Compositions targeting au-rich elements of FGF21 gene and GDF15 gene and methods of use thereof

Abstract

This disclosure generally relates to methods and compositions for functional genetic modifications at selected genomic sites, such as the FGF21 gene or the GDF15 gene. This disclosure further provides compositions and methods for genomic editing of AU-rich elements in the FGF21 and / or GDF15 genes for treating obesity-related diseases and disorders.

Description

COMPOSITIONS TARGETING AU-RICH ELEMENTS OF FGF21 GENE AND GDF15 GENE AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of United States Provisional Patent Application No. 63 / 716,884, filed November 6, 2024, the contents of which are herein incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] The disclosure is directed to the field of genetic editing and genomic engineering. More particularly, the present disclosure is directed to compositions and methods for targeted genetic modification and modulating expression of a target nucleic acid sequence and applications thereof.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 5, 2025, is named “000218-0155-W01-SL.xml,” is 63,347 bytes in size.BACKGROUND

[0004] Genome editing refers to strategies and techniques for the targeted, specific modification of the genetic information (genome) of living organisms. Genome engineering is an active field of research because of the wide range of possible applications, particularly in the area of human health, e.g., to correct a gene carrying a harmful mutation or to explore the function of a gene. Early technologies developed to insert a transgene into a living cell were often limited by the random nature of the insertion location of the new sequence into the genome. Common genome editing strategies allow a specific area of the DNA to be modified, thereby increasing the precision of the correction or insertion compared to earlier technologies. While these platforms offer a greater degree of reproducibility and decreased levels of unintended effects from random insertions and deletions in the genome, limitations remain.SUMMARY

[0005] This disclosure provides compositions and methods for genomic editing of AU-rich elements in the FGF21 and / or GDF15 genes for treating obesity-related diseases and disorders.

[0006] In one aspect, the present disclosure provides compositions comprising a) a first guide RNA (gRNA) comprising a first targeting sequence set forth in SEQ ID NOs: 1-3; b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or nuclease domain thereof; and d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or nuclease domain thereof.

[0007] In another aspect, the present disclosure provides compositions a) a first guide RNA (gRNA) comprising a first targeting sequence set forth in SEQ ID NOs: 6-10; b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NOs: 11-18; c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or nuclease domain thereof; and d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or nuclease domain thereof.

[0008] In one aspect, the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, and / or the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25. In another aspect, the polynucleotide encoding the first fusion protein is an mRNA comprising the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 26, and / or the polynucleotide encoding the first fusion protein is an mRNA comprising the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 26. In one aspect, the mRNA comprises a 5’-cap.

[0009] In one aspect, the C-terminus of the first fusion protein further comprises a linker comprising the sequence set forth in SEQ ID NO: 21 between the first inactivated Cas9 domain, or nuclease domain thereof and the first Clo051 domain, or nuclease domain thereof, and / or the C-terminus of the second fusion protein further comprises a linker comprising the sequence set forth in SEQ ID NO: 21 between the second inactivated Cas9 domain, or nuclease domain thereof and the second Clo051 domain, or nuclease domain thereof.

[0010] In one aspect, the first inactivated Cas9 domain, or nuclease domain thereof is derived from a Streptococcus pyogenes Cas9 polypeptide, and / or the second inactivated Cas9 domain, or nuclease domain thereof is derived from a Streptococcus pyogenes Cas9 polypeptide.In one aspect, the first gRNA comprises a spacer sequence and a scaffold sequence isolated from Streptococcus pyogenes, and / or the second gRNA comprises a spacer sequence and a scaffold sequence isolated from Streptococcus pyogenes. In one aspect, the scaffold sequence comprises the nucleic acid sequence of SEQ ID NO: 35.

[0011] In one aspect, the first gRNA comprises one or more chemical modifications of a ribonucleotide, a ribonucleotide base, or a phosphodiester bond, and / or the second gRNA comprises one or more chemical modifications of a ribonucleotide, a ribonucleotide base, or a phosphodiester bond. In one aspect, the at least one chemically modified phosphodiester bond is a phosphorothioate bond.

[0012] In one aspect, the 5’ and / or 3’ terminus of the first gRNA comprises at least two consecutive phosphorothioate bonds, and / or the 5’ and / or 3’ terminus of the second gRNA comprises at least two consecutive phosphorothioate bonds. In another aspect, the 5’ and / or 3’ terminus of the first gRNA comprises at least one 2’ O-Me chemical modification, and / or the 5’ and / or 3’ terminus of the second gRNA comprises at least one 2’ O-Me chemical modification.

[0013] In one aspect, a composition of the present disclosure is encapsulated in at least one lipid nanoparticle (LNP) comprising about 50% of HBC365, about 38% of cholesterol by moles, about 10% of DSPC by moles, and about 2% of DMG-PEG2000 by moles. In some aspects, the ratio of lipid to RNA molecule in the at least one nanoparticle is about 50: 1 (w / w) and the total lipid is 25 nM.

[0014] In one aspect, provided herein are methods of increasing serum concentrations of FGF21 protein in a subject, comprising administering to the subject a therapeutically effective amount of an FGF21 gene targeting composition of the present disclosure.

[0015] In one aspect, provided herein are methods of treating a disease or disorder mediated by FGF21 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FGF21 gene targeting composition of the present disclosure.

[0016] In one aspect, provided herein are methods of treating a disease or disorder mediated by GDF15 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GDF15 gene targeting composition of the present disclosure.

[0017] In one aspect, provided are methods of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GDF15 gene targeting composition of the present disclosure.

[0018] In another aspect, provided herein are methods of modifying the genomes of a population of cells comprising contacting a population of cells with a composition of the present disclosure. The present disclosure also provides a population of cells modified according to the methods of the present disclosure.

[0019] All documents cited herein, including any cross referenced or related patent or application, are hereby incorporated herein by reference in its entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A is a graph showing secreted GDF15 protein expression in HepG2 cells following editing with compositions comprising Cas-CLOVER mRNA (2.5 pg) and gRNA pairs targeting GDF15 gene (1 pg). On the y-axis, secreted GDF15 protein concentrations (ng / ml) are shown and the x-axis shows the gRNA pairs that were tested (GDF15 guide Pairs #1-20). A guide RNA pair targeting the unrelated KLKB1 gene was used as a control. FIG. 1B is a graph showing the relative fold change in the expression of GDF15 mRNA in HepG2 cells following editing with compositions comprising Cas-CLOVER mRNA (2.5 pg) and gRNA pairs targeting the GDF15 gene (1 pg). On the y-axis, relative fold increases of GDF15 mRNA compared to the control are shown and the x-axis shows the gRNA pairs that were tested (GDF15 guide Pairs #1-20). A guide RNA pair targeting the unrelated KLKB1 gene was used as a control.DETAILED DESCRIPTION

[0021] The present disclosure provides compositions and methods for genetically modifying a genome to include a polynucleotide insertion, deletion and / or a substitution into chromosomal DNA that: a) enhances the mRNA stability and increases the protein expression of FGF21genes orb) enhances the mRNA stability and increases the protein expression of GDF15 genes. In particular, the present disclosure overcomes problems associated with currently available technologies by providing methods for efficient modification of cellular genomes. These methods may be useful to treat a wide range of metabolic disorders by introducing polynucleotide insertions, deletions, and / or substitutions in the FGF27 gene, a key mediator of a cluster of obesity-related metabolic complications, or the GDF15 gene, a key mediator of several diseases, including obesity.Fibroblast growth factor 21 (FGF21)

[0022] Fibroblast growth factor 21 (FGF21) is a stress-inducible hormone that has important roles in regulating energy balance and glucose and lipid homeostasis through a heterodimeric receptor complex comprising FGF receptor 1 (FGFR1) and P-klotho. FGF21 has shown pharmacological benefits on a cluster of obesity-related metabolic complications, including a reduction in fat mass and alleviation of hyperglycemia, insulin resistance, dyslipidemia, cardiovascular disorders, type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH; For a review, see Geng, L. et al, Nat Rev Endocrinol 16, 654-667 (2020). https: / / doi.org / 10.1038 / s41574-020-0386-0).

[0023] An exemplary human FGF21 coding sequence comprising the flanking 5’-UTR and 3’-UTR sequences is shown in SEQ ID NO: 19, wherein the 5’-UTR sequence is underlined, the FGF21 coding sequence is shown in bold font; the 3’-UTR is shown in italics with the 3 AU-rich elements highlighted in italicized bold font and a polyadenylation signal highlighted in italicized underlined font:GAGGCTTCCAAGGCAGGATACTTGTGTCTCAGATGCGGTCGCTTCTTTCATACAGCAATTGCCGCCTT GCTGAGGATCAAGGAACCTCAGTGTCAGATCACGCCCTCCCCCCAAACTTAGAAATTCAGATGGGGCG CAGAAATTTCTCTTGTTCTGCGTGATCTGCATAGATGGTCCAAGAGGTGGTTTTTCCAGGAGCCCAGC ACCCCTCCTCCCTCCGACTCAGACCCAGGAGTCTGGCCCTCCATTGAAAGGACCCCAGGTTACATCAT CCATTCAGGCTGCCCTTGCCACGATGGAATTCTGTAGCTCCTGCCAAATGGGTCAAATATCATGGTTC AGGCGCAGGGAGGGTGATTGGGCGGGCCTGTCTGGGTATAAATTCTGGAGCTTCTGCATCTATCCCAA AAAACAAGGGTGTTCTGTCAGCTGAGGATCCAGCCGAAAGAGGAGCCAGGCACTCAGGCCACCTGAGT CTACTCACCTGGACAACTGGAATCTGGCACCAATTCTAAACCACTCAGCTTCTCCGAGCTCACACCCC GGAGATCACCTGAGGACCCGAGCCATTGATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGT GGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCT CTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGC CCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGC AGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAG CGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACA AGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCC GCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAG CATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGAAGCCAGAGGC7G777AC7A7GA CATCTCCTCTTTATTTATTAGGTTATTTATCTTATTTATTTTTTTATTTTTCTTACTTGAGATAATAA AGAGTTCCAGAGGAGGATAA (SEQ IDNO: 19)

[0024] An analysis of the mRNA sequence of the FGF21 transcript revealed that the 3’-UTR, which contributes to FGF21 mRNA stability, comprises a number of AU-rich elements (AREs), which generally comprise a core ATTTA pentamer. The presence of AREs in an mRNA sequence has been reported to contribute to mRNA degradation; however, mutations of the pentamer sequence, e.g., AUCUA, or complete deletion of these elements can result in increased mRNA stability and potentially increased protein expression levels. Increased mRNA stability and increased expression levels of FGF1 protein could beneficially lead to reduced obesity-related metabolic complications.Growth Differentiation Factor 15 (GDF15)

[0025] Growth Differentiation Factor 15 (GDF15) is a member of the Glial cell-derived neurotropic factor (GDNF) family contributing to multiple roles in several diseases, including cancer, cardiovascular disease, and obesity (see, e.g, review by Baek & Eling, Pharmacol Ther. 2019 Jun; 198: 46-58. published online 2019 Feb 18. doi: 10.1016 / j.pharmthera.2019.02.008.)

[0026] Human GDF15 protein is synthesized as pro-GDF15, which then dimerizes through cysteine residues to form pro-GDF15 dimer. It is then cleaved at an RXXR (where X is any amino acid) site, forming a 112 amino acid C-terminal dimeric protein and a pro-peptide. The mature dimeric protein is secreted into the extracellular matrix and can be found in human blood. Experimental evidence has confirmed that the secreted mature dimer exhibits biological activity.

[0027] The circulating serum levels of only the mature GDF15 protein can be easily measured and are normally very low in humans; and increasing serum levels of GDF15 protein has been proposed as a strategy for treating obesity and promoting weight loss.

[0028] An exemplary human GDF15 coding sequence comprising the flanking 5’-UTR and 3’-UTR sequences is shown in SEQ ID NO: 20, wherein the 5’-UTR sequence is underlined, the GDF15 coding sequence is shown in bold font; the 3’-UTR is shown in italics with the 3 AU-rich elements highlighted in italicized bold font and a polyadenylation signal highlighted in italicized underlined font:CTGAGGCCCAGAAATGTGCCCTAGCTTTACTAGGAGCGCCCCCACCTAAAGATCCTCCCCCTAAATAC ACCCCCAGACCCCGCCCAGCTGTGGTCATTGGAGTGTTTACTCTGCAGGCAGGGGGAGGAGGGCGGGA CTGAGCAGGCGGAGACGGACAAAGTCCGGGGACTATAAAGGCCGGTCCGGCAGCATCTGGTCAGTCCC AGCTCAGAGCCGCAACCTGCACAGCCATGCCCGGGCAAGAACTCAGGACGGTGAATGGCTCTCAGATG CTCCTGGTGTTGCTGGTGCTCTCGTGGCTGCCGCATGGGGGCGCCCTGTCTCTGGCCGAGGCGAGCCG CGCAAGTTTCCCGGGACCCTCAGAGTTGCACTCCGAAGACTCCAGATTCCGAGAGTTGCGGAAACGCT ACGAGGACCTGCTAACCAGGCTGCGGGCCAACCAGAGCTGGGAAGATTCGAACACCGACCTCGTCCCG GCCCCTGCAGTCCGGATACTCACGCCAGAAGTGCGGCTGGGATCCGGCGGCCACCTGCACCTGCGTAT CTCTCGGGCCGCCCTTCCCGAGGGGCTCCCCGAGGCCTCCCGCCTTCACCGGGCTCTGTTCCGGCTGT CCCCGACGGCGTCAAGGTCGTGGGACGTGACACGACCGCTGCGGCGTCAGCTCAGCCTTGCAAGACCC CAGGCGCCCGCGCTGCACCTGCGACTGTCGCCGCCGCCGTCGCAGTCGGACCAACTGCTGGCAGAATC TTCGTCCGCACGGCCCCAGCTGGAGTTGCACTTGCGGCCGCAAGCCGCCAGGGGGCGCCGCAGAGCGC GTGCGCGCAACGGGGACCACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACACGGTCCGCGCG TCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGACCATGTGCAT CGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAGCCTGCACCGCC TGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAA AAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATATG AGCAGTCCTGGTCCTTCCACTGTGCACCTGCGCGGAGGACGCGACCTCAGTTGTCCTGCCCTGTGGAA TGGGCTCAAGGTTCCTGAGACACCCGATTCCTGCCCAAACAGCTGTATTTATATAAGTCTGTTATTTA TTATTAATTTATTGGGGTGACCTTCTTGGGGACTCGGGGGCTGGTCTGATGGAACTGTGTATTTATTT AAAACTCTGGTGATAAAAATAAAGCTGTCTGAACTGTTC (SEQ ID NO: 20)

[0029] Similar to FGF21, an analysis of the mRNA sequence of the GDF15 transcript revealed that the 3’-UTR sequence, which contributes to GDF15 mRNA stability, comprises a number of AU-rich elements (AREs). In this instance, mutation of, or a complete deletion of, these AREs in the 3’-UTR results in increased mRNA stability and increased protein expression levels, which could be therapeutically relevant to addressing disorders such as obesity.

[0030] The genome editing systems of the present disclosure can also be encapsulated in one or more Lipid Nanoparticles (LNPs). In certain embodiments, an LNP composition of the present disclosure can deliver a genome editing system of this disclosure to a target cell or tissue.Genome Editing Systems

[0031] The genome editing systems of this disclosure can include two or more fusion proteins (e.g., Cas-CLOVER) and two or more gRNAs having a targeting domain that is complementary to a sequence in or near the target region. In certain embodiments, the AU-rich elements (AREs) of the FGF21 gene are targeted for disruption. In certain embodiments, the AREs of the GDF15 gene are targeted for disruption. In certain embodiments, the AREs of the 3’-UTR of the GDF15 gene and FGF21 gene are targeted for disruption. In certain embodiments, genome editing systems disclosed herein may be used to introduce a polynucleotide insertion, deletion and / or a substitution in the targeted region.

[0032] The present disclosure overcomes problems associated with current technologies by providing compositions comprising genetically engineered fusion molecules (e.g., Cas-CLOVER) for targeted reduction or elimination of gene products in a cell for use in in vivo gene therapy. The compositions comprising genetically engineered fusion molecules of the disclosure are useful for the treatment of diseases or disorders, including genetic diseases. Nonlimiting examples of diseases or disorders include insulin resistance, dyslipidemia, cardiovascular disorders, type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), weight loss and obesity. In some embodiments, administration of the compositions disclosed herein leads to a reduction in fat mass and / or alleviation of hyperglycemia in a subject. Accordingly, methods of making genetically engineered fusion molecules and pharmaceutical formulations thereof (e.g., lipid nanoparticle formulations) for use in in vivo delivery are also provided. As a non-limiting example, the administration of the compositions of the disclosure could enhance FGF21 protein expression and / or GDF15 protein expression to a level sufficient to provide therapeutic efficacy for the treatment of a wide range of genetic disorders.Methods for Targeted Genome Editing at Selected LocusGene Editing Compositions and Methods

[0033] The present disclosure provides a gene editing composition as well as a cell comprising the gene editing composition. The gene editing composition can comprise a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof can comprise one or more of a CRISPR / Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.

[0034] In some aspects, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some aspects, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. Modified nucleic acids can include, but are not limited to, 5-methoxy uridine (5moU), Nl-methyl pseudouridine (mely), pseudouridine (Y), 5-methylcytidine (5-MeC). In some aspects, the non-naturally occurring nucleic acid molecule can comprise at least one non-naturallyoccurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some aspects, the nucleic acid molecule can be a modified nucleic acid molecule. In some aspects, the modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art. In some aspects, the nucleic acid molecule may be a circular DNA molecule, such as, but not limited to, a DNA plasmid. In some aspects, the nucleic acid molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid. In some aspects, the nucleic acid molecule can be a DoggyBone DNA molecule. In some aspects, the nucleic acid molecule can be a DNA nanoplasmid.

[0035] In some embodiments, a nucleic acid molecule of the present disclosure can be at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb in length.

[0036] A method for directing proteins to a specific locus in a genome of an organism is also disclosed herein. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.Exemplary dCas9-Clo051 (Cas-CLOVER) fusion proteins

[0037] The nuclease or the nuclease domain thereof can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein comprising a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0038] The disclosure provides compositions comprising a Cas9 operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a Cas9. A Cas9 of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0039] The disclosure provides compositions comprising an inactivated Cas9 (dSaCas9) operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dSaCas9). An inactivated Cas9 (dSaCas9) construct of the disclosure can comprise an effector comprising a type IIS endonuclease. A dSaCas9 can comprise the amino acid sequence of SEQ ID NO: 27, which includes a D10A and an N580A mutation relative to wildtype Cas9 to inactivate the catalytic site.

[0040] The disclosure provides compositions comprising an inactivated Cas9 (dCas9) operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of, or consisting of, a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). An inactivated Cas9 (dCas9) construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0041] The dCas9 can be isolated or derived from Streptococcus pyogenes. The dCas9 can comprise a dCas9 with substitutions at amino acid positions 10 and 840 of the wildtype sequence, which inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A. The dCas9 can comprise the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0042] In some embodiments, the C-terminus of the dCas9 or nuclease domain thereof, is joined to the N-terminus of the Clo051 polypeptide or nuclease domain thereof via peptide linker sequence selected from GGGGS (SEQ ID NO: 21).

[0043] An exemplary Clo051 nuclease domain comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 30. In some aspects, the Clo051 nuclease domain comprises at least one amino acid substitution. In some aspects, the amino acid substitution is in the alpha-helix-loop domain of the Clo051 nuclease. In some aspects, the amino acid substitution is at position 35, 37, 60, 92, 98, 100 or 146 of SEQ ID NO: 30. In some aspects, the amino acid substitution is at position 37 of SEQ ID NO: 30. In some aspects, the amino acid substitution is at positions 37 and 92 of SEQ ID NO: 30.

[0044] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 31. The exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 32. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0045] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 33. The exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 34. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0046] An exemplary dCas9-Clo051 fusion (Cas-CLOVER) fusion protein of the disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the dCas9-Clo051 fusion protein of the disclosure comprises at least two nuclear localization sequences. In some embodiments, the NLS is located on the N-terminal end of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is located on the C-terminal end of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, the NLS is located on the N-terminal end and at the C-terminal end of the dCas9-Clo051 fusion protein (“NLS-dCas9-Clo051-NLS” or “wildtype Cas-CLOVER” or “dspCas9 Ca-CLOVER”).

[0047] The NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas-CLOVER”) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 22.

[0048] Cas-CLOVER v2 amino acid sequence (NLS amino acid sequence is bolded and underlined; linker is bolded and italicized) MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEY GFKGRHLGGSRKPDGIVYSTTLEDNFGI IVDTKAYSEGYSLPISQADEMERYVRENSNRDEE VNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNWNLLLGAEKIRS GEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE ETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSS (SEQ ID NO: 22).

[0049] The nucleic acid encoding the NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas-CLOVER”) fusion protein can be DNA or RNA. In some embodiments, a dCas9-Clo051 fusion protein comprising two NLS regions is encoded by an mRNA sequence comprising, consisting essentially of or consisting of SEQ ID NO: 23 or a DNA sequence comprising, consisting essentially of or consisting of SEQ ID NO: 24.

[0050] NLS-dCas9-Clo051-NLS mRNA sequence auggcucccaagaagaagcggaaggucGAGGGCAUCAAGAGCAACAUCAGCCUGCUGAAGGA CGAGCUGAGAGGCCAGAUCAGCCACAUCUCCCACGAGUACCUGAGCCUGAUCGACCUGGCCU UCGACcccAAGCAGAACCGGCUGUUCGAGAUGAAGGUGCUGGAACUGCUGGUCAACGAGUAC GGCUUCAAGGGCAGACACCUCGGCGGCAGCAGAAAGCCUGAUGGCAUCGUGUACAGCACCAC ACUCGAGGACAACUUCGGCAUCAUCGUGGACACCAAGGCCUACAGCGAGGGCUACAGCCUGC CUAUCUCUCAGGCCGACGAGAUGGAAAGAUACGUGCGCGAGAACAGCAACCGCGACGAGGAA GUGAAC C C CAACAAGUGGUGGGAGAACUUCAGC GAGGAAGUCAAAAAGUACUACUUC GUGUU CAUCAGCGGCAGCUUUAAGGGCAAGUUCGAGGAACAGCUGCGGCGGCUGUCUAUGACCACAG GCGUUAACGGCAGCGCCGUGAACGUGGUCAAUCUGCUGCUGGGCGCCGAGAAGAUUAGAAGCGGCGAGAUGACCAUCGAGGAACUGGAACGGGCCAUGUUCAACAACAGCGAGUUCAUCCUGAA GUACggcggaggcggcagcgacaagaaguacucuaucggacuggccaucggcaccaacucug uuggaugggccgugaucaccgacgaguacaaggugcccagcaagaaauucaaagugcugggc aacaccgaccggcacagcaucaagaagaaucugaucggcgcccugcuguucgacucuggcga aacagccgaagccaccagacugaagagaaccgccagacggcgguacaccagaagaaagaacc ggaucugcuaccugcaagagaucuucagcaacgagauggccaagguggacgacagcuucuuc cacagacuggaagaguccuuccugguggaagaggacaagaagcacgagcggcaccccaucuu cggaaauaucguggacgagguggccuaccacgagaaguaccccaccaucuaccaccugagaa agaaacugguggacagcaccgacaaggccgaccugcgacugaucuaucuggcccuggcucac augaucaaguuccggggccacuuccugaucgagggcgaccugaauccugacaacuccgacgu ggacaagcuguucauccagcuggugcagaccuacaaucagcuguucgaagagaaucccauca acgccucuggcguggacgccaaagccauccugucugccagacugagcaagagcagacggcug gaaaaccugaucgcucagcugcccggcgagaagaagaauggccuguucggcaaccugauugc ccugucucugggccugacaccuaacuucaaguccaacuucgaucuggccgaggaugccaaac ugcagcuguccaaggacaccuacgacgacgaccuggauaaccugcuggcccagaucggcgau caguacgccgacuuguuucuggccgccaagaaccugucugacgccauccugcugagcgacau ccugagagugaacaccgagaucacaaaggccccucugagcgccucuaugaucaagagauacg acgagcaccaccaggaucugacccugcugaaagcucucgucaggcagcagcugccagagaag uacaaagagauuuucuucgaccagagcaagaacggcuacgccggcuacauugauggcggagc cagccaagaggaauucuacaaguucaucaagcccauccucgagaagauggacggcacagagg aacugcucgugaagcugaacagagaggaccugcugcggaagcagcggaccuucgacaauggc ucuaucccucaccagauccaccugggagagcugcacgccauucugcggagacaagaggacuu uuacccauuccugaaggacaaccgggaaaagauugagaagauccugaccuucaggauccccu acuacgugggaccacuggccagaggcaauagcagauucgccuggaugaccagaaagagcgag gaaaccaucacacccuggaacuucgaagaggugguggacaagggcgccagcgcucaguccuu caucgagcggaugaccaauuucgacaagaaucugcccaacgagaaagugcugcccaagcacu cccugcuguacgaguacuucaccguguacaacgagcugaccaaagugaaauacgugaccgag ggaaugagaaagcccgccuuucuguccggcgagcagaaaaaggccaucguggaucugcuguu caagaccaaccggaaagugaccgugaagcagcugaaagaggacuacuucaagaaaaucgagu gcuucgacuccguggaaaucagcggcguggaagaucgguucaaugccagccugggcacauac cacgaucugcugaaaauuaucaaggacaaggacuuccuggacaacgaggaaaacgaggacau ccuugaggacaucgugcugacccugacacuguucgaggacagagagaugaucgaggaaaggc ugaaaacauacgcccaccuguucgacgacaaagucaugaagcaacugaagcggcggcgcuac acaggcuggggcagacugucuagaaagcugaucaacggcauccgggacaagcaguccggcaa gaccauccuggacuuucugaaguccgacggcuucgccaacagaaacuucaugcagcugauuc acgacgacagccucaccuucaaagaggacauucagaaggcccagguuuccggccagggcgau ucucugcacgagcacauugccaaucuggccggcucucccgccauuaagaagggcauucugca gacagugaaagugguggaugagcuggucaaagugauggggagacacaagcccgagaacaucg ugaucgaaauggccagagagaaccagaccacacagaagggccagaagaacucccgcgagaga augaagcggaucgaagagggaaucaaagagcuggggagccagauccugaaagaacaccccgu ggaaaacacccagcugcagaacgagaagcuguaccuguacuaccuccagaacggccgggaua uguacguggaccaagagcuggacaucaaccgccugagcgacuacgauguggacgcuaucgug ccccagucuuuucugaaagaugacuccaucgacaacaaggugcugaccagaagcgauaagaa ccggggcaagagcgacaacgugcccucugaagaggucgugaagaagaugaagaacuacuggc gacagcugcugaacgccaagcugauuacccagcggaaguucgauaaccugaccaaggccgag agaggcggccugucugaacuggauaaggccggcuucaucaagagacagcugguggaaacccg gcagaucaccaaacacguggcacagauucuggacucccggaugaacaccaaauacgaugaga acgacaaacugauccgggaagugaaagucaucacccugaaguccaagcugguguccgauuuc cggaaggauuuccaguucuacaaagugcgggaaaucaacaacuaccaucacgcccacgacgccuaccugaaugccguuguuggaacagcccugaucaagaaguaucccaagcuggaaagcgagu ucguguacggcgacuacaagguguacgacgugcggaagaugaucgccaagagcgagcaagag auuggaaaggcuaccgccaaauacuucuucuacuccaacaucaugaacuuuuucaagacaga gaucacccucgccaacggcgagaucagaaagcggccucugaucgagacaaacggcgaaaccg gcgagauugugugggauaagggcagagacuuugccacagugcggaaggugcucagcaugccc caagugaauaucgugaaaaagaccgaggugcagacaggcggcuucagcaaagaguccauucu gccuaagcggaacuccgacaagcugaucgcccggaagaaggacugggaccccaagaaauacg gcggcuucgauagcccuaccguggccuauucugugcuggugguggccaaaguggaaaaggga aaguccaagaagcucaagagcgucaaagaacuccugggcaucaccaucauggaacgguccag cuucgagaagaacccuaucgacuuucuggaagccaagggcuacaaagaagucaagaaggacc ugaucaucaagcuccccaaguacagccuguucgagcuggaaaauggccggaagcggaugcug gcuucugcuggcgaacugcagaagggaaacgaacuggcccugccuagcaaauaugugaacuu ccuguaccuggccagccacuaugagaagcugaagggcagccccgaggacaaugagcagaagc agcuuuucgucgagcagcacaagcacuaccuggacgagaucaucgagcagaucuccgaguuc uccaagagagugauccuggccgacgccaaccuggacaagguucuguccgccuacaacaagca ccgggauaagcccaucagagagcaggccgagaauaucauccaccuguuuacccugaccaacc ugggagccccugccgccuucaaguacuucgacaccaccaucgaccggaagcgcuacaccagc accaaagaagugcuggacgccacacugauccaccagagcaucaccggccuguacgagacacg gaucgaucugucucagcuuggaggcgacggcagcccuaagaagaagagaaagguuuccagcu aauaa (SEQ ID NO: 23).

[0051] A cell comprising the gene editing composition can express the gene editing composition stably or transiently.Exemplary mutant Cas-CLOVER fusion proteins

[0052] In some aspects, NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) comprises at least one amino acid substitution. In some aspects, the amino acid substitution is located in the Clo051 domain of the NLS-dCas9-Clo051-NLS.

[0053] In some aspects, the NLS-dCas9-Clo051-NLS of SEQ ID NO: 22 can comprise at least one substitution at amino acid positions 42, 44, 67, 105, 107 or 153. In some aspects, the amino acid substitutions are F42E, F42D, S44E, S44P, R67E, I105Q, Q107A, Q107E, Q107H, Q107D and / or K153D. In some aspects, the amino acid substitution is S44P.

[0054] An exemplary S44P mutant NLS-dCas9-Clo051-NLS (“S44P Cas-CLOVER”, or “S44P CC”, or “S44P”, or “Cas-CLOVERv3”, or “CCv3”) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 25.

[0055] Cas-CLOVER v3 amino acid sequence (NLS amino acid sequence is bolded and underlined; linker is bolded and italicized) MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDPKQNRLFEMKVLELLVNEY GFKGRHLGGSRKPDGIVYSTTLEDNFGI IVDTKAYSEGYSLPISQADEMERYVRENSNRDEE VNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNWNLLLGAEKIRS GEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE ETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSS (SEQ ID NO: 25)

[0056] The Cas-CLOVER v3 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 26. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0057] Cas-CLOVER v3 mRNA sequence acauuugcuucugacacaacuguguucacLiagcaaccLicaaacagacaccaagcLiLigccaccaLiggcLicccaaga agaagcggaaggucGACGGCAUCAAGAGCAACAUCAGCCUGCUGAAGGACGAGCUGAGAGGCCAGAUCAGCCACA UCUCCCACGAGUACCUGAGCCUGAUCGACCUGGCCUUCGACcccAAGCAGAACCGGCUGUUCGAGAUGAAGGUGC UGGAACUGCUGGUCAACGAGUACGGCUUCAAGGGCAGACACCUCGGCGGCAGCAGAAAGCCUGAUGGCAUCGUGU ACAGCACCACACUCGAGGACAACUUCGGCAUCAUCGUGGACACCAAGGCCUACAGCGAGGGCUACAGCCUGCCUA UCUCUCAGGCCGACGAGAUGGAAAGAUACGUGCGCGAGAACAGCAACCGCGACGAGGAAGUGAACCCCAACAAGU GGUGGGAGAACUUCAGCGAGGAAGUCAAAAAGUACUACUUCGUGUUCAUCAGCGGCAGCUUUAAGGGCAAGUUCG AGGAACAGCUGCGGCGGCUGUCUAUGACCACAGGCGUUAACGGCAGCGCCGUGAACGUGGUCAAUCUGCUGCUGG GCGCCGAGAAGAUUAGAAGCGGCGAGAUGACCAUCGAGGAACUGGAACGGGCCAUGUUCAACAACAGCGAGUUCA UCCUGAAGUACggcggaggcggcagcgacaagaagLLacLLCLLaLLcggacLLggccaLLcggcaccaacLLCLLgLLLLggau gggccgLLgaLLcaccgacgagLLacaaggugcccagcaagaaaLLLLcaaagLLgcLLgggcaacaccgaccggcacagca ucaagaagaaLLCLLgaLLcggcgcccLLgcLLgLLLLcgacLLCLLggcgaaacagccgaagccaccagacugaagagaaccg ccagacggcggLLacaccagaagaaagaaccggaLLCLLgcLLaccLLgcaagagaucLLLLcagcaacgagaLLggccaagg LLggacgacagcLLLLCLLLLCcacagacLLggaagaguccLLLLCCLLggLLggaagaggacaagaagcacgagcggcacccca ucuucggaaaLLaLLcgLLggacgaggLLggccLLaccacgagaagLLaccccaccaucLLaccaccLLgagaaagaaacLLgg uggacagcaccgacaaggccgaccugcgacugaLLCLLaLLCLLggcccLLggcLLcacaLLgaLLcaagLLUccggggccacLL uccugaucgagggcgaccugaauccLLgacaacLLCcgacgLLggacaagcLLgLLLLcauccagcLLggLLgcagaccLLaca aucagcuguucgaagagaaucccaLLcaacgccLLCLLggcgLLggacgccaaagccauccLLgLLCLLgccagacLLgagca agagcagacggcLLggaaaaccLLgaLLcgcLLcagcLLgcccggcgagaagaagaaLLggccLLgLLLLcggcaaccugaLLLLg cccugucucugggccugacaccuaacuucaagLLCcaacLLLLcgaLLCLLggccgaggaLLgccaaacLLgcagcLLgucca aggacaccuacgacgacgaccuggauaaccLLgcLLggcccagaLLcggcgaLLcagLLacgccgacLLugLLLLLLCLLggccg ccaagaaccugucugacgccauccLLgcLLgagcgacaLLCCLLgagagugaacaccgagaLLcacaaaggccccLLCLLgagcgccucuaugaucaagagauacgacgagcaccaccaggaucugacccugcugaaagcucucgucaggcagcagc ugccagagaaguacaaagagauuuucuucgaccagagcaagaacggcuacgccggcuacauugauggcggagcca gccaagaggaauucuacaaguucaucaagcccauccucgagaagauggacggcacagaggaacugcucgugaagc ugaacagagaggaccugcugcggaagcagcggaccuucgacaauggcucuaucccucaccagauccaccugggag agcugcacgccauucugcggagacaagaggacuuuuacccauuccugaaggacaaccgggaaaagauugagaaga uccugaccuucaggauccccuacuacgugggaccacuggccagaggcaauagcagauucgccuggaugaccagaa agagcgaggaaaccaucacacccuggaacuucgaagaggugguggacaagggcgccagcgcucaguccuucaucg agcggaugaccaauuucgacaagaaucugcccaacgagaaagugcugcccaagcacucccugcuguacgaguacu ucaccguguacaacgagcugaccaaagugaaauacgugaccgagggaaugagaaagcccgccuuucuguccggcg agcagaaaaaggccaucguggaucugcuguucaagaccaaccggaaagugaccgugaagcagcugaaagaggacu acuucaagaaaaucgagugcuucgacuccguggaaaucagcggcguggaagaucgguucaaugccagccugggca cauaccacgaucugcugaaaauuaucaaggacaaggacuuccuggacaacgaggaaaacgaggacauccuugagg acaucgugcugacccugacacuguucgaggacagagagaugaucgaggaaaggcugaaaacauacgcccaccugu ucgacgacaaagucaugaagcaacugaagcggcggcgcuacacaggcuggggcagacugucuagaaagcugauca acggcauccgggacaagcaguccggcaagaccauccuggacuuucugaaguccgacggcuucgccaacagaaacu ucaugcagcugauucacgacgacagccucaccuucaaagaggacauucagaaggcccagguuuccggccagggcg auucucugcacgagcacauugccaaucuggccggcucucccgccauuaagaagggcauucugcagacagugaaag ugguggaugagcuggucaaagugauggggagacacaagcccgagaacaucgugaucgaaauggccagagagaacc agaccacacagaagggccagaagaacucccgcgagagaaugaagcggaucgaagagggaaucaaagagcugggga gccagauccugaaagaacaccccguggaaaacacccagcugcagaacgagaagcuguaccuguacuaccuccaga acggccgggauauguacguggaccaagagcuggacaucaaccgccugagcgacuacgauguggacgcuaucgugc cccagucuuuucugaaagaugacuccaucgacaacaaggugcugaccagaagcgauaagaaccggggcaagagcg acaacgugcccucugaagaggucgugaagaagaugaagaacuacuggcgacagcugcugaacgccaagcugauua cccagcggaaguucgauaaccugaccaaggccgagagaggcggccugucugaacuggauaaggccggcuucauca agagacagcugguggaaacccggcagaucaccaaacacguggcacagauucuggacucccggaugaacaccaaau acgaugagaacgacaaacugauccgggaagugaaagucaucacccugaaguccaagcugguguccgauuuccgga aggauuuccaguucuacaaagugcgggaaaucaacaacuaccaucacgcccacgacgccuaccugaaugccguug uuggaacagcccugaucaagaaguaucccaagcuggaaagcgaguucguguacggcgacuacaagguguacgacg ugcggaagaugaucgccaagagcgagcaagagauuggaaaggcuaccgccaaauacuucuucuacuccaacauca ugaacuuuuucaagacagagaucacccucgccaacggcgagaucagaaagcggccucugaucgagacaaacggcg aaaccggcgagauugugugggauaagggcagagacuuugccacagugcggaaggugcucagcaugccccaaguga auaucgugaaaaagaccgaggugcagacaggcggcuucagcaaagaguccauucugccuaagcggaacuccgaca agcugaucgcccggaagaaggacugggaccccaagaaauacggcggcuucgauagcccuaccguggccuauucug ugcuggugguggccaaaguggaaaagggaaaguccaagaagcucaagagcgucaaagaacuccugggcaucacca ucauggaacgguccagcuucgagaagaacccuaucgacuuucuggaagccaagggcuacaaagaagucaagaagg accugaucaucaagcuccccaaguacagccuguucgagcuggaaaauggccggaagcggaugcuggcuucugcug gcgaacugcagaagggaaacgaacuggcccugccuagcaaauaugugaacuuccuguaccuggccagccacuaug agaagcugaagggcagccccgaggacaaugagcagaagcagcuuuucgucgagcagcacaagcacuaccuggacg agaucaucgagcagaucuccgaguucuccaagagagugauccuggccgacgccaaccuggacaagguucuguccg ccuacaacaagcaccgggauaagcccaucagagagcaggccgagaauaucauccaccuguuuacccugaccaacc ugggagccccugccgccuucaaguacuucgacaccaccaucgaccggaagcgcuacaccagcaccaaagaagugc uggacgccacacugauccaccagagcaucaccggccuguacgagacacggaucgaucugucucagcuuggaggcg acggcagcccuaagaagaagagaaagguuuccagcuaauaaggcggccgcccucgccccggaccugcccucccgc caggugcacccaccugcaauaaaugcagcgaagccgggagaauucccucgccccggaccugcccucccgccaggu gcacccaccugcaauaaaugcagcgaagccgggagcggccgcggauccccggguaccgaauucgauaucucuaua gugucaccuaaauuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaA (SEQ ID NO: 26)gRNAs

[0058] As used herein, the term “guide sequence” or “spacer” in the context of a Cas-CLOVER system or a CRISPR-Cas9 system, comprises any polynucleotide molecule having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. The guide sequence may comprise both RNA and DNA polynucleotides. The guide sequence may form a duplex with a target sequence. The duplex may be a DNA duplex, an RNA duplex, or an RNA / DNA duplex. The terms “guide molecule”, “guide RNA”, “gRNA”, “single guide RNA” and “sgRNA” are used interchangeably herein to refer to RNA-based molecules that are capable of forming a complex with a Cas-CLOVER or a CRISPR-Cas protein and comprises a guide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of the complex to the target nucleic acid sequence. The guide molecule or guide RNA may encompass RNA-based molecules having one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides), as described herein. The guide sequence may also partially comprise RNA and DNA-based nucleotides in which the molecule is chimeric for RNA and DNA nucleobases (e.g., containing either ribose or deoxyribose sugars).

[0059] The term “target region”, “target sequence” or “protospacer” as used interchangeably herein refers to the region of the target gene or genomic target site, to which the Cas-CLOVER system or the CRISPR / Cas9-based system targets. The Cas-CLOVER or the CRISPR / Cas9-based system may include at least two gRNAs, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The Cas-CLOVER system may include at least two gRNAs, wherein the gRNAs target different DNA sequences. The target sequence or protospacer is generally followed by a protospacer adj acent motif (PAM) sequence at the 3' end of the protospacer. Different Type II CRISPR systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

[0060] The guide RNA or the guide RNA of a Cas-CLOVER protein or a CRISPR-Cas protein may comprise a tracr-mate sequence (encompassing a “direct repeat” in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system). In some embodiments, the Cas-CLOVER or the CRISPR-Cas system or complex as described herein does not comprise and / or does not rely on the presence of a tracr sequence. In certain embodiments, the guide molecule may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.

[0061] In some embodiments, the guide RNA comprises a guide sequence and a scaffold sequence. In some embodiments, the scaffold sequence is isolated from Streptococcus pyogenes. In some embodiments, the Streptococcus pyogenes scaffold sequence comprises the nucleic acid sequence:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUUUU (SEQ ID NO: 35).

[0062] In certain embodiments, the guide sequence or spacer length of the guide molecules is 15 to 50 nucleotides in length. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides in length. In certain embodiments, the spacer length is from 15 to 17 nucleotides in length, from 17 to 20 nucleotides in length, from 20 to 24 nucleotides in length, from 23 to 25 nucleotides in length, from 24 to 27 nucleotides in length, from 27-30 nucleotides in length, from 30-35 nucleotides in length, or greater than 35 nucleotides in length.

[0063] In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 nucleotides in length.

[0064] In some embodiments, the sequence of the guide molecule (direct repeat and / or spacer) is selected to reduce the degree of secondary structure within the guide molecule. In some embodiments, about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008,Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0065] As described herein, the Cas-CLOVER system and the CRISPR / Cas9 system utilizes one or more targeting gRNAs that provides the targeting of the Cas-CLOVER system and the CRISPR / Cas9-based system. The gRNA may be a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The sgRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA: tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid.

[0066] In some embodiments, the gRNA targets an FGF21 gene locus (e.g., the FGF21 open reading frame (ORF) and between 0-1000 bp upstream of the ORF). In some embodiments, the gRNA targets a GDF15 gene locus (e.g., the GDF15 ORF and between 0-1000 bp upstream of the ORF). In some embodiments, the gRNA targets a 3’-UTR of an FGF21 gene locus (e.g., between 0-1000 bp upstream of the FGF21 ORF). In some embodiments, the gRNA targets a 3’-UTR of a GDF15 gene locus (e.g., between 0-1000 bp upstream of the GDF15 ORF). In some embodiments, the gRNA targets AU-rich elements in a FGF21 gene locus or a GDF15 gene locus. In some embodiments, the gRNA targets AU-rich elements in the 3’-UTR of a FGF21 gene locus. In some embodiments, the gRNA targets AU-rich elements in the 3’-UTR of a GDF15 gene locus. In some embodiments, the gRNA targets a region between 0-50 bp, 0- 100 bp, 0-150 bp, 0-200 bp, or 0-250 bp upstream or downstream of one or more AU-rich element of a FGF21 or GDF15 ORF. In some embodiments, the gRNA targets a region between 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0-350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0-800 bp, 0-850 bp, 0-900 bp, 0-950 bp or 0-1000 bp upstream of the transcription start site of an FGF21 or GDF15 ORF. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp or about 1500 bp upstream of the FGF21 or GDF15 ORF.

[0067] A gRNA can be divided into a target binding region (also referred to herein as a “targeting sequence”) and a Cas9 binding region. The target binding region hybridizes with atarget region in a target gene or intergenic region. In some embodiments, the target gene is an FGF21 gene or GDF15 gene. Methods for designing such target binding regions are known in the art, see, e.g., Doench et al., Nat Biotechnol. (2014) 32: 1262-7; and Doench et al., Nat Biotechnol. (2016) 34: 184-91, incorporated by reference herein in their entirety. Design tools are available at, e.g., Feng Zhang lab's target Finder, Michael Boutros lab's Target Finder (E-CRISP), RGEN Tools (Cas-OF Finder), CasFinder, and CRISPR Optimal Target Finder. In certain embodiments, the target binding region can be between about 15 and about 50 nucleotides in length (about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length). In certain embodiments, the target binding region can be between about 19 and about 21 nucleotides in length. In one embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0068] In one embodiment, the target binding region is complementary, e.g., completely complementary, to the target region in the target gene. In one embodiment, the target binding region is substantially complementary to the target region in the target gene. In one embodiment, the target binding region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are not complementary to the target region in the target gene.

[0069] Exemplary gRNAs of the disclosure include but are not limited to sequences for targeting an FGF21 gene locus, sequences for targeting a GDF15 gene locus, and sequences for targeting AU-rich elements in the 3’-UTR of an FGF21 gene locus or the 3’-UTR of a GDF 15 gene locus.

[0070] In some embodiments, the first gRNA (also referred to as “left gRNA”) binds to a target sequence at the 5’ terminus of the target gene locus and the second gRNA (also referred to as “right gRNA”) binds to a target sequence at the 3’ terminus of the target gene locus.

[0071] Exemplary FGF21 gene gRNAs of the disclosure comprise, consist essentially of, or consists of the target sequences as shown in Table 1 and are not conserved in cynomolgus monkeys. In some embodiments, each sequence of Table 1 comprises the following modifications: 2'-O-m ethyl analogs (OMe) on the first three bases and last three positions, and 3' phosphorothioate internucleotide linkages (PS) between the first three bases and between the last two bases.Table 1. Exemplary FGF21 gene gRNAs of the disclosure

[0072] Exemplary GDF15 gene gRNAs of the disclosure comprise, consist essentially of, or consist of the target sequences as shown in Table 2. In some embodiments, a GDF15 gene gRNA sequence of Table 2 comprises the following modifications: 2'-O-methyl analogs (OMe) on the first three bases and last three positions, and 3' phosphorothioate internucleotide linkages (PS) between the first three bases and between the last two bases.Table 2. Exemplary GDF15 gene gRNAs of the disclosuregRNA Modifications

[0073] The activity, stability, or other characteristics of gRNAs can be altered through the incorporation of certain modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory, it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into cells. Those of skill in the art will be aware of certain cellular responses commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can include induction of cytokine expression and release and cell death, may be reduced or eliminated altogether by the modifications presented herein.

[0074] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5 ' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5,1or 1-2 nucleotides of the 3' end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Cpfl gRNA, and / or a targeting domain of a gRNA.

[0075] As one example, the 5' end of a gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5)ppp(5)G cap analog, a m7G(5)ppp(5)G cap analog, or a 3'-0-Me- m7G(5)ppp(5)G anti reverse cap analog (ARCA)), as shown below;

[0076] The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.

[0077] Along similar lines, the 5' end of the gRNA can lack a 5' triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5' triphosphate group.

[0078] Another common modification involves the addition, at the 3' end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract. The polyA tract can be added to a gRNA during chemical synthesis, following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase), or in vivo by means of a polyadenylation sequence, as described in Maeder.

[0079] It should be noted that the modifications described herein can be combined in any suitable manner, e.g, a gRNA, whether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can include either or both of a 5' cap structure or cap analog and a 3' polyA tract.

[0080] Guide RNAs can be modified at a 3' terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:

[0082] wherein “U” can be an unmodified or modified uridine.

[0083] The 3' terminal U ribose can be modified with a 2'3 ' cyclic phosphate as shown below:

[0085] wherein “U” can be an unmodified or modified uridine.

[0086] Guide RNAs can contain 3' nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein. In some embodiments, adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.

[0087] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2' OH-group is replaced by a group selected from H, — OR, — R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, — SH, — SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano ( — CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2'-sugar modified, such as, 2'-O-methyl, 2'-O-methoxy ethyl, or 2'-Fluoro modified including, e.g., 2'-F or 2'-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2'-O-methyl, thymidine (T), 2'-F or 2'-O-methyl, guanosine (G), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

[0088] Guide RNAs can also include “locked” nucleic acids (LNA) in which the 2' OH-group can be connected, e.g., by a Cl -6 alkylene or Cl -6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, including without limitation methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0089] In certain embodiments, a gRNA can include a modified nucleotide that is multicyclic (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3'^2')).

[0090] Generally, gRNAs include the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2' position, other sites are amenable to modification, including the 4' position. In certain embodiments, a gRNA comprises a 4'-S, 4'-Se or a 4'-C-aminomethyl-2'-O-Me modification.

[0091] In certain embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.

[0092] In some embodiments, the gRNA comprises one or more chemical modifications of a ribonucleotide, a ribonucleotide base, or a phosphodiester bond. In some embodiments, the one or more chemical modifications comprises at least one chemically modified phosphodiester bond. In some embodiments, the at least one chemically modified phosphodiester bond is a phosphorothioate bond.

[0093] In some embodiments, the gRNA comprises three phosphorothioate bonds at the 5’ terminus of the gRNA. In some embodiments, the gRNA comprises two phosphorothioate bonds at the 3 ’ -terminus of the gRNA. In some embodiments, the gRNA comprises a 2’ O-Me chemical modification at the 3 ’-terminus of the gRNA.Exemplary gRNA sequences

[0094] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 1. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 1.

[0095] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 2. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 2.

[0096] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 3. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 3.

[0097] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 4. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 4.

[0098] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 5. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 5.

[0099] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 6. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 6.

[0100] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 7. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 7.

[0101] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 8. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 8.

[0102] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 9. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 9.

[0103] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 10. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 10.

[0104] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 11. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 11.

[0105] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 12. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 12.

[0106] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 13. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 13.

[0107] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 14. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 14.

[0108] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 15. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 15.

[0109] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 16. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 16.

[0110] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 17. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 17.

[0111] In some embodiments, a gRNA comprises a nucleotide sequence at least 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 18. In some embodiments, a gRNA comprises a nucleotide sequence of SEQ ID NO: 18.Exemplary gRNA Compositions

[0112] In certain compositions of the disclosure, a gRNA composition comprises a first gRNA and a second gRNA. In some embodiments, the first gRNA comprises a first targeting sequence. In some embodiments, the second gRNA comprises a second targeting sequence.

[0113] In certain embodiments, the first and second gRNA of the composition target AU-rich elements in the 3’-UTR of the FGF21 gene. It will be apparent to a person of skill in the art that the gRNA sequences present in Table 2 can be freely combined to achieve optimal targeting of the construct.

[0114] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 1 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 4.

[0115] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 1 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 5.

[0116] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 2 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 4.

[0117] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 2 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 5.

[0118] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 3 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 4.

[0119] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 3 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 5.

[0120] In certain embodiments, the first and second gRNA of the composition target AU-rich elements in the 3’-UTR of the GDF15 gene.

[0121] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 6 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 11.

[0122] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 6 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 12.

[0123] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 6 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 13.

[0124] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 7 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 11.

[0125] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 7 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 12.

[0126] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 7 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 13.

[0127] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 8 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 14.

[0128] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 8 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 15.

[0129] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 8 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 16.

[0130] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 8 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 17.

[0131] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 9 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 14.

[0132] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 9 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 15.

[0133] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 9 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 16.

[0134] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 9 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 17.

[0135] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 10 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 14.

[0136] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 10 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 15.

[0137] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 10 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 16.

[0138] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 10 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 17.

[0139] In some embodiments, the composition comprises a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 10 and a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 18.Exemplary Cas-CLOVER and gRNA Compositions

[0140] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in PCT Application Numbers PCT / US2016 / 037922, PCT / US2018 / 066941, PCT / US2017 / 054799, U. S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U. S. Patent No. 10,415,024, each of which are incorporated herein by reference in its entirety for examples of Cas-CLOVER constructs and other gene editing compositions that may be used in the methods described herein.

[0141] In one aspect, provided are compositions comprising: a) a first guide RNA (gRNA) and a polynucleotide encoding a first fusion protein comprising: a first mutant Cas9 (dCas9) polypeptide or an inactivated nuclease domain thereof and a first Clo051 polypeptide or a nuclease domain thereof and b) a second gRNA and a polynucleotide encoding a second fusion protein comprising: a second mutant Cas9 (dCas9) polypeptide or an inactivated nuclease domain thereof and second a Clo051 polypeptide or a nuclease domain thereof. Preferably, the first fusion protein is expressed in a cell and forms a complex with the first gRNA and the second fusion protein is expressed in a cell and forms a complex with the second gRNA.

[0142] In some embodiments, the first gRNA comprises a first targeting sequence comprising the nucleic acid sequence selected from SEQ ID NOs: 1-3; and the second gRNA comprises a second targeting sequence comprising the nucleic acid sequence of SEQ ID NOs: 4 or 5. In some embodiments the first gRNA comprises a first targeting sequence comprising the nucleic acid sequence selected from SEQ ID NOs: 6-10; and the second gRNA comprises a second targeting sequence comprising the nucleic acid sequence of SEQ ID NOs: 11-18.

[0143] In some embodiments, a composition of the disclosure comprises (a) a first guide RNA (gRNA) comprising a first targeting sequence set forth in SEQ ID NOs: 1-3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0144] In some embodiments, (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NOs: 6-10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NOs: 11-18; (c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0145] In some embodiments, the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, both the first fusion protein and the second fusion protein comprise the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25.

[0146] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO:22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0147] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0148] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0149] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a secondpolynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0150] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0151] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0152] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivatedCas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0153] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0154] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0155] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0156] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0157] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0158] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0159] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0160] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0161] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0162] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO:22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0163] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0164] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0165] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a secondpolynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0166] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0167] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0168] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivatedCas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0169] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0170] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0171] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0172] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 18; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0173] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0174] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0175] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0176] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0177] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0178] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ IDNO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0179] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0180] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0181] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) asecond polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0182] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0183] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0184] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a secondinactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0185] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0186] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0187] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0188] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0189] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0190] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0191] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0192] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0193] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0194] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ IDNO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0195] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0196] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0197] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) asecond polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0198] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0199] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 18; (c) a first polynucleotide encoding a first fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide encoding a second fusion protein comprising the polypeptide sequence set forth in SEQ ID NO: 25, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0200] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a secondinactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0201] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0202] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0203] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0204] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0205] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0206] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0207] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0208] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0209] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0210] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusionprotein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0211] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0212] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0213] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) asecond polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0214] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0215] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0216] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a secondinactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0217] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0218] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0219] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0220] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0221] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0222] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0223] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0224] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0225] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0226] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 18; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusionprotein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0227] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0228] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 1; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 23, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0229] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) asecond polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0230] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 2; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0231] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0232] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 3; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 5; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a secondinactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0233] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0234] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0235] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 6; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0236] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 11; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0237] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 12; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0238] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 7; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 13; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0239] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNAcomprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0240] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0241] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0242] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 8; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusionprotein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0243] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0244] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0245] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) asecond polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0246] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0247] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 9; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0248] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 14; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a secondinactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0249] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0250] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 15; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0251] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 16; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0252] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 17; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0253] In some embodiments, a composition of the disclosure comprises (a) a first gRNA comprising a first targeting sequence set forth in SEQ ID NO: 10; (b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 18; (c) a first polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or a nuclease domain thereof; and (d) a second polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 26, encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or a nuclease domain thereof.

[0254] In certain compositions of the disclosure, the composition comprises a first gRNA, a first polynucleotide encoding the first fusion protein (e.g., Cas-CLOVER), a second gRNA and a second fusion protein or a second polynucleotide encoding a second fusion protein (e.g., Cas-CLOVER). In certain embodiments, the first gRNA comprises a first targeting sequence and the second gRNA comprises a second targeting sequence.

[0255] In some embodiments, the first gRNA and the first fusion protein, once expressed in a cell, form a complex that localizes at the 5’ terminus of the target DNA. In some embodiments, the second gRNA and the second fusion protein form a complex that localizes at the 3’ terminus of the target DNA. In some embodiments, the first and second gRNA are capable of targeting the fusion protein to a target region.

[0256] In some embodiments, the first fusion protein, the second fusion protein, or both the first and the second fusion protein comprises a dCas9 derived from an S. pyogenes Cas9 polypeptide.

[0257] In certain embodiments, the compositions are designed to target and edit AU-rich sequences in the 3’-UTR of the FGF21 gene.

[0258] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 1, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0259] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO:1, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0260] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0261] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0262] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0263] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22 a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0264] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 1, a first fusion protein comprising the polypeptidesequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0265] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO:1, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0266] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0267] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0268] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0269] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25 a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0270] In certain embodiments, the compositions are designed to target and edit AU-rich sequences in the 3’-UTR of the GDF15 gene.

[0271] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0272] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0273] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0274] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0275] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0276] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0277] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0278] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0279] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0280] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0281] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0282] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0283] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0284] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0285] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0286] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0287] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0288] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0289] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 22, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 18 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0290] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0291] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0292] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0293] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0294] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0295] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0296] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0297] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0298] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0299] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0300] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0301] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0302] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0303] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0304] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0305] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0306] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0307] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0308] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first fusion protein comprising the polypeptide sequence of SEQ ID NO: 25, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 18 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 25.

[0309] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 1, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0310] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO:1, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0311] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0312] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0313] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0314] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0315] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 1, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0316] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO:1, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0317] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0318] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 2, a first nucleic acid comprising the sequence of SEQ IDNO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0319] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 4 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0320] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 3, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein a second gRNA comprising a nucleotide sequence of SEQ ID NO: 5 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0321] In certain embodiments, the compositions are designed to target and edit AU-rich sequences in the 3’-UTR of the GDF15 gene.

[0322] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0323] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0324] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0325] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ IDNO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0326] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 22.

[0327] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0328] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0329] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0330] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0331] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence ofSEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0332] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0333] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0334] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0335] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0336] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0337] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0338] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0339] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0340] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 18 and a second nucleic acid comprising the sequence of SEQ ID NO: 23 encoding a second fusion protein.

[0341] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0342] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0343] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 6, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0344] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ IDNO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 11 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0345] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 12 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0346] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 7, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 13 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0347] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0348] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0349] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0350] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 8, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence ofSEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0351] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0352] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0353] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0354] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 9, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0355] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 14 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0356] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 15 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0357] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 16 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0358] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 17 and a second nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a second fusion protein.

[0359] In some embodiments, the composition comprises a first gRNA comprising a nucleotide sequence of SEQ ID NO: 10, a first nucleic acid comprising the sequence of SEQ ID NO: 26 encoding a first fusion protein, a second gRNA comprising a nucleotide sequence of SEQ ID NO: 18 and a second fusion protein comprising the polypeptide sequence of SEQ ID NO: 2.Delivery of gRNAs and Genetic Editing Compositions

[0360] The compositions described herein can be delivered to a cell using any suitable method known in the art or described herein. For example, the gRNAs and polynucleotides encoding a fusion protein can be present on a transposon or a vector. The compositions, transposons, and vectors can be delivered using, for example, lipid nanoparticles.Lipid Nanoparticles

[0361] The compositions of the present disclosure may be encapsulated in at least one lipid nanoparticle comprising at least one cationic lipid. In some aspects, a cationic lipid can be a bioreducible ionizable cationic lipid. In some aspects, the compositions of the present disclosure can be formulated in a lipid nanoparticle.

[0362] In some aspects, a lipid nanoparticle can further comprise at least one structural lipid. In some aspects, a lipid nanoparticle can further comprise at least one phospholipid. In some aspects, a lipid nanoparticle can further comprise at least one PEGylated lipid.

[0363] Accordingly, the compositions of the present disclosure may be encapsulated in at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one cationic lipid, at least one composition of the present disclosure, at least one structural lipid, at least one phospholipid and at least one PEGylated lipid. In some aspects, the lipid nanoparticlecan comprise lipid and one or more nucleic acids of the present disclosure at a specified ratio (weight / weight).

[0364] The compositions disclosed herein can also be delivered to cells or target tissues using one or more lipid nanoparticle compositions and methods of making the same, as described in PCT Application No. PCT / US2023 / 061005 and PCT Publication No. WO 2022 / 182792, which is incorporated herein by reference in its entirety for examples of lipid nanoparticles that may be used to deliver the compositions disclosed herein to their target.Bioreducible Ionizable Cationic Lipids

[0365] In some aspects, a cationic lipid can be a bioreducible ionizable cationic lipid. Accordingly, the compositions of the present disclosure can be encapsulated in at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid.

[0366] As used herein, the term “bioreducible ionizable cationic lipid” is used in its broadest sense to refer to a cationic lipid comprising: at least one tertiary amine, at least one disulfide group, at least one group comprising a bond that is susceptible to cleavage by thioesterification, and further comprising at least two saturated or unsaturated hydrocarbon chains. Exemplary bioreducible ionizable cationic lipids include, but are not limited to, those described in Akita et al., (2020) Biol. Phar. Bull. 43:1617 - 1625, the contents of which are incorporated herein by reference in their entirety.

[0367] Additional exemplary bioreducible ionizable cationic lipids and methods of preparing such lipids useful in the methods of the present disclosure include those disclosed in International Patent Application No. PCT / JP2016 / 052690, published as WO / 2016 / 121942 and International Patent Application No. PCT / JP2019 / 012302, published as WO / 2019 / 188867, the contents of each of which are incorporated herein by reference in their entirety for examples of lipid nanoparticles that may be used to deliver the compositions disclosed herein to their target.

[0368] Accordingly, compositions of the present disclosure may be encapsulated in at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises any one of the bioreducible ionizable cationic lipids put forth in WO / 2016 / 121942 and WO / 2019 / 188867.

[0369] The bioreducible ionizable cationic lipids of the present disclosure are biodegradable, thereby allowing the bioreducible ionizable cationic lipids to be broken down and metabolized in an animal. Without wishing to be bound by theory, this bioreducibility advantageously lessens cationic lipid-associated cytotoxicity.

[0370] In some aspects of the compositions and methods of the present disclosure, a bioreducible ionizable cationic lipid for use in the LNP compositions can be ssPalmO-Ph-P4C2. As would be appreciated by the skilled artisan, ssPalmO-Ph-P4C2 has the following structure:(Formula I)

[0371] As would be appreciated by the skilled artisan, ssPalmO-Ph-P4C2 can also be referred to as Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe and ssPalmO-Ph. Accordingly, ssPalmO-Ph-P4C2, Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe and ssPalmO-Ph are used interchangeably herein to refer to the bioreducible ionizable cationic lipid with the chemical structure put forth in Formula I. Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles. Poly(histidine) (e.g., poly(L-histidine)), is a pH-sensitive polymer due to the imidazole ring providing an electron lone pair on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties through protonation-deprotonation. In particular, at certain pHs, poly(histidine)-containing triblock copolymers may assemble into a micelle with positively charged poly(histidine) units on the surface, thereby enabling complexing with the negatively charged gene editing molecule(s). Using these nanoparticles to bind and release proteins and / or nucleic acids in a pH-dependent manner may provide an efficient and selective mechanism to perform a desired gene modification. In particular, this micelle-based delivery system provides substantial flexibility with respect to the charged materials, as well as a large payload capacity, and targeted release of the nanoparticle payload. In one example, site-specific cleavage of the double stranded DNA is enabled by delivery of a composition disclosed herein using the poly(histidine)-based micelles. Without wishing to be bound by a particular theory, it is believed that in the micelles that are formed by the various triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidine) blocks on the ends to form one or more surrounding layer.

[0372] Without wishing to be bound by theory, three specific segments of ssPalmO-Ph-P4C2 are hypothesized to facilitate its biodegradation. First, the tertiary amine of each piperidinering is an acidic pH-responsive cation-charging unit. Upon endocytosis, the tertiary amine moieties become positively charged in response to the acidic, intracellular endosomal compartment. These are now able to interact and destabilize the membrane and this leads to endosomal escape. Once in the cytosol, the disulfide bond is susceptible to reduction by glutathione generating two free sulfhydryl groups. The resulting high concentration of free thiols further leads to nucleophilic reaction and the particle undergoes self-degradation / collapse via thioesterification and releases the payload in the cytosol. This is defined as HyPER or Hydrolysis accelerated by the intra-Particle Enrichment of Reactant and potentially eliminates the potentially toxic side effects of cationic lipids in general.

[0373] In an aspect, the disclosure provides triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block may be poly(ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example triblock copolymer that can be used is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design.

[0374] Diblock copolymers that can be used as intermediates for making triblock copolymers can have hydrophilic biocompatible poly(ethylene oxide) (PEO), which is chemically synonymous with PEG, coupled to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(ortho esters), poly(peptides), poly(phosphazenes) and poly(saccharides), including but not limited by poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(s-caprolactone) (PCL), and poly (trimethylene carbonate) (PTMC). Polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailablity, and prolonged blood circulatory half-lives.

[0375] Polymeric vesicles, polymersomes and poly(Histidine)-based micelles, including those that comprise triblock copolymers, and methods of making the same, are described in further detail in U. S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U. S. Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO 2019 / 126589, each of which is incorporated herein by reference in its entirety for examples of lipid nanoparticles that may be used to deliver the compositions disclosed herein to their target.

[0376] In some aspects, the composition is encapsulated in at least one lipid nanoparticle comprising: about 40.75% of a terpene lipidoid compound by moles, about 51.75% ofcholesterol by moles, about 5% of DOPC by moles, and about 2.5% of DMG-PEG2000 by moles, wherein a polynucleotide encoding the mutant Cas-Clover is an RNA molecule, and wherein the ratio of lipid to RNA molecule in the at least one nanoparticle is about 120: 1 (w / w).

[0377] In some aspects, the terpene lipidoid compound is HMA-404 and has the following structure:

[0378] Accordingly, in some aspects, the composition is encapsulated in at least one lipid nanoparticle comprising: about 40.75% of HMA-404 by moles, about 51.75% of cholesterol by moles, about 5% of DOPC by moles, and about 2.5% of DMG-PEG2000 by moles, wherein a polynucleotide encoding the mutant Cas-CLOVER is a RNA molecule, and wherein the ratio of lipid to RNA molecule in the at least one nanoparticle is about 120: 1 (w / w).

[0379] Accordingly, in some aspects, the composition is encapsulated in at least one lipid nanoparticle comprising: about 50% of the lipidoid HBC365 by moles, about 38.0% of cholesterol by moles, about 10% of DSPC by moles, and about 2.0% of DMG-PEG2000 by moles, wherein a polynucleotide encoding the mutant Cas-CLOVER is a RNA molecule, and wherein the ratio of lipid to RNA molecule in the at least one nanoparticle is about 50: 1 (w / w).

[0380] The lipidoid HBC365 is disclosed in international Patent Application No. PCT / US2024 / 012245 and has the structure:

[0381] In some aspects, a composition of the present disclosure is encapsulated in at least one lipid nanoparticle comprising: about 54% of SS-OP by moles, about 35% of cholesterol by moles, about 5% of DOPC by moles, about 5% of DSPC by moles, and about 1% of DMG-PEG2000 by moles. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle can be about 100:1 (w / w).

[0382] In some aspects, a composition of the present disclosure is encapsulated in at least one lipid nanoparticle comprising: about 50% HBC365 by moles, about 38% cholesterol by moles, about 10% DSPC by moles, and about 2% DMG-PEG2000 by moles. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle can be about 50: 1 (w / w).In some embodiments, the LNP comprises a total lipid concentration of abound 25 nM. In some aspects, a lipid nanoparticle can comprise lipid and nucleic acid at a specified ratio (weight / weight). In some aspects, the lipid to nucleic acid ratio is 50: 1.

[0383] In some aspects, an mRNA of the present disclosure further comprises a 5’-CAP. In some aspects, an mRNA molecule can be capped using any method and / or capping moiety known in the art. An mRNA molecule can be capped with m7G(5')ppp(5')G moiety. A m7G(5')ppp(5')G moiety is also referred to herein as a “CapO”. An mRNA molecule can be capped with a CleanCap® moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. A CleanCap® moiety can comprise a m7(3’OMeG)(5’)ppp(‘5)m6(2’OMeA)pG (CleanCap® M6) moiety. An mRNA moleculecan be capped with an anti-reverse cap analog (ARCA®) moiety. An ARCA® moiety can comprise a m7(3'-0-methyl)G(5')ppp(5')G moiety. An mRNA molecule can be capped with a CleanCap® 3'0Me moiety (CleanCap®+ARCA®). In some aspects, an mRNA molecule can comprise a 5 CleanCap® AG moiety. In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0384] As described herein, the LNPs encapsulating the compositions of the present disclosure that comprise at least one bioreducible ionizable cationic lipid advantageously exhibit significantly reduced toxicity in animals as compared to LNPs comprising non-bioreducible ionizable cationic lipids. In particular, administration of the LNPs of the present disclosure surprisingly does not result in any body weight loss. In some aspects, certain LNPs of the present disclosure are sufficiently non-toxic that animals administered the LNPs actually gain body weight, even when administered amounts of LNPs that exceed the lethal dose of LNP comprising non-bioreducible ionizable cationic lipids.LNP Components

[0385] The LNPs disclosed herein may comprise one or more structural lipids, one or more phospholipids, and / or one or more pegylated lipids.Structural Lipids

[0386] In some aspects, a structural lipid can be a steroid. In some aspects, a structural lipid can be a sterol. In some aspects, a structural lipid can comprise cholesterol. In some aspects, a structural lipid can comprise ergosterol. In some aspects, a structural lipid can be a phytosterol.Phospholipid

[0387] As used herein, the term “phospholipid” is used in its broadest sense to refer to any amphiphilic molecule that comprises a polar (hydrophilic) headgroup comprising phosphate and two hydrophobic fatty acid chains.

[0388] In some aspects of the LNPs of the present disclosure, a phospholipid can comprise di ol eoy Iphosphati dy 1 ethanol amine (DOPE).

[0389] In some aspects of the LNPs of the present disclosure, a phospholipid can comprise DOPC (l,2-Dioleoyl-sn-glycero-3-phosphocholine).

[0390] In some aspects of the LNPs of the present disclosure, a phospholipid can comprise DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine).PEGylated Lipid

[0391] As used herein, the term “PEGylated lipid” is used to refer to any lipid that is modified (e.g., covalently linked to) at least one polyethylene glycol molecule. In some aspects, a PEGylated lipid can comprise l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000, hereafter referred to as DMG-PEG2000.Cells and Modified Cells of the Disclosure

[0392] In another aspect, provided herein are cells that are modified using the compositions and methods disclosed herein. In preferred embodiments, the cells are modified to increase the expression of FGF21 and / or GDF15.

[0393] Cells and modified cells of the disclosure can be mammalian cells. The cells and modified cells are human cells. In some embodiments, the cells can comprise liver hepatocytes. In some embodiments, the hepatocytes may be modified ex vivo and subsequently transplanted into a subject in need thereof. In some embodiments, the cell is not a germ cell. In some embodiments, the cell is not a human germ cell.

[0394] Cells that have been altered ex vivo according to this disclosure can be manipulated (e.g., expanded, passaged, frozen, differentiated, de-differentiated, transduced with a transgene, etc.) prior to their delivery to a subject. The cells can be delivered to a subject from which they are obtained (in an “autologous” transplant), or to a recipient who is immunologically distinct from a donor of the cells (in an “allogeneic” transplant).

[0395] The compositions disclosed herein are preferably delivered in the form of a nucleic acid molecule comprising the first and / or second gRNA a the nucleic acid sequence encoding the first and / or second fusion protein, but can be implemented or delivered to the cells in any suitable format, including as a ribonucleoprotein complex, as separated protein and nucleic acid components, and / or as nucleic acids encoding the components of the genome editing system.In Vivo Modification of Cells

[0396] The disclosure provides a method of modifying a population of cells comprising contacting the population of cells with the compositions of the disclosure (e.g., a composition comprising a nucleic acid sequence encoding a first Cas-CLOVER fusion protein and first gRNA, and a nucleic acid sequence encoding a second Cas-CLOVER fusion protein and second gRNA compositions). Once delivered to the cell, the first gRNA preferably forms acomplex with the first fusion protein, and the second gRNA forms a complex with the second fusion protein. The complexes may then associate with the target sequence in the cell’s DNA and the nucleases can generate an indel between the first targeting sequence and the second targeting sequence, producing a modified population of cells.

[0397] In some embodiments, the method of modifying a population of cells comprises modifying the genomes of a population of cells. In some embodiments, the method of modifying the genome of a population of cells comprises contacting the population of cells with a composition of the present disclosure. In some embodiments the first and second fusion proteins are expressed by each cell of the population, wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in each cell of the population and the second gRNA specifically binds to a second strand of a second double-stranded DNA target sequence in each cell of the population. In some embodiments, the first and second fusion proteins introduces a modification into the genome of one or more cells in the population. In some embodiments, the modification is an indel between the first and second target sequences.

[0398] In some aspects, the population of cells is contacted and modified in vivo. In some aspects, the population of cells is contacted and modified in vitro.

[0399] In some aspects, the targeting sequence is in the FGF21 gene. In some aspects, the targeting sequence is one or more AU-rich element(s) of the FGF21 gene. In some embodiments, the AU-rich element is located in the 3’-UTR of the FGF21 mRNA. In some embodiments, the indel is generated in the AU-rich element of the FGF21 gene. In some embodiments, the indel causes a mutation or a deletion of the AU-rich element of the FGF21 gene resulting in enhanced FGF21 protein expression.

[0400] In some aspects, the targeting sequence is at the GDF15 gene. In some aspects, the targeting sequence is one or more AU-rich element(s) of the GDF15 gene. In some embodiments, the AU-rich element is located in the 3’-UTR of the GDF15 mRNA. In some embodiments, the indel is generated at the AU-rich element of the GDF15 gene. In some embodiments, the indel causes a mutation or deletion of the AU-rich element of the GDF15 gene resulting in enhanced GDF15 protein expression.

[0401] In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% ofthe target cells, e.g., liver hepatocytes, include an indel between the first targeting sequence and the second targeting sequence. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which 20-90%, 30-80%, 40-70%, or 50-60% of the target cells include an indel between the first targeting sequence and the second targeting sequence. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the target cells include an indel between the first targeting sequence and the second targeting sequence. In some embodiments, the first targeting sequence, the second targeting sequence, or the first and second targeting sequence is at the FGF21 gene. In some embodiments, the first targeting sequence, the second targeting sequence, or the first and second targeting sequence is at the GDF15 gene.

[0402] In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells comprise an edited FGF21 or GDF15 gene. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which 15-90%, 30-80%, 40-70%, or 50-60% of cells include edited FGF21 or GDF15 gene. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells comprise an edited FGF21 or GDF15 gene. In some embodiments, the disclosure relates to compositions including a plurality of cells generated by the method disclosed herein, in which at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells comprise an edited FGF21 or GDF15 gene. In some embodiments, the disclosure relates to compositions including a plurality of cells generated by the method disclosed herein, in which about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells comprise an edited FGF21 or GDF15 gene. In some embodiments, the disclosure relates to compositions including a plurality of cells generated by the method disclosed herein, in which 15-90%, 30-80%, 40-70%, or 50-60% of the cells comprise an edited FGF21 or GDF15 gene.

[0403] In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase of FGF21 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is about a 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% increase of FGF21 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is a 15%-99%, 30-95%, 40-90%, or 60-80% increase of FGF21 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 99%, or 100% have increased FGF21 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 99%, or 100% have increased FGF21 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is a 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold increase of FGF21 protein relative to an unmodified population of cells.

[0404] In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase of secreted GDF15 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is about a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase of secreted GDF15 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is a 15-99%, 30-95%, 40-90%, or 60-80% increase of GDF15 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 99%, or 100% have an increase of GDF15 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which at about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 99%, or 100% have an increase of GDF15 protein relative to an unmodified population of cells. In some embodiments, the disclosure relates to a plurality of cells generated by the method disclosed herein, in which there is a 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold increase in GDF15 protein relative to an unmodified population of cells.

[0405] The unmodified population of cells may be, for example, a population of cells prior to modification by one or gene editing compositions of the present disclosure, or a population of cells that did not receive a gene composition of the present disclosure.Formulations, Dosages and Modes of Administration

[0406] Genome editing systems, or cells altered or manipulated using such systems, can be administered to subjects by any suitable mode or route, whether local or systemic. Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intramarrow, intrarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Components administered systemically can be modified or formulated to target, e.g., HSCs, hematopoietic stem / progenitor cells, or erythroid progenitors or precursor cells.

[0407] Local modes of administration include, by way of example, intramarrow injection into the trabecular bone or intrafemoral injection into the marrow space, and infusion into the portal vein. In certain embodiments, significantly smaller amounts of the components (compared with systemic approaches) can exert an effect when administered locally (for example, directly into the bone marrow) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

[0408] Administration can be provided as a periodic bolus (for example, intravenously) or as a continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag or implantable pump). Components can be administered locally, for example, by continuous release from a sustained release drug delivery device.

[0409] In addition, components can be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material that releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems can be useful, however, the choice of the appropriate system will depend upon the rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic.However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.

[0410] Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymerspolyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.

[0411] Poly(lactide-co-glycolide) microsphere can also be used. Typically, the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein. In some embodiments, genome editing systems, system components and / or nucleic acids encoding system components, are delivered with a block copolymer such as a poloxamer or a poloxamine.Methods of Using the Compositions of the Disclosure

[0412] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of the composition or pharmaceutical composition. In one aspect,the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0413] The disclosure provides a method for modulating or treating at least one disease or disorder in a cell, tissue, organ, animal or subject by upregulating serum FGF21 protein concentrations. Preferably, the disease or disorder is obesity or complications associated with obesity.

[0414] In some embodiments, the disclosure provides methods for increasing the concentration ofFGF21 protein in a biological sample from a subject comprising administering to the subject, a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first double-stranded DNA target sequence and the second double-stranded DNA target sequence target sequence. In some embodiments, the indel results in an increase in the concentration of the FGF21 protein in the biological sample from the subject.

[0415] In some embodiments, the disclosure provides methods for increasing the concentration of GDF15 protein in a biological sample from a subject comprising administering to the subject, a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second double-stranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first double-stranded DNA target sequence and the second double-stranded DNA target sequence target sequence. In some embodiments, the indelresults in an increase in the concentration of the GDF15 protein in the biological sample from the subject.

[0416] In some embodiments, the biological sample is a bodily fluid. In some embodiments, the bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof. For example, the bodily fluid can be urine, blood, serum, or cerebrospinal fluid. In some embodiments, the biological sample is a tissue sample from the subject.

[0417] In some embodiments, the disclosure provides methods for increasing the serum concentration of FGF21 protein in a subject comprising administering to the subject, a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first double-stranded DNA target sequence and the second double-stranded DNA target sequence target sequence. In some embodiments, the indel results in an increase in serum concentration of the FGF21 protein in the subject.

[0418] In some embodiments, the disclosure provides methods for increasing the serum concentration of GDF15 protein in a subject comprising administering to the subject, a therapeutically effective amount of a composition of the present disclosure. In some embodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second double-stranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first double-stranded DNA target sequence and the second double-stranded DNA target sequence target sequence. In some embodiments, the indel results in an increase in serum concentration of the GDF15 protein in the subject.

[0419] Levels of FGF21 and / or GDF15 in a biological sample from a subject may be determined using any suitable method known in the art or described herein (e.g., ELISA or Western Blotting). The serum levels can be determined at any suitable time point, e.g, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4, weeks or 1, 2, 3, 4, 5, or 6 months after administration of the composition disclosed herein.

[0420] The disclosure provides a method for modulating or treating at least one disease or disorder in a cell, tissue, organ, animal or subject by upregulating serum GDF15 protein concentrations. The disclosure also provides a method for modulating or treating at least one disease or disorder in a cell, tissue, organ, animal or subject by upregulating serum FGF21 protein concentrations. Preferably, the methods disclosed herein improve one or more obesity-related metabolic complications, including insulin resistance, dyslipidemia, cardiovascular disorders, type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), nonalcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH). In some embodiments, a method disclosed herein results in a reduction in fat mass and alleviation of hyperglycemia.

[0421] The compositions of the disclosure may be used to treat a disease or disorder by use of a therapeutic transgene encoding an exogenous nucleic acid sequence or exogenous amino acid sequence. For certain diseases or disorders, the therapeutic transgene can include the FGF21 gene or the GDF15 gene. The FGF21 and GFD15 coding sequences, including 5’-UTR and 3’-UTR sequences, are shown in SEQ ID NOs: 19 and 20, respectively. TheFGF27 gene or the GDF15 gene may be edited to mutate or delete one or more AU-rich elements in the 3’-UTR to improve mRNA stability and / or protein expression. In some embodiments, a composition of the present disclosure is for use in modifying an FGF21 gene. In some embodiments, a composition of the present disclosure is for use in modifying a GDF15 gene.

[0422] In some embodiments, the disclosure provides methods treating a disease or disorder mediated by FGF21 in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure. In someembodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first double-stranded DNA target sequence and the second double-stranded DNA target sequence target sequence.

[0423] In some embodiments, the indel results in the reduction or alleviation of one or more symptoms of the disease or disorder in the subject. In some embodiments, the reduction or alleviation of one or more symptoms of the disease or disorder is a reduction in fat mass and / or alleviation of one or more of hyperglycaemia, insulin resistance, dyslipidaemia, cardiovascular disorders, type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0424] In some embodiments, the disclosure provides methods treating a disease or disorder mediated by GDF15 in a subject in need thereof comprising administering to the subject, a therapeutically effective amount of a composition of the present disclosure. In some embodiments the disease or disorder is obesity. In some embodiments, the first and second fusion proteins are expressed in at least one cell of the subject. In some embodiments the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject and the second gRNA specifically binds to a second strand of a second double-stranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an insertion or deletion (“indel”) between the first doublestranded DNA target sequence and the second double-stranded DNA target sequence target sequence. In some embodiments, the indel results in the reduction or alleviation of one or more symptoms of obesity in the subject. In some embodiments, the reduction or alleviation of one or more symptoms of obesity in the subject is weight loss.

[0425] In some embodiments, the disclosure provides methods of treating obesity in a subject comprising administering to the subject, a therapeutically effective amount of a compositionof the present disclosure. In some embodiments, the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein. In some embodiments, the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject. In some embodiments, the second gRNA specifically binds to a second strand of a second double-stranded DNA target sequence in the at least one cell of the subject. In some embodiments, the first fusion protein and the second fusion protein introduce an indel between the first double-stranded DNA target sequence and the double stranded DNA target sequence target sequence. In some embodiments, the method results in reduction or alleviation of one or more symptoms of obesity, optionally wherein the method results in weight loss.Definitions

[0426] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0427] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0428] The disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated"polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0429] Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and / or repeat units in target vectors that can subsequently be assembled into a final destination vector. Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector. The disclosure provides polypeptides produced by this method as well as nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced by this modular approach.

[0430] The term “binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0431] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

[0432] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0433] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0434] “Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0435] The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.

[0436] A “target site” or “target sequence” or “targeting sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0437] Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

[0438] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or moremutations, substitutions, deletions, or insertions that do not naturally occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally occur, rendering the entire nucleic acid sequence non-naturally occurring.

[0439] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotide sequences are contemplated herein.

[0440] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits the calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U. S. Patent No. 4,554,101, incorporated fully herein by reference.

[0441] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example, immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0442] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables 3, 4, or 5 below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions that have been introduced bymodification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 3.Table 3 — Conservative Substitutions I

[0443] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N. Y. (1975), pp. 71-77) as set forth in Table 4.Table 4 — Conservative Substitutions II

[0444] Alternately, exemplary conservative substitutions are set out in Table 5.Table 5 — Conservative Substitutions III

[0445] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitutions.

[0446] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally occur, rendering the entire amino acid sequence non-naturally occurring.

[0447] As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms "identical" or "identity" when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of a single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0448] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequences naturally associated with a target gene or a host cell into which it is introduced.

[0449] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequences not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non-naturally occurring genome location.

[0450] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. "Introducing" is intended to present to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs intobacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0451] As used herein, the term “isolated” or the like refers to a cell, or a population of cells, which has been separated from its original environment, i.e., the environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist. The term includes a cell that is removed from some or all components as it is found in its natural environment, for example, tissue, or biopsy. The term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, culture, cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated cells include partially pure cells, substantially pure cells and cells cultured in a medium that is non-naturally occurring. Isolated cells may be obtained from separating the desired cells, or populations thereof, from other substances or cells in the environment, or from removing one or more other cell populations or subpopulations from the environment. As used herein, the term “purify” or the like refers to increased purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0452] A “therapeutically sufficient amount” or a “therapeutically effective amount”, as used herein, includes within its meaning a non-toxic but sufficient and / or effective amount of the particular therapeutic and / or pharmaceutical composition to which it is referring to provide a desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the patient's general health, the patient's age and the stage and severity of the condition. In particular embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with a disease or condition of the subject being treated.EXAMPLESEXAMPLE 1: Design and Synthesis of gRNA Pairs Targeting AU-Rich Elements in the 3’-UTR of the Human FGF21 Gene

[0453] A set of 6 gRNA pairs targeting AU-rich elements within the 3’-UTR of the human FGF21 gene were designed and the targeting sequence of each gRNA is shown in Table 1.

[0454] The 6 gRNA pairs comprising the targeting sequences of Table 1 were synthesized, purified, and resuspended in RNAse free water and stored at -70°C until use.EXAMPLE 2: Design and Synthesis of gRNA Pairs Targeting AU-Rich Elements in the 3’-UTR of the Human GDF15 Gene

[0455] A set of 20 gRNA pairs targeting AU-rich elements within the 3’-UTR of the human GDF 15 gene were designed and the targeting sequence of each gRNA is shown in Table 2.

[0456] The 20 gRNA pairs comprising the targeting sequences of Table 2 were synthesized, purified, and resuspended in RNAse free water and stored at -70°C until use.EXAMPLE 3: In Vitro Cas-CLO VER Editing of AU-Rich Elements of Human FGF21 Gene in Huh7 Cell Line

[0457] A subset of the gRNA pairs targeting AU-rich elements of the human FGF21 gene, gRNA Pairs #F3 and #F4 (Table 1), were screened in the Huh7 cell line to determine the percent of Cas-CLO VER editing of the FGF21 locus for each gRNA pair. A gRNA pair targeting the KLKB1 locus was used as a negative control.

[0458] Briefly, 150,000 Huh7 cells were transfected with 2.5 pg of an mRNA encoding Cas-CLOVER v3.0 (full length mRNA sequence (SEQ ID NO: 26) encodes Cas-CLO VER v3.0 (SEQ ID NO: 25)) and 1 pg of each gRNA pair using lipofectamine (MessengerMax™, Thermo-Fisher), and cultured in DMEM medium supplemented with 10% FBS. After 72 hours, the supernatant was harvested to determine secreted FGF21 protein concentrations, the cells were lysed and total RNA was isolated from the cell extracts using an RNeasy Plus Mini Kit (Qiagen) in accordance with the manufacturer’s instructions.

[0459] The amount of FGF21 mRNA was quantified by quantitative polymerase chain reaction (qPCR), and the relative fold change in the expression levels of FGF21 mRNA compared to mock treated samples was determined. The results are shown in Table 6.Table 6: Relative Fold Expression of FGF21 mRNA in Mock and Edited Cells

[0460] As shown in Table 6, editing the AU-rich elements in the FGF21 3’-UTR sequence results in an increase in the relative expression of FGF21 mRNA with FGF21 Pair #F4, exhibiting a 5-fold increase in FGF21 mRNA.

[0461] In addition, the amount of FGF21 protein present in the supernatants of collected samples was determined by ELISA (Thermo Fisher Scientific), and the results (pg / ml) are shown in Table 7.Table 7: FGF21 Secreted Protein Levels in Mock and Edited Cell Supernatants

[0462] As shown in Table 7, FGF21 Pair #F4 exhibited a modest increase in FGF21 protein levels in the supernatant of treated samples.

[0463] Genomic DNA was isolated from the cell extracts using a Monarch Genomic DNA Purification Kit (New England Biolabs®) in accordance with the manufacturer’s instructions. The percentage of indels at the FGF27 locus was determined by amplicon sequencing (amplicon-seq) for each of the gRNA pairs.

[0464] Briefly, genomic DNA samples were subjected to PCR amplification using DNA primers flanking the edit site at the FGF21 gene that further contain Illumina partial adapters. The resulting PCR amplicons underwent a second PCR reaction using primers containing Illumina P5 and P7 sequences (Illumina Corp.) and a unique index sequence (New England Biolabs®). The final amplicons were pooled at equimolar concentrations and analyzed using a Miseq benchtop sequencer following standard procedures for amplicon-seq according to the manufacturer (Illumina Corp). Sequence data were analyzed using a CRISPResso2 program to determine the frequency of indels in each sample. The results are shown in Table 8.Table 8. Percent Indels at FGF21 locus using Cas-CLOVER and targeted gRNA pairs

[0465] As shown in Table 8, FGF21 Pairs #F3 and #F4 were capable of editing the AU-rich element target sequences in Huh7 cells resulting 15.7 and 36.3 percent indels at the FGF21 locus.

[0466] Without wishing to be bound by theory, these data demonstrate that even though the FGF21 targeting gRNAs were capable of editing between only 15 (#F3) and 36 (#F4) percent indels at the FGF21 locus, FGF21 Pair #F4 was capable of increasing mRNA and secreted FGF21 protein levels inHuh7 cells which could be beneficial for treating a wide range of metabolic disorders.EXAMPLE 4: In Vitro Cas-CLOVER Editing of AU-Rich Elements Located in the 3’-UTR of the Human GDF15 Gene in HepG2 Cell Line

[0467] The gRNA pairs targeting AU-rich elements of the human GDF15 gene, #G1 -#G20, were screened in the HepG2 hepatocyte cell line to determine the relative amount of secreted GDF15 protein and GDF15 mRNA expression relative to control for each of the 20 gRNA pairs. A gRNA pair targeting the KLKB1 locus was used as a negative control. Briefly, 250,000 HepG2 cells were transfected with 2.5 pg of an mRNA encoding Cas-CLOVER v3.0 (full length mRNA sequence (SEQ ID NO: 26) encodes Cas-CLOVER v3.0 (SEQ ID NO: 25)) and 1 pg of gRNA pairs using lipofectamine (MessengerMax™, Thermo-Fisher), and cultured in EMEM medium supplemented with 10% FBS. After 72 hours, the medium was removed, cells were re-seeded into fresh media, and the cells were cultured for an additional 48 hours. After that time, the supernatant was harvested to determine secreted GDF15 protein concentrations, the cells were lysed and total RNA was isolated from cell extracts using an RNeasy Plus Mini Kit (Qiagen) in accordance with the manufacturer’s instructions.

[0468] The amount of GDF15 mRNA was quantified by qPCR, and the relative fold change in expression levels of GDF15 mRNA compared to KLKB1 control samples was determined. The results are shown in FIG. IB.

[0469] As shown in FIG. IB, several gRNA pairs exhibited a 2-4-fold increase in relative fold expression of GDF15 mRNA in HepG2 cells. gRNA Pairs #G11-20, which comprise the L04 (SEQ ID NO: 9) or L05 (SEQ ID NO: 10) left gRNA, appear to show the greatest increase in relative fold expression change of GDF15 mRNA.

[0470] In addition, the amount of GDF15 protein present in the supernatants of collected samples was determined by ELISA (R& D Systems), and the results (ng / ml) are shown in FIG. 1A.

[0471] As shown in FIG. 1 A, several gRNA pairs exhibited 3-4-fold increase in secreted GDF15 protein levels. Similar to the increased relative expression of GDF15 mRNA, gRNA Pairs #G11-G20, which comprise the L04 (SEQ ID NO: 9) or L05 (SEQ ID NO: 10) left gRNA, appear to show the greatest increase in secreted GDF15 protein levels.

[0472] Without wishing to be bound by theory, the data taken together demonstrate that editing of the AU-rich elements located in the 3’-UTR of the GDF15 gene can increase GDF15 mRNA expression and amounts of secreted GDF15 protein in HepG2 cells.EXAMPLE 5: Preparation of 5’-Capped mRNA Encoding Cas-CLOVER for Encapsulation in LNP Compositions

[0473] The following is a non-limiting example demonstrating the preparation of an exemplary mRNA encoding Cas-CLOVER that may be incorporated in LNP compositions for use in methods of the present disclosure.

[0474] The DNA plasmid “pRTb_Cas-CLOVERv3” encodes Cas-CLOVERv3 (amino acid sequence - SEQ ID NO: 27). The DNA plasmid “pRTb_Cas-CLOVERv3” encodes a Cas-CLOVERv3 (SEQ ID NO: 25) comprising an N-terminal SV40 nuclear localization signal (NLS) and containing the 5’UTR of the human beta-globin gene (HBB) and the 3’-UTR of the human cytochrome b-245 alpha chain gene (CYBA). This plasmid was used as a template for in vitro transcription reactions to produce mRNA encoding Cas-CLOVERv3 further comprising a 5'-CAP (full length mRNA sequence - SEQ ID NO: 26).

[0475] Briefly, approximately 300 pg of supercoiled pRTb_Cas-CLOVERv3 was added to a 15 mL conical comprising 300 pL CutSmart® Buffer, 60 pL of the restriction enzyme Bbsl-HF in 3000pL total volume. The plasmid DNA was linearized by incubating at 37°C overnight to ensure complete digestion.

[0476] The linearized plasmid was purified using a DNA QIAquick PCR purification kit according to the manufacturer's instructions, and the purified DNA was eluted in 900 pL ofnuclease-free water. The DNA concentration and purity of the eluate were determined using a NanoDrop® microvolume spectrophotometer in accordance with the manufacturer's instructions.

[0477] The purified plasmid was used as a DNA template to produce mRNA using the custom in vitro transcription mMESSAGE mMACHINE™ T7 Transcription Kit in accordance with internal, quality-controlled manufacturing batch records. Briefly, 100 mM stocks of the nucleotides GTP, ATP, CTP, NlMeTTP (Nl-Methylpseudouridine-5'-Triphosphate ), and CleanCap® Reagent AG (m7G(5')ppp(5')(2'OMeA)pG; Trilink®) were prepared. 1,485 pL each of ATP, UTP, and 5MeC and 1,188 pL each of GTP and CleanCap® Reagent AG were blended.

[0478] For the in vitro transcription (IVT) reaction, 153 pg of linearized pRTb Cas-CLOVERv3 DNA, 1,800 pL of 10X T7 RXN Buffer, 1,800 pL of T7 Enzyme mix, and 6,831 pL of the NTP and cap blend were added to a 50 mL conical (18,000 pL final volume) and incubated at 37°C for 3 hours. A 900 pL aliquot of DNasel enzyme was added and the tube further incubated at 37°C for 15 min to degrade the DNA template.

[0479] A poly(A) tail was post-enzymatically added to the 3' end of the 5'-CleanCap®-Cas-CLOVER-NlMeT mRNA. 18 mL of 5X E. coli Poly(A) Polymerase (E-PAP) Buffer, 9 mL of 25 mM MnCh, 9 mL ATP Solution, and 3,000 pL E-PAP, were added to the IVT reaction (90,000 pL total volume), and incubated at 37°C for 1 hour. The bulk E-PAP reaction was subsequently divided into three 125 mL PETG bottles in 30 mL aliquots.

[0480] The 5'-CleanCap®-Cas-CLOVER-poly(A)- NlMe'P mRNA was purified using a RNeasy Maxi Purification Kit according to the manufacturer's instructions. Briefly, a working stock of Buffer RLT (Qiagen) was formulated using 178.2 mL of Buffer RLT with 1.8 mL of 2-mercaptoethanol (BME). 52.2 mL of the BME+RLT solution and 37.8 mL of 100% EtOH were added to each 30 mL mRNA aliquot. The purified mRNA product was eluted in 52.5 mL of nuclease-free water, and the bulk product was stored at -80°C. The DNA linearization, IVT, and mRNA purification process is repeated until the target yield is reached.

[0481] Bulk mRNA lots were analyzed using gel electrophoresis before combination in a 500 mL PETG bottle and sampled for concentration readings using the NanoDrop®. Lithium Chloride 5X was added to the pooled mRNA in the amount of 1 / 3 of the total volume of the mRNA, then divided into equal 40 mL aliquots in 50 mL conical tubes and incubated at -20°C for 45 minutes. Directly following incubation, the conical tubes were centrifuged at 14,000 x g for 30 minutes at 4°C. The mRNA pellet is washed using 70% EtOH three times.

[0482] The washed mRNA pellets are dried, and then resuspended in nuclease-free water. The mRNA concentration was determined using the NanoDrop®, and additional nuclease-free water was added as necessary to further dilute the product to the target concentration. The mRNA is sterile filtered using a 0.22 pm PES SteriCup® Filter before the final mRNA concentration and purity are measured on the NanoDrop®.EXAMPLE 6: Preparation of an LNP Composition Comprising a 5’-Capped mRNA Encoding Cas-CLOVER and a gRNA Pair

[0483] The following is a non-limiting example that provides exemplary methods for formulating an LNP composition comprising a 5’-capped mRNA encoding Cas-CLOVER v3.0 (SEQ ID NO: 26) and a FGF21 gRNA pair for use in combination to edit the AU-rich elements of the 3-UTR of the FGF21 gene.

[0484] Individual 25 mg / ml stock solutions were prepared by solubilizing the lipids in 200-proof HPLC-grade ethanol and stock solutions were stored at -80° C until formulated. At the time of formulation, the lipid stock solutions were briefly allowed to equilibrate to room temperature and then placed on a hot plate maintained at a temperature range of 50-55°C. Subsequently, the hot lipid stock solutions were combined to yield desired final molar percentages.

[0485] A 1 mg / ml solution of the 5’CleanCap®-Nl-CC mRNA (Cas-CLOVER v3.0; SEQ ID NO: 26) to be incorporated into the LNPs was added to 150 mM sodium acetate buffer (pH 5.2) to form a stock solution and kept on ice. A 1 mg / ml solution of the gRNA Pair dissolved in RNAse free water to be incorporated into the LNPs was added to 150 mM sodium acetate buffer (pH 5.2) to form a gRNA stock solution and kept on ice. mRNA and gRNA stock solutions were mixed at a 3: 1 ratio to form a nucleic acid stock solution. The lipid phase was mixed with the aqueous mRNA / gRNA phase inside a microfluidic chip using a NanoAssemblr® instrument according to the manufacturer's instructions to form LNP compositions comprising encapsulated Cas-CLOVER mRNAs and a targeting gRNA pair. NanoAssemblr® process parameters for mRNA encapsulation were at a flow rate of 20 ml / min and at a lipid: RNA ratio (v / v) of 1:3.

[0486] The resultant Cas-CLOVER mRNA-gRNA pair LNP compositions were then transferred to a Repligen Float-A-Lyzer® dialysis device having a molecular weight cut off(MWCO) of 8-10kDa and processed by dialysis against 25 mM sodium acetate (dialysate: dialysis buffer volume at least 1:200 v / v), pH 5.5 overnight at 4°C (or alternatively room temperature for at least 4 hours), to remove the 25% ethanol and achieve a complete buffer exchange. Where applicable, the LNP compositions were further concentrated using an Amicon® Ultra-4 centrifugal filter unit, MWCO-30kDa spun at -4100 x g in an ultracentrifuge. Sucrose was added to a final concentration of 5% (w / v) to the mRNA LNPs which were then stored at 4°C or frozen at -80°C until further use. The average particle size diameter of the LNPs ranged from approximately 85-105 nm.Ill

Claims

CLAIMS1. A composition comprising:(a) a first guide RNA (gRNA) comprising a first targeting sequence set forth in SEQ IDNOs: 1-3;(b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5;(c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or nuclease domain thereof; and(d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or nuclease domain thereof.

2. A composition comprising:(a) a first guide RNA (gRNA) comprising a first targeting sequence set forth in SEQ IDNOs: 6-10;(b) a second gRNA comprising a second targeting sequence set forth in SEQ ID NOs: 11-18;(c) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first inactivated Cas9 domain, or nuclease domain thereof, and a first Clo051 domain, or nuclease domain thereof; and(d) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second inactivated Cas9 domain, or nuclease domain thereof, and a second Clo051 domain, or nuclease domain thereof.

3. The composition of claim 1, wherein(a) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(b) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(c) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(d) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(e) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4; or(f) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5.

4. The composition of claim 2, wherein(a) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(b) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(c) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(d) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(e) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(f) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(g) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(h) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(i) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(j) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(k) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(l) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(m) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(n) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(o) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(p) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(q) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(r) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17; or(s) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 18.

5. The composition of any one of claims 1-4, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, and / orthe second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25.

6. The composition of any one of claims 1-5, wherein the polynucleotide encoding the first fusion protein is an mRNA comprising the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 26, and / orthe polynucleotide encoding the second fusion protein is an mRNA comprising the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 26.

7. The composition of claim 6, wherein the mRNA comprises a 5 ’-cap.

8. The composition of any one of claims 1-7, wherein the first inactivated Cas9 domain, or nuclease domain thereof is derived from a Streptococcus pyogenes Cas9 polypeptide, and / orthe second inactivated Cas9 domain, or nuclease domain thereof is derived from a Streptococcus pyogenes Cas9 polypeptide.

9. The composition of any one of claims 1-8, wherein the first gRNA comprises a spacer sequence and a scaffold sequence isolated from Streptococcus pyogenes, and / orthe second gRNA comprises a spacer sequence and a scaffold sequence isolated from Streptococcus pyogenes.

10. The composition of claim 9, wherein the scaffold sequence comprises the nucleic acid sequence of SEQ ID NO: 35.

11. The composition of any one of claims 1-10, wherein the C-terminus of the first fusion protein further comprises a linker comprising the sequence set forth in SEQ ID NO: 21 between the first inactivated Cas9 domain, or nuclease domain thereof and the first Clo051 domain, or nuclease domain thereof, and / orthe C-terminus of the second fusion protein further comprises a linker comprising the sequence set forth in SEQ ID NO: 21 between the second inactivated Cas9 domain, or nuclease domain thereof and the second Clo051 domain, or nuclease domain thereof.

12. The composition of any one of claims 1-11, wherein the first gRNA comprises one or more chemical modifications of a ribonucleotide, a ribonucleotide base, or a phosphodiester bond, and / orthe second gRNA comprises one or more chemical modifications of a ribonucleotide, a ribonucleotide base, or a phosphodiester bond.

13. The composition of claim 12, wherein the one or more chemical modifications comprises a phosphorothioate bond.

14. The composition of claim 12 or claim 13, wherein the 5’ and / or 3’ terminus of the first gRNA comprises at least two consecutive phosphorothioate bonds, and / orthe 5’ and / or 3’ terminus of the second gRNA comprises at least two consecutive phosphorothioate bonds.

15. The composition of any one of claims 12-14, wherein the 5’ and / or 3’ terminus of the first gRNA comprises at least one 2’ O-Me chemical modification, and / orthe 5’ and / or 3’ terminus of the second gRNA comprises at least one 2’ O-Me chemical modification.

16. The composition of any one of claims 1-15, wherein the first and second polynucleotides comprise the sequence set forth in SEQ ID NO: 23, and wherein(a) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(b) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(c) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(d) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(e) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(f) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(g) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(h) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(i) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(j) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(k) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(l) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(m) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(n) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(o) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(p) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(q) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(r) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(s) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(t) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(u) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(v) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(w) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(x) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17; or(y) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 18.

17. The composition of any one of claims 1-15, wherein the first and second polynucleotides comprise the sequence set forth in SEQ ID NO: 26, and wherein(a) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(b) the first gRNA comprises the sequence set forth in SEQ ID NO: 1 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(c) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(d) the first gRNA comprises the sequence set forth in SEQ ID NO: 2 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(e) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 4;(f) the first gRNA comprises the sequence set forth in SEQ ID NO: 3 and the second gRNA comprises the sequence set forth in SEQ ID NO: 5;(g) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(h) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(i) the first gRNA comprises the sequence set forth in SEQ ID NO: 6 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(j) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 11;(k) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 12;(l) the first gRNA comprises the sequence set forth in SEQ ID NO: 7 and the second gRNA comprises the sequence set forth in SEQ ID NO: 13;(m) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(n) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(o) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(p) the first gRNA comprises the sequence set forth in SEQ ID NO: 8 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(q) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(r) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(s) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(t) the first gRNA comprises the sequence set forth in SEQ ID NO: 9 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17;(u) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 14;(v) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 15;(w) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 16;(x) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 17; or(y) the first gRNA comprises the sequence set forth in SEQ ID NO: 10 and the second gRNA comprises the sequence set forth in SEQ ID NO: 18.

18. The composition of any one of claims 1- 17, wherein the composition is encapsulated in at least one LNP comprising:about 50% of HBC365, about 38% of cholesterol by moles, about 10% of DSPC by moles, and about 2% of DMG-PEG2000 by moles;wherein the ratio of lipid to RNA molecule in the at least one nanoparticle is about 50: 1 (w / w) and the total lipid is 25 nM.

19. A method of increasing serum concentrations of FGF21 protein in a subject, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1, 3, or 5-18,wherein the first and second fusion proteins are expressed in at least one cell of the subject,wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein,wherein the first gRNA specifically binds to a first strand of a double-stranded DNA target sequence in the at least one cell of the subject,wherein the second gRNA specifically binds to a second strand of a double-stranded DNA target sequence in the at least one cell of the subject,wherein the first fusion protein and the second fusion protein introduce an insertion or deletion (indel) between the first strand of the double-stranded DNA target sequence and the second strand of the double stranded DNA target sequence target sequence.

20. A method of increasing serum concentrations of GDF15 protein in a subject, comprising administering to the subject a therapeutically effective amount the composition of any one of claims 2, 4, or 5-18,wherein the first and second fusion proteins are expressed in at least one cell of the subject,wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein,wherein the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject,wherein the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject,wherein the first fusion protein and the second fusion protein introduce an indel between the first double-stranded DNA target sequence and the second double stranded DNA target sequence target sequence.

21. The method of claim 19, wherein the indel mutates or deletes one or more AU-rich sequences in the 3-UTR of the FGF21 gene.

22. The method of claim 20, wherein the indel mutates or deletes one or more AU-rich sequences in the 3’-UTR of the GDF15 gene.

23. A method of treating a disease or disorder mediated by FGF21 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1, 3, or 5-18,wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein,wherein the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject,wherein the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject,wherein the first fusion protein and the second fusion protein introduce an indel between the first double-stranded DNA target sequence and the second double stranded DNA target sequence target sequence.

24. The method of claim 23, wherein the method results in the reduction or alleviation of one or more symptoms of the disease or disorder is a reduction in fat mass and / or alleviation of one or more of hyperglycaemia, insulin resistance, dyslipidaemia, cardiovascular disorders, type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

25. A method of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 2, 4, or 5-18,wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein,wherein the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in the at least one cell of the subject,wherein the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in the at least one cell of the subject,wherein the first fusion protein and the second fusion protein introduce an indel between the first double-stranded DNA target sequence and the double stranded DNA target sequence target sequence.

26. The method of claim 25, wherein the method results in reduction or alleviation of one or more symptoms of obesity, optionally wherein the method results in weight loss.

27. A method of modifying the genomes of a population of cells, comprising contacting the population of cells with the composition of any one of claims 1-18,wherein the first and second fusion proteins are expressed by each cell of the population,wherein the first gRNA is bound to the first fusion protein and the second gRNA is bound to the second fusion protein,wherein the first gRNA specifically binds to a first strand of a first double-stranded DNA target sequence in each cell of the population, andwherein the second gRNA specifically binds to a second strand of a second doublestranded DNA target sequence in each cell of the population.

28. The method of claim 27, wherein the first fusion protein and the second fusion protein introduces a modification into the genome of one or more cells in the population.

29. The method of claim 28, wherein the modification is an indel between the first strand of the double-stranded DNA target sequence and the second strand of the double stranded DNA target sequence target sequence.

30. The method of claim 29, wherein the indel mutates or deletes one or more AU-rich sequences in the 3-UTR of the FGF21 gene or the GDF15 gene.

31. A population of cells modified according to the method of any one of claims 27-30.

32. The population of cells of claim 31, wherein the population of cells has increased FGF21 protein or GDF15 protein relative to an unmodified population of cells.

33. The population of cells of claim 31 or 32, wherein at least 15% of the cells in the population of cells include an indel at the FGF21 or GDF15 locus.

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