Usher syndrome-related gene modification

Abstract

The disclosure relates to compositions, systems, and methods related to repair RNA sequences, as well as related systems for trans-splicing a target nucleic acid using a repair RNA sequence. Various compositions and systems are described herein, including compositions comprising a repair RNA sequence comprising one or more exons and / or introns, a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing. The compositions, systems, and methods described herein are suitable for targeting one or more Usher syndrome-associated genes, and treating Usher syndrome.

Description

[0001] USHER SYNDROME-RELATED GENE MODIFICATION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 519,830, filed on August 15, 2023, U.S. Provisional Application No. 63 / 602,531, filed on November 24, 2023, and U.S. Provisional Application No.: 63 / 554,908, filed on February 16, 2024, the entire contents of which are incorporated herein.

[0004] FIELD

[0005] The disclosure relates to compositions, systems, and methods for trans-splicing a target nucleic acid comprising a repair RNA.

[0006] DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0007] The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy named “AMR-012PC_134241-5012_Sequence Listing,” which was created on August 15, 2024 and is 1,961,984 bytes in size, the contents of which are incorporated herein by reference in their entirety.

[0008] BACKGROUND

[0009] Usher syndrome is a condition typically characterized by partial or total hearing loss and vision loss that worsens over time. Usher syndrome encompasses a triad of sensory dysfunction: retinitis pigmentosa, congenital auditory deficits and variable vestibular function. Patients are placed into one of three categories (type I, II or III) based on the severity of their phenotype. These types are distinguished by the severity of hearing loss, the presence or absence of balance problems, and the age at which signs and symptoms appear. The types are further divided into subtypes based on their genetic cause. Type I Usher syndrome is generally recognized as the most severe form: profound deafness is usually present from birth and sight loss occurs early in development. Balance is usually affected for type II Usher Syndrome (USH2), which generally manifests as a milder early onset hearing loss and sight loss progresses during adolescence. Type II Usher patients represent about 50% of Usher cases. The rarest form is type III, which can start with a loss of night vision around puberty and hearing loss develops in late childhood. To date, clinicians have explored several approaches to treating Usher Syndrome, such as the use of dual vector systems, or suboptimal CRISPR tools that can fit within a single AAV vector to target Usher Syndrome. Tools outside of the CRISPR domain have also been investigated, such as stem cell therapies and antisense oligonucleotides. However, all of these approaches remain problematic due to a combination of unwanted off target effects, lack of efficiency, or limited applicability.

[0010] Accordingly, there is a need for new and reliable genetic therapies without off-target effects, and related methods, e.g., for treating Usher Syndrome.

[0011] SUMMARY

[0012] Therefore, the present disclosure provides, in aspects, a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing.

[0013] Additionally, the present disclosure provides, in aspects, a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor.

[0014] In embodiments, the one or more exons is or comprises one or more exons of a target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of a target nucleic acid molecule. In embodiments, the one or more exons is or comprises one or more exons of the target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of the target nucleic acid molecule.

[0015] In embodiments, the repRNA comprises one or more binding motifs that direct, and / or hybridizes, the repRNA to a target nucleic acid molecule. In embodiments, the one or more binding motifs indirectly or directly bind to, and / or hybridize to, a target nucleic acid molecule. In embodiments, the one or more binding motifs comprises a sequence that is antisense to a target nucleic acid molecule. In embodiments, the one or more binding motifs hybridizes to an exon and / or an intron, or a fragment thereof, of the target nucleic acid. In embodiments, the one or more binding motifs hybridizes to a fragment of an exon of the target nucleic acid. In embodiments, the one or more binding motifs hybridizes to a fragment of an intron of the target nucleic acid. In embodiments, the one or more binding motifs comprises about 10-300 nucleotides, about 15- 300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0016] In embodiments, the one or more binding motifs is within about 10-500 nucleotides, about 15-500 nucleotides, about 20-500 nucleotides, about 30-500 nucleotides, about 40-500 nucleotides, about 50-500 nucleotides, or about 60-500 nucleotides, or about 70-500 nucleotides, or about 80-500 nucleotides, or about 90-500 nucleotides, about 100-500 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, or about 300-400 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 nucleotides, at least about 400 nucleotides, or at least about 500 nucleotides of a sequence that binds to, and / or hybridizes to, a RNA-binding polypeptide.

[0017] In embodiments, the composition or system disclosed herein comprises one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the sequence that binds to the RNA-binding polypeptide assembles into a secondary structure suitable for interaction with the RNA-binding polypeptide. In embodiments, the secondary structure is or comprises a hairpin. In embodiments, the secondary structure is or comprises a stem, internal loop, multibranch loop, or a pseudoknot. In embodiments, the RNA-binding protein is a viral protein.

[0018] In embodiments, the RNA-binding polypeptide is any RNA-binding polypeptide, optionally wherein the RNA-binding polypeptide is selected from MS2 coat protein (MCP), PP7 coat protein, PRR1, Hgall, a Qbeta coat protein, the IN protein, SLBP (Stem-Loop Histone MRNA Binding Protein), and M protein, or a variant thereof. In embodiments, the RNA-binding protein is MS2. In embodiments, the RNA-binding protein is a PP7 coat protein. In embodiments, the RNA- binding protein is PRR1. In embodiments, the RNA-binding protein is Hgall. In embodiments, the RNA-binding protein is Qbeta coat protein. In embodiments, the RNA-binding protein is the IN protein, or the SLBP protein. In embodiments, the RNA-binding protein is the M protein.

[0019] In embodiments, the one or more binding motifs comprises a recognition sequence for ribonucleoprotein (RNP) complex formation.

[0020] In embodiments, the composition or system further comprises a protein that forms an RNP or is within an RNP, with the one or more binding motifs (e.g., without limitation snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP. In embodiments, the one or more binding motifs (e.g., without limitation snRNA or snoRNA), protein that forms or is within an RNP, a protein within the RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates RNP activity, and / or leads to attenuation of RNA modifying activity as compared to an unmodified form, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation. In embodiments, the one or more binding motifs (e.g., without limitation snRNA or snoRNA), protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that increases, stimulates, or enhances RNP activity, or enhances RNA modifying activity as compared to an unmodified form, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation.

[0021] In embodiments, the composition or system comprises a repair RNA (repRNA) and / or a protein that forms or is within an RNP, with the one or more binding motifs (e.g., without limitation snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme. In embodiments, the composition or system comprises a repair RNA (repRNA) and / or a protein that forms or is within an RNP, with the one or more binding motifs (e.g., without limitation snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme when the present methods are undertaken in cis or trans, as described herein. In embodiments, cleavage is initiated from RNPs that are formed on the repRNA, or from RNPs that are formed in cis or trans.

[0022] In embodiments, the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide, which is herein described as a “grepRNA”.

[0023] In embodiments, composition or system comprises a splice acceptor. In embodiments, composition or system comprises a splice donor.

[0024] In embodiments, the (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in cis or trans, or are suitable for being provided in cis or trans. In embodiments, the (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in trans, or are suitable for being provided in trans. In embodiments, these elements are under the control of one or more promoters. In embodiments, these elements are under the control of different promoters. In embodiments, these elements are operably linked, but separated by a cleavable sequence (e.g., a self-cleaving ribozyme). In embodiments, (i) multiple populations of repRNA are under the control of different promoters or (ii) the repRNA and another system member under control of different promoters.

[0025] In embodiments, the one or more binding motifs comprises a sequence from a small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises a sequence from the snRNA or the snoRNA. In embodiments, the snRNAs comprises Ul, U2, U3, U4, U5, U6, U7, U8, U9, U10 or Ul 1, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the snRNA or snoRNA is selected from any one of SEQ ID NOs: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions, optionally wherein any one of SEQ ID NOs: 144-802 form an RNP complex, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0026] In embodiments, the snRNA or snoRNA target one or more exonic splicing enhancers (ESEs), one or more intronic splicing enhancers (ISEs), one or more exonic splicing silencers (ESSs), and / or one or more intronic splicing silencers (ISSs). In embodiments, the snRNA or snoRNA comprise a modification to include at least one or more exonic splicing enhancers (ESEs), at least one or more intronic splicing enhancers (ISEs), at least one or more exonic splicing silencers (ESSs), and / or at least one or more intronic splicing silencers (ISSs).

[0027] In embodiments, the composition or system comprises a repair RNA (repRNA) and a small RNA that induces cleavage in an RNA. In embodiments, the small RNA that induces cleavage in an RNA is one or more of an siRNA, small hairpin RNA (shRNA), U7 snRNA, a U1 snRNA, a U2 snRNA, a U4 snRNA, a U4atac snRNA, a U5 snRNA, a U6 snRNA, a U6atac snRNA, a Ul l snRNA, aU12 snRNA, and an antisense oligonucleotide (ASO). In embodiments, the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates activity as compared to an unmodified form. In embodiments, the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that increases, stimulates, or enhances activity as compared to an unmodified form. In embodiments, the composition or system comprises a repair RNA (repRNA) and a small RNA that induces cleavage in an RNA when the present methods are undertaken in cis or trans, as described herein.

[0028] In embodiments, the snRNA or snoRNA comprises a N6-Methyl adenosine (M6A) modification. In embodiments, the snRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the snRNA or snoRNA is modified to comprise at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence. In embodiments, the snRNA or snoRNA is modified to not comprise M6A sites. In embodiments, the repRNA comprises at least one or more M6A sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence. In embodiments, the repRNA comprises no M6A sites.

[0029] In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans-splicing efficiency of a target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a off-target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans-splicing efficiency of a off-target RNA as compared to an unmodified form.

[0030] In embodiments, the repRNA comprises a ESS, ESE, ISS, and / or ISE sequence. In embodiments, the repRNA targets one or more of ESS, ESE, ISS, and / or ISE. In embodiments, an interaction, modulation and / or binding to one or more of ESS, ESE, ISS, and / or ISE reduces or ablates interaction, modulation and / or binding of the one or more of the ESS, ESE, ISS, and / or ISE with a target. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences. In embodiments, the exon sequences with ESE and ESS sequences increase or decrease trans- splicing efficiency to an RNA target as compared to an unmodified form. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences increase or decrease trans-splicing efficiency to an RNA off-target as compared to an unmodified form. In embodiments, the repRNA comprises at least one or more G4 structures. In embodiments, the repRNA comprises at least one or more G4 structures sequester SD / SA motifs. In embodiments, the G4 structure is unwound, such as by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., endogenous target or exogenously delivered trigger RNA). In embodiments, the G4 structure decreases off-targets as compared to an unmodified form.

[0031] In embodiments, the repRNA comprises a modification comprising at least one or more scaffolding sequences. In embodiments, the at least one or more scaffolding sequences mediates (e.g., recruits) phase condensate-like formation and / or improves local concentrations of repRNAs compared to an unmodified form, and other targeted proteins and / or RNA. In embodiments, the repRNA comprises a modification comprising at least one or more sequences to target the repRNA to the promoter of the target gene of interest, or to proximal condensates that may contain the promoter. In embodiments, the one or more sequences comprises an enhancer RNA, snRNA and / or snoRNA sequences.

[0032] In embodiments, the repRNA comprises a modification. In embodiments, the modification improves interaction and localization to a DNA sequence of the non-template strand of the target gene as compared to an unmodified form. In embodiments, the DNA sequence of the non-template strand of the target gene is the promoter, intron, exon, or enhancer. In embodiments, the modification improves interaction and localization to the DNA sequence of the non-template strand of the target gene through protein-directed (e g. transcription factor, dCas, ZNF, or other RBP) or nucleotide-directed (e.g., R-loop) methods as compared to an unmodified form.

[0033] In embodiments, the repRNA comprises a modification comprising additional RNA elements. In embodiments, the additional RNA elements improve subnuclear localization to nuclear speckles for enhanced trans-splicing efficiency as compared to an unmodified form. In embodiments, the additional RNA element comprise NEAT1 and / or MALAT1, or a fragment thereof. In embodiments, the additional RNA element comprises a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the repRNA comprises a modification to enable targeting to site of transcription of target RNAs. In embodiments, the repRNA comprises a modification comprising 5’ UTR or 3’ UTR modifications. In embodiments, the modification comprising 5’ UTR or 3’ UTR modifications alter intracellular or intranuclear localization based on interactions with endogenous or exogenously supplied molecules (e.g., RNA G4 interactions with transcription factors or other proteins that are localized to specific cellular compartments).

[0034] In embodiments, the repRNA comprises a modification in the 5’ UTR of the repRNA. In embodiments, the modification in the 5’ UTR of the repRNA increases stability as compared to an unmodified form. In embodiments, the modification in the 5’ UTR of the repRNA decreases stability as compared to an unmodified form. In embodiments, the modification in the 5’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form. In embodiments, the repRNA comprises a modification in the 3’ UTR of the repRNA. In embodiments, the modification in the 3’ UTR of the repRNA increases stability as compared to an unmodified form. In embodiments, the modification in the 3’ UTR of the repRNA decreases stability as compared to an unmodified form. In embodiments, the modification in the 3’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form.

[0035] In embodiments, the repRNA comprises a modification comprising modifying the repRNA to comprise a G4 structure that mediates recruitment of splicing-associated RBPs.

[0036] In embodiments, the repRNA comprises a modification comprising at least one or more toehold switches in the repRNA. In embodiments, the at least one or more toehold switches in the repRNA conditionally activate or deactivate (e.g., SD / SA occlusion, binding motif occlusion, or RBP occlusion) upon detection of an endogenous or exogenously supplied target RNA.

[0037] In embodiments, the repRNA comprises a modification comprising at least one or more complementary riboregulators in repRNAs (in cis). In embodiments, the at least one or more complementary riboregulators in repRNAs (in cis) occlude splice donor (SD) site and reduce off- target trans-splicing.

[0038] In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in cis). In embodiments, the at least one or more self- complementary riboregulators in repRNAs (in cis) occlude splice acceptor (SA) site and reduce off-target trans-splicing. In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in trans). In embodiments, the at least one or more self-complementary riboregulators in repRNAs (in trans) occlude splice donor (SD) site and reduce off-target trans-splicing.

[0039] In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in trans). In embodiments, the at least one or more self-complementary riboregulators in repRNAs (in trans) occlude splice acceptor (SA) site and reduce off-target trans-splicing.

[0040] In embodiments, the repRNA comprises a modification comprising at least one or more binding motifs. In embodiments, the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site occlusion (SA, SD, ISS, ISE, ESE, and ESS) as compared to an unmodified form.

[0041] In embodiments, the repRNA comprises a modification to enable induction of trans-splicing in response to a stimulus as compared to an unmodified form. In embodiments, the repRNA comprises a modification to turn off or decrease trans-splicing in response to a stimulus as compared to an unmodified form.

[0042] In embodiments, the repRNA comprises a modification to enable small molecule induction of trans-splicing as compared to an unmodified form. In embodiments, the repRNA comprises a modification to repress small molecule induction of trans-splicing as compared to an unmodified form.

[0043] In embodiments, the repRNA comprises a modification to enable light induction of trans-splicing.

[0044] In embodiments, the repRNA comprises a modification comprising at least one or more motifs that are bound and regulated by light-sensitive proteins.

[0045] In embodiments, the snRNA or snoRNA comprises a sequence at the 3’ untranslated region (3’UTR). In embodiments, the sequence at the 3’ untranslated region (3’UTR) increases trans- splicing efficiency as compared to an unmodified form. In embodiments, the sequence is from the MALAT1 gene. In embodiments, the sequence is a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0046] In embodiments, the RNP assembles on the repRNA and / or the target. In embodiments, the RNP assembles on the repRNA. In embodiments, the RNP assembles on the target. In embodiments, the RNP sterically occludes and inhibits cis-splicing.

[0047] In embodiments, the repRNA comprises a minimal intron. In embodiments, the minimal intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

[0048] In embodiments, the repRNA further comprises a guide RNA (gRNA). In embodiments, the gRNA hybridizes to the target nucleic acid molecule. In embodiments, the gRNA directs the repRNA to a target nucleic acid molecule. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0049] In embodiments, the composition or system disclosed herein comprises an endonuclease, wherein the endonuclease is a CRISPR-Cas enzyme.

[0050] In embodiments, the Cas is a type I. In embodiments, the Cas is a type I- A, e.g., without limitation Cas8a or Cas5. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-B, e.g., without limitation Cas8b. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-C, e.g., without limitation Cas8c. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-D, e.g., without limitation CaslOd. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-E, e.g., without limitation Csel or Cse2. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-F, e.g., without limitation Csyl, Csy2, or Csy3. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-G, e.g., without limitation GSU0054. In embodiments, the Cas is a type I. In embodiments, the Cas type I is without limitation, Cas3.

[0051] In embodiments, the Cas is a type II. In embodiments, the Cas is a type II- A, e.g., without limitation Csn2. In embodiments, the Cas is a type II. In embodiments, the Cas is a type II-B, e.g., without limitation Cas4. In embodiments, the Cas is a type II. In embodiments, the Cas is a type II-C. In embodiments, the Cas is a type II. In embodiments, the Cas type II is without limitation Cas 9.

[0052] In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-A, e.g., without limitation Csm2. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-B, e.g., without limitation Cmr5. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-C, e.g., without limitation CaslO or Csxl l. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-D, e.g., without limitation CsxlO. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-E. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-F. In embodiments, the Cas is a type III. In embodiments, the Cas type III is without limitation Cas 10.

[0053] In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-A. In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-B. In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-C. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-A, e.g., without limitation Casl2a (Cpfl). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-B, e.g., without limitation Casl2b (C2cl). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-C, e.g., without limitation Casl2c (C2c3). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-D, e.g., without limitation Casl2d (CasY). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-E, e.g., without limitation Casl2e (CasX). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-F, e.g., without limitation Casl2f (Casl4, or C2cl0). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-G, e.g., without limitation Casl2g. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-H, e.g., without limitation Casl2h. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-I, e.g., without limitation Casl2i. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-K, e g., without limitation Casl2k (C2c5). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-U, e.g., without limitation C2c4, C2c8, or C2c9. In embodiments, the Cas is a type V. In embodiments, the Cas type V is without limitation Cas 12. In embodiments, the Cas is a type VI.

[0054] In embodiments, the Cas is a type VI-A, e.g., without limitation Casl3a (C2c2). In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VLB, e.g., without limitation Cas 13b. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VLC, e.g., without limitation Casl3c. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-D, e.g., without limitation Casl3d. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-X, e g., without limitation Casl3x. l. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI- Y. In embodiments, the Cas is a type VI. In embodiments, the Cas type VI is without limitation Cas 13.

[0055] In embodiments the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5, CaslO or Csxl l, CsxlO, Csfl, Cas9, Csn2, Cas4, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (Casl4, C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), C2c4, C2c8, C2c9, Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, or Casl3x.l.

[0056] In embodiments, the composition or system comprises a CRISPR-Cas enzyme, e.g., a protein and / or nucleic acid thereof. In embodiments, the composition or system further comprises a repair RNA (repRNA) and a CRISPR-Cas enzyme. In embodiments, the CRISPR / Cas system comprises a guide RNA (gRNA) and a repRNA in cis or trans capable of Cas protein binding and transsplicing. In embodiments, the CRISPR-Cas enzyme is active, e.g., catalytically active. In embodiments, the CRISPR-Cas enzyme is inactive, e.g., catalytically inactive, e.g., “dead”. In embodiments, the composition or system comprises an intronic sequence comprising a sequence which interacts with or is suitable for interacting with the CRISPR-Cas enzyme. In embodiments, the composition or system further comprises a repair RNA (repRNA) and a CRISPR-Cas enzyme when the present methods are undertaken in cis or trans, as described herein. In embodiments, the composition or system disclosed herein comprises an RNA sequence that interacts with an active or catalytically inactive endonuclease.

[0057] In embodiments, the gRNA is associated with, or suitable for associating with, one or more endonucleases. In embodiments, the endonuclease comprises one or more mutations to reduce catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations. In embodiments, the endonuclease comprising one or more mutations increases catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form. In embodiments, the endonuclease comprises an amino acid sequence of one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications.

[0058] In embodiments, the repRNA is operably linked to one or more sequences that are antisense to the target nucleic acid molecule. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is not operably linked to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is operably linked to one or more sequences that bind to, and / or hybridize to, an RNA- binding polypeptide. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

[0059] In embodiments, the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the repRNA is operably linked to one or more recognition sequences for RNP complex formation.

[0060] In embodiments, the repRNA is provided in cis to one or more recognition sequences for RNP complex formation. In embodiments, the repRNA is not operably linked to one or more recognition sequences for RNP complex formation. In embodiments, the repRNA is provided in trans to one or more recognition sequences for RNP complex formation.

[0061] In embodiments, the repRNA is operably linked to one or more gRNAs. In embodiments, the repRNA is provided in cis to one or more gRNAs. In embodiments, the repRNA is not operably linked to one or more gRNAs. In embodiments, the repRNA is provided in trans to one or more gRNAs.

[0062] In embodiments, the repRNA further comprises a ribozyme site. In embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In embodiments, the ribozyme site is a HDV ribozyme site. In embodiments, the ribozyme site is a twister ribozyme site. In embodiments, the ribozyme site is upstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is downstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme cleaves the target. In embodiments, the ribozyme is a trans-cleaving ribozyme.

[0063] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 3’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 3’ end of the repRNA.

[0064] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the snRNA or snoRNA is modified to comprise at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence. In embodiments, the snRNA or snoRNA is modified to not comprise M6A sites. In embodiments, the repRNA comprises at least one or more M6A sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence. In embodiments, the repRNA comprises no M6A sites.

[0065] In embodiments, the composition or system further comprises at least one pre-rRNA stemloop. In embodiments, the at least one pre-rRNA stemloop removes either the 5 ’cap or 3’ polyA tail.

[0066] In embodiments, the repRNA comprises at least one or more snRNA or snoRNA sequences. In embodiments, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In embodiments, the repRNA comprises an artificial smU7 system. In embodiments, the artificial smU7 system stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 5’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 5’ end of the snRNA or snoRNA stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 3 ’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 3’ end of the snRNA or snoRNA stabilizes the repRNA.

[0067] In embodiments, there are a plurality of repRNAs under the control of the same, different, or a plurality of promoters. In embodiments, the repRNA and one or more other components of the present system are under the control of the same or different promoters.

[0068] In embodiments, the repRNA comprises alternative promoters. In embodiments, the repRNA comprises at least one or more alternative Pol II promoters. In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-m ethylguanosine) or TMG (tri -methyl uanosine). In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) stabilize the repRNA.

[0069] In embodiments, the repRNA comprises at least one or more circularized 5’ replacement splice donor (SD) repRNAs. In embodiments, the at least one or more circularized 5’ replacement splice donor (SD) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 5’ replacement (SD) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs. In embodiments, the repRNA comprising at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 3’ replacement (SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In embodiments, the at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized internal replacement (SD + SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

[0070] In embodiments, the composition or system targets, or is suitable for targeting, one or more Usher syndrome-associated genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system targets, or is suitable for targeting, one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets or is suitable for replacing c. 2299delG and / or C.2276G > T of USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more Usher syndrome-associated genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

[0071] In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof.

[0072] In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more genes selected from one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting c.2299delG and / or C.2276G > T of USH2A, or a pre-mRNA sequence thereof.

[0073] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0074] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0075] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0076] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0077] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0078] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0079] In embodiments, the endonuclease is linked, associated, and / or fused with the RNA-binding protein. In embodiments, the endonuclease is linked with the RNA-binding protein via a linker. In embodiments, the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.

[0080] In embodiments, the linker is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, whereinnis 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS)n(n=l-4) (SEQ ID NO: 65), (Gly)s (SEQ ID NO: 66), (Gly)6(SEQ ID NO: 67), (EAAAK)n (n=l-3) (SEQ ID NO: 68), A(EAAAK)nA (n = 2- 5) (SEQ ID NO: 69), AEAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

[0081] In embodiments, the repRNA comprises the splice donor. In embodiments, the repRNA comprises the splice acceptor.

[0082] In embodiments, the repRNA comprises an exon of the target nucleic acid. In embodiments, the repRNA comprises an intron of the target nucleic acid.

[0083] In embodiments, the repRNA comprises one or more non-natural introns. In embodiments, the target nucleic acid is a pre-mRNA transcript molecule.

[0084] In embodiments, the target nucleic acid is one or more one or more Usher syndrome-associated genes, or a fragment thereof, or a pre-mRNA sequence thereof. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the target nucleic acid is one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof, bearing a c.2299delG and / or C.2276G > T mutation.

[0085] In embodiments, the hybridization is mediated by perfect sequence complementarity to one strand of a target nucleic acid molecule. In embodiments, the hybridization is mediated by partial sequence complementarity to one strand of a target nucleic acid molecule.

[0086] In embodiments, the repRNA mediates trans-splicing production of a corrected and / or wild-type USH2A nucleic acid gene transcript. In embodiments, the composition or system substantially prevents or eliminates cis-splicing of a nucleic acid.

[0087] In embodiments, the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0088] In embodiments, the composition or system disclosed herein further comprises a viral vector or a non-viral vector. In embodiments, the viral vector is or comprises an AAV, optionally wherein the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

[0089] In embodiments, the composition or system disclosed herein further comprises a lipid nanoparticle (LNP), liposome, lipoplex, or polymeric nanoparticle. In embodiments, the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids, and lipoids.

[0090] In embodiments, the composition or system components are nucleic acids. In embodiments, the composition or system components comprise a DNA molecule or an RNA molecule. In embodiments, the RNA is or comprises mRNA or modified mRNA (mmRNA). In embodiments, the DNA molecule is or comprises a vector or plasmid. In embodiments, the nucleic acid comprises a codon optimized sequence. In embodiments, the nucleic acid comprises one or more modifications. In embodiments, the modifications are one or more of base modifications and backbone modifications.

[0091] In embodiments, disclosed herein is a cell comprising a nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, or the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein.

[0092] In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell. In embodiments, the cell is an immortalized cell. In embodiments, the cell is harvested from a subject.

[0093] In embodiments, disclosed herein is a pharmaceutical composition comprising the composition or system nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and a pharmaceutically acceptable carrier.

[0094] In embodiments, disclosed herein is a kit comprising a container comprising the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and instructions for use in trans-splicing a nucleic acid.

[0095] In embodiments, disclosed herein is a method for targeted trans-splicing of a USH2A pre-mRNA in a cell, comprising contacting the cell with the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein.

[0096] In embodiments, disclosed herein is a method of treating a patient who has a condition associated with a mutation in a USH2A gene, the method comprising administering a therapeutically effective amount of composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein.

[0097] In embodiments, disclosed herein is a method of treating, ameliorating or preventing a condition associated with a mutation in a USH2A gene, comprising: contacting a cell with the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and administering an effective amount of the cell to the subject.

[0098] In embodiments, disclosed herein is a method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, comprising administering an effective amount of the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, to the subject.

[0099] In embodiments, disclosed herein is a method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject, in need thereof, comprising: contacting a cell with the composition or system of any one of claims 1-111, the nucleic acid of any one of claims 116- 122, the viral vector of any one of claims 112-113, the lipid nanoparticle of claim 114-115, or the cell of any one of claims 123-128, and administering an effective amount of the cell to the subject.

[0100] In embodiments, the cell is derived from the subject.

[0101] In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the Usher syndrome is Usher syndrome type I. In embodiments, the Usher syndrome is Usher syndrome type II. In embodiments, the Usher syndrome is Usher syndrome type III.

[0102] In embodiments, the method targets one or more Usher syndrome-associated genes. In embodiments, the method targets one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In embodiments, the method targets one or more of USH2A, GPR98, and DFNB31. In embodiments, the method targets USH2A.

[0103] In embodiments, the method corrects a mutation or defect in one or more Usher syndrome- associated genes. In embodiments, the method corrects a mutation or defect in one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN 1. In embodiments, the method corrects a mutation or defect in one or more of USH2A, GPR98, and DFNB31. In embodiments, the method corrects a mutation or defect in USH2A.

[0104] In embodiments, the method causes trans-splicing of one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In embodiments, the method causes trans-splicing of one or more of USH2A, GPR98, and DFNB31. In embodiments, the method causes trans-splicing of USH2A.

[0105] In embodiments, the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa. In embodiments, the method treats, ameliorates or prevents hearing reduction or loss. In embodiments, the method treats, ameliorates or prevents vision reduction or loss. In embodiments, the method treats, ameliorates or prevents one or more of night blindness and loss or reduction of peripheral vision.

[0106] In various embodiments, the composition disclosed herein, or a system for targeting a nucleic acid for trans-splicing system disclosed herein, comprises a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing. In embodiments, the trans-splicing system comprises a splice donor, a splice acceptor, and replaces an internal exon. In embodiments, the repRNA is operably linked to the RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule or the gRNA.

[0107] In aspects, the present disclosure provides a system for targeting a nucleic acid for trans-splicing, the system comprising: (a) an endonuclease of any one of the embodiments disclosed herein, and optionally an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide that associates with the endonuclease; and (c) a repair RNA (repRNA) sequence, comprising: (i) one or more exons and / or introns; (ii) a splice donor and / or splice acceptor.

[0108] In embodiments, the RNA molecule is a gRNA.

[0109] In embodiments, the endonuclease is not linked, associated, and / or fused with an RNA binding protein.

[0110] In embodiments, the repRNA is not operably linked to one or more gRNAs. In embodiments, the repRNA is provided in trans to one or more gRNAs.

[0111] In embodiments, the repRNA further comprises a ribozyme site. In embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In embodiments, the ribozyme site is a HDV ribozyme site. In embodiments, the ribozyme site is a twister ribozyme site. In embodiments, the ribozyme site is upstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is downstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme cleaves the target. In embodiments, the ribozyme is a trans-cleaving ribozyme.

[0112] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 3’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 3’ end of the repRNA.

[0113] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the snRNA or snoRNA is modified to comprise at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence. In embodiments, the snRNA or snoRNA is modified to not comprise M6A sites. In embodiments, the repRNA comprises at least one or more M6A sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence. In embodiments, the repRNA comprises no M6A sites.

[0114] In embodiments, the composition or system further comprises at least one pre-rRNA stemloop. In embodiments, the at least one pre-rRNA stemloop removes either the 5 ’cap or 3’ polyA tail.

[0115] In embodiments, the repRNA comprises at least one or more snRNA or snoRNA sequences. In embodiments, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In embodiments, the repRNA comprises an artificial smU7 system. In embodiments, the artificial smU7 system stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 5’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 5’ end of the snRNA or snoRNA stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 3 ’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 3’ end of the snRNA or snoRNA stabilizes the repRNA. In embodiments, there are a plurality of repRNAs under the control of the same, different, or a plurality of promoters. In embodiments, the repRNA and one or more other components of the present system are under the control of the same or different promoters.

[0116] In embodiments, the repRNA comprises alternative promoters. In embodiments, the repRNA comprises at least one or more alternative Pol II promoters. In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-m ethylguanosine) or TMG (tri-methylguanosine). In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) stabilize the repRNA.

[0117] In embodiments, the repRNA comprises at least one or more circularized 5’ replacement splice donor (SD) repRNAs. In embodiments, the at least one or more circularized 5’ replacement splice donor (SD) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 5’ replacement (SD) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs. In embodiments, the at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 3’ replacement (SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In embodiments, the at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized internal replacement (SD + SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

[0118] In aspects, the present disclosure provides a system for targeting a nucleic acid for trans-splicing, the system comprising: (a) an endonuclease of any one of claims 1-108 and an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; and (b) a repair RNA (repRNA) sequence, comprising: (i) one or more exons and / or introns; (ii) a splice donor and / or splice acceptor. In embodiments, the RNA molecule is a gRNA. In embodiments, the endonuclease is not linked, associated, and / or fused with an RNA binding protein. In embodiments, the repRNA is operably linked to one or more gRNAs.

[0119] In embodiments, the composition comprises a gRNA, repRNA, and a Cas endonuclease operably linked to a single promoter or a bidirectional promoter.

[0120] In embodiments, the gRNA and repRNA are located on a first side of the bidirectional promoter, and the Cas endonuclease is located on a second side of the bidirectional promoter.

[0121] In aspects, the present disclosure provides a method of slowing, reducing, or ablating transcription of a target gene, and / or stimulating, enhancing, or increasing the Pol II stalling / release. In embodiments, this method increases trans-splicing, without wishing to be bound by theory, by forcing G4 or similarly bulky RNA-RNA motif formation downstream of the repRNA’ s binding motif as compared to an unmodified form.

[0122] In aspects, the present disclosure provides compositions, systems, and / or methods for separate delivery of at least one repRNA capable of trans-splicing at least one other repRNA (e.g., daisychain) and / or an endogenous RNA target, e.g., for multi -kilobase edits larger than the cargo capacity of a single AAV. In embodiments, this permits single AAV delivery, e.g., by effectively reducing the need for a cargo size that is greater than AAV loading capacity.

[0123] In aspects, the present disclosure provides a method for modifying and converting an endogenous RNA, such as and without limitation a pre-mRNA, mRNA, IncRNA, into a repRNA for trans- splicing.

[0124] In embodiments, the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any one of SEQ ID NOs: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0125] In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid, wherein the USH2A target nucleic acid is selected from exon 13 (SEQ ID NO: 2023) or intron 13 SEQ ID NO: 2024. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 or SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about

[0126] 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a nontargeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 to any one of SEQ ID NOs: 804-2022 is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0127] In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1 .0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans- splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, or 2.5-3.0 wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 to any one of SEQ ID NOs: 804-2022 is greater than a fold change of about 0.5, 1.0, 1.5, 2.0,

[0128] 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 to any one of SEQ ID NOs: 804- 2022 is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5- 7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, or 2.5-3.0, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0129] In embodiments, the binding motif binds to a position of intron 13 of aUSH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2024. In embodiments, the binding motif binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,

[0130] 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,

[0131] 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,

[0132] 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,

[0133] 950, 960, 970, 980, 990, or about 1000.

[0134] In embodiments, the binding motif binds to a position of intron 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2024. In embodiments, the binding motif binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,

[0135] 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,

[0136] 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,

[0137] 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,

[0138] 990, or about 1000 nucleotides from the splice donor site in intron 13 of the USH2A target nucleic acid. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024) that is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,

[0139] 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,

[0140] 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,

[0141] 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,

[0142] 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000 nucleotides from the splice donor site in intron 13 of USH2A. In embodiments, the binding motif binds to a position of exon 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2023. In embodiments, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from about position -10, -20, -30, -40, -50, -60, -70, or about -80. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position -10, -20, -30, -40, -50, -60, -70, or about -80.

[0143] In embodiments, the binding motif binds to a position of exon 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2023. In embodiments, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected relative to a splice donor site, without limitation, from about position -10, -20, -30, -40, -50, -60, -70, or about -80. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected relative to a splice donor site, without limitation, from about position -10, -20, -30, -40, -50, -60, -70, or about -80.

[0144] In embodiments, the binding motif is selected from SEQ ID NO: 804, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NO: 804 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif is SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16, or relative to the splice donor site, about position 5 to 20, about 8 to 16, or about 12 nucleotides from the splice donor site in intron 13 of USH2A. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 12. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position ofintron 13 of the USH2 A target nucleic acid (SEQ IDNO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from position 12.

[0145] In embodiments, the binding motif is selected from SEQ IDNOs: 804-818, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-818 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130, or relative to the splice donor site, about position 5 to 20, about 8 to 16, or about 12 nucleotides from the splice donor site in intron 13 of USH2A. In embodiments, the binding motif is selected from SEQ ID NOs: 804- 818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position ofintron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from position position 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123.

[0146] In embodiments, the binding motif is selected from SEQ ID NOs: 804-883, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-883 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0147] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1019, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-1019 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0148] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1788, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-1788 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a nontargeting rate.

[0149] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1788 and binds to about position 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20,

[0150] 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86, 102, 66, 5,

[0151] 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34,

[0152] 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 5, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22,6, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69, 7, 2, 8, 102, 92,6, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104,26, 20, 36, 9, 27, 35, 138, 121, 170, 92, 36, 890, 104, 12, 870, 14, 38, 34, 34, 69, 72, 11, 72, 16,7, 136, 143, 91, 38, 1, 21, 137, 83, 61, 20, 36, 104, 29, 8, 30, 5, 84, 30, 106, 78, 16, 148, 9, 25,44, 87, 36, 74, 44, 9, 34, 14, 32, 4, 6, 35, 34, 76, 44, 77, 73, 2, 6, 2, 138, 30, 850, 62, 5, 137, 12,9, 39, 40, 23, 79, 141, 74, 30, 540, 130, 14, 15, 20, 23, 12, 77, 146, 10, 8, 2, 13, 42, 35, 28, 730,4, 143, 62, 4, 41, 24, 37, 92, 10, 840, 29, 8, 45, 138, 660, 38, 11, 20, 39, 90, 90, 107, 31, 135, 37,1, 22, 2, 142, 37, 4, 6, 40, 87, 38, 140, 38, 59, 6, 29, 139, 15, 9, 145, 640, 15, 131, 13, 141, 2, 26,8, 106, 4, 32, 62, 21, 14, 100, 820, 60, 8, 3, 33, 12, 22, 7, 59, 76, 47, 9, 30, 59, 730, 860, 26, 143,30, 540, 32, 27, 54, 136, 36, 17, 10, 22, 36, 250, 590, 155, 150, 12, 105, 81, 36, 8, 850, 34, 35,10, 52, 840, 22, 21, 18, 91, 4, 820, 52, 29, 28, 660, 31, 63, 85, 880, 18, 18, 78, 890, 640, 155, 34,3, 4, 81, 890, 78, 28, 24, 14, 22, 20, 24, 70, 210, 24, 147, 153, 10, 34, 139, 18, 19, 0, 16, 470,40, 14, 220, 630, 71, 26, 22, 23, 15, 28, 105, 93, 85, 14, 420, 146, 31, 8, 39, 37, 35, 16, 10, 530,, 78, 21, 22, 47, 60, 28, 8, 77, 0, 19, 78, 85, 20, 660, 21, 4, 45, 4, 12, 5, 24, 154, 31, 390, 470, 4,5, 8, 7, 6, 152, 140, 220, 55, 1, 25, 29, 4, 3, 27, 14, 6, 190, 45, 24, 13, 46, 18, 12, 46, 32, 151,10, 7, 20, 350, 3, 40, 620, 39, 0, 880, 30, 22, 155, 24, 0, 157, 32, 1, 8, 142, 14, 440, 160, 16, 64,30, 870, 147, 160, 240, 17, 33, 8, 2, 68, 39, 33, 141, 137, 76, 2, 19, 400, 76, 860, 58, 19, 23, 8,60, 360, 7, 8, 51, 820, 14, 12, 1, 190, 6, 26, 450, 16, 150, 52, 1, 47, 18, 24, 27, 210, 18, 57, 36,3, 650, 4, 80, 70, 32, 26, 16, 46, 150, 29, 17, 250, 600, 35, 56, 60, 48, 23, 37, 19, 0, 154, 0, 180,7, 26, 4, 830, 25, 58, 149, 410, 7, 152, 49, 0, 160, 380, 600, 16, 56, 590, 0, 28, 440, 58, 2, 630,3, 158, 31, 2, 650, 16, 240, 84, 4, 57, 330, 20, 16, 70, 9, 62, 360, 26, 26, 17, 153, 50, 14, 350, 2,6, 68, 52, 200, 154, 18, 32, 153, 27, 3, 0, 53, 460, 560, 600, 1, 159, 16, 120, 18, 0, 230, 28, 730,60, 54, 1, 30, 72, 105, 158, 390, 390, 540, 48, 880, 370, 32, 17, 4, 49, 36, 18, 8, 500, 50, 26, 2,3, 610, 25, 6, 200, 145, 4, 3, 450, 55, 16, 80, 570, 6, 460, 670, 17, 152, 76, 560, 16, 71, 20, 74,56, 14, 7, 74, 0, 220, 550, 18, 15, 530, 18, 350, 21, 450, 400, 720, 2, 66, 0, 0, 630, 500, 33, 14, 4,70, 6, 68, 2, 370, 151, 80, 50, 54, 180, 7, 0, 16, 560, 74, 158, 22, 900, 0, 1, 420, 180, 31, 60, 70,70, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151,, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0,57, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550,5, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25, or relative to the splice donor site, about position 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118,

[0153] 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97,

[0154] 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110,

[0155] 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128,

[0156] 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86, 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69,

[0157] 7, 2, 8, 102, 92, 36, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104, 126, 20, 36, 9, 27, 35, 138, 121, 170, 92, 36, 890, 104, 12, 870, 14, 38, 34, 34, 69, 72, 11, 72, 16, 67, 136, 143, 91, 38, 1, 21, 137, 83, 61, 20, 36, 104, 29, 8, 30, 5, 84, 30, 106, 78, 16, 148, 9, 25, 144, 87, 36, 74, 44, 9, 34, 14, 32, 4, 6, 35, 34, 76, 44, 77, 73, 2, 6, 2, 138, 30, 850, 62, 5, 137, 12, 89, 39, 40, 23, 79, 141, 74, 30, 540, 130, 14, 15, 20, 23, 12, 77, 146, 10, 8, 2, 13, 42, 35, 28, 730, 24, 143, 62, 4, 41, 24, 37, 92, 10, 840, 29, 8, 45, 138, 660, 38, 11, 20, 39, 90,

[0158] 90, 107, 31, 135, 37, 61, 22, 2, 142, 37, 4, 6, 40, 87, 38, 140, 38, 59, 6, 29, 139, 15, 9, 145, 640, 15, 131, 13, 141, 2, 26, 18, 106, 4, 32, 62, 21, 14, 100, 820, 60, 8, 3, 33, 12, 22, 7, 59, 76, 47, 9, 30, 59, 730, 860, 26, 143, 230, 540, 32, 27, 54, 136, 36, 17, 10, 22, 36, 250, 590, 155, 150, 12, 105, 81, 36, 8, 850, 34, 35, 210, 52, 840, 22, 21, 18, 91, 4, 820, 52, 29, 28, 660, 31, 63, 85, 880, 18, 18, 78, 890, 640, 155, 34, 93, 4, 81, 890, 78, 28, 24, 14, 22, 20, 24, 70, 210, 24, 147, 153, 10, 34, 139, 18, 19, 0, 16, 470, 140, 14, 220, 630, 71, 26, 22, 23, 15, 28, 105, 93, 85, 14, 420, 146, 31,

[0159] 8, 39, 37, 35, 16, 10, 530, 6, 78, 21, 22, 47, 60, 28, 8, 77, 0, 19, 78, 85, 20, 660, 21, 4, 45, 4, 12, 5, 24, 154, 31, 390, 470, 4, 25, 8, 7, 6, 152, 140, 220, 55, 1, 25, 29, 4, 3, 27, 14, 6, 190, 45, 24, 13, 46, 18, 12, 46, 32, 151, 410, 7, 20, 350, 3, 40, 620, 39, 0, 880, 30, 22, 155, 24, 0, 157, 32, 1, 8, 142, 14, 440, 160, 16, 64, 530, 870, 147, 160, 240, 17, 33, 8, 2, 68, 39, 33, 141, 137, 76, 2, 19, 400, 76, 860, 58, 19, 23, 8, 860, 360, 7, 8, 51, 820, 14, 12, 1, 190, 6, 26, 450, 16, 150, 52, 1, 47, 18, 24, 27, 210, 18, 57, 36, 33, 650, 4, 80, 70, 32, 26, 16, 46, 150, 29, 17, 250, 600, 35, 56, 60, 48, 23, 37, 19, 0, 154, 0, 180, 27, 26, 4, 830, 25, 58, 149, 410, 7, 152, 49, 0, 160, 380, 600, 16, 56, 590, 0, 28, 440, 58, 2, 630, 53, 158, 31, 2, 650, 16, 240, 84, 4, 57, 330, 20, 16, 70, 9, 62, 360, 26, 26, 17, 153, 50, 14, 350, 2, 26, 68, 52, 200, 154, 18, 32, 153, 27, 3, 0, 53, 460, 560, 600, 1, 159, 16, 120, 18, 0, 230, 28, 730, 360, 54, 1, 30, 72, 105, 158, 390, 390, 540, 48, 880, 370, 32, 17, 4, 49, 36, 18, 8, 500, 50, 26, 2, 23, 610, 25, 6, 200, 145, 4, 3, 450, 55, 16, 80, 570, 6, 460, 670, 17, 152, 76, 560, 16, 71, 20, 74, 156, 14, 7, 74, 0, 220, 550, 18, 15, 530, 18, 350, 21, 450, 400, 720, 2, 66, 0, 0, 630, 500, 33, 14, 4, 670, 6, 68, 2, 370, 151, 80, 50, 54, 180, 7, 0, 16, 560, 74, 158, 22, 900, 0, 1, 420, 180, 31, 60, 70, 270, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151, 7, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0, 157, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25.

[0160] The details of one or more examples of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings, detailed description of several examples, and also from the appended claims. The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0161] DESCRIPTION OF THE DRAWINGS

[0162] FIG. 1 is a non-limiting schematic showing the components and assembly of the USH2A transsplicing system, the mechanism of splice editing, and the trans-spliced product from a RNA transsplicing reporter assay.

[0163] FIG. 2 is an image showing a plasmid with the target sequences of the USH2A gene, which includes exon 13, and intron 13, and the location of binding motifs, allowing for the replacement of USH2A exon 13 via 5’ trans-splicing. FIG. 3 is an image showing the addition of a ribozyme to the target splice donor in a RNA transsplicing reporter assay.

[0164] FIG. 4 is a graph showing the percent of trans-splicing editing activity, as measured by the percent of GFP positive cells, following transient transfection with the USH2A trans-splicing system either having a (i) 5’ repRNA (i.e., as used herein “repRNA” is equivalent to an “RNA sequence that binds to the RNA-binding polypeptide”), and a reporter having a sequence motif for the indicated binding motif (first bar from the left in each set on the x-axis); (ii) 5’ repRNA with an HDV ribozyme and a reporter having a sequence motif for the indicated binding motif (second bar from the left in each set on the x-axis); or (iii) only the repRNA having a sequence motif for the indicated binding motif (third bar from the left in each set on the x-axis).

[0165] FIG. 5 is a graph showing the percent of trans-splicing editing activity, as measured by the percent of GFP positive cells, following integrated transfection with the USH2A trans-splicing system having a (i) reporter; (ii) a repRNA with a binding motif; (iii) a Cas protein; and / or (iv) a Cas gRNA. In FIG. 5, “NT” refers to a random non-targeting polynucleotide control (SEQ ID NO: 138). In FIG. 5, the numbers 2 and 5 in the second row refer to the binding motifs (SEQ ID NOs: 132, and 135, respectively) and the numbers 1, 2, and 3 in the last row refer to the Cas guide RNAs (SEQ ID NOs: 139-141, respectively).

[0166] FIG. 6 is a graph showing 5’ editing as applied to a USH2A reporter, with gRNAs targeting intron 12, vs a non-targeting guide where activity is driven by the presence of the repair RNA. In FIG. 6, “NT” refers to a random non-targeting polynucleotide control. The endonuclease repeat to formulate the guide RNAs was SEQ ID NO: 30.

[0167] FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D are images showing the protein sizes of the Casl3K2F system for SEQ ID NO: 1 (Casl3K2Fl), SEQ ID NO: 2 (Casl3K2F2), SEQ ID NO: 3 (Casl3K2F3), SEQ ID NO: 4 (Casl3K2F5), SEQ ID NO: 80 (Casl3K2F7), SEQ ID NO: 81 (Casl3K2F8), SEQ ID NO: 82 (Casl3K2F9), SEQ ID NO: 83 (Casl3K2F10), SEQ ID NO: 84 (Casl3K2Fl l), SEQ ID NO: 85 (Casl3K2F12), SEQ ID NO: 86 (Casl3K2F13), and SEQ ID NO: 87 (Casl 3K2F14). The red or black arrows in FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D indicate the placement of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain in each protein. FIG. 8 is a percent identity matrix of SEQ ID NO: 6 (Casl3X.l), SEQ ID NO: 7 (Casl3bt3), SEQ ID NO: 8 (Casl3bt2), SEQ ID NO: 9 (Casl3btl), SEQ ID NO: 10 (Casl3bt8), SEQ ID NO: 11 (Casl3X.2), SEQ ID NO: 12 (Casl3bt9), SEQ ID NO: 13 (Casl3btl l), SEQ ID NO: 14

[0168] (Casl3bt5), SEQ ID NO: 15 (Casl3btl0), SEQ ID NO: 16 (Casl3btl5), SEQ ID NO: 17

[0169] (Casl3bt7), SEQ ID NO: 18 (Casl3bt6), SEQ ID NO: 19 (Casl3btl4), SEQ ID NO: 20

[0170] (Casl3Y.3), SEQ ID NO: 21 (Casl3btl2), SEQ ID NO: 22 (Casl3Y. l), SEQ ID NO: 23

[0171] (Casl3bt4), SEQ ID NO: 24 (Casl3btl6), SEQ ID NO: 25 (Casl3Y.5), and SEQ ID NO: 26 (Casl3Y.4).

[0172] FIG. 9 is an image showing a maximum likelihood phylogenetic tree of SEQ ID NO: 6 (Casl3X.1), SEQ ID NO: 7 (Casl3bt3), SEQ ID NO: 8 (Casl3bt2), SEQ ID NO: 9 (Casl3btl), SEQ ID NO: 10 (Casl3bt8), SEQ ID NO: 11 (Casl3X.2), SEQ ID NO: 12 (Casl3bt9), SEQ ID NO: 13 (Casl3btl l), SEQ ID NO: 14 (Casl3bt5), SEQ ID NO: 15 (Casl3btlO), SEQ ID NO: 16

[0173] (Casl3btl5), SEQ ID NO: 17 (Casl3bt7), SEQ ID NO: 18 (Casl3bt6), SEQ ID NO: 19

[0174] (Casl3btl4), SEQ ID NO: 20 (Casl3Y.3), SEQ ID NO: 21 (Casl3btl2), SEQ ID NO: 22

[0175] (Casl3Y.l), SEQ ID NO: 23 (Casl3bt4), SEQ ID NO: 24 (Casl3btl6), SEQ ID NO: 25

[0176] (Casl3Y.5), and SEQ ID NO: 26 (Casl3Y.4).

[0177] FIG. 10 is a graph showing screening data from a set of binding motif (BM) sequences (i.e., SEQ ID NOs: 804-2022) in the repair RNA (repRNA) and their ability to target USH2A.

[0178] DETAILED DESCRIPTION

[0179] The present disclosure provides, inter alia, compositions, systems, and methods related to a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for transsplicing.

[0180] The present disclosure is based, in part, on the discovery of compositions comprising a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for transsplicing, and methods of use thereof. The present disclosure is also based, in part, on the discovery of a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns of a target nucleic acid molecule; and (b) a splice donor and / or splice acceptor, and methods of use thereof.

[0181] Usher Syndrome

[0182] Usher syndrome is inherited as an autosomal recessive condition characterized by hearing loss or deafness and progressive vision loss. The loss of vision is caused by retinitis pigmentosa (RP), which affects the layer of light-sensitive tissue at the retina. Without wishing to be bound by theory, vision loss occurs as the light-sensing cells of the retina gradually deteriorate.

[0183] Three major types of Usher syndrome, designated as types I (subtypes IA through IG), II (subtypes IIA, IIB, and IIC), and III, have been identified. These types are distinguished by their severity and the age when signs and symptoms appear.

[0184] Subjects with Usher Syndrome type I are born profoundly deaf and begin to lose their vision in the first decade of life. They also exhibit balance difficulties and learn to walk slowly as children, due to problems in their vestibular system.

[0185] Usher Syndrome type II is a heterogeneous autosomal recessive disorder characterized by progressive retinitis pigmentosa and sensorineural hearing deficiencies, resulting in deaf-blindness in patients. Subjects with Usher II are generally have a reduced ability to hear rather than deaf, and their hearing does not degrade over time. They do not seem to have noticeable problems with balance. They typically begin to lose their vision in the second decade of life, and may preserve some vision into middle age.

[0186] Subjects with Usher syndrome III experience a progressive loss of hearing, and roughly half have balance difficulties. Mutations in CLRN1, have been linked to Usher syndrome type III. CLRN1 encodes clarin-1, a protein involved in the development and maintenance of the inner ear and retina.

[0187] Several genes have been implicated or associated with Usher syndrome, which include mutations in the genes: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. Mutations in any one of these genes alters gene expression or protein function and can lead to Usher syndrome. The genes play roles in the development and maintenance of hair cells, which have sensory functionalities in the inner ear that help transmit sound and motion signals to the brain. In the retina, these genes are also involved in determining the structure and function of light-sensing cells called rods and cones. In some cases, the exact role of these genes in hearing and vision is unknown. Most of the mutations responsible for Usher syndrome lead to a loss or reduction of hair cells in the inner ear and a gradual loss or reduction of rods and cones in the retina. Degeneration of these sensory cells causes hearing loss, balance problems, and vision loss characteristic of this condition.

[0188] Usher syndrome type II can be caused by mutations in, e.g., USH2A, ADGRV1, WHRN, GPR98 (also called VLGR1) gene, and DFNB31. Usher syndrome type III can be caused by mutations in e g , CLRN1.

[0189] The protein encoded by the USH2A gene, usherin, is located in the supportive tissue in the inner ear and retina. Usherin is involved in the development and maintenance of these structures. WHRN mutations are associated with Usher syndrome type 2D or non-syndromic deafness (DFNB31). The associated phenotypes are dependent on the mutation location within the two predominantly expressed variants (long and short). Variants in ADGRV1 are a cause of Usher syndrome type 2, and the associated phenotype is less known.

[0190] In embodiments, present disclosure provides, a composition comprising a repRNA sequence comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing. In embodiments, present disclosure provides, a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In embodiments, the composition or system targets, or is suitable for targeting, one or more Usher syndrome-associated genes. In embodiments, the composition or system targets, or is suitable for targeting, one or more Usher syndrome-associated genes. In embodiments, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system targets, or is suitable for targeting, one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, USH2A, or a pre- mRNA sequence thereof. In embodiments, the composition or system targets, or is suitable for targeting, exon 13 of USH2A, or a pre-mRNA sequence thereof.

[0191] In embodiments, the composition or system targets or is suitable for targeting c.2299delG and / or C.2276G > T of USH2A, or a pre-mRNA sequence thereof.

[0192] In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more Usher syndrome-associated genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in one or more genes selected from one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting a mutation or defect in exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the composition or system is suitable for correcting c.2299delG and / or C.2276G > T of USH2A, or a pre-mRNA sequence thereof.

[0193] In embodiments, the target nucleic acid of the composition or system disclosed herein is the target nucleic acid is one or more one or more Usher syndrome-associated genes, or a fragment thereof, or a pre-mRNA sequence thereof. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the target nucleic acid of the composition or system disclosed herein is one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof, bearing a c.2299delG and / or C.2276G > T mutation. In embodiments, the composition comprising the repRNA sequence, or the system for transsplicing a target nucleic acid comprising a repRNA is for use in treating a disease, such as Usher Syndrome type II.

[0194] In embodiments, disclosed herein is a method for targeted trans-splicing of a USH2A pre-mRNA in a cell, comprising contacting the cell with the composition or system of any one of the embodiments and / or aspects disclosed herein, nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein.

[0195] In embodiments, the present disclosure provides a method of treating a patient who has a condition associated with a mutation in a USH2A gene, the method comprising administering a therapeutically effective amount of the composition or system of any one of the embodiments and / or aspects disclosed herein, nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein.

[0196] In embodiments, disclosed herein is a method of treating, ameliorating or preventing a condition associated with a mutation in a USH2A gene, comprising: contacting a cell with the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and administering an effective amount of the cell to the subject.

[0197] In embodiments, the present disclosure provides a method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, comprising administering an effective amount of the composition or system of any one of the embodiments and / or aspects disclosed herein, nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, to the subject. In embodiments, the present disclosure provides a method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject, in need thereof, comprising: (a) contacting a cell with the composition or system of any one of the embodiments and / or aspects disclosed herein, nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and (b) administering an effective amount of the cell to the subject.

[0198] In embodiments, the cell is derived from the subject.

[0199] In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the Usher syndrome is Usher syndrome type I. In embodiments, the Usher syndrome is Usher syndrome type II. In embodiments, the Usher syndrome is Usher syndrome type III.

[0200] In embodiments, the method targets one or more Usher syndrome-associated genes. In embodiments, the method targets one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In embodiments, the method targets one or more of USH2A, GPR98, and DFNB31. In embodiments, the method targets USH2A.

[0201] In embodiments, the method corrects a mutation or defect in one or more Usher syndrome- associated genes.

[0202] In embodiments, the method corrects a mutation or defect in one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN 1. In embodiments, the method corrects a mutation or defect in one or more of USH2A, GPR98, and DFNB31. In embodiments, the method corrects a mutation or defect in USH2A.

[0203] In embodiments, the method causes trans-splicing of one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In embodiments, the method causes trans-splicing of one or more of USH2A, GPR98, and DFNB31. In embodiments, the method causes trans-splicing of USH2A.

[0204] In embodiments, the method treats, ameliorates or prevents hearing reduction or loss.

[0205] In embodiments, the method treats, ameliorates or prevents vision reduction or loss. In embodiments, the method treats, ameliorates or prevents one or more of night blindness and loss or reduction of peripheral vision.

[0206] In various embodiments, the “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested)). In embodiments, the subject is a human.

[0207] Repair RNA, and Guide RNAs

[0208] The present disclosure provides, in aspects, a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing. Additionally, the present disclosure provides, in aspects, a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In embodiments, the one or more exons is or comprises one or more exons of a target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of a target nucleic acid molecule. In embodiments, the one or more exons is or comprises one or more exons of the target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of the target nucleic acid molecule.

[0209] In embodiments, the repRNA further comprises a guide RNA (gRNA). In embodiments, the gRNA hybridizes to the target nucleic acid molecule. In embodiments, the gRNA directs the repRNA to a target nucleic acid molecule. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 1 1, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0210] In embodiments, the gRNA is associated with, or suitable for associating with, one or more endonucleases. In embodiments, the endonuclease comprises one or more mutations to reduce catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations. In embodiments, the one or more mutations increase catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form. In embodiments, the endonuclease comprises an amino acid sequence of one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications.

[0211] In embodiments, the repRNA is operably linked to one or more sequences that are antisense to the target nucleic acid molecule. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is not operably linked to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is operably linked to one or more sequences that bind to, and / or hybridize to, an RNA- binding polypeptide. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

[0212] In embodiments, the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the repRNA is operably linked to one or more recognition sequences for RNP complex formation.

[0213] In embodiments, the repRNA is provided in cis to one or more recognition sequences for RNP complex formation. In embodiments, the repRNA is not operably linked to one or more recognition sequences for RNP complex formation. In embodiments, the repRNA is provided in trans to one or more recognition sequences for RNP complex formation.

[0214] In embodiments, the repRNA is operably linked to one or more gRNAs. In embodiments, the repRNA is provided in cis to one or more gRNAs. In embodiments, the repRNA is not operably linked to one or more gRNAs. In embodiments, the repRNA is provided in trans to one or more gRNAs.

[0215] In embodiments, the one or more binding motifs comprises a recognition sequence for RNP complex formation.

[0216] In embodiments, the composition or system further comprises a protein that forms an RNP or is within an RNP, with one or more binding motifs (e.g., without limitation the snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP. In embodiments, the one or more binding motifs (e.g., without limitation snRNA or snoRNA), protein that forms or is within an RNP, a protein within the RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates RNP activity, and / or leads to attenuation of RNA modifying activity as compared to an unmodified form, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation. In embodiments, the one or more binding motifs (e.g., without limitation snRNA or snoRNA), protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that increases, stimulates, or enhances RNP activity, or enhances RNA modifying activity as compared to an unmodified form, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation as compared to an unmodified form. In embodiments, the snRNA, snoRNA, protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises at least one or more pseudouridylation sites. In embodiments, the snRNA, snoRNA, protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises no pseudouridylation sites. In embodiments, the repRNA comprises at least one or more pseudouridylation sites. In embodiments, the repRNA comprises no pseudouridylation sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more pseudouridylation sites than the number of pseudouridylation sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence.

[0217] In embodiments, the composition or system comprises a repair RNA (repRNA) and / or a protein that forms or is within an RNP, with the one or more binding motifs (e.g., without limitation snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme. In embodiments, the composition or system comprises a repair RNA (repRNA) and / or a protein that forms or is within an RNP, with the one or more binding motifs (e.g., without limitation snRNA or snoRNA), or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme when the present methods are undertaken in cis or trans, as described herein. In embodiments, cleavage is initiated from RNPs that are formed on the repRNA, or from RNPs that are formed in cis or trans.

[0218] In embodiments, the one or more binding motifs comprises a sequence from a small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises a sequence from the snRNA or the snoRNA. In embodiments, the snoRNA comprises or is aH / ACA snoRNA. In embodiments, the snoRNA comprises or is a C / D snoRNA.

[0219] In embodiments, the snRNA or snoRNA target one or more exonic splicing enhancers (ESEs), one or more intronic splicing enhancers (ISEs), one or more exonic splicing silencers (ESSs), and / or one or more intronic splicing silencers (ISSs). In embodiments, the snRNA or snoRNA comprise a modification to include at least one or more exonic splicing enhancers (ESEs), at least one or more intronic splicing enhancers (ISEs), at least one or more exonic splicing silencers (ESSs), and / or at least one or more intronic splicing silencers (ISSs).

[0220] In embodiments, the composition or system comprises a repair RNA (repRNA) and a small RNA that induces cleavage in an RNA. In embodiments, the small RNA that induces cleavage in an RNA is one or more of an siRNA, small hairpin RNA (shRNA), U7 snRNA, a U1 snRNA, a U2 snRNA, a U4 snRNA, a U4atac snRNA, a U5 snRNA, a U6 snRNA, a U6atac snRNA, a UH snRNA, aU12 snRNA, and an antisense oligonucleotide (ASO). In embodiments, the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates activity as compared to an unmodified form. In embodiments, the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that increases, stimulates, or enhances activity as compared to an unmodified form. In embodiments, the composition or system comprises a repair RNA (repRNA) and a small RNA that induces cleavage in an RNA when the present methods are undertaken in cis or trans, as described herein.

[0221] In embodiments, the snRNA or snoRNA comprises a N6-Methyladenosine (M6A) modification. In embodiments, the snRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the snRNA or snoRNA is modified to comprise at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence. In embodiments, the snRNA or snoRNA is modified to not comprise M6A sites. In embodiments, the repRNA comprises at least one or more M6A sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence. In embodiments, the repRNA comprises no M6A sites.

[0222] In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans-splicing efficiency of a target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a off-target RNA as compared to an unmodified form. In embodiments, the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences. In embodiments, the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans-splicing efficiency of a off-target RNA as compared to an unmodified form.

[0223] In embodiments, the repRNA comprises a ESS, ESE, ISS, and / or ISE sequence. In embodiments, the repRNA targets one or more of ESS, ESE, ISS, and / or ISE. In embodiments, an interaction, modulation and / or binding to one or more of ESS, ESE, ISS, and / or ISE reduces or ablates interaction, modulation and / or binding of the one or more of the ESS, ESE, ISS, and / or ISE with a target. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences. In embodiments, the exon sequences with ESE and ESS sequences increase or decrease trans- splicing efficiency to an RNA target as compared to an unmodified form. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences. In embodiments, the repRNA comprises exon sequences with ESE and ESS sequences increase or decrease trans-splicing efficiency to an RNA off-target as compared to an unmodified form. In embodiments, the repRNA comprises at least one or more G4 structures. In embodiments, the repRNA comprises at least one or more G4 structures sequester SD / SA motifs. In embodiments, the G4 structure is unwound, such as by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., endogenous target or exogenously delivered trigger RNA). In embodiments, the G4 structure decreases off-targets as compared to an unmodified form.

[0224] In embodiments, the repRNA comprises a modification comprising at least one or more scaffolding sequences. In embodiments, the at least one or more scaffolding sequences mediates (e.g., recruits) phase condensate-like formation and / or improves local concentrations of repRNAs compared to an unmodified form, and other targeted proteins and / or RNA. In embodiments, the repRNA comprises a modification comprising at least one or more sequences to target the repRNA to the promoter of the target gene of interest, or to proximal condensates that may contain the promoter. In embodiments, the one or more sequences comprises an enhancer RNA, snRNA and / or snoRNA sequences.

[0225] In embodiments, the repRNA comprises a modification. In embodiments, the modification improves interaction and localization to a DNA sequence of the non-template strand of the target gene as compared to an unmodified form. In embodiments, the DNA sequence of the non-template strand of the target gene is the promoter, intron, exon, or enhancer. In embodiments, the modification improves interaction and localization to the DNA sequence of the non-template strand of the target gene through protein-directed (e g. transcription factor, dCas, ZNF, or other RBP) or nucleotide-directed (e.g., R-loop) methods as compared to an unmodified form.

[0226] In embodiments, the repRNA comprises a modification comprising additional RNA elements. In embodiments, the additional RNA elements improve subnuclear localization to nuclear speckles for enhanced trans-splicing efficiency as compared to an unmodified form. In embodiments, the additional RNA element comprise NEAT1 and / or MALAT1, or a fragment thereof. In embodiments, the additional RNA element comprises a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the repRNA comprises a modification to enable targeting to site of transcription of target RNAs. In embodiments, the repRNA comprises a modification comprising 5’ UTR or 3’ UTR modifications. In embodiments, the modification comprising 5’ UTR or 3’ UTR modifications alter intracellular or intranuclear localization based on interactions with endogenous or exogenously supplied molecules (e.g., RNA G4 interactions with transcription factors or other proteins that are localized to specific cellular compartments).

[0227] In embodiments, the repRNA comprises a modification in the 5’ UTR of the repRNA. In embodiments, the modification in the 5’ UTR of the repRNA increases stability as compared to an unmodified form. In embodiments, the modification in the 5’ UTR of the repRNA decreases stability as compared to an unmodified form. In embodiments, the modification in the 5’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form. In embodiments, the repRNA comprises a modification in the 3’ UTR of the repRNA. In embodiments, the modification in the 3’ UTR of the repRNA increases stability as compared to an unmodified form. In embodiments, the modification in the 3’ UTR of the repRNA decreases stability as compared to an unmodified form. In embodiments, the modification in the 3’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form.

[0228] In embodiments, the repRNA comprises a modification comprising modifying the repRNA to comprise a G4 structure that mediates recruitment of splicing-associated RBPs. In embodiments, the repRNA comprises a modification comprising at least one or more toehold switches in the repRNA. In embodiments, the at least one or more toehold switches in the repRNA conditionally activate or deactivate (e.g., SD / SA occlusion, binding motif occlusion, or RBP occlusion) upon detection of an endogenous or exogenously supplied target RNA.

[0229] In embodiments, the repRNA comprises a modification comprising at least one or more complementary riboregulators in repRNAs (in cis). In embodiments, the at least one or more complementary riboregulators in repRNAs (in cis) occlude splice donor (SD) site and reduce off- target trans-splicing.

[0230] In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in cis). In embodiments, the at least one or more self- complementary riboregulators in repRNAs (in cis) occlude splice acceptor (SA) site and reduce off-target trans-splicing.

[0231] In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in trans). In embodiments, the at least one or more self-complementary riboregulators in repRNAs (in trans) occlude splice donor (SD) site and reduce off-target trans-splicing.

[0232] In embodiments, the repRNA comprises a modification comprising at least one or more self- complementary riboregulators in repRNAs (in trans). In embodiments, the at least one or more self-complementary riboregulators in repRNAs (in trans) occlude splice acceptor (SA) site and reduce off-target trans-splicing.

[0233] In embodiments, the repRNA comprises a modification comprising at least one or more binding motifs. In embodiments, the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site occlusion (SA, SD, ISS, ISE, ESE, and ESS) as compared to an unmodified form.

[0234] In embodiments, the repRNA comprises a modification to enable induction of trans-splicing in response to a stimulus as compared to an unmodified form. In embodiments, the repRNA comprises a modification to turn off or decrease trans-splicing in response to a stimulus as compared to an unmodified form. In embodiments, the repRNA comprises a modification to enable small molecule induction of trans-splicing as compared to an unmodified form. In embodiments, the repRNA comprises a modification to repress small molecule induction of trans-splicing as compared to an unmodified form.

[0235] In embodiments, the repRNA comprises a modification to enable light induction of trans-splicing.

[0236] In embodiments, the repRNA comprises a modification comprising at least one or more motifs that are bound and regulated by light-sensitive proteins.

[0237] In embodiments, the snRNA or snoRNA comprises a sequence at the 3’ untranslated region (3’UTR). In embodiments, the sequence at the 3’ untranslated region (3’UTR) increase trans- splicing efficiency as compared to an unmodified form. In embodiments, the sequence is from the MALAT1 gene. In embodiments, the sequence is a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0238] In embodiments, the RNP assembles on the repRNA and / or the target. In embodiments, the RNP assembles on the repRNA. In embodiments, the RNP assembles on the target. In embodiments, the RNP sterically occludes and inhibits cis-splicing.

[0239] In embodiments, the repRNA comprises a minimal intron. In embodiments, the minimal intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

[0240] In embodiments, the snRNAs comprises Ul, U2, U3, U4, U5, U6, U7, U8, U9, U10 or U11, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1 , or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the snRNA or snoRNA is selected from any one of SEQ ID NOs: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions, optionally wherein any one of SEQ ID NOs: 144-802 form an RNP complex, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0241] In embodiments, the repRNA further comprises a ribozyme site. In embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In embodiments, the ribozyme site is a HDV ribozyme site. In embodiments, the ribozyme site is a twister ribozyme site. In embodiments, the ribozyme site is upstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is downstream of the one or more exons and / or introns of the repRNA. In embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor the repRNA. In embodiments, the ribozyme cleaves the target. In embodiments, the ribozyme is a trans-cleaving ribozyme.

[0242] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves at the 3’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 5’ end of the repRNA. In embodiments, the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 3’ end of the repRNA.

[0243] In embodiments, the repRNA comprises a M6A modification when the present methods are undertaken in cis or trans, as described herein. In embodiments, the snRNA or snoRNA is modified to comprise at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to comprise at least 1 , 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence. In embodiments, the snRNA or snoRNA is modified to not comprise M6A sites. In embodiments, the repRNA comprises at least one or more M6A sites. In embodiments, the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence. In embodiments, the repRNA comprises no M6A sites.

[0244] In embodiments, the composition or system further comprises at least one pre-rRNA stemloop. In embodiments, the at least one pre-rRNA stemloop removes either the 5’cap or 3’ polyA tail.

[0245] In embodiments, the repRNA comprises at least one or more snRNA or snoRNA sequences. In embodiments, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In embodiments, the repRNA comprises an artificial smU7 system. In embodiments, the artificial smU7 system stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 5’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 5’ end of the snRNA or snoRNA stabilizes the repRNA. In embodiments, the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 3 ’ end of the snRNA or snoRNA. In embodiments, the pseudoknot at the 3’ end of the snRNA or snoRNA stabilizes the repRNA.

[0246] In embodiments, there are a plurality of repRNAs under the control of the same, different, or a plurality of promoters. In embodiments, the repRNA and one or more other components of the present system are under the control of the same or different promoters.

[0247] In embodiments, the repRNA comprises alternative promoters. In embodiments, the repRNA comprises at least one or more alternative Pol II promoters. In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-m ethylguanosine) or TMG (tri-methylguanosine). In embodiments, the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) stabilize the repRNA.

[0248] In embodiments, the repRNA comprises at least one or more circularized 5’ replacement splice donor (SD) repRNAs. In embodiments, the at least one or more circularized 5’ replacement splice donor (SD) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 5’ replacement (SD) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs. In embodiments, the at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized 3’ replacement (SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases. In embodiments, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In embodiments, at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In embodiments, the repRNA comprising one or more circularized internal replacement (SD + SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

[0249] Methods of Use

[0250] In aspects, the present disclosure provides a method of modifying a nucleic acid in a cell using a method of exon skipping, comprising contacting the cell with the composition of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, the cell of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein.

[0251] In aspects, the present disclosure provides a method of modifying a nucleic acid in a cell in a subject in need thereof, using a method of exon skipping, comprising administering an effective amount of the composition of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, the cell of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein.

[0252] In embodiments, the cell is induced to skip over faulty sections of pre-mRNA molecules by interfering with mRNA splicing. In embodiments, the methods produce a truncated, but functional, protein despite the presence of a mutation. In embodiments, the method of exon skipping involves binding an oligonucleotide (e.g. without limitation the present RNA molecule) to a splice site in a pre-mRNA molecule. In embodiments, when the pre-mRNA that is bound by the oligonucleotide is processed into a mature mRNA, the corresponding exon is skipped over, which, for example, restores a disrupted reading frame caused by the mutation. In embodiments, the exon skipping allows translation of an internally-deleted, but substantially functional protein.

[0253] In embodiments, the present exon skipping methods comprise creating a single-strand or doublestrand break in a gene. In embodiments, the single-strand or double-strand break causes persistent altered splicing of the gene. In embodiments, the altered splicing results in expression of a truncated protein which lacks at least the polypeptide sequence corresponding to an exon containing the mutation. In embodiments, the single-strand or double-strand break removes a splice acceptor site or produces a non-functional splice acceptor site in or near an exon of the gene or removes a splice donor site or produces a non-functional splice donor site in or near an exon of the gene.

[0254] In aspects, the present disclosure provides use of the composition of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, the cell of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein in the manufacture of a medicament for the treating, ameliorating or preventing of a disease or disorder.

[0255] In aspects, the present disclosure provides a method of detecting and / or quantifying a nucleic acid in a sample, comprising contacting the sample with a composition of any one of the embodiments and / or aspects disclosed herein.

[0256] In embodiments, the nucleic acid is a target and / or reporter nucleic acid. In embodiments, the method comprises detection of a reporter signal, the reporter signal being generated upon endonuclease cleavage. In embodiments, the reporter signal is a fluorescent signal. In embodiments, the endonuclease has collateral cleavage activity.

[0257] In aspects, the present disclosure provides a variety of methods (e.g., with a composition of any one of the embodiments and / or aspects disclosed herein). For example, a composition of any one of the embodiments and / or aspects disclosed herein can be used to (i) modify (e.g., cleave, e.g., nick; methylate; and the like) target nucleic acid (DNA or RNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid e.g., for purposes of isolation, labeling, imaging, tracking, and the like); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like.

[0258] In embodiments, the present disclosure provides a method of modifying a target nucleic acid. In embodiments, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with (a) a composition of the present disclosure (e.g. an endonuclease and / or a chimeric protein of any one of the embodiments and / or aspects disclosed herein); and (b) one or more (e.g., two) RNAs of any one of the embodiments and / or aspects disclosed herein. In embodiments, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a composition of the present disclosure (e.g. an endonuclease and / or a chimeric protein of any one of the embodiments and / or aspects disclosed herein); and b) one or more (e.g, two) RNAs of any one of the embodiments and / or aspects disclosed herein; and c) a donor nucleic acid (e.g., a donor template). In embodiments, the contacting step is carried out in a cell in vitro. In embodiments, the contacting step is carried out in a cell in vivo. In embodiments, the contacting step is carried out in a cell ex vivo.

[0259] It is to be understood that, in embodiments, while a method of binding may result in nothing more than binding of the target nucleic acid, in other embodiments, the method can have different final results (e.g., the method can result in modification of the target nucleic acid, e.g., cleavage / methylation / and the like, modulation of transcription from the target nucleic acid; modulation of translation of the target nucleic acid; genome editing; modulation of a protein associated with the target nucleic acid; isolation of the target nucleic acid; and the like).

[0260] For examples of suitable methods, see, for example, Jinek et al., Science. 2012 Aug 17;337(6096): 816-21 ; Chylinski et al., RNA Biol. 2013 May; 10(5)726-37; Ma et al., Biomed Res Int. 2013 ;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al, Cell. 2013 Feb 28; 152(5): 1173-83 ; Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et al., Genome Res. 2013 Oct 31; Chen et al., Nucleic Acids Res. 2013 Nov l;41(20):el9; Cheng et al., Cell Res. 2013 Oct;23(10): 1163-71; Cho et al., Genetics. 2013 Nov; 195(3): 1177-80; DiCarlo et al., Nucleic Acids Res. 2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods. 2013 Oct;10(10):1028-34; Ebina et al., Sci Rep. 2013;3:2510; Fujii et al, Nucleic Acids Res. 2013 Nov l;41(20):el87; Hu et al., Cell Res. 2013 Nov;23(l 1): 1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov l;41(20):el88; Larson et al., Nat Protoc. 2013 Nov;8(l l):2180-96; Mali et. at., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et al., Genesis. 2013 Dec;51(12):835-43; Ran et al., Nat Protoc. 2013 Nov;8(l l):2281-308; Ran et al., Cell. 2013 Sep 12; 154(6): 1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15514-5; Xie et al., Mol Plant. 2013 Oct 9; Yang et al., Cell. 2013 Sep 12; 154(6): 1370- 9; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896;

[0261] 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702;

[0262] 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231;

[0263] 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556;

[0264] 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830;

[0265] 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458;

[0266] 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959;

[0267] 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated by reference in its entirety.

[0268] In embodiments, the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.

[0269] In embodiments, the terms “contact a target nucleic acid” and “contacting a target nucleic acid”, for example, encompass all methods for contacting the target nucleic acid. For example, a present polypeptide (e.g. of the present endonuclease and / or chimeric protein) is provided to a cell as protein, RNA (encoding the present polypeptide), or DNA (encoding the present polypeptide); while a present RNA can be provided as a RNA or as a nucleic acid encoding the RNA. As such, when, for example, performing a method in a cell (e.g., inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo), a method that includes contacting the target nucleic acid encompasses the introduction into the cell of any or all of the components in their active / final state (e.g., in the form of a protein(s) for a polypeptide; in the form of a protein for a chimeric polypeptide; in the form of an RNA in embodiments for the present RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a present polypeptide or a present chimeric polypeptide, nucleic acid(s) comprising nucleotide sequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotide sequence encoding a donor template, and the like). Because the methods can also be performed in vitro outside of a cell, a method that includes contacting a target nucleic acid, (unless otherwise specified) encompasses contacting outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, or inside of a cell ex vivo.

[0270] In embodiments, the method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a present locus, e.g., a nucleic acid comprising a nucleotide sequence encoding a present polypeptide as well as nucleotide sequences of about 1 kilobase (kb) to 5 kb in length surrounding the present-encoding nucleotide sequence from a cell (e.g., in embodiments, a cell that in its natural state (the state in which it occurs in nature) comprises a present locus) comprising a present locus, where the target cell does not normally (in its natural state) comprise a present locus. However, one or more spacer sequences, encoding guide sequences for the encoded crRNA(s), can be modified such that one or more target sequences of interest are targeted. Thus, for example, in embodiments, a method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a present locus, e.g., a nucleic acid obtained from a source cell (e.g., in embodiments, a cell that in its natural state (the state in which it occurs in nature) comprises a present locus), where the nucleic acid has a length of from 100 nucleotides (nt) to 5 kb in length (e.g., from 100 nt to 500 nt, from 500 nt to 1 kb, from 1 kb to 1.5 kb, from 1.5 kb to 2 kb, from 2 kb to 2.5 kb, from 2.5 kb to 3 kb, from 3 kb to 3.5 kb, from 3.5 kb to 4 kb, or from 4 kb to 5 kb in length) and comprises a nucleotide sequence encoding a present polypeptide. As noted above, in some such embodiments, one or more spacer sequences, encoding guide sequences for the encoded crRNA(s), can be modified such that one or more target sequences of interest are targeted. In embodiments, the method comprises introducing into a target cell: i) a present locus; and ii) a donor DNA template. In embodiments, the target nucleic acid is in a cell- free composition in vitro. In embodiments, the target nucleic acid is present in a target cell. In embodiments, the target nucleic acid is present in a target cell, where the target cell is a eukaryotic cell. In embodiments, the target nucleic acid is present in a target cell, where the target cell is a mammalian cell. In embodiments, the target nucleic acid is present in a target cell, where the target cell is a plant cell.

[0271] Trans-Splicing System and Binding Motifs

[0272] In embodiments, the present disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor.

[0273] Additionally, the present disclosure provides a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing.

[0274] In embodiments, the trans-splicing system comprises a splice donor, a splice acceptor, and the trans-splicing system is suitable for replacing an internal exon in a gene.

[0275] In embodiments, the trans-splicing system comprises a repair template comprising a splice donor and / or a splice acceptor. In embodiments, the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor. In embodiments, the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor, and the trans-splicing system and / or repair template is suitable for replacing an internal exon in a gene. In embodiments, there is provided a method of trans-splicing an exon in a target nucleic acid, e.g. a pre-mRNA in a cell, the method comprising contacting the cell with the trans-splicing system disclosed herein, wherein the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor. In embodiments, the present disclosure provides a method of trans-splicing an exon in a target nucleic acid, e.g. a pre-mRNA in a cell in a subject in need thereof, comprising administering an effective amount of the trans-splicing system disclosed herein, wherein the trans- splicing system comprises a repair template comprising a splice donor and a splice acceptor.

[0276] In embodiments, the trans-splicing system further comprises a repair template lacking a splice donor and / or a splice acceptor. In embodiments, the trans-splicing system is suitable for splicing of a target nucleic acid comprising a splice donor or a splice acceptor site. In embodiments, the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice acceptor site. In embodiments, the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice donor site. In embodiments, the trans-splicing system comprises is regulated or regulatable by a small molecule. In various embodiments, the small molecule is selected from abscisic acid (ABA), rapamycin (or rapalog), FK506, Cyclosporine A, FK1012, Gibberellin3-AM, FKCsA, AP1903 / AP20187, and auxin. In embodiments, the pre-mRNA is at an intron-exon junction or exon-intron junctions.

[0277] In embodiments, the trans-splicing system comprises a pre-trans-splicing (PTS) molecule, wherein the PTS molecule comprises: i) one or more guideRNAs (gRNAs) that target a pre-mRNA; ii) an intronic sequence having a splice signal; and iii) a donor sequence encoding a gene product of a gene of interest, or portion thereof. In embodiments, the gRNA or gRNAs are within the PTS and processed by the endonuclease. In embodiments, the gRNA or gRNAs are within the PTS and are not processed by the endonuclease. In embodiments, the gRNA or gRNAs are not within the PTS. In embodiments, the PTS is the repRNA.

[0278] In embodiments, the trans-splicing system comprises a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing. In embodiments, the trans-splicing system comprises a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In embodiments, the repRNA comprises one or more binding motifs that direct, and / or hybridizes, the repRNA to a target nucleic acid molecule. In embodiments, the repRNA further comprises a guide RNA (gRNA). In embodiments, the gRNA hybridizes to the target nucleic acid molecule. In embodiments, the gRNA directs the repRNA to a target nucleic acid molecule.

[0279] In embodiments, the repRNA is operably linked to one or more sequences that are antisense to the target nucleic acid molecule. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is not operably linked to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is operably linked to one or more sequences that bind to, and / or hybridize to, an RNA- binding polypeptide. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide, which is herein described as a “grepRNA”.

[0280] In embodiments, the grepRNA comprises a repair RNA and a gRNA. In embodiments, “grepRNA” is used interchangeably with “PTS”.

[0281] In embodiments, composition or system comprises a splice acceptor. In embodiments, composition or system comprises a splice donor.

[0282] In embodiments, the (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in cis or trans, or are suitable for being provided in cis or trans. In embodiments, the (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in trans, or are suitable for being provided in trans. In embodiments, these elements are under the control of one or more promoters. In embodiments, these elements are under the control of different promoters. In embodiments, these elements are operably linked, but separated by a cleavable sequence (e.g., a self-cleaving ribozyme). In embodiments, (i) multiple populations of repRNA are under the control of different promoters or (ii) the repRNA and another system member under control of different promoters.

[0283] In embodiments, the one or more exons is or comprises one or more exons of a target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of a target nucleic acid molecule. In embodiments, the one or more exons is or comprises one or more exons of the target nucleic acid molecule. In embodiments, the one or more introns is or comprises one or more introns of the target nucleic acid molecule.

[0284] In embodiments, the repRNA comprises one or more binding motifs that direct, and / or hybridizes, the repRNA to a target nucleic acid molecule. In embodiments, the one or more binding motifs indirectly or directly bind to, and / or hybridize to, a target nucleic acid molecule. In embodiments, the one or more binding motifs comprises a sequence that is antisense to a target nucleic acid molecule. In embodiments, the one or more binding motifs hybridizes to an exon and / or an intron, or a fragment thereof, of the target nucleic acid. In embodiments, the one or more binding motifs hybridizes to a fragment of an exon of the target nucleic acid. In embodiments, the one or more binding motifs hybridizes to a fragment of an intron of the target nucleic acid. In embodiments, the one or more binding motifs comprises about 10-300 nucleotides, about 15- 300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0285] In embodiments, the one or more binding motifs is within about 10-500 nucleotides, about 15-500 nucleotides, about 20-500 nucleotides, about 30-500 nucleotides, about 40-500 nucleotides, about 50-500 nucleotides, or about 60-500 nucleotides, or about 70-500 nucleotides, or about 80-500 nucleotides, or about 90-500 nucleotides, about 100-500 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, or about 300-400 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 nucleotides, at least about 400 nucleotides, or at least about 500 nucleotides of a sequence that binds to, and / or hybridizes to, a RNA-binding polypeptide.

[0286] In embodiments, the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any one of SEQ ID NOs: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid, wherein the USH2A target nucleic acid is selected from exon 13 (SEQ ID NO: 2023) or intron 13 SEQ ID NO: 2024. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 or SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about

[0287] 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a nontargeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 to any one of SEQ ID NOs: 804-2022 is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0288] In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans- splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, or 2.5-3.0 wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 to any one of SEQ ID NOs: 804-2022 is greater than a fold change of about 0.5, 1.0, 1.5, 2.0,

[0289] 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 to any one of SEQ ID NOs: 804- 2022 is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5- 7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, or 2.5-3.0, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0290] In embodiments, the binding motif binds to a position of intron 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2024. In embodiments, the binding motif binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,

[0291] 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,

[0292] 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,

[0293] 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,

[0294] 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,

[0295] 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,

[0296] 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,

[0297] 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,

[0298] 950, 960, 970, 980, 990, or about 1000.

[0299] In embodiments, the binding motif binds to a position of intron 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2024. In embodiments, the binding motif binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,

[0300] 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,

[0301] 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,

[0302] 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,

[0303] 990, or about 1000 nucleotides from the splice donor site in intron 13 of the USH2A target nucleic acid. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024) that is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,

[0304] 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,

[0305] 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,

[0306] 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000 nucleotides from the splice donor site in intron 13 of USH2A.

[0307] In embodiments, the binding motif binds to a position of exon 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2023. In embodiments, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from about position -10, -20, -30, -40, -50, -60, -70, or about -80. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position -10, -20, -30, -40, -50, -60, -70, or about -80.

[0308] In embodiments, the binding motif binds to a position of exon 13 of a USH2A target nucleic acid. In embodiments, the USH2A target nucleic acid is SEQ ID NO: 2023. In embodiments, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected relative to a splice donor site, without limitation, from about position -10, -20, -30, -40, -50, -60, -70, or about -80. In embodiments, the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected relative to a splice donor site, without limitation, from about position -10, -20, -30, -40, -50, -60, -70, or about -80.

[0309] In embodiments, the binding motif is selected from SEQ ID NO: 804, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NO: 804 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif is SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16, or relative to the splice donor site, about position 5 to 20, about 8 to 16, or about 12 nucleotides from the splice donor site in intron 13 of USH2A. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 12. In embodiments, the binding motif is selected from SEQ ID NO: 804 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from position 12.

[0310] In embodiments, the binding motif is selected from SEQ IDNOs: 804-818, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-818 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from about position 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130, or relative to the splice donor site, about position 5 to 20, about 8 to 16, or about 12 nucleotides from the splice donor site in intron 13 of USH2A. In embodiments, the binding motif is selected from SEQ ID NOs: 804- 818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123. In embodiments, the binding motif is selected from SEQ ID NOs: 804-818 and binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from position position 38, 43, 95, 108, 1 10, 111, 112, 113, 1 15, 118, or 123.

[0311] In embodiments, the binding motif is selected from SEQ ID NOs: 804-883, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-883 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0312] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1019, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-1019 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

[0313] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1788, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions. In embodiments, the binding motif of SEQ ID NOs: 804-1788 has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a nontargeting rate.

[0314] In embodiments, the binding motif is selected from SEQ ID NOs: 804-1788 and binds to about position 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 8, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 1 19, 2, 86, 102, 66, 5,6, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34,8, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116,5, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22,6, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69, 7, 2, 8, 102, 92,6, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104,26, 20, 36, 9, 27, 35, 138, 121, 170, 92, 36, 890, 104, 12, 870, 14, 38, 34, 34, 69, 72, 11, 72, 16,7, 136, 143, 91, 38, 1, 21, 137, 83, 61, 20, 36, 104, 29, 8, 30, 5, 84, 30, 106, 78, 16, 148, 9, 25,44, 87, 36, 74, 44, 9, 34, 14, 32, 4, 6, 35, 34, 76, 44, 77, 73, 2, 6, 2, 138, 30, 850, 62, 5, 137, 12,9, 39, 40, 23, 79, 141, 74, 30, 540, 130, 14, 15, 20, 23, 12, 77, 146, 10, 8, 2, 13, 42, 35, 28, 730,4, 143, 62, 4, 41, 24, 37, 92, 10, 840, 29, 8, 45, 138, 660, 38, 11, 20, 39, 90, 90, 107, 31, 135, 37,1, 22, 2, 142, 37, 4, 6, 40, 87, 38, 140, 38, 59, 6, 29, 139, 15, 9, 145, 640, 15, 131, 13, 141, 2, 26,8, 106, 4, 32, 62, 21, 14, 100, 820, 60, 8, 3, 33, 12, 22, 7, 59, 76, 47, 9, 30, 59, 730, 860, 26, 143,30, 540, 32, 27, 54, 136, 36, 17, 10, 22, 36, 250, 590, 155, 150, 12, 105, 81, 36, 8, 850, 34, 35,10, 52, 840, 22, 21, 18, 91, 4, 820, 52, 29, 28, 660, 31, 63, 85, 880, 18, 18, 78, 890, 640, 155, 34,3, 4, 81, 890, 78, 28, 24, 14, 22, 20, 24, 70, 210, 24, 147, 153, 10, 34, 139, 18, 19, 0, 16, 470,40, 14, 220, 630, 71, 26, 22, 23, 15, 28, 105, 93, 85, 14, 420, 146, 31, 8, 39, 37, 35, 16, 10, 530,, 78, 21, 22, 47, 60, 28, 8, 77, 0, 19, 78, 85, 20, 660, 21, 4, 45, 4, 12, 5, 24, 154, 31, 390, 470, 4,5, 8, 7, 6, 152, 140, 220, 55, 1, 25, 29, 4, 3, 27, 14, 6, 190, 45, 24, 13, 46, 18, 12, 46, 32, 151,10, 7, 20, 350, 3, 40, 620, 39, 0, 880, 30, 22, 155, 24, 0, 157, 32, 1, 8, 142, 14, 440, 160, 16, 64,30, 870, 147, 160, 240, 17, 33, 8, 2, 68, 39, 33, 141, 137, 76, 2, 19, 400, 76, 860, 58, 19, 23, 8,60, 360, 7, 8, 51, 820, 14, 12, 1, 190, 6, 26, 450, 16, 150, 52, 1, 47, 18, 24, 27, 210, 18, 57, 36,3, 650, 4, 80, 70, 32, 26, 16, 46, 150, 29, 17, 250, 600, 35, 56, 60, 48, 23, 37, 19, 0, 154, 0, 180,7, 26, 4, 830, 25, 58, 149, 410, 7, 152, 49, 0, 160, 380, 600, 16, 56, 590, 0, 28, 440, 58, 2, 630,3, 158, 31, 2, 650, 16, 240, 84, 4, 57, 330, 20, 16, 70, 9, 62, 360, 26, 26, 17, 153, 50, 14, 350, 2,6, 68, 52, 200, 154, 18, 32, 153, 27, 3, 0, 53, 460, 560, 600, 1, 159, 16, 120, 18, 0, 230, 28, 730,60, 54, 1, 30, 72, 105, 158, 390, 390, 540, 48, 880, 370, 32, 17, 4, 49, 36, 18, 8, 500, 50, 26, 2,3, 610, 25, 6, 200, 145, 4, 3, 450, 55, 16, 80, 570, 6, 460, 670, 17, 152, 76, 560, 16, 71, 20, 74,56, 14, 7, 74, 0, 220, 550, 18, 15, 530, 18, 350, 21, 450, 400, 720, 2, 66, 0, 0, 630, 500, 33, 14, 4,70, 6, 68, 2, 370, 151, 80, 50, 54, 180, 7, 0, 16, 560, 74, 158, 22, 900, 0, 1, 420, 180, 31, 60, 70,70, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151, 7, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0, 157, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25, or relative to the splice donor site, about position 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118,

[0315] 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97,

[0316] 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110,

[0317] 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128,

[0318] 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86, 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69,

[0319] 7, 2, 8, 102, 92, 36, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104, 126, 20, 36, 9, 27, 35, 138, 121, 170, 92, 36, 890, 104, 12, 870, 14, 38, 34, 34, 69, 72, 11, 72, 16, 67, 136, 143, 91, 38, 1, 21, 137, 83, 61, 20, 36, 104, 29, 8, 30, 5, 84, 30, 106, 78, 16, 148, 9, 25, 144, 87, 36, 74, 44, 9, 34, 14, 32, 4, 6, 35, 34, 76, 44, 77, 73, 2, 6, 2, 138, 30, 850, 62, 5, 137, 12, 89, 39, 40, 23, 79, 141, 74, 30, 540, 130, 14, 15, 20, 23, 12, 77, 146, 10, 8, 2, 13, 42, 35, 28, 730, 24, 143, 62, 4, 41, 24, 37, 92, 10, 840, 29, 8, 45, 138, 660, 38, 11, 20, 39, 90,

[0320] 90, 107, 31, 135, 37, 61, 22, 2, 142, 37, 4, 6, 40, 87, 38, 140, 38, 59, 6, 29, 139, 15, 9, 145, 640, 15, 131, 13, 141, 2, 26, 18, 106, 4, 32, 62, 21, 14, 100, 820, 60, 8, 3, 33, 12, 22, 7, 59, 76, 47, 9, 30, 59, 730, 860, 26, 143, 230, 540, 32, 27, 54, 136, 36, 17, 10, 22, 36, 250, 590, 155, 150, 12, 105, 81, 36, 8, 850, 34, 35, 210, 52, 840, 22, 21, 18, 91, 4, 820, 52, 29, 28, 660, 31, 63, 85, 880, 18, 18, 78, 890, 640, 155, 34, 93, 4, 81, 890, 78, 28, 24, 14, 22, 20, 24, 70, 210, 24, 147, 153, 10, 34, 139, 18, 19, 0, 16, 470, 140, 14, 220, 630, 71, 26, 22, 23, 15, 28, 105, 93, 85, 14, 420, 146, 31,

[0321] 8, 39, 37, 35, 16, 10, 530, 6, 78, 21, 22, 47, 60, 28, 8, 77, 0, 19, 78, 85, 20, 660, 21, 4, 45, 4, 12, 5, 24, 154, 31, 390, 470, 4, 25, 8, 7, 6, 152, 140, 220, 55, 1, 25, 29, 4, 3, 27, 14, 6, 190, 45, 24, 13, 46, 18, 12, 46, 32, 151, 410, 7, 20, 350, 3, 40, 620, 39, 0, 880, 30, 22, 155, 24, 0, 157, 32, 1, 8, 142, 14, 440, 160, 16, 64, 530, 870, 147, 160, 240, 17, 33, 8, 2, 68, 39, 33, 141, 137, 76, 2, 19, 400, 76, 860, 58, 19, 23, 8, 860, 360, 7, 8, 51 , 820, 14, 12, 1, 190, 6, 26, 450, 16, 150, 52, 1 , 47, 18, 24, 27, 210, 18, 57, 36, 33, 650, 4, 80, 70, 32, 26, 16, 46, 150, 29, 17, 250, 600, 35, 56, 60, 48, 23, 37, 19, 0, 154, 0, 180, 27, 26, 4, 830, 25, 58, 149, 410, 7, 152, 49, 0, 160, 380, 600, 16, 56, 590, 0, 28, 440, 58, 2, 630, 53, 158, 31, 2, 650, 16, 240, 84, 4, 57, 330, 20, 16, 70, 9, 62, 360, 26, 26, 17, 153, 50, 14, 350, 2, 26, 68, 52, 200, 154, 18, 32, 153, 27, 3, 0, 53, 460, 560, 600, 1, 159, 16, 120, 18, 0, 230, 28, 730, 360, 54, 1, 30, 72, 105, 158, 390, 390, 540, 48, 880, 370, 32, 17, 4, 49, 36, 18, 8, 500, 50, 26, 2, 23, 610, 25, 6, 200, 145, 4, 3, 450, 55, 16, 80, 570, 6, 460, 670, 17, 152, 76, 560, 16, 71, 20, 74, 156, 14, 7, 74, 0, 220, 550, 18, 15, 530, 18, 350, 21, 450, 400, 720, 2, 66, 0, 0, 630, 500, 33, 14, 4, 670, 6, 68, 2, 370, 151, 80, 50, 54, 180, 7, 0, 16, 560, 74, 158, 22, 900, 0, 1, 420, 180, 31, 60, 70, 270, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151, 7, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0, 157, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25.

[0322] In embodiments, the binding motif is any one of those of Table 5.

[0323] In embodiments, the composition or system disclosed herein comprises one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide. In embodiments, the sequence that binds to the RNA-binding polypeptide assembles into a secondary structure suitable for interaction with the RNA-binding polypeptide. In embodiments, the secondary structure is or comprises a hairpin. In embodiments, the secondary structure is or comprises a stem, internal loop, multibranch loop, or a pseudoknot.

[0324] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0325] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1 , or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0326] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0327] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0328] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0329] In embodiments, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

[0330] In embodiments, the composition or system disclosed herein comprises an endonuclease, wherein the endonuclease is a CRISPR-Cas enzyme.

[0331] In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-A, e.g., without limitation Cas8a or Cas5. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-B, e.g., without limitation Cas8b. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-C, e.g., without limitation Cas8c. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-D, e.g., without limitation CaslOd. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-E, e.g., without limitation Csel or Cse2. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-F, e.g., without limitation Csyl, Csy2, or Csy3. In embodiments, the Cas is a type I. In embodiments, the Cas is a type I-G, e.g., without limitation GSU0054. In embodiments, the Cas is a type I. In embodiments, the Cas type I is without limitation, Cas3.

[0332] In embodiments, the Cas is a type II. In embodiments, the Cas is a type II- A, e.g., without limitation Csn2. In embodiments, the Cas is a type II. In embodiments, the Cas is a type II-B, e.g., without limitation Cas4. In embodiments, the Cas is a type II. In embodiments, the Cas is a type II-C. In embodiments, the Cas is a type II. In embodiments, the Cas type II is without limitation Cas 9.

[0333] In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-A, e.g., without limitation Csm2. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-B, e g., without limitation Cmr5. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-C, e.g., without limitation CaslO or Csxl l. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-D, e.g., without limitation CsxlO. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-E. In embodiments, the Cas is a type III. In embodiments, the Cas is a type III-F. In embodiments, the Cas is a type III. In embodiments, the Cas type III is without limitation Cas 10.

[0334] In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-A. In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-B. In embodiments, the Cas is a type IV. In embodiments, the Cas is a type IV-C.

[0335] In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-A, e.g., without limitation Casl2a (Cpfl). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-B, e g., without limitation Casl2b (C2cl). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-C, e.g., without limitation Casl2c (C2c3). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-D, e.g., without limitation Casl2d (CasY). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-E, e.g., without limitation Casl2e (CasX). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-F, e.g., without limitation Casl2f (Casl4, or C2cl0). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-G, e.g., without limitation Casl2g. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-H, e g., without limitation Casl2h. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-I, e.g., without limitation Casl2i. In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-K, e g., without limitation Casl2k (C2c5). In embodiments, the Cas is a type V. In embodiments, the Cas is a type V-U, e.g., without limitation C2c4, C2c8, or C2c9. In embodiments, the Cas is a type V. In embodiments, the Cas type V is without limitation Cas 12. In embodiments, the Cas is a type VI.

[0336] In embodiments, the Cas is a type VI-A, e.g., without limitation Casl3a (C2c2). In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-B, e.g., without limitation Cas 13b. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-C, e.g., without limitation Casl3c. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-D, e.g., without limitation Casl3d. In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI-X, e g., without limitation Casl3x.l . In embodiments, the Cas is a type VI. In embodiments, the Cas is a type VI- Y. In embodiments, the Cas is a type VI. In embodiments, the Cas type VI is without limitation Cas 13.

[0337] In embodiments the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5, CaslO or Csxl l, CsxlO, Csfl, Cas9, Csn2, Cas4, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (Casl4, C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), C2c4, C2c8, C2c9, Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, or Casl3x.l.

[0338] In embodiments, the composition or system disclosed herein comprises an RNA sequence that interacts with an active or catalytically inactive endonuclease.

[0339] In embodiments, the gRNA is associated with, or suitable for associating with, one or more endonucleases. In embodiments, the endonuclease comprises one or more mutations to reduce catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations. In embodiments, the one or more mutations increase catalytic activity relative to an unmutated form. In embodiments, the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form. In embodiments, the endonuclease comprises an amino acid sequence of one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1 -4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications.

[0340] In embodiments, the composition or system disclosed herein comprises an endonuclease disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NOs: 106-130, or fragments or variants thereof), linked, associated, and / or fused to an RNA-binding polypeptide. In embodiments, the RNA-binding protein is a viral protein. In embodiments, the RNA-binding polypeptide is any RNA-binding polypeptide, optionally wherein the RNA-binding polypeptide is selected from MS2 coat protein (MCP), PP7 coat protein, PRR1, Hgall, a Qbeta coat protein, the IN protein, SLBP (Stem-Loop Histone MRNA Binding Protein), and M protein, or a variant thereof. In embodiments, the RNA-binding protein is MS2. In embodiments, the RNA-binding protein is a PP7 coat protein. In embodiments, the RNA-binding protein is PRR1. In embodiments, the RNA-binding protein is Hgall. In embodiments, the RNA-binding protein is Qbeta coat protein. In embodiments, the RNA-binding protein is the IN protein, or the SLBP protein. In embodiments, the RNA-binding protein is the M protein.

[0341] In embodiments, the composition or system disclosed herein comprises an inactive Casl3K2F endonuclease fused to an RBP. In embodiments, the composition or system disclosed herein comprises an inactive Casl3K2F endonuclease fused to MS2 (“dCasl3K2F-MS2”). In embodiments, the composition or system disclosed herein comprises an inactive Casl3K2F endonuclease fused to PP7 (“dCasl3K2F-PP7”).

[0342] In embodiments, the trans-splicing nucleic acid template comprises the splice donor.

[0343] In embodiments, the target nucleic acid comprises a splice acceptor.

[0344] In embodiments, disclosed herein is a method for targeted trans-splicing of a pre-mR A in a cell, comprising contacting the cell with the system of any one of the embodiments disclosed herein.

[0345] In embodiments, RNA molecule comprising a sequence complementary to one strand of a target nucleic molecule is a guide RNA (gRNA).

[0346] In embodiments, the endonuclease is linked, associated, and / or fused with the RNA-binding protein. In embodiments, the composition or system substantially prevents or eliminates cis-splicing of a nucleic acid.

[0347] In embodiments, trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0348] In embodiments, disclosed herein is a method for targeted trans-splicing of a pre-mRNA in a cell, comprising contacting the cell with the system of any one of the embodiments disclosed herein.

[0349] Linkers

[0350] In embodiments, the endonuclease is linked, associated, and / or fused with the RNA-binding protein. In embodiments, the endonuclease is linked with the RNA-binding protein via a linker. In embodiments, the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids. In embodiments, the linker is substantially comprised of glycine and serine residues. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS)n (n=l-4) (SEQ ID NO: 65), (Gly)s (SEQ ID NO: 66), (Gly)6(SEQ ID NO: 67), (EAAAK)n (n=l-3) (SEQ ID NO: 68), A(EAAAK)nA (n = 2-5) (SEQ ID NO: 69), AEAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

[0351] Target Nucleic Acid

[0352] In embodiments, the repRNA comprises the splice donor. In embodiments, the repRNA comprises the splice acceptor.

[0353] In embodiments, the repRNA comprises an exon of the target nucleic acid. In embodiments, the repRNA comprises an intron of the target nucleic acid. In embodiments, the repRNA comprises one or more non-natural introns. In embodiments, the target nucleic acid is a pre-mRNA transcript molecule.

[0354] In embodiments, the target nucleic acid is one or more one or more Usher syndrome-associated genes, or a fragment thereof, or a pre-mRNA sequence thereof. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the target nucleic acid is one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is exon 13 of USH2A, or a pre-mRNA sequence thereof. In embodiments, the target nucleic acid is USH2A, or a pre-mRNA sequence thereof, bearing a c.2299delG and / or C.2276G > T mutation.

[0355] In embodiments, the target nucleic acid is referred to as a target sequence. In embodiments, the target nucleic acid is or comprises a sequence of contiguous nucleotides present in a target RNA or target DNA. In embodiments, a stretch of contiguous nucleotides refers to a string of nucleotides that are covalently linked and immediately adjacent to one another. In embodiments, the target nucleic acid is or comprises at least about 10, 20, 30, 40, 50, 75, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500, 5,000, 7,500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 nucleotides in length. In embodiments, the target nucleic acid is or comprises less than about 300,000, 250,000, 200,000, 100,000, 150,000, or 50,000 nucleotides in length. In embodiments, the target nucleic acid is or comprises about 5-10, about 5-15, about 5-20, about 10- 20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50-150, about 50-200, about 50-250, about 50-300, about 100- 200, about 100-300, about 200-300, about 100-400, about 100-500, about 100-600, about 100-700, about 100-800, about 100-900, about 100-1000, about 200-400, about 200-500, about 200-600, about 200-700, about 200-800, about 200-900, about 200-1000, about 300-400, about 300-500, about 300-600, about 300-700, about 300-800, about 300-900, about 300-1000, about 300-400, about 300-500, about 300-600, about 300-700, about 300-800, about 300-900, about 300-1000, about 400-500, about 400-600, about 400-700, about 400-800, about 400-900, about 400-1000, about 500-600, about 500-700, about 500-800, about 500-900, about 500-1000, about 1,000-5,000, about 1 ,000-10,000, about 1,000-15,000, about 1,000-20,000, about 1 ,000-25,000, about 1,000- 30,000, about 1,000-35,000, about 1,000-40,000, about 1,000-45,000, about 1,000-50,000, about 2,000-5,000, about 2,000-10,000, about 2,000-15,000, about 2,000-20,000, about 2,000-25,000, about 2,000-30,000, about 2,000-35,000, about 2,000-40,000, about 2,000-45,000, about 2,000- 50,000, about 3,000-5,000, about 3,000-10,000, about 3,000-15,000, about 3,000-20,000, about 3,000-25,000, about 3,000-30,000, about 3,000-35,000, about 3,000-40,000, about 3,000-45,000, about 3,000-50,000, about 4,000-5,000, about 4,000-10,000, about 4,000-15,000, about 4,000- 20,000, about 4,000-25,000, about 4,000-30,000, about 4,000-35,000, about 4,000-40,000, about 4,000-45,000, about 4,000-50,000, about 5,000-10,000, about 5,000-15,000, about 5,000-20,000, about 5,000-25,000, about 5,000-30,000, about 5,000-35,000, about 5,000-40,000, about 5, GOO- 45, 000, about 5,000-50,000, about 10,000-15,000, about 10,000-20,000, about 10,000-25,000, about 10,000-30,000, about 10,000-35,000, about 10,000-40,000, about 10,000-45,000, about 10,000-50,000, about 15,000-20,000, about 15,000-25,000, about 15,000-30,000, about 15, GOO- 35, 000, about 15,000-40,000, about 15,000-45,000, about 15,000-50,000, about 20,000-25,000, about 20,000-30,000, about 20,000-35,000, about 20,000-40,000, about 20,000-45,000, about 20,000-50,000, about 25,000-30,000, about 25,000-35,000, about 25,000-40,000, about 25, GOO- 45, 000, about 25,000-50,000, about 30,000-35,000, about 30,000-40,000, about 30,000-45,000, about 30,000-50,000, about 35,000-40,000, about 35,000-45,000, about 35,000-50,000, about 40,000-45,000, or about 45,000-50,000 nucleotides in length.

[0356] In embodiments, the target nucleic acid is 10-50,000 nucleotides in length, e.g., 10-45,000, 10-

[0357] 40,000, 10-35,000, 10-30,000, 10-20,000, 11-45,000, 11-40,000, 11-35,000, 11-30,000, 11-

[0358] 20,000, 12-45,000, 12-40,000, 12-35,000, 12-30,000, 12-25,000, 12-20,000, 13-45,000, 13-

[0359] 40,000, 13-35,000, 13-30,000, 13-25,000, 13-20,000, 14-45,000, 14-40,000, 14-35,000, 14-

[0360] 30,000, 14-25,000, 14-20,000, 15-45,000, 15-40,000, 15-35,000, 15-30,000, 15-25,000, 15-

[0361] 20,000, 16-45,000, 16-40,000, 16-35,000, 16-30,000, 16-25,000, 16-20,000, 17-45,000, 17-

[0362] 40,000, 17-35,000, 17-30,000, 17-25,000, 17-20,000, 18-45,000, 18-40,000, 18-35,000, 18-

[0363] 30,000, 18-25,000, 18-20,000, 19-45,000, 19-40,000, 19-35,000, 19-30,000, 19-25,000, 19-

[0364] 20,000, e.g, 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, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or 50,000 nucleotides in length.

[0365] In embodiments, the target nucleic acid is or comprises ssRNA. In embodiments, the target nucleic acid is or comprises dsRNA. In embodiments, the target nucleic acid is or comprises ssDNA. In embodiments, the target nucleic acid is or comprises dsDNA. In embodiments, the target nucleic acid is about 2 to about 6 nucleotides upstream of a PAM sequence.

[0366] In embodiments, the target nucleic acid is proximal to an exon. In embodiments, the target nucleic acid is upstream of an exon. In embodiments, the target nucleic acid is downstream of an exon. In embodiments, the target nucleic acid overlaps with an exon.

[0367] In embodiments, the target nucleic acid is proximal to an intron. In embodiments, the target nucleic acid is upstream of an intron. In embodiments, the target nucleic acid is downstream of an intron. In embodiments, the target nucleic acid overlaps with an intron.

[0368] Hybridization and / or Binding

[0369] In embodiments, the present disclosure provides a composition comprising a repair RNA (repRNA) sequence, comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing.

[0370] In embodiments, the present disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor.

[0371] In embodiments, the hybridization is mediated by perfect sequence complementarity to one strand of a target nucleic acid molecule. In embodiments, the hybridization is mediated by partial sequence complementarity to one strand of a target nucleic acid molecule.

[0372] In embodiments, the endonuclease comprises an amino acid sequence of one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications.

[0373] In embodiments, the RNA-binding polypeptide and / or endonuclease bind to the target nucleic molecule. In embodiments, the composition or system substantially prevents or eliminates cis-splicing of a nucleic acid.

[0374] In embodiments, trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0375] In embodiments, the guide ribonucleic structure (i) comprises (a) a CRISPR RNA (crRNA) suitable for hybridizing to a target nucleic acid molecule and / or (b) a transactivating CRISPR RNA (tracrRNA) suitable for interacting with the endonuclease or (ii) lacks a (a) a crRNA suitable for hybridizing to a target nucleic acid molecule and / or (b) a tracrRNA suitable for interacting with the endonuclease.

[0376] In embodiments, the endonuclease repeat sequences to formulate the guide RNA are selected from Table 3 below.

[0377] Table 3: Endonuclease repeat sequences to formulate the guide RNA

[0378] In embodiments, the guide RNA is or comprises a sequence of SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 28 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 29 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 30 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 31 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 90 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 91 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 92 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 93 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 94 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 95 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 96 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the guide RNA is or comprises a sequence of SEQ ID NO: 97 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0379] In embodiments, the endonuclease is a polypeptide of one of SEQ ID NOs: 1-4, SEQ ID NOs: SO- 89, and / or SEQ ID NOs: 106-130, or a fragment or a variant thereof, or a nucleic acid polypeptide of one of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof. In embodiments, the endonuclease is guided to a target nucleic acid using a guide RNA selected from SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, and / or the endonuclease is associated with a guide RNA selected from SEQ ID NOs: 28- 31, and / or SEQ ID NOs: 90-97, or a fragment or variant thereof.

[0380] In embodiments, the endonuclease is SEQ ID NO: 1, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0381] In embodiments, the endonuclease is SEQ ID NO: 2, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0382] In embodiments, the endonuclease is SEQ ID NO: 3, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0383] In embodiments, the endonuclease is SEQ ID NO: 4, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0384] In embodiments, the endonuclease is SEQ ID NO: 80, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0385] In embodiments, the endonuclease is SEQ ID NO: 81, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0386] In embodiments, the endonuclease is SEQ ID NO: 82, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0387] In embodiments, the endonuclease is SEQ ID NO: 83, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0388] In embodiments, the endonuclease is SEQ ID NO: 84, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0389] In embodiments, the endonuclease is SEQ ID NO: 85, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0390] In embodiments, the endonuclease is SEQ ID NO: 86, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0391] In embodiments, the endonuclease is SEQ ID NO: 87, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0392] In embodiments, the endonuclease is SEQ ID NO: 88, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0393] In embodiments, the endonuclease is SEQ ID NO: 89, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0394] In embodiments, the endonuclease is SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97, or a fragment or variant thereof.

[0395] In embodiments, the endonuclease is SEQ ID NO: 1, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28.

[0396] In embodiments, the endonuclease is SEQ ID NO: 2, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 29.

[0397] In embodiments, the endonuclease is SEQ ID NO: 3, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 30.

[0398] In embodiments, the endonuclease is SEQ ID NO: 4, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 31.

[0399] In embodiments, the endonuclease is SEQ ID NO: 80, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90.

[0400] In embodiments, the endonuclease is SEQ ID NO: 81, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90

[0401] In embodiments, the endonuclease is SEQ ID NO: 82, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90.

[0402] In embodiments, the endonuclease is SEQ ID NO: 83, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 91.

[0403] In embodiments, the endonuclease is SEQ ID NO: 84, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 92.

[0404] In embodiments, the endonuclease is SEQ ID NO: 85, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 93, or SEQ ID NO: 94.

[0405] In embodiments, the endonuclease is SEQ ID NO: 86, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28. In embodiments, the endonuclease is SEQ ID NO: 87, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28.

[0406] In embodiments, the endonuclease is SEQ ID NO: 88, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 95, or SEQ ID NO: 96.

[0407] In embodiments, the endonuclease is SEQ ID NO: 89, or any one of SEQ ID NOs: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 97.

[0408] In embodiments, the RNA molecule is or comprises at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In embodiments, the RNA molecule is or comprises less than about 300, 250, 200, 100, 150, or 50 nucleotides in length. In embodiments, the RNA molecule is or comprises about 5-10, about 5-15, about 5-20, about 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50-150, about 50-200, about 50-250, about 50-300, about 100-200, about 100-300, or about 200-300 nucleotides in length.

[0409] In embodiments, the RNA molecule is 10-50 nucleotides in length, e.g., 10-45, 10-40, 10-35, 10- 30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0410] In embodiments, the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, in conjunction with an RNA containing a repeat having at least about 70% identity to one or more of SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97. In embodiments, the composition has least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or has about 1 to about 20 nucleotide modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In embodiments, the sequence comprises at least one HEPN domain, or fragments or variants thereof In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.

[0411] Nucleic Acid Modifications

[0412] In embodiments, a nucleic acid disclosed herein has one or more modifications, e.g., a base modification, a backbone modification, and the like, to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2’, the 3’, or the 5’ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3’ to 5’ phosphodiester linkage.

[0413] In embodiments, a suitable nucleic acid modification include, but are not limited to: 2’0 methyl modified nucleotides, 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap e.g., a 7-methylguanylate cap (m7G)). A 2’-O-Methyl modified nucleotide (also referred to as 2’-0-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2’-O-Methyl RNA. Without wishing to be bound by theory, this modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically about 5 to about 10-fold less susceptible to Dnases than DNA. 2’ Fluoro modified nucleotides (e.g., 2’ Fluoro bases) have a fluorine modified ribose which increases binding affinity I and also confers some relative nuclease resistance when compared to native RNA. LNA bases have a modification to the ribose backbone that locks the base in the C3’-endo position, which favors RNA A-type helix duplex geometry. Without wishing to be bound by theory, this modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3 ’-end. Applications have been described ranging from antisense oligos to hybridization probes to SNP detection and allele specific PCR. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. In embodiments, the number of LNAs incorporated into a single oligo is 10 bases or less. The phosphorothioate (PS) bond e.g., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). Without wishing to be bound by theory, this modification renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5’- or 3 ’-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.

[0414] Modified Backbones and Modified Inter nucleoside Linkages

[0415] In embodiments, the present nucleic acids, optionally containing modifications, include nucleic acids containing modified backbones or non-natural internucleoside linkages. In embodiments, nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0416] In embodiments, oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 ’-alkylene phosphonates, 5 ’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 ’-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 ’-5’ linkages, 2’ -5’ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage. Suitable oligonucleotides having inverted polarity comprise a single 3’ to 3’ linkage at the 3’-most internucleotide linkage i.e., a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.

[0417] In embodiments, the present nucleic acids comprise one or more phosphorothioate and / or heteroatom internucleoside linkages, in particular -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -0-P(=0)(0H)-0-CH2-). MMI type intemucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677, the disclosure of which is incorporated herein by reference in its entirety. Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.

[0418] Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside 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; riboacetyl 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.

[0419] Mimetics

[0420] In embodiments, the present nucleic acid is in the form of or comprises a nucleic acid mimetic. In embodiments, the term “mimetic” as it is applied to polynucleotides is intended to include polynucleotides where only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar- backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to nitrogen atoms of the amide portion of the backbone.

[0421] In embodiments, the present nucleic acid is or comprises a mimetic based on morpholino units (morpholino nucleic acid), having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.

[0422] In embodiments, the present nucleic acid is a mimetic is in the form of or comprises cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA / RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595- 8602, the disclosure of which is incorporated herein by reference in its entirety). In general, the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA / RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.

[0423] In embodiments, the present nucleic acid comprises Locked Nucleic Acids (LNAs) in which the 2’ -hydroxyl group is linked to the 4’ carbon atom of the sugar ring thereby forming a 2’-C, 4’-C- oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (- CH2-), group bridging the 2’ oxygen atom and the 4’ carbon atom wherein n is i or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10° C), stability towards 3’-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Set. U.S.A., 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).

[0424] Modified Sugar Moieties

[0425] In embodiments, the present nucleic acid comprises one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: 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 may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or 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 the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2’- methoxy ethoxy (2’-O-CH2CH2OCH3, also known as 2’-O-(2 -methoxy ethyl) or 2’-M0E) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group. A further suitable modification includes 2’- dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2’-DMA0E, as described in examples hereinbelow, and 2’-dimethylaminoethoxy ethoxy (also known in the art as 2’-O-dimethyl-amino-ethoxy-ethyl or 2’-DMAEOE), i.e., 2’-O-CH2-O-CH2-N(CH3)2.

[0426] Other suitable sugar substituent groups include methoxy (-O-CH3), aminopropoxy (—0 CH2 CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2— CH=CH2) and fluoro (F). 2’ -sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2’- arabino modification is 2’-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3’ position of the sugar on the 3’ terminal nucleoside or in 2’-5’ linked oligonucleotides and the 5’ position of 5’ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0427] Base Modifications and Substitutions

[0428] In embodiments, the present nucleic acid comprises one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions. In embodiments, as used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CHs) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3’,2’:4,5)pyrrolo(2,3- d)pyrimidin-2-one).

[0429] In embodiments, heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Certain nucleobases are, without wishing to be bound by theory, useful for increasing the binding affinity of an oligomeric compound. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability and are suitable base substitutions, e.g., when combined with 2’-O-methoxyethyl sugar modifications.

[0430] Conjugates

[0431] In embodiments, the present compositions are chemically linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. In embodiments, the moieties or conjugates include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.

[0432] In embodiments, the conjugate may include a Protein Transduction Domain or PTD (also known as a CPP - cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and / or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle (e.g., the nucleus). In embodiments, a PTD is covalently linked to the 3’ end of an exogenous polynucleotide. In embodiments, a PTD is covalently linked to the 5’ end of an exogenous polynucleotide.

[0433] Kits

[0434] In aspects, the present disclosure provides a kit comprising a container comprising the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, the cell of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein, and instructions for use in trans-splicing a nucleic acid.

[0435] In embodiments, the present disclosure provides kits for carrying out the methods described herein. In embodiments, the kit comprises a composition described herein, a recombinant expression vector, a delivery system, and / or a pharmaceutical composition described herein, optionally further with a reagent for reconstitution and / or dilution.

[0436] General Features of the Compositions and Systems

[0437] In embodiments, the composition further comprises a viral vector. In embodiments, the viral vector is or comprises an AAV. In embodiments, the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9. In embodiments, the composition further comprises a non-viral vector.

[0438] In embodiments, the present compositions take the form of, or delivery of the present compositions is effected using, a nanoparticle, e.g., any particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles suitable for use in delivering the present compositions to a target cell have a diameter of about 500 nm or less, e.g., from about 25 nm to about 35 nm, from about 35 nm to about 50 nm, from about 50 nm to about 75 nm, from about 75 nm to about 100 nm, from about 100 nm to about 150 nm, from about 150 nm to about 200 nm, from about 200 nm to about 300 nm, from about 300 nm to about 400 nm, or from about 400 nm to about 500 nm. In embodiments, nanoparticles suitable for use in delivering the present compositions to a target cell have a diameter of from about 25 nm to about 200 nm. In embodiments, nanoparticles suitable for use in delivery have a diameter of about 100 nm or less. In embodiments, nanoparticles suitable for use in delivery have a diameter of from about 35 nm to about 60 nm.

[0439] In embodiments, the composition further comprises a lipid nanoparticle (LNP) liposomes, lipoplexes or polymeric nanoparticle. In embodiments, the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids, and lipoids. In embodiments there is provided a particle, e.g., a delivery particle comprising lipid or lipidoid and hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance where the cationic lipid comprises l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2- ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and / or where the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or where the particle further comprises cholesterol e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5).

[0440] In embodiments, a liposome is used to deliver a composition of the present disclosure to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid fdm is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2- distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

[0441] In embodiments, the composition is in the form of a lipoplex. Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also, as a result of their charge, they interact with the cell membrane. Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.

[0442] In embodiments, the composition is in the form of a polyplex. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

[0443] In embodiments, the composition is in the form of a dendrimer, a highly branched macromolecule with a spherical shape may be also be used to genetically modify stem cells. The surface of the dendrimer particle may be functionalized to alter its properties. In particular, it is possible to construct a cationic dendrimer (e.g., one with a positive surface charge). When in the presence of genetic material such as a DNA plasmid, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination, the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.

[0444] In aspects, the composition or system components are nucleic acids. In embodiments, the RNA is or comprises mRNA or modified mRNA (mmRNA). In embodiments, the DNA molecule is or comprises a vector or plasmid. In embodiments, the nucleic acid comprises a codon optimized sequence. In embodiments, the nucleic acid comprises one or more modifications. In embodiments, the modifications are one or more of base modifications and backbone modifications.

[0445] In embodiments, sugar-based particles may be used, for example GalNAc, can be used to deliver a composition of the present disclosure to a target cell.

[0446] In aspects, the present disclosure provides a viral vector comprising the nucleic acid of any one of the embodiments and / or aspects disclosed herein. In aspects, the present disclosure provides an expression vector comprising the nucleic acid of any one of the embodiments and / or aspects disclosed herein.

[0447] In embodiments, the expression vector is selected from viral expression vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94: 10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In embodiments, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In embodiments, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In embodiments, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0448] In embodiments, the viral vector is or comprises an AAV. In embodiments, the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

[0449] In embodiments, the composition further comprises a VLP, e.g. a structure that in at least one attribute resembles a virus, but which has not been demonstrated to be infectious. In embodiments, the VLP is a nonreplicating, noninfectious viral shell that contains a viral capsid but lacks all or part of the viral genome, in particular, the replicative components of the viral genome. In embodiments, the VLP is composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface, and structural proteins (e.g., VP1, VP2). In embodiments, the VLP resembles the structure of a bacteriophage, being non-repl icative and noninfectious, and lacking at least the gene or genes coding for the replication machinery of the bacteriophage, and also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. In embodiments, the VLP comprises a polypeptide that promotes or is suitable for VLP delivery, including, without limitation, a retroviral gag polyprotein comprising a matrix polypeptide, a capsid polypeptide, and a nucleocapsid polypeptide (optionally with one or more heterologous protease cleavage sites (e.g. TEV cleavage site, a PreScission (fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage site, a human rhinovirus 3C protease cleavage site, an enterokinase cleavage site, an Epstein-Barr virus protease cleavage site, a cathepsin D cleavage site, and / or a thrombin cleavage site) between one or both of: the matrix polypeptide and the capsid polypeptide; and the capsid polypeptide and the nucleocapsid polypeptide, e.g., a lentiviral gag polyprotein, e.g., a bovine immunodeficiency virus gag polyprotein, a murine leukemia virus (MLV) a gag protein, a simian immunodeficiency virus gag polyprotein, a feline immunodeficiency virus gag polyprotein, a human immunodeficiency virus gag polyprotein, an equine infection anemia virus gag polyprotein, and a caprine arthritis encephalitis virus gag polyprotein or a gag polyprotein of an alpha retrovirus, a beta retrovirus, a gamma retrovirus, a delta retrovirus, an epsilon retrovirus, or a spumavirus. In embodiments, the polypeptide that promotes or is suitable for VLP delivery is co-delivered with a protease to promote cleavage of the chimeric protein. In embodiments, the cleavage of the chimeric protein occurs between the endonuclease and the polypeptide that promotes or is suitable for VLP delivery. In embodiments, the protease is fused to a polypeptide that promotes or is suitable for VLP delivery.

[0450] Depending on the host / vector system utilized, in embodiments, any of a number of transcription and / or translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like may be used in the expression vector. In embodiments, a nucleotide sequence encoding a present RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In embodiments, a nucleotide sequence encoding a present protein, or a present fusion polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In embodiments, the transcriptional control element is a promoter. In embodiments, the promoter is a constitutively active promoter. In embodiments, the promoter is a regulatable promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is a cell type-specific promoter. In embodiments, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in embodiments, the transcriptional control element is functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, and the like). In embodiments, eukaryotic promoters (promoters functional in a eukaryotic cell) include EFla, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0451] In embodiments, a nucleotide sequence encoding a present RNA and / or a present fusion polypeptide is operably linked to an inducible promoter. In embodiments, a nucleotide sequence encoding a present RNA and / or a present chimeric protein is operably linked to a constitutive promoter.

[0452] In embodiments, the promoter is derived from viruses and can therefore be referred to as viral promoters, or they are derived from any organism, including prokaryotic or eukaryotic organisms. In embodiments, the promoter is used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter (e.g. is or comprising SEQ ID NO: 76, or a variant thereof) such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, -97 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31(17)), a human Hl promoter (Hl), and the like.

[0453] In embodiments, a nucleotide sequence encoding a present RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an Hl promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another PolIII promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA). This is because a string of Ts (e.g., about 5 Ts) in DNA can act as a terminator for polymerase III (PolIII). Thus, in order to ensure transcription of an RNA in a eukaryotic cell it may be desirable to modify the sequence encoding the RNA to eliminate runs of Ts. In embodiments, a nucleotide sequence encoding a present protein (e.g., a disclosed protein or chimeric protein) is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EFla promoter, an estrogen receptor- regulated promoter, and the like).

[0454] In embodiments, the inducible promoter includes, but are not limited to, one of T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline- regulated promoter, Steroid-regulated promoter, Metal -regulated promoter, estrogen receptor- regulated promoter, and the like. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; estrogen and / or an estrogen analog; IPTG; and the like. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g, heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0455] In embodiments, the promoter is a spatially restricted promoter (e.g, cell type specific promoter, tissue specific promoter, and the like) such that in a multi-cellular organism, the promoter is active (e.g., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g, eukaryotic cell; prokaryotic cell).

[0456] In embodiments, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), and the like), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, and the like), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, and the like), metal regulated promoters (e.g., metallothionein promoter systems, and the like), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, and the like), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, and the like), light regulated promoters, synthetic inducible promoters, and the like.

[0457] In embodiments, the vector contains a ribosome binding site for translation initiation and a transcription terminator. In embodiments, the vector includes appropriate sequences for amplifying expression. In embodiments, the vector includes nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag (e.g., GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)), fluorescent protein, and the like) that are fused to the present protein, e.g., at the N- or C- terminus or between the N- or C- terminus, thus resulting in a fusion present polypeptide.

[0458] In embodiments, methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a present protein and / or a present RNA, and the like) into a host cell are known in the art, and any convenient method is used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0459] In embodiments, introducing the recombinant expression vector into cells can occur in any culture media and under any culture conditions that promote the survival of the cells. In embodiments, introducing the recombinant expression vector into a target cell is carried out in vivo or ex vivo. In embodiments, introducing the recombinant expression vector into a target cell is carried out in vitro.

[0460] In embodiments, the present protein (e.g, endonuclease, chimeric protein) is provided as a nucleic acid. In embodiments, the present protein (e.g, endonuclease, chimeric protein) is provided as RNA. In embodiments, the present protein (e.g., endonuclease, chimeric protein) is provided as DNA. In embodiments, the RNA is generated by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the present protein). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, and the like).

[0461] Methods of introducing a nucleic acid into a host cell are known in the art, and any convenient method can be used to introduce a present composition into a target cell e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0462] In embodiments, nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g., Angel and Yanik (2010) PloS ONE 5(7): el 1756, and the commercially available TRANSMESSENGER reagents from Qiagen, STEMFECT RNA Transfection Kit from Stemgent, and TRANSIT-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer et al. (2008) PNAS 105(50): 19821-19826.

[0463] In aspects, the present disclosure provides a lipid nanoparticle comprising the nucleic acid of any one of the embodiments and / or aspects disclosed herein.

[0464] In aspects, the present disclosure provides a cell comprising a nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, or the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein.

[0465] In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell. In embodiments, the cell is an immortalized cell. In embodiments, the cell is harvested from a subject.

[0466] In embodiments, the cell derived from the subject is derived from a biological sample. In embodiments, the biological sample comprises a biopsy, tissue or bodily fluid. In embodiments, the biological sample comprises one or more of tumor cells, cultured cells, stem cells, and differentiated cells. In embodiments, biological sample refers to a sample obtained or derived from a source of interest (e.g., a cell), as described herein. In embodiments, a source of interest comprises an organism, such as an animal or human. In embodiments, a biological sample is a biological tissue or fluid. Non-limiting examples of biological samples include bone marrow, blood, blood cells, ascites, (tissue or fine needle) biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, swabs e.g., skin swabs, vaginal swabs, oral swabs, and nasal swabs), washings or lavages such as a ductal lavages or broncheoalveolar lavages, aspirates, scrapings, specimens (e.g., bone marrow specimens, tissue biopsy specimens, and surgical specimens), feces, other body fluids, secretions, and / or excretions, and cells therefrom, and the like.

[0467] In embodiments, the present disclosure provides a modified cell comprising a composition of the present disclosure. In embodiments, the present disclosure provides a modified cell comprising a composition of the present disclosure, where the modified cell is a cell that does not normally comprise a composition of the present disclosure. In embodiments, the present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a composition of the present disclosure. In embodiments, there is provided a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding a composition of the present disclosure. In embodiments, there is provided a genetically modified cell that is genetically modified with a recombinant expression vector comprising a composition of the present disclosure.

[0468] In embodiments, the cells are primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; and the like. In embodiments, a cell that serves as a recipient for a composition of the present disclosure is referred to as a “host cell” or a “target cell.” In embodiments, the host cell or a target cell can be a recipient of a composition or system of the present disclosure. A host cell or a target cell can be a recipient of a RNP of the present disclosure. A host cell or a target cell can be a recipient of a single component of a k system of the present disclosure.

[0469] Non-limiting examples of cells (target cells) include: a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, and the like), an algal cell, (e.g, Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g, a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e. , fruit fly, cnidarian, echinoderm, nematode, and the like), a cell from a vertebrate animal (e.g, fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g, an ungulate (e.g, a pig, a cow, a goat, a sheep); a rodent (e.g, a rat, a mouse); a non-human primate; a human; a feline (e.g, a cat); a canine (e.g, a dog); and the like), and the like. In embodiments, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0470] In embodiments, the cell is an in vitro cell (e.g, established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). In embodiments, the cell is in vivo cell (e.g., a cell in an individual). In embodiments, the cell is an isolated cell. In embodiments, the cell is a cell inside of an organism. In embodiments, the cell is an organism. In embodiments, the cell is a cell in a cell culture (e.g., in vitro cell culture). In embodiments, the cell is in a collection of cells. In embodiments, the cell is a prokaryotic cell or derived from a prokaryotic cell. Cell culture (e.g, in vitro cell culture). In embodiments, the cell is cell in a collection of cells. In embodiments, the cell is a bacterial cell or can be derived from a bacterial cell. In embodiments, the cell is an archaeal cell or derived from an archaeal cell. In embodiments, the cell is an eukaryotic cell or derived from a eukaryotic cell. In embodiments, the cell is a plant cell or derived from a plant cell. In embodiments, the cell is an animal cell or derived from an animal cell. In embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In embodiments, the cell is a mammalian cell or derived from a mammalian cell. In embodiments, the cell is a rodent cell or derived from a rodent cell. In embodiments, the cell is a human cell or derived from a human cell. In embodiments, the cell is a microbe cell or derived from a microbe cell. In embodiments, the cell is a fungal cell or derived from a fungal cell. In embodiments, the cell is an insect cell. In embodiments, the cell is an arthropod cell. In embodiments, the cell is a protozoan cell.

[0471] In embodiments, the suitable cells include a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g, an oocyte, a sperm, an oogonia, a spermatogonia, and the like); a somatic cell, e.g., a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, and the like.

[0472] In embodiments, the suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0473] In embodiments, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In embodiments, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In embodiments, the immune cell is a cytotoxic T cell. In embodiments, the immune cell is a helper T cell. In embodiments, the immune cell is a regulatory T cell (Treg).

[0474] In embodiments, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells. Adult stem cells are resident in differentiated tissue but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like. Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In embodiments, the stem cell is a human stem cell. In embodiments, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In embodiments, the stem cell is a non-human primate stem cell. In embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+and CD3’. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0475] In embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0476] In embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0477] In embodiments, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g, Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, com stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (Chinese mustard), gailon, galanga (siam, Thai ginger), garlic, ginger root, gobo, greens, Hanover salad greens, huauzontle, Jerusalem artichokes, jicama, kale greens, kohlrabi, lamb’s quarters (quilete), lettuce (bibb), lettuce (Boston), lettuce (Boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (Russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled / ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, Swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[0478] In embodiments, the cell is an arthropod cell. For example, the cell can be a cell of a sub-order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Che / icerala, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Elolometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0479] In embodiments, the cell is an insect cell. For example, in embodiments, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

[0480] In aspects, the present disclosure provides a pharmaceutical composition comprising the composition or system of any one of the embodiments and / or aspects disclosed herein, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the viral vector of any one of the embodiments and / or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and / or aspects disclosed herein, or the cell of any one of the embodiments and / or aspects disclosed herein, and a pharmaceutically acceptable carrier.

[0481] The compositions described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds ), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

[0482] Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxy benzoate, naphthalene-2 -benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne- 1,4- di carb oxy late, hexyne- 1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p- bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthal ene-1 -sulfonate, naphthalene-2-sulfonate, naphthalene-l,5-sulfonate, xylenesulfonate, and tartarate salts.

[0483] The term “pharmaceutically acceptable salt” also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH- lower alkyl amines), such as mono-; bis, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkydamines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl- D-glucamine; and amino acids such as arginine, lysine, and the like.

[0484] In embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

[0485] In various embodiments, the present invention pertains to pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable carrier or excipient. Any pharmaceutical compositions described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

[0486] In various embodiments, pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In embodiments, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington ’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds.’ 19thed. 1995), incorporated herein by reference.

[0487] The present invention includes the described pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any inventive pharmaceutical composition (and / or additional therapeutic agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powder, frozen suspension, desiccated powder, or any other form suitable for use. In embodiments, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In embodiments, the pharmaceutical composition is formulated in the form of a soft-gel capsule. In a further embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In embodiments, the pharmaceutical composition is formulated as a liquid.

[0488] Where necessary, the inventive pharmaceutical compositions (and / or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.

[0489] The formulations comprising the inventive pharmaceutical compositions (and / or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

[0490] In various embodiments, any pharmaceutical compositions (and / or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

[0491] Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically. Administration can be local or systemic. In embodiments, the administration is by parenteral injection. The mode of administration can be left to the discretion of the practitioner and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream. In embodiments, the compositions described herein is formulated in accordance with routine procedures as a composition adapted for oral administration. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving any compositions described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A timedelay material such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In embodiments, the excipients are of pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.

[0492] Dosage forms suitable for parenteral administration (e.g, intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g, lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0493] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany , NJ) or phosphate buffered saline (PB S). The carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.

[0494] The compositions provided herein, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0495] Any inventive pharmaceutical compositions (and / or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled-or sustained-release of one or more active ingredients using, for example, hydropropyl cellulose, hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.

[0496] Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

[0497] I l l In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249: 1527-1533) may be used.

[0498] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by fdtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0499] Endonucleases (Protein, Nucleic Acid, and System)

[0500] The present disclosure provides, in aspects, a composition comprising an endonuclease comprising a sequence, or a fragment or variant thereof, having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).

[0501] The present disclosure provides, in aspects, a composition comprising an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).

[0502] In embodiments, one or more HEPN domains are located according to the positions of the arrows in FIG. 7 A, FIG. 7B, FIG. 7C, and FIG. 7D and / or by reference to Table 1 or Table 2. In embodiments, the endonuclease recognizes a PAM. In embodiments, the endonuclease recognizes a plurality of PAMs (e.g., about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 PAMs). In embodiments, the PAM sequence is about 1 to about 20, or about 2 to about 12, or about 2 to about 6, or about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 nucleotides in length.

[0503] In embodiments, the endonuclease (or chimeric protein) comprises one or more mutations to reduce catalytic activity relative to an unmutated form. In embodiments, the one or more mutations to reduce catalytic activity relative to an unmutated form are in one or more HEPN domains of the present endonucleases. One of skill in the art may select the one or more mutations to reduce catalytic activity relative to an unmutated form by reference, e.g., to FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D and / or by reference to Table 1 or Table 2 and / or by reference to structural information about other endonucleases known in the art, e.g. Slaymaker, et al., “High-Resolution Structure of Casl3b and Biochemical Characterization of RNA Targeting and Cleavage “2019, Cell Reports 26, 3741-3751, Zhang et al., “Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Casl3d” Cell 175(1): 212-22, 2018 (each hereby incorporated by reference in their entireties), and the like.

[0504] In embodiments, the endonuclease (or chimeric protein) comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form. In embodiments, the one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form are in one or more HEPN domains of the present endonucleases. One of skill in the art may select the one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form by reference, e.g., to FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D and / or by reference to Table 1 or Table 2 and / or by reference to structural information about other endonucleases known in the art, e.g. Slaymaker, et al., “High-Resolution Structure of Cast 3b and Biochemical Characterization of RNA Targeting and Cleavage “2019, Cell Reports 26, 3741-3751, Zhang et al., “Structural Basis for the RNA- Guided Ribonuclease Activity of CRISPR-C...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a repair RNA (repRNA) sequence, comprising:(a) one or more exons and / or introns;(b) a splice donor and / or splice acceptor, wherein the repRNA is suitable for trans-splicing.

2. A system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising:(a) one or more exons and / or introns; and(b) a splice donor and / or splice acceptor.

3. The composition of claim 1, wherein the one or more exons is or comprises one or more exons of a target nucleic acid molecule.

4. The composition of claim 1, wherein the one or more introns is or comprises one or more introns of a target nucleic acid molecule.

5. The system of claim 2, wherein the one or more exons is or comprises one or more exons of the target nucleic acid molecule.

6. The system of claim 2, wherein the one or more introns is or comprises one or more introns of the target nucleic acid molecule.

7. The composition or system of any one of claims 1-6, wherein the repRNA comprises one or more binding motifs that direct, and / or hybridizes, the repRNA to a target nucleic acid molecule.

8. The composition or system of claim 7, wherein the one or more binding motifs indirectly or directly bind to, and / or hybridize to, a target nucleic acid molecule.

9. The composition or system of any one of claims 7-8, wherein the one or more binding motifs comprises a sequence that is antisense to a target nucleic acid molecule.

10. The composition or system of any one of claims 7-9, wherein the one or more binding motifs hybridizes to an exon and / or an intron, or a fragment thereof, of the target nucleic acid.

11. The composition or system of any one of claims 7-10, wherein the one or more binding motifs hybridizes to a fragment of an exon of the target nucleic acid.

12. The composition or system of any one of claims 7-11, wherein the one or more binding motifs hybridizes to a fragment of an intron of the target nucleic acid, optionally wherein the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate, optionally wherein the binding motif binds to a position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, or about 1000 nucleotides from the splice donor site in intron 13 of the USH2A target nucleic acid, optionally wherein the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024) that is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000 nucleotides from the splice donor site in intron 13 of USH2A.

13. The composition or system of any one of claims 7-12, wherein the one or more binding motifs comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides, optionally comprising the polynucleotide sequence of any one of SEQ NOs: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions, or optionally comprising the polynucleotide sequence of SEQ ID NO: 804, or any one of SEQ ID NOs: 804-818, or any one of SEQ ID NOs: 804-883, or any one of SEQ ID NOs: 804-1019, or any one of SEQ ID NOs: 804-1788.

14. The composition or system of any one of claims 7-13 wherein the one or more binding motifs is within about 10-500 nucleotides, about 15-500 nucleotides, about 20-500 nucleotides, about 30-500 nucleotides, about 40-500 nucleotides, about 50-500 nucleotides, or about 60-500 nucleotides, or about 70-500 nucleotides, or about 80-500 nucleotides, or about 90-500 nucleotides, about 100-500 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, or about 300-400 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at leastabout 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 nucleotides, at least about 400 nucleotides, or at least about 500 nucleotides of a sequence that binds to, and / or hybridizes to, a RNA-binding polypeptide.

15. The composition or system of any one of claims 1-14, comprising one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

16. The composition or system of claim 15, wherein the sequence that binds to the RNA- binding polypeptide assembles into a secondary structure suitable for interaction with the RNA-binding polypeptide.

17. The composition or system of claim 16, wherein the secondary structure is or comprises a hairpin.

18. The composition or system of any one of claims 15-16, wherein the secondary structure is or comprises a stem, internal loop, multibranch loop, or a pseudoknot.

19. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is a viral protein.

20. The composition or system of any one of claims 14-18, wherein the RNA-binding polypeptide is any RNA-binding polypeptide, optionally wherein the RNA-binding polypeptide is selected from MS2 coat protein (MCP), PP7 coat protein, PRR1, Hgall, a Qbeta coat protein, the IN protein, SLBP (Stem-Loop Histone MRNA Binding Protein), and M protein, or a variant thereof.

21. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is MS2.

22. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is a PP7 coat protein.

23. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is PRRl.

24. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is Hgall.

25. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is Qbeta coat protein.

26. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is the IN protein, or the SLBP protein.

27. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is the M protein.

28. The composition or system of any one of claims 7-27, wherein the one or more binding motifs comprises a recognition sequence for ribonucleoprotein (RNP) complex formation.

29. The composition or system of any one of claims 7-28, wherein the one or more binding motifs comprises a sequence from a small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises a sequence from the snRNA or the snoRNA, optionally wherein the snRNA, snoRNA, protein that forms or is within the RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates RNP activity, and / or leads to attenuation of RNA modifying activity as compared to an unmodified form, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation, as compared to an unmodified form.

30. The composition or system of claim 29, wherein the snRNA, snoRNA, protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises at least one or more pseudouridylation sites.

31. The composition or system of claim 29, wherein the snRNA, snoRNA, protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within theRNP comprises no pseudouridylation sites.

32. The composition or system of any one of claims 1 -31, wherein the repRNA comprises at least one or more pseudouridylation sites.

33. The composition or system of any one of claims 1-31, wherein the repRNA comprises no pseudouridylation sites.

34. The composition or system of any one of claims 1-32, wherein the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more pseudouridylation sites than the number of pseudouridylation sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence.

35. The composition or system of claim 1-34, wherein the composition or system comprises a repair RNA (repRNA) and / or a protein that forms an RNP or is within an RNP, with the snRNA or snoRNA, or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme.

36. The composition or system of any one of claims 30-35, wherein the snRNA, snoRNA, protein that forms or is within an RNP, and / or a nucleic acid encoding the protein that forms or is within the RNP comprises a modification or mutation that increases, stimulates, or enhances RNP activity, or enhances RNA modifying activity, the RNP activity optionally being selected from cleavage, nucleic acid processing, pseudouridylation, and / or methylation as compared to an unmodified form.

37. The composition or system of claim 36, wherein the composition or system comprises a repair RNA (repRNA) and / or a protein that forms an RNP or is within an RNP, with the snRNA or snoRNA, or a nucleic acid encoding the protein that forms, or is within, the RNP and / or a small RNA that induces cleavage in an RNA and / or a CRISPR-Cas enzyme in cis or trans.

38. The composition or system of claim 37, wherein the cleavage is initiated from RNPs that are formed on the repRNA, or from RNPs that are formed in cis or trans.

39. The composition or system of any one of claims 1 -38, wherein the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide (e.g., a “grepRNA”).

40. The composition or system of any one of claims 1-39, wherein the composition or system comprises a splice acceptor; optionally wherein the composition or system comprises a splice donor.

41. The composition or system of any one of claims 1-40, wherein the composition or system comprises (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in cis or trans, or are suitable for being provided in cis or trans.

42. The composition or system of any one of claims 1-40, wherein the (a) at least one intronic sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exonic sequence are provided in trans, or are suitable for being provided in trans.

43. The composition or system of any one of claims 1-42, wherein the elements are under the control of one or more promoters.

44. The composition or system of any one of claims 1-42, wherein the elements are under the control of different promoters.

45. The composition or system of any one of claims 1-44, wherein the elements are operably linked, but separated by a cleavable sequence (e.g., a self-cleaving ribozyme).

46. The composition or system of any one of claims 1-45, wherein (i) multiple populations of repRNA are under the control of different promoters or (ii) the repRNA and another system member under control of different promoters.

47. The composition or system of any one of claims 29-46, wherein the snRNAs comprises Ul, U2, U3, U4, U5, U6, U7, U8, U9, U10 or Ul l, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

48. The composition or system of any one of claims 29-47, wherein the snRNA or snoRNA is selected from any one of SEQ ID NOs: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions, optionally wherein any one of SEQ ID NOs: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions form an RNP complex.

49. The composition or system of any one of claims 1-48, wherein the repRNA further comprises a guide RNA (gRNA). optionally wherein the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that attenuates, weakens, reduces, decreases, or ablates activity as compared to an unmodified form, or optionally wherein the composition or system comprises a repair RNA (repRNA) and the small RNA comprises a modification or mutation that increases, stimulates, or enhances activity as compared to an unmodified form, or optionally wherein the composition or system comprises a repair RNA (repRNA) and a small RNA that induces cleavage in an RNA in cis or trans; optionally wherein the snRNA comprises a M6A modification; optionally wherein the snRNA comprises a M6A modification in cis or trans.

50. The composition or system of any one of claims 1-49, wherein the repRNA further comprises a ribozyme site; optionally wherein the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), glmS ribozyme site, twister ribozyme site, or a variant thereof;51. The composition or system of claim 50, wherein the ribozyme site is a HDV ribozyme site.

52. The composition or system of claim 50, wherein the ribozyme site is a twister ribozyme site.

53. The composition or system of any one of claims 50-52, wherein the ribozyme site is upstream of the one or more exons and / or introns of the repRNA.

54. The composition or system of any one of claims 50-52, wherein the ribozyme site is downstream of the one or more exons and / or introns of the repRNA.

55. The composition or system of any one of claims 50-54, wherein the ribozyme site is upstream of the splice donor and / or splice acceptor the repRNA.

56. The composition or system of any one of claims 50-55, wherein the ribozyme site is downstream of the splice donor and / or splice acceptor the repRNA.

57. The composition or system of any one of claims 50-56, wherein the ribozyme cleaves the target.

58. The composition or system of any one of claims 50-57, wherein the ribozyme is a transcleaving ribozyme.

59. The composition or system of any one of claims 1-58, wherein the repRNA comprises a M6A modification; optionally wherein the repRNA comprises a M6A modification in cis or trans.

60. The composition or system of any one of claims 29-59, wherein the snRNA or snoRNA is modified to comprise at least one or more M6A sites.

61. The composition or system of any one of claims 29-60, wherein the snRNA or snoRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the snRNA or snoRNA or (ii) an exonic sequence.

62. The composition or system of any one of claims 29-59, wherein the snRNA or snoRNA is modified to not comprise M6A sites.

63. The composition or system of any one of claims 1 -62, wherein the repRNA comprises at least one or more M6A sites.

64. The composition or system of any one of claims 1-63, wherein the repRNA is modified to comprise at least 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than the number of M6A sites in (i) an unmodified state of the repRNA or (ii) an exonic sequence.

65. The composition or system of any one of claims 1-62, wherein the repRNA comprises no M6A sites.

66. The composition or system of any one of claims 1-65, wherein the repRNA comprises a ribozyme site that cleaves at the 5’ end of the repRNA.

67. The composition or system of any one of claims 1-65, wherein the repRNA comprises a ribozyme site that cleaves at the 3’ end of the repRNA.

68. The composition or system of any one of claims 1-67, wherein the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 5’ end of the repRNA.

69. The composition or system of any one of claims 1-68, wherein the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 3’ end of the repRNA.

70. The composition or system of any one of claims 1-69, wherein the composition or system further comprises at least one pre-rRNA stemloop.

71. The composition or system of claim 70, wherein the at least one pre-rRNA stemloop removes either the 5 ’cap or 3’ poly A tail.

72. The composition or system of any one of claims 1-71, wherein the repRNA comprises at least one or more snRNA or snoRNA sequences.

73. The composition or system of claim 72, wherein the at least one or more snRNA or snoRNA sequences stabilize the repRNA.

74. The composition or system of any one of claims 1-73, wherein the repRNA comprises an artificial smU7 system; optionally wherein the artificial smU7 system stabilizes the repRNA.

75. The composition or system of any one of claims 72-74, wherein the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 5’ end of the snRNA or snoRNA.

76. The composition or system of claim 75, wherein the pseudoknot at the 5’ end of the snRNA or snoRNA stabilizes the repRNA.

77. The composition or system of claim 75, wherein the least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 3’ end of the snRNA or snoRNA.

78. The composition or system of 77, wherein the pseudoknot at the 3’ end of the snRNA or snoRNA stabilizes the repRNA.

79. The composition or system of any one of claims 1-78, wherein there are a plurality of repRNAs under the control of the same, different, or a plurality of promoters.

80. The composition or system of any one of claims 1-79, wherein the repRNA and one or more other components of the present system are under the control of the same or different promoters.

81. The composition or system of any one of claims 1-80, wherein the repRNA comprises alternative promoters.

82. The composition or system of claim 81, wherein the repRNA comprises at least one or more alternative Pol II promoters.

83. The composition or system of claim 82, wherein the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-m ethylguanosine) or TMG (trimethylguanosine), optionally wherein the one or more alternative Pol II promoters cap the 5’ end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) stabilize the repRNA.

84. The composition or system of any one of claims 1 -83, wherein the repRNA comprises at least one or more circularized 5’ replacement splice donor (SD) repRNAs, optionally wherein the repRNA comprises at least one or more circularized 5’ replacement splice donor (SD) repRNAs stabilize the repRNA.

85. The composition or system of claim 84, wherein the repRNA comprising one or more circularized 5’ replacement (SD) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

86. The composition or system of claim 84 or 85, wherein the repRNA comprises at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs, optionally wherein the at least one or more circularized 3’ replacement splice acceptor (SA) repRNAs stabilizes the repRNA.

87. The composition or system of claim 86, wherein the repRNA comprising one or more circularized 3’ replacement (SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

88. The composition or system of any one of claims 1-87, wherein the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs, optionally wherein the at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA.

89. The composition or system of claim 88, wherein the repRNA comprising one or more circularized internal replacement (SD + SA) repRNAs improves stability as compared to an unmodified form and is resistant to exonucleases.

90. The composition or system of any one of claims 49-89, wherein the gRNA hybridizes to the target nucleic acid molecule.

91. The composition or system of any one of claims 49-90, wherein the gRNA directs the repRNA to a target nucleic acid molecule.

92. The composition or system of any one of claims 49-91, wherein the guide RNA is or comprises a sequence of SEQ ID NOs: 28-31, and / or SEQ ID NOs: 90-97, or a fragmentor variant thereof, or a nucleic acid sequence having at least about 70%, or at least about75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about99% identity thereto.

93. The composition or system of any one of claims 49-92, wherein the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60- 300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

94. The composition or system of any one of claims 1-93, comprising an endonuclease, wherein the endonuclease is a CRISPR-Cas enzyme, optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-A, optionally wherein the Cas is Cas8a or Cas5; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-B, optionally wherein the Cas is Cas8b; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-C, optionally wherein the Cas is Cas8c; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-D, optionally wherein the Cas is CaslOd; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-E, optionally wherein the Cas is Csel or Cse2; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-F, optionally wherein the Cas is Csyl, Csy2, or Csy3; optionally wherein the Cas is a type I; optionally wherein the Cas is a type I-G, optionally wherein the Cas is GSU0054; optionally wherein the Cas is a type I; optionally wherein the Cas type I is without limitation, Cas3; or optionally wherein the Cas is a type II; optionally wherein theCas is a type II-A, optionally wherein the Cas is Csn2; optionally wherein the Cas is a type II; optionally wherein the Cas is a type II-B, optionally wherein the Cas is Cas4; optionally wherein the Cas is a type II; optionally wherein the Cas is a type II-C; optionally wherein the Cas is a type II; optionally wherein the Cas type II is without limitation Cas 9; or optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-A, optionally wherein the Cas is Csm2; optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-B, optionally wherein the Cas is Cmr5; optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-C, optionally wherein the Cas is CaslO or Csxl 1; optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-D, optionally wherein the Cas is CsxlO; optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-E; optionally wherein the Cas is a type III; optionally wherein the Cas is a type III-F; optionally wherein the Cas is a type III; optionally wherein the Cas type III is without limitation Cas 10; or optionally wherein the Cas is a type IV; optionally wherein the Cas is a type IV-A; optionally wherein the Cas is a type IV; optionally wherein the Cas is a type IV-B; optionally wherein the Cas is a type IV; optionally wherein the Cas is a type IV-C; or optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-A, optionally wherein the Cas is Casl2a (Cpfl); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-B, optionally wherein the Cas is Casl2b (C2cl); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-C, optionally wherein the Cas is Casl2c (C2c3); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-D, optionally wherein the Cas is Casl2d (CasY); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-E, optionally wherein the Cas is Casl2e (CasX); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-F, optionally wherein the Cas is Casl2f (Casl4, or C2cl0); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-G, optionally wherein the Cas is Casl2g; optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-H, optionally wherein the Cas is Casl2h; optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-I, optionally wherein the Cas is Casl2i; optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-K, optionally wherein the Cas is Casl2k (C2c5); optionally wherein the Cas is a type V; optionally wherein the Cas is a type V-U, optionally wherein the Cas isC2c4, C2c8, or C2c9; optionally wherein the Cas is a type V; optionally wherein the Cas type V is without limitation Cas 12; optionally wherein the Cas is a type VI; or optionally wherein the Cas is a type VI- A, optionally wherein the Cas is Cas 13a (C2c2); optionally wherein the Cas is a type VI; optionally wherein the Cas is a type VI-B, optionally wherein the Cas is Casl3b; optionally wherein the Cas is a type VI; optionally wherein the Cas is a type VI-C, optionally wherein the Cas is Casl3c; optionally wherein the Cas is a type VI; optionally wherein the Cas is a type VI-D, optionally wherein the Cas is Casl3d; optionally wherein the Cas is a type VI; optionally wherein the Cas is a type VI-X, optionally wherein the Cas is Casl3x.1; optionally wherein the Cas is a type VI; optionally wherein the Cas is a type VI-Y; optionally wherein the Cas is a type VI; optionally wherein the Cas type VI is without limitation Cas 13; or optionally wherein the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5, CaslO or Csxl l, CsxlO, Csfl, Cas9, Csn2, Cas4, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (Casl4, C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), C2c4, C2c8, C2c9, Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, or Casl3x.l.

95. The composition or system of any one of claims 1-94, comprising an RNA sequence that interacts with an active or catalytically inactive endonuclease.

96. The composition or system of any one of claims 49-95, wherein the gRNA is associated with, or suitable for associating with, one or more endonucleases.

97. The composition or system of any one of claims 94-96, wherein the endonuclease comprises one or more mutations to reduce catalytic activity relative to an unmutated form.

98. The composition or system of any one of claims 94-97, wherein the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form.

99. The composition or system of any one of claims 94-98, wherein the endonuclease comprises one or more mutations; optionally wherein the one or more mutations increase catalytic activity relative to an unmutated form.

100. The composition or system of any one of claims 94-98, wherein the endonuclease comprises one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form.

101. The composition or system of any one of claims 94-100, wherein the endonuclease comprises an amino acid sequence of one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80- 89, and / or SEQ ID NOs: 106-130, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4, SEQ ID NOs: 80-89, and / or SEQ ID NOs: 106-130, or having about 1 to about 20 amino acid modifications.

102. The composition or system of any one of claims 1-101, wherein the repRNA is operably linked to one or more sequences that are antisense to the target nucleic acid molecule.

103. The composition or system of any one of claims 1-102, wherein the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule.

104. The composition or system of any one of claims 1-103, wherein the repRNA is not operably linked to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule.

105. The composition or system of any one of claims 1-104, wherein the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, the target nucleic acid molecule.

106. The composition or system of any one of claims 1-105, wherein the repRNA is operably linked to one or more sequences that bind to, and / or hybridize to, an RNA-binding polypeptide.

107. The composition or system of any one of claims 1-106, wherein the repRNA is provided in cis to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

108. The composition or system of any one of claims 1-105, wherein the repRNA is not operably to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

109. The composition or system of any one of claims 1 -105, wherein the repRNA is provided in trans to one or more sequences that bind to, and / or hybridize to, a RNA-binding polypeptide.

110. The composition or system of any one of claims 1-109, wherein the repRNA is operably linked to one or more recognition sequences for ribonucleoprotein (RNP) complex formation.

111. The composition or system of any one of claims 1-109, wherein the repRNA is provided in cis to one or more recognition sequences for ribonucleoprotein (RNP) complex formation.

112. The composition or system of any one of claims 1-109, wherein the repRNA is not operably linked to one or more recognition sequences for ribonucleoprotein (RNP) complex formation.

113. The composition or system of any one of claims 1-109, wherein the repRNA is provided in trans to one or more recognition sequences for ribonucleoprotein (RNP) complex formation.

114. The composition or system of any one of claims 1-113, wherein the repRNA is operably linked to one or more gRNAs.

115. The composition or system of any one of claims 1-113, wherein the repRNA is provided in cis to one or more gRNAs.

116. The composition or system of any one of claims 1-113, wherein the repRNA is not operably linked to one or more gRNAs.

117. The composition or system of any one of claims 1-113, wherein the repRNA is provided in trans to one or more gRNAs.

118. The composition or system of any one of claims 29-117, wherein the snRNA or snoRNA target one or more exonic splicing enhancers (ESEs), one or more intronic splicingenhancers (ISEs), one or more exonic splicing silencers (ESSs), and / or one or more intronic splicing silencers (ISSs).

119. The composition or system of claim 29-118, wherein the snRNA or snoRNA comprise a modification to include at least one or more exonic splicing enhancers (ESEs), at least one or more intronic splicing enhancers (ISEs), at least one or more exonic splicing silencers (ESSs), and / or at least one or more intronic splicing silencers (ISSs).

120. The composition or system of any one of claims 1-119, further comprising a small RNA that induces cleavage in an RNA optionally selected from one or more of an siRNA, small hairpin RNA (shRNA), U7 snRNA, a U1 snRNA, a U2 snRNA, a U4 snRNA, a U4atac snRNA, a U5 snRNA, a U6 snRNA, a U6atac snRNA, a U11 snRNA, a U12 snRNA, and an antisense oligonucleotide (ASO).

121. The composition or system of any one of claims 1-119, wherein the composition or system targets, or is suitable for targeting, one or more Usher syndrome-associated genes, optionally wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

122. The composition or system of any one of claims 1-121, wherein the composition or system targets, or is suitable for targeting, one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof.

123. The composition or system of claim 122, wherein the composition or system targets, or is suitable for targeting, one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof.

124. The composition or system of any one of claims 1-123, wherein the composition or system targets, or is suitable for targeting, USH2A, or a pre-mRNA sequence thereof.

125. The composition or system of any one of claims 1-124, wherein the composition or system targets, or is suitable for targeting, exon 13 of USH2A, or a pre-mRNA sequence thereof.

126. The composition or system of any one of claims 1-125, wherein the composition or system targets or is suitable for replacing c. 2299delG and / or C.2276G > T of USH2A, or a pre- mRNA sequence thereof.

127. The composition or system of any one of claims 1-126, wherein the composition or system is suitable for correcting a mutation or defect in one or more Usher syndrome-associated genes.

128. The composition or system of any one of claims 121-127, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

129. The composition or system of any one of claims 1-128, wherein the composition or system is suitable for correcting a mutation or defect in one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof.

130. The composition or system of claim 129, wherein the composition or system is suitable for correcting a mutation or defect in one or more genes selected from one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof.

131. The composition or system of claim 130, wherein the composition or system is suitable for correcting a mutation or defect in USH2A, or a pre-mRNA sequence thereof.

132. The composition or system of any one of claims 128-131, wherein the composition or system is suitable for correcting a mutation or defect in exon 13 of USH2A, or a pre-mRNA sequence thereof.

133. The composition or system of any one of claims 128-132, wherein the composition or system is suitable for correcting c.2299delG and / or C.2276G > T of USH2A, or a pre- mRNA sequence thereof.

134. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

135. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

136. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

137. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

138. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

139. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionallyhaving at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

140. The composition or system of any one of claims 94-139, wherein the endonuclease is linked, associated, and / or fused with the RNA-binding protein.

141. The composition or system of any one of claims 94-140, wherein the endonuclease is linked with the RNA-binding protein via a linker.

142. The composition or system of claim 141, wherein the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.

143. The composition or system of any one of claims 141-142, wherein the linker is substantially comprised of glycine and serine residues.

144. The composition or system of any one of claims 141-143, wherein the linker is (GGS)n, whereinnis 1, or 2, or 3, or 4, or 5.

145. The composition or system of any one of claims 141-144, wherein the linker is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS)n (n=l-4) (SEQ ID NO: 65), (Gly)s (SEQ ID NO: 66), (Gly)6(SEQ ID NO: 67), (EAAAK)n (n=l-3) (SEQ ID NO: 68), A(EAAAK)nA (n = 2-5) (SEQ ID NO: 69), AEAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

146. The composition or system of any one of claims 1 -145, wherein the repRNA comprises the splice donor.

147. The composition or system of any one of claims 1-145, wherein the repRNA comprises the splice acceptor.

148. The composition or system of any one of claims 1-147, wherein the repRNA comprises an exon of the target nucleic acid.

149. The composition or system of any one of claims 1-148, wherein the repRNA comprises an intron of the target nucleic acid.

150. The composition or system of any one of claims 1-149, wherein the repRNA comprises one or more non-natural introns.

151. The composition or system of any one of claims 2-150, wherein the target nucleic acid is a pre-mRNA transcript molecule.

152. The composition or system of any one of claims 1-151, wherein the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences.

153. The composition or system of claim 152, wherein the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a target RNA as compared to an unmodified form.

154. The composition or system of claim 152 or 153 wherein the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences.

155. The composition or system of any one of claims 152-154, wherein the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans-splicing efficiency of a target RNA as compared to an unmodified form.

156. The composition or system of any one of claims 152-155, wherein the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences.

157. The composition or system of any one of claims 152-156, wherein the at least one intronic spacer sequence comprising at least one ISE and ESS sequences increases trans-splicing efficiency of a off-target RNA as compared to an unmodified form.

158. The composition or system of any one of claims 152-156, wherein the repRNA comprises at least one intronic spacer sequence comprising at least one ISE and ESS sequences.

159. The composition or system of any one of claims 152-156 or 158, wherein the at least one intronic spacer sequence comprising at least one ISE and ESS sequences decreases trans- splicing efficiency of a off-target RNA as compared to an unmodified form.

160. The composition or system of any one of claims 1-159, wherein the repRNA comprises a ESS, ESE, ISS, and / or ISE sequence.

161. The composition or system of claim 160, wherein the repRNA targets one or more of ESS, ESE, ISS, and / or ISE.

162. The composition or system of any one of claims 152-161, wherein interaction, modulation and / or binding to one or more of ESS, ESE, ISS, and / or ISE reduces or ablates interaction, modulation and / or binding of the one or more of the ESS, ESE, ISS, and / or ISE with a target.

163. The composition or system of any one of claims 1-162, wherein the repRNA comprises exon sequences with ESE and ESS sequences.

164. The composition or system of claim 163, wherein the exon sequences with ESE and ESS sequences increase or decrease trans-splicing efficiency to an RNA target as compared to an unmodified form.

165. The composition or system of claim 163, wherein the repRNA comprises exon sequences with ESE and ESS sequences.

166. The composition or system of claim 165, wherein the repRNA comprises exon sequences with ESE and ESS sequences increase or decrease trans-splicing efficiency to an RNA off- target as compared to an unmodified form.

167. The composition or system of any one of claims 1-166, wherein the repRNA comprises at least one or more G4 structures.

168. The composition or system of claim 167, wherein the repRNA comprises at least one or more G4 structures sequester SD / SA motifs.

169. The composition or system of claim 167 or 168, wherein the G4 structure is unwound, such as by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., endogenous target or exogenously delivered trigger RNA).

170. The composition or system of claims 167-169, wherein the G4 structure decreases off- targets as compared to an unmodified form.

171. The composition or system of any one of claims 1-170, wherein the repRNA comprises a modification comprising at least one or more scaffolding sequences.

172. The composition or system of claim 171, wherein the at least one or more scaffolding sequences mediates (e.g., recruits) phase condensate-like formation and / or improves local concentrations of repRNAs compared to an unmodified form, and other targeted proteins and / or RNA.

173. The composition or system of any one of claims 1-172, wherein the repRNA comprises a modification comprising at least one or more sequences to target the repRNA to the promoter of the target gene of interest, or to proximal condensates that may contain the promoter.

174. The composition or system of claim 173, wherein the one or more sequences comprises an enhancer RNA, snRNA and / or snoRNA sequences.

175. The composition or system of any one of claims 1-174, wherein the repRNA comprises a modification, optionally wherein the modification improves interaction and localization to a DNA sequence of the non-template strand of the target gene as compared to an unmodified form.

176. The composition or system of claim 175, wherein the DNA sequence of the non-template strand of the target gene is the promoter, intron, exon, or enhancer.

177. The composition or system of claim 175 or 176, wherein the modification improves interaction and localization to the DNA sequence of the non-template strand of the target gene through protein-directed (e.g. transcription factor, dCas, ZNF, or other RBP) or nucleotide-directed (e.g., R-loop) methods as compared to an unmodified form.

178. The composition or system of any one of claims 1-177, wherein the repRNA comprises a modification comprising additional RNA elements, optionally wherein the additional RNA elements improve subnuclear localization to nuclear speckles for enhanced trans-splicing efficiency as compared to an unmodified form.

179. The composition or system of claim 178, wherein the additional RNA element comprise NEAT1 and / or MALAT1, or a fragment thereof.

180. The composition or system of claim 179, wherein the additional RNA element comprises a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

181. The composition or system of any one of claims 1-180, wherein the repRNA comprises a modification to enable targeting to site of transcription of target RNAs.

182. The composition or system of any one of claims 1-181, wherein the repRNA comprises a modification comprising 5’ UTR or 3’ UTR modifications; optionally wherein the modification comprising 5’ UTR or 3’ UTR modifications alter intracellular or intranuclear localization based on interactions with endogenous or exogenously supplied molecules (e.g., RNA G4 interactions with transcription factors or other proteins that are localized to specific cellular compartments).

183. The composition or system of any one of claims 1-182, wherein the repRNA comprises a modification in the 5’ UTR of the repRNA.

184. The composition or system of claim 183, wherein the modification in the 5’ UTR of the repRNA increases stability as compared to an unmodified form.

185. The composition or system of claim 183, wherein the modification in the 5’ UTR of the repRNA decreases stability as compared to an unmodified form.

186. The composition or system of claims 183-185, wherein the modification in the 5’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form.

187. The composition or system of any one of claims 1-186, wherein the repRNA comprises a modification in the 3’ UTR of the repRNA.

188. The composition or system of claim 187, wherein the modification in the 3’ UTR of the repRNA increases stability as compared to an unmodified form.

189. The composition or system of claim 188, wherein the modification in the 3’ UTR of the repRNA decreases stability as compared to an unmodified form.

190. The composition or system of claims 187-189, wherein the modification in the 3’ UTR of the repRNA increases or decreases translation efficiency as compared to an unmodified form.

191. The composition or system of any one of claims 1-190, wherein the repRNA comprises a modification comprising modifying the repRNA to comprise a G4 structure that mediates recruitment of splicing-associated RBPs.

192. The composition or system of any one of claims 1-191, wherein the repRNA comprises a modification comprising at least one or more toehold switches in the repRNA.

193. The composition or system of claim 192, wherein the at least one or more toehold switches in the repRNA conditionally activate or deactivate (e.g., SD / SA occlusion, binding motifocclusion, or RBP occlusion) upon detection of an endogenous or exogenously supplied target RNA.

194. The composition or system of any one of claims 1-193, wherein the repRNA comprises a modification comprising at least one or more complementary riboregulators in repRNAs (in cis).

195. The composition or system of claim 194, wherein the at least one or more complementary riboregulators in repRNAs (in cis) occlude splice donor (SD) site and reduce off-target trans-splicing.

196. The composition or system of any one of claims 1-195, wherein the repRNA comprises a modification comprising at least one or more self-complementary riboregulators in repRNAs (in cis).

197. The composition or system of claim 196, wherein the at least one or more self- complementary riboregulators in repRNAs (in cis) occlude splice acceptor (SA) site and reduce off-target trans-splicing.

198. The composition or system of any one of claims 1-197, wherein the repRNA comprises a modification comprising at least one or more self-complementary riboregulators in repRNAs (in trans).

199. The composition or system of claim 198, wherein the at least one or more self- complementary riboregulators in repRNAs (in trans) occlude splice donor (SD) site and reduce off-target trans-splicing.

200. The composition or system of any one of claims 1-199, wherein the repRNA comprises a modification comprising at least one or more self-complementary riboregulators in repRNAs (in trans).

201. The composition or system of claim 200, wherein the at least one or more self- complementary riboregulators in repRNAs (in trans) occlude splice acceptor (SA) site and reduce off-target trans-splicing.

202. The composition or system of any one of claims 1-201, wherein the repRNA comprises a modification comprising at least one or more binding motifs.

203. The composition or system of claim 202, wherein the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site occlusion (SA, SD, ISS, ISE, ESE, and ESS) as compared to an unmodified form.

204. The composition or system of any one of claims 1-203, wherein the repRNA comprises a modification to enable induction of trans-splicing in response to a stimulus as compared to an unmodified form.

205. The composition or system of any one of claims 1-204, wherein the repRNA comprises a modification to turn off or decrease trans-splicing in response to a stimulus as compared to an unmodified form.

206. The composition or system of any one of claims 1-205, wherein the repRNA comprises a modification to enable small molecule induction of trans-splicing as compared to an unmodified form.

207. The composition or system of any one of claims 1-205, wherein the repRNA comprises a modification to repress small molecule induction of trans-splicing as compared to an unmodified form.

208. The composition or system of any one of claims 1-207, wherein the repRNA comprises a modification to enable light induction of trans-splicing.

209. The composition or system of any one of claims 1-208, wherein the repRNA comprises a modification comprising at least one or more motifs that are bound and regulated by lightsensitive proteins.

210. The composition or system of any one of claims 1-209, wherein the snRNA or snoRNA comprises a sequence at the 3’ untranslated region (3’UTR), optionally wherein the sequence at the 3’ untranslated region (3’UTR) increases trans-splicing efficiency as compared to an unmodified form.211 . The composition or system of claim 210, wherein the sequence is from the MALAT1 gene.

212. The composition or system of claim 211, wherein the sequence is a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

213. The composition or system of any one of claims 1-212, wherein the RNP assembles on the repRNA and / or the target.

214. The composition or system of any one of claims 1-212, wherein the RNP assembles on the repRNA.

215. The composition or system of any one of claims 1-212, wherein the RNP assembles on the target.

216. The composition or system of any one of claims 1-215, wherein the RNP sterically occludes and inhibits cis-splicing.

217. The composition or system of any one of claims 1-216, wherein the repRNA comprises a minimal intron.

218. The composition or system of claim 217, wherein the minimal intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

219. The composition or system of any one of claims 2-218, wherein the target nucleic acid is one or more one or more Usher syndrome-associated genes, or a fragment thereof, or a pre- mRNA sequence thereof.

220. The composition or system of claim 219, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

221. The composition or system of claim 220, wherein the target nucleic acid is one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or a pre-mRNA sequence thereof.

222. The composition or system of claim 221, wherein the target nucleic acid is one or more genes selected from USH2A, GPR98, and DFNB31, or a pre-mRNA sequence thereof.

223. The composition or system of any one of claims 219-222, wherein the target nucleic acid is USH2A, or a pre-mRNA sequence thereof.

224. The composition or system of any one of claims 219-223, wherein the target nucleic acid is exon 13 of USH2A, or a pre-mRNA sequence thereof.

225. The composition or system of any one of claims 219-224, wherein the target nucleic acid is USH2A, or a pre-mRNA sequence thereof, bearing a c.2299delG and / or C.2276G > T mutation.

226. The composition or system of any one of claims 8-225, wherein the hybridization is mediated by perfect sequence complementarity to one strand of a target nucleic acid molecule.

227. The composition or system of any one of claims 8-225, wherein the hybridization is mediated by partial sequence complementarity to one strand of a target nucleic acid molecule.

228. The composition or system of any one of claims 1-227, wherein the repRNA mediates trans-splicing production of a corrected and / or wild-type USH2A nucleic acid gene transcript.

229. The composition or system of any one of claims 1 -228, wherein the composition or system substantially prevents or eliminates cis-splicing of a nucleic acid.

230. The composition or system of any one of claims 2-229, wherein the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

231. The composition or system of any one of claims 1-230, further comprising a viral vector or a non-viral vector.

232. The composition or system of claim 231, wherein the viral vector is or comprises an AAV, optionally wherein the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

233. The composition or system of any one of claims 1-232, further comprising a lipid nanoparticle (LNP), liposome, lipoplex, or polymeric nanoparticle.

234. The composition or system of claim 233, wherein the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids, and lipoids.

235. The composition or system of any one of claims 1-234, wherein the composition or system components are nucleic acids.

236. The composition or system of any one of claims 1-235, wherein the composition or system components comprise a DNA molecule or an RNA molecule.

237. The composition or system of claim 236, wherein the RNA is or comprises mRNA or modified mRNA (mmRNA).

238. The composition or system of claim 236, wherein the DNA molecule is or comprises a vector or plasmid.

239. The composition or system of any one of claims 235-237, wherein the nucleic acid comprises a codon optimized sequence.

240. The composition or system of any one of claims 235-239, wherein the nucleic acid comprises one or more modifications.

241. The composition or system of claim 240, wherein the modifications are one or more of base modifications and backbone modifications.

242. A cell comprising a nucleic acid of any one of claims 235-241, the viral vector of claims 231-232, or the lipid nanoparticle of claims 233-234.

243. The cell of claim 242, wherein the cell is a eukaryotic cell.

244. The cell of claim 242, wherein the cell is a mammalian cell.

245. The cell of claim 242, wherein the cell is a human cell.

246. The cell of claim 242, wherein the cell is an immortalized cell.

247. The cell of claim 242, wherein the cell is harvested from a subject.

248. A pharmaceutical composition comprising the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and a pharmaceutically acceptable carrier.

249. A kit comprising a container comprising the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and instructions for use in trans-splicing a nucleic acid.

250. A method for targeted trans-splicing of a USH2A pre-mRNA in a cell, comprising contacting the cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247.251 . A method for targeted trans-splicing a pre-mRNA in a cell, comprising contacting the cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247.

252. The method of claim 250 or 251, wherein the method further comprises slowing, reducing, or ablating transcription of a target gene, and / or stimulating, enhancing, or increasing the Pol II stalling / release.

253. The method of any one of claims 250-252, wherein the method increases trans-splicing by forcing G4 or similarly bulky RNA-RNA motif formation downstream of the repRNA’s binding motif as compared to an unmodified form.

254. The method of any one of claims 250-253, wherein the method further comprises modifying and converting an endogenous RNA, such as and without limitation a pre- mRNA, mRNA, IncRNA, into a repRNA for trans-splicing.

255. A method of treating a patient who has a condition associated with a mutation in a USH2A gene, the method comprising administering a therapeutically effective amount of the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233- 234, or the cell of any one of claims 242-247.

256. A method of treating, ameliorating or preventing a condition associated with a mutation in a USH2A gene, comprising:(a) contacting a cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and(b) administering an effective amount of the cell to the subject.

257. A method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, comprising administering an effective amount of the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, theviral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, to the subject.

258. A method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject, in need thereof, comprising:(a) contacting a cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and(b) administering an effective amount of the cell to the subject.

259. The method of any one of claims 250-258, wherein the cell is derived from the subject.

260. The method of any one of claims 250-259, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

261. The method of claim 260, wherein the Usher syndrome is Usher syndrome type I.

262. The method of claim 260, wherein the Usher syndrome is Usher syndrome type II.

263. The method of claim 260, wherein the Usher syndrome is Usher syndrome type III.

264. The method of any one of claims 250-263, wherein the method targets one or more Usher syndrome-associated genes.

265. The method of claim 264, wherein the method targets one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

266. The method of claim 265, wherein the method targets one or more of USH2A, GPR98, and DFNB31.

267. The method of claim 265 or 266, wherein the method targets USH2A.

268. The method of any one of claims 250-267, wherein the method corrects a mutation or defect in one or more Usher syndrome-associated genes.

269. The method of claim 250-268, wherein the method corrects a mutation or defect in one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

270. The method of claim 269, wherein the method corrects a mutation or defect in one or more of USH2A, GPR98, and DFNB31.

271. The method of claim 270, wherein the method corrects a mutation or defect in USH2A.

272. The method of any one of claims 250-271, wherein the method causes trans-splicing of one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

273. The method of claim 272, wherein the method causes trans-splicing of one or more of USH2A, GPR98, and DFNB31.

274. The method of claim 273, wherein the method causes trans-splicing of USH2A.

275. The method of any one of claims 250-274, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa, hearing reduction or loss, night blindness, and loss or reduction of peripheral vision.

276. The method of any one of claims 250-275, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa and loss or reduction of peripheral vision.

277. The method of any one of claims 250-276, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa.

278. The method of any one of claims 250-277, wherein the method treats, ameliorates or prevents hearing reduction or loss.

279. The method of any one of claims 250-278, wherein the method treats, ameliorates or prevents vision reduction or loss.

280. The method of any one of claims 250-279, wherein the method treats, ameliorates or prevents one or more of night blindness and loss or reduction of peripheral vision.

281. The composition or system of any one of claims 1-280, further comprising a repair RNA (repRNA) sequence.

282. The composition of claim 281, wherein the trans-splicing system comprises a splice donor, a splice acceptor, and replaces an internal exon.

283. The composition of claim 282, wherein the repRNA is operably linked to the RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule or the gRNA.

284. A system for targeting a nucleic acid for trans-splicing, the system comprising:(a) an endonuclease of any one of claims 1-241 and optionally an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule;(b) an RNA-binding polypeptide that associates with the endonuclease; and(c) a repair RNA (repRNA) sequence, comprising:(i) one or more exons and / or introns;(ii) a splice donor and / or splice acceptor.

285. The system of claim 284, wherein the RNA molecule is a gRNA.

286. The system of claim 284 or 285, wherein the endonuclease is not linked, associated, and / or fused with an RNA binding protein.

287. A system for targeting a nucleic acid for trans-splicing, the system comprising:(a) an endonuclease of any one of claims 1-241 and an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; and(b) a repair RNA (repRNA) sequence, comprising:(i) one or more exons and / or introns;(ii) a splice donor and / or splice acceptor.

288. The system of claim 287, wherein the RNA molecule is a gRNA.

289. The system of claim 287 or 288, wherein the endonuclease is not linked, associated, and / or fused with an RNA binding protein.

290. The composition of any one of claims 1-241, wherein the composition comprises a gRNA, repRNA, and a Cas endonuclease operably linked to a single promoter or a bidirectional promoter.

291. The composition of claim 290, wherein the gRNA and repRNA are located on a first side of the bidirectional promoter, and the Cas endonuclease is located on a second side of the bidirectional promoter.

292. A kit comprising a container comprising the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and instructions for use in trans-splicing a nucleic acid.

293. A method for targeted trans-splicing of a USH2A pre-mRNA in a cell, comprising contacting the cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247.

294. A method of treating a patient who has a condition associated with a mutation in a USH2A gene, the method comprising administering a therapeutically effective amount of the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233- 234, or the cell of any one of claims 242-247.

295. A method of treating, ameliorating or preventing a condition associated with a mutation in a USH2A gene, comprising:(a) contacting a cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and(b) administering an effective amount of the cell to the subject.

296. A method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, comprising administering an effective amount of the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, to the subject.

297. A method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject, in need thereof, comprising:(a) contacting a cell with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, and(b) administering an effective amount of the cell to the subject.

298. The method of any one of claims 295 or 297, wherein the cell is derived from the subject.

299. The method of any one of claims 295-298, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

300. The method of claim 299, wherein the Usher syndrome is Usher syndrome type I.

301. The method of claim 299, wherein the Usher syndrome is Usher syndrome type II.

302. The method of claim 299, wherein the Usher syndrome is Usher syndrome type III.

303. The method of any one of claims 295-302, wherein the method targets one or more Usher syndrome-associated genes.

304. The method of claim 303, wherein the method targets one or more genes selected from CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRNl .

305. The method of claim 303 or 304, wherein the method targets one or more of USH2A, GPR98, and DFNB31.

306. The method of claim 305, wherein the method targets USH2A.

307. The method of any one of claims 295-306, wherein the method corrects a mutation or defect in one or more Usher syndrome-associated genes.

308. The method of any one of claims 295-307, wherein the method corrects a mutation or defect in one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

309. The method of claim 308, wherein the method corrects a mutation or defect in one or more of USH2A, GPR98, and DFNB31.

310. The method of claim 309, wherein the method corrects a mutation or defect in USH2A.

311. The method of any one of claims 295-310, wherein the method causes trans-splicing of one or more genes selected from USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

312. The method of claim 311, wherein the method causes trans-splicing of one or more of USH2A, GPR98, and DFNB31.

313. The method of claim 312, wherein the method causes trans-splicing of USH2A.

314. The method of any one of claims 295-313, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa, hearing reduction or loss, night blindness, and loss or reduction of peripheral vision.

315. The method of any one of claims 295-314, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa and loss or reduction of peripheral vision.

316. The method of any one of claims 295-315, wherein the method treats, ameliorates or prevents one or more symptoms of retinitis pigmentosa.

317. The method of any one of claims 295-316, wherein the method treats, ameliorates or prevents hearing reduction or loss.

318. The method of any one of claims 295-317, wherein the method treats, ameliorates or prevents vision reduction or loss.

319. The method of any one of claims 295-318, wherein the method treats, ameliorates or prevents one or more of night blindness and loss or reduction of peripheral vision.

320. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the binding motif has a molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 or SEQ ID NO: 2024 that is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a non-targeting rate.

321. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein, the binding motif is selected from any one of SEQ ID NOs: 804-2022.

322. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the molecular activity for binding an USH2A target nucleic acid of SEQ ID NO: 2023 or SEQ ID NO: 2024 to any one of SEQ ID NOs: 804-2022 is greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the fold change is measured from a trans-splicing editing rate relative to a nontargeting rate.

323. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291 , the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the binding motif binds to an intronic position of an USH2A target nucleic acid of SEQ ID NO: 2024 that is at about position 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000, optionally wherein the binding motif is selected from any one of SEQ ID NOs: 804- 2022 and binds to a position intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024) that is selected relative to a splice donor site, without limitation, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, or about 1000 nucleotides from the splice donor site in intron 13 ofUSH2A.

324. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the binding motif binds to intron 13 of USH2A.

325. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the binding motif binds to an exonic position of an USH2A target that is at about position 0, -10, -20, -30, -40, -50, -60, -70, or about -80.

326. The composition or system of any one of claims 1-230, claims 281-283 or claims 290-291, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231- 232, the lipid nanoparticle of claim 233-234, or the cell of any one of claims 242-247, the pharmaceutical composition of claim 248, the kit of claim 249 or claim 292, the method of any one of claims 250-280 or claims 293-319, the system of any one of claims 284-289, wherein the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from about position -10, -20, -30, - 40, -50, -60, -70, or about -80, optionally wherein the binding motif is selected from any one of SEQ ID NOs: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected relative to a splice donor site, without limitation, from about position -10, -20, -30, -40, -50, -60, -70, or about -80.

327. A repRNA polynucleotide comprising a binding motif directed against a target nucleic acid, the target nucleic acid being intron 13 or human USH2A or exon 13 of human USH2A.

328. The repRNA of claim 327, wherein the exon 13 of human USH2A is or comprises the polynucleotide sequence of SEQ ID NO: 2023 and / or intron 13 of human USH2A is or comprises the polynucleotide sequence of SEQ ID NO: 2024.

329. The repRNA of claim 327 or 328, wherein the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any one of SEQ ID NOs: 804- 2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and / or or having about 1 to about 20 (e.g. about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitutions, additions, or deletions.

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