WO2023154807A2 - Compositions et procédés de modulation d'épissage de pré-arnm - Google Patents

Compositions et procédés de modulation d'épissage de pré-arnm Download PDF

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WO2023154807A2
WO2023154807A2 PCT/US2023/062301 US2023062301W WO2023154807A2 WO 2023154807 A2 WO2023154807 A2 WO 2023154807A2 US 2023062301 W US2023062301 W US 2023062301W WO 2023154807 A2 WO2023154807 A2 WO 2023154807A2
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exon
seq
sequence
rna
mrna
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PCT/US2023/062301
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WO2023154807A3 (fr
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Ranjan BATRA
Rea LARDELLI MARKMILLER
Daniela ROTH
Angeline TA
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Locanabio, Inc.
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Publication of WO2023154807A2 publication Critical patent/WO2023154807A2/fr
Publication of WO2023154807A3 publication Critical patent/WO2023154807A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the disclosure is directed to molecular biology, gene therapy, and compositions and methods for modifying expression and activity of RNA molecules.
  • RNA splicing wherein precursor messenger RNA (pre-mRNA) is processed into mature messenger RNA (mRNA) via the removal of intron sequences and the joining of exon sequences, is essential for the production of functional proteins.
  • pre-mRNA precursor messenger RNA
  • mRNA mature messenger RNA
  • Examples of monogenic disorders where splicing mutations, mutations within the spliceosome, and/or frameshift mutations lead to diseases include, but are not limited to, Usher syndrome type IIA, autosomal recessive retinitis pigmentosa (RP39), Duchenne muscular dystrophies (DMD), Becker Muscular Dystrophy (BMD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Leber congenital amaurosis (LCA), Early infantile epileptic encephalopathy-6 (EIEE6), Spinal muscular atrophy (SMA), and Huntington’s disease (HD), Dravet Syndrome, Frontal Temporal Dementia (MAPT), Parkinson Disease (PINK1).
  • Usher syndrome type IIA autosomal recessive retinitis pigmentosa
  • DMD Duchenne muscular dystrophies
  • BMD Becker Muscular Dystrophy
  • ALS amyotrophic lateral sclerosis
  • Alzheimer’s disease Leber congenital
  • splicing regions including exonic and intronic sequences
  • Splicing modulation can resolve many of these disorders.
  • inducing exon inclusion/exclusion can be used to resolve mutations that introduce frameshifts and missense mutations.
  • compositions were engineered to target exonic and intronic sequences having disease-causing mutations. These compositions can modulate RNA splicing, in some cases causing mutation-bearing exons to be skipped, leading to alternately processed mRNA that, when expressed, at least partially restores the function and activity of the disease-implicated protein.
  • the disclosure provides gene therapy compositions and methods comprising RNA-targeting compositions capable of modulating RNA splicing.
  • methods of using RNA-targeting compositions to treat subjects with diseases and disorders linked to improper splicing wherein splicing modulation can be used to restore function are provided herein.
  • the disclosure provides a pre-mRNA sequence binding composition
  • a nucleic acid sequence encoding (a) an RNA-binding polypeptide or portion thereof, and (b) one or more cognate nucleic acid guide RNA, wherein each of the one or more cognate nucleic acid guide RNA are capable of binding a target pre-mRNA sequence
  • the target pre-mRNA sequence comprises an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BP A), o a poly pyrimidine tract (PPY), exon splicing silencer (ESS) motif, or an intron splicing silencer (ISS) motif.
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • BP A branchpoint adenosine
  • PPY poly pyrimidine tract
  • ESS ex
  • the disclosure provides a pre-mRNA sequence binding composition
  • a pre-mRNA sequence binding composition comprising a nucleic acid sequence encoding a PUF or PUMBY polypeptide capable of binding a target pre-mRNA sequence; wherein the target pre-mRNA sequence comprises an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BPA), o a polypyrimidine tract (PPY), exon splicing silencer (ESS) motif, or an intron splicing silencer (ISS) motif.
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • BPA branchpoint adenosine
  • PPY polypyrimidine tract
  • ESS exon splicing silencer
  • ISS intron splicing silencer
  • the 5’ splice site is a splice donor site.
  • the 3’ splice site is a splice acceptor site.
  • the composition comprises at least two cognate nucleic acid guides.
  • the polypeptide of is a fusion protein.
  • the fusion protein comprises a peptide capable of modulating splicing.
  • the peptide capable of modulating splicing binds RNA.
  • the peptide capable of modulating splicing is RBFOX1, RMP38, hnRNPAl, or AF2.
  • the RNA-binding polypeptide is a catalytically inactivated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) polypeptide.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the Cas polypeptide is a catalytically inactivated or dead Casl3d (dCas!3d) polypeptide.
  • the Casl3d polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, or SEQ ID NO: 12307.
  • the Casl3d polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396 or SEQ ID NO: 397.
  • the PUF or PUMBY comprises an amino acid sequence set forth in SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, or SEQ ID NO: 389.
  • the target pre-mRNA sequence comprises a sequence encoding for USH2A.
  • the sequence encoding for USH2A comprises a mutation.
  • the mutation in USH2A occurs in exon 13.
  • the pre-mRNA sequence comprises SEQ ID NO: 467.
  • the cognate nucleic acid guide RNA comprises any one of SEQ ID NOs 403-412.
  • the target pre-mRNA sequence comprises a sequence encoding for DMD.
  • the sequence encoding for DMD comprises a mutation.
  • the mutation in DMD occurs in at least one of exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, and exon 55.
  • the pre-mRNA sequences comprises SEQ ID NO: 471.
  • the cognate nucleic acid guide RNA comprises any one of SEQ ID NOs 422-437, 12272-12276, 12285, 12288-12306, or 12311-12312.
  • the target pre-mRNA sequence comprises a sequence encoding for MAPT.
  • the sequence encoding for MAPT comprises a mutation.
  • the mutation in MAPT occurs in exon 10.
  • the pre-mRNA sequences comprises SEQ ID NO: 479.
  • the cognate nucleic acid guide RNA comprises any one of SEQ ID NOs 438-440.
  • a PUMBY protein binds a MAPT exon 10 target sequence set forth in any one of SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, of SEQ ID NO: 394.
  • the nucleic acid sequence comprises at least one promoter.
  • the at least one promoter is a constitutive promoter or a tissue-specific promoter.
  • the at least one promoter is selected from the group consisting of a tCAG promoter, an EFS promoter, and an EFS-UBB promoter.
  • the nucleic acid sequence comprises two promoters.
  • a first promoter controls expression of the one or more cognate nucleic acid guide RNA and a second promoter controls expression of the RNA- binding polypeptide or portion thereof.
  • the disclosure provides a vector comprising the composition of any embodiment of the disclosure.
  • the vector is selected from the group consisting of: adeno-associated virus, retrovirus, lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex, polymersome, polyplex, and dendrimer.
  • the disclosure provides a cell comprising the vector of any embodiment of the disclosure.
  • the disclosure provides a method of modulating pre-mRNA splicing comprising administering a composition comprising a nucleic acid sequence encoding (a) an RNA- binding polypeptide or portion thereof, and (b) one or more cognate nucleic acid guides, wherein each of the one or more cognate nucleic acid guides are capable of binding a target pre-mRNA sequence.
  • the disclosure provides a method of modulating pre-mRNA splicing comprising administering a composition comprising a nucleic acid sequence encoding a non-guided RNA-binding polypeptide or portion thereof comprising a PUF or PUMBY protein capable of binding a target pre-mRNA sequence.
  • the pre-mRNA sequence comprises an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BPA), or a polypyrimidine tract (PPY).
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • BPA branchpoint adenosine
  • PPY polypyrimidine tract
  • the 5’ splice site is a splice donor site.
  • the 3’ splice site is a splice acceptor site.
  • the pre-mRNA sequence comprises an exon splicing silencer (ESS) motif or an intron splicing silencer (ISS) motif.
  • binding the pre-mRNA sequence prevents inclusion of the bound sequence into an mRNA sequence. In some embodiments, binding the pre-mRNA sequence promotes inclusion of the bound sequence into an mRNA sequence. In some embodiments, binding the pre-mRNA sequence produces a truncated mRNA sequence. In some embodiments, the mRNA sequence is expressed into a protein.
  • the protein expressed from the mRNA is expressed at an increased level compared to a wild-type mRNA. In some embodiments, the protein expressed from the mRNA is expressed at a decreased level compared to a wild-type mRNA. In some embodiments, the protein expressed from the protein has increased activity compared to a wild-type mRNA. In some embodiments, the protein expressed from the protein has decreased activity compared to a wild-type mRNA.
  • the disclosure provides a method of treating a disease or disorder associated with a gene mutation, wherein the mutation occurs in an exon of a pre-mRNA sequence, comprising administering a composition of any embodiment of the disclosure.
  • the target pre-mRNA sequence comprises a sequence encoding for USH2A.
  • the sequence encoding for USH2A comprises a mutation.
  • the mutation in USH2A occurs in exon 13.
  • the target pre-mRNA sequence comprises a sequence encoding for DMD.
  • the sequence encoding for DMD comprises a mutation.
  • the mutation in DMD occurs in at least one of exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, and exon 55.
  • the disease or disorder is Duchenne muscular dystrophy, amyotrophic lateral sclerosis, or Usher syndrome type II.
  • FIG. 1 A is a schematic overview of mRNA splicing depicting: I) a precursor mRNA (pre-mRNA) comprising three exonic sequences and two corresponding intron sequences linking the exons; II) in order to be processed the pre-mRNA is spliced by a protein complex comprising the spliceosome, including U1 and U2, and splicing regulator factors (SR); III) splicing removed the two intronic sequences leading to a mature mRNA that can then be translated in the cell.
  • pre-mRNA precursor mRNA
  • SR splicing regulator factors
  • FIG. IB (I-II) is a schematic depiction of the use of CRISPR-Cas nucleoprotein complexes to target specific exonic and intronic sequences to block components of the splicing machinery leading to alternative splicing.
  • FIG. 2 is a schematic depicting a mutated exon 13 in USH2A leading to improper splicing and the expression of no protein or non-functional protein leading to Usher’s syndrome type IIA.
  • Specific targeting of exon 13 of USH2A leads to the skipping of exon 13 during mRNA processing producing a shortened, but functional Usherin protein, resulting in the treatment and correction of Usher’s syndrome type IIA.
  • FIG. 3A is a schematic showing exons 12, 13, and 14 of a human USH2A minigene pre-mRNA. Also depicted are CRISPR-Cas guide RNAs, gl-g8, targeting distinct regions of the pre-mRNA including exon 13 and intronic sequences.
  • FIG. 3B is a tape station image depicting the effect of human USH2A pre-mRNA targeting dCasRX complexes on USH2A minigene splicing, wherein the various complexes each bear guides gl-g8.
  • the gel depicts an 860 basepair (bp) band corresponding to a transcript comprising exons 12, 13, and 14.
  • a second 208 bp band is depicted demonstrating the skipping of exon 13.
  • the NT (non-targeting) control lane depicts very little exon 13 skipping, whereas each of guides gl-g8 lead to amplified exon 13 skipping.
  • FIG. 3C is a bar graph depicting the fraction of human USH2A exon 13 excluded by dCasRX complexes comprising guides gl-g8.
  • NT is the non-targeting control.
  • Guide g7 results in almost complete exclusion or skipping of exon 13.
  • FIG. 3D shows dose-dependent exon 13 skipping upon delivery of USH2A pre- mRNA targeting dCas!3d complexes tested with guides g7, g9 and glO targeting human USHA2A exon 13, showing dose dependent Exon 13 skipping.
  • FIG. 3E is a series of tape station images depicting exclusion of exon 13 of human USH2A using vectors expressing guide RNA, g7, g9, glO, or a non-targeting guide RNA.
  • Unitary plasmids were transfected at varying doses and RNA was harvested at 48 hr.
  • FIG. 4A is a schematic showing exons 12, 13, and 14 of a human USH2A minigene pre-mRNA. Also depicted is CRISPR-Cas guide RNA, g7, targeting exon 13.
  • FIG.4B is a tape station depicting the effect of human USH2A pre-mRNA targeting dCasRX complexes on USH2A minigene splicing, wherein the various complexes each include guide g7.
  • the gel depicts an 860 base pair (bp) band corresponding to a transcript comprising exons 12, 13, and 14.
  • a second 208 bp band is depicted demonstrating the skipping of exon 13.
  • FIG. 5A is a bar graph depicting cas!3d activity of Casl3d variants bearing point mutations to inactivate or diminish their catalytic activity. Activity is assessed by measuring relative levels of fluorescence. Cast 3d variants were tested with a guide targeting a GFP reporter and relative levels of GFP were evaluated compared to a control non-targeting guide RNA.
  • FIG. 5B is a bar graph depicting cas!3d activity of active Casl3d variants to serve as a control to the inactivated variants in FIG. 5 A. Activity is assessed by measuring relative levels of fluorescence. Cast 3d variants with a guide targeting a GFP reporter and relative levels of GFP were evaluated compared to a control non-targeting guide RNA
  • FIG. 6A is a schematic that depicts the targeting sequences of guides g9 and glO, which partially overlap with guide g7 and also target the ESE of exon 13 of USH2A.
  • FIG. 6B is a tape station image depicting that AAV vector constructs expressing dSeq212 and guides g7, g9 or glO, targeting the ESE of exon 13 co-transfected with the human USH2A minigene are functional in excluding exon 13 of USH2A minigene.
  • RNA was extracted, cDNA was synthesized and PCR was performed as in FIG 3.
  • FIG. 7A is a schematic of a mouse USH2A minigene pre-mRNA and mouse guide RNAs, mgl and mg4-mg9, that target different sites of exon 12 of the mouse USH2A as well as intronic sequences.
  • FIG. 7B is a tape station image depicting the effect of mouse USH2A pre-mRNA targeting dCasRX complexes on USH2A minigene splicing, wherein the various complexes each bear guides mg4-mg9.
  • the gel depicts a 770 basepair (bp) band corresponding to a transcript comprising exons 11, 12, and 13.
  • a second approximately 350 bp band is depicted corresponding to partial exon 12 exclusion.
  • a third 128 bp band is depicted demonstrating the skipping of exon 12.
  • FIG. 8A is a tape station image depicting exon 12 skipping of mouse USH2A using AAV vector comprising Casl3d-expresion constructs that co-express both mgl and mg7 in tandem array.
  • FIG. 8B is a series of microscope images depicting splice junctions of mouse USH2A minigene in cosm6 cells transfected with 250 ng of guides mgl and mg7, 500 ng dCasRx and 50 ng mouse minigene in 12 well format. Cells were trypsinized and seeded in Matrigel-coated chamber slides 24 hr before fixation with PF A.
  • Cells not treated with minigene show no signal from either set of probes
  • cells treated with a non-targeting (NT) guide show signal only from exon 1 l/exonl2 splice junction
  • cells treated with both mgl and mg7 show reduced signal for the exon 11/exon 12 splice junction and presence of the signal from exon 11/exon 13 splice junction (indicative of exon 12 skipping).
  • FIG. 9A is a schematic depicting an exemplary AAV vector construct encoding mgl and mg7 guide RNAs under the control of a human U6 (hU6) promoter and a dCasl3d-NLS (dCasl3d tethered to a nuclear localization signal) under the control of an EFS promoter for the targeting of exon 12 of mouse USH2A.
  • hU6 human U6
  • dCasl3d-NLS dCasl3d tethered to a nuclear localization signal
  • FIG. 9B is a schematic depicting an exemplary AAV vector construct encoding a g7 guide RNA under the control of a human U6 (hU6) promoter and a dCasl3d-NLS (dCasl3d tethered to a nuclear localization signal) under the control of a cone/rod specific promoter for the targeting of exon 13 of human USH2A.
  • hU6 human U6
  • dCasl3d-NLS dCasl3d tethered to a nuclear localization signal
  • FIG. 10 is a series of tape station images depicting exclusion of exon 12 of mouse USH2A using vectors expressing dual guide RNA, mgl and mg7.
  • Unitary plasmids were constructed expressing dSeq212 under EFS or EFS-UBB promoters and mgl and mg7 either under separate hU6 promoters or as a tandem array.
  • Unitary plasmids were transfected at varying doses with the mouse USH2A minigene and RNA was harvested at 48 hr. Splicing assays were performed as described above.
  • FIG. 11 A is a schematic depicting the targeting of exon 51 of endogenous DMD with guide RNA.
  • FIG. 1 IB a series of tape station images depicting the impact of guides g2-g7 with either dCasRx or dSeq!89 on exon 51 skipping.
  • the gel image depicts a 322 bp band corresponding to a transcript comprising exons 50, 51 and 52.
  • a second 89 bp band is depicted corresponding to a transcript comprising exons 50 and 52, having skipped exon 51.
  • FIG. 12 is a schematic depicting a multi-exon skipping approach to restore dystrophin levels by skipping exons 45-50 or exons 46-50.
  • FIG. 13 A is a schematic depicting guides that target exons 45 and 51 of DMD.
  • FIG. 13B is a tape station image depicting DMD exon skipping following treatment with dCasRX and guide RNAs targeting exons 45 and 51.
  • FIG. 14 is a tape station image image depicting the impact of guides gl-g3, targeting exon 10 of a human MAPT minigene, complexed with dCasRx.
  • the image depicts a 197 bp band corresponding to a transcript comprising exon 10.
  • a second 104 bp band is also depicted corresponding to the absence of exon 10, indicative of exon 10 skipping.
  • JO and JP are MAPT minigenes for exon 10 skipping.
  • FIG. 15 is tape station image depicting the impact of targeting exon 10 of a human MAPT minigene with PUMBY proteins.
  • a positive control is dCasRx complexed with guide g3 from FIG. 14.
  • the gel image depicts a 197 bp band corresponding to a transcript comprising exon 10.
  • a second 104 bp band is also depicted corresponding to the absence of exon 10, indicative of exon 10 skipping.
  • JO is a MAPT minigene for exon 10 skipping.
  • FIG. 16A is a schematic depicting a strategy to knockdown a transcript such as SOD1 by introduction of a PTC (premature termination codon) through exon skipping.
  • the image shows SOD1 cDNA comprising exons 1-5.
  • Orange reverse arrows demonstrate where guides target exon sequences of SOD1.
  • Forward arrows depict predicted enhancer sequences within the exon sequences.
  • FIG. 16B is a schematic depicting exons 1-5 of endogenous SOD1 and guide RNAs targeting exon 2 and exon 3.
  • FIG. 16C is a tape station image depicting the impact of guides gl-g4, targeting exons 2 and 3 of endogenous SOD1, complexed with dCasRx.
  • the gel depicts a 224 bp band corresponding to a transcript comprising exons 1-4.
  • a second 154 bp band is also depicted corresponding to the absence of exon 3, indicative of exon 3 skipping.
  • a third 127 bp band is also depicted corresponding to the absence of exon 2, indicative of exon 2 skipping.
  • a fourth 57 bp band is also depicted corresponding to the absence of exons 2 and 3, indicative of exon 2 and 3 skipping. Skipping of exons 2 and/or 3 is predicted to lead to NMD (nonsense mediated decay).
  • FIG. 17 is a schematic depicting an LRRK3 cDNA comprising exons 1-4.
  • FIG. 18A is an exemplary vector schematic expressing green fluorescent protein (GFP).
  • FIG. 18B is a series of immunofluorescent microscope images of mice retinal cryosections following subretinal injection of AAV5, 8, 9 or RhlO vectors encoding GFP.
  • FIG. 18C is a vector schematic depicting a AAV8-U6-mgl/mg7-dCAsl3d vector.
  • U6 human U6 promoter
  • mgl mouse USH2A exon 13 guide 1
  • mg7 mouse USH2A exon 13 guide 7.
  • FIG. 18D is a series of immunofluorescent RNAscope microscope images and BaseScope images of mice retinal cryosections injected with control vector or AAV8-U6- mgl/mg7-dCAsl3d vector.
  • RNAscope probes are specific for USH2A.
  • BaseScope probes were specific for spliced (Exonl 1/13) and intact (Exonl 1/12) mRNA
  • FIG. 18E is a bar graph depicting percent exon 12 exclusion following treatment with control vector or AAV8-U6-mgl/mg7-dCAsl3d vector.
  • FIG. 19A is vector schematic depicting a AAV8-U6-mgl/mg7-dCAsl3d vector.
  • FIG. 19B is a series of immunofluorescent microscope images. Cone photoreceptors were immunolabeled with the anti-cone opsin Antibody (Ab). Rod photoreceptors were immunolabeled with the anti-rhodopsin Ab. T Cells were immunolabeled using the anti-CD3 Ab.
  • FIG. 20 is a schematic depicting exon skipping of exon 51 in DMD.
  • FIG. 21 is a schematic depicting that therapies designed to skip multiple exons may benefit around 65% of DMD patients.
  • FIG. 22A is a schematic showing exon 51 of a human DMD pre-mRNA and CRISPR-Cas guide RNAs targeting distinct regions of the pre-mRNA including ESE sequences.
  • FIG. 22B is a series of tape station images depicting the impact of guides from FIG. 22A with dSeq212/4aa mutation on exon 51.
  • the gel image depicts a band corresponding to a transcript comprising exon 51.
  • a second band is depicted corresponding to a transcript having skipped exon 51.
  • FIG. 22C is a graph depicting the percentage of exon 51 excluded for different exon 51 targeting guides.
  • FIG. 23 A schematic showing exon 51 of a human DMD pre-mRNA and CRISPR- Cas guide RNAs targeting distinct regions of the pre-mRNA including ESE sequences.
  • FIG. 23B is a series of tape station images depicting the impact of multiple guides from FIG. 23A with dSeq212/4aa mutation on exon 51. In some cases, multi-targeting was performed with two guides targeting distinct sites of exon 51.
  • the gel image depicts a band corresponding to a transcript comprising exon 51. A second band is depicted corresponding to a transcript having skipped exon 51.
  • FIG. 23C is a graph depicting percentage of exon 51 excluded for different exon 51 targeting guides alone or in combination with another exon 51 guide.
  • FIG. 24 is a tape station image and a diagram depicting additional DMD exons for targeting to induce further DMD exon skipping.
  • FIG. 25A is a tape station image depicting the impact of A03278 AAV_hU6-36DR- g38-36DR-g42(tandem)_CMV-dSeq212-4aa-NLS-WPRE3 (dCasl3d dSeq212 complexed with guides g38 and g42), targeting exon splice enhancers in DMD exon 51 in human myotubes with a deletion of exon 52. The deletion of exon 52 results in no dystrophin protein expression.
  • a 496 bp band represents the exon 51 included DMD transcript.
  • a 263 bp band corresponds to the absence of exon 51 and 52, indicative of exon 51 and 52 skipping.
  • FIG. 25B is a graph depicting percentage of exon 51 skipping upon treatment with A03278.
  • FIG. 25C is a series of immunofluorescence images depicting dystrophin expression. Restoration of dystrophin expression is observed upon A03278 treatment compared to an untreated control which expresses no dystrophin protein due to deletion of exon 52.
  • FIG. 26A is a schematic showing exon 7 and intron 7-8 of a human SMN2 minigene pre-mRNA and guides gl-g5 targeting the intronic region.
  • FIG. 26B is an SMN2 minigene comprising exon 6, exon 7, and exon 8. Guides from FIG. 26A are depicted targeting the intronic ISS sequence.
  • FIG. 26C is a tape station image depicting the impact of guides gl-g5 targeting the intronic ISS between exons 7 and 8 of SMN2, complexed with dCas!3d, on mRNA splicing.
  • a 218bp band is depicted corresponding to exon 6 and exon 8, with exon 7 being skipped.
  • a second 272 bp band is also depicted corresponding to the exons 6, 7, and 8 indicative of the inclusion of exon 7.
  • FIG. 26D is a graph depicting percentage of exon 7 inclusion upon treatment with dCas!3d and multiple guides described in FIG.26A.
  • the disclosure provides gene therapy compositions comprising polypeptides, polynucleotides, or nucleoprotein complexes capable of binding target pre-mRNA sequences and modulating RNA splicing and mRNA processing.
  • vectors comprising the pre-mRNA binding compositions of the disclosure.
  • a “pre-mRNA sequence”, or precursor mRNA is a single-stranded RNA sequence comprising exonic and intronic sequences synthesized by transcription of DNA that has not yet undergone processing to become messenger RNA (mRNA).
  • the pre- mRNA sequence comprises a mutation.
  • the mutation is a base-pair change, a deletion, an insertion, or a frame-shift mutation.
  • the mutation in the pre- mRNA sequence produces an mRNA sequence that encodes for a protein that is one or more of: not expressed, expressed poorly, truncated, misfolded, catalytically inactive, or catalytically hyperactive.
  • the mutation occurs in an exonic region.
  • the pre-mRNA sequence comprises at least one of: an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BP A), and a polypyrimidine tract (PPY).
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • a 5’ splice site is a splice donor site.
  • the 3’ splice site is a splice acceptor site.
  • the pre-mRNA sequence comprises an exon splicing silencer (ESS) motif or an intron splicing silencer (ISS) motif.
  • RNA processing occurs in the nucleus and comprises modifying pre- mRNA to include 5 ’capping, 3’ poly adenylation, and RNA splicing.
  • RNA splicing involves removing intronic sequences, which do not code for proteins, from the transcript and rejoining the exonic sequences to form a mature mRNA sequence that codes for a given protein sequence that is expressed or translated in the cell.
  • RNA splicing is performed by the spliceosome, a large RNA-protein complex comprised of small nuclear ribonucleoproteins (snRNP) which assemble at intronic 5’ and 3’ splice sites on the pre-mRNA to remove intronic sequences and ligate the exonic sequences to form the mature mRNA sequence (FIG. 1 A).
  • Components of the spliceosome include, but are not limited to, Ul, U2, U4, U5, and U6 snRNPs.
  • Further components include splicing regulatory factors such as splicing factor 1 (SF1), splicing factor U2AF 35 kd (U2AF35), and splicing factor U2AF 65 kd (U2AF65).
  • compositions of the disclosure comprise sequence specific RNA binding proteins, nucleic acids, and nucleoprotein complexes, that bind intronic and exonic sequences of pre-mRNA sequences.
  • the binding of the pre-mRNA by these sequences blocks, inhibits, or prevents components of the spliceosome from assembling at the blocked site thereby preventing inclusion of the bound exon into the mature mRNA sequence.
  • the resultant modified mRNA sequence lacks the skipped exon or exons.
  • the transcribed mRNA can produce a polypeptide lacking the amino acids that are encoded by the skipped exon or exons.
  • compositions of the disclosure, and methods of using the compositions comprise sequence specific RNA binding proteins, nucleic acids, and nucleoprotein complexes, that bind intronic and exonic sequences of pre-mRNA sequences.
  • the binding of the pre-mRNA by these sequences promotes inclusion of exons into mature mRNA sequences that are otherwise skipped during processing.
  • exon inclusion is promoted by specifically binding an exon splicing silencer (ESS) motif or an intron splicing silencer (ISS) motif.
  • ESS exon splicing silencer
  • ISS intron splicing silencer
  • RNA binding proteins, nucleic acids, and nucleoprotein complexes of the disclosure bind a pre-mRNA sequence comprising at least one of an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BPA), a polypyrimidine tract (PPY), an exon splicing silencer (ESS) motif, or an intron splicing silencer (ISS) motif.
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • BPA branchpoint adenosine
  • PY polypyrimidine tract
  • ESS exon splicing silencer
  • ISS intron splicing silencer
  • the disclosure provides RNA-guided RNA-binding systems.
  • the RNA-guided RNA-binding system is an RNase Cas-based RNA-guided RNA-binding polypeptide.
  • a nucleic acid sequence encodes an RNA- guided RNA-binding polypeptide which is an RNase Cas protein (or a deactivated RNase Cas protein).
  • the nucleic acid sequence further comprises a gRNA sequence comprising a spacer sequence which binds to a pre-mRNA sequence of the disclosure and a direct repeat (DR) sequence which binds to the RNase Cas protein.
  • DR direct repeat
  • a Casl3d system is catalytically active, in which case, the Cas 13d nucleoprotein complex cleaves and destroys a pre-mRNA sequence.
  • a Casl3d system is catalytically inactive, in which case, the Casl3d nucleoprotein complex binds the pre-mRNA sequence.
  • the catalytically inactive Cast 3d nucleoprotein complex binds the pre-mRNA sequence and modifies pre-mRNA splicing and processing. In some aspects, this splicing modification results in the inclusion of exclusion of one or more exons in the mature mRNA sequence.
  • a Casl3d comprises a catalytically inactive Casl3d fused to an endonuclease which is capable of cleaving the target pre-mRNA or mRNA sequence.
  • the endonuclease is an active RNase.
  • Exemplary endonucleases with RNase activity can be found herein, and these include, for example, a domain from a ZC3H12A zinc-finger (also referred herein as El 7) or a PIN endonuclease.
  • the RNase Cas protein is a Casl3 protein.
  • the Casl3 protein is a Casl3d protein.
  • the Casl3d protein is a deactivated RNase Cas 13d protein (dCas!3d).
  • the dCas!3d protein is a fusion protein comprising 1) dCas!3d and 2) a polypeptide encoding a protein or fragment thereof having nuclease activity.
  • the dCas!3d protein is a fusion protein comprising 1) dCas!3d and 2) a nuclease domain of ZC3H12A, a zinc-finger endonuclease, (referred to as E17 herein).
  • the Cas configuration comprises a signal sequence(s) such as NLS(s) and/or NES(s).
  • the dCas!3d is linked to E17 via a linker sequence.
  • the linker sequence is VDTANGS.
  • the nucleic acid sequence encoding the Casl3d or dCas!3d fusion proteins are operably linked to at least one promoter sequence.
  • the promoter sequence comprises an enhancer and/or an intron.
  • the promoter sequence is a constitutive promoter such as, EIFla or its truncated form, the EFS promoter sequence, or the full length or truncated (tCAG) promoter sequence or the EFS/UBB promoter sequence.
  • the promoter is a tissue specific promoter such as the neuron specific synapsin promoter sequence.
  • the nucleic acid sequence comprises a first promoter sequence that controls expression of a Cas 13d protein or Cas 13d fusion protein and a second promoter sequence that controls expression of the at least one guide RNA sequence.
  • the sequence encoding the RNA-guided RNA binding protein comprises a sequence isolated or derived from a protein with no DNA nuclease activity.
  • the sequence encoding the RNA-guided RNA binding protein disclosed herein comprises a sequence isolated or derived from a CRISPR Cas protein.
  • the CRISPR Cas protein is not a Type II CRISPR Cas protein.
  • the CRISPR Cas protein is not a Cas9 protein.
  • the sequence encoding the RNA-guided RNA binding protein comprises a Type VI CRISPR Cas protein or portion thereof.
  • the Type VI CRISPR Cas protein comprises a Cas 13 protein or portion thereof.
  • Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, bacteria or archaea.
  • Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, Leptotrichia wadei, Listeria seeligeri serovar l/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954), Lachnospiraceae bacterium, Clostridium aminophilum DSM 10710, Camobacterium gallinarum DSM 4847, Paludibacter propioni cigenes WB4, Listeria weihenstephanensis FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL M6-0635 (Listeria newyorkensis), Leptotrichia wadei F0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442 and Corynebacterium ulcerans.
  • Exemplary Cas 13 proteins of the disclosure may be DNA nuclease inactivated.
  • Exemplary Cas 13 proteins of the disclosure include, but are not limited to, Casl3a, Casl3b, Casl3c, Casl3d and orthologs thereof.
  • Exemplary Casl3b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively.
  • the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a Casl3d protein.
  • Cas 13d is an effector of the type VI-D CRISPR-Cas systems.
  • the Cas 13d protein is an RNA-guided RNA endonuclease enzyme that can cut or bind RNA.
  • the Cas 13d protein can include one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains.
  • the Casl3d protein can include either a wild-type or mutated HEPN domain.
  • the Casl3d protein includes a mutated HEPN domain that cannot cut RNA but can process guide RNA.
  • the Cas 13d protein does not require a protospacer flanking sequence. Also see WO Publication No. W02019/040664 & US2019/0062724, which is incorporated herein by reference in its entirety, for further examples and sequences of Cas 13d protein, without limitation.
  • Casl3d sequences of the disclosure include without limitation SEQ ID NOS: 1-296 of WO 2019/040664, so numbered herein and included herewith.
  • Yan et al. (2016) Mol Cell. 70(2):327-339 (doi: 10.1016/j.molcel.2018.02.2018) and Konermann et al. (2016) Cell 173(3):665-676 (doi: 10. 1016/j . cell/2018.02.033) have described Casl3d proteins and both of which are incorporated by reference herein in their entireties.
  • gRNA guide RNA
  • sgRNA single guide RNA
  • Guide RNAs (gRNAs) of the disclosure may comprise a spacer sequence and a “direct repeat” (DR) sequence.
  • a guide RNA is a single guide RNA (sgRNA) comprising a contiguous spacer sequence and DR sequence.
  • the spacer sequence and the DR sequence are not contiguous.
  • the gRNA comprises a DR sequence.
  • DR sequences refer to the repetitive sequences in the CRISPR locus (naturally-occurring in a bacterial genome or plasmid) that are interspersed with the spacer sequences.
  • a guide RNA comprises a direct repeat (DR) sequence and a spacer sequence.
  • a sequence encoding a guide RNA or single guide RNA of the disclosure comprises or consists of a spacer sequence and a DR sequence, that are separated by a linker sequence.
  • the linker sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of nucleotides (nt) in between.
  • the linker sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of nucleotides in between.
  • the DR sequence is a Cas 13d DR sequence.
  • the gRNA that hybridizes with the one or more target RNA molecules in a Cas 13d-mediated manner includes one or more direct repeat (DR) sequences, one or more spacer sequences, such as, e.g., one or more sequences comprising an array of DR-spacer-DR-spacer.
  • DR direct repeat
  • spacer sequences such as, e.g., one or more sequences comprising an array of DR-spacer-DR-spacer.
  • a plurality of gRNAs are generated from a single array, wherein each gRNA can be different, for example target different RNAs or target multiple regions of a single RNA, or combinations thereof.
  • an isolated gRNA includes one or more direct repeat sequences, such as an unprocessed (e.g., about 36 nt) or processed DR (e.g., about 30 nt).
  • a gRNA can further include one or more spacer sequences specific for (e.g., is complementary to) the target RNA.
  • multiple polIII promoters can be used to drive multiple gRNAs, spacers and/or DRs.
  • a guide array comprises a DR (about 36nt)-spacer (about 30nt)-DR (about 36nt)-spacer (about 30nt).
  • Guide RNAs (gRNAs) of the disclosure may comprise non-naturally occurring nucleotides.
  • a guide RNA of the disclosure or a sequence encoding the guide RNA comprises or consists of modified or synthetic RNA nucleotides.
  • RNA nucleotides include, but are not limited to, pseudouridine ( ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G), hypoxanthine, xanthine, xanthosine, 7- methylguanine, 5, 6-Dihydrouracil, 5 -methylcytosine, 5 -methylcytidine, 5- hydropxymethylcytosine, isoguanine, and isocytosine.
  • pseudouridine ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G), hypoxanthine, xanthine, xanthosine, 7- methylguanine, 5, 6-Dihydrouracil, 5 -methylcytosine, 5 -methylcytidine, 5- hydropxymethylcytosine, isoguanine, and isocytosine.
  • Guide RNAs (gRNAs) of the disclosure may bind modified RNA within a target sequence.
  • guide RNAs (gRNAs) of the disclosure may bind modified or mutated (e.g., pathogenic) RNA.
  • exemplary epigenetically or post- transcriptionally modified RNA include, but are not limited to, 2’-O-Methylation (2’-0Me) (2’-O-methylation occurs on the oxygen of the free 2’-OH of the ribose moiety), N6- methyladenosine (m6A), and 5 -methylcytosine (m5C).
  • a guide RNA of the disclosure comprises at least one sequence encoding a non-coding C/D box small nucleolar RNA (snoRNA) sequence.
  • the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the target sequence of the RNA molecule comprises at least one 2’-OMe.
  • the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the at least one sequence that is complementary to the target RNA comprises a box C motif (RUGAUGA SEQ ID NO: 375) and a box D motif (CUGA SEQ ID NO: 376).
  • Spacer sequences of the disclosure bind to the target sequence of an RNA molecule. In some embodiments, spacer sequences of the disclosure bind to pathogenic target RNA. In some embodiments, spacer sequences of the disclosure bind pre-mRNA sequences. In some embodiments, spacer sequences of the disclosure bind intronic sequences of pre-mRNA. In some embodiments, spacer sequences of the disclosure bind exonic sequences of pre-mRNA. [0116] In some embodiments of the compositions of the disclosure, the sequence comprising the gRNA comprises a spacer sequence that specifically binds to the target RNA sequence.
  • the spacer sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in between of complementarity to the target RNA sequence. In some embodiments, the spacer sequence has 100% complementarity to the target RNA sequence. In some embodiments, the spacer sequence comprises or consists of 20 nucleotides.
  • the spacer sequence comprises or consists of 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotide, 30 nucleotides, 31 nucleotides or 32 nucleotides.
  • the spacer sequence comprises or consists of 26 nucleotides.
  • the spacer sequence is non-processed and comprises or consists of 30 nucleotides.
  • the non-processed spacer sequence comprises or consists of 30-36 nucleotides.
  • DR sequences of the disclosure bind the Cas polypeptide of the disclosure. Upon binding of the spacer sequence of the gRNA to the target RNA sequence, the Cas protein bound to the DR sequence of the gRNA is positioned at the target RNA sequence.
  • a DR sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99%, 100% or any percentage in between identity to a DR sequence disclosed herein. .
  • DR sequences of the disclosure comprise a secondary structure or a tertiary structure.
  • Exemplary secondary structures include, but are not limited to, a helix, a stem loop, a bulge, a tetraloop and a pseudoknot.
  • Exemplary tertiary structures include, but are not limited to, an A-form of a helix, a B-form of a helix, and a Z-form of a helix.
  • Exemplary tertiary structures include, but are not limited to, a twisted or helicized stem loop. Exemplary tertiary structures include, but are not limited to, a twisted or helicized pseudoknot.
  • DR sequences of the disclosure comprise at least one secondary structure or at least one tertiary structure. In some embodiments, DR sequences of the disclosure comprise one or more secondary structure(s) or one or more tertiary structure(s).
  • a guide RNA or a portion thereof selectively binds to a tetraloop motif in an RNA molecule of the disclosure.
  • a target sequence of an RNA molecule comprises a tetraloop motif.
  • the tetraloop motif is a “GRNA” motif comprising or consisting of one or more of the sequences of GAAA, GUGA, GCAA or GAGA.
  • a guide RNA or a portion thereof that binds to a target sequence of an RNA molecule hybridizes to the target sequence of the RNA molecule.
  • a guide RNA or a portion thereof that binds to a first RNA binding protein or to a second RNA binding protein covalently binds to the first RNA binding protein or to the second RNA binding protein.
  • a guide RNA or a portion thereof that binds to a first RNA binding protein or to a second RNA binding protein non-covalently binds to the first RNA binding protein or to the second RNA binding protein.
  • a guide RNA or a portion thereof comprises or consists of between 10 and 100 nucleotides, inclusive of the endpoints.
  • a spacer sequence of the disclosure comprises or consists of between 10 and 30 nucleotides, inclusive of the endpoints.
  • a spacer sequence of the disclosure comprises or consists of 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, or 40 nucleotides.
  • the spacer sequence of the disclosure comprises or consists of 20 nucleotides.
  • the spacer sequence of the disclosure comprises or consists of 21 nucleotides.
  • the spacer sequence of the disclosure comprises or consists of 26 nucleotides.
  • Guide molecules generally exist in various states of processing.
  • an unprocessed guide RNA is 36nt of DR followed by 30-32 nt of spacer.
  • the guide RNA is processed (truncated/modified) by Cas 13d itself or other RNases into the shorter "mature" form.
  • an unprocessed guide sequence is about, or at least about 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more nucleotides (nt) in length.
  • a processed guide sequence is about 40 to 150 nt (such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 nt).
  • an unprocessed spacer is about 28-40 nt long (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nt) while the mature (processed) spacer can be about 10 to 30 nt, 10 to 25 nt, 14 to 25 nt, 20 to 22 nt, or 14-30 nt (such as 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, or 35 nt).
  • an unprocessed DR is about 36 nt (such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 nt), while the processed DR is about 30 nt (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt).
  • a DR sequence is truncated by 1-10 nucleotides (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, to 10 nucleotides at e.g., the 5’ end in order to be expressed as mature pre-processed guide RNAs.
  • a guide RNA or a portion thereof does not comprise a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • a guide RNA or a portion thereof comprises a sequence complementary to a protospacer flanking sequence (PF S).
  • PF S protospacer flanking sequence
  • the first RNA binding protein may comprise a sequence isolated or derived from a Cast 3 protein.
  • the first RNA binding protein may comprise a sequence encoding a Cast 3 protein or an RNA- binding portion thereof.
  • the guide RNA or a portion thereof does not comprise a sequence complementary to a PFS.
  • the spacer sequence which binds the target pre-mRNA sequence comprises or consists of about 20-40 nucleotides.
  • a gRNA comprises one or more spacer sequences.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to pre-mRNA encoding genes including USH2A (usherin), DMD (dystrophin), SOD1 (superoxide dismutase 1), LRRK2 (Leucine-rich repeat kinase 2), MAPT (microtubule-associated protein tau), CEP290 (Centrosomal protein of 290 kDa), SCN1 A (sodium voltage-gated channel alpha subunit 1), SMN1 (Survival of motor neuron 1), SMN2 (survival of motor neuron 2), or HTT (huntingtin).
  • USH2A usherin
  • DMD distrophin
  • SOD1 superoxide dismutase 1
  • LRRK2 Leucine-rich repeat kinase 2
  • MAPT microtubule-associated protein tau
  • CEP290 Certrosomal protein of 290 kDa
  • SCN1 A sodium voltage-gated channel alpha sub
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a USH2A pre-mRNA.
  • the sequence encoding for USH2A comprises a mutation.
  • the USH2A pre-mRNA is human.
  • the USH2A pre-mRNA is murine.
  • the gRNA spacer sequences of the disclosure specifically bind exon 13 of human USH2A pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind the ESE motif of exon 13 of human USH2A pre- mRNA.
  • the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 13 of human USH2A pre-mRNA.
  • USH2A gRNA spacer sequences of the disclosure are selected from those listed in Table 1.
  • the gRNA spacer sequence targeting human USH2A is at least one of SEQ ID NO: 528-1413.
  • the gRNA spacer sequences of the disclosure specifically bind exon 12 of murine USH2A pre-mRNA. In some embodiments, the gRNA spacer sequences of the disclosure bind the ESE motif of exon 12 of murine USH2A pre-mRNA. In some embodiments, the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 12 of murine USH2A pre-mRNA. In some aspects, murine USH2A gRNA spacer sequences of the disclosure are selected from those listed in Table 2.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a DMD pre-mRNA.
  • the sequence encoding for DMD comprises a mutation.
  • the DMD pre-mRNA is human.
  • the gRNA spacer sequences of the disclosure specifically bind at least one of exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, and exon 51 of human DMD pre-mRNA.
  • the gRNA spacer sequences of the disclosure specifically bind exon 51 of human DMD pre- mRNA.
  • the gRNA spacer sequences of the disclosure bind the ESE motif of exon 51 of human DMD pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 51 of human DMD pre- mRNA.
  • DMD pe-mRNA gRNA spacer sequences of the disclosure are selected from those listed in Table 3.
  • the gRNA spacer sequence targeting human DMD pre-mRNA is at least one of SEQ ID NO: 1414-12271.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a MAPT pre-mRNA.
  • the sequence encoding for MAPT comprises a mutation.
  • the MAPT pre-mRNA is human.
  • the gRNA spacer sequences of the disclosure specifically bind exon 10 of human MAPT pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind the ESE motif of exon 10 of human MAPT pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 10 of human MAPT pre-mRNA.
  • MAPT pre-mRNA gRNA spacer sequences of the disclosure are selected from those listed in Table 4.
  • gRNA spacer sequences of the disclosure specifically bind to a SOD1 pre-mRNA.
  • the sequence encoding for SOD1 comprises a mutation.
  • the SOD1 pre-mRNA is human.
  • gRNA spacer sequences of the disclosure specifically bind at least one of exon 2 and exon 3 of human SOD1 pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind the ESE motif of exon 2 or exon 3 of human SOD1 pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 2 or exon 3 of human SOD1 pre-mRNA.
  • SOD1 pre-mRNA gRNA spacer sequences of the disclosure are selected from those listed in Table 5.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a LRRK2 pre-mRNA.
  • the sequence encoding for LRRK2 comprises a mutation.
  • the LRRK2 pre-mRNA is human.
  • the gRNA spacer sequences of the disclosure specifically bind at least one of exon 2 and exon 3 of human LRRK2 pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind the ESE motif of exon 2 or exon 3 of human LRRK2 pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind intronic sequences 5’ and 3’ of exon 2 or exon 3 of human LRRK2 pre-mRNA.
  • LRRK2 pre-mRNA gRNA spacer sequences of the disclosure are selected from those listed in Table 6.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a SMN2 pre-mRNA.
  • the sequence encoding for SMN2 comprises a mutation.
  • the SMN2 pre-mRNA is human.
  • gRNA spacer sequences of the disclosure specifically bind an intronic region of the SMN2 pre-mRNA.
  • the gRNA spacer sequences of the disclosure bind the ISS motif of an intron of SMN2.
  • SMN2-targeting gRNA spacer sequences of the disclosure promote inclusion of an exon that is excluded during pre-mRNA splicing due to the presence of a mutation or SNP.
  • the ISS motif occurs between exon 7 and exon 8.
  • SMN2 pre-mRNA gRNA spacer sequences of the disclosure are selected from those listed in Table B.
  • gRNAs correspond to target RNA molecules and an RNA-guided RNA binding protein.
  • the gRNAs correspond to an RNA-guided RNA binding fusion protein, wherein the fusion protein comprises first and second RNA binding proteins.
  • the RNA- binding protein is a deactivated RNA-binding protein, e.g., a deactivated Cas or catalytic dead Cas protein.
  • the first RNA-binding protein in the fusion protein is a deactivated RNA-binding protein, e.g., a deactivated Cas or catalytic dead Cas protein.
  • the sequence encoding the first RNA binding protein is positioned 5’ of the sequence encoding the second RNA binding protein. In some embodiments, along a sequence encoding the fusion protein, the sequence encoding the first RNA binding protein is positioned 3’ of the sequence encoding the second RNA binding protein.
  • the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule. In some embodiments, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a protein capable of selectively binding an RNA molecule and not binding a DNA molecule, a mammalian DNA molecule or any DNA molecule. In some embodiments, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule and inducing a break in the RNA molecule.
  • the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule, inducing a break in the RNA molecule, and not binding a DNA molecule, a mammalian DNA molecule or any DNA molecule. In some embodiments, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a protein capable of binding an RNA molecule, inducing a break in the RNA molecule, and neither binding nor inducing a break in a DNA molecule, a mammalian DNA molecule or any DNA molecule.
  • the sequence encoding the RNA-guided RNA binding protein comprises a sequence isolated or derived from a protein with no DNA nuclease activity.
  • the sequence encoding the RNA-guided RNA binding protein disclosed herein comprises a sequence isolated or derived from a CRISPR Cas protein.
  • the CRISPR Cas protein is not a Type II CRISPR Cas protein.
  • the CRISPR Cas protein is not a Cas9 protein.
  • the sequence encoding the RNA-guided RNA binding protein comprises a Type VI CRISPR Cas protein or portion thereof.
  • the Type VI CRISPR Cas protein comprises a Cas 13 protein or portion thereof.
  • Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, a bacteria or an archaea.
  • Exemplary Cas 13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, Leptotrichia wadei, Listeria seeligeri serovar l/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954), Lachnospiraceae bacterium, Clostridium aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Paludibacter propionicigenes WB4, Listeria weihenstephanensis FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL M6-0635 (Listeria newyorkensis), Leptotrichia wadei F0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442 and Corynebacterium ulcerans.
  • Exemplary Casl3 proteins of the disclosure may be DNA nuclease inactivated.
  • Exemplary Cast 3 proteins of the disclosure include, but are not limited to, Casl3a, Casl3b, Casl3c, Casl3d and orthologs thereof.
  • Exemplary Casl3b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively.
  • Exemplary Casl3a proteins include, but are not limited to:
  • Exemplary wild type Casl3a proteins of the disclosure may comprise or consist of the amino acid sequence of SEQ ID NO: 338.
  • Exemplary Casl3b proteins include, but are not limited to:
  • Exemplary wild type Bergeyella zoohelcum ATCC 43767 Casl3b (BzCasl3b) proteins of the disclosure may comprise or consist of the amino acid sequence of SEQ ID NO: 339.
  • the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a Casl3d protein.
  • Cast 3d is an effector of the type VI-D CRISPR-Cas systems.
  • the Cast 3d protein is an RNA-guided RNA endonuclease enzyme that can cut or bind RNA.
  • the Cast 3d protein can include one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains.
  • the Casl3d protein can include either a wild-type or mutated HEPN domain.
  • the Casl3d protein includes a mutated HEPN domain that cannot cut RNA but can process guide RNA.
  • the Cast 3d protein does not require a protospacer flanking sequence. Also see WO Publication No. WO2019/040664 & US2019/0062724, which is incorporated herein by reference in its entirety, for further examples and sequences of Cast 3d protein, without limitation.
  • Casl3d sequences of the disclosure include without limitation SEQ ID NOS: 1-296 of WO 2019/040664, so numbered herein and included herewith.
  • SEQ ID NO: 1 is an exemplary Casl3d sequence from Eubacterium siraeum containing a HEPN site.
  • SEQ ID NO: 2 is an exemplary Casl3d sequence from Eubacterium siraeum containing a mutated HEPN site.
  • SEQ ID NO: 3 is an exemplary Casl3d sequence from uncultured Ruminococcus sp. containing a HEPN site.
  • SEQ ID NO: 4 is an exemplary Casl3d sequence from uncultured Ruminococcus sp. containing a mutated HEPN site.
  • SEQ ID NO: 5 is an exemplary Casl3d sequence from Gut_metagenome_contig2791000549.
  • SEQ ID NO: 6 is an exemplary Casl3d sequence from Gut_metagenome_contig855000317
  • SEQ ID NO: 7 is an exemplary Casl3d sequence from Gut_metagenome_contig3389000027.
  • SEQ ID NO: 8 is an exemplary Casl3d sequence from Gut_metagenome_contig8061000170.
  • SEQ ID NO: 9 is an exemplary Casl3d sequence from Gut_metagenome_contigl509000299.
  • SEQ ID NO: 10 is an exemplary Casl3d sequence from Gut_metagenome_contig9549000591.
  • SEQ ID NO: 11 is an exemplary Casl3d sequence from Gut_metagenome_contig71000500.
  • SEQ ID NO: 12 is an exemplary Casl3d sequence from human gut metagenome.
  • SEQ ID NO: 13 is an exemplary Casl3d sequence from Gut_metagenome_contig3915000357.
  • SEQ ID NO: 14 is an exemplary Casl3d sequence from Gut_metagenome_contig4719000173.
  • SEQ ID NO: 15 is an exemplary Casl3d sequence from Gut_metagenome_contig6929000468.
  • SEQ ID NO: 16 is an exemplary Casl3d sequence from Gut_metagenome_contig7367000486.
  • SEQ ID NO: 17 is an exemplary Casl3d sequence from Gut_metagenome_contig7930000403.
  • SEQ ID NO: 18 is an exemplary Casl3d sequence from Gut_metagenome_contig993000527.
  • SEQ ID NO: 19 is an exemplary Casl3d sequence from Gut_metagenome_contig6552000639.
  • SEQ ID NO: 20 is an exemplary Casl3d sequence from Gut_metagenome_contigll932000246.
  • SEQ ID NO: 21 is an exemplary Casl3d sequence from Gut_metagenome_contigl2963000286.
  • SEQ ID NO: 22 is an exemplary Casl3d sequence from Gut_metagenome_contig2952000470.
  • SEQ ID NO: 23 is an exemplary Casl3d sequence from Gut_metagenome_contig451000394.
  • SEQ ID NO: 24 is an exemplary Casl3d sequence from Eubacterium_siraeum_DSM_15702.
  • SEQ ID NO: 25 is an exemplary Casl3d sequence from gut_metagenome_P 19E0k2120140920, _c369000003.
  • SEQ ID NO: 26 is an exemplary Casl3d sequence from Gut_metagenome_contig7593000362.
  • SEQ ID NO: 27 is an exemplary Casl3d sequence from Gut_metagenome_contigl2619000055.
  • SEQ ID NO: 28 is an exemplary Casl3d sequence from Gut_metagenome_contigl405000151.
  • SEQ ID NO: 29 is an exemplary Casl3d sequence from Chicken_gut_metagenome_c298474.
  • SEQ ID NO: 30 is an exemplary Casl3d sequence from Gut_metagenome_contigl516000227.
  • SEQ ID NO: 31 is an exemplary Casl3d sequence from Gut_metagenome_contigl838000319.
  • SEQ ID NO: 32 is an exemplary Casl3d sequence from Gut metagenome contigl 3123000268.
  • SEQ ID NO: 33 is an exemplary Casl3d sequence from Gut_metagenome_contig5294000434.
  • SEQ ID NO: 34 is an exemplary Casl3d sequence from Gut_metagenome_contig6415000192.
  • SEQ ID NO: 35 is an exemplary Casl3d sequence from Gut_metagenome_contig6144000300.
  • SEQ ID NO: 36 is an exemplary Casl3d sequence from Gut_metagenome_contig9118000041.
  • SEQ ID NO: 37 is an exemplary Casl3d sequence from Activated_sludge_metagenome_transcript_124486.
  • SEQ ID NO: 38 is an exemplary Casl3d sequence from Gut_metagenome_contig 1322000437.
  • SEQ ID NO: 39 is an exemplary Casl3d sequence from Gut_metagenome_contig4582000531.
  • SEQ ID NO: 40 is an exemplary Casl3d sequence from Gut_metagenome_contig9190000283.
  • SEQ ID NO: 41 is an exemplary Casl3d sequence from Gut_metagenome_contigl709000510.
  • SEQ ID NO: 42 is an exemplary Casl3d sequence from
  • SEQ ID NO: 43 is an exemplary Casl3d sequence from Gut_metagenome_contig3833000494.
  • SEQ ID NO: 44 is an exemplary Casl3d sequence from Activated sludge metagenome transcript l 17355.
  • SEQ ID NO: 45 is an exemplary Casl3d sequence from Gut_metagenome_contigll061000330.
  • SEQ ID NO: 46 is an exemplary Casl3d sequence from Gut_metagenome_contig338000322 from sheep gut metagenome.
  • SEQ ID NO: 47 is an exemplary Casl3d sequence from human gut metagenome.
  • SEQ ID NO: 48 is an exemplary Casl3d sequence from Gut_metagenome_contig9530000097.
  • SEQ ID NO: 49 is an exemplary Casl3d sequence from Gut_metagenome_contigl750000258.
  • SEQ ID NO: 50 is an exemplary Casl3d sequence from Gut_metagenome_contig5377000274.
  • SEQ ID NO: 51 is an exemplary Casl3d sequence from gut_metagenome_P 19E0k2120140920_c248000089.
  • SEQ ID NO: 52 is an exemplary Casl3d sequence from Gut_metagenome_contigll400000031.
  • SEQ ID NO: 53 is an exemplary Casl3d sequence from Gut_metagenome_contig7940000191.
  • SEQ ID NO: 54 is an exemplary Casl3d sequence from Gut_metagenome_contig6049000251.
  • SEQ ID NO: 55 is an exemplary Casl3d sequence from Gut_metagenome_contigl 137000500.
  • SEQ ID NO: 56 is an exemplary Casl3d sequence from Gut_metagenome_contig9368000105.
  • SEQ ID NO: 57 is an exemplary Casl3d sequence from Gut_metagenome_contig546000275.
  • SEQ ID NO: 58 is an exemplary Casl3d sequence from Gut_metagenome_contig7216000573.
  • SEQ ID NO: 59 is an exemplary Casl3d sequence from Gut_metagenome_contig4806000409.
  • SEQ ID NO: 60 is an exemplary Casl3d sequence from Gut_metagenome_contigl0762000480.
  • SEQ ID NO: 61 is an exemplary Casl3d sequence from Gut_metagenome_contig4114000374.
  • SEQ ID NO: 62 is an exemplary Casl3d sequence from Ruminococcus flavcfacicns _FD1.
  • SEQ ID NO: 63 is an exemplary Casl3d sequence from Gut_metagenome_contig7093000170.
  • SEQ ID NO: 64 is an exemplary Casl3d sequence from Gut metagenome contigll 113000384.
  • SEQ ID NO: 65 is an exemplary Casl3d sequence from Gut_metagenome_contig6403000259.
  • SEQ ID NO: 66 is an exemplary Casl3d sequence from Gut_metagenome_contig6193000124.
  • SEQ ID NO: 67 is an exemplary Casl3d sequence from Gut_metagenome_contig721000619.
  • SEQ ID NO: 68 is an exemplary Casl3d sequence from Gut_metagenome_contigl666000270.
  • SEQ ID NO: 69 is an exemplary Casl3d sequence from Gut_metagenome_contig2002000411.
  • SEQ ID NO: 70 is an exemplary Casl3d sequence from Ruminococcus albus.
  • SEQ ID NO: 71 is an exemplary Casl3d sequence from Gut metagenome contigl 3552000311.
  • SEQ ID NO: 72 is an exemplary Casl3d sequence from Gut_metagenome_contigl0037000527.
  • SEQ ID NO: 73 is an exemplary Casl3d sequence from Gut_metagenome_contig238000329.
  • SEQ ID NO: 74 is an exemplary Casl3d sequence from Gut_metagenome_contig2643000492.
  • SEQ ID NO: 75 is an exemplary Casl3d sequence from Gut_metagenome_contig874000057.
  • SEQ ID NO: 76 is an exemplary Casl3d sequence from Gut_metagenome_contig4781000489.
  • SEQ ID NO: 77 is an exemplary Casl3d sequence from Gut_metagenome_contigl2144000352.
  • SEQ ID NO: 78 is an exemplary Casl3d sequence from Gut_metagenome_contig5590000448.
  • SEQ ID NO: 79 is an exemplary Casl3d sequence from Gut_metagenome_contig9269000031.
  • SEQ ID NO: 80 is an exemplary Casl3d sequence from Gut_metagenome_contig8537000520.
  • SEQ ID NO: 81 is an exemplary Casl3d sequence from Gut_metagenome_contigl845000130.
  • SEQ ID NO: 82 is an exemplary Casl3d sequence from gut_metagenome_P13E0k2120140920_c3000072.
  • SEQ ID NO: 83 is an exemplary Casl3dsequence from gut metagenome P 1 E0k2120140920 _cI000078.
  • SEQ ID NO: 84 is an exemplary Casl3d sequence from Gut_metagenome_contigl2990000099.
  • SEQ ID NO: 85 is an exemplary Casl3d sequence from Gut_metagenome_contig525000349.
  • SEQ ID NO: 86 is an exemplary Casl3d sequence from Gut_metagenome_contig7229000302.
  • SEQ ID NO: 87 is an exemplary Casl3d sequence from Gut_metagenome_contig3227000343.
  • SEQ ID NO: 88 is an exemplary Casl3d sequence from Gut_metagenome_contig7030000469.
  • SEQ ID NO: 89 is an exemplary Casl3d sequence from Gut_metagenome_contig5149000068.
  • SEQ ID NO: 90 is an exemplary Casl3d sequence from Gut_metagenome_contig400200045.
  • SEQ ID NO: 91 is an exemplary Casl3d sequence from Gut_metagenome_contigl0420000446.
  • SEQ ID NO: 92 is an exemplary Casl3d sequence from new_flavefaciens_strain_XPD3002 (CasRx).
  • SEQ ID NO: 93 is an exemplary Casl3d sequence from M26_Gut_metagenome_contig698000307.
  • SEQ ID NO: 94 is an exemplary Casl3d sequence from M36_Uncultured_ Eubacteri u/??_sp_TS28_c40956.
  • SEQ ID NO: 95 is an exemplary Casl3d sequence from M12_gut_metagenome_P25C0k2120140920 _c 134000066.
  • SEQ ID NO: 96 is an exemplary Casl3d sequence from human gut metagenome.
  • SEQ ID NO: 97 is an exemplary Casl3d sequence from MIO gut metagenome _P25C90k21201 40920_c2800004 1.
  • SEQ ID NO: 98 is an exemplary Casl3d sequence from 30 Ml
  • SEQ ID NO: 99 is an exemplary Casl3d sequence from gut_metagenome_P25C0k2120140920_c32000045.
  • SEQ ID NO: 100 is an exemplary Casl3d sequence from M13_gut_metagenome _P23C7k2120140920 _c3000067.
  • SEQ ID NO: 101 is an exemplary Cas 13d sequence from M5_gut_metagenome_P18E90k2120140920.
  • SEQ ID NO: 102 is an exemplary Cas 13d sequence from M21_gut_metagenome_P18E0k2120140920.
  • SEQ ID NO: 103 is an exemplary Cas 13d sequence from 7_gut_metagenome _P38C7k2120 1 40920_c484 1 000003.
  • SEQ ID NO: 104 is an exemplary Cas 13d sequence from Ruminococcus bicirculans .
  • SEQ ID NO: 105 is an exemplary Casl3d sequence.
  • SEQ ID NO: 106 is an exemplary Cas 13d consensus sequence.
  • SEQ ID NO: 107 is an exemplary Casl3d sequence from M18_gut_metagenome _P22EOk2120140920_c3395000078.
  • SEQ ID NO: 108 is an exemplary Cas 13d sequence from Ml 7_gut_metagenome_P22E90k2120140920_cl 14.
  • SEQ ID NO: 109 is an exemplary Cas 13d sequence from
  • Ruminococcus _sp_CAG51 Ruminococcus _sp_CAG51.
  • SEQ ID NO: 110 is an exemplary Casl3d sequence from gut metagenome Pl lE90k2120 1 40920_c43000123.
  • SEQ ID NO: 111 is an exemplary Cas 13d sequence from M6_gut_metagenome_P13E90k2120 1 40920_c7000009.
  • SEQ ID NO: 112 is an exemplary Cas 13d sequence from M19_gut_metagenome_Pl 7E90k2120140920.
  • SEQ ID NO: 113 is an exemplary Casl3d sequence from gut_metagenome_P17E0k2120140920, _c87000043.
  • SEQ ID NO: 114 is an exemplary human codon optimized Eubacterium siraeum Casl3d nucleic acid sequence.
  • SEQ ID NO: 115 is an exemplary human codon optimized Eubacterium siraeum Casl3d nucleic acid sequence with a mutant HEPN domain.
  • SEQ ID NO: 116 is an exemplary human codon-optimized Eubacterium siraeum Cas 13d nucleic acid sequence with N-terminalNLS.
  • SEQ ID NO: 117 is an exemplary human codon-optimized Eubacterium siraeum Casl3d nucleic acid sequence with N- and C-terminal NLS tags.
  • SEQ ID NO: 118 is an exemplary human codon-optimized uncultured Ruminococcus sp. Casl3d 30 nucleic acid sequence.
  • SEQ ID NO: 119 is an exemplary human codon-optimized uncultured Ruminococcus sp. Cas 13d nucleic acid sequence with a mutant HEPN domain.
  • SEQ ID NO: 120 is an exemplary human codon-optimized uncultured Ruminococcus sp. Casl3d nucleic acid sequence with N-terminal NLS.
  • SEQ ID NO: 121 is an exemplary human codon-optimized uncultured Ruminococcus sp. Cas 13d nucleic acid sequence with N- and C-terminal NLS tags.
  • SEQ ID NO: 122 is an exemplary human codon-optimized uncultured Ruminococcus flavefaciens FD1 Cas 13d nucleic acid sequence.
  • SEQ ID NO: 123 is an exemplary human codon-optimized uncultured Ruminococcus flavefaciens FD1 Casl3d nucleic acid sequence with mutated HEPN domain.
  • SEQ ID NO: 124 is an exemplary Casl3d nucleic acid sequence from Ruminococcus bicirculans .
  • SEQ ID NO: 125 is an exemplary Casl3d nucleic acid sequence from Eubacterium siraeum.
  • SEQ ID NO: 126 is an exemplary Casl3d nucleic acid sequence from
  • SEQ ID NO: 127 is an exemplary Casl3d nucleic acid sequence from
  • SEQ ID NO: 128 is an exemplary Casl3d nucleic acid sequence from Ruminococcus flavefaciens XPD.
  • SEQ ID NO: 129 is an exemplary consensus DR nucleic acid sequence for E. siraeum Casl3d.
  • SEQ ID NO: 130 is an exemplary consensus DR nucleic acid sequence for Rum. Sp. Casl3d.
  • SEQ ID NO: 131 is an exemplary consensus DR nucleic acid sequence for Rum. Flavefaciens strain XPD3002 Casl3d (CasRx).
  • SEQ ID NOS: 132-137 are exemplary consensus DR nucleic acid sequences.
  • SEQ ID NO: 138 is an exemplary 50% consensus sequence for seven full-length Casl3d orthologues.
  • SEQ ID NO: 139 is an exemplary Casl3d nucleic acid sequence from Gut metagenome P1EO.
  • SEQ ID NO: 140 is an exemplary Casl3d nucleic acid sequence from Anaerobic digester.
  • SEQ ID NO: 141 is an exemplary Casl3d nucleic acid sequence from Ruminococcus sp. CAG:57.
  • SEQ ID NO: 142 is an exemplary human codon-optimized uncultured Gut metagenome P1EO Casl3d nucleic acid sequence.
  • SEQ ID NO: 143 is an exemplary human codon-optimized Anaerobic Digester Cast 3d nucleic acid sequence.
  • SEQ ID NO: 144 is an exemplary human codon-optimized Ruminococcus flavefaciens XPD Cast 3d nucleic acid sequence.
  • SEQ ID NO: 145 is an exemplary human codon-optimized Ruminococcus albus Casl3d nucleic acid sequence.
  • SEQ ID NO: 146 is an exemplary processing of the Ruminococcus sp. CAG:57 CRISPR array.
  • SEQ ID NO: 147 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 148 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 147).
  • SEQ ID NO: 149 is an exemplary Casl3d protein sequence from contig tpg
  • SEQ ID NOS: 150-152 are exemplary consensus DR nucleic acid sequences (goes with SEQ ID NO: 149).
  • SEQ ID NO: 153 is an exemplary Casl3d protein sequence from contig tpg
  • SEQ ID NO: 154 is an exemplary consensus DRnucleic acid sequence (goes with SEQ ID NO: 153).
  • SEQ ID NO: 155 is an exemplary Casl3d protein sequence from contig OGZC01000639.1 (human gut metagenome assembly).
  • SEQ ID NOS: 156-177 are exemplary consensus DR nucleic acid sequences (goes with SEQ ID NO: 155).
  • SEQ ID NO: 158 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 159 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 158).
  • SEQ ID NO: 160 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 161 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 160).
  • SEQ ID NO: 162 is an exemplary Casl3d protein sequence from contig emblOGDFOl 008514.1 1 (human gut metagenome assembly).
  • SEQ ID NO: 163 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 162).
  • SEQ ID NO: 164 is an exemplary Casl3d protein sequence from contig emb 10GPN01002610.1 (human gut metagenome assembly).
  • SEQ ID NO: 165 is an exemplary consensus DRnucleic acid sequence (goes with SEQ ID NO: 164).
  • SEQ ID NO: 166 is an exemplary Casl3d protein sequence from contig NFIR01000008. 1 (Eubacterium sp. An3, from chicken gut metagenome).
  • SEQ ID NO: 167 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 166).
  • SEQ ID NO: 168 is an exemplary Casl3d protein sequence from contig NFLV01000009.1 (Eubacterium sp. Anil from chicken gut metagenome).
  • SEQ ID NO: 169 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 168).
  • SEQ ID NOS: 171-174 are an exemplary Casl3d motif sequences.
  • SEQ ID NO: 175 is an exemplary Casl3d protein sequence from contig OJMMO 1002900 human gut metagenome sequence.
  • SEQ ID NO: 176 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 175).
  • SEQ ID NO: 177 is an exemplary Casl3d protein sequence from contig ODAI011611274.1 gut metagenome sequence.
  • SEQ ID NO: 178 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 177).
  • SEQ ID NO: 179 is an exemplary Casl3d protein sequence from contig
  • SEQ ID NO: 180 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 179).
  • SEQ ID NO: 181 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 182 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 181).
  • SEQ ID NO: 183 is an exemplary Casl3d protein sequence from contig
  • SEQ ID NO: 184 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 183).
  • SEQ ID NO: 185 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 186 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 185).
  • SEQ ID NO: 187 is an exemplary Casl3d protein sequence from contig emb
  • SEQ ID NO: 188 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 187).
  • SEQ ID NO: 189 is an exemplary Casl3d protein sequence from contig e- k87_l 1092736.
  • SEQ ID NOS: 190-193 are exemplary consensus DR nucleic acid sequences (goes with SEQ ID NO: 189).
  • SEQ ID NO: 194 is an exemplary Casl3d sequence from Gut_metagenome_contig6893000291.
  • SEQ ID NOS: 195-197 are exemplary Casl3d motif sequences.
  • SEQ ID NO: 198 is an exemplary Casl3d protein sequence from Ga0224415_l 0007274.
  • SEQ ID NO: 199 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 198).
  • SEQ ID NO: 200 is an exemplary Casl3d protein sequence from EMGJ0003641.
  • SEQ ID NO: 202 is an exemplary Casl3d protein sequence from Ga0129306_1000735.
  • SEQ ID NO: 201 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 200).
  • SEQ ID NO: 202 is an exemplary Casl3d protein sequence from Ga0129306_1000735.
  • SEQ ID NO: 203 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 203
  • SEQ ID NO: 204 is an exemplary Casl3d protein sequence from GaO129317_l 008067.
  • SEQ ID NO: 205 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 204).
  • SEQ ID NO: 206 is an exemplary Casl3d protein sequence from Ga0224415_l 0048792.
  • SEQ ID NO: 207 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 206).
  • SEQ ID NO: 208 is an exemplary Cast 3d protein sequence from 160582958 _gene49834.
  • SEQ ID NO: 209 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 208).
  • SEQ ID NO: 210 is an exemplary Casl3d protein sequence from 250twins_35838_GL0110300.
  • SEQ ID NO: 211 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 210).
  • SEQ ID NO: 212 is an exemplary Casl3d protein sequence from 250twins_36050_GLOI58985.
  • SEQ ID NO: 213 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 212).
  • An exemplary direct repeat sequence for Casl3d Seq212 comprises the nucleic acid sequence: TAGCCCTGCAGTAAGGCAGGGTTCTAAGAC (SEQ ID NO: 12279).
  • SEQ ID NO: 214 is an exemplary Casl3d protein sequence from 31009_GL0034153.
  • SEQ ID NO: 215 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 214).
  • SEQ ID NO: 216 is an exemplary Casl3d protein sequence from 530373_GL0023589.
  • SEQ ID NO: 217 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 216).
  • SEQ ID NO: 218 is an exemplary Casl3d protein sequence from BMZ-1 1B GL0037771.
  • SEQ ID NO: 219 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 218).
  • SEQ ID NO: 220 is an exemplary Casl3d protein sequence from BMZ-1 1B GL0037915.
  • SEQ ID NO: 221 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 220).
  • SEQ ID NO: 222 is an exemplary Cast 3d protein sequence from BMZ- 1 1B GL00696 1 7.
  • SEQ ID NO: 223 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 222).
  • SEQ ID NO: 224 is an exemplary Casl3d protein sequence from DLF014 GL0011914.
  • SEQ ID NO: 225 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 224).
  • SEQ ID NO: 226 is an exemplary Casl3d protein sequence from EYZ- 362B GL0088915.
  • SEQ ID NO: 227-228 are exemplary consensus DR nucleic acid sequences (goes with SEQ ID NO: 226).
  • SEQ ID NO: 229 is an exemplary Casl3d protein sequence from Ga0099364 10024192.
  • SEQ ID NO: 230 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 229).
  • SEQ ID NO: 231 is an exemplary Casl3d protein sequence from
  • GaO 187910 10006931 GaO 187910 10006931.
  • SEQ ID NO: 232 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 231).
  • SEQ ID NO: 233 is an exemplary Casl3d protein sequence from Ga0187910_10015336.
  • SEQ ID NO: 234 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 233).
  • SEQ ID NO: 235 is an exemplary Casl3d protein sequence from Ga0187910_l 0040531.
  • SEQ ID NO: 236 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 23).
  • SEQ ID NO: 237 is an exemplary Casl3d protein sequence from Ga0187911_10069260.
  • SEQ ID NO: 238 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 237).
  • SEQ ID NO: 239 is an exemplary Casl3d protein sequence from MH0288_GL0082219.
  • SEQ ID NO: 240 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 239).
  • SEQ ID NO: 241 is an exemplary Casl3d protein sequence from O2.UC29- 0_GL0096317.
  • SEQ ID NO: 242 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 241).
  • SEQ ID NO: 243 is an exemplary Casl3d protein sequence from PIG- 014_GL0226364.
  • SEQ ID NO: 244 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 243).
  • SEQ ID NO: 245 is an exemplary Casl3d protein sequence from PIG- 018_GL0023397.
  • SEQ ID NO: 246 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 245).
  • SEQ ID NO: 247 is an exemplary Casl3d protein sequence from PIG- 025_GL0099734.
  • SEQ ID NO: 248 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 247).
  • SEQ ID NO: 249 is an exemplary Casl3d protein sequence from PIG- 028_GL0185479.
  • SEQ ID NO: 250 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 249).
  • SEQ ID NO: 251 is an exemplary Casl3d protein sequence from - Ga0224422_10645759.
  • SEQ ID NO: 252 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 251).
  • SEQ ID NO: 253 is an exemplary Casl3d protein sequence from ODAI chimera.
  • SEQ ID NO: 254 is an exemplary consensus DR nucleic acid sequence (goes with SEQ ID NO: 253).
  • SEQ ID NO: 255 is an HEPN motif.
  • SEQ ID NOs: 256 and 257 are exemplary Casl3d nuclear localization signal amino acid and nucleic acid sequences, respectively.
  • SEQ ID NOs: 258 and 260 are exemplary SV40 large T antigen nuclear localization signal amino acid and nucleic acid sequences, respectively.
  • SEQ ID NO: 259 is a dCas9 target sequence.
  • SEQ ID NO: 261 is an artificial Eubacterium siraeum nCasl array targeting ccdB.
  • SEQ ID NO: 262 is a full 36 nt direct repeat.
  • SEQ ID Nos: 263-266 are spacer sequences.
  • SEQ ID NO: 267 is an artificial uncultured Ruminoccus sp. nCasl array targeting ccdB.
  • SEQ ID NO: 268 is a full 36 nt direct repeat.
  • SEQ ID Nos: 269-272 are spacer sequences.
  • SEQ ID NO: 273 is a ccdB target RNA sequence.
  • SEQ ID Nos: 274-277 are spacer sequences.
  • SEQ ID NO: 278 is a mutated Casl3d sequence, NLS-Ga_0531(trunc)-NLS-
  • SEQ ID NO: 279 is a mutated Casl3d sequence, NES-Ga_0531(trunc)-NES-HA. This mutant has a deletion of the non-conservedN-terminus.
  • SEQ ID NO: 280 is a full-length Casl3d sequence, NLS-R&Casl3d-NLS-HA.
  • SEQ ID NO: 281 is a mutated Casl3d sequence, NLS-RfxCasl3d(del5)-NLS- HA. This mutant has a deletion of amino acids 558-587.
  • SEQ ID NO: 282 is a mutated Casl3d sequence, NLS-RfxCasl3d(del5.12)-NLS- HA. This mutant has a deletion of amino acids 558-587 and 953-966.
  • SEQ ID NO: 283 is a mutated Casl3d sequence, NLS-RfxCasl3d(del5.13)-NLS- HA. This mutant has a deletion of amino acids 376-392 and 558-587.
  • SEQ ID NO: 284 is a mutated Casl3d sequence, NLS-RfxCasl3d(del5.12+5.13)- NLS-HA. This mutant has a deletion of amino acids 376-392, 558-587, and 953-966.
  • SEQ ID NO: 285 is a mutated Casl3d sequence, NLS-RfxCasl3d(dell3)-NLS- HA. This mutant has a deletion of amino acids 376-392.
  • SEQ ID NO: 286 is an effector sequence used to edit expression of ADAR2.
  • Amino acids 1 to 969 are dRfxCasl3, aa 970 to 991 are an NLS sequence, and amino acids 992 to 1378 are ADAR2DD.
  • SEQ ID NO: 287 is an exemplary HIV NES protein sequence.
  • SEQ ID NOS: 288-291 are exemplary Casl3d motif sequences.
  • SEQ ID NO: 292 is Casl3d ortholog sequence MH_4866.
  • SEQ ID NO: 293 is an exemplary Casl3d protein sequence from 037 - _emblOIZA01000315.ll
  • SEQ ID NO: 294 is an exemplary Casl3d protein sequence from PIG- 022 GL002635 1.
  • SEQ ID NO: 295 is an exemplary Casl3d protein sequence from PIG- 046_GL0077813.
  • SEQ ID NO: 296 is an exemplary Casl3d protein sequence from pig chimera.
  • SEQ ID NO: 297 is an exemplary nuclease-inactive or dead Casl3d (dCasl3d) protein sequence from Ruminococcus flavefaciens XPD3002 (CasRx)
  • SEQ ID NO: 298 is an exemplary Casl3d protein sequence.
  • SEQ ID NO: 299 is an exemplary Casl3d protein sequence from (contig tpg
  • SEQ ID NO: 300 is an exemplary Casl3d direct repeat nucleotide sequence from Casl3d (contig tpg
  • SEQ ID NO: 301 is an exemplary Casl3d protein contig emb
  • SEQ ID NO: 303 is an exemplary CasM protein from Eubacterium siraeum.
  • SEQ ID NO: 304 is an exemplary CasM protein from Ruminococcus sp., isolate 2789STDY5834971.
  • SEQ ID NO: 305 is an exemplary CasM protein from Ruminococcus bicirculans.
  • SEQ ID NO: 306 is an exemplary CasM protein from Ruminococcus sp., isolate 2789STDY5608892.
  • SEQ ID NO: 307 is an exemplary CasM protein from Ruminococcus sp. CAG:57.
  • SEQ ID NO: 308 is an exemplary CasM protein from Ruminococcus flavefciciens FD-1.
  • SEQ ID NO: 309 is an exemplary CasM protein from Ruminococcus albus strain KH2T6.
  • SEQ ID NO: 310 is an exemplary CasM protein from Ruminococcus flavefaciens strain XPD3002.
  • SEQ ID NO: 311 is an exemplary CasM protein from Ruminococcus sp., isolate 2789STDY5834894.
  • SEQ ID NO: 312 is an exemplary RtcB homolog.
  • SEQ ID NO: 313 is an exemplary WYL from Eubacterium siraeum + C-terminal NLS.
  • SEQ ID NO: 314 is an exemplary WYL from Ruminococcus sp.isolate 2789STDY5834971 + C-term NLS.
  • SEQ ID NO: 315 is an exemplary WYL from Ruminococcus bicirculans + C-term NLS.
  • SEQ ID NO: 316 is an exemplary WYL from Ruminococcus sp. isolate 2789STDY5608892 + C-term NLS.
  • SEQ ID NO: 317 is an exemplary WYL from Ruminococcus sp. CAG:57 + C-term NLS.
  • SEQ ID NO: 318 is an exemplary WYL from Ruminococcus flavefaciens FD-1 + C- termNLS.
  • SEQ ID NO: 319 is an exemplary WYL from Ruminococcus albus strain KH2T6 + C-term NLS.
  • SEQ ID NO: 320 is an exemplary WYL from Ruminococcus flavefaciens strain XPD3002 + C-term NLS.
  • SEQ ID NO: 321 is an exemplary RtcB from Eubacterium siraeum + C-term NLS.
  • SEQ ID NO: 322 is an exemplary direct repeat sequence of Ruminococcus flavefaciens XPD3002 Casl3d (CasRx).
  • Exemplary wild type Casl3d proteins of the disclosure may comprise or consist of the amino acid sequence SEQ ID NO: 92 or SEQ ID NO: 298 (Casl3d protein also known as CasRx).
  • An exemplary direct repeat sequence of Ruminococcus flavefaciens XPD3002 Casl3d comprises the nucleic acid sequence:
  • AACCCCTACCAACTGGTCGGGGTTTGAAAC (SEQ ID NO: 302).
  • the disclosure provides Casl3d protein variants comprising deactivating mutations to produce deactivated Casl3d (dCas!3d) variants.
  • deactivating mutations reduce or eliminate the nuclease activity of Casl3d.
  • dCas!3d variants comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations to a Casl3d sequence that reduce or eliminate the nuclease activity of the Casl3d thereby producing a dCas!3d variant of the Casl3d protein.
  • Deactivated Cast 3d variants retain the full ability to bind guide RNAs.
  • Deactivated Casl3d variants also retain RNA-binding activity.
  • SEQ ID NO: 397 is an exemplary dCasl3d protein, dSeq42.
  • dSeq42 comprises an H841A mutation of the Casl3d protein comprising SEQ ID NO: 42.
  • a nucleic acid sequence encoding SEQ ID NO: 397 is set forth in SEQ ID NO: 461.
  • An exemplary direct repeat sequence for Casl3d Seq42 comprises the nucleic acid sequence:
  • SEQ ID NO: 396 is an exemplary dCas!3d protein, dSeq212.
  • dSeq212 comprises a H919A mutation of the Casl3d protein comprising SEQ ID NO: 212.
  • a nucleic acid sequence encoding SEQ ID NO: 396 is set forth in SEQ ID NO: 395.
  • SEQ ID NO: 398 is an exemplary dCas!3d protein, dSeq208.
  • dSeq208 comprises a H901A mutation of the Casl3d protein comprising SEQ ID NO: 208.
  • SEQ ID NO: 399 is an exemplary dCas!3d protein, dSeq!79.
  • dSeq!79 comprises a H840A mutation of the Casl3d protein comprising SEQ ID NO: 179.
  • SEQ ID NO: 400 is an exemplary dCas!3d protein, dSeq!98.
  • dSeq!98 comprises a H841A mutation of the Casl3d protein comprising SEQ ID NO: 198.
  • SEQ ID NO: 401 is an exemplary dCas!3d protein, dSeq!89.
  • dSeq!89 comprises a H892A mutation of the Casl3d protein comprising SEQ ID NO: 189.
  • SEQ ID NO: 402 is an exemplary dCas!3d protein, dCasRX.
  • dCasRX comprises an R238A, H243A, R857A, and H862A mutation of the Casl3d protein comprising SEQ ID NO: 92.
  • a nucleic acid sequence encoding SEQ ID NO: 402 is set forth in SEQ ID NO: 463.
  • SEQ ID NO: 12307 is an exemplary dCas!3d protein, dSeq212 4aa.
  • a nucleic acid sequence encoding SEQ ID NO: 12307 is set forth in SEQ ID NO: 12278.
  • compositions of the disclosure bind a target RNA sequence.
  • the target RNA sequence comprises a pre-mRNA sequence.
  • the target pre- mRNA sequence comprises an exonic sequence to be skipped.
  • the target pre-mRNA comprises a sequence motif corresponding to a spacer sequence of the guide RNA corresponding to the RNA-guided RNA-binding protein.
  • one or more spacer sequences are used to target one or more target sequences.
  • multiple spacers are used to target multiple target RNAs.
  • Such target RNAs can be different target sites within the same RNA molecule or can be different target sites within different RNA molecules. Spacer sequences can also target non-coding RNA.
  • multiple promoters e.g., Pol III promoters
  • the binding of the target RNA(s) or target sequence motif(s) induces altered RNA splicing during mRNA processing.
  • the altered RNA splicing involved the targeted exon to be skipped and not incorporated into the mature mRNA.
  • the sequence motif of the target RNA is a signature of a disease or disorder.
  • a sequence motif of the disclosure may be isolated or derived from a sequence of foreign or exogenous sequence found in a genomic sequence, and therefore translated into an mRNA molecule of the disclosure or a sequence of foreign or exogenous sequence found in an RNA sequence of the disclosure.
  • a target pre-mRNA sequence of the disclosure may comprise, consist of, be situated by, or be associated with a mutation in an endogenous sequence that causes a disease or disorder.
  • the mutation may comprise or consist of a sequence substitution, inversion, deletion, insertion, transposition, or any combination thereof.
  • the deletion is a single nucleotide. In some embodiments, the deletion can be one or more nucleotides. In some embodiments, the deletion is an exonic or intronic sequence. In some embodiments, the deletion comprises an intronic and exonic sequence.
  • the insertion is a single nucleotide. In some embodiments, the insertion can be one or more nucleotides. In some embodiments, the insertion occurs in an exonic or intronic sequence. In some embodiments, the insertion occurs in an intronic and exonic sequence.
  • a target pre-mRNA sequence of the disclosure may comprise or consist of a repeated sequence. In some embodiments, the repeated sequence may be associated with a microsatellite instability (MSI). MSI at one or more loci results from impaired DNA mismatch repair mechanisms of a cell of the disclosure.
  • a hypervariable sequence of DNA may be transcribed into an mRNA of the disclosure comprising a target sequence comprising or consisting of the hypervariable sequence.
  • a target pre-mRNA sequence of the disclosure may comprise or consist of a biomarker.
  • the biomarker may indicate a risk of developing a disease or disorder.
  • the biomarker may indicate a healthy gene (low or no determinable risk of developing a disease or disorder.
  • the biomarker may indicate an edited gene.
  • Exemplary biomarkers include, but are not limited to, single nucleotide polymorphisms (SNPs), sequence variations or mutations, epigenetic marks, splice acceptor sites, exogenous sequences, heterologous sequences, and any combination thereof.
  • SNPs single nucleotide polymorphisms
  • a target pre-mRNA sequence of the disclosure may comprise or consist of a secondary, tertiary or quaternary structure.
  • the secondary, tertiary or quaternary structure may be endogenous or naturally occurring.
  • the secondary, tertiary or quaternary structure may be induced or non-naturally occurring.
  • the secondary, tertiary or quaternary structure may be encoded by an endogenous, exogenous, or heterologous sequence.
  • a target sequence of an RNA molecule comprises or consists of between 2 and 1,000 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 2 and 100 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 2 and 50 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 2 and 20 nucleotides or nucleic acid bases, inclusive of the endpoints.
  • the target sequence of an RNA molecule comprises or consists of between 20-30 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of about 26 nucleotides or nucleic acid bases, inclusive of the endpoints.
  • a target sequence of an RNA molecule is continuous.
  • the target sequence of an RNA molecule is discontinuous.
  • the target sequence of an RNA molecule may comprise or consist of one or more nucleotides or nucleic acid bases that are not contiguous because one or more intermittent nucleotides are positioned in between the nucleotides of the target sequence.
  • a target sequence of an RNA molecule is naturally occurring.
  • the target sequence of an RNA molecule is non-naturally occurring.
  • Exemplary non-naturally occurring target sequences may comprise or consist of sequence variations or mutations, chimeric sequences, exogenous sequences, heterologous sequences, chimeric sequences, recombinant sequences, sequences comprising a modified or synthetic nucleotide or any combination thereof.
  • a target sequence of an RNA molecule binds to a guide RNA of the disclosure. In some embodiments of the compositions and methods of the disclosure, one or more target sequences of an RNA molecule binds to one or more guide RNA spacer sequences of the disclosure. [0458] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule binds to a first RNA binding protein of the disclosure.
  • compositions of the disclosure comprise a gRNA comprising a spacer sequence that specifically binds to a pre-mRNA sequence comprising a mutation resulting in a disease or disorder.
  • the mutation occurs in an exon of the pre-mRNA.
  • the mutation occurs in an intron of the pre-mRNA.
  • the pre- mRNA sequence comprises at least one of: an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BP A), and a polypyrimidine tract (PPY).
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • a 5’ splice site is a splice donor site.
  • the 3’ splice site is a splice acceptor site.
  • the pre-mRNA sequence comprises an exon splicing silencer (ESS) motif or an intron splicing silencer (ISS) motif.
  • the spacer which binds the target pre-mRNA sequence comprises or consists of about 20-30 nucleotides.
  • a gRNA comprises one or more spacer sequences.
  • Exemplary gRNA spacer sequences of the disclosure can be configured to specifically bind to an RNA sequence encoding any gene in a subject’s genome. Exemplary gRNA spacer sequences of the disclosure can be configured to specifically bind to a pre- mRNA sequence encoding any gene, or derived from any gene, in a subject’s genome.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a USH2A pre-mRNA.
  • the sequence encoding for USH2A comprises a mutation.
  • the USH2A pre-mRNA is human.
  • the USH2A pre-mRNA is murine.
  • the gRNA spacer sequence targets an USH2A sequence comprising a c.2299delG mutation.
  • the nucleic acid sequence encoding wild-type human USH2A mRNA comprises or consists of SEQ ID NO: 465 or SEQ ID NO: 466.
  • the nucleic acid sequence encoding wild-type human USH2A pre-mRNA comprises or consists of SEQ ID NO: 467.
  • the amino acid sequence encoding human usherin comprises or consists of SEQ ID NO: 468.
  • the amino acid sequence encoding human usherin comprises a C759F mutation.
  • the nucleic acid sequence encoding wild-type murine USH2A mRNA comprises or consists of SEQ ID NO: 486.
  • the amino acid sequence encoding murine usherin comprises or consists of SEQ ID NO: 488.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a DMD pre-mRNA.
  • the sequence encoding for DMD comprises a mutation.
  • the DMD pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human DMD mRNA comprises or consists of SEQ ID NO: 470. In some embodiments, the nucleic acid sequence encoding wild-type human DMD pre-mRNA comprises or consists of SEQ ID NO: 471. In some embodiments, the nucleic acid sequence encoding human DMD comprises a deletion. In some embodiments, the deletion is a single nucleotide. In some embodiments, the deletion can be one or more nucleotides. In some embodiments, the deletion is an exonic or intronic sequence. In some embodiments, the deletion comprises an intronic and exonic sequence.
  • the deletion occurs across a region comprising one or more of exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, and exon 55 and any introns upstream downstream of therebetween of these exons.
  • the amino acid sequence encoding human dystrophin comprises or consists of SEQ ID NO: 472.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a MAPT pre-mRNA.
  • the sequence encoding for MAPT comprises a mutation.
  • the MAPT pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human MAPT mRNA comprises or consists of SEQ ID NO: 478. In some embodiments, the nucleic acid sequence encoding MAPT comprises a mutation in exon 10 or intron 10. In some embodiments, the nucleic acid sequence encoding wild-type human MAPT pre-mRNA comprises or consists of SEQ ID NO: 479. In some embodiments, the amino acid sequence encoding human tau comprises or consists of SEQ ID NO: 480.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a SOD1 pre-mRNA.
  • the sequence encoding for SOD1 comprises a mutation.
  • the SOD1 pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human SOD1 mRNA comprises or consists of SEQ ID NO: 482.
  • the nucleic acid sequence encoding wild-type human SOD1 pre-mRNA comprises or consists of SEQ ID NO: 483.
  • the amino acid sequence encoding human superoxide dismutase 1 (SOD1) comprises or consists of SEQ ID NO: 484.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a LRRK2 pre-mRNA.
  • the sequence encoding for LRRK2 comprises a mutation.
  • the LRRK2 pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human LRRK2 mRNA comprises or consists of SEQ ID NO: 474.
  • the nucleic acid sequence encoding wild-type human LRRK2 pre-mRNA comprises or consists of SEQ ID NO: 475.
  • the amino acid sequence encoding Leucine-rich repeat kinase 2 (LRRK2) comprises or consists of SEQ ID NO: 476.
  • the amino acid sequence encoding human LRRK2 comprises a mutation comprising one or more of G2019S, R1441G, R1441C, R1441H, Y1699C, and I2020T.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a SMN1 pre-mRNA.
  • the sequence encoding for SMN1 comprises a mutation.
  • the SMN1 pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human SMN1 mRNA comprises or consists of SEQ ID NO: 12324. In some embodiments, the nucleic acid sequence encoding wild-type human SMN1 gene is set forth in ENST00000380707.9 . In some embodiments, the amino acid sequence encoding human survival motor neuron 1 (SMN1) comprises or consists of SEQ ID NO: 12322.
  • Exemplary gRNA spacer sequences of the disclosure specifically bind to a SMN2 pre-mRNA.
  • the sequence encoding for SMN2 comprises a mutation.
  • the SMN2 pre-mRNA is human.
  • the nucleic acid sequence encoding wild-type human SMN2 mRNA comprises or consists of SEQ ID NO: 12325.
  • the nucleic acid sequence encoding wild-type human SMN2 gene is set forth in ENST00000380743.9.
  • the amino acid sequence encoding human survival motor neuron 2 (SMN2) comprises or consists of SEQ ID NO: 12323.
  • pre-mRNA-targeting compositions are packaged as AAV vectors.
  • pre-mRNA-targeting Casl3d compositions are packaged as AAV vectors.
  • pre-mRNA-targeting PUF/PUMBY compositions are packaged as AAV vectors.
  • RNA-targeting Casl3d vectors comprise a nucleic acid sequence encoding at least one guide RNA under control of a promoter and a sequence encoding a casl3d peptide under the control of a second promoter.
  • an exemplary pre-mRNA-targeting Casl3d composition comprises from 5’ to 3’: a) a promoter, b) a cast 3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, c) a promoter, and d) a sequence encoding dCas!3d.
  • the composition further comprises a 5’ ITR sequence and a 3’ ITR sequence.
  • an exemplary pre-mRNA-targeting Casl3d composition comprises from 5’ to 3’: a) a promoter, b) a first cas!3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, c) a second cas!3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, d) a promoter, and e) a sequence encoding dCas!3d.
  • the composition further comprises a 5’ ITR sequence and a 3’ ITR sequence.
  • an exemplary pre-mRNA-targeting Casl3d composition comprises from 5’ to 3’: a) a first promoter, b) a first cas!3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, c) a second promoter, d) a second cast 3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, e) a promoter, and I) a sequence encoding dCas!3d.
  • the composition further comprises a 5’ ITR sequence and a 3’ ITR sequence.
  • an exemplary pre-mRNA-targeting PUF composition comprises from 5’ to 3’: a) a promoter, b) a sequence encoding a PUF protein. In some embodiments, the composition further comprises a 5’ ITR sequence and a 3’ ITR sequence.
  • an exemplary pre-mRNA-targeting Casl3d composition includes additional elements such as signal sequences, regulatory sequences, linkers, tags, and poly A sequences.
  • a pre-mRNA-targeting Casl3d composition comprises from 5’ to 3’: a) a human U6 promoter, b) a cast 3d gRNA, wherein the gRNA comprises i) a direct repeat sequence and ii) a RNA targeting spacer sequence, c) a promoter, d) a kozak sequence, an e) NLS sequence, I) a linker sequence, g) a sequence encoding dCas!3d, h) a linker sequence, i) an NLS sequence, j) a linker sequence, k) a tag sequence, and 1) a poly A sequence.
  • the composition further comprises a 5’ ITR sequence and a 3’ ITR sequence.
  • An "AAV vector” as used herein refers to a vector comprising, consisting essentially of, or consisting of one or more nucleic acid molecules and one or more AAV inverted terminal repeat sequences (ITRs).
  • the nucleic acid molecule encodes for a repeat targeting protein and/or composition of the disclosure.
  • AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides the functionality of rep and cap gene products, for example, by transfection of the host cell.
  • AAV vectors contain a promoter, at least one nucleic acid that may encode at least one protein or RNA, and/or an enhancer and/or a terminator within the flanking ITRs that is packaged into the infectious AAV particle.
  • the encapsidated nucleic acid portion may be referred to as the AAV vector genome.
  • Plasmids containing AAV vectors may also contain elements for manufacturing purposes, e.g., antibiotic resistance genes, origin of replication sequences etc., but these are not encapsidated and thus do not form part of the AAV particle.
  • an AAV vector can comprise at least one nucleic acid molecule encoding a repeat targeting composition of the disclosure.
  • an AAV vector can comprise at least one regulatory sequence.
  • an AAV vector can comprise at least one AAV inverted terminal (ITR) sequence.
  • ITR AAV inverted terminal
  • an AAV vector can comprise a first ITR sequence and a second ITR sequence.
  • an AAV vector can comprise at least one promoter sequence.
  • an AAV vector can comprise at least one enhancer sequence.
  • an AAV vector can comprise at least one polyA sequence.
  • an AAV vector can comprise at least one linker sequence.
  • an AAV vector of the disclosure can comprise at least one nuclear localization signal, or nuclear export signal and or both.
  • an AAV vector of the disclosure will contain a WPRE (woodchuck hepatitis virus post-trancriptional regulatory element).
  • an AAV vector of the disclosure can comprise a Cas protein, peptide, or fragment thereof.
  • an AAV vector of the disclosure can comprise an endonuclease protein, peptide, or fragment thereof.
  • an AAV vector of the disclosure can comprise a guide RNA, in some cases a pre-mRNA targeting guide RNA.
  • AAV vectors of the disclosure can comprise a fusion protein comprising one or more elements of the disclosure, including, but not limited to, an RNA-targeting protein (such as a Cas, PUF, or PUMBY) and an endonuclease.
  • fusion proteins of the AAV vector can further comprise a linker amino acid sequence between the one or more elements of the disclosure.
  • an AAV vector can comprise a first AAV ITR sequence, a promoter sequence, a RNA-targeting composition nucleic acid molecule, a regulatory sequence and a second AAV ITR sequence.
  • an AAV vector can comprise, in the 5’ to 3’ direction, a first AAV ITR sequence, a promoter sequence, a trans gene nucleic acid molecule, and a second AAV ITR sequence.
  • a vector comprises a guide RNA of the disclosure.
  • the vector comprises at least one guide RNA of the disclosure.
  • the vector comprises one or more guide RNA(s) of the disclosure.
  • the vector comprises two or more guide RNAs of the disclosure.
  • the vector comprises three guide RNAs.
  • the vector comprises four guide RNAs.
  • the vector comprises a PolIII promoter, one or multiple guides, a PolII promoter, the Cas protein, a regulatory element and a poly A.
  • the vector further comprises a guided or non-guided RNA-binding protein of the disclosure.
  • the vector further comprises an RNA-binding fusion protein of the disclosure.
  • the fusion protein comprises a first RNA binding protein and a second RNA binding protein.
  • the RNA-guided RNA-binding systems comprising an RNA-binding protein and a gRNA are in a single vector.
  • the single vector comprises the RNA-guided RNA-binding systems which are Cas 13d RNA-guided RNA- binding systems or catalytic deactivated Cas 13d (dCas!3d) RNA-guided RNA-binding systems.
  • the single vector comprises the Casl3d RNA-guided RNA-binding systems which are CasRx or dCasRx RNA-guided RNA-binding systems.
  • the single vector comprises a non-guided RNA-binding system comprising a PUF or PUMBY-based protein.
  • the single vector comprises anon- guided RNA-binding system comprising a PUF or PUMBY-based protein fused with a nuclease domain from ZC3H12A, such as El 7.
  • the single vector comprises a dCas!3d RNA-binding system fused with a nuclease domain from ZC3H12A, such as El 7 (SEQ ID NO: 359).
  • the nuclease domain from ZC3H12A, El 7 can comprise, consist essentially of, or consist of the amino acid sequence: GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFL ERGHTDITVF VP S WRKEQPRPDVPITDQHILRELEKKKILVFTP SRRVGGKRVVCYDD RFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGR HGPSLDNFLRKKPLTLE (SEQ ID NO: 12308).
  • the nuclease domain from ZC3H12A, El 7 can comprise, consist essentially of, or consist of the amino acid sequence: SGPCGEKPVLEASPTMSLWEFEDSHSRQGTPRPGQELAAEEASALELQMKVDFFRKL GYSSTEIHSVLQKLGVQADTNTVLGELVKHGTATERERQTSPDPCPQLPLVPRGGGT PKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFLERGH TDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIV KLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGP SLDNFLRKKPLTLEHRKQPCPYGRKCTYGIKCRFFHPERPSCPQRSVADELRANALLS PPRAPSKDKNGRRPSPSSQSQS
  • a first vector comprises a guide RNA of the disclosure and a second vector comprises an RNA- binding protein or RNA-binding fusion protein of the disclosure.
  • the first vector comprises at least one guide RNA of the disclosure.
  • the first vector comprises one or more guide RNA(s) of the disclosure.
  • the first vector comprises two or more guide RNA(s) of the disclosure.
  • the fusion protein comprises a first RNA binding protein and a second RNA binding protein.
  • the first vector and the second vector are identical vectors or vector serotypes.
  • the first vector and the second vector are not identical vectors or vector serotypes.
  • the RNA-binding systems capable of targeting pre-mRNA sequences are in a single vector.
  • vectors refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • the vector is a lentivirus (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector.
  • Vectors are capable of autonomous replication in a host cell into which they are introduced such as e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors and other vectors such as, e.g., non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors such as e.g., expression vectors
  • Common expression vectors are often in the form of plasmids.
  • recombinant expression vectors comprise a nucleic acid provided herein such as e.g., a guide RNA which can be expressed from a DNA sequence, and a nucleic acid encoding a Cas 13d protein, in a form suitable for expression of a protein in a host cell.
  • Recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence such as e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell. Certain embodiments of a vector depend on factors such as the choice of the host cell to be transformed, and the level of expression desired.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein such as, e.g., CRISPR transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.
  • a vector of the disclosure is a viral vector.
  • the viral vector comprises a sequence isolated or derived from a retrovirus.
  • the viral vector comprises a sequence isolated or derived from a lentivirus.
  • the viral vector comprises a sequence isolated or derived from an adenovirus.
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the viral vector is replication incompetent.
  • the viral vector is isolated or recombinant.
  • the viral vector is self- complementary.
  • Adeno-associated virus refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae.
  • Adeno-associated virus is a single-stranded DNA virus that grows in cells in which certain functions are provided by a co-infecting helper virus.
  • General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).
  • the degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs).
  • ITRs inverted terminal repeat sequences
  • the similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types.
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy.
  • AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible.
  • AAV AAV genome encapsidation
  • some or all of the internal approximately 4.3 kb of the genome encoding replication and structural capsid proteins, rep-cap
  • the rep and cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized.
  • AAV- infected cells are not resistant to superinfection.
  • Recombinant AAV (rAAV) genomes of the invention comprise, consist essentially of, or consist of a nucleic acid molecule encoding a pre-mRNA targeting composition (such as a guided RNA-binding compositions such as a Cast 3d polypeptide or a non-guided RNA- binding composition such as a PUF or PUMBY polypeptide) and one or more AAV ITRs flanking the nucleic acid molecule.
  • a pre-mRNA targeting composition such as a guided RNA-binding compositions such as a Cast 3d polypeptide or a non-guided RNA- binding composition such as a PUF or PUMBY polypeptide
  • AAV ITRs flanking the nucleic acid molecule such as a pre-mRNA targeting composition
  • a guided RNA-binding compositions such as a Cast 3d polypeptide or a non-guided RNA- binding composition such as a PUF or PUMBY polypeptide
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO), AAV11 or AAV12.
  • the AAV serotype is AAVrh.74.
  • the AAV vector comprises a modified capsid.
  • the AAV vector is an AAV2-Tyr mutant vector.
  • the AAV vector comprises a capsid with a non-tyrosine amino acid at a position that corresponds to a surface-exposed tyrosine residue in position Tyr252, Tyr272, Tyr275, Tyr281, Tyr508, Tyr612, Tyr704, Tyr720, Tyr730 or Tyr673 of wild-type AAV2. See also WO 2008/124724 incorporated herein in its entirety.
  • the AAV vector comprises an engineered capsid.
  • AAV vectors comprising engineered capsids include without limitation, AAV2.7m8, AAV9.7m8, AAV22tYF, and AAV8 Y733F).
  • the capsid is a ubiquitination resistant capsid.
  • the ubiquitination capsid is an AAV2 capsid comprising tyrosine (Y) and serine (S) mutations.
  • the AAV2 capsid comprises Y, S and threonine (T) mutations.
  • the AAV2 capsid includes, without limitation, AAV2 capsid mutants such as T455V, T491V, T550V, T659V, Y444+500+730F, and Y444+500+730F+T491V.
  • the viral vector is replication incompetent.
  • the viral vector is isolated or recombinant (rAAV).
  • the viral vector is self-complementary (scAAV).
  • an AAV inverted terminal repeat sequence can comprise any AAV ITR sequence known in the art.
  • an AAV ITR sequence can comprise or consist of an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV3 ITR sequence, an AAV4 ITR sequence, an AAV 5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAVrhlO ITR sequence, an AAV11 ITR sequence, an AAV 12 ITR sequence, an AAV 13 ITR sequence, or an AAVrh74 ITR sequence.
  • the ITR sequence can comprise a modified AAV ITR sequence.
  • an AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 458 or 459.
  • a modified ITR sequence can comprise consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 458 or 459.
  • a first AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 458 and a second AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 459.
  • the first AAV ITR sequence is positioned at the 5’ of a AAV vector.
  • the second AAV ITR sequence is positioned at the 3’ of a AAV vector.
  • a vector of the disclosure is a non-viral vector.
  • the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
  • the vector is an expression vector or recombinant expression system.
  • the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.
  • an expression vector, viral vector or non-viral vector provided herein includes without limitation, an expression control element.
  • An “expression control element” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene.
  • Exemplary expression control elements include but are not limited to promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements may be constitutive, inducible, repressible, or tissue- specific, for example.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled.
  • Non-limiting exemplary promoters include a Pol III promoter such as, e.g., U6 and Hl promoters and/or a Pol II promoter e.g., SV40, CMV (optionally including the CMV enhancer), RSV (Rous Sarcoma Virus LTR promoter (optionally including RSV enhancer), CBA (hybrid CMV enhancer/ chicken B-actin), CAG (hybrid CMV enhancer fused to chicken B-actin), truncated CAG, Cbh (hybrid CBA), EF-la (human elongation factor alpha- 1) or EFS (short intron-less EF-1 alpha), PGK (phosphoglycerol kinase), CEF (chi
  • a Pol III promoter such as, e.g., U6 and Hl promoters and/or a Pol II promoter e.g., SV40, CMV (optionally including the CMV enhancer), R
  • Enhancer is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription.
  • Non-limiting exemplary enhancers and posttranscriptional regulatory elements include the CMV enhancer, MCK enhancer, R-U5’ segment in LTR of HTLV-1, SV40 enhancer, the intron sequence between exons 2 and 3 of rabbit B-globin, and Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus
  • a nucleic acid sequence encoding a WPRE element is set forth in SEQ ID NO: 456.
  • an intron is used to enhance promoter activity such as a UBB intron.
  • the UBB intron is used with an EFS promoter.
  • Tissue-specific ocular promoters include, without limitation, human blue opsin (e.g., HB570 and HB569), human red cone opsin (e.g., PR2.1, PR1.7, PRO.5, 3LCR-PRO.5), human green red opsin (G1.7p), human rhodopsin kinase (RHOK/GRK), human interphotoreceptor retinoid binding protein (hIRBP), human retinal pigment epithelial (hRPE65p, NA65p, SynPIII), bestrophinl promoter (VMD2), human gamma-synuclein promoter (hSNCGp), synapsin-1 (Synl), human neurofilament heavy gene promoter (Nefh), human cone arrestin (e.g.,
  • tissue-specific ocular promoters are capable of driving expression to a retinal cell such as, without limitation, a photoreceptor cell, a retinal pigment epithelial cell, a ganglion cell, an amacrine cell, a bipolar cell, a horizontal cell, a Muller glial cell, a rod cell, or a cone cell.
  • a retinal cell such as, without limitation, a photoreceptor cell, a retinal pigment epithelial cell, a ganglion cell, an amacrine cell, a bipolar cell, a horizontal cell, a Muller glial cell, a rod cell, or a cone cell.
  • an expression vector, viral vector or non-viral vector includes without limitation, vector elements such as an IRES or 2A peptide sites for configuration of “multicistronic” or “polycistronic” or “bicistronic” or tricistronic” constructs, i.e., having double or triple or multiple coding areas or exons, and as such will have the capability to express from mRNA two or more proteins from a single construct.
  • Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA.
  • the two strategies most widely used for constructing multicistronic configurations are through the use of an IRES or a 2A self-cleaving site.
  • an “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs.
  • an IRES is an RNA element that allows for translation initiation in a cap-independent manner.
  • self-cleaving peptides or “sequences encoding self-cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such selfcleaving peptides include without limitation, T2A, and P2A peptides or other sequences encoding the self-cleaving peptides.
  • Exemplary vector configurations comprise a promoter or regulatory sequence (promoter/ enhancer combination) driving the expression of the nucleic acid encoding the RNA-targeting Casl3d-based system.
  • a vector configuration comprises a promoter driving expression of the RNA-guided Cas RNase RNA-binding protein, or dCas protein fusion in operable linkage with a second promoter driving expressing of a cognate gRNA.
  • the vector configuration comprises a linker and one or more tags.
  • vectors comprising guide RNA sequences of the disclosure comprises a promoter sequence to drive expression of the guide RNA.
  • a vector comprising a guide RNA sequence of the disclosure comprises a promoter sequence to drive expression of the guide RNA.
  • the promoter to drive expression of the guide RNA is a constitutive promoter.
  • the promoter sequence is an inducible promoter.
  • the promoter is a sequence is a tissue-specific and/or cell-type specific promoter.
  • the promoter is a hybrid or a recombinant promoter.
  • the promoter is a promoter capable of expressing the guide RNA in a mammalian cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA in a human cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA and restricting the guide RNA to the nucleus of the cell. In some embodiments, the promoter is a human RNA polymerase promoter or a sequence isolated or derived from a sequence encoding a human RNA polymerase promoter. In some embodiments, the promoter is a U6 promoter or a sequence isolated or derived from a sequence encoding a U6 promoter.
  • the U6 promoter is a human U6 promoter. In some embodiments, the promoter is a human tRNA promoter or a sequence isolated or derived from a sequence encoding a human tRNA promoter. In some embodiments, the promoter is a human valine tRNA promoter or a sequence isolated or derived from a sequence encoding a human valine tRNA promoter.
  • a promoter to drive expression of the guide RNA further comprises a regulatory element.
  • a vector comprising a promoter sequence to drive expression of the guide RNA further comprises a regulatory element.
  • a regulatory element enhances expression of the guide RNA.
  • Exemplary regulatory elements include, but are not limited to, an enhancer element, an intron, an exon, or a combination thereof.
  • a vector of the disclosure comprises one or more of a sequence encoding a guide RNA, a promoter sequence to drive expression of the guide RNA and a sequence encoding a regulatory element. In some embodiments of the compositions of the disclosure, the vector further comprises a sequence encoding a fusion protein of the disclosure.
  • a promoter driving expression of one or more gRNA of the disclosure is a human U6 promoter. In some aspects, a nucleic acid sequence encoding a human U6 promoter is set forth in SEQ ID NO: 448.
  • a promoter driving expression of one or more RNA-binding polypeptides of the disclosure is an EFS promoter.
  • a nucleic acid sequence encoding an EFS promoter is set forth in SEQ ID NO: 451.
  • a promoter driving expression of one or more RNA-binding polypeptides of the disclosure is an EFS-UBB promoter.
  • a nucleic acid sequence encoding an EFS-UBB promoter is set forth in SEQ ID NO: 450.
  • a nucleic acid sequence encoding an EFS-UBB promoter is set forth in SEQ ID NO: 453.
  • a promoter driving expression of one or more RNA-binding polypeptides of the disclosure is a CMV promoter.
  • a nucleic acid sequence encoding a CMV promoter is set forth in SEQ ID NO: 12277.
  • a nucleic acid sequence encoding a polyA sequence is set forth in SEQ ID NO: 457.
  • the vector is a viral vector.
  • the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.
  • the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors.
  • the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from 4.5 kb to 4.75 kb.
  • exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV2-Tyr mutant vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, an AAVrh.74, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64Rl vector, and a modified AAV.rh64Rl vector, an AAV
  • the lentiviral vector is an integrase-competent lentiviral vector (ICLV).
  • the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral- based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • Lentiviral vectors are well-known in the art (see, e.g., Trono D.
  • exemplary lentiviral vectors that may be used in any of the herein described compositions, systems, methods, and kits can include a human immunodeficiency virus (HIV) 1 vector, a modified human immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mangabey simian immunodeficiency virus (SIVSM) vector, a modified sooty mangabey simian immunodeficiency virus (SIVSM) vector, a African green monkey simian immunodeficiency virus (SIVAGM) vector, a modified African green monkey simian immunodeficiency virus (SIVAGM) vector, an equine infectious an HIV 1 vector
  • a modified human immunodeficiency virus (HIV) 1 vector a human immunodeficiency virus (HIV) 2 vector
  • AAV vectors comprising RNA- binding polypeptides capable of binding pre-mRNA sequences.
  • AAV vectors of the disclosure comprise Casl3d constructs capable of binding target RNA sequences disclosed herein.
  • murine USH2A exon 12 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 7.
  • the sequence encoding the vector of Table 7 comprises SEQ ID NO: 508.
  • murine USH2A exon 12 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 8.
  • the sequence encoding the vector of Table 8 comprises SEQ ID NO: 509.
  • Table 8 AAV Vector A01271 targeting murine USH2A exon 12
  • murine USH2A exon 12 RNA-targeting Casl3d vectors comprise from 5’ to 3 ’ a nucleic acid sequence comprising the elements set forth in Table 9.
  • the sequence encoding the vector of Table 9 comprises SEQ ID NO: 510.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’a nucleic acid sequence comprising the elements set forth in Table 10.
  • the sequence encoding the vector of Table 10 comprises SEQ ID NO: 511.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 11.
  • the sequence encoding the vector of Table 11 comprises SEQ ID NO: 512.
  • Table 11 AAV Vector A01293 targeting human USH2A exon 13 polyA signal SEQ ID NO: 457
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 12.
  • sequence encoding the vector of Table 12 comprises SEQ ID NO:
  • Table 12 AAV Vector A01296 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 13.
  • the sequence encoding the vector of Table 13 comprises SEQ ID NO: 514.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 14.
  • the sequence encoding the vector of Table 14 comprises SEQ ID NO: 490.
  • Table 14 AAV Vector A01361 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 15.
  • the sequence encoding the vector of Table 15 comprises SEQ ID NO: 491.
  • Table 15 AAV Vector A01362 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 16.
  • the sequence encoding the vector of Table 16 comprises SEQ ID NO: 492.
  • Table 16 AAV Vector A01363 targeting human USH2A exon 13 [0531]
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 17.
  • the sequence encoding the vector of Table 17 comprises SEQ ID NO: 493.
  • Table 17 AAV Vector A01364 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 18.
  • the sequence encoding the vector of Table 18 comprises SEQ ID NO: 494.
  • Table 18 AAV Vector A01365 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 19.
  • the sequence encoding the vector of Table 19 comprises SEQ ID NO: 495.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 20.
  • the sequence encoding the vector of Table 20 comprises SEQ ID NO: 496.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 21.
  • the sequence encoding the vector of Table 21 comprises SEQ ID NO: 497.
  • Table 21 AAV Vector A01368 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 22.
  • the sequence encoding the vector of Table 22 comprises SEQ ID NO: 498.
  • Table 22 AAV Vector A01369 targeting human USH2A exon 13 [0537]
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 23.
  • the sequence encoding the vector of Table 23 comprises SEQ ID NO: 499.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 24.
  • the sequence encoding the vector of Table 24 comprises SEQ ID NO: 500.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 25.
  • the sequence encoding the vector of Table 25 comprises SEQ ID NO: 501.
  • Table 25 AAV Vector A01372 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 26.
  • the sequence encoding the vector of Table 26 comprises SEQ ID NO: 502.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 27.
  • the sequence encoding the vector of Table 27 comprises SEQ ID NO: 503.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 28.
  • the sequence encoding the vector of Table 28 comprises SEQ ID NO: 504.
  • Table 28 AAV Vector A01375 targeting human USH2A exon 13
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 29.
  • the sequence encoding the vector of Table 29 comprises SEQ ID NO: 505.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 30.
  • the sequence encoding the vector of Table 30 comprises SEQ ID NO: 506.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 31.
  • the sequence encoding the vector of Table 31 comprises SEQ ID NO: 507.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 32.
  • the sequence encoding the vector of Table 32 comprises SEQ ID NO: 515.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 33.
  • the sequence encoding the vector of Table 33 comprises SEQ ID NO: 516.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 34.
  • the sequence encoding the vector of Table 34 comprises SEQ ID NO: 517.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 35.
  • the sequence encoding the vector of Table 35 comprises SEQ ID NO: 518.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 36.
  • the sequence encoding the vector of Table 36 comprises SEQ ID NO: 519.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 37.
  • the sequence encoding the vector of Table 37 comprises SEQ ID NO: 520.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 38.
  • the sequence encoding the vector of Table 38 comprises SEQ ID NO: 521.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 39.
  • the sequence encoding the vector of Table 39 comprises SEQ ID NO: 522.
  • human USH2A exon 13 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 40.
  • the sequence encoding the vector of Table 40 comprises SEQ ID NO: 523.
  • Table 40 AAV Vector A01501 targeting human USH2A exon 13
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 41.
  • the sequence encoding the vector of Table 41 comprises SEQ ID NO: 524.
  • Table 41 AAV Vector A01511 targeting human DMD exon 51
  • human DMD exon 45 RNA-targeting Cast 3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 42.
  • the sequence encoding the vector of Table 42 comprises SEQ ID NO: 525.
  • Table 42 AAV Vector A01512 targeting human DMD exon 45
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 43.
  • the sequence encoding the vector of Table 43 comprises SEQ ID NO: 526.
  • Table 43 AAV Vector A01513 targeting human DMD exon 51
  • human DMD exon 45 RNA-targeting Cast 3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table 44.
  • the sequence encoding the vector of Table 44 comprises SEQ ID NO: 527.
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AA.
  • the sequence encoding the vector of Table AA comprises SEQ ID NO: 12280.
  • Table AA AAV Vector A02636 targeting human DMD exon 51
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AB.
  • the sequence encoding the vector of Table AB comprises SEQ ID NO: 12281.
  • Table AB AAV Vector A02637 targeting human DMD exon 51
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AC.
  • the sequence encoding the vector of Table AC comprises SEQ ID NO: 12282.
  • Table AC AAV Vector A02638 targeting human DMD exon 51
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AD.
  • the sequence encoding the vector of Table AD comprises SEQ ID NO: 12283.
  • Table AD AAV Vector A02639 targeting human DMD exon 51
  • human DMD exon 51 RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AE.
  • the sequence encoding the vector of Table AE comprises SEQ ID NO: 12284.
  • Table AE AAV Vector A02640 targeting human DMD exon 51
  • human DMD RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AF.
  • the sequence encoding the vector of Table AF comprises SEQ ID NO: 12286.
  • Table AF AAV Vector A03278 targeting human DMD exon 51 exon splicing enhancers
  • human DMD RNA-targeting Cast 3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AG.
  • the sequence encoding the vector of Table AG comprises SEQ ID NO: 12310.
  • human DMD RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AH.
  • the sequence encoding the vector of Table AH comprises SEQ ID NO: 12313.
  • Table AH AAV Vector A03276 targeting human DMD exon 51 exon splicing enhancers
  • human DMD RNA-targeting Casl3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table Al.
  • the sequence encoding the vector of Table Al comprises SEQ ID NO: 12315.
  • human DMD RNA-targeting Cast 3d vectors comprise from 5’ to 3’ a nucleic acid sequence comprising the elements set forth in Table AJ.
  • the sequence encoding the vector of Table AJ comprises SEQ ID NO: 12316.
  • nucleic acid sequences encoding RNA-targeting Casl3d compositions of the disclosure are codon optimized nucleic acid sequences.
  • the codon optimized sequence encoding an RNA-targeting Casl3d composition exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased translation in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • a codon optimized nucleic acid sequence encoding an RNA- targeting Casl3d composition exhibits increased stability. In some aspects, a codon optimized nucleic acid sequence encoding an RNA-targeting Casl3d composition exhibits increased stability through increased resistance to hydrolysis. In some embodiments, the codon optimized sequence encoding an RNA-targeting Casl3d composition exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased stability relative to a wildtype or non-codon optimized nucleic acid sequence.
  • the codon optimized sequence encoding an RNA-targeting Casl3d composition exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased resistance to hydrolysis in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • a codon optimized nucleic acid sequence encoding an RNA- targeting Casl3d composition can comprise no donor splice sites.
  • a codon optimized nucleic acid sequence encoding an RNA-targeting dCasl3d protein can comprise no more than about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight, or about nine, or about ten donor splice sites.
  • a codon optimized nucleic acid sequence encoding an RNA-targeting Casl3d composition comprises at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer donor splice sites as compared to a non-codon optimized nucleic acid sequence encoding the RNA-targeting Cast 3d composition.
  • the removal of donor splice sites in the codon optimized nucleic acid sequence can unexpectedly and unpredictably increase expression of the RNA-targeting dCasl3d protein in vivo, as cryptic splicing is prevented.
  • cryptic splicing may vary between different subjects, meaning that the expression level of the RNA-targeting dCasl3d protein comprising donor splice sites may unpredictably vary between different subjects. Such unpredictability is unacceptable in the context of human therapy.
  • the codon optimized nucleic acid sequences which lacks donor splice sites, unexpectedly and surprisingly allows for increased expression of the RNA- targeting dCasl3d protein in human subjects and regularizes expression of the RNA-targeting dCasl3d protein across different human subjects.
  • a codon optimized nucleic acid sequence encoding an RNA- targeting Casl3d composition can have a GC content that differs from the GC content of the non-codon optimized nucleic acid sequence encoding the RNA-targeting Casl3d composition.
  • the GC content of a codon optimized nucleic acid sequence encoding a RNA-targeting Casl3d composition is more evenly distributed across the entire nucleic acid sequence, as compared to the non-codon optimized nucleic acid sequence encoding the RNA-targeting Casl3d protein.
  • the codon optimized nucleic acid sequence exhibits a more uniform melting temperature (“Tm”) across the length of the transcript.
  • Tm melting temperature
  • a codon optimized nucleic acid sequence encoding an RNA- targeting Casl3d composition can have fewer repressive microRNA target binding sites as compared to the non-codon optimized nucleic acid sequence encoding the RNA-targeting Casl3d composition.
  • a codon optimized nucleic acid sequence encoding an RNA-targeting Casl3d composition can have at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least ten fewer repressive microRNA target binding sites as compared to the non-codon optimized nucleic acid sequence the RNA-targeting Cast 3d composition.
  • the codon optimized nucleic acid sequence encoding an RNA-targeting Cast 3d composition unexpectedly exhibits increased expression in a human subject.
  • the composition comprises a sequence encoding a target RNA-binding fusion protein comprising
  • the composition comprises a sequence encoding a target RNA-binding fusion protein comprising (a) a sequence encoding a first RNA-binding polypeptide or portion thereof; and optionally (b) a sequence encoding a second RNA-binding polypeptide.
  • the first RNA-binding polypeptide binds a target pre-mRNA
  • the second RNA-binding polypeptide comprises a polypeptide capable of modulating RNA splicing.
  • the first RNA-binding polypeptide binds a target RNA
  • the second RNA-binding polypeptide comprises RNA- nuclease activity.
  • polypeptides capable of modulating RNA splicing include, but are not limited to, components of the spliceosome, RNA Binding Fox-1 Homolog 1 (RBFOX1), RNA Binding Motif Protein 38 (RMP38), heterogeneous nuclear ribonucleoprotein Al (hnRNPAl), and alternative splicing factor-1 (ASF1).
  • RBFOX1 RNA Binding Fox-1 Homolog 1
  • RMP38 RNA Binding Motif Protein 38
  • hnRNPAl heterogeneous nuclear ribonucleoprotein Al
  • ASF1 alternative splicing factor-1
  • a target RNA-binding fusion protein is an RNA-guided target RNA-binding fusion protein.
  • RNA-guided target RNA-binding fusion proteins comprise at least one RNA-binding polypeptide which corresponds to a gRNA which guides the RNA- binding polypeptide to target RNA.
  • RNA-guided target RNA-binding fusion proteins include without limitation, RNA-binding polypeptides which are CRISPR/Cas-based RNA-binding polypeptides or portions thereof.
  • a target RNA-binding protein of the disclosure comprises a signal sequence.
  • a target RNA-binding protein comprises one or more signal sequences.
  • the signal sequence(s) is a nuclear localization sequence (NLS), nuclear export signal (NES) or a combination thereof.
  • the tag sequence comprises a nuclear localization sequence (NLS).
  • the NLS sequence comprises a sequence listed in table 45.
  • the NLS signal sequence is a human NLS.
  • the human NLS signal sequence is a human pRB-NLS or a human pRB-NLS (extended version).
  • a nucleic acid sequence encoding an SV40 NLS is set forth in SEQ ID NO: 449. In some embodiments, a nucleic acid sequence encoding an SV40 NLS is set forth in SEQ ID NO: 460.
  • the signal sequence comprises one or more NES sequences.
  • the one or more NES sequence comprises a sequence listed in Table 46.
  • a target RNA-binding protein of the disclosure comprises a tag sequence.
  • the tag sequence is a FLAG tag.
  • the FLAG tag sequence is DYKDDDDK (SEQ ID NO: 366).
  • a target RNA-binding protein comprises a linker sequence.
  • the linker sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of amino acids in between. In some embodiments, the linker sequence comprises a linker sequence listed in Table 47.
  • a target RNA-binding protein is not an RNA-guided target RNA-binding protein and as such comprises at least one RNA-binding polypeptide which is capable of binding a target RNA without a corresponding gRNA sequence.
  • Such non-guided RNA-binding polypeptides include, without limitation, at least one RNA-binding protein or RNA-binding portion thereof which is a PUF (Pumilio and FBF homology family) protein. This type RNA-binding polypeptide can be used instead of a gRNA-guided RNA binding protein such as CRISPR/Cas.
  • the unique RNA recognition mode of PUF proteins (named for Drosophila Pumilio and C.
  • the PUF domain of human Pumilio 1 also known in the art, binds tightly to cognate RNA sequences and its specificity can be modified. It contains eight PUF modules that recognize eight consecutive RNA bases with each module recognizing a single base. Since two amino acid side chains in each module recognize the Watson-Crick edge of the corresponding base and determine the specificity of that module, a PUF protein can be designed to specifically bind most 8 to 16-nt RNA. Wang etal., Nat Methods. 2009; 6(11) : 825-830. See also WO2012/068627 which is incorporated by reference herein in its entirety.
  • PurnHD is a modified version of the WT Pumilio protein that exhibits programmable binding to arbitrary 8-base sequences of RNA.
  • Each of the eight units of PurnHD can bind to all four RNA bases, and the RNA bases flanking the target sequence do not affect binding. See also the following for art-recognized RNA-binding rules of PUF design: Filipovska A, Razif MF, Nygard KK, & Rackham O. A universal code for RNA recognition by PUF proteins.
  • human PUM1 (1186 amino acids) contains an RNA-binding domain (RBD) in the C-terminus of the protein (also known as Pumilio homology domain PUM-HD amino acid 828-amino acid 1175) and that PUFs are based on the RBD of human PUM1.
  • RBD RNA-binding domain
  • amino acids 12, 13, and 16 are important for RNA binding with 12 and 16 responsible for RNA base recognition.
  • Amino acid 13 stacks with RNA bases and can be modified to tune specificity and affinity.
  • the PUF design may maintain amino acid 13 as human PUMl’s native residue.
  • amino acid 13 for stacking
  • amino acid 13 will be engineered with an H and in other embodiments, will be engineered with a Y.
  • stacking residues may be modified to improve binding and specificity.
  • Recognition occurs in reverse orientation as N- to C-terminal PUF recognizes 3’ to 5’ RNA.
  • PUF engineering of 8 modules (8PUF) as known in the art, mimics a human protein.
  • An exemplary 8-mer RNA recognition (8PUF) would be designed as follows: R1’-R1-R2-R3-R4-R5-R6-R7-R8-R8’.
  • an 8PUF is used as the RBD.
  • a variation of the 8PUF design is used to create a 14-mer RNA recognition (14PUF) RBD, 15-mer RNA recognition (15PUF) RBD, or a 16- mer RNA recognition (16PUF) RBD.
  • the PUF can be engineered to comprise a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14- mer, 15-mer, 16-mer, 24-mer, 30-mer, 36-mer, or any number of modules between. Shinoda et al., 2018; Criscuolo et al., 2020. See also US Patent 9,580,714 which is incorporated herein in its entirety.
  • the fusion protein comprises at least one RNA-binding protein or RNA-binding portion thereof which is a PUMBY (Pumilio-based assembly) protein.
  • RNA-binding protein PumHD which has been widely used in native and modified form for targeting RNA, has been engineered into a protein architecture designed to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (i.e., Pumby, for Pumilio- based assembly) are concatenated in chains of varying composition and length, to bind desired target RNAs.
  • PUMBY is a more simple and modular form of PumHD, in which a single protein unit of PumHD is concatenated into arrays of arbitrary size and binding sequence specificity.
  • the specificity of such Pumby-RNA interactions is high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence.
  • the RNA binding protein comprises a Pumilio and FBF (PUF) protein. In some embodiments, the RNA binding protein comprises a Pumilio-based assembly (PUMBY) protein.
  • PAF Pumilio and FBF
  • PUMBY Pumilio-based assembly
  • RNA-binding proteins or RNA-binding portions thereof is a PPR protein.
  • PPR proteins proteins with pentatricopeptide repeat (PPR) motifs derived from plants
  • PPR proteins are nuclear- encoded and exclusively controlled at the RNA level organelles (chloroplasts and mitochondria), cutting, translation, splicing, RNA editing, genes specifically acting on RNA stability.
  • PPR proteins are typically a motif of 35 amino acids and have a structure in which a PPR motif is about 10 contiguous amino acids.
  • the combination of PPR motifs can be used for sequence-selective binding to RNA.
  • PPR proteins are often comprised of PPR motifs of about 10 repeat domains.
  • PPR domains or RNA-binding domains may be configured to be catalytically inactive. WO 2013/058404 incorporated herein by reference in its entirety.
  • the fusion protein disclosed herein comprises a linker between the at least two RNA-binding polypeptides.
  • the linker is a peptide linker.
  • the linker is VDTANGS (SEQ ID NO: 341).
  • the peptide linker comprises one or more repeats of the tri-peptide GGS. In other embodiments, the linker is a non-peptide linker.
  • the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co- poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • POE polyoxyethylene
  • polyurethane polyphosphazene
  • polysaccharides dextran
  • polyvinyl alcohol polyvinylpyrrolidones
  • polyvinyl ethyl ether polyacryl amide
  • polyacrylate polycyanoacrylates
  • lipid polymers chitins, hyaluronic acid
  • the at least one RNA-binding protein does not require multimerization for RNA-binding activity.
  • the at least one RNA- binding protein is not a monomer of a multimer complex.
  • a multimer protein complex does not comprise the RNA binding protein.
  • the at least one of RNA-binding protein selectively binds to a target sequence within the RNA molecule.
  • the at least one RNA-binding protein does not comprise an affinity for a second sequence within the RNA molecule.
  • the at least one RNA-binding protein does not comprise a high affinity for or selectively bind a second sequence within the RNA molecule.
  • the at least one RNA-binding protein comprises between 2 and 1300 amino acids, inclusive of the endpoints.
  • the at least one RNA-binding protein of the proteins disclosed herein further comprises a sequence encoding a nuclear localization signal (NLS).
  • a nuclear localization signal (NLS) is positioned at the N-terminus of the RNA binding protein.
  • the at least one RNA-binding protein comprises an NLS at a C-terminus of the protein.
  • the at least one RNA-binding protein further comprises a first sequence encoding a first NLS and a second sequence encoding a second NLS.
  • the first NLS or the second NLS is positioned at the N-terminus of the RNA-binding protein.
  • the at least one RNA-binding protein comprises the first NLS or the second NLS at a C-terminus of the protein. In some embodiments, the at least one RNA-binding protein further comprises an NES (nuclear export signal) or other peptide tag or secretory signal. In one embodiment, the tag is a FLAG tag.
  • the second RNA-binding polypeptide is operably configured to the first RNA-binding polypeptide at the C-terminus of the first RNA-binding polypeptide. In some embodiments, the second RNA-binding polypeptide is operably configured to the first RNA-binding polypeptide at the N-terminus of the first RNA-binding polypeptide [0596] In some embodiments, nucleic acid sequences encoding PUF or PUMBY proteins of the disclosure are codon optimized nucleic acid sequences.
  • the codon optimized sequence encoding a PUF or PUMBY protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased expression in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • nucleic acid sequences encoding PUF or PUMBY proteins of the disclosure are codon optimized nucleic acid sequences.
  • the codon optimized sequence encoding a PUF or PUMBY protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased translation in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein exhibits increased stability. In some aspects, a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein exhibits increased stability through increased resistance to hydrolysis. In some embodiments, the codon optimized sequence encoding a PUF or PUMBY protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased stability relative to a wild-type or non-codon optimized nucleic acid sequence.
  • the codon optimized sequence encoding a PUF or PUMBY protein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased resistance to hydrolysis in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein can comprise no donor splice sites. In some aspects, a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein can comprise no more than about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight, or about nine, or about ten donor splice sites.
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein comprises at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer donor splice sites as compared to a non-codon optimized nucleic acid sequence encoding the PUF or PUMBY protein.
  • the removal of donor splice sites in the codon optimized nucleic acid sequence can unexpectedly and unpredictably increase expression of the PUF or PUMBY protein in vivo, as cryptic splicing is prevented.
  • cryptic splicing may vary between different subjects, meaning that the expression level of the PUF or PUMBY protein comprising donor splice sites may unpredictably vary between different subjects. Such unpredictability is unacceptable in the context of human therapy.
  • the codon optimized nucleic acid sequences which lacks donor splice sites, unexpectedly and surprisingly allows for increased expression of the PUF or PUMBY protein in human subjects and regularizes expression of the PUF or PUMBY protein across different human subjects.
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein can have a GC content that differs from the GC content of the non-codon optimized nucleic acid sequence encoding the PUF or PUMBY protein.
  • the GC content of a codon optimized nucleic acid sequence encoding a PUF protein is more evenly distributed across the entire nucleic acid sequence, as compared to the non-codon optimized nucleic acid sequence encoding the PUF or PUMBY protein.
  • the codon optimized nucleic acid sequence exhibits a more uniform melting temperature (“Tm”) across the length of the transcript.
  • Tm melting temperature
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein can have fewer repressive microRNA target binding sites as compared to the non-codon optimized nucleic acid sequence encoding the PUF or PUMBY protein.
  • a codon optimized nucleic acid sequence encoding a PUF or PUMBY protein can have at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least ten fewer repressive microRNA target binding sites as compared to the non-codon optimized nucleic acid sequence the PUF protein.
  • the codon optimized nucleic acid sequence encoding a PUF or PUMBY protein unexpectedly exhibits increased expression in a human subject.
  • PUMBY proteins of the disclosure can target human MAPT. In some embodiments, PUMBY proteins of the disclosure can target and bind exon 10 of a human MAPT. In some embodiments, PUMBY proteins of the disclosure bind UACCAAAGGUGCAG (SEQ ID NO: 390) of exon 10 of a MAPT pre-mRNA. In some embodiments, a MAPT exon 10 targeting PUMBY, PUMBY HO, comprises the amino acid sequence:
  • a MAPT exon 10 targeting PUMBY, PUMBY HO comprises the amino acid sequence: MPKKKRKVEDPKKKRKVDVGRSRLLEDFRNNRYPNLQLREIAGHTEQLVQDQYGS YVIEHVLEHGRPEDKSKIVAEIRGHTEQLVQDQYGCYVIQHVLEHGRPEDKSKIVAEI RGHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGHTEQLVQDQYGSYVIEHVL EHGRPEDKSKIVAEIRGHTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRGHTEQL VQDQYGSYVIEHVLEHGRPEDKSKIVAEIRGHTEQL VQDQYGSYVIEHVLEHGRPEDKSKIVAEIRGHTEQL VQDQYGSYVIEHVLEHGRPEDKSKIVAEIRGHTEQL VQDQYGSYVIE
  • PUMBY proteins of the disclosure can target human MAPT. In some embodiments, PUMBY proteins of the disclosure can target and bind exon 10 of a human MAPT. In some embodiments, PUMBY proteins of the disclosure bind AUAAGAAGCUGGAU (SEQ ID NO: 391) of exon 10 of a MAPT pre-mRNA. In some embodiments, a MAPT exon 10 targeting PUMBY, PUMBY HQ, comprises the amino acid sequence:
  • PUMBY proteins of the disclosure can target human MAPT. In some embodiments, PUMBY proteins of the disclosure can target and bind exon 10 of a human MAPT. In some embodiments, PUMBY proteins of the disclosure bind CUGGAUCUUAGCAA (SEQ ID NO: 392) of exon 10 of a MAPT pre-mRNA. In some embodiments, a MAPT exon 10 targeting PUMBY, PUMBY HR, comprises the amino acid sequence:
  • PUMBY proteins of the disclosure can target human MAPT. In some embodiments, PUMBY proteins of the disclosure can target and bind exon 10 of a human MAPT. In some embodiments, PUMBY proteins of the disclosure bind CGUCCCGGGAGGCG (SEQ ID NO: 393) of exon 10 of a MAPT pre-mRNA. In some embodiments, a MAPT exon 10 targeting PUMBY, PUMBY HS, comprises the amino acid sequence:
  • PUMBY proteins of the disclosure can target human MAPT. In some embodiments, PUMBY proteins of the disclosure can target and bind exon 10 of a human MAPT. In some embodiments, PUMBY proteins of the disclosure bind GAGGCGGCAGUGUG (SEQ ID NO: 394) of exon 10 of a MAPT pre-mRNA. In some embodiments, a MAPT exon 10 targeting PUMBY, PUMBY HT, comprises the amino acid sequence:
  • a MAPT exon 10 targeting PUMBY, PUMBY HT comprises the amino acid sequence:
  • GVDLG (SEQ ID NO: 389).
  • nucleic acid sequences encoding the gene therapy compositions or RNA-targeting systems for use in gene transfer and expression techniques described herein, including modulating RNA splicing. It should be understood, although not always explicitly stated that the sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein.
  • They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions.
  • Specific polypeptide sequences are provided as examples of particular embodiments. Modifications to the sequences to amino acids with alternate amino acids that have similar charge.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand.
  • an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.
  • nucleic acid sequences e.g., polynucleotide sequences
  • exemplary Cas sequences such as e.g., a nucleic acid sequence encoding SEQ ID NO: 92 (Casl3d known as CasRx) or the nucleic acid sequence encoding SEQ ID NO: 298 (Casl3d known as CasRx), are codon optimized for expression in human cells. Codon optimization refers to the fact that different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type.
  • nucleic acid sequences coding for, e.g., a Cas protein can be generated.
  • such a sequence is optimized for expression in a host or target cell, such as a host cell used to express the Cas protein or a cell in which the disclosed methods are practiced (such as in a mammalian cell, e.g., a human cell).
  • Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a Cas protein (such as one encoding a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type protein) that takes advantage of the codon usage preferences of that particular species.
  • the Cas proteins disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest.
  • a Cas nucleic acid sequence is optimized for expression in human cells, such as one having at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to its corresponding wild-type or originating nucleic acid sequence.
  • an isolated nucleic acid molecule encoding at least one Cas protein (which can be part of a vector) includes at least one Cas protein coding sequence that is codon optimized for expression in a eukaryotic cell, or at least one Cas protein coding sequence codon optimized for expression in a human cell.
  • a codon optimized Cas coding sequence has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating sequence.
  • a eukaryotic cell codon optimized nucleic acid sequence encodes a Cas protein having at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating protein.
  • a variety of clones containing functionally equivalent nucleic acids may be routinely generated, such as nucleic acids which differ in sequence but which encode the same Cas protein sequence. Silent mutations in the coding sequence result from the degeneracy (i.e. , redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue.
  • leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3. sup. rd Edition, W.H. 5 Freeman and Co., NY).
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.
  • a cell of the disclosure is a prokaryotic cell.
  • a cell of the disclosure is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell.
  • the cell is a non-human mammalian cell such as a nonhuman primate cell.
  • a cell of the disclosure is a somatic cell. In some embodiments, a cell of the disclosure is a germline cell. In some embodiments, a germline cell of the disclosure is not a human cell. [0618] In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a stem cell. In some embodiments, a cell of the disclosure is an embryonic stem cell. In some embodiments, an embryonic stem cell of the disclosure is not a human cell. In some embodiments, a cell of the disclosure is a multipotent stem cell or a pluripotent stem cell. In some embodiments, a cell of the disclosure is an adult stem cell. In some embodiments, a cell of the disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, a cell of the disclosure is a hematopoietic stem cell (HSC).
  • iPSC induced pluripotent stem cell
  • HSC hematopoietic stem cell
  • a somatic cell of the disclosure is a neuronal cell.
  • a cell or cells of a patient treated with compositions disclosed herein include, without limitation, central nervous system (neurons), peripheral nervous system (neurons), peripheral motor neurons, and/or sensory neurons.
  • a neuronal cell is a glial cell.
  • a somatic cell of the disclosure is a muscle cell.
  • a muscle cell of the disclosure is a myoblast or a myocyte.
  • a muscle cell of the disclosure is a cardiac muscle cell, skeletal muscle cell or smooth muscle cell.
  • a muscle cell of the disclosure is a striated cell.
  • a cell or cells of a patient treated with compositions disclosed herein include, without limitation, skeletal muscle (developing and mature muscle fibers and satellite cells), neuromuscular junction, cardiomyocytes, smooth muscle cells, peripheral nervous system (neurons), peripheral motor neurons, and/or sensory neurons.
  • a somatic cell is an ocular cell.
  • An ocular cell includes, without limitation, comeal epithelial cells, keratyocytes, retinal pigment epithelial (RPE) cells, lens epithelial cells, iris pigment epithelial cells, conjunctival fibroblasts, non-pigmented ciliary epithelial cells, trabecular meshwork cells, ocular choroid fibroblasts, conjunctival epithelial cells,
  • an ocular cell is a retinal cell or a comeal cell.
  • a retinal cell is a photoreceptor cell or a retinal pigment epithelial cell.
  • a retinal cell is a ganglion cell, an amacrine cell, a bipolar cell, a horizontal cell, a Muller glial cell, a rod cell, or a cone cell.
  • a somatic cell of the disclosure is a fibroblast or an epithelial cell.
  • an epithelial cell of the disclosure forms a squamous cell epithelium, a cuboidal cell epithelium, a columnar cell epithelium, a stratified cell epithelium, a pseudostratified columnar cell epithelium or a transitional cell epithelium.
  • an epithelial cell of the disclosure forms a gland including, but not limited to, a pineal gland, a thymus gland, a pituitary gland, a thyroid gland, an adrenal gland, an apocrine gland, a holocrine gland, a merocrine gland, a serous gland, a mucous gland and a sebaceous gland.
  • an epithelial cell of the disclosure contacts an outer surface of an organ including, but not limited to, a lung, a spleen, a stomach, a pancreas, a bladder, an intestine, a kidney, a gallbladder, a liver, a larynx or a pharynx.
  • an epithelial cell of the disclosure contacts an outer surface of a blood vessel or a vein.
  • a somatic cell of the disclosure is a primary cell.
  • a somatic cell of the disclosure is a cultured cell.
  • a somatic cell of the disclosure is in vivo, in vitro, ex vivo or in situ.
  • a somatic cell of the disclosure is autologous or allogeneic.
  • the disclosure provides a method of modulating pre-mRNA splicing in a subject.
  • the disclosure further provides a method of modulating pre-mRNA splicing comprising administering an RNA-binding composition comprising a nucleic acid sequence encoding a polypeptide, polynucleotide, or nucleoprotein complex capable of binding a target pre-mRNA sequence.
  • the disclosure further provides a method of modulating pre-mRNA splicing comprising administering at least one RNA-binding composition comprising a nucleic acid sequence encoding a polypeptide, polynucleotide, or nucleoprotein complex capable of binding a target pre-mRNA sequence.
  • the method comprises at least 2, at least 3, at least, 4, at least 5, at least 6, at least 7, at least 8, at least 8, or at least 10 RNA binding compositions, wherein each RNA binding composition targets a different pre-mRNA sequence.
  • the disclosure further provides a method of modulating pre-mRNA splicing comprising administering a composition comprising a nucleic acid sequence encoding (a) an RNA-binding polypeptide or portion thereof, and (b) one or more cognate nucleic acid guide RNAs, wherein each of the one or more cognate nucleic acid guide RNAs are capable of binding a target pre-mRNA sequence.
  • the disclosure further provides a method of modulating pre-mRNA splicing comprising administering a composition comprising a nucleic acid sequence encoding a nonguided RNA-binding polypeptide or portion thereof comprising a PUF or PUMBY protein capable of binding a target pre-mRNA sequence.
  • the disclosure provides a method of modifying the level of expression of a gene of the disclosure under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or RNA-binding fusion protein (or a portion thereof) to the pre-mRNA molecule.
  • the RNA-binding composition binds an intronic or exonic sequence of the pre-mRNA.
  • the intronic or exonic sequence comprises an an exon splicing enhancer (ESE) motif, an intronic splicing enhancer (ISE) motif, a 5’ splice site, a 3’ splice site, a branchpoint adenosine (BP A), or a polypyrimidine tract (PPY).
  • ESE exon splicing enhancer
  • ISE intronic splicing enhancer
  • BP A branchpoint adenosine
  • PPY polypyrimidine tract
  • the 5’ splice site is a splice donor site.
  • the 3’ splice site is a splice acceptor site.
  • the RNA-binding composition when bound to the pre-mRNA sequence prevents inclusion of the bound sequence into an mRNA sequence.
  • exonic sequences bound by RNA- binding compositions of the disclosure can be skipped.
  • a skipped exonic sequence is one that is not incorporated into the mature mRNA.
  • the RNA-binding composition when bound to the pre-mRNA sequence prevents inclusion of the bound sequence into an mRNA sequence. In some aspects, this results in a truncation of the mature mRNA sequence. In some aspects, the truncated mature mRNA sequence is either not-expressed or expressed at a diminished level.
  • the intronic or exonic sequence bound by the RNA-binding composition comprises an exon splicing silencer (ESS) motif or an intron splicing silencer (ISS) motif.
  • ESS exon splicing silencer
  • ISS intron splicing silencer
  • mutated genes and mRNA sequences of the disclosure yield an expressed protein with no activity or diminished activity.
  • the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or the fusion protein (or a portion thereof) to the RNA molecule.
  • the mature mRNA lacking a skipped exon of the disclosure when expressed, produced a protein with increased activity.
  • the protein activity is increased 1- fold or greater.
  • the level of activity is increased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10- fold. In another embodiment, the level of activity is increased 10-fold or greater. In another embodiment, the level of activity is increased between 10-fold and 20-fold. In another embodiment, the level of activity is increased 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16- fold, 17-fold, 18-fold, 19-fold, or 20-fold.
  • mutated genes and mRNA sequences of the disclosure lead to no expression, very little expression, or reduced expression of a protein encoded by the mutated gene or mRNA sequence.
  • the disclosure provides a method of modifying the expression of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or the fusion protein (or a portion thereof) to the RNA molecule.
  • the mature mRNA lacking a skipped exon of the disclosure when expressed, leads to increased expression of the protein encoded by the mRNA.
  • the level of protein expression is increased 1- fold or greater.
  • the level of protein expression is increased 2-fold, 3 -fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold. In another embodiment, the level of protein expression is increased 10-fold or greater. In another embodiment, the level of protein expression is increased between 10-fold and 20-fold. In another embodiment, the level of protein expression is increased 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.
  • mutated genes and mRNA sequences of the disclosure yield an expressed protein with increased activity or hyperactivity.
  • the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or the fusion protein (or a portion thereof) to the RNA molecule.
  • the mature mRNA lacking a skipped exon of the disclosure when expressed, produced a protein with decreased activity.
  • the protein activity is decreased 1- fold or greater.
  • the level of activity is decreased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10- fold. In another embodiment, the level of activity is decreased 10-fold or greater. In another embodiment, the level of activity is decreased between 10-fold and 20-fold. In another embodiment, the level of activity is decreased 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16- fold, 17-fold, 18-fold, 19-fold, or 20-fold.
  • mutated genes and mRNA sequences of the disclosure lead to increased expression or overexpression of a protein encoded by the mutated gene or mRNA sequence.
  • the disclosure provides a method of modifying the expression of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or the fusion protein (or a portion thereof) to the RNA molecule.
  • the mature mRNA lacking a skipped exon of the disclosure when expressed, leads to decreased expression of the protein encoded by the mRNA.
  • the level of protein expression is decreased 1- fold or greater.
  • the level of protein expression is decreased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold. In another embodiment, the level of protein expression is decreased 10-fold or greater. In another embodiment, the level of protein expression is decreased between 10-fold and 20-fold. In another embodiment, the level of protein expression is decreased 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.
  • the disclosure provides a method of modifying level of expression of a gene or an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting the composition of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule.
  • the cell is in vivo, in vitro, ex vivo or in situ.
  • the composition of the disclosure comprises a vector comprising a guide RNA of the disclosure and an RNA-binding protein or fusion protein of the disclosure.
  • the vector is an AAV.
  • the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule.
  • the cell is in vivo, in vitro, ex vivo or in situ.
  • the composition comprises a vector comprising composition comprising a guide RNA of the disclosure and an RNA-binding fusion protein of the disclosure.
  • the vector is an AAV.
  • the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the disclosure.
  • the disclosure provides a method of treating monogenic diseases or disorders.
  • Monogenic diseases or disorders are diseases caused by variation in a single gene. In some cases, the variation is at least one mutation to the gene. In some cases, the monogenic disease or disorder arises from more than one mutation in the gene.
  • Methods of the disclosure can be configured to modulate splicing of any gene in the genome of a subject.
  • the gene is a protein-expressing gene.
  • the gene is USH2A, DMD, SOD1, LRRK2, MAPT, CEP290, SCN1A, SMN1, SMN2, and HTT.
  • the monogenic disease or disorder is associated with USH2A.
  • the USH2A-associated monogenic disease or disorder is Usher Syndrome type II or Retinitis Pigmentosa 39.
  • the monogenic disease or disorder is associated with DMD.
  • the DMD-associated monogenic disease or disorder is Duchenne Muscular Dystrophy or Becker Muscular Dystrophy.
  • Duchenne s muscular dystrophy is caused by mutations in the DMD gene.
  • DMD is the largest gene in the human genome having 79 exons separated by introns that are up to 250kb.
  • DMD encodes dystrophin protein, which stabilized the plasma membrane in striated muscle. Exon skipping to restore frame (caused by exon 50 deletion frameshift) reduces the severity of DMD to that exhibited by Becker Muscular Dystrophy patients. See FIG. 20.
  • Duchenne Muscular Dystrophy is caused by large frame-shifting deletions in the DMD gene, leading to insufficient levels of dystrophin protein. Common disease-causing mutations include whole exon 50 or exon 52 deletions.
  • dCas!3d with mutated HEPN domains can be programmed to bind exon splicing enhancers (ESEs) and/or other splicing regulatory domains in DMD pre-mRNAs to prevent inclusion of exon 51 during normal pre- mRNA splicing.
  • ESEs exon splicing enhancers
  • Cast 3d technology permits expression of multiple guides expressed as a tandem array, which may increase the efficiency and/or specificity of single exon 51 skipping and enable multi-exon skipping, which could be applied to -65% of DMD patients. See FIG. 21.
  • monogenic disease or disorder is associated with SOD1.
  • the SOD 1 -associated monogenic disease or disorder is amyotrophic lateral sclerosis (ALS).
  • monogenic disease or disorder is associated with LRRK2.
  • the LRRK2-associated monogenic disease or disorder is Parkinson’s disease or Crohn’s disease.
  • monogenic disease or disorder is associated with MAPT.
  • the MAPT-associated monogenic disease or disorder is Alzheimer’s disease.
  • the monogenic disease or disorder is associated with SMN1 or SMN2.
  • the SMN2 or SMN1 -associated monogenic disease or disorder is spinal muscular atrophy (SMA).
  • a subject of the disclosure is female. In some embodiments of the methods of the disclosure, a subject of the disclosure is male. In some embodiments, a subject of the disclosure has two XX or XY chromosomes. In some embodiments, a subject of the disclosure has two XX or XY chromosomes and a third chromosome, either an X or a Y.
  • a subject of the disclosure is a neonate, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the disclosure, a subject of the disclosure is 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 31 days old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months old.
  • a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of years or partial years in between of age.
  • a subject of the disclosure is a mammal. In some embodiments, a subject of the disclosure is a non-human mammal.
  • a subject of the disclosure is a human.
  • a therapeutically effective amount comprises a single dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises at least one dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises one or more dose(s) of a composition of the disclosure.
  • a therapeutically effective amount eliminates a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount reduces a severity of a sign or symptom of the disease or disorder.
  • a therapeutically effective amount eliminates the disease or disorder.
  • a therapeutically effective amount prevents an onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount improves a prognosis for the subject.
  • a composition of the disclosure is administered to the subject via intracerebral administration. In some embodiments, the composition of the disclosure is administered to the subject by an intrastriatal route. In some embodiments, the composition of the disclosure is administered to the subject by a stereotaxic injection or an infusion. In some embodiments, the composition is administered to the brain. In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject locally.
  • compositions disclosed herein are formulated as pharmaceutical compositions.
  • pharmaceutical compositions for use as disclosed herein may comprise a protein(s) or a polynucleotide encoding the protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose
  • compositions of the disclosure may be formulated for routes of administration, such as e.g., oral, enteral, topical, transdermal, intranasal, and/or inhalation; and for routes of administration via injection or infusion such as, e.g., intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and/or parenteral administration.
  • the compositions of the present disclosure are formulated for intracerebral or intrastriatal administration.
  • FIG. 5 A depicts the activity of each dCasl3d as measured by their relative ability to cleave Cast 3d.
  • FIG. 5B depicts the nuclease activity of active Cast 3d variants that do not bear the deactivating mutations.
  • HEK293 cells were seeded at 20K cells/well in 96-well plates. 100 ng of Casl3d variant, 100 ng of guide RNA and 10 ng of pLenti-GFP were transfected. Each condition was plated in 4 replicates. A plate reader was used to measure GFP at a wavelength of 479 nm. Values were background subtracted (the average of 4 media-only wells) before being normalized to non-targeting (NT). Error bars indicate standard deviation.
  • Example 2 Exon 13 skipping of human USH2A [0664] Patients with USH2A syndrome experience deafness and retinal degeneration. A single base-pair deletion, c.2299 delG, in exon 13 is the most common mutation in USH2A gene, resulting in a truncated and non-functional Usherin protein. There are an estimated 2,000 - 4,000 patients in the United States with the c.2299 delG mutation and approximately 16,000 patients with Exon 13 mutations. Usherin localizes to connecting cilia of mammalian photoreceptors and its absence leads to a ciliary defect and cell death.
  • CRISPR Cas RNA guides targeting intronic sequences flanking exon 13 and exon 13 of USH2A pre-mRNA were designed and detailed in Table 49 and depicted in FIG. 3 A.
  • HEK293T cells were transfected with 500 ng of guideexpressing plasmid and 500 ng of dCasRx-expressing plasmid and 50 ng of human USH2A minigene. 48 hours after transfection, RNA was harvested, cDNA was synthesized, 25 cycles of PCR was performed. 500 ng of RNA was subjected to reverse transcription using qScript cDNA synthesis kit.
  • 1 pl of cDNA was used in a 25 cycle, 25 pl GoTaq PCR reaction with oligos annealing to exons 12 and 14 with annealing at 55C and 1 minute extension.
  • the PCR product was run on a DI 000 tape (Agilent).
  • the fraction excluded depicted in FIG. 3C and correlating to the amount of exon 13 excluded (i.e. skipped) in the resulting USH2A transcripts, is calculated by dividing the intensity of the peak by the sum of the intensities of from the band representing exon 13 included and the band representing exon 13 excluded.
  • FIG. 3D is a bar graph depicting dose-dependent exon 13 skipping upon delivery of USH2A pre-mRNA targeting dCasl3d complexes with guides g7, g9 and glO targeting human USHA2A exon 13, showing dose dependent Exon 13 skipping.
  • FIG. 3E is a series of tape station images depicting exclusion of exon 13 of human USH2A using vectors expressing guide RNA, g7, g9, glO, or a non-targeting guide RNA. Unitary plasmids were transfected at varying doses and RNA was harvested at 48 hr.
  • FIG. 6B is a gel depicting that AAV vector constructs expressing dSeq212 and guides g7, g9 or glO, targeting the ESE of exon 13, were co-transfected with the human USH2A minigene. Guides g7, g9, and glO induce robust exon 13 skipping.
  • Example 3 Impact of inactivated Casl3 nuclease variants on USH2A exon 13 skipping
  • the impact of single point mutant Casl3d orthologs on exon skipping was evaluated.
  • the casl3d orthologs, dSeq42, dSeql79, dSeql89, dSeql98, dSeq212, and dSeq208, were transfected with Cas 13 d-relevant guide g7 (from Table 49) and the degree of USH2A exon 13 skipping was measured (FIGs 4A and 4B).
  • RNA 500 ng was used in a 10 pL cDNA reaction. 1 pL of cDNA was used in a 25 pL GoTaq PCR with exon 12 fwd and exon 14 rev primers, with annealing at 55C, 1 minute extension, and 25 cycles. DNA was run on a DI 000 tape.
  • FIG. 6A is an alignment of human USH2A exon 13 and mouse USH2A exon 12. Accordingly, the approach to skipping of exon 12 of USH2A in mice is analogous to the approach to skipping human USH2A exon 13.
  • FIG. 7B is a tape station image depicting the effect of mouse USH2A pre-mRNA targeting dCasRX complexes on USH2A minigene splicing, wherein the various complexes each bear mgl and an additional guide: mg4-mg9. Combination of guides mgl and mg7 targeting exon 12 ESE and the 5’ end of exon 12, respectively, induced robust exon 12 skipping.
  • FIG. 8A is a gel image demonstrating the robust skipping achieved by combining guides.
  • FIG. 8B is a series of microscope images detecting splice junctions of mouse USH2A minigene in cosm6 cells transfected with 250 ng of guides mgl and mg7, 500 ng dCasRx and 50 ng mouse minigene in 12 well format. Cells were trypsinized and seeded in chamber slides 24 hr before fixation with PF A.
  • Example 6 Development of AAV vectors for delivering exon skipping dCasl3d complexes
  • AAV vectors were developed for delivery of USH2A exon skipping complexes to both mice and human.
  • mice For mice, an exemplary AAV vector construct encoding mgl and mg7 guide RNAs under the control of a human U6 (hU6) promoter and a dCasl3d-NLS (dCasl3d tethered to a nuclear localization signal) under the control of an EFS promoter for the targeting of exon 12 of mouse USH2A is depicted in FIG. 9A.
  • hU6 human U6
  • dCasl3d-NLS dCasl3d tethered to a nuclear localization signal
  • an exemplary AAV vector construct encoding a g7 guide RNA under the control of a human U6 (hU6) promoter and a dCasl3d-NLS (dCasl3d tethered to a nuclear localization signal) under the control of a cone/rod specific promoter for the targeting of exon 13 of human USH2A is depicted in FIG. 9B.
  • mice USH2A exon 12 skipping was assessed by transfecting cells with AAV vectors at doses of 1 pg, 500 ng, 250 ng, or 100 ng.
  • AAV vectors expressing dual guide RNAs, mgl and mg7 were assessed.
  • Vector A01270 SEQ ID NO: 598 containing dSeq212 under an EFS promoter and guides mgl and mg7 each controlled by their own hU6 promoter.
  • Vector A01271 SEQ ID NO: 509 containing dSeq212 under an EFS-UBB promoter and guides mgl and mg7 each controlled by their own hU6 promoter.
  • Vector A01285 (SEQ ID NO: 510) containing dSeq212 under an EFS-UBB promoter and guides mgl and mg7 under the control of a single hU6 promoter.
  • FIG. 10 depicts splicing modulation of exon 12 of mouse USH2A .
  • Unitary plasmids were transfected at varying doses and RNA was harvested at 48 hr.
  • Cells were transfected with 1 pg, 500 ng, 250 ng, 100 ng of AAV vector (total DNA transfected: 1 pg per condition) and 50 ng mouse minigene for all conditions.
  • Example 8 Exon skipping in DMD for treating Duchenne Muscular Dystrophies
  • Duchenne muscular dystrophy is caused by mutations in the DMD gene DMD is the largest gene in human genome; 79 exons separated by introns that are up to 250 kb. DMD encodes dystrophin protein, which stabilizes the plasma membrane in striated muscle. A hotspot of mutations is found between exons 43 and 55. Distinct mutations in DMD lead to different types of Duchenne Muscular Dystrophy as detailed in Table 51.
  • Exon 51 skipping was evaluated using dCasRx and dSeql 89 with guides from table 52.
  • Guide g5 (Table 52) induced robust exon 51 skipping of a minigene comprising exogenous DMD exons 50, 51, and 52 (FIG. 11B).
  • Example 9 Multiple guides to target multiple exon skipping in DMD
  • Guide 45 sequence TGCCGCTGCCCAATGCCATCCTGGAG (SEQ ID NO: 436).
  • Guide 51 sequence CCTCTGTGATTTTATAACTTGATCAA (SEQ ID NO: 437).
  • Example 10 Exon 10 skipping in human MAPT minigene using dCasRx
  • MAPT which is the gene encoding the tau protein
  • tauopathies including but not limited to Alzheimer's disease, progressive supranuclear palsy, corticobasal syndrome, some frontotemporal dementias, and chronic traumatic encephalopathy. Skipping exons bearing tauopathy-associated mutations offers a means to restoring functional levels of tau in subjects.
  • CRISPR Cas RNA guides targeting exon 10 of MAPT pre-mRNA were designed and detailed in Table 54.
  • MAPT minigene JO expressing exon 9, 10 and 11 of MAPT separated by truncated introns were used to evaluate exon skipping using dCasRx with guides gl-g3. g3 induced robust exon 10 skipping using MAPT minigene (FIG. 14). MAPT minigene JP was also used to show exon 10 skipping.
  • Example 11 Exon 10 skipping in human MAPT minigene using PUMBYs
  • PUMBYs were developed and evaluated for their ability to induce exon 10 skipping in MAPT JO minigene (FIG. 15).
  • the gel image depicts a 197 bp band corresponding to a transcript comprising exon 10.
  • a second 104 bp band is also depicted corresponding to the absence of exon 10, indicative of exon 10 skipping. All PUMBYs evaluated induced at least partial exon 10 skipping, with PUMBY HR and HS producing the most robust exon 10 skipping.
  • Example 12 Splicing mediated knockdown of SOD1 and LRRK2
  • FIG. 16A is a schematic depicting a SOD1 cDNA comprising exons 1-5.
  • FIG. 17 is a schematic depicting an LRRK3 cDNA comprising exons 1-4.
  • CRISPR Cas RNA guides targeting exons 3 and 2 of SOD1 pre-mRNA were designed and detailed in Table 55.
  • FIG. 16C The impact of guides gl-g4, targeting exons 2 and 3 of endogenous SOD1, complexed with dCasRx is shown in FIG. 16C.
  • the gels depict a 224 bp band corresponding to a transcript comprising exons 1-4.
  • a second 154 bp band is also depicted corresponding to the absence of exon 3, indicative of exon 3 skipping.
  • a third 127 bp band is also depicted corresponding to the absence of exon 2, indicative of exon 2 skipping.
  • a fourth 57 bp band is also depicted corresponding to the absence of exons 2 and 3, indicative of exon 2 and 3 skipping. Skipping of exons 2 and/or 3 is will introduce a premature termination codon that is predicted to lead to nonsense-mediated mRNA decay (NMD).
  • NMD nonsense-mediated mRNA decay
  • Example 13 AAV8-dCasl3d tandem guide vector drives robust Cas 13d expression and exon 12 skipping in mouse photoreceptors
  • AAV5, 8, 9 or RhlO vectors encoding GFP were delivered by subretinal injection to wt BALB/C mice at le9 vg/eye (FIG. 18A). Histology was performed 4-weeks post injection using 12 pm retinal cryosections. Native EGFP expression was compared across serotypes to assess spread and photoreceptor targeting. Sub-retinal delivery of AAV serotypes identified AAV8 as the most effective for rod/cone photoreceptor transduction and vector spread (FIG. 18B).
  • AAV8-U6-mgl/mg7-dCAsl3d vector was manufactured in-house and delivered by sub retinal injection to wt BALB/C mice at le9 vg/eye (FIG. 18C). Histology was performed 4-weeks post injection.
  • dCas!3d mRNA expression was assessed by RNAscope probes specific for Ush2a (FIG. 18D).
  • Ush2a splicing was assessed by duplex BaseScope with probes for spliced (Exonll/13) and intact (Exonl l/12) mRNA (FIG. 18D - FIG. 18E).
  • Example 14 Sub retinal delivery of AAV8-dCAS13d+mgl/mg7 in mouse retina shows preservation of retinal structure and minimal T-cell infiltration
  • AAV8-U6-mgl/mg7-dCAsl3d vector was manufactured in-house and delivered by sub-retinal injection to wt BALB/C mice at le9 vg/eye (FIG. 19A). Immunohistology was performed 4-weeks post injection on 12 pm retinal cryosections. Retina were imaged using immunofluorescence microscopy (FIG. 19B). Cone photoreceptors were immunolabeled with the anti-cone opsin Antibody (Ab). Rod photoreceptors were immunolabeled with the antirhodopsin Ab. T Cells were immunolabeled using the anti-CD3 Ab.
  • Example 15 Identification and Use of Multi-targeting guides for efficient and specific DMD exon 51 skipping
  • Guide screen identifies targeting regions that result in exon 51 skipping:
  • dSeq212 (4aa mutation) programmed with g5, gl7, g38, g42 result in reproducible exon 51 skipping. Multiple exon 51 targeting regions (enriched with predicted SRSF binding sites) can be targeted by dSeq212 to result in efficient exon 51 skipping. Experiment performed in duplicate, exon 49 fwd, exon 53 reverse, 35x cycles, single PCR optimization. See FIGs 22A-22C. Combining guides that target different ESEs leads to enhanced exon 51 skipping:
  • Multi -targeting guide pair combinations utilizing guide numbers 5, 17, 38, and 42 were selected: g5/gl7, g5/g42, g38/gl7, g38/g42 and compared to single guide targeting. See FIG. 23 A. Lower levels of exon skipping were observed with single guides. Total guide transfected with 500ng (single targeting 500ng; dual targeting 250ng of each guide), single PCR was performed with exon 49 fwd and exon 53 reverse for 35 cycles at an optimal temperature of 58 degrees Celsius. Guide combos g5/gl7, g5/g42, g38/gl7, g38/g42 which target different ESEs in exon 51 lead to enhanced exon 51 skipping. See FIG. 23B and 23C. Guide combos for DMD-targeting systems:
  • Multi-targeting guide pair combinations such as g5/gl7, g5/g42, g38/gl7, g38/g42 and dSEQ212 (4aa mutation) systems are delivered to achieve higher levels of exon 51 skipping.
  • Example 16 Multi-exon skipping (exons 45 through 55) are used to treat 65% of DMD patients
  • Example 17 Multiple guide targeting of exon 51 of DMD leads to restoration of dystrophin expression
  • exon 52 from DMD leads to no dystrophin protein expression. Restoring dystrophin expression can lead to the improvement of symptoms or treatment of Duchenne muscular dystrophy. Without wishing to be bound by theory, skipping exon 51 of the exon 52 deletion DMD gene can restore dystrophin protein expression levels.
  • AAV vector A03278 comprising dCas!3d dSeq212 having 4aa mutation is programmed to bind two exon splicing enhancers in exon 51 of human DMD. The 2 guides are expressed from a tandem array, under the hU6 promoter.
  • Myotubes having DMD lacking exon 52 were transduced with A03278 for 7 days before harvest and RNA extraction (for cDNA synthesis and semi-quantitative PCR) and cell fixation (for IF).
  • FIG. 25A demonstrates that treatment of myotubes with the A03278 vector results in efficient exon 51 skipping. As measured by semi-quantitative PCR, 60% exon 51 skipping is observed relative to a control sample (FIG. 25B).
  • DMD lacking exon 52 leads to no dystrophin expression (FIG. 25C).
  • exon 51 skipping Upon treatment with the vector, thereby inducing exon 51 skipping, dystrophin protein expression is rescued.
  • Example 18 dCasl3d programmed to target an intronic splicing silencer in intron 7 of SMN2 leads to increased inclusion of exon 7
  • Treatment for SMA includes the restoration of protein expression from SMN2 when SMN1 is dysfunctional.
  • Exon 7 of SMN2 is normally excluded during pre-mRNA splicing due to a SNP in an exon splicing enhancer.
  • Engineering the inclusion of exon 7 will restore expression from SMN2.
  • dCas!3d constructs comprising guides targeting to promote exon inclusion we designed guides that would bind and block an intronic splicing silencer in intron 7 of SMN2 (Table B and FIG. 26 A) and tested them on a mini gene of SMN2 (FIG. 26B).
  • (Semi quantitative PCR was performed and products run on a DI 000 tape (FIG. 26C).
  • the bottom band represents exon 7 excluded and the top band represents exon 7 included.
  • the amount of exon 7 inclusion was quantified by PCR (FIG. 26D) and demonstrated that that guides 1, 2 and 3 result in an increase of exon 7 inclusion.

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Abstract

L'invention concerne des compositions pouvant cibler des séquences de pré-ARNm introniques et exoniques qui modulent l'épissage d'ARNm en excluant ou incluant la séquence de pré-ARNm liée dans l'ARNm mature.
PCT/US2023/062301 2022-02-09 2023-02-09 Compositions et procédés de modulation d'épissage de pré-arnm WO2023154807A2 (fr)

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