primary neuron cells of two developmental vector candidate designs (Candidate 1 and 2), compared to the guide RNAexpression from the research vector design and a vector design with a single expression cassette at various AAV doses (MOI).

[0155] FIG. 41B provides a bar graph of the guide RNA expression in mouse primary neuron cells from each of the expression cassettes in a developmental vector design (Candidate 1). Each of the expression cassette guide RNA expressions (Cassette 1 and Cassette 2) was measured at low (500), middle (5000), and high (50000) AAV doses (MOI).

[0156] FIG. 41C provides a graph of guide RNA expression in human iPSC-derived neuron cells of two developmental vector candidate designs (Candidate 1 and 2) at various AAV doses (MOI).

[0157] FIG. 41D provides a bar graph of the guide RNA expression in human iPSC-derived neuron cells from each of the expression cassettes in a developmental vector design (Candidate 1). Each of the expression cassette guide RNA expressions (Cassette 1 and Cassette 2) was measured at low (10000), middle (50000), and high (100000) AAV doses (MOI).DETAILED DESCRIPTION

[0158] Vectors with multiple expression cassettes may be beneficial for increasing RNA payload expression by providing additional copies of a polynucleotide encoding an RNA payload per each vector delivered. Variation in the read orientation (i.e., forward or reverse) of each expression cassette in a vector with a plurality of expression cassettes may influence the expression of an RNA payload and frequency of unwanted events, such as recombination. For example, by orienting expression cassettes with opposing read orientations, expression of an RNA payload may be increased, and the frequency of recombination events may be decreased. Provided herein are various polynucleotides (e.g., viral vectors) comprising a plurality of expression cassettes encoding small RNA payloads that are designed to reduce the frequency of unwanted events as well as methods of making and using those viral vectors.

[0159] The present disclosure provides viral vectors with a plurality of expression cassettes for expressing RNA payloads. The expression cassettes described herein may be engineered for increased expression of the encoded RNA payload sequence. In some embodiments, certain elements of the expression cassette, such as enhancer sequences, core promoter sequences, or transcriptional termination sequences, may be engineered for enhanced payload expression. These sequence elements may be engineered from various endogenous promoters, such as Ul, U6, or U7 promoters, for increased payload expression. The individual sequence elements of the expression cassette may be engineered to enhance expression of the encoded RNA payload.Expression Cassette Multitude

[0160] The present disclosure provides polynucleotides (e.g., viral vectors) with a plurality of expression cassettes for expressing RNA payloads e.g., small RNA payloads. A plurality of expression cassettes can include two or more expression cassettes, three or more expression cassettes, four or more expression cassettes, five or more expression cassettes, six or more expression cassettes, seven or more expression cassettes, eight or more expression cassettes, nine or more expression cassettes, or ten or more expression cassettes. In some embodiments, the plurality of expression cassette sequences comprises two expression cassette sequences, three expression cassette sequences, four expression cassette sequences, five expression cassette sequences, six expression cassette sequences, seven expression cassette sequences, eight expression cassette sequences, nine expression cassette sequences, or ten expression cassette sequences. For example, a polynucleotide (e.g., a viral vector) with a plurality of expression cassettes may be a polynucleotide with two expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with three expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with four expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with five expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with six expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with seven expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with eight expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with nine expression cassettes. In another example, a polynucleotide with a plurality of expression cassettes may be a polynucleotide with ten expression cassettes. Each expression cassette of the plurality of expression cassettes can each independently include a promoter sequence, a payload sequence, and a transcription termination sequence. The payload sequence can be under the transcriptional control of the promoter sequence.

[0161] When a plurality of expression cassettes is included in a polynucleotide, the arrangement of the plurality of expression cassettes can be designed for enhanced expression. For example, the arrangement of the plurality of expression cassettes can be designed to increase the expression of the small RNA payload. The plurality of expression cassettes of a polynucleotide may be arranged in the polynucleotide relative to the 5’ and 3’ ends of the polynucleotide. For example, a first expression cassette may be included in the polynucleotide with a second expression cassette included on the 3’ end of the first expression cassette. In anotherembodiment, a third expression cassette could be included on the 3’ end of the second expression cassette. In another embodiment, a fourth expression cassette could be included on the 3’ end of the third expression cassette.

[0162] Each expression cassette within the polynucleotide can have a read directionality relative to the 5’ to 3’ orientation of the sequence of the polynucleotide. As used herein, the term “read directionality” refers to the direction of transcription for a given expression cassette. Forward read directionality can be used to describe that the direction of transcription is from 5 ’ to 3 ’ whereas the reverse read directionality can be used to describe that the direction of transcription is 3 to 5’. In some embodiments, an expression cassette having a reverse read directionality can include the antisense strand of the expression cassette.

[0163] In some embodiments, all of the expression cassettes in the plurality of expression cassettes in a polynucleotide can have the same read directionality. In some embodiments, more than one expression cassette in a polynucleotide can have the same read directionality. In some embodiments, at least one expression cassette in a polynucleotide can have a different read directionality compared to other expression cassettes in the polynucleotide.

[0164] In some embodiments, a polynucleotide can include two expression cassettes with a first expression cassette and a second expression cassette. In one embodiment, the first expression cassette has the read directionality of forward and the second expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse and the second expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward and the second expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse and the second expression cassette has the read directionality of reverse.

[0165] A “bidirectional” vector orientation as used herein is used to describe a vector orientation where a first expression cassette and a second expression cassette have read directions that are not the same. For example, a bidirectional vector orientation may include a vector orientation wherein the first expression cassette has a read directionality of forward and the second expression cassette has the read directionality of reverse. In another example, a bidirectional vector orientation may include a vector orientation wherein the first expression cassette has a read directionality of reverse and the second expression cassette has the read directionality of forward.

[0166] A “tandem” vector orientation as used herein is used to describe a vector orientation where a first expression cassette and a second expression cassette have read directions that are the same. For example, a tandem vector orientation may include a vector orientation wherein thefirst expression cassette has a read directionality of forward and the second expression cassette has a read directionality of forward. In another example, a tandem vector orientation may include a vector orientation wherein the first expression cassette has a read directionality of reverse and the second expression cassette has a read directionality of reverse. A tandem vector orientation may refer to a vector orientation wherein the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward.

[0167] In some embodiments, the polynucleotides can include three expression cassettes with a first expression cassette, a second expression cassette and a third expression cassette. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, and the third expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, and the third expression cassette has the read directionality of reverse.

[0168] In some embodiments, the polynucleotide can include four expression cassettes with a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette. FIG. 4A provides exemplary arrangements of four expression cassettes. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality offorward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of forward, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse. In oneembodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of forward. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of forward, the third expression cassette has the read directionality of reverse, and the fourth expression cassette has the read directionality of reverse. In one embodiment, the first expression cassette has the read directionality of reverse, the second expression cassette has the read directionality of reverse, the third expression cassette has the read directionality of forward, and the fourth expression cassette has the read directionality of reverse.

[0169] In some embodiments, the read directionality of each of the two or more expression cassettes is selected such that the expression cassette does not read into each other. For example, an expression cassette with a forward read directionality may not be placed 5 ’ of an expression cassette with a reverse read directionality within the polynucleotide. As another example, an expression cassette with a reverse read directionality may not be placed 3’ of an expression cassette with a forward read directionality within the polynucleotide. Non-limiting examples of configurations with four expression cassettes that do not read into each other are provided in FIG. 4B.

[0170] In some embodiments, each of the expression cassettes can have a different promoter sequence. In some embodiments, two or more of the expression cassettes can have the same promoter sequences. In some embodiments, the first expression cassette and the second expression cassette in a polynucleotide with two expression cassettes can have a different promoter sequence. In one embodiment, the first expression cassette and the second expression cassette in a polynucleotide with two expression cassettes can have the same promoter sequence. In some embodiments, the first expression cassette, the second expression cassette and the third expression cassette in a polynucleotide with three expression cassettes each comprise a different promoter sequence. In some embodiments, at least two of the three expression cassettes in a polynucleotide with three expression cassettes can have the same promoter sequence. In some embodiments, the first expression cassette, the second expression cassette, the third expression cassette and fourth expression cassette in a vector with four expression cassettes can each have a different promoter sequence. In some embodiments, at least two of the four expression cassettes in a vector with four expression cassettes can have the same promoter sequence.Promoter Sequences, Termination Sequences, and Hairpin Sequences

[0171] A vector comprising a plurality of expression cassettes of the present disclosure may include an expression cassette that has a promoter sequence, an RNA payload coding sequence, a termination sequence, and a hairpin sequence (e.g., a non-recruitment hairpin). The promoter may recruit transcription factors, polymerases (e.g., RNA polymerase II or RNA polymerase III), or other transcriptional machinery to promote transcription of the RNA payload. For example, the vector comprising a plurality of expression cassettes may promote transcription of a guide RNA for RNA editing, a guide RNA for DNA editing, a tracrRNA, an siRNA, an shRNA, or a miRNA, or an antisense oligonucleotide). In some embodiments, the promoter may be engineered for increased expression of the RNA payload under transcriptional control of the promoter. The termination sequence may enhance termination of transcription and promote transcriptional turnover, increasing transcription of the payload. In some embodiments, the termination sequence may be engineered for enhanced expression of the RNA payload.Sequence elements within the promoter or termination sequence (e.g., transcription factor binding sequences, transcription initiation sequences, termination sequences, or combinations thereof) may be engineered for enhanced payload expression. The sequence elements may be interchangeable with sequence elements from endogenous RNA promoters, such as Ul, U6, or U7 promoters.

[0172] An expression cassette may be engineered from an endogenous sequence. For example, an expression cassette may be engineered from an endogenous Ul, U2, U3, U4, U5, U6, or U7 sequence. The endogenous sequence may be from any organism, including human, mouse, or other mammals. In some embodiments, an expression cassette may comprise a promoter engineered from an endogenous promoter, such as an endogenous Ul , U2, U3, U4, U5, U6, or U7 promoter. In some embodiments, an expression cassette may comprise a transcriptional termination sequence engineered from an endogenous transcriptional termination sequence, such as an endogenous Ul, U2, U3, U4, U5, U6, or U7 transcriptional termination sequence. Examples of sequence elements that may be inserted or substituted into an expression cassette are provided in TABLE 1. In some embodiments, the present disclosure provides for a vector comprising a plurality of expression cassettes and where each expression cassette has a distinct promoter. Further, said plurality of expression cassettes can have distinct transcriptional terminators. For example, a vector provided herein may have two expression cassettes. The first expression cassette may have a first promoter and a first transcriptional terminator. The second expression cassette may have a second promoter and a second transcriptional terminator. The first promoter and the second promoter may be different. The first transcriptional terminator and second transcriptional terminator may be different. In some cases, it is advantageous for thepromoter and terminator sequences to differ from expression cassette to expression cassette in vectors comprising a plurality of expression cassettes. This may be because having repetitive sequences within a single vector genome can lead to unwanted effects such as recombination, which can result in improper, diminished, or abolished expression of the therapeutic payload.TABLE 1 - Exemplary Sequence Elements

[0173] In some embodiments, a vector comprising a plurality of expression cassettes can have a promoter (e.g., a DNA sequence encoding a promoter sequence) for enhanced expression of an RNA payload that may have at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91. In some embodiments, a promoter sequence may enhance transcription of an RNA payload. The promoter sequence may be positioned upstream of the payload sequence. In some embodiments, the DNA sequence encoding a promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 67. In some embodiments, at least one of the DNA sequences encoding a promoter sequence of the plurality of expression cassettes in a multi-expression cassette vector comprises at least 80% sequence identity to SEQ ID NO: 5.

[0174] In some embodiments, a vector comprising a plurality of expression cassettes can additionally have a transcriptional termination sequence (e.g., a DNA sequence encoding a transcriptional termination sequence), including an engineered termination sequence. The termination sequence may enhance expression of a payload (e.g., a small RNA payload) encoded by the expression cassette. In some embodiments, the termination sequence may have at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89. In some embodiments, a termination sequence, also referred to as a terminator, may enhance transcription of an RNA payload. The termination sequence may be positioned downstream of the payload sequence. In some embodiments, the DNA sequence encoding a transcription termination comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.

[0175] The small nuclear RNA (snRNA) hairpin sequence (e.g., a non-recruitment hairpin sequence) may enhance expression of a payload (e.g., a small RNA payload) encoded by the expression cassette. In some embodiments, the snRNA hairpin sequence (e.g., a non-recruitment hairpin sequence) may have at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 83 or SEQ ID NO: 84. In some embodiments, a snRNA hairpin sequence (e.g., a non-recruitment hairpin sequence) may enhance transcription of an RNA payload. The snRNA hairpin sequence (e.g., a non-recruitment hairpin sequence) may be positioned downstream of the payload sequence. In some embodiments, a snRNA hairpin sequence is paired with an sm- binding sequence (e.g., an SmOPT sequence). In some embodiments, an SmOPT and snRNA hairpin sequence may be encoded by a DNA sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92.Multi-Expression Cassette Vector Architecture

[0176] In some embodiments, the present disclosure provides a vector comprising a plurality of expression cassettes. A vector comprising a plurality of expression cassettes may comprise a first pair of a promoter sequence and a terminator sequence (also referred to as a promoterterminator pair) and a second promoter-terminator pair. The first promoter-terminator pair maybe different than the second promoter-terminator pair. The first promoter-terminator pair and second promoter-terminator pair may be arranged in the same or different read orientations. The first promoter-terminator pair, the second promoter-terminator pair, or both may comprise a promoter sequence of SEQ ID NO: 5 paired with a terminator sequence of SEQ ID NO: 11; a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13; a promoter sequence of SEQ ID NO: 8 paired with a terminator sequence of SEQ ID NO: 15; or a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13. A vector comprising a plurality of expression cassettes may comprise a first pair of a promoter sequence and a terminator sequence, a second pair of a promoter sequence and a terminator sequence, and a third pair of a promoter sequence and a terminator sequence. The first promoterterminator pair, the second promoter-terminator pair, and the third promoter-terminator pair may be different. The first promoter-terminator pair, second promoter-terminator pair, third promoter-terminator pair, or a combination thereof may be arranged in the same or different read orientations. The first promoter-terminator pair, second promoter-terminator pair, third promoter-terminator pair, or a combination thereof, may comprise a promoter sequence of SEQ ID NO: 5 paired with a terminator sequence of SEQ ID NO: 11 ; a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13; a promoter sequence of SEQ ID NO: 8 paired with a terminator sequence of SEQ ID NO: 15; or a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13. A vector comprising a plurality of expression cassettes may comprise a first promoter-terminator pair, a second promoterterminator pair, a third promoter-terminator pair, and a fourth promoter-terminator pair. The first promoter-terminator pair, the second promoter-terminator pair, the third promoter-terminator pair, and the fourth promoter-terminator pair may be different. The first promoter-terminator pair, second promoter-terminator pair, third promoter-terminator pair, fourth promoterterminator pair, or a combination thereof may be arranged in the same or different read orientations. The first promoter-terminator pair, second promoter-terminator pair, third promoter-terminator pair, fourth promoter-terminator pair, or a combination thereof may comprise a promoter sequence of SEQ ID NO: 5 paired with a terminator sequence of SEQ ID NO: 11 ; a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13; a promoter sequence of SEQ ID NO: 8 paired with a terminator sequence of SEQ ID NO: 15; or a promoter sequence of SEQ ID NO: 6 paired with a terminator sequence of SEQ ID NO: 13.

[0177] In some embodiments, the present disclosure provides a vector comprising two expression cassettes. In such a vector, the first expression cassette has a first promoter selected from TABLE 1 and a first terminator selected from TABLE 1 and the second expressioncassette has a second promoter selected from TABLE 1, which is different from the first promoter, and a second terminator selected from TABLE 1, which is different from the first terminator. For example, a vector comprising two expression cassettes may have a first expression cassette with a first promoter comprising SEQ ID NO: 5 and a first terminator comprising SEQ ID NO: 11 and a second expression cassette with a second promoter comprising SEQ ID NO: 6 and a second terminator comprising SEQ ID NO: 13. In another example, a vector comprising two expression cassettes may have a first expression cassette with a first promoter comprising SEQ ID NO: 5 and a first terminator comprising SEQ ID NO: 34 and a second expression cassette with a second promoter comprising SEQ ID NO: 6 and a second terminator comprising SEQ ID NO: 35.

[0178] In some embodiments, a multi-expression cassette vector may comprise a DNA sequence encoding a promoter sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5 and a DNA sequence encoding a transcription termination sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79. In some embodiments, a multi-expression cassette vector may comprise a DNA sequence encoding a promoter sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67 and a DNA sequence encoding a transcription termination sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0179] In some embodiments, a multi-expression cassette vector may comprise a DNA sequence encoding a promoter sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5 and a DNA sequence encoding a promoter sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67. In some embodiments, a multi-expression cassette vector may comprise a DNA sequence encoding a transcription termination sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79 and a DNA sequence encoding a transcription termination sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0180] In some embodiments, a multi-expression cassette vector may comprise an expression cassette that comprises a DNA sequence encoding a promoter sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to SEQ ID NO: 5, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and a DNA sequence encoding a transcription termination sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79.

[0181] In some embodiments, a multi-expression cassette vector may comprise an expression cassette that comprises a DNA sequence encoding a promoter sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and DNA sequence encoding a transcription termination sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

[0182] In some embodiments, a multi-expression cassette vector may comprise a first expression cassette that comprises: a DNA sequence encoding a promoter sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and a DNA sequence encoding a transcription termination sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78; and a second expression cassette comprising: DNA sequence encoding a promoter sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and a DNA sequence encoding a transcription termination sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the first expression cassette and the second expression cassette are oriented in a tandem read orientation.

[0183] In some embodiments, a multi-expression cassette vector may comprise a first expression cassette that comprises: a DNA sequence encoding a promoter sequence of the firstexpression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and a DNA sequence encoding a transcription termination sequence of the first expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79; and a second expression cassette comprising: a DNA sequence encoding a promoter sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and a DNA sequence encoding a transcription termination sequence of the second expression cassette comprising at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the first expression cassette and the second expression cassette are oriented in a tandem read orientation.TABLE 2: Particular Vector Constructs Comprising a First Expression Cassette and aSecond Expression Cassette

[0184] In some embodiments, the first expression cassette and the second expression cassette as set out in TABLE 2 each independently comprise a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence. In some embodiments, the first expression cassette and the second expression cassette as set out in TABLE 2 are orientated in a tandem read orientation. In some embodiments, the DNA sequence encoding the promoter sequence, comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequences identified in TABLE 2. In some embodiments, the DNA sequence encoding the SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequences identified in TABLE 2. In some embodiments, the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequences identified in TABLE 2.

[0185] In some embodiments, the present disclosure provides a vector comprising three expression cassettes. In such a vector, the first expression cassette has a first promoter selected from TABLE 1 and a first terminator selected from TABLE 1, the second expression cassette has a second promoter selected from TABLE 1, which is different from the first promoter, and a second terminator selected from TABLE 1, which is different from the first terminator, and thethird expression cassette has a third promoter selected from TABLE 1, which is different from the first promoter and the second promoter, and a third terminator selected from TABLE 1, which is different from the first terminator and the second terminator. For example, a vector comprising four expression cassettes may have a first expression cassette with a first promoter comprising SEQ ID NO: 5 and a first terminator comprising SEQ ID NO: 11; a second expression cassette with a second promoter comprising SEQ ID NO: 6 and a second terminator comprising SEQ ID NO: 13; a third expression cassette with a third promoter comprising SEQ ID NO: 8 and a third terminator comprising SEQ ID NO: 15. In another example, a vector comprising four expression cassettes may have a first expression cassette with a first promoter comprising SEQ ID NO: 5 and a first terminator comprising SEQ ID NO: 11; a second expression cassette with a second promoter comprising SEQ ID NO: 6 and a second terminator comprising SEQ ID NO: 13; a third expression cassette with a third promoter comprising SEQ ID NO: 9 and a third terminator comprising SEQ ID NO: 16.

[0186] In some embodiments, the present disclosure provides a vector comprising four expression cassettes. In such a vector, the first expression cassette has a first promoter selected from TABLE 1 and a first terminator selected from TABLE 1, the second expression cassette has a second promoter selected from TABLE 1, which is different from the first promoter, and a second terminator selected from TABLE 1, which is different from the first terminator, the third expression cassette has a third promoter, which is different from the first promoter and the second promoter, and a third terminator selected from TABLE 1, which is different from the first terminator and the second terminator, and the fourth expression cassette has a fourth promoter selected from TABLE 1, which is different from the first promoter, the second promoter, and the third promoter, and a fourth terminator selected from TABLE 1, which is different from the first terminator, the second terminator, and the third terminator. For example, a vector comprising four expression cassettes may have a first expression cassette with a first promoter comprising SEQ ID NO: 5 and a first terminator comprising SEQ ID NO: 11; a second expression cassette with a second promoter comprising SEQ ID NO: 6 and a second terminator comprising SEQ ID NO: 13; a third expression cassette with a third promoter comprising SEQ ID NO: 8 and a third terminator comprising SEQ ID NO: 15; and a fourth expression cassette with a fourth promoter comprising SEQ ID NO: 9 and a fourth terminator comprising SEQ ID NO: 16.

[0187] In some embodiments, the present disclosure provides extended sequence elements (e.g., extended promoter sequences, extended termination sequences, or extended hairpin sequences) for engineering of ideal expression cassette size, vector genome size, or both. A sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may beextended by the addition of nucleotides on the 5’ end of the sequence element, the 3’ end of the sequence element, or a combination thereof. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended by the addition of nucleotides on the 5 ’ end of the sequence element. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended by the addition of nucleotides on the 3 ’ end of the sequence element. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended by the addition of nucleotides on the 5 ’ end and 3 ’ end of the sequence element.

[0188] A sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended for engineering of ideal vector size (e.g., an AAV genome size such as a scAAV genome size) of the present disclosure. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended to achieve a final vector genome size of at least 1.2 kb. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended to achieve a final vector genome size of at least 1 .0 kb and no greater than 1 .4 kb, at least 1 .1 kb and no greater than 1.4 kb, at least 1.2 kb and no greater than 1.4 kb, or at least 1.2 kb and no greater than 1 .3 kb.

[0189] In some embodiments a promoter sequence (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91) may be extended for engineering of ideal expression cassette size, vector genome size, or both. In some embodiments, a promoter sequence (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91) may be extended to a total sequence length of 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, or 450 nucleotides. In some embodiments, a promoter sequence (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91) may be extended to a total sequence length of 300 nucleotides. In some embodiments, a promoter sequence (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91) may be extended to a total sequence length of 350 nucleotides. In some embodiments, a promoter sequence (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91) may be extended to a total sequence length of 400 nucleotides. For example, a promoter sequence of SEQ ID NO: 6 or SEQ ID NO: 66 may be extended to a total sequence length of 400 nucleotides (e.g., a promoter sequence of SEQ ID NO: 67).

[0190] In some embodiments a termination sequence (e.g., any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89) may be extended for engineering of ideal expression cassette size, vector genome size, or both. In some embodiments, a termination sequence (e.g., any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89) may be extended to a total sequence length of 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, or 300 nucleotides. In some embodiments, a termination sequence (e.g., any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89) may be extended to a total sequence length of 250 nucleotides. In some embodiments, a termination sequence (e.g., any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89) may be extended to a total sequence length of 200 nucleotides. For example, a terminator sequence of SEQ ID NO: 34 may be extended to a total sequence length of 200 nucleotides (e.g., a terminator sequence of SEQ ID NO: 78). For example, a terminator sequence of SEQ ID NO: 35 may be extended to a total sequence length of 200 nucleotides (e.g., a terminator sequence of SEQ ID NO: 79). For example, a terminator sequence of SEQ ID NO: 82 may be extended to a total sequence length of 200 nucleotides (e.g., a terminator sequence of SEQ ID NO: 80). For example, a terminator sequence of SEQ ID NO: 11 or SEQ ID NO: 12 may be extended to a total sequence length of 199 nucleotides (e.g., a terminator sequence of SEQ ID NO: 81).

[0191] A sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended for engineering of ideal size of an expression cassette of the present disclosure. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended to achieve a final expression cassette length of at least 600 nucleotides. In some embodiments, a sequence element (e.g., a promoter sequence, a termination sequence, or a hairpin sequence) may be extended to achieve a final expression cassette length of at least 500 and no greater than 700 nucleotides, at least 550 and no greater than 750 nucleotides, at least 550 and no greater than 650 nucleotides, or at least 575 and no greater than 625 nucleotides. For example, an expression cassette of the current disclosure may comprise an extended promoter sequence with a length of 300 nucleotides, a guide RNA sequence with a length of 100 nucleotides, and an extended termination sequence with a length of 250 nucleotides resulting in a total expression cassette length of 650 nucleotides.

[0192] A vector comprising a plurality of expression cassettes may comprise one or more extended sequence elements (e.g., extended promoter sequences, extended terminationsequences, or extended hairpin sequences) for engineering of ideal expression cassette size, vector genome size, or both. In some embodiments, a vector may comprise one or more extended promoter sequences and one or more extended terminator sequences. In some embodiments, a vector may comprise two extended promoter sequences and two extended termination sequences. For example, a vector comprising two expression cassettes may comprise two extended promoter sequences each with a sequence length of 300 nucleotides, two guide RNA sequences each with a sequence length of 100 nucleotides, and two extended termination sequences each with a sequence length of 250 nucleotides resulting in a total vector genome size of greater than 1.2 kb.Payloads

[0193] The vectors comprising a plurality of expression cassettes of the present disclosure may encode an RNA payload under transcriptional control of a promoter (e.g., an engineered promoter). In some embodiments, the RNA payload may encode a small RNA payload such as a guide sequence (e.g., for RNA or DNA editing), a tracrRNA, an siRNA, an shRNA, a miRNA, an antisense oligonucleotide (e.g., for expression knockdown), a structural element (e.g., an RNA hairpin), or combinations thereof. Provided herein are engineered RNA payloads and polynucleotides encoding the same; as well as compositions comprising said engineered RNA payloads or said polynucleotides. As used herein, the term “engineered” in reference to an RNA payload or polynucleotide encoding the same refers to a non-naturally occurring 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.Guide RNA Pay loads for RNA Editing

[0194] The vectors comprising a plurality of expression cassettes described herein may be used to enhance expression of engineered guide RNAs and engineered polynucleotides encoding the same for site-specific, selective editing of a target RNA via an RNA editing entity or 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.

[0195] An engineered guide RNA, as described herein, may comprise a targeting domain with complementarity to a target RNA described herein. As such, a guide RNA can be engineered to site-specifically / selectively target and hybridize to a particular target RNA, thus facilitating editing of specific nucleotide in the target RNA via an RNA editing entity or a biologically active fragment thereof. The targeting domain can include a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or 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.

[0196] Hybridization of the target RNA and the targeting domain of the guide RNA may produce 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, may 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 residue (e.g., an adenosine residue), 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.

[0197] In some examples, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target 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.

[0198] In some examples, a target RNA of an engineered guide RNA of the present disclosure can be a pre-mRNA or mRNA. 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.Targeting Domain

[0199] 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.”

[0200] In some cases, a targeting domain of an engineered guide allows the engineered guide to target an RNA sequence through base pairing, such as Watson Crick base pairing. In some examples, the targeting sequence can be located at either the N-terminus or C-terminus of the engineered guide. In some cases, the targeting sequence can be located at both termini. The targeting sequence can be of any length. In some cases, the targeting sequence can be at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, or up to about 200 nucleotides in length. In some cases, the targeting sequence can be no greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 200 nucleotides in length. In some examples, an engineered guide comprises a targeting sequence that can be from about 60 to about 500, from about 60 to about 200, from about 75 to about 100, from about 80 to about 200, from about 90 to about 120, or from about 95 to about 115 nucleotides in length. In some examples, an engineered guide RNA comprises a targeting sequence that can be about 100 nucleotides in length.

[0201] In some cases, a targeting domain comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to a target RNA. In some cases, a targeting sequence comprises less than 100% complementarity to a target RNA sequence. For example, a targeting sequence and aregion of a target RNA that can be bound by the targeting sequence can have a single base mismatch.

[0202] The targeting sequence can have sufficient complementarity to a target RNA to allow for hybridization of the targeting sequence to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 50 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 60 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 70 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 80 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 90 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 100 nucleotides or more to the target RNA. In some embodiments, antisense complementarity refers to non-contiguous stretches of sequence. In some embodiments, antisense complementarity refers to contiguous stretches of sequence.

[0203] In some embodiments, hybridization of the targeting sequence to the target RNA to form a guide-target RNA scaffold may manifest a latent structural feature. For example, a latent structural feature may comprise a symmetric bulge, an asymmetric bulge, a symmetric internal loop, an asymmetric internal loop, or combinations thereof. In some embodiments, the latent structural feature may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 unpaired nucleotides on the target RNA side. In some embodiments, the latent structural feature may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 unpaired nucleotides on the guide RNA side.

[0204] In some embodiments an engineered guide RNA for RNA editing may have at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 25. For example, an engineered guide RNA of SEQ ID NO: 24 may be used to target PMP22. In another example, an engineered guide RNA of SEQ ID NO: 25 may be used to target SNCA.Engineered Guide RNAs Having a Recruitment Domain

[0205] 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, asubject engineered guide can facilitate editing of a base of a nucleotide of in a target sequence of a target RNA that results in modulating the expression of a polypeptide encoded by the target RNA. In some instances, modulation can be increased or decrease expression of the polypeptide. 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 an RNA editing entity (e.g., ADAR or APOBEC). In order to facilitate editing, an engineered polynucleotide of the disclosure can recruit an RNA editing entity (e.g., ADAR or APOBEC). Various RNA editing entity recruiting domains can be utilized. In some examples, a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), an Alu sequence, or, in the case of recruiting APOBEC, an APOBEC recruiting domain.

[0206] 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 hybridizes to a target sequence of 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, or 80 nucleotides in length. In some cases, a recruiting 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.

[0207] In some embodiments, a recruiting domain comprises a GluR2 sequence or functional fragment thereof. In some cases, a GluR2 sequence can be recognized by an RNA editing entity, such as an ADAR or biologically active fragment thereof. In some embodiments, a GluR2 sequence can be a non-naturally occurring sequence. In some cases, a GluR2 sequence can be modified, for example for enhanced recruitment. In some embodiments, a GluR2 sequence can comprise a portion of a naturally occurring GluR2 sequence and a synthetic sequence.

[0208] 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: 93). 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: 51. 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: 51.

[0209] 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.

[0210] 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 engineered guide RNAs. In some cases, a recruiting domain can be on an N- terminus, middle, or C-terminus of an engineered guide RNA. 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.Engineered Guide RNAs with Latent Structure

[0211] 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 latent structure. “Latent structure” refers to a structural feature that substantially forms upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed, and the latent structure provided in the guide RNA is, thus, unmasked.

[0212] A double stranded RNA (dsRNA) substrate may be 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.”

[0213] 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.

[0214] 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 or APOBEC). 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 or APOBEC).

[0215] 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 or APOBEC) 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 featurescan comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge, symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, or any combination thereof.

[0216] 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).

[0217] A guide-target RNA scaffold may be 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 is an A / C mismatch. An A / C mismatch can comprise a C in an engineered guide RNA of the present disclosure opposite an A in a target RNA. An A / C mismatch can comprise an A in an engineered guide RNA of the present disclosure opposite a C in a target RNA. A G / G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA.

[0218] 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.

[0219] 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 canrefer 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.

[0220] 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.

[0221] 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.

[0222] A hairpin of the present disclosure can be of any length. In an aspect, a hairpin can be from about 10-500 or more nucleotides. In some cases, a hairpin can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,492, 493, 494, 495, 496, 497, 498, 499, 500 or more nucleotides. In other cases, a hairpin can also comprise 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to190, 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 to280, 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 to370, 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 to460, 10 to 470, 10 to 480, 10 to 490, or 10 to 500 nucleotides.

[0223] 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. A bulge can change the secondary or tertiarystructure of the guide-target RNA scaffold. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair - a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a mismatch. Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an internal loop. In some embodiments, the guide-target RNA scaffold of the present disclosure has 2 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 4 bulges. Thus, a bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0224] 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.

[0225] 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. A symmetrical bulge is formed when the same number of nucleotides is present on each side of the bulge. For example, a symmetrical bulge in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 2 nucleotides on the target RNA side of the guidetarget RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 3nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0226] 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 guidetarget RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the 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 2nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide -target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the 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.

[0227] In some cases, a structural feature can be an internal loop. As disclosed herein, an internal loop refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guidetarget 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 onthe 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.

[0228] 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.

[0229] An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. A symmetrical internal loop is formed when the same number of nucleotides is present on each side of the internal loop. For example, a symmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 8 nucleotides on the target RNA side of the guide -target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 9 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 10 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 15 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 15 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 20 nucleotides on the engineered guide RNA side of the 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 guide-target RNA scaffold target and 30 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 40 nucleotides on the engineered guide RNA side of the 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 theguide-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 the engineered guide RNA side of the guide-target RNA scaffold target and 110 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 120 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold target and 120 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 130 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 130 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 140 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 140 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the engineered guide RNA side of the guidetarget 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 guide-target 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 targetand 400 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 450 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold target and 450 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 500 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 600 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 600 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered guide RNA side of the guidetarget RNA scaffold target and 700 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 800 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 800 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 900 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 900 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 1000 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0230] An asymmetrical internal loop is formed when a different number of nucleotides is present on each side of the internal loop. For example, an asymmetrical internal loop in a guidetarget RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.

[0231] 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. Anasymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNAscaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNAscaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide -target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-targetRNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 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 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-targetRNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guidetarget RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-targetRNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the 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 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-targetRNA 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 guidetarget RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guidetarget RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guidetarget RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guidetarget RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the 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 guidetarget RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0232] 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 or focus an RNA editing entity (e.g., ADAR) and direct its activity towards 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 group consisting 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.

[0233] Guide RNAs of the present disclosure may further comprise a macro-footprint. In some embodiments, the macro-footprint comprises a barbell macro-footprint. A micro-footprint can serve to guide or focus an RNA editing enzyme and direct its activity towards the target adenosine to be edited. A “barbell” as described herein refers to a pair of internal loop latent structural features 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 ofbarbell internal loops on opposing sides of the micro-footprint, which in turn comprises at least one structural feature that facilitates editing of a specific target RNA.

[0234] 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 micro-footprint 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. Thus, inclusion of barbell structures can provide a facile method of improving editing of guide RNAs previously selected to facilitate editing of a target RNA of interest. For example, macro-footprints (e.g., barbell macrofootprints) 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 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.

[0235] 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 micro-footprint sequence, additional latent structures can be incorporated that flank either end of the macrofootprint 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 macrofootprint sequence can comprise a barbell macro-footprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop.

[0236] 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 internal loop 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.

[0237] 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, 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 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.

[0238] 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 to150, 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 to150, 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 to175, 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, 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 base pairs.Additional Engineered Guide RNA Components

[0239] 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 sequences.

[0240] 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.

[0241] 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.

[0242] 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 an engineered 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 aphosphorus-oxygen bond. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be modified into a phosphoester with a phosphorus-containing moiety.

[0243] 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 ribozyme 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.

[0244] 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 that provides 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 engineeredpolynucleotide 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 ribozyme 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.

[0245] 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 23S rRNA, a GIRI branching ribozyme, a leadzyme, a group II intron, a hairpin ribozyme, a VS ribozyme, a CPEB3 ribozyme, a CoTC ribozyme, or a group I intron. In some cases, the selfcleaving 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, primers can 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.

[0246] 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, anengineered 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.

[0247] 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 23 S 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.

[0248] 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.

[0249] 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 been re-engineered to alter splicing at desired disease targets. Replacing a portion of the U7 snRNA which naturally hybridizes to the spacer element of histone pre-mRNA (e.g., the first 18 nucleotides of the U7 snRNA) with a short targeting (or antisense) sequence of a disease gene, may redirect 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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. While AAV-2 / 1 based vectors expressing an appropriately modified murine U7 gene along with its natural promoter and 3' elements have enabled high efficiency gene transfer into the skeletal muscle and complete dystrophin rescue by covering and skipping mouse Dmd exon 23, the engineered polynucleotides as described herein (whether directly administered or administered via, for example, AAV vectors) can facilitate editing of target RNA by a deaminase.

[0254] 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.

[0255] 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 exon skipping 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.

[0256] 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. An expression cassette or polynucleotide as described herein may comprise one or moreU7 snRNA hairpin sequences, SmOPT sequences, or combinations thereof. For example, an expression cassette or polynucleotide as described herein may comprise two U7 snRNA hairpin sequences and two SmOPT sequences. 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: 17) or RNA: UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU (SEQ ID NO: 18). In some cases, a mouse U7 hairpin sequence comprises CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 19) or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU (SEQ ID NO: 20). In some cases, a hairpin sequence may comprise SEQ ID NO: 83 or SEQ ID NO: 84. In some embodiments, the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 21) or RNA: AAUUUUUGGAG (SEQ ID NO: 22). In some embodiments, an RNA payload may comprise a guide RNA, a U7 hairpin sequence (e.g., a human or a mouse U7 hairpin sequence), an SmOPT sequence, or a combination thereof. For example, an RNA payload may comprise a sequence of AATTTTTGGAGCAGGTTTTCTGACTTCGGTCGGAAAACCCCTCCCAATTTCACTGGT CTACAATGAAAGCAAAACAGTTCTCTTCCCCGCTCCCCGGTGTGTGAGAGGGGCTTT GATCCTTCTCTGGTTTCCTAGGAAACGCGTATGTG (SEQ ID NO: 23). 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. An expression cassette or polynucleotide as described herein may comprise one or more sequences comprising an SmOPT sequence linked to a U7 sequence. For example, an expression cassette or polynucleotide as described herein may comprise two sequences each comprising an SmOPT sequence linked to a 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.Guide RNA Pay loads for DNA Editing

[0257] The vectors comprising a plurality of expression cassettes described herein may be used to enhance expression of RNA components for site-specific, selective editing of a target DNA via a DNA editing entity or a biologically active fragment thereof. An RNA component for sitespecific DNA editing may comprise a guide RNA, a transactivating CRISPR RNA (tracrRNA), a single guide RNA, or engineered polynucleotides encoding the same. An engineered guide RNA, as described herein, may comprise a sequence with complementarity to a target DNA described herein. As such, a guide RNA can be engineered to site-specifically / selectively target and hybridize to a particular target DNA, thus facilitating editing of specific nucleotide in the target DNA via a DNA editing entity or a biologically active fragment thereof. DNA editingmay be facilitated by a nuclease, such as a Cas nuclease. In some embodiments, the Cas nuclease may be a Cas9, a Cas 12, or a Cas 14.

[0258] In some embodiments, an engineered guide RNA hybridizes to a sequence of the target DNA. In some embodiments, part of the engineered guide RNA hybridizes to the sequence of the target DNA. The part of the engineered guide RNA that hybridizes to the target DNA is of sufficient complementary to the sequence of the target DNA for hybridization to occur. In some embodiments, the guide RNA may comprise a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to a target DNA. A guide RNA encoded by an expression cassette of the present disclosure may comprise a length of from about 15 to about 70 nucleotides, from about 40 to about 70 nucleotides, or from about 70 to about 100 nucleotides. In some embodiments, the region of the guide RNA that hybridizes to the target may comprise a length of from about 18 to about 44 nucleotides.

[0259] In some examples, an engineered guide RNA can facilitate editing of a base of a nucleotide of in a target sequence of a target DNA that results in modulating the expression of a gene encoded by the target DNA. In some instances, modulation can be increased or decrease expression of the gene. 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 DNA by a DNA editing entity (e.g., a Cas nuclease).

[0260] In some embodiments, the vectors comprising a plurality of expression cassettes described herein may be used to enhance expression of transactivating crRNAs (tracrRNAs) and engineered polynucleotides encoding the same for editing of a target DNA via a DNA editing entity or a biologically active fragment thereof. The tracrRNA may bind to and activate a DNA editing enzyme (e.g., a Cas nuclease). A tracrRNA encoded by an expression cassette of a vector of the present disclosure may comprise a length of from about 75 to about 100 nucleotides.

[0261] In some embodiments, the expression cassettes described herein may be used to enhance expression of a single guide RNA and engineered polynucleotides encoding the same for editing of a target DNA via a DNA editing entity or a biologically active fragment thereof. The single guide RNA may comprise a region that binds to and activates a DNA editing enzyme (e.g., a Cas nuclease) and a region that hybridizes to the sequence of the target DNA. The part of the single guide RNA that hybridizes to the target DNA is of sufficient complementary to the sequence of the target DNA for hybridization to occur. In some embodiments, the single guide RNA may comprise a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to a target DNA. A single guide RNA encoded by an expression cassette of the present disclosuremay comprise a length of from about 80 to about 120 nucleotides. In some embodiments, the region of the single guide RNA that hybridizes to the target may comprise a length of from about 18 to about 44 nucleotides.Other RNA-Targeting Oligonucleotides

[0262] The vectors comprising a plurality of expression cassettes described herein may be used to enhance expression of other engineered RNA-targeting oligonucleotides, including antisense oligonucleotides, siRNAs, shRNAs, and miRNAs, and engineered polynucleotides encoding the same that hybridizes to a target RNA (e.g., a target mRNA or a target pre-mRNA). An engineered oligonucleotide, as described herein, may comprise a targeting domain with complementarity to a target RNA described herein. As such, an oligonucleotide can be engineered to target and hybridize to a particular target RNA, thus altering expression of a polypeptide encoded by the target RNA.

[0263] In some embodiments, the engineered oligonucleotide (e.g., antisense oligonucleotide, siRNA, shRNA, or miRNA) of the present disclosure hybridizes to a sequence of the target RNA. In some embodiments, part of the engineered oligonucleotide (e.g., a targeting domain) hybridizes to the sequence of the target RNA. The part of the engineered oligonucleotide that hybridizes to the target RNA is of sufficient complementary to the sequence of the target RNA for hybridization to occur. A targeting sequence can also be referred to as a “targeting domain” or a “targeting region.” In some embodiments, binding of the engineered oligonucleotide to the target RNA may recruit additional components, such as RISC components.

[0264] In some cases, a targeting domain of an engineered oligonucleotide allows the engineered oligonucleotide to target an RNA sequence through base pairing, such as Watson Crick base pairing. In some examples, the targeting sequence can be located at either the N- terminus or C-terminus of the engineered oligonucleotide. In some cases, the targeting sequence can be located at both termini. The targeting sequence can be of any length. In some cases, the targeting sequence can be at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or up to about 200 nucleotides in length. In some cases, the targeting sequence can be no greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, or 200 nucleotides in length. In some examples, an engineered oligonucleotide comprises a targeting sequence that can be from about 60 to about 500, from about 60 to about 200, from about 75 to about 100, from about 80 to about 200, from about 90 to about 120, or from about 95 to about 115 nucleotides in length. In some examples, an engineered oligonucleotide comprises a targeting sequence that can be about 100 nucleotides in length.

[0265] In some cases, a targeting domain comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to a target RNA. In some cases, a targeting sequence comprises less than 100% complementarity to a target RNA sequence. For example, a targeting sequence and a region of a target RNA that can be bound by the targeting sequence can have a single base mismatch.

[0266] The targeting sequence can have sufficient complementarity to a target RNA to allow for hybridization of the targeting sequence to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 50 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 60 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 70 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 80 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 90 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 100 nucleotides or more to the target RNA. In some embodiments, antisense complementarity refers to non-contiguous stretches of sequence. In some embodiments, antisense complementarity refers to contiguous stretches of sequence.Chemical Modifications

[0267] 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, 11, 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 chemicalmodification. 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).

[0268] 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’ O-methyl RNA, methylphosphonate, phospho diester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 ’-triphosphate, 5 -methylcytidine-5 ’-triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof.

[0269] 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,I I, 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 I I, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150. 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.

[0270] In some embodiments, a chemical modification can also be a phosphorothioate substitute. In some cases, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and a modification of internucleotide 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 can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases.

[0271] 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.

[0272] 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 ribosephosphate 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 3.TABLE 3 - Exemplary Chemical ModificationModification of the Phosphate Backbone

[0273] 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 alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n- pentyl, isopentyl, or neopentyl). An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. As used herein, “aryl” 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 alkynyl 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, tetrahydro furanyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. “Heteroaryl” may refer to a monovalent radical of aheteroaromatic 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.

[0274] 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 phospho triesters. 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 stereogenic 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 non-bridging oxygens can also include the replacement of the nonbridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). In some embodiments, the phosphate linker can also be modified byreplacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). In some cases, the replacement can occur at either or both of the linking oxygens.

[0275] 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-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0276] In some cases, backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and can be used in any combination. Other non-phosphate linkages may also be used.

[0277] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and / or enhance their stability in vivo.

[0278] 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.

[0279] 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; peptidenucleic 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 phosphoro thioate linkages.

[0280] In some cases, substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts. It 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.

[0281] 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 followinggroups: 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 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.

[0282] 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., alkyl or aryl).

[0283] In some cases, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.Replacement of Phosphate Moiety

[0284] 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 alkyl phosphonates including 3 ’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 ’-amino phosphoramidate and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In some cases, the phosphate or modified phosphate linkage between two nucleotides can be through a 3 ’-5’ linkage or a 2 ’-5’ linkage, and the linkage contains inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’.Substitution of Phosphate Group

[0285] 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, thio formacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino .Modification of the Ribophosphate Backbone

[0286] 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

[0287] 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 intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxy" -2’ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2’ hydroxyl group modification can include (LNA, in which the 2’ hydroxyl can be connected, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH ; 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(CH2CH2NH)nCH2CH2-amino (wherein amino can be, e.g., as described herein), NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which can be optionally substituted with e.g., an amino as described herein. In some instances, the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the T position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack 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 and modifiednucleotides 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 (BN A).Modification of a Constituent of the Ribose Sugar

[0288] 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’-O- methoxy-ethyl (2’-MOE), 2’-fluoro, 2 ’-aminoethyl, 2’-deoxy-2’-fuloarabinou-cleic acid, 2'- deoxy, 2'-O-methyl, 3'-phosphorothioate, 3'-phosphonoacetate (PACE), or 3'- phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the constituent of the ribose sugar comprises unnatural nucleic acid. In some instances, the unnatural nucleic acids include modifications at the 5 ’-position and the 2’-position of the sugar ring, such as 5’-CH2-substituted 2’-O-protected nucleosides. In some cases, unnatural nucleic acids include amide linked nucleoside dimers that can be prepared for incorporation into oligonucleotides. In some cases, the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH3 and a 5’-(S)- CH3. Unnatural nucleic acids can include 2 ’-substituted 5’-CH2 (or O) modified nucleosides. Unnatural nucleic acids can include 5’-methylenephosphonate DNA and RNA monomers, and dimers. Unnatural nucleic acids can include 5 ’-phosphonate monomers having a 2 ’-substitution and other modified 5 ’-phosphonate monomers. Unnatural nucleic acids can include 5 ’-modified methylenephosphonate monomers. Unnatural nucleic acids can include analogs of 5’ or 6’- phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and / or 6’-position.Unnatural nucleic acids can include 5 ’-phosphonate deoxyribonucleoside monomers and dimers having a 5 ’-phosphate group. Unnatural nucleic acids can include nucleosides having a 6’- phosphonate group wherein the 5’ or / and 6 ’-position may be unsubstituted or substituted with athio-tert-butyl group (SC(CH )3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof).

[0289] 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)(R.2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof.

[0290] 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-RNAZDNA chimeras. For example, a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O-ethyleneglycol-like ribonucleosides.

[0291] 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)nOCH3,- 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, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving thepharmacodynamic 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 Ci- C10 alkyl.

[0292] 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’-(CH2)2-O-2’ (ENA); 4’-CH(CH3)-O- 2’ and 4’-CH(CH2OCH3)-O-2’, and analogs thereof; 4’-C(CH3)(CH3)-O-2’and analogs thereof.Modifications on the Base of Nucleotide

[0293] 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.

[0294] In some embodiments, the chemical modification described herein can comprise modifying an uracil. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified uracil. Exemplary chemically modified uracil can include pseudouridine, pyridin-4-one ribonucleoside, 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza- uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 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 methylester, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5 -carboxyhydroxymethyluridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5 -methylaminomethyluridine, 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, 1- 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 -methyldihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N 1 - methyl-pseudouridine, 3-(3-amino-3-carboxypropyl) uridine, l-methyl-3-(3-amino-3- carboxypropy pseudouridine, 5-(isopentenylaminomethyl) uridine, 5-(isopentenylaminomethy])- 2-thio-uridine, a-thio-uridine, 2’-O-methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl- pseudouridine, 2-thio-2’-O-methyl-uridine, 5-methoxycarbonylmethyl-2’-O-methyl -uridine, 5- carbamoylmethyl-2 ’ -O-methyl-uridine, 5 -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-( l-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

[0295] 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, pseudo isocytidine, 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-pseudo isocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio-l-methyl- 1 -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- 1-methyl-pseudoisocytidine, lysidine, a- thio-cytidine, 2’-O- methyl-cytidine, 5,2’-O-dimethyl-cytidine, 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.

[0296] 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-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl- adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl) adenosine , 2-methylthio- N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyl-adenosine, N6- threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6- threonylcarbamoyl-adenosine, N6, N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl- adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7- methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl- adenosine, N6, 2’-O-dimethyl-adenosine, N6-Methyl-2’-deoxyadenosine, N6, N6, 2’-O- trimethyl-adenosine, 1 ,2’-O-dimethyl-adenosine, 2’-O-ribosyladenosine (phosphate) (Ar(p)), 2- amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F- adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0297] In some embodiments, the chemical modification described herein can comprise modifying a guanine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified guanine. Exemplary chemically modified guanine can include inosine, 1 -methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undemriodified hydroxywybutosine, 7- deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7- cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1- methyl-guanosine, N2-methyl-guanosine, N2, N2-dimethyl-guanosine, N2, 7-dimethyl- guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- meththio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio- guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O- methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2, 7-dimethyl-2’-O-methyl-guanosine, 2’- O-methyl-inosine, 1 , 2 ’-O-dimethyl- inosine, 6-O-phenyl-2’ -deoxyinosine, 2’-O-ribosylguanosine, 1 -thio-guanosine, 6-O-methyguanosine, O6-Methyl-2’ -deoxy guanosine, 2’-F- ara-guanosine, and 2’-F-guanosine.

[0298] In some cases, the chemical modification of the engineered guide RNA can include introducing or substituting a nucleic acid analog or an unnatural nucleic acid into the engineered guide RNA. In some embodiments, nucleic acid analog can be any one of the chemically modified nucleic acid described herein. Exemplary nucleic acid analog can be found in PCT / US2021 / 034272, PCT / US2015 / 025175, PCT / US2014 / 050423, PCI7US2016 / 067353, PCT / US2018 / 041503, PCT / US 18 / 041509, PCI7US2004 / 011786, or PCT / US2004 / 011833, all of which are expressly incorporated by reference in their entireties. In some cases, the chemically modified nucleotide described herein can include a variant of guanosine, uridine, adenosine, thymidine, and cytosine, including any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytidine that has been altered chemically, for example by acetylation, methylation, hydroxylation. Exemplary chemically modified nucleotide can include 1 -methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl- guanosine, 2,6-diaminopurine, 2 ’-amino-2’ -deoxyadenosine, 2 ’-amino-2’ -deoxy cytidine, 2’- amino-2’ -deoxy guanosine, 2 ’-amino-2 ’-deoxyuridine, 2-amino-6-chloropurineriboside, 2- aminopurine-riboside, 2’-araadenosine, 2’-aracytidine, 2’-arauridine, 2’-azido-2’- deoxyadenosine, 2 ’-azido-2’ -deoxy cytidine, 2’-azido-2’-deoxyguanosine, 2’-azido-2’- deoxyuridine, 2-chloroadenosine, 2’-fluoro-2’-deoxyadenosine, 2 ’-fluoro-2’ -deoxycytidine, 2’- fluoro-2’ -deoxyguanosine, 2’-fluoro-2’-deoxyuridine, 2 ’-fluoro thymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2’-O-methyl-2-aminoadenosine, 2’-O-methyl-2’-deoxyadenosine, 2’-O-methyl-2’-deoxycytidine, 2 ‘-O-methyl-2’- deoxyguanosine, 2,-O-methyl-2’-deoxyuridine, 2 ’-O-methyl-5 -methyluridine, 2’-O- methylinosine, 2’-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4- acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine, 5- aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5 -bromouridine, 5-carboxymethylaminomethyl- 2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-chloro-ara-cytosine, 5 -fluoro-uridine, 5- iodouridine, 5-methoxycarbonylmethyl-uridine, 5 -methoxy-uridine, 5-methyl-2-thio-uridine, 6- Azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurineriboside, 6-mercapto- guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2’-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto- guanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D-mannosyl-queosine, dihydrouridine, inosine, N 1 -methyladenosine, N6-([6-ami nohexyl] carbamoylmethyl)-adenosine, N6- isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine, uracil-5 -oxyacetic acid, uracil-5 -oxyacetic acid methyl ester,wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 2-amino-6-chloropurineriboside-5 ’ -triphosphate, 2-aminopurine -riboside-5 ’ -triphosphate, 2-aminoadenosine-5 ’-triphosphate, 2’-amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5 ’- triphosphate, 2-thiouridine-5 ’-triphosphate, 2 ’-fluorothymidine-5 ’-triphosphate, 2’-O-methyl- inosine-5 ’-triphosphate, 4-thiouridine-5 ’-triphosphate, -aminoallylcytidine-5 ’-triphosphate, 5- aminoallyluridine-5 ’ -triphosphate, 5 -bromocytidine-5 ’ -triphosphate, 5 -bromouridine-5 ’ - triphosphate, 5 -bromo-2 ’ -deoxycytidine-5 ’ -triphosphate, 5 -bromo-2 ’-deoxyuridine-5 ’ - triphosphate, 5 -iodocytidine- ’-triphosphate, 5-iodo-2’-deo...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A polynucleotide comprising a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence, and the first and second expression cassette sequences are different sequences.

2. The polynucleotide of claim 1, wherein the first expression cassette sequence and the second expression cassette sequence are orientated in a tandem read orientation.

3. The polynucleotide of claim 1 or claim 2, wherein the DNA sequence encoding a promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 67.

4. The polynucleotide of any one of claims 1-3, wherein the DNA sequence encoding a transcription termination comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.

5. The polynucleotide of any one of claims 1 -4, wherein the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92.

6. The polynucleotide of any one of claims 1-5, wherein: the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, and the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79.

7. The polynucleotide of any one of claims 1-6, wherein: the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

8. The polynucleotide of any one of claims 1-7, wherein: the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, and the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67.

9. The polynucleotide of any one of claims 1-8, wherein: the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

10. The polynucleotide of any one of claims 1-9, wherein: the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5,the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78 or SEQ ID NO: 79.11 . The polynucleotide of any one of claims 1-10, wherein: the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79 or SEQ ID NO: 80.

12. The polynucleotide of any one of claims 1-11, wherein: the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78, the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, andthe DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79.

13. The polynucleotide of any one of claims 1-12, wherein: the DNA sequence encoding a promoter sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, the DNA sequence encoding a transcription termination sequence of the first expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79, the DNA sequence encoding a promoter sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67, the DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 92, and the DNA sequence encoding a transcription termination sequence of the second expression cassette sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80.

14. A polynucleotide comprising a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising a small RNA payload, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences.

15. The polynucleotide of claim 14, wherein the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of reverse.

16. The polynucleotide of claim 14, wherein the first expression cassette sequence has a read directionality of reverse and the second expression cassette sequence has a read directionality of forward.

17. The polynucleotide of claim 14, wherein the first expression cassette sequence has a read directionality of forward and the second expression cassette sequence has a read directionality of forward.

18. The polynucleotide of any one of claims 14-17, wherein the plurality of expression cassette sequences comprises two expression cassette sequences, three expression cassette sequences, four expression cassette sequences, five expression cassette sequences, six expression cassette sequences, seven expression cassette sequences, eight expression cassette sequences, nine expression cassette sequences, or ten expression cassette sequences.

19. The polynucleotide of any one of claims 14-18, wherein the small RNA payload comprises an engineered guide RNA sequence.

20. The polynucleotide of any one of claims 1-19, wherein the engineered guide RNA sequence is capable of hybridizing to a target sequence.21 . The polynucleotide of any one of claims 1-20, wherein the engineered guide RNA sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% reverse complementary to the target sequence.

22. The polynucleotide of any one of claims 1-21, wherein the engineered guide RNA sequence is capable of forming a guide-target RNA scaffold comprising one or more structural features upon hybridization to a target sequence.

23. The polynucleotide of claim 22, wherein the one or more structural features comprise a bulge, a mismatch, an internal loop, a hairpin, or combinations thereof.

24. The polynucleotide of claim 22 or claim 23, wherein the one or more structural features comprises the bulge, and wherein the bulge is a symmetric bulge.

25. The polynucleotide of any one of claims 22-24, wherein the one or more structural features comprises the bulge, and wherein the bulge is an asymmetric bulge.

26. The polynucleotide of any one of claims 22-25, wherein the one or more structural features comprises the internal loop, and wherein the internal loop is a symmetric internal loop.

27. The polynucleotide of any one of claims 22-26, wherein the one or more structural features comprises the internal loop, and wherein the internal loop is an asymmetric internal loop.

28. The polynucleotide of any one of claims 22-27, wherein the one or more structural features comprises the hairpin, and wherein the hairpin is a recruitment hairpin or a nonrecruitment hairpin.

29. The polynucleotide of any one of claims 22-28, wherein the guide-target RNA scaffold comprises one or more wobble base pairs.

30. The polynucleotide of claim 29, wherein the one or more of the wobble base pairs are GU wobble base pairs.

31. The polynucleotide of any one of claims 20-30, wherein the engineered guide RNA sequence comprises at least one base pair mismatch relative to the target sequence.

32. The polynucleotide of any one of claims 20-31 , wherein the target sequence comprises an adenosine residue.

33. The polynucleotide of any one of claims 20-32, wherein the target sequence is an RNA sequence.

34. The polynucleotide of claim 33, wherein the RNA sequence is a mRNA or a pre-mRNA.

35. The polynucleotide of any one of claims 20-34, wherein the target sequence comprises a G to A mutation relative to a wild type sequence.

36. The polynucleotide of any one of claims 20-35, wherein the target sequence comprises a missense mutation or a nonsense mutation relative to a wild type sequence.

37. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes a- synuclein (SNCA).

38. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes peripheral myelin protein 22 (PMP22).

39. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes double homeobox 4 (DUX4).

40. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes leucine rich repeat kinase 2 (LRRK2).

41. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes Tau (MAPT).

42. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes ATP-binding cassette sub-family A member 4 (ABCA4).

43. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes alpha-1 antitrypsin (SERPINA1).

44. The polynucleotide of any one of claims 20-36, wherein the target sequence encodes methyl CpG binding protein 2 (MECP2).

45. The polynucleotide of any one of claims 1-44, wherein the engineered guide RNA sequence is not less than 20 nucleotide residues and not more than 500 nucleotide residues long.

46. The polynucleotide of any one of claims 1-45, wherein the engineered guide RNA sequence is not less than 60 and not more than 100 residues long.

47. The polynucleotide of any one of claims 1-46, wherein the engineered guide RNA sequence is not less than 80 and not more than 120 residues long.

48. The polynucleotide of any one of claims 1-47, wherein the engineered guide RNA sequence is not less than 100 and not more than 140 residues long.

49. The polynucleotide of any one of claims 1-48, wherein the engineered guide RNA sequence is not less than 130 and not more than 170 residues long.

50. The polynucleotide of any one of claims 14-49, wherein the promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91.

51. The polynucleotide of any one of claims 14-50, wherein the transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

52. The polynucleotide of any one of claims 14-51, wherein the payload sequence further comprises an Sm binding sequence or a hairpin sequence.

53. The polynucleotide of claim 52, wherein the hairpin sequence comprises a U7 hairpin.

54. The polynucleotide of any one of claims 1-53, wherein the first expression cassette sequence, the second expression cassette sequence, or each expression cassette of the plurality of expression cassette sequences independently has a length of not less than 1300 nucleotide residues and not more than 2160 nucleotide residues.

55. The polynucleotide of any one of claims 14-54, wherein the first expression cassette sequence, the second expression cassette sequence, or each expression cassette of the plurality of expression cassette sequences independently comprises at least 80% sequence identity to a U1 sequence or a U7 sequence.

56. The polynucleotide of claim 55, wherein the U1 sequence is a mouse U1 sequence or a human U1 sequence.

57. The polynucleotide of claim 55 or claim 56, wherein the U7 sequence is a mouse U7 sequence or a human U7 sequence.

58. A viral vector encoding the polynucleotide of any one of claims 1-57.

59. The viral vector of claim 58, wherein the viral vector is an adeno-associated viral vector.

60. The viral vector of claim 59, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV- DJ / 9, AAV1 / 2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, variants or derivatives thereof, and combinations thereof.

61. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-57 or the viral vector of any one of claims 58-60 and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

62. A method of expressing an engineered guide RNA in a cell, the method comprising:(i) delivering a composition comprising a polynucleotide to a cell, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequences; and(ii) expressing the engineered guide RNA (gRNA) sequence in the cell.

63. A method of editing a target sequence, the method comprising:(i) delivering a composition comprising a polynucleotide to a cell encoding a target sequence, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence;at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequences;(ii) expressing the engineered guide RNA (gRNA) sequence in the cell;(iii) forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA (gRNA) sequence to the target sequence;(iv) recruiting an editing enzyme to the target sequence; and(v) editing the target sequence with the editing enzyme.

64. A method of expressing an engineered guide RNA sequence in a cell, the method comprising:(i) delivering a composition comprising a polynucleotide to a cell, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences; and(ii) expressing the engineered guide RNA sequence in the cell.

65. A method of editing a target sequence, the method comprising:(i) delivering a composition comprising a polynucleotide to a cell encoding a target sequence, wherein the polynucleotide comprises a plurality of expression cassette sequencescomprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences;(ii) expressing the engineered guide RNA sequence in the cell;(iii) forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA sequence to the target sequence;(iv) recruiting an editing enzyme to the target sequence; and(v) editing the target sequence with the editing enzyme.

66. A method of administering a polynucleotide to a subject with a disease, the method comprising:(i) administering to the subject a composition comprising the polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5;the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequence;(ii) delivering the polynucleotide to a cell of the subject; and(iii) expressing the engineered guide RNA (gRNA) sequence in the cell.

67. A method of treating a disease in a subject, the method comprising:(i) administering to the subject a composition comprising a polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first expression cassette sequence and the second expression cassette sequence each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; at least one of the DNA sequences encoding a promoter sequence comprises at least 80% sequence identity to SEQ ID NO: 5; the first expression cassette sequence and the second expression cassette sequence are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward; the DNA sequence encoding an engineered guide RNA (gRNA) sequence of the first expression cassette sequence and the second expression cassette sequence encodes the same engineered guide RNA (gRNA) sequence; and the first and second expression cassette sequences are different sequence;(ii) delivering the polynucleotide to a cell of the subject; and(iii) expressing the engineered guide RNA (gRNA) sequence in the cell, thereby treating the disease.

68. A method of administering a polynucleotide to a subject with a disease, the method comprising:(i) administering to the subject a composition comprising the polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences;(ii) delivering the therapeutic polynucleotide to a cell of the subject; and(iii) expressing the engineered guide RNA sequence in the cell.

69. A method of treating a disease in a subject, the method comprising:(i) administering to the subject a composition comprising a polynucleotide, wherein the polynucleotide comprises a plurality of expression cassette sequences comprising a first expression cassette sequence and a second expression cassette sequence, wherein: the first, second, and plurality of expression cassette sequences each independently comprise: a promoter sequence, a payload sequence under transcriptional control of the promoter sequence, the payload sequence comprising an engineered guide RNA sequence, and a transcription termination sequence; the first, second, and plurality of expression cassette sequences are each independently arranged in a 5 ’ to 3 ’ orientation to have a read directionality of forward or reverse; and the first and second expression cassette sequences are different sequences;(ii) delivering the therapeutic polynucleotide to a cell of the subject; and(iii) expressing the engineered guide RNA sequence in the cell, thereby treating the disease.

70. The method of any one of claims 62-69, wherein the cell is in a central nervous system tissue.

71. The method of any one of claims 62-69, wherein the cell is in a liver tissue, muscle tissue, ocular tissue, retinal tissue, heart tissue, skeletal muscle tissue, or kidney tissue.

72. The method of any one of claims 62-71, wherein the composition is the pharmaceutical composition of claim 61.

73. The method of any one of claims 62-72, wherein the composition comprises the polynucleotide of any one of claims 1-57 or the viral vector of any one of claims 58-60.

74. The method of any one of claims 66-73, wherein the disease is a synucleinopathy, Parkinson’s disease, Lewy body dementia, multiple system atrophy, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, Yuan-Hare 1-Lupski syndrome, a tauopathy, Alzheimer’s disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, autism, traumatic brain injury, Dravet syndrome, Crohn’s disease, muscular dystrophy, B-cell leukemia, Dejerine-Sottas disease, Stargardt disease, alpha-1 antitrypsin deficiency, Tay-Sachs disease, cystic fibrosis, liposomal acid lipase deficiency, or Gaucher disease.

75. The method of any one of claims 62, 64, or 66-74, wherein the engineered guide RNA sequence hybridizes to a target sequence, and wherein the cell encodes the target sequence.

76. The method of claim 75, further comprising forming a guide-target RNA scaffold upon hybridization of the engineered guide RNA to the target sequence, recruiting an editing enzyme to the target sequence, and editing the target sequence with the editing enzyme.

77. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes a-synuclein (SNCA).

78. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes peripheral myelin protein 22 (PMP22).

79. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes double homeobox 4 (DUX4).

80. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes leucine rich repeat kinase 2 (LRRK2).

81. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodesTau (MAPT).

82. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes ATP-binding cassette sub-family A member 4 (ABCA4).

83. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes alpha-1 antitrypsin (SERPINA1).

84. The method of any one of claims 63, 65, or 70-76, wherein the target sequence encodes methyl CpG binding protein 2 (MECP2).

85. The method of any one of claims 63, 65, or 70-84, wherein the target sequence comprises a mutation relative to a wild type sequence.

86. The method of claim 85, wherein editing the target sequence corrects the mutation in the target sequence.

87. The method of claim 85 or claim 86, wherein the mutation is a missense mutation.

88. The method of claim 85 or claim 86, wherein the mutation is a nonsense mutation.

89. The method of any one of claims 85-88, wherein the mutation is a G to A mutation.

90. The method of any one of claims 85-89, wherein the mutation is associated with the disease.

91. The method of any one of claims 63, 65, or 70-76, wherein editing the target sequence comprises editing an untranslated region of the target sequence.

92. The method of claim 91 , wherein the untranslated region is a 5 ’ untranslated region or a 3 ’ untranslated region.

93. The method of claim 92, wherein the 3 ’ untranslated region is a polyadenylation sequence.

94. The method of any one of claims 63, 65, or 70-93, wherein editing the target sequence comprises editing a translation initiation site.

95. The method of any one of claims 63, 65, or 70-94, wherein editing the target sequence alters expression of the target sequence.

96. The method of claim 95, wherein editing the target sequence increases expression of the target sequence.

97. The method of claim 95, wherein editing the target sequence decreases expression of the target sequence.

98. The method of any one of claims 63, 65, or 70-97, wherein the editing enzyme comprises an ADAR, an APOBEC, or a Cas nuclease.

99. The method of claim 98, wherein the ADAR comprises AD ARI, ADAR2, or a combination thereof.

100. The method of any one of claims 63, 65, or 70-99, wherein the target sequence comprises RNA or DNA.101 . The method of any one of claims 63, 65, or 70-100, wherein the target sequence is a mRNA or a pre-mRNA.

102. The method of any one of claims 63, 65, or 70-101, wherein editing the target sequence comprises deamidating a nucleotide of the target sequence.

103. The method of any one of claims 63, 65, or 70-102, wherein the target sequence is edited with an efficiency of at least 10%, at least 20%, or at least 25%.

104. A method of increasing a vector genome integrity of a multi-expression cassette vector comprising a first expression cassette and a second expression cassette, the method comprising: a) generating a sequence of the second expression cassette by altering a sequence of the first expression cassette, wherein the first expression cassette and the second expression cassette each independently comprise: a DNA sequence encoding a promoter sequence, a DNA sequence encoding an engineered guide RNA (gRNA) sequence under transcriptional control of the promoter sequence, a DNA sequence encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence, and a DNA sequence encoding a transcription termination sequence; b) generating the multi-expression cassette vector by combining the sequence of the first expression cassette and the sequence of the second expression cassette; and c) increasing the vector genome integrity as compared to a vector comprising a first expression cassette and a second expression cassette that each have the sequence of the first expression cassette.

105. The method of claim 104, wherein the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a promoter sequence.

106. The method of claim 104 or claim 105, wherein the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a transcription termination sequence.

107. The method of any one of claims 104-106, wherein the sequence of the first expression cassette and the sequence of the second expression cassette have different DNA sequences encoding a SmOPT and a small nuclear RNA (snRNA) processing hairpin sequence.

108. The method of any one of claims 104-107, wherein the DNA sequence encoding a promoter sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 9, SEQ ID NO: 66 - SEQ ID NO: 76, SEQ ID NO: 90, or SEQ ID NO: 91.

109. The method of any one of claims 104-108, wherein the DNA sequence encoding a transcription termination sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NO: 10 - SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 77 - SEQ ID NO: 82, or SEQ ID NO: 85 - SEQ ID NO: 89.

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