Compositions for the treatment of amyotrophic lateral sclerosis

Abstract

Provided herein are compositions, systems, and methods comprising effector proteins for treating spinal muscular atrophy. These effector proteins may be characterized as CRISPRassociated (Cas) proteins. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification of the SOD1 gene.

Description

Cooley Ref: MABI-044 / 03WO 344183-2319 COMPOSITIONS FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 509,919, filed June 23, 2023; U.S. Provisional Application 63 / 514,848, filed July 21, 2023; and U.S. Provisional Application 63 / 584,246, September 21, 2023, the contents of each are herein incorporated by reference in their entireties. SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (MABI_044_03WO_SeqList_ST26.xml; Size: 985,972 bytes; and Date of Creation: June 17, 2024) are herein incorporated by reference in its entirety. BACKGROUND

[0003] Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive neurodegenerative disease characterized by the degeneration of motor neurons. Familial ALS (fALS) is typically a result of mutations in the SOD1 gene which encodes for the superoxide dismutase enzyme (SOD1). SOD1 mutations may result in the misfolding of superoxide dismutase enzyme. Sporadic ALS (sALS) may also be a result of an SOD1 mutation.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins), sometimes referred to as a CRISPR / Cas system, were first identified in certain bacterial species and are now understood to form part of a prokaryotic acquired immune system. CRISPR / Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequence- specific manner. Native systems contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets. The discovery of CRISPR / Cas systems has revolutionized the field of genomic manipulation and engineering, and therapeutic applications of these systems are being explored. SUMMARY

[0005] The instant disclosure describes compositions, systems, kits, and methods that are useful for modifying the SOD1 gene. Compositions, systems, kits, and methods described herein comprise a CRISPR system or a use thereof. Such compositions, systems, kits, and ^Cooley Ref: MABI-044 / 03WO 344183-2319 methods may be useful for the treatment of ALS. Compositions, systems, kits, and methods disclosed herein may produce a double stranded break in an SOD1 gene comprising a mutation, thereby disrupting expression and reducing production of a pathogenic SOD1 variant. In some instances, the pathogenic SOD1 variant is a misfolded protein relative to wildtype SOD1. By way of non-limiting example, the mutation may be a missense mutation. Non-limiting examples of a missense mutation in SOD1 is G93A in exon 4. Compositions, systems, kits, and methods that do not cleave an SOD1 gene, but otherwise modify its expression, e.g., through epigenetic silencing are also described.

[0006] In some embodiments, the present disclosure provides systems comprising: (a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of a superoxide dismutase 1 (SOD1) gene, wherein the first region is located 5’ of the second region; and optionally, an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

[0007] In some embodiments, the present disclosure provides compositions comprising: (a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of a superoxide dismutase 1 (SOD1) gene, wherein the first region is located 5’ of the second region; and optionally, an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

[0008] In some embodiments, the present disclosure provides kits comprising: (a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of a superoxide dismutase 1 (SOD1) gene, wherein the first region is located 5’ of the second region; and optionally, an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

[0009] In some embodiments, the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1. In some embodiments, the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 9. In some embodiments, the protein binding sequence comprises a nucleotide ^Cooley Ref: MABI-044 / 03WO 344183-2319 sequence selected from SEQ ID NOs: 506, 513, 514, 1063, and combinations thereof. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515, 1045, or 1047.

[0010] In some embodiments, the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2. In some embodiments, the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 10. In some embodiments, the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516, 1041, 1042, 1044, or 1046.

[0011] In some embodiments, the effector protein is a dCas protein. In some embodiments, the dCas protein comprises a heterologous polypeptide. In some embodiments, the dCas protein is covalently linked to the heterologous polypeptide. In some embodiments, the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor. In some embodiments, the effector protein is linked to a nuclear localization signal.

[0012] In some embodiments, the system, composition, or kit comprises the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in a single composition. In some embodiments, the system, composition, or kit comprises the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in separate compositions, separate solutions, separate containers, or a combination thereof. In some embodiments, the nucleic acid encoding the effector protein is a messenger RNA. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0013] In some embodiments, the system, composition, or kit comprises a lipid or a lipid nanoparticle.

[0014] In some embodiments, the guide nucleic acid comprises at least one chemical modification, optionally wherein the at least one chemical modification is selected from a 2’- O-Methyl sugar modification, a 2’-fluoro sugar modification, a phosphorothioate internucleoside linkage, and a combination thereof.

[0015] In some embodiments, the system, composition, or kit comprises a nucleic acid expression vector, wherein the nucleic acid expression vector comprises the nucleic acid encoding the effector protein, the DNA molecule encoding the guide nucleic acid, or a combination thereof. In some embodiments, the nucleic acid expression vector is an adenoviral associated viral (AAV) vector. In some embodiments, the nucleic acid expression vector comprises or encodes a donor nucleic acid. In some embodiments, the system, composition, or kit further comprises a donor nucleic acid.

[0016] In some embodiments, the present disclosure provides a nucleic acid expression vector encoding (a) a guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of an SOD1 gene, wherein the first region is located 5’ of the second region; and optionally, an effector protein that binds the protein binding sequence of the guide nucleic acid.

[0017] In some embodiments, the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1. In some embodiments, the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 9. In some embodiments, the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 506, 513, 514, 1063, and combinations thereof. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515, 1045, or 1047.

[0018] In some embodiments, the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2. In some embodiments, the target sequence is adjacent ^Cooley Ref: MABI-044 / 03WO 344183-2319 to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 10. In some embodiments, the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516, 1041, 1042, 1044, or 1046.

[0019] In some embodiments, the effector protein is a dCas protein. In some embodiments, the dCas protein comprises a third region encoding a heterologous polypeptide. In some embodiments, the dCas protein is covalently linked to the heterologous polypeptide upon expression of the nucleic acid expression vector. In some embodiments, the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor.

[0020] In some embodiments, the expression vector is an adenoviral associated viral (AAV) vector.

[0021] In some embodiments, the present disclosure provides an expression cassette comprising, from 5’ to 3’: a first inverted terminal repeat (ITR); a first promoter sequence operably linked to a nucleic acid sequence encoding a guide RNA wherein the guide RNA comprises: a first region comprising a protein binding sequence; and a second region comprising a spacer sequence that is complementary to a target sequence of a SOD1 gene, wherein the spacer sequence selected from SEQ ID NOs: 1-505; a second promoter sequence operably linked to a nucleic acid sequence encoding an effector protein; a poly(A) signal; and a second ITR. In some embodiments, the expression cassette further comprises a WPRE sequence located between the nucleic acid sequence encoding an effector protein and the poly(A) signal. In some embodiments, the first promoter is a U6 promoter. In some embodiments, the second promoter is a CK8E promoter or a SPC5 promoter. In some embodiments, the poly(A) signal is a bGH or an hGH poly(A) signal. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:516. In some embodiments, the effector protein comprises the amino acid substitution of L26R relative to SEQ ID NO: 516. In some embodiments, the effector protein comprises SEQ ID NO: 1046. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:515. In some embodiments, the effector protein ^Cooley Ref: MABI-044 / 03WO 344183-2319 comprises the amino acid substitution of D220R relative to SEQ ID NO: 515. In some embodiments, the effector protein comprises SEQ ID NO: 1047. In some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector comprising an expression cassette described herein.

[0022] In some embodiments, the present disclosure provides a cell comprising or modified by a system or nucleic acid expression vector described herein. In some embodiments, the cell is a human cell. In some embodiments, the cell is a motor neuron. In some embodiments, the present disclosure provides a population of cell described herein.

[0023] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a system or a nucleic acid expression vector described herein, and a pharmaceutical acceptable carrier.

[0024] In some embodiments, the present disclosure provides a method of modifying an SOD1 gene, comprising contacting the SOD1 gene with a system, a nucleic acid expression vector, or a pharmaceutical composition described herein. In some embodiments, the method comprises contacting a cell that comprises the SOD1 gene with a system, a nucleic acid expression vector, or a pharmaceutical composition described herein. In some embodiments, an amount of a superoxide dismutase (SOD1) protein in the cell is reduced relative to the amount of the SOD1 protein in the cell prior to contacting the cell with a system, a nucleic acid expression vector, or a pharmaceutical composition described herein.

[0025] In some embodiments, the present disclosure provides a method of treating or preventing amyotrophic lateral sclerosis in a human subject in need thereof, the method comprising administering to the human subject components of a system, a nucleic acid expression vector, or a pharmaceutical composition described herein. In some embodiments, the subject harbors a mutation in the SOD1 gene.

[0026] In some embodiments, the present disclosure provides a fusion protein or nucleic acid encoding the fusion protein, wherein the fusion protein comprises an effector protein and a base editing enzyme, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515; and the base editing enzyme comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1062.

[0027] In some embodiments, the effector protein comprises the amino acid substitutions of D220R and E335Q relative to SEQ ID NO: 515. In some embodiments, the fusion protein ^Cooley Ref: MABI-044 / 03WO 344183-2319 comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1048. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 1048.

[0028] In some embodiments, the present disclosure provides a system comprising (a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a targeting sequence that is complementary to a target sequence of an SOD1 gene, wherein the targeting sequence comprises a spacer sequence comprising a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1, wherein the first region is located 5’ of the second region; and (b) a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

[0029] In some embodiments, the present disclosure provides a system comprising a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 534-796; and a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1048.

[0030] In some embodiments, the present disclosure provides a fusion protein comprising an effector protein sequence and a base editing enzyme sequence, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516; and the base editing enzyme comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1062.

[0031] In some embodiments, the effector protein comprises the amino acid substitutions of L26K and E567Q relative to SEQ ID NO: 516. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1049. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 1049.

[0032] In some embodiments, the present disclosure provides a system comprising (a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a targeting sequence that is complementary to a target sequence of an SOD1 gene, wherein the targeting sequence comprises a spacer sequence comprising a nucleotide sequence ^Cooley Ref: MABI-044 / 03WO 344183-2319 that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2, wherein the first region is located 5’ of the second region; and (b) a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

[0033] In some embodiments, the present disclosure provides a system comprising a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038; and a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1049. BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 depicts in vivo gene editing of PCSK9 in muscle tissues using AAV9-A4 delivery of CasPhi.12 and CasM.265466 variants. INCORPORATION BY REFERENCE

[0035] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. DETAILED DESCRIPTION OF THE INVENTION

[0036] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0038] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. 1. Definitions

[0039] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings: ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0040] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0041] The terms, “or” and “and / or,” as used herein, include any, and all, combinations of one or more of the associated listed items.

[0042] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0043] The terms, “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0044] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0045] The terms, “% identical,” “% identity,” and “percent identity,” or grammatical equivalents thereof, refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG.

[0046] The term, “base editing enzyme,” as used herein, refers to a protein, polypeptide or fragment thereof that is capable of catalyzing the chemical modification of a nucleobase of a deoxyribonucleotide or a ribonucleotide. Such a base editing enzyme, for example, is capable of catalyzing a reaction that modifies a nucleobase that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleobase to a different nucleobase, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). A base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0047] The term, “base editor,” as used herein, refers to a fusion protein comprising a base editing enzyme linked to an effector protein. The base editing enzyme may be referred to as a fusion partner. The base editing enzyme can differ from a naturally occurring base editing enzyme. It is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein. Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.

[0048] The term, “dCas protein,” as used herein, also referred to as a “catalytically inactive effector protein,” refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally- occurring effector protein may be a wildtype protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.

[0049] The term, “cis cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by an effector protein complexed with a guide nucleic acid (e.g., an RNP complex), wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide nucleic acid.

[0050] The terms, “complementary” and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T or U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5’- to 3’-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3’- to its 5’-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5’- to its 3’-end. Each nucleotide in a ^Cooley Ref: MABI-044 / 03WO 344183-2319 double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0051] The terms, “cleave,” “cleaving,” and “cleavage,” as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[0052] The term, “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen.

[0053] The terms, “CRISPR RNA” or “crRNA,” as used herein, refer to a type of guide nucleic acid, wherein the nucleic acid is RNA comprising a first sequence that is capable of interacting with an effector protein either directly (by being bound by an effector protein) or indirectly (e.g., by hybridization with a second nucleic acid molecule that can be bound by an effector, such as a tracrRNA); and a second sequence that hybridizes to a target sequence of a target nucleic acid. In some embodiments, the first sequence is referred to as a repeat sequence and the second sequence is referred to as a spacer sequence. The first sequence and the second sequence are directly connected to each other or by a linker.

[0054] The term, “disrupt,” as used herein, refers to reducing or abolishing a function of a gene regulatory element by altering or modifying the nucleotide sequence of the gene regulatory element or the nucleotide sequence located in proximity (e.g., less than 200 linked nucleotides) to the gene regulatory element. In some embodiments, the gene regulatory element is a splicing-regulatory element. In some embodiments, the original function of the gene regulatory element is repressing exonic splicing. In some embodiments, there is an increased inclusion of an exon region in a mature mRNA after the disruption.

[0055] The term, “donor nucleic acid,” as used herein, refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0056] The term, “dual nucleic acid system,” as used herein, refers to a system that uses a transactivated or transactivating tracrRNA-crRNA duplex complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence selective manner.

[0057] The term, “effector protein,” as used herein, refers to a protein, polypeptide, or peptide that is capable of interacting with a guide nucleic acid to form a complex (e.g., a RNP complex), wherein the complex interacts with a target nucleic acid. A complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is linked to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid. A non-limiting example of an effector protein modifying a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout. Herein, reference to an effector protein includes reference to a nucleic acid encoding the effector protein, unless indicated otherwise.

[0058] The term, “engineered modification,” as used herein, refers to a structural change of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence, such as chemical modification of one or more nucleobases; or a chemical change to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition, or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro- transcription, cloning, enzymatic, or chemical cleavage, etc. In some embodiments, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions within the nucleic acid.

[0059] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. “Operably linked” refers to a juxtaposition wherein the components so described are ^Cooley Ref: MABI-044 / 03WO 344183-2319 in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence (or the coding sequence can also be said to be operably linked to the promoter) if the promoter affects its transcription or expression.

[0060] The term, “fusion protein,” as used herein, refers to a protein comprising at least two heterologous polypeptides. The fusion protein may comprise one or more effector proteins and fusion partners. In some embodiments, an effector protein and fusion partner are not found connected to one another as a native protein or complex that occurs together in nature.

[0061] The term, “functional domain,” as used herein, refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid modification, nucleic acid cleavage, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity.

[0062] The terms, “fusion partner” or “fusion partner protein,” as used herein, refer to a protein, polypeptide or peptide that is linked to an effector protein or capable of being proximal to an effector protein. In some embodiments, a fusion partner that is capable of being proximal to an effector protein is a fusion partner that is capable of binding a guide nucleic acid, wherein the effector protein is also capable of binding the guide nucleic acid. In some embodiments, a fusion partner directly interacts with (e.g., binds to / by) an effector protein. In some embodiments, a fusion partner indirectly interacts with an effector protein (e.g., through another protein or moiety). The fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein.

[0063] The term, “genetic disease,” as used herein, refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.

[0064] The term, “guide nucleic acid,” as used herein, refers to a nucleic acid comprising: a first nucleotide sequence that is capable of being non-covalently bound by an effector protein; and a second nucleotide sequence that hybridizes to a target nucleic acid. When in a complex with one or more polypeptides described herein (e.g., an RNP complex), a guide nucleic acid can impart sequence selectivity to the complex when the complex interacts with a target nucleic acid. The first sequence may be referred to herein as a repeat sequence. The second sequence may be referred to herein as a spacer sequence. The term, “guide nucleic acid,” may be used interchangeably herein with the term “guide RNA” (gRNA) however it is understood that guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a ^Cooley Ref: MABI-044 / 03WO 344183-2319 combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0065] The term, “heterologous,” as used herein with reference to a nucleic acid or polypeptide, means a nucleic acid or protein that is not found in nature with a reference nucleic acid or protein, respectively. In some instances, the heterologous polypeptide or nucleotide sequence is not found covalently bound to the reference nucleic acid or protein. In some instances, the heterologous polypeptide or nucleotide sequence is not found non-covalently interacting with the reference nucleic acid or protein. In some instances, the heterologous polypeptide or nucleotide sequence is not expressed by the same species as that which expresses the reference nucleic acid or protein. In some embodiments, compositions and systems comprise a fusion protein that comprises an effector protein and a fusion partner protein, wherein the fusion partner protein is heterologous to the effector protein. These fusion partner proteins may be referred to as a “heterologous protein.” A protein that is heterologous to the effector protein may be a protein that is not covalently linked via an amide bond to the effector protein in nature. In some embodiments, a heterologous protein is not encoded by a species that encodes the effector protein. In some embodiments, the heterologous protein exhibits an activity (e.g., enzymatic activity) when it is linked to the effector protein. In some embodiments, the heterologous protein exhibits increased or reduced activity (e.g., enzymatic activity) when it is linked to the effector protein, relative to when it is not linked to the effector protein. In some embodiments, the heterologous protein exhibits an activity (e.g., enzymatic activity) that it does not exhibit when it is linked to the effector protein. A guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid.

[0066] The term, “linked,” when used in reference to biopolymers (e.g., nucleic acids, polypeptides) refers to being covalently connected. In some embodiments, two polymers are linked by at least a covalent bond. In some embodiments, two nucleic acids are linked by at least one nucleotide. In some embodiments, two nucleic acids are linked by at least one amino acid. The terms “fused” and “linked” are used interchangeably herein.

[0067] The term, “linker,” as used herein, refers to a covalent bond or molecule that links a first polypeptide to a second polypeptide (e.g., by an amide bond, or one or more amino acids) ^Cooley Ref: MABI-044 / 03WO 344183-2319 or a first nucleic acid to a second nucleic acid (e.g., by a phosphodiester bond, or one or more nucleotides).

[0068] The term, “modified target nucleic acid,” as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein. In some cases, the modification is an alteration in the sequence of the target nucleic acid. In some cases, the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target nucleic acid.

[0069] The terms, “non-naturally occurring” and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and / or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non- limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0070] The term, “nucleic acid expression vector,” as used herein, refers to a nucleic acid that can be used to express a nucleic acid of interest.

[0071] The term, “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[0072] The term, “nuclease activity,” as used herein, refers to the catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0073] The term, “pharmaceutically acceptable excipient, carrier or diluent,” as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system. Such a substance can be included for the purpose of long- ^Cooley Ref: MABI-044 / 03WO 344183-2319 term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0074] The term, “protospacer adjacent motif (PAM),” as used herein, refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. In some embodiments, a PAM sequence is required for a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex) to hybridize to and edit the target nucleic acid. In some embodiments, the complex does not require a PAM to edit the target nucleic acid.

[0075] In some embodiments, the term “region” as used herein may be used to describe a portion of, or all of, a corresponding sequence, for example, a spacer region is understood to comprise a portion of or all of a spacer sequence.

[0076] The term, “regulatory element,” used herein, refers to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., a guide nucleic acid) or a coding sequence (e.g., effector proteins, fusion proteins, and the like) and / or regulate translation of an encoded polypeptide.

[0077] The term, “repeat sequence,” as used herein, refers to a sequence of nucleotides in a guide nucleic acid that is capable of, at least partially, interacting with an effector protein.

[0078] The terms, “ribonucleotide protein complex” and “RNP,” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes “ribonucleotides” it is understood that the one or more nucleic acid may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0079] The terms, “RuvC” and “RuvC domain,” as used herein, refer to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain embodiments, of processing a pre-crRNA. In some embodiments, the RuvC domain is located near the C- terminus of the effector protein. A single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term ^Cooley Ref: MABI-044 / 03WO 344183-2319 “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (ebi.ac.uk / interpro / ). For example, a RuvC-like domain may be a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons

[0080] The term, “sample,” as used herein, generally refers to something comprising a target nucleic acid. In some embodiments, the sample is a biological sample, such as a biological fluid or tissue sample. In some embodiments, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.

[0081] The terms, “single guide nucleic acid”, “single guide RNA” and “sgRNA,” as used herein, in the context of a single nucleic acid system, refers to a guide nucleic acid, wherein the guide nucleic acid is a single polynucleotide chain having all the required sequence for a functional complex with an effector protein (e.g., being bound by an effector protein, including in some embodiments activating the effector protein, and hybridizing to a target nucleic acid, without the need for a second nucleic acid molecule). For example, an sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary RNA sequence, a repeat sequence, a spacer sequence and optionally a linker). In some embodiments, an sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence.

[0082] The term, “single nucleic acid system,” as used herein, refers to a system that uses a guide nucleic acid complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence specific manner, and wherein the guide nucleic acid is capable of non-covalently interacting with the one or more polypeptides described herein, and wherein the guide nucleic acid is capable of hybridizing with a target sequence of the target nucleic acid. A single nucleic acid system lacks a duplex of a guide nucleic acid as hybridized to a second nucleic acid, wherein in such a duplex the second nucleic acid, and not the guide nucleic acid, is capable of interacting with the effector protein.

[0083] The term, “spacer sequence,” as used herein, refers to a nucleotide sequence in a guide nucleic acid that is capable of, at least partially, hybridizing to an equal length portion of a sequence (e.g., a target sequence) of a target nucleic acid.

[0084] The term, “subject,” as used herein, refers to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, ^Cooley Ref: MABI-044 / 03WO 344183-2319 for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some embodiments, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0085] The term, “target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).

[0086] The terms, “target nucleic acid sequence” and “target sequence,” as used herein, when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to an equal length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[0087] The term, “trans cleavage,” as used herein, in the context of cleavage (e.g., hydrolysis of a phosphodiester bond) of one or more target nucleic acids or non-target nucleic acids, or both, by an effector protein that is complexed with a guide nucleic acid and the target nucleic acid. Trans cleavage activity may be triggered by the hybridization of a guide nucleic acid to a target nucleic acid. The effector may cleave a target strand as well as non-target strand, wherein the target nucleic is a double stranded nucleic acid. Trans cleavage of the target nucleic acid may occur away from (e.g., not within or directly adjacent to) the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.

[0088] The terms, “trans-activating RNA”, “transactivating RNA” and “tracrRNA,” refer to a transactivating or transactivated nucleic acid in a dual nucleic acid system that is capable of hybridizing, at least partially, to a crRNA to form a tracrRNA-crRNA duplex, and of interacting with an effector protein to form a complex (e.g., an RNP complex).

[0089] The terms, “transactivating”, “trans-activating”, “trans-activated”, “transactivated” and grammatical equivalents thereof, as used herein, in the context of a dual nucleic acid system refers to an outcome of the system, wherein a polypeptide is enabled to have a binding and / or nuclease activity on a target nucleic acid, by a tracrRNA or a tracrRNA-crRNA duplex.

[0090] The term, “transcriptional activator,” as used herein, refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0091] The term “transcriptional repressor,” as used herein, refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid.

[0092] The term, “transgene,” as used herein, refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell. A transgene is meant to include (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced. A donor nucleic acid can comprise a transgene. The cell in which transgene expression occurs can be a target cell, such as a host cell.

[0093] The terms, “treatment” and “treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0094] The term, “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single- stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. Non-limiting examples of viruses or viral particles that can deliver a viral vector include retroviruses (e.g., lentiviruses and Ȗ- retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A viral vector ^Cooley Ref: MABI-044 / 03WO 344183-2319 delivered by such viruses or viral particles may be referred to by the type of virus to deliver the viral vector (e.g., an AAV viral vector is a viral vector that is to be delivered by an adeno- associated virus). A viral vector referred to by the type of virus to be delivered by the viral vector can contain viral elements (e.g., nucleotide sequences) necessary for packaging of the viral vector into the virus or viral particle, replicating the virus, or other desired viral activities. A virus containing a viral vector may be replication competent, replication deficient or replication defective. 2. Introduction

[0095] The present disclosure describes compositions, systems, and methods for modifying the SOD1 gene. The SOD1 gene encodes the SOD1 protein, which is also referred to ALS, ALS1, HEL-S-44, IPOA, SOD, hSod1, superoxide dismutase 1, and STAHP. In some embodiments, compositions and systems comprise a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises a first region comprising a protein binding sequence and a second region comprising a spacer sequence that is complementary to a target sequence of an SOD1 gene. In some embodiments, the first region is located 5’ of the second region. In some embodiments, the protein binding sequence comprises a repeat sequence. In some embodiments, the composition or system comprises an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

[0096] The present disclosure describes compositions, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in a single composition. In some embodiments, the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in a single solution. The present disclosure describes systems, also referred to as kits, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in separate solutions, containers, vectors, or compositions.

[0097] In some embodiments, the compositions and systems comprise guide nucleic acids that are capable of hybridizing to a target sequence of the SOD1 gene. In some embodiments, compositions and systems comprise an effector protein that is capable of binding a portion of the guide nucleic acid. In some embodiments, the effector protein is a CRISPR-associated (Cas) protein. In general, Cas proteins bind and / or modify nucleic acids in a sequence-specific ^Cooley Ref: MABI-044 / 03WO 344183-2319 manner. Cas proteins with guide nucleic acids may modify DNA at a precise target location in the genome of a wide variety of cells and organisms, allowing for precise and efficient editing of DNA sequences of interest (e.g., the SOD1 gene). In some embodiments, the present disclosure provides methods for treating a disease (e.g., amyotrophic lateral sclerosis (ALS)) by modifying one or more target genes (e.g., the SOD1 gene). In some embodiments, ALS is familial ALS (fALS).

[0098] Compositions and systems disclosed herein are not naturally occurring. In general, an effector protein and a guide nucleic acid refer to an effector protein and a guide nucleic acid, respectively, that are not found in nature. In some embodiments, systems and compositions herein comprise at least one non-naturally occurring component. For example, compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some embodiments, compositions and systems comprise at least two components that do not naturally occur together. For example, compositions and systems may comprise a guide nucleic acid comprising a repeat sequence and a spacer sequence which do not naturally occur together. For example, a guide nucleic acid may comprise a sequence of a naturally-occurring repeat region and a spacer region that is complementary to a naturally-occurring eukaryotic sequence. The guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism. A guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. Also, by way of example, composition and systems may comprise a guide nucleic acid and an effector protein that do not naturally occur together. Conversely, and for clarity, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.

[0099] In some embodiments, a guide nucleic acid is a crRNA, wherein the crRNA comprises a repeat sequence and a spacer sequence that is complementary to a eukaryotic target sequence. In some embodiments, a guide nucleic acid may comprise a repeat sequence, an intermediary sequence, and a spacer sequence coupled by one or more linker sequences. In some embodiments, the guide nucleic acid comprises two heterologous sequences arranged in an ^Cooley Ref: MABI-044 / 03WO 344183-2319 order or proximity that is not observed in nature. Therefore, guide nucleic acid compositions described herein are not naturally occurring. 3. Guide Nucleic Acids

[0100] The compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid. Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid. In general, guide nucleic acids comprise two heterologous sequences arranged in an order or proximity that is not observed in nature.

[0101] In general, guide nucleic acids, also referred to as guide RNAs (gRNAs), comprise a nucleotide sequence. Such nucleotide sequence may be described as a nucleotide sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid. Similarly, disclosure of the nucleotide sequences described herein also discloses a complementary nucleotide sequence, a reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which can be a nucleotide sequence for use in a guide nucleic acid. In some embodiments, a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alterations can include a nucleotide substitution, a nucleotide deletion, or a nucleotide insertion.

[0102] A guide nucleic acid of the present disclosure may comprise one or more of the following: a) an RNA nucleobase; b) a DNA nucleobase; c) a modified nucleobase; d) a modified sugar; and e) a modified backbone. Modified nucleobases, sugars and backbones are described in greater detail herein. A guide nucleic acid may be chemically synthesized or recombinantly produced by any suitable methods. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.

[0103] In general, a guide nucleic acid comprises a first nucleotide sequence that is capable of being non-covalently bound by an effector protein and a second nucleotide sequence that hybridizes to a target nucleic acid. The first nucleotide sequence may comprise or be referred ^Cooley Ref: MABI-044 / 03WO 344183-2319 to as a protein binding sequence. In some embodiments, the first nucleotide sequence is located 5’ to the second nucleotide sequence. In some embodiments, the second nucleotide sequence is located 5’ to the first nucleotide sequence. In some embodiments, the first nucleotide and second nucleotide sequences are linked either by a covalent bond (e.g., a phosphodiester bond) or linker (e.g., one or more nucleotides) The protein binding sequence may comprise a repeat sequence, an intermediary sequence, a handle sequence, or a combination thereof. In some embodiments, the first nucleotide sequence comprises a repeat sequence. In some embodiments, the first nucleotide sequence comprises an intermediary sequence and a repeat sequence. In some embodiments, the first nucleotide sequence comprises a handle sequence. In some embodiments, an effector protein binds to at least a portion of the first nucleotide sequence. In some embodiments, the second nucleotide sequence comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g., has complementarity with, or can hybridize to a target sequence in) a target nucleic acid (e.g., the SOD1 gene). Although the term may imply that a gRNA consists of RNA, in some embodiments, a gRNA may comprise one or more deoxyribonucleotides and / or a deoxyribonucleotide nucleobase (e.g., thymine). However, the majority of the nucleotides in a guide nucleic acid (at least 50%) are ribonucleotides.

[0104] Modifications can further include changing of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a nucleobase base modification, a backbone modification, a sugar modification, a phosphorothioate internucleotide linkage, or combinations thereof. In some embodiments, the modifications can be of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid. In some embodiments, uridines can be exchanged for pseudouridines (e.g., 1N-Methyl-Pseudouridine). In some embodiments, all uridines can be exchanged for 1N-Methyl-Pseudouridine. In this application, U can represent uracil or 1N-Methyl-Pseudouridine.

[0105] Guide nucleic acids may form complexes as described herein. For example, a guide nucleic acid may hybridize to another nucleic acid, such as a target nucleic acid, or a portion thereof. In some embodiments, guide nucleic acids may complex with an effector protein. In such embodiments, a guide nucleic acid-effector protein complex may be described herein as an RNP. In some embodiments, when in a complex, at least a portion of the complex may bind, recognize, and / or hybridize to a target nucleic acid (e.g., a target sequence in the SOD1 gene). For example, when a guide nucleic acid and an effector protein are complexed to form an RNP, at least a portion of the guide nucleic acid hybridizes to a target sequence in a target nucleic ^Cooley Ref: MABI-044 / 03WO 344183-2319 acid (e.g., the SOD1 gene). Those skilled in the art in reading the below specific examples of guide nucleic acids as used in RNPs described herein, will understand that in some embodiments, a RNP may hybridize to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and / or recognize a target nucleic acid or sequence contained therein (e.g., PAM) or to modify and / or recognize non-target sequences depending on the guide nucleic acid, and in some embodiments, the effector protein, used.

[0106] In some embodiments, a guide nucleic acid may comprise or form intramolecular secondary structure (e.g., hairpins, stem-loops, etc.). In some embodiments, a guide nucleic acid comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the guide nucleic acid comprises a pseudoknot (e.g., a secondary structure comprising a stem, at least partially, hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a guide nucleic acid comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the guide nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0107] In some embodiments, the compositions, systems, and methods of the present disclosure comprise two or more guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 9, 10 or more guide nucleic acids), and / or uses thereof. Multiple guide nucleic acids may target an effector protein to different locations in the target nucleic acid by hybridizing to different target sequences. In some embodiments, a first guide nucleic acid may hybridize within a location of the target nucleic acid that is different from where a second guide nucleic acid may hybridize the target nucleic acid. In some embodiments, the first loci and the second loci of the target nucleic acid may be located at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides apart. In some embodiments, the first loci and the second loci of the target nucleic acid may be located between 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900 or 900 and 1000 nucleotides apart.

[0108] In some embodiments, the first loci and / or the second loci of the target nucleic acid are located in an intron of a gene (e.g., an intron of the SOD1 gene). In some embodiments, the first loci and / or the second loci of the target nucleic acid are located in an exon of a gene (e.g., ^Cooley Ref: MABI-044 / 03WO 344183-2319 an exon of the SOD1 gene). In some embodiments, the first portion and / or the second portion of the target nucleic acid are located on either side of an exon and cutting at both sites results in deletion of the exon. In some embodiments, upon removal of a sequence between two guide nucleic acids, the wild-type reading frame may be restored, e.g., by a polymerase, resulting in at least a partially functional protein. In some embodiments, composition, systems, and methods comprise a donor nucleic acid that may be inserted in replacement of a deleted or cleaved sequence of the target nucleic acid. In some embodiments, compositions, systems, and methods comprising multiple guide nucleic acids or uses thereof comprise multiple effector proteins, wherein the effector proteins may be identical, non-identical, or combinations thereof.

[0109] In some embodiments, a guide nucleic acid comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In general, a guide nucleic acid comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides.

[0110] In some embodiments, the guide nucleic acid comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In general, a guide nucleic acid comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, a guide nucleic acid comprises at least 25 linked nucleotides. A guide nucleic acid may comprise 10 to 50 linked nucleotides. In some embodiments, the guide nucleic acid comprises or consists essentially of about 12 to about 80 linked nucleotides, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19 , about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleotides. In some embodiments, the guide nucleic acid comprises about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides.

[0111] In some embodiments, a length of a guide nucleic acid is about 30 to about 120 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 ^Cooley Ref: MABI-044 / 03WO 344183-2319 to about 60 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, or about 125 linked nucleotides.

[0112] In some embodiments, guide nucleic acids comprise elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. Such elements may be one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.).

[0113] In some embodiments, guide nucleic acids comprise one or more linkers connecting different nucleotide sequences as described herein. A linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. A linker may be any suitable linker, examples of which are described herein.

[0114] Guide nucleic acids may comprise deoxyribonucleotides, ribonucleotides, or a combination thereof. In some embodiments, a guide nucleic acid comprises a ribonucleotide with a thymine nucleobase. Guide nucleic acids may comprise a chemically modified nucleobase or phosphate backbone. Guide nucleic acids may be referred to herein as a guide RNA (gRNA). However, a guide RNA is not limited to ribonucleotides, but may comprise deoxyribonucleotides and other chemically modified nucleotides. A guide nucleic acid may comprise a non-naturally occurring guide nucleic acid, including a guide nucleic acid that is designed to contain a chemical or biochemical modification.

[0115] In some embodiments, effector proteins are targeted by a guide nucleic acid (e.g., a guide RNA) to a specific location in the target nucleic acid where they exert locus-specific nucleotide modification or gene regulation. Non-limiting examples of gene regulation include blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and / or modifying local chromatin (e.g., modifying the target nucleic acid or modifying a protein associated with the target nucleic acid). The guide RNA may bind to a target nucleic acid (e.g., a single strand of a target nucleic acid) or a portion thereof, an amplicon thereof, or a portion thereof. By way of non-limiting example, a guide nucleic acid ^Cooley Ref: MABI-044 / 03WO 344183-2319 may bind to a portion of a gene associated with a genetic disorder, or an amplicon thereof, as described herein.

[0116] Exemplary guide nucleic acids that may be used with systems, compositions, and methods described herein comprise a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 534-1038. Spacer Sequences

[0117] Guide nucleic acids described herein generally comprise one or more spacer regions represented by a spacer sequence. The terms, “spacer region,” “targeting sequences,” and “spacer sequence” may be used interchangeably. In some embodiments, a spacer region is capable of hybridizing to a target sequence of a target nucleic acid. In some embodiments, a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g., a target sequence) of a target nucleic acid. Exemplary hybridization conditions are described herein. In some embodiments, the spacer region may function to direct an RNP complex comprising the guide nucleic acid to the target nucleic acid for detection and / or modification of the target nucleic acid. A spacer sequence may be complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein described herein.

[0118] The spacer sequence of a guide nucleic acid is generally complementary to a target sequence of a target nucleic acid. It is understood that the spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. However, there is sufficient complementarity between the spacer sequence and the target sequence in order for the spacer sequence to hybridize to the target sequence. The spacer sequence of a guide nucleic acid may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a target sequence of a target nucleic acid.

[0119] In some embodiments, the spacer region comprises 5-50 linked nucleotides in length. In some embodiments, the spacer region is 15-28 linked nucleotides in length. In some embodiments, the spacer region is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16- 22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length. In some embodiments, the spacer region is 18-24 linked nucleotides in length. In some embodiments, the spacer region is at least 15 linked nucleotides in length. In some embodiments, the spacer region is at least 16, 18, 20, or 22 linked nucleotides in length. In ^Cooley Ref: MABI-044 / 03WO 344183-2319 some embodiments, the spacer region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer region is at least 17 linked nucleotides in length. In some embodiments, the spacer region is at least 18 linked nucleotides in length. In some embodiments, the spacer region is at least 20 linked nucleotides in length. In some embodiments, the spacer region is 17 linked nucleotides in length. In some embodiments, the spacer region is 18 linked nucleotides in length. In some embodiments, the spacer region is 19 linked nucleotides in length. In some embodiments, the spacer region is 20 linked nucleotides in length. In some embodiments, the spacer region comprises at least 15 contiguous nucleotides that are complementary to the target nucleic acid.

[0120] In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequences. Linkers may be any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions.

[0121] In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid (e.g., the SOD1 gene). A spacer sequence is capable of hybridizing to an equal length portion of a target nucleic acid. In some embodiments, a spacer sequence comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the SOD1 gene. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are capable of hybridizing to the target sequence. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to the target sequence.

[0122] In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical, complementary to, or reverse complementary to a target sequence of the SOD1 gene. In some embodiments, the target sequence is within an exon region of the SOD1 gene. In some embodiments, the target sequence is within an intron region of the SOD1 gene. In some embodiments, the target sequence is spans and exon and intron of the ^Cooley Ref: MABI-044 / 03WO 344183-2319 SOD1 gene. In some embodiments, the target sequence is located in a regulatory element of the SOD1 gene. In some embodiments, the regulatory element is a promoter.

[0123] In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a sequence in exon 1 of the SOD1 gene. In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a sequence in exon 2 of the SOD1 gene. In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a sequence in exon 3 of the SOD1 gene. In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a sequence in exon 4 of the SOD1 gene. In some embodiments, a spacer sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a sequence in exon 5 of the SOD1 gene.

[0124] In some embodiments, a spacer sequence is complementary to a target sequence that is adjacent to a PAM in the SOD1 gene. In some embodiments, a spacer sequence is complementary to a target sequence that is within 0-10 nucleotides of a PAM in the SOD1 gene. In some embodiments, the PAM is NTTN. In some embodiments, the PAM is TNTR. In some embodiments, the PAM is VNTR. In some embodiments, the PAM is TNTY. In some embodiments, N represents any nucleotide; V represents A, C, or G; Y represents C or T; and R represents A or G. In some embodiments, the PAM is selected from: AATA, AATG, ACTA, ACTG, AGTA, AGTG, ATTA, ATTG, CATA, CATG, CCTA, CCTG, CGTA, CGTG, CTTA, CTTG, GATA, GATG, GCTA, GCTG, GGTA, GGTG, GTTA, GTTG, TATA, TATC, TATG, TATT, TCTA, TCTC, TCTG, TCTT, TGTA, TGTC, TGTG, TGTT, TTTA, TTTC, TTTG, and TTTT. In some embodiments, the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT.

[0125] TABLE 1 and TABLE 2 provide illustrative spacer sequences targeting the SOD1 gene for use with the compositions, systems, and methods of the disclosure. In particular, TABLE 1 provides spacer sequences suitable for use in the combination with an effector protein of SEQ ID NO: 515 or variants thereof (e.g., variants provided in TABLES 5 and 7). In particular, TABLE 2 provides spacer sequences suitable for use in the combination with an effector protein of SEQ ID NO: 516 or variants thereof (e.g., variants provided in TABLES 6 and 7). In some embodiments, the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100% identical to a spacer sequence selected from TABLE 1 and TABLE 2. In some embodiments, spacer sequences comprise at ^Cooley Ref: MABI-044 / 03WO 344183-2319 least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of a sequence selected from any one of SEQ ID NOs: 1-505.

[0126] Guide nucleic acids disclosed herein may target various regions of the SOD1 gene. In some embodiments, the spacer sequences are complementary to a target sequence in exon 1 of SOD1. In some embodiments, the spacer sequences hybridize to a target sequence in exon 1 of SOD1. By way of non-limiting example, spacer sequences that are complementary to exon 1 of SOD1 include SEQ ID NOs: 1-4, 70-88, 203-204, 264-272, and 390-398. In some embodiments, the spacer sequences are complementary to a target sequence in exon 2 of SOD1. In some embodiments, the spacer sequences hybridize to a target sequence in exon 2 of SOD1. By way of non-limiting example, spacer sequences that are complementary to exon 2 of SOD1 include SEQ ID NOs: 5-7, 89-105, 205-208, 273-280, and 399-407. In some embodiments, the spacer sequences are complementary to a target sequence in exon 3 of SOD1. In some embodiments, the spacer sequences hybridize to a target sequence in exon 3 of SOD1. By way of non-limiting example, spacer sequences that are complementary to exon 3 of SOD1 include SEQ ID NOs: 8-12, 106-111, 209-214, 281-289, and 408-416. In some embodiments, the spacer sequences are complementary to a target sequence in exon 4 of SOD1. In some embodiments, the spacer sequences hybridize to a target sequence in exon 4 of SOD1. By way of non-limiting example, spacer sequences that are complementary to exon 4 of SOD1 include SEQ ID NOs: 13-20, 112-129, 215-223, 290-297, and 417-425. In some embodiments, the spacer sequences are complementary to a target sequence in exon 5 of SOD1. In some embodiments, the spacer sequences hybridize to a target sequence in exon 5 of SOD1. By way of non-limiting example, spacer sequences that are complementary to exon 5 of SOD1 include SEQ ID NOs: 21-69, 130-202, 224-263, 298-389, and 426-505.

[0127] In some embodiments, the spacer sequence comprises one or more nucleobase alterations at one or more positions in any one of the sequences of TABLE 1 and TABLE 2. Alternative nucleobases can be any one or more of A, C, G, T or U, or a deletion, or an insertion. In some embodiments, the U is pseudouracil. By way of non-limiting example, a guanine nucleobase could be replaced with the nucleobase of any one of a cytosine, adenosine, thymine, and uracil. In some instance, the spacer sequence comprises only one nucleobase alterations relative to a sequence of TABLE 1 and TABLE 2. In some instance, the spacer sequence comprises not more than 1, not more than 2, nor more than 3, or not more than 4 nucleobase alterations relative to a sequence of TABLE 1 and TABLE 2. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0128] In some embodiments, compositions and systems comprise an effector protein or nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a guide nucleic acid comprising a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a spacer sequence selected from TABLE 1. In some embodiments, compositions and systems comprise an effector protein or nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NOs: 515, 1045, or 1047; and a guide nucleic acid comprising a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence in the SOD1 gene, wherein the target sequence is adjacent to a PAM of NNTN. In some embodiments, the PAM is TNTR. In some embodiments, the PAM is VNTR. In some embodiments, the PAM is TNTY. In some embodiments, the PAM is selected from: AATA, AATG, ACTA, ACTG, AGTA, AGTG, ATTA, ATTG, CATA, CATG, CCTA, CCTG, CGTA, CGTG, CTTA, CTTG, GATA, GATG, GCTA, GCTG, GGTA, GGTG, GTTA, GTTG, TATA, TATC, TATG, TATT, TCTA, TCTC, TCTG, TCTT, TGTA, TGTC, TGTG, TGTT, TTTA, TTTC, TTTG, and TTTT.

[0129] In some embodiments, compositions and systems comprise an effector protein or nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046; and a guide nucleic acid comprising a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a spacer sequence selected from TABLE 2. In some embodiments, compositions and systems comprise an effector protein or nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NOs: 516, 1041, 1042, 1044, or 1046; and a guide nucleic acid comprising a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence in the SOD1 gene, wherein the target sequence is adjacent to a PAM of NTTN. In some embodiments, the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT. ^Cooley Ref: MABI-044 / 03WO 344183-2319 Nucleic acid linkers

[0130] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers. In some embodiments, the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the guide nucleic acid comprises two or more linkers. In some embodiments, at least two or more linkers are the same. In some embodiments, at least two or more linkers are not same.

[0131] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5’-GAAA-3’ (SEQ ID NO: 1063).

[0132] In some embodiments, a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences. In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker. Repeat Sequences

[0133] Guide nucleic acids described herein may comprise one or more repeat sequences. A protein binding sequence of a guide nucleic acid may comprise a repeat sequence. A protein binding sequence of a guide nucleic acid may consist of a repeat sequence. In some embodiments, effector proteins are capable of non-covalently binding a repeat sequence. In some embodiments, a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid. In some embodiments, a repeat sequence comprises a nucleotide sequence that may interact with an effector protein. In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex). In some embodiments, the repeat sequence may also be referred to as a “protein-binding segment.”

[0134] In some embodiments, the repeat sequence is between 10 and 50, 12 and 48, 14 and 46, 16 and 44, and 18 and 42 nucleotides in length. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0135] In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3’ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and / or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence, which may be a direct link or by any suitable linker, examples of which are described herein. In some embodiments, the linker comprises SEQ ID NO: 1063.

[0136] In some embodiments, the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex). In some embodiments, the two sequences are not directly linked and hybridize to form a stem loop structure. In some embodiments, the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp). In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence may include a bulge. In some embodiments, the repeat sequence comprises a hairpin or stem-loop structure, optionally at the 5’ portion of the repeat sequence. In some embodiments, a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary. In some embodiments, such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity). In some embodiments, a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).

[0137] TABLE 3 provides illustrative repeat sequences for use with the compositions and methods of the disclosure. In some embodiments, the repeat sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences as set forth in TABLE 3.

[0138] In some embodiments, compositions, systems, and methods described herein comprise a guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 65%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%, or 100% identical to a sequence selected from TABLE 3. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0139] In some embodiments, compositions disclosed herein comprises a spacer sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences as set forth in TABLE 1 and a repeat sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 506. In some embodiments, compositions disclosed herein comprises a spacer sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences as set forth in TABLE 2, and a repeat sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064.^The combination of spacer and repeat sequences provided in SEQ ID NOs: 534-1038 are provided for illustrative purposes. It should be understood that these guides can comprise any of the repeat sequences disclosed herein (e.g., any one of SEQ ID NOs: 507-512 and 1064). For example, in some embodiments, the guide sequence comprises a spacer sequence selected from any one of SEQ ID NOs: 264-505 with a repeat sequence selected from any one of SEQ ID NOs: 507-512 and 1064. Intermediary sequences

[0140] Guide nucleic acids described herein may comprise an intermediary sequence. In some embodiments, the protein binding sequence comprises an intermediary sequence. In general, an intermediary sequence used in the present disclosure is not transactivated or transactivating. An intermediary sequence may also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides instead of or in addition to ribonucleotides, and / or modified bases. In general, the intermediary sequence non-covalently binds to an effector protein. In some embodiments, the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.

[0141] The terms, “intermediary RNA,” “intermediary RNA sequence,” and “intermediary sequence” as used herein, in a context of a single nucleic acid system, refers to a nucleotide sequence in a handle sequence, wherein the intermediary RNA sequence is capable of, at least partially, being non-covalently bound to an effector protein to form a complex (e.g., an RNP complex). An intermediary RNA sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein.

[0142] In some embodiments, a length of the intermediary sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the intermediary sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked ^Cooley Ref: MABI-044 / 03WO 344183-2319 nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.

[0143] An intermediary sequence may also comprise or form a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and / or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). An intermediary sequence may comprise from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region of the intermediary sequence does not hybridize to the 3’ region.

[0144] In some embodiments, the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may interact with an intermediary sequence comprising a single stem region or multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, an intermediary sequence comprises 1, 2, 3, 4, 5 or more stem regions.

[0145] In some embodiments, compositions and systems comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047; and a guide nucleic acid comprising an intermediary sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the sequence: ACAGCUUAUUUGGAAGCUGAAAUGUGAGGUUUAUAACACUCACAAGAAUCCU (SEQ ID NO: 513). In some embodiments, an intermediary sequence comprises at least 5, at ^Cooley Ref: MABI-044 / 03WO 344183-2319 least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 45, or at least 50 contiguous nucleotides of SEQ ID NO: 513. Handle sequences

[0146] In some embodiments, compositions, systems, and methods described herein comprise a guide nucleic acid, wherein the guide nucleic acid comprises a handle sequence. In some embodiments, the handle sequence comprises a protein binding sequence. In some embodiments, the handle sequence is the protein binding sequence. In some embodiments, the handle sequence comprises an intermediary sequence. In some embodiments, the handle sequence comprises a repeat sequence. In some embodiments, the intermediary sequence is at the 5’-end of the handle sequence. In some embodiments, the repeat sequence is at the 3’- end of the handle sequence. In some embodiments, the handle sequence comprises a linker sequence. In some embodiments, the linker sequence links the intermediary sequence and the repeat sequence. A non-limiting example of a linker sequence is 5’-GAAA-3’ (SEQ ID NO: 1063). In some embodiments, the intermediary sequence is 5’ to the repeat sequence. In some embodiments, the intermediary sequence is 5’ to the linker. In some embodiments, the intermediary sequence is 3’ to the repeat sequence. In some embodiments, the intermediary sequence is 3’ to the linker. In some embodiments, the repeat sequence is 3’ to the linker. In some embodiments, the repeat sequence is 5’ to the linker.

[0147] In some embodiments, compositions and systems comprise an sgRNA. In some embodiments, the sgRNA comprises a handle sequence. The handle sequence of the sgRNA may comprise a hairpin region. The handle sequence of the sgRNA may comprise a linker and a repeat sequence. The hairpin region may be positioned 5’ of the linker sequence and / or repeat sequence. The hairpin region may be positioned 3’ of the linker sequence and / or repeat sequence. The hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop structure linking the first sequence and the second sequence.

[0148] In some embodiments, an effector protein may recognize a secondary structure of a handle sequence. In some embodiments, at least a portion of the handle sequence interacts with an effector protein described herein. Accordingly, in some embodiments, at least a portion of the intermediary sequence interacts with the effector protein described herein. In some embodiments, both, at least a portion of the intermediary sequence and at least a portion of the repeat sequence, interacts with the effector protein. In general, the handle sequence is capable of interacting (e.g., non-covalent binding) with an effector protein described herein. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0149] The term, “handle sequence,” as used herein, refers to a sequence of nucleotides in a single guide RNA (sgRNA), that is: 1) capable of being non-covalently bound by an effector protein and 2) connects the portion of the sgRNA capable of being non-covalently bound by an effector protein to a nucleotide sequence that is hybridizable to a target nucleic acid. In general, the handle sequence comprises an intermediary RNA sequence, that is capable of being non-covalently bound by an effector protein. In some embodiments, the handle sequence further comprises a repeat sequence. In such embodiments, the intermediary RNA sequence or a combination of the intermediary RNA and the repeat sequence is capable of being non- covalently bound by an effector protein.

[0150] In some embodiments, the handle sequence of an sgRNA comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the sgRNA comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize an sgRNA comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the sgRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0151] A handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a length of the handle sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.

[0152] In some embodiments, the length of a handle sequence in an sgRNA is not greater than 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides. In some embodiments, the length of a handle sequence in an ^Cooley Ref: MABI-044 / 03WO 344183-2319 sgRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 66 to 105 linked nucleotides, 67 to 105 linked nucleotides, 68 to 105 linked nucleotides, 69 to 105 linked nucleotides, 70 to 105 linked nucleotides, 71 to 105 linked nucleotides, 72 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is 40 to 70 nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides.

[0153] In some embodiments, a handle sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the sequence: ACAGCUUAUUUGGAAGCUGAAAUGUGAGGUUUAUAACACUCACAAGAAUCCU GAAAAAGGAUGCCAAAC (SEQ ID NO: 514). In some embodiments, a handle sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 45, or at least 50 contiguous nucleotides of SEQ ID NO: 514. Such a handle sequence may be useful in a guide nucleic acid that is to be used with an effector protein that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047.

[0154] In some embodiments, the sequences in any one of TABLES 1-3 can be modified. In some embodiments, nucleic acids (e.g., nucleic acids encoding effector proteins, engineered guide nucleic acids, or nucleic acids encoding engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2’O-methyl modified nucleotides (e.g., 2’-O- Methyl (2’OMe) sugar modifications); 2’ fluoro modified nucleotides (e.g., 2’-fluoro (2’-F) sugar modifications); locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7- methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates , thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage; phosphorothioate and / or heteroatom internucleoside linkages, such as -CH2-NH-O- CH2-, -CH2-N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2- ^Cooley Ref: MABI-044 / 03WO 344183-2319 O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -O-P(=O)(OH)-O-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester internucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH2 component parts; and combinations thereof. In some embodiments, the modification includes at least one phosphorothioate (PS) linkage. In some embodiments, the modification includes at least one 2’-O-Methyl oligonucleotide (OMe). In some embodiments, the modification includes at least one locked nucleic acid (LNA). In some embodiments, the modification includes at least one phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, the modification includes at least one or more peptide nucleic acid (PNA). In some embodiments, the first 3 and last 3 amino acids are O-Me modified, and the first 3 and last 2 linkages are phosphorothioate linkages. In some embodiments, the sequence is modified mN*mN*mN*NNN....NNNmN*mN*mN where m is a 2' O-Me modified sugar moiety and the * denotes a PS linkage. Single Guide Nucleic Acid Systems

[0155] In some embodiments, compositions, systems, and methods described herein comprise a single guide nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid. In some embodiments, a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In some embodiments, a second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In the single nucleic acid system, the guide nucleic acid forms a complex with the effector protein, and the effector protein is not transactivated by the guide nucleic acid. In other words, activity of effector protein does not require binding to a second or intermediary guide nucleic acid molecule. Exemplary guide nucleic acids for a single nucleic acid system are crRNAs or sgRNAs. crRNA

[0156] In some embodiments, a single guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is the crRNA. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and ^Cooley Ref: MABI-044 / 03WO 344183-2319 the SR of the crRNA comprises a spacer sequence. In some embodiments, the FR consists of a repeat sequence. In some embodiments, the spacer sequence follows the repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence precedes the repeat sequence in a 5’ to 3’ direction. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker (e.g., one or more nucleotides).

[0157] In some embodiments, a crRNA is useful as a single guide nucleic acid system for compositions, methods, and systems described herein or as part of a single guide nucleic acid system for compositions, methods, and systems described herein. In such embodiments, a single guide nucleic acid system comprises a guide nucleic acid comprising a crRNA wherein, a repeat sequence of a crRNA is capable of causing a crRNA to interact with an effector protein. In some embodiments, a single guide nucleic acid system comprises a guide nucleic acid comprising a crRNA linked to another nucleotide sequence that is capable of being non- covalently bound by an effector protein. In some embodiments, a crRNA is sufficient to form complex with an effector protein (e.g., to form an RNP) through the repeat sequence and direct the effector protein to a target nucleic acid sequence through the spacer sequence.

[0158] In some embodiments, compositions and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046; and a guide nucleic acid that consists essentially of a crRNA. In some embodiments, the crRNA comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038. In some embodiments, the crRNA consists of a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038.

[0159] A crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a crRNA comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In some embodiments, a crRNA comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about ^Cooley Ref: MABI-044 / 03WO 344183-2319 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. sgRNA

[0160] In some embodiments, a guide nucleic acid comprises a single guide RNA (sgRNA). In some embodiments, the guide nucleic acid is an sgRNA. In some embodiments, an sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary RNA sequence, a repeat sequence, a spacer sequence, and optionally a linker). In some embodiments, an sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence. The combination of a spacer sequence (e.g., a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid) with a handle sequence may be referred to herein as a single guide RNA (sgRNA), wherein the spacer sequence and the handle sequence are covalently linked. In some embodiments, the spacer sequence and handle sequence are linked by a phosphodiester bond. In some embodiments, the spacer sequence and handle sequence are linked by one or more linked nucleotides. In some embodiments, a guide nucleic acid may comprise a spacer sequence, a repeat sequence, or handle sequence, or a combination thereof. In some embodiments, the handle sequence may comprise a portion of, or all of, a repeat sequence. In general, an sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence.

[0161] In some embodiments, the compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g., a single nucleic acid system), wherein the guide RNA is an sgRNA. An sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. An sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and / or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). Such a sequence can be contained within a handle sequence as described herein.

[0162] In some embodiments, an sgRNA comprises one or more of one or more of a handle sequence, an intermediary sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof. For example, an sgRNA comprises a handle sequence and a spacer ^Cooley Ref: MABI-044 / 03WO 344183-2319 sequence; an intermediary sequence and a crRNA; an intermediary sequence, a repeat sequence and a spacer sequence; and the like.

[0163] In some embodiments, an sgRNA comprises an intermediary sequence and an crRNA. In some embodiments, an intermediary sequence is 5’ to a crRNA in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and crRNA. In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0164] In some embodiments, an sgRNA comprises a handle sequence and a spacer sequence. In some embodiments, a handle sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked handle sequence and spacer sequence. In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0165] In some embodiments, an sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence. In some embodiments, an intermediary sequence is 5’ to a repeat sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and repeat sequence. In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. In some embodiments, a repeat sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked repeat sequence and spacer sequence. In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0166] An exemplary handle sequence in an sgRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region does not hybridize to the 3’ region. In some embodiments, the 3’ region is covalently linked to a spacer sequence (e.g., through a ^Cooley Ref: MABI-044 / 03WO 344183-2319 phosphodiester bond). In some embodiments, the 5’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).

[0167] In some embodiments, compositions and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 515, 1045, or 1047; and a guide nucleic acid that comprises an sgRNA. In some embodiments, the sgRNA comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 534-796. In some embodiments, the sgRNA consists of a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 534-796. Dual Nucleic Acid Systems

[0168] In some embodiments, compositions, systems, and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector protein or a nucleotide sequence encoding the one or more effector protein, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid. In the dual nucleic acid system having a complex of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA. In other words, in a dual nucleic acid system, activity of effector protein requires binding to a tracrRNA molecule.

[0169] In some embodiments, a repeat hybridization sequence is at the 3’ end of a tracrRNA sequence. In some embodiments, a repeat hybridization sequence may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.

[0170] A tracrRNA and / or tracrRNA-crRNA duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRNA. In some embodiments, the secondary structure modifies activity of the effector protein on a target ^Cooley Ref: MABI-044 / 03WO 344183-2319 nucleic acid. In some embodiments, the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the secondary structure comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a secondary structure comprising multiple stem regions. In some embodiments, nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary structure comprises one or more loops. In some embodiments, the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops. 4. Effector Proteins

[0171] In some embodiments, compositions provided herein comprise one or more effector proteins. In some embodiments, compositions and systems described herein comprise an effector protein that is similar to a naturally occurring effector protein. The effector protein may lack a portion of the naturally occurring effector protein. The effector protein may comprise an engineered mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature.

[0172] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein.

[0173] In some embodiments, the effector protein is a programmable nuclease (e.g., a CRISPR-associated (Cas) protein) that modifies a target sequence in a target nucleic acid. In some embodiments, the effector protein is a programmable nuclease that modifies a region of the nucleic acid that is near, but not within, to the target sequence. Effector proteins may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and ^Cooley Ref: MABI-044 / 03WO 344183-2319 single-stranded DNA (ssDNA). Effector proteins may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof.

[0174] An effector protein may function as a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., a dimer or a multimer). An effector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of another functional activity (e.g., modifying a target nucleic acid).

[0175] In some embodiments, the effector protein is a Type V Cas protein. In some embodiments, the effector protein is CasPhi.12 or a variant thereof. In some embodiments, the effector protein is CasM.265466 or a variant thereof. A CasPhi.12 is around half of the size of Cas9, and CasM.265466 is around one third of the size of Cas9. The smaller sizes of CasPhi.12 and CasM.265466 make them ideal to be packaged together with their corresponding guide RNAs into a single AAV vector, thus overcoming the drawbacks of dual AAV vector systems.

[0176] TABLEs 5 and 6 provides illustrative amino acid sequences of effector proteins. In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences as set forth in TABLEs 4-7. In some embodiments, the amino acid sequence of an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences as set forth in TABLEs 4-7. In some embodiments, the effector protein consists of an amino acid sequence selected from the sequences as set forth in TABLE 4-7.

[0177] In some embodiments, compositions, systems, and methods comprise an effector protein or uses thereof, wherein the amino acid sequence of the effector protein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, at least about 520, at least about 540, at least about 560, at least about 580, at least about 600, at least about 620, at least about 640, at least about 660, at least about 680, or at least about 700 contiguous amino acids of a sequence in TABLEs 4-7. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0178] In some embodiments, compositions, systems, and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to a sequence recited in TABLEs 4-7. In some embodiments, an amino acid alteration comprises a deletion of an amino acid. In some embodiments, an amino acid alteration comprises an insertion of an amino acid. In some embodiments, an amino acid alteration comprises a conservative amino acid substitution. In some embodiments, an amino acid alteration comprises a non-conservative amino acid substitution. In some embodiments, one or more amino acid alterations comprises a combination of one or more conservative amino acid substitutions and one or more non- conservative amino acid substitutions. When describing a conservative amino acid substitution herein, reference is made to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, when describing a non-conservative alteration (e.g., non-conservative substitution), reference is made to the replacement of one amino acid residue for another that does not have a related side chain. It is understood that genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gln (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl. Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gln (Q). Amino acids may be related by sulfur- containing side chains: Cys (C) and Met (M).

[0179] In some embodiments, effector proteins are engineered variants of CasM.265466 (SEQ ID NO: 515) and CasPhi.12 (SEQ ID NO: 516). Engineered variants of CasM.265466 (SEQ ID NO: 515) and CasPhi.12 (SEQ ID NO: 516) may comprise amino acid substitutions relative to SEQ ID NO: 515 and SEQ ID NO: 516, respectively. Exemplary amino acid substitutions are described in TABLE 5 and TABLE 6. The amino acid substitutions in TABLE 7 can be combined with any of the substitutions listed in either TABLE 5 or TABLE 6. The amino acid substitutions in TABLE 5 and TABLE 6 may be combined with other amino acid alterations described herein. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0180] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is at a position selected from K58, I80, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 515, with the exception of at least one amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is a position selected from K58, I80, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the amino acid substitution is selected from K58X, I80X, T84X, K105X, N193X, C202X, S209X, G210X, A218X, D220X, E225X, C246X, N286X, M295X, M298X, A306X, Y315X, and Q360X, wherein X is selected from R, K, and H.

[0181] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D220R / A306K and D220R / K250N and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 515, with the exception of at least one amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D220R / A306K, D220R / K250N, D220R / E335Q and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[0182] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, wherein the polypeptide comprises at least one ^Cooley Ref: MABI-044 / 03WO 344183-2319 amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 515, with the exception of at least one amino acid substitution relative to SEQ ID NO: 515, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof. In some aspects, these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein. TABLE 5 provides the exemplary amino acid alterations relative to SEQ ID NO: 515 useful in compositions, systems, and methods described herein.

[0183] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 515, with the exception of two amino acid substitutions at D220 and E335 relative to SEQ ID NO: SEQ ID NO: 515. In some embodiments, the amino acid substitutions are D220R and E335Q. In some embodiments, the engineered effector protein comprises or consists of SEQ ID NO: 1045.

[0184] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, and comprises the amino acid substitution of D220R relative to SEQ ID NO: 515. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, and comprises the amino acid substitutions of D220R and K250N relative to SEQ ID NO: 515. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 515, and comprises the amino acid substitutions of D220R and any one of A306K, A306H, and A306R, relative to SEQ ID NO: 515.

[0185] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is at a position selected from I2, T5, K15, R18, H20, S21, L26, N30, E33, E34, A35, K37, K38, R41, N43, Q54, Q79R, K92E, K99R, S108, E109, H110, G111, D113, T114, P116, K118, E119, A121, N132, K135, Q138, V139, N148, L149, E157, E164, E166, E170, Y180, L182, Q183, K184, S186, K189, S196, S198, K200, I203, S205, K206, Y207, H208, N209, Y220, S223, E258, K281, K348, N355, S362, N406, K435, I471, I489, Y490, F491, D495, K496, K498, K500, D501, V502, K504, S505, D506, V521, N568, S579, Q612, S638, F701, P707, ^Cooley Ref: MABI-044 / 03WO 344183-2319 and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is at a position selected from I2, T5, K15, R18, H20, S21, L26, N30, E33, E34, A35, K37, K38, R41, N43, Q54, Q79R, K92E, K99R, S108, E109, H110, G111, D113, T114, P116, K118, E119, A121, N132, K135, Q138, V139, N148, L149, E157, E164, E166, E170, Y180, L182, Q183, K184, S186, K189, S196, S198, K200, I203, S205, K206, Y207, H208, N209, Y220, S223, E258, K281, K348, N355, S362, N406, K435, I471, I489, Y490, F491, D495, K496, K498, K500, D501, V502, K504, S505, D506, V521, N568, S579, Q612, S638, F701, P707, and a combination thereof. In some embodiments, the amino acid substitution is selected from I2X, T5X, K15X, R18X, H20X, S21X, L26X, N30X, E33X, E34X, A35X, K37X, K38X, R41X, N43X, Q54X, Q79RX, K92EX, K99RX, S108X, E109X, H110X, G111X, D113X, T114X, P116X, K118X, E119X, A121X, N132X, K135X, Q138X, V139X, N148X, L149X, E157X, E164X, E166X, E170X, Y180X, L182X, Q183X, K184X, S186X, K189X, S196X, S198X, K200X, I203X, S205X, K206X, Y207X, H208X, N209X, Y220X, S223X, E258X, K281X, K348X, N355X, S362X, N406X, K435X, I471X, I489X, Y490X, F491X, D495X, K496X, K498X, K500X, D501X, V502X, K504X, S505X, D506X, V521X, N568X, S579X, Q612X, S638X, F701X, P707X, wherein X is selected from R, K, and H.

[0186] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 516 wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, I471T, S579R, F701R, P707R, K189P, S638K, Q54R, Q79R, Y220S, N406K, E119S, K92E, K435Q, N568D, and V521T, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is selected from T5R, L26R, L26K, A121Q, V139R, S198R, S223P, E258K, I471T, S579R, F701R, P707R, K189P, S638K, Q54R, Q79R, Y220S, N406K, E119S, K92E, K435Q, N568D, and V521T, and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[0187] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein the polypeptide comprises at least one ^Cooley Ref: MABI-044 / 03WO 344183-2319 amino acid substitution relative to SEQ ID NO: 516 wherein the amino acid substitution is selected from L26K / A121Q, L26R / A121Q, K99R / L149R, K99R / N148R, L149R / H208R, S362R / L26R L26R / N148R, L26R / H208R, N30R / N148R, L26R / K99R, L26R / P707R, L26R / L149R, L26R / N30R, L26R / N355R, L26R / K281R, L26R / S108R, L26R / K348R, T5R / V139R, I2R / V139R, K99R / S186R, L26R / A673G, L26R / Q674R, S579R / L26K, F701R / E258K, T5R / L26K, L26R / K435Q, L26K / E567Q, L26R / G685R, L26R / Q674K, L26R / P699R, L26R / T70E, L26R / Q232R, L26R / T252R, L26R / P679R, L26R / E83K, L26R / E73P, L26R / K248E, L26R, T5R / S223P, S579R / S223P, L26R / S223P, T5R / A121Q, L26R / A696R, S198R / I471T, L26R / N153R, L26R / E682R, L26R / D703R, Q612R / L26K, L26R / I471T, K348R / L26K, S579R / I471T, L26R / V228R, T5R / S638K, S579R / K189P, S579R / E258K, L26R / K260R, L26R / S638K, S579R / Y220S, T5R / I471T, L26R / F233R, L26R / V521T, F701R / A121Q, L26R / G361R, S198R / E258K, L26R / S472R, T5R / Y220S, L26R / A150K, L26R / S684R, L26R / E157R, L26R / K248R, F701R / L26K, S198R / N406K, S198R / Y220S, S198R / S638K, S198R / V521T, S579R / A121Q, K348R / Y220S, S198R / K189P, L26R / E242R, L26R / K678R, T5R / N406K, L26R / I158K, T5R / V521T, L26R / N259R, L26R / K257R, L26R / K256R, T5R / K189P, L26R / C405R, S579R / V521T, S579R / N406K, T5R / K92E, T5R / E258K, L26R / I97R, S579R / S638K, T5R / K435Q, F701R / S638K, L26R / L236R, F701R / I471T, Q612R / S223P, F701R / S223P, S198R / E119S, S579R / K92E, L26R / E715R, Q612R / I471T, F701R / Y220S, S198R / S223P, and L26R / K266R, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is selected from L26K / A121Q, L26R / A121Q, K99R / L149R, K99R / N148R, L149R / H208R, S362R / L26R L26R / N148R, L26R / H208R, N30R / N148R, L26R / K99R, L26R / P707R, L26R / L149R, L26R / N30R, L26R / N355R, L26R / K281R, L26R / S108R, L26R / K348R, T5R / V139R, I2R / V139R, K99R / S186R, L26R / A673G, L26K / E567Q, L26R / Q674R, S579R / L26K, F701R / E258K, T5R / L26K, L26R / K435Q, L26R / G685R, L26R / Q674K, L26R / P699R, L26R / T70E, L26R / Q232R, L26R / T252R, L26R / P679R, L26R / E83K, L26R / E73P, L26R / K248E, L26R, T5R / S223P, S579R / S223P, L26R / S223P, T5R / A121Q, L26R / A696R, S198R / I471T, L26R / N153R, L26R / E682R, L26R / D703R, Q612R / L26K, L26R / I471T, K348R / L26K, S579R / I471T, L26R / V228R, T5R / S638K, S579R / K189P, S579R / E258K, L26R / K260R, L26R / S638K, S579R / Y220S, T5R / I471T, L26R / F233R, L26R / V521T, F701R / A121Q, L26R / G361R, S198R / E258K, L26R / S472R, T5R / Y220S, L26R / A150K, L26R / S684R, L26R / E157R, L26R / K248R, F701R / L26K, S198R / N406K, S198R / Y220S, ^Cooley Ref: MABI-044 / 03WO 344183-2319 S198R / S638K, S198R / V521T, S579R / A121Q, K348R / Y220S, S198R / K189P, L26R / E242R, L26R / K678R, T5R / N406K, L26R / I158K, T5R / V521T, L26R / N259R, L26R / K257R, L26R / K256R, T5R / K189P, L26R / C405R, S579R / V521T, S579R / N406K, T5R / K92E, T5R / E258K, L26R / I97R, S579R / S638K, T5R / K435Q, F701R / S638K, L26R / L236R, F701R / I471T, Q612R / S223P, F701R / S223P, S198R / E119S, S579R / K92E, L26R / E715R, Q612R / I471T, F701R / Y220S, S198R / S223P, and L26R / K266R, and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[0188] In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least two amino acid substitutions relative to SEQ ID NO: 516, wherein the amino acid substitutions comprise L26K / E567Q. In some embodiments, the polypeptide comprises or consists of SEQ ID NO: 1044.

[0189] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 516 wherein the amino acid substitution is selected from E157A, E164A, E164L, E166A, E166I, E170A, I489A, I489S, Y490S, Y490A, F491A, F491S, F491G, D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S, D501R, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is selected from E157A, E164A, E164L, E166A, E166I, E170A, I489A, I489S, Y490S, Y490A, F491A, F491S, F491G, D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S, D501R, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A, and a combination thereof. In some embodiments, these engineered effector proteins comprise a nickase activity.

[0190] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein amino acids S478-S505 have been deleted. In some embodiments, the effector protein is an engineered effector protein that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein amino acids S478-S505 have been deleted and replaced with SDLYIERGGDPRDVHQQVETKPKGKRKSEIRILKIR (SEQ ID NO: 1039) or SDYIVDHGGDPEKVFFETKSKKDKTKRYKRR (SEQ ID NO: 1040). In some ^Cooley Ref: MABI-044 / 03WO 344183-2319 embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 1041. In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or is 100% identical to SEQ ID NO: 1042.

[0191] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 516 wherein the amino acid substitution is selected from D369A, D369N, D658A, D658N, E567A, E567Q, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 516, with the exception of at least one amino acid substitution relative to SEQ ID NO: 516, wherein the amino acid substitution is selected from D369A, D369N, D658A, D658N, E567A, E567Q, and a combination thereof. In some aspects, these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein. TABLE 6 provides the exemplary amino acid alterations relative to SEQ ID NO: 516 useful in compositions, systems, and methods described herein.

[0192] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516, and comprises the amino acid substitution selected from L26R, L26K, I471T, and any combination thereof relative to SEQ ID NO: 516. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516 and comprises the amino acid substitutions of: L26R or L26K, and A673G relative to SEQ ID NO: 516. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 516 and comprises the amino acid substitutions of: L26R or L26K, and I471T, relative to SEQ ID NO: 516.

[0193] Exemplary sequences of engineered effector proteins are provided in TABLE 7.

[0194] In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLEs 4-7, wherein the effector protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions relative to the sequence selected from TABLEs 4-7. In some ^Cooley Ref: MABI-044 / 03WO 344183-2319 embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLEs 4-7, wherein the effector protein comprises 1 to 10, 10 to 20, 20 to 30, or 30 to 40 conservative amino acid substitutions relative to the sequence selected from TABLEs 4-7. In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLEs 4-7, wherein the effector protein comprises not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-conservative amino acid substitutions relative to the sequence selected from TABLEs 4-7.

[0195] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% similar to any one of the sequences selected from TABLEs 4-7. An amino acid sequence of the effector protein is similar to the reference amino acid sequence, when a value that is calculated by dividing a similarity score by the length of the alignment. The similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89:10915–10919 (1992)) that is transformed so that any value ^ 1 is replaced with +1 and any value ^ 0 is replaced with 0. For example, an Ile (I) to Leu (L) substitution is scored at +2.0 by the BLOSUM62 similarity matrix, which in the transformed matrix is scored at +1. This transformation allows the calculation of percent similarity, rather than a similarity score. Alternately, when comparing two full protein sequences, the proteins can be aligned using pairwise MUSCLE alignment. Then, the % similarity can be scored at each residue and divided by the length of the alignment. For determining % similarity over a protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, if a substitution could be treated as conservative with any of the amino acids in that position of the multilevel consensus sequence, +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence QIQ would receive three points. This is because in the transformed BLOSUM62 matrix, each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1. For each position, the highest score is used when calculating ^Cooley Ref: MABI-044 / 03WO 344183-2319 similarity. The % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given the parameters matrix = BLOSUM62 and threshold ^ 1.

[0196] In some cases, the effector proteins comprise a RuvC domain. In some embodiments, the RuvC domain may be defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multiple RuvC subdomains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary amino acid sequence of the effector protein but form a RuvC domain once the protein is produced and folds. In many cases, effector proteins comprise a recognition domain with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. An effector protein may comprise a zinc finger domain.

[0197] An effector protein may be small, which may be beneficial for nucleic acid detection or editing (for example, the effector protein may be less likely to adsorb to a surface or another biological species due to its small size). The smaller nature of these effector proteins may allow for them to be more easily packaged and delivered with higher efficiency in the context of genome editing and more readily incorporated as a reagent in an assay. In some embodiments, the length of the effector protein is less than 400 linked amino acid residues. In some embodiments, the length of the effector protein is less than 1200 linked amino acid residues. In some embodiments, the length of the effector protein is less than 900 linked amino acid residues. In some embodiments, the length of the effector protein is less than 800 linked amino acid residues. In some embodiments, the length of the effector protein is less than 500 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 1200 linked amino acids. In some embodiments, the length of the effector protein is about 400 to about 800 linked amino acid residues. In some embodiments, the length of the effector protein is about 650 to about 750 linked amino acids.

[0198] In some embodiments, the effector protein is a Cas protein other than a Cas9 protein. Cas9 proteins include, but are not limited to, Neisseria meningitidis (NmCas9); Streptococcus pyogenes (SpCas9) including SpCas9-HF1, eSpCas9; Staphylococcus aureus (SaCas9) including SaCas9-HF, KKHSaCas9, microABE1744; Streptococcus thermophilus (StCas9); Staphylococcus lugdunensis (SluCas9); Francisella novicida (FnCas9); Campylobacter jejuni (CjCas9); Staphylococcus auricularis (SauriCas9); Streptococcus canis (ScCas9); ^Cooley Ref: MABI-044 / 03WO 344183-2319 Staphylococcus lugdunensis (SluCas9); Staphylococcus lugdunensis Cas9 (SluCas9); Staphylococcus lutrae Cas9 (SlutrCas9); Staphylococcus haemolyticus Cas9 (ShaCas9); Staphylococcus microti (SmiCas9); Staphylococcus pasteuri (SpaCas9); HypaCas9, FokI- Fused dCas9, and xCas9.

[0199] In some embodiments, the effector protein is linked to a nuclear localization signal (NLS). In some embodiments, compositions and systems described herein may comprise a NLS sequence that is adjacent to the N terminal of the effector protein or that is adjacent to the C terminal of the effector protein, or both. In some embodiments, effector proteins are linked to an endosomal escape polypeptide (EEP). In certain embodiments, the nucleotide sequence encoding the effector protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.

[0200] TABLE 8 provides exemplary nuclear localization sequences and endosomal escape polypeptide sequences. In TABLE 8, X is any naturally occurring amino acid, and ^D / E is any naturally occurring amino acid except Asp or Glu. Protospacer Adjacent Motif (PAM) Sequences

[0201] Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 10, 20, 30, 40 or 50 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to a target sequence. In some embodiments, systems, compositions, and methods comprise a guide nucleic acid or use thereof, wherein the guide nucleic acid comprises a spacer sequence that is complementary to a target sequence that is adjacent to a PAM sequence.

[0202] In some embodiments, the PAM is 5’-NNTN-3’, wherein N = any nucleic acid. In some embodiments, the PAM is 5’-TNTR-3’, wherein N = any nucleic acid and wherein R = a purine nucleic acid (i.e., A or G). Non-limiting examples of 5’-TNTR-3’ PAM sequences are disclosed in TABLE 9. In some embodiments, the effector protein recognizes a PAM sequence as shown in TABLE 9, wherein: N represents any nucleotide; V represents A, C, or G; Y represents C or T; and R represents A or G. In some embodiments, the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 9. In some embodiments, the PAM is selected from: AATA, AATC, AATG, AATT, ACTA, ACTC, ACTG, ACTT, AGTA, AGTG, AGTC, AGTG, AGTT, ATTA, ATTC, ATTG, ^Cooley Ref: MABI-044 / 03WO 344183-2319 ATTT, CATA, CATC, CATG, CATT, CCTA, CCTC, CCTG, CCTT, CGTA, CGTC, CGTG, CGTT, CTTA, CTTC, CTTG, CTTT, GATA, GATC, GATG, GATT, GCTA, GCTC, GCTG, GCTT, GGTA, GGTC, GGTG, GGTT, GTTA, GTTC, GTTG, GTTT, TATA, TATC, TATG, TATT, TCTA, TCTC, TCTG, TCTT, TGTA, TGTC, TGTG, TGTT, TTTA, TTTC, TTTG, and TTTT. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047.

[0203] In some embodiments, the PAM is 5’-NTTN-3’, wherein N = any nucleic acid. Exemplary PAM sequences are disclosed in TABLE 10. In some embodiments, the effector protein recognizes a PAM sequence as shown in TABLE 10. In some embodiments, the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 10. In some embodiments, the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT. In some embodiments, the effector protein^comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046. Nuclease-dead effector proteins

[0204] In some embodiments, the effector protein is a dCas protein, also referred to as a catalytically inactive effector protein^and / or “dead” (abbreviated by “d”) effector protein in combination (e.g., fusion) with a polypeptide comprising recombinase activity. Although an effector protein normally has nuclease activity, in some embodiments, an effector protein does not have nuclease activity. In some embodiments, a dCas protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99%, but not 100% identical to a sequence recited in TABLE 4. In some embodiments, a dCas protein of the systems and compositions described herein comprises at least one amino acid alteration relative to a sequence recited in TABLE 5 or TABLE 6.

[0205] Catalytically inactive effector proteins may comprise a modified form of a wildtype counterpart. The modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the effector protein. In such embodiments, the catalytically inactive effector protein may ^Cooley Ref: MABI-044 / 03WO 344183-2319 also be referred to as a catalytically reduced effector protein. For example, a nuclease domain (e.g., RuvC domain) of an effector protein can be deleted or mutated so that it is no longer functional or comprises reduced nuclease activity. The modified form of the effector protein may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart. The modified form of an effector protein may have no substantial nucleic acid-cleaving activity. When an effector protein is a modified form that has no substantial nucleic acid-cleaving activity, it may be referred to as enzymatically inactive and / or dead. A dead effector polypeptide (e.g., catalytically inactive effector protein) may bind to a target nucleic acid but may not cleave the target nucleic acid. A dead effector polypeptide (e.g., catalytically inactive effector protein) may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid.

[0206] In some embodiments, a nuclease-dead effector protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515, and wherein the effector protein further comprises one or more alterations selected from D237A, D418A, D418N, E335A, and E335Q. In some embodiments, a nuclease-dead effector protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to SEQ ID NO: 515, and wherein the effector protein further comprises one or more alterations selected from D237A, D418A, D418N, E335A, and E335Q.

[0207] In some embodiments, a nuclease-dead effector protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516, and wherein the effector protein further comprises one or more alterations selected from D369A, D369N, E567A, E567Q, D658A and D658N. In some embodiments, a nuclease-dead effector protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to SEQ ID NO: 516, and wherein the effector protein further comprises one or more alterations selected from D369A, D369N, E567A, E567Q, D658A and D658N. ^Cooley Ref: MABI-044 / 03WO 344183-2319 Fusion Proteins

[0208] In some embodiments, compositions, systems, and methods comprise a fusion protein, a fusion partner, or uses thereof. A fusion protein generally comprises an effector protein and a fusion partner. In some embodiments, the fusion partner comprises a polypeptide or peptide that is linked to the effector protein. In some embodiments, the fusion partner is not linked to the effector protein, but is brought into proximity of the effector protein by other means. By way of non-limiting example, a fusion partner protein may comprise a peptide that binds an aptamer of a guide nucleic acid, wherein the effector protein is also capable of binding the guide nucleic acid, the guide nucleic acid thereby bringing the fusion partner into proximity of the effector protein. In some embodiments, the fusion partner is capable of binding or being bound by an effector protein. In some embodiments, the fusion partner and the effector protein are both capable of binding or being bound by an additional protein or moiety, the additional protein or moiety thereby bringing the fusion partner into proximity of the effector protein. In some embodiments, the fusion protein is a heterologous peptide or polypeptide as described herein. In some embodiments, the amino terminus of the fusion partner is linked to the carboxy terminus of the effector protein. In some embodiments, the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker. In some embodiments, the fusion partner is not an effector protein as described herein. In some embodiments, the fusion partner comprises a second effector protein or a multimeric form thereof. Accordingly, in some embodiments, the fusion protein comprises more than one effector protein. In such embodiments, the fusion protein can comprise at least two effector proteins that are same. In some embodiments, the fusion protein comprises at least two effector proteins that are different. In some embodiments, the multimeric form is a homomeric form. In some embodiments, the multimeric form is a heteromeric form. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins comprising the effector protein described herein and a fusion partner.

[0209] In some embodiments, a fusion partner imparts some function or activity to a fusion protein that is not provided by an effector protein. Such activities may include but are not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g., pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ^Cooley Ref: MABI-044 / 03WO 344183-2319 ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity, modification of a polypeptide associated with target nucleic acid (e.g., a histone), and / or signaling activity.

[0210] In some embodiments, a fusion partner may provide signaling activity. In some embodiments, a fusion partner may inhibit or promote the formation of multimeric complex of an effector protein. In an additional example, the fusion partner may directly or indirectly edit a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid. In some embodiments, the fusion partner may interact with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid. In other embodiments, the fusion partner may modify proteins associated with a target nucleic acid. In some embodiments, a fusion partner may modulate transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid. In yet another example, a fusion partner may directly or indirectly inhibit, reduce, activate, or increase expression of a target nucleic acid.

[0211] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA comprising a repeat sequence that is at least 90% identical to any one of SEQ ID NOs: 507-512 and 1064 and a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 2.

[0212] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA comprising a repeat sequence that is at least 95% identical to any one of SEQ ID NOs: 507-512 and 1064 and a spacer sequence that is at least 95% identical to any one of the sequences recited in TABLE 2.

[0213] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA comprising a repeat sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and a spacer sequence selected from any one of the sequences recited in TABLE 2. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0214] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA sequence that is at least 90% identical to any one of SEQ ID NOs: 797-1038.

[0215] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA sequence that is at least 95% identical to any one of SEQ ID NOs: 797-1038.

[0216] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising SEQ ID NOs: 516, 1041, 1042, 1044, or 1046 and a guide RNA sequence comprising any one of SEQ ID NOs: 797- 1038.

[0217] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a guide RNA comprising a repeat sequence that is at least 90% identical to SEQ ID NOs: 506 and a spacer sequence that is at least 90% identical to any one of the sequences recited in TABLE 1.

[0218] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a guide RNA comprising a repeat sequence that is at least 95% identical to SEQ ID NO: 506 and a spacer sequence that is at least 95% identical to any one of the sequences recited in TABLE 1.

[0219] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising SEQ ID NOs: 515, 1045, or 1047 and a guide RNA comprising a repeat sequence of SEQ ID NO: 506 and a spacer sequence selected from any one of the sequences recited in TABLE 1.

[0220] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a guide RNA sequence that is at least 90% identical to any one of SEQ ID NOs: 534-796.

[0221] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising an amino acid sequence ^Cooley Ref: MABI-044 / 03WO 344183-2319 that is at least 95% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a guide RNA sequence that is at least 95% identical to any one of SEQ ID NOs: 534-796.

[0222] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising SEQ ID NOs: 515, 1045, or 1047 and a guide RNA sequence comprising any one of SEQ ID NOs: 534-796. Nucleic Acid Modification Activity

[0223] In some embodiments, fusion partners have enzymatic activity that modifies a nucleic acid, such as a target nucleic acid. In some embodiments, the target nucleic acid may comprise or consist of a ssRNA, dsRNA, ssDNA, or a dsDNA. Examples of enzymatic activity that modifies the target nucleic acid include, but are not limited to: nuclease activity, which comprises the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids, such as that provided by a restriction enzyme, or a nuclease (e.g., FokI nuclease); methyltransferase activity such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1); DNA repair activity; DNA damage (e.g., oxygenation) activity; deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer forming activity; integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase); transposase activity; recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity; and glycosylase activity.

[0224] In some embodiments, fusion partners target a ssRNA, dsRNA, ssDNA, or a dsDNA. In some embodiments, fusion partners target ssRNA. Non-limiting examples of fusion partners for targeting ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and / or C to U editing enzymes); helicases; and RNA-binding proteins. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0225] It is understood that a fusion partner may include an entire protein, or in some embodiments, may include a fragment of the protein (e.g., a functional domain). In some embodiments, the functional domain binds or interacts with a nucleic acid, such as ssRNA, including intramolecular and / or intermolecular secondary structures thereof (e.g., hairpins, stem-loops, etc.). The functional domain may interact transiently or irreversibly, directly, or indirectly. In some embodiments, a functional domain comprises a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid mutating, nucleic acid modifying, nucleic acid cleaving, protein binding or combinations thereof. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity.

[0226] Accordingly, fusion partners may comprise a protein or domain thereof selected from: endonucleases (e.g., RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus); SMG5 and SMG6; domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFIIm); exonucleases such as XRN-1 or Exonuclease T; deadenylases such as HNT3; protein domains responsible for nonsense mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (e.g., CID1 and terminal uridylate transferase); and other suitable domains that affect nucleic acid modifications.

[0227] In some embodiments, an effector protein is a fusion protein, wherein the effector protein is linked to a chromatin-modifying enzyme. In some embodiments, the fusion protein chemically modifies a target nucleic acid, for example by methylating, demethylating, or acetylating the target nucleic acid in a sequence specific or non-specific manner. Base editors

[0228] In some embodiments, fusion partners edit a nucleobase of a target nucleic acid. Fusion proteins comprising such a fusion partner and an effector protein may be referred to as base editors. Such a fusion partner may be referred to as a base editing enzyme. In some embodiments, a base editor comprises a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant. In some embodiments, a base editor may be a fusion protein comprising a base editing enzyme linked to an effector protein. In some ^Cooley Ref: MABI-044 / 03WO 344183-2319 embodiments, the amino terminus of the fusion partner protein is linked to the carboxy terminus of the effector protein by the linker. In some embodiments, the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker. The base editor may be functional when the effector protein is coupled to a guide nucleic acid. The base editor may be functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein). Also, by way of non- limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.

[0229] In some embodiments, base editors are capable of catalyzing editing (e.g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). In some embodiments, a base editing enzyme, and therefore a base editor, is capable of converting an existing nucleobase to a different nucleobase, such as: an adenine (A) to guanine (G); cytosine (C) to thymine (T); cytosine (C) to guanine (G); uracil (U) to cytosine (C); guanine (G) to adenine (A); hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). In some embodiments, base editors edit a nucleobase on a ssDNA. In some embodiments, base editors edit a nucleobase on both strands of dsDNA. In some embodiments, base editors edit a nucleobase of an RNA.

[0230] In some embodiments, a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase. In some embodiments, upon binding to its target locus in the target nucleic acid (e.g., a DNA molecule), base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop”. In some embodiments, DNA bases within the R-loop are edited by the base editor having the deaminase enzyme activity. In some embodiments, base editors for improved efficiency in eukaryotic cells comprise a catalytically inactive effector protein that may generate a nick in the non-edited strand, inducing repair of the non-edited strand using the edited strand as a template.

[0231] In some embodiments, a base editing enzyme comprises a deaminase enzyme. Exemplary deaminases are described in US20210198330, WO2021041945, WO2021050571A1, and WO2020123887, all of which are incorporated herein by reference in their entirety. Exemplary deaminase domains are described WO2018027078 and WO2017070632, and each are hereby incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al., Nature, 533, 420-424 (2016); ^Cooley Ref: MABI-044 / 03WO 344183-2319 Gaudelli et al., Nature, 551, 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018- 0059-l, which are hereby incorporated by reference in their entirety. In some embodiments, the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein. In some embodiments, base editors comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)). In some embodiments, the fusion partner is a deaminase, e.g., ADAR1 / 2, ADAR-2, AID, or any function variant thereof.

[0232] In some embodiments, a base editor is a cytosine base editor (CBE). In some embodiments, the CBE may convert a cytosine to a thymine. In some embodiments, a cytosine base editing enzyme may accept ssDNA as a substrate but may not be capable of cleaving dsDNA, as linked to a catalytically inactive effector protein. In some embodiments, when bound to its cognate DNA, the catalytically inactive effector protein of the CBE may perform local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single-stranded bubble. In some embodiments, the catalytically inactive effector protein generated ssDNA R-loop may enable the CBE to perform efficient and localized cytosine deamination in vitro. In some embodiments, deamination activity is exhibited in a window of about 4 to about 10 base pairs. In some embodiments, fusion to the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which may enable the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies. In some embodiments, the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2018) Nature Biotechnology 36:848-846; Komor et al. (2016) Nature 533:420-424; Koblan et al. (2021) “Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning,” Nature Biotechnology; Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al. (2021) Nature Communications 12:1384, all incorporated herein by reference.

[0233] In some embodiments, CBEs comprise a uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG). In some embodiments, base excision repair (BER) of U•G in DNA is initiated by a UNG, which recognizes a U•G mismatch and cleaves the glyosidic bond between a uracil and a deoxyribose backbone of DNA. In some embodiments, BER results in the ^Cooley Ref: MABI-044 / 03WO 344183-2319 reversion of the U•G intermediate created by the first CBE back to a C•G base pair. In some embodiments, the UNG may be inhibited by fusion of a UGI. In some embodiments, the CBE comprises a UGI. In some embodiments, a C-terminus of the CBE comprises the UGI. In some embodiments, the UGI is a small protein from bacteriophage PBS. In some embodiments, the UGI is a DNA mimic that potently inhibits both human and bacterial UNG. In some embodiments, the UGI inhibitor is any protein or polypeptide that inhibits UNG. In some embodiments, the CBE may mediate efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C•G base pair to a T•A base pair through a U•G intermediate. In some embodiments, the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.

[0234] In some embodiments, a CBE nicks a non-edited DNA strand. In some embodiments, the non-edited DNA strand nicked by the CBE biases cellular repair of a U•G mismatch to favor a U•A outcome, elevating base editing efficiency. In some embodiments, a APOBEC1– nickase–UGI fusion efficiently edits in mammalian cells, while minimizing frequency of non- target indels. In some embodiments, base editors do not comprise a functional fragment of the base editing enzyme. In some embodiments, base editors do not comprise a function fragment of a UGI, where such a fragment may be capable of excising a uracil residue from DNA by cleaving an N-glycosidic bond.

[0235] In some embodiments, the fusion protein further comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI). In some embodiments, the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG. In some embodiments, the npUGI is a small molecule derived from uracil. Examples of small molecule non-protein uracil-DNA glycosylase inhibitors, fusion proteins, and Cas- CRISPR systems comprising base editing activity are described in WO2021087246, which is incorporated by reference in its entirety.

[0236] In some embodiments, a cytosine base editing enzyme, and therefore a cytosine base editor, is a cytidine deaminase. In some embodiments, the cytidine deaminase base editor is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety. Non-limiting exemplary cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, ^Cooley Ref: MABI-044 / 03WO 344183-2319 saBE4, and saBE4-Gam as described in WO2021163587, WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.

[0237] In some embodiments, a base editor is a cytosine to guanine base editor (CGBE). A CGBE may convert a cytosine to a guanine.

[0238] In some embodiments, a base editor is an adenine base editor (ABE). An ABE may convert an adenine to a guanine. In some embodiments, an ABE converts an A•T base pair to a G•C base pair. In some embodiments, the ABE converts a target A•T base pair to G•C in vivo or in vitro. In some embodiments, ABEs provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations. In some embodiments, ABEs provided herein enable correction of pathogenic SNPs (~47% of disease-associated point mutations). In some embodiments, the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine. In some embodiments, inosine exhibits the base- pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation. Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. Non-limiting exemplary ABEs suitable for use herein include: ABE7, ABE8.1m, ABE8.2m, ABE8.3m, ABE8.4m, ABE8.5m, ABE8.6m, ABE8.7m, ABE8.8m, ABE8.9m, ABE8.10m, ABE8.11m, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8.18m, ABE8.19m, ABE8.20m, ABE8.21m, ABE8.22m, ABE8.23m, ABE8.24m, ABE8.1d, ABE8.2d, ABE8.3d, ABE8.4d, ABE8.5d, ABE8.6d, ABE8.7d, ABE8.8d, ABE8.9d, ABE8.10d, ABE8.11d, ABE8.12d, ABE8.13d, ABE8.14d, ABE8.15d, ABE8.16d, ABE8.17d, ABE8.18d, ABE8.19d, ABE8.20d, ABE8.21d, ABE8.22d, ABE8.23d, and ABE8.24d. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The CRISPR Journal 4:2:169- 177, incorporated herein by reference. In some embodiments, the adenine deaminase is an adenine deaminase described by Koblan et al. (2018) Nature Biotechnology 36:848-846, incorporated herein by reference. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871.

[0239] In some embodiments, the ABE is ABE8e and comprises an amino acid sequence that is at least at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1062. In some embodiments, the ABE is ABE8e and comprises or consists of SEQ ID NO: 1062. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0240] In some embodiments, the present disclosure provides a fusion protein comprising an effector protein described herein and a base editing enzyme described herein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an effector protein and a base editing enzyme. In some embodiments, the fusion protein comprises, from N- terminus to C-terminus, a base editing enzyme and an effector protein. In some embodiments, the base editing enzyme is ABE8e.

[0241] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 515, 1045, or 1047 and a base editing enzyme comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1062. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of any one of SEQ ID NOs: 515, 1045, 1047 and a base editing enzyme comprising or consisting of SEQ ID NO: 1062. In some embodiments, the fusion protein comprises a linker sequence comprising GSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSGGGS SEQ ID NO: 1043. In some embodiments, the fusion protein comprises an amino acid sequence that is at least at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1048. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 1048.

[0242] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 516, 1041, 1042, 1044, or 1046 and a base editing enzyme comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1062. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of any one of SEQ ID NOs: 516, 1041, 1042, 1044, 1046 and a base editing enzyme comprising or consisting of SEQ ID NO: 1062. In some embodiments, the fusion protein comprises a linker sequence comprising SEQ ID NO: 1043. In some embodiments, the fusion protein comprises an amino acid sequence that is at least at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1049. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 1049.

[0243] Exemplary base editing enzyme and base editor fusion proteins are provided in TABLE 11. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0244] In some embodiments, an adenine base editing enzyme of an ABE is an adenosine deaminase. Non-limiting exemplary adenosine base editing enzymes suitable for use herein include ABE9. In some embodiments, the ABE comprises an engineered adenosine deaminase enzyme capable of acting on ssDNA. The engineered adenosine deaminase enzyme may be an adenosine deaminase variant that differs from a naturally occurring deaminase. Relative to the naturally occurring deaminase, the adenosine deaminase variant may comprise one or more amino acid alteration, including a V82S alteration, a T166R alteration, a Y147T alteration, a Y147R alteration, a Q154S alteration, a Y123H alteration, a Q154R alteration, or a combination thereof.

[0245] In some embodiments, a base editor comprises a deaminase dimer. In some embodiments, the base editor further comprising a base editing enzyme and an adenine deaminase (e.g., TadA). In some embodiments, the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA*9). In some embodiments, the adenosine deaminase is a TadA*8 variant (e.g., any one of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24 as described in WO2021163587 and WO2021050571, which are each hereby incorporated by reference in its entirety). In some embodiments, the base editor comprises a base editing enzyme linked to TadA by a linker (e.g., wherein the base editing enzyme is linked to TadA at N-terminus or C-terminus by a linker).

[0246] In some embodiments, a base editing enzyme is a deaminase dimer comprising an ABE. In some embodiments, the deaminase dimer comprises an adenosine deaminase. In some embodiments, the deaminase dimer comprises TadA linked to a suitable adenine base editing enzyme including an: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), BtAPOBEC2, and variants thereof. In some embodiments, the adenine base editing enzyme is linked to amino-terminus or the carboxy-terminus of TadA.

[0247] In some embodiments, RNA base editors comprise an adenosine deaminase. In some embodiments, ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine. In some embodiments, RNA base editors comprise an effector protein that is activated by or binds RNA.

[0248] In some embodiments, base editors are used to treat a subject having or a subject suspected of having a disease related to a gene of interest. In some embodiments, base editors are useful for treating a disease or a disorder caused by a point mutation in a gene of interest. In some embodiments, compositions, systems, and methods described herein comprise a base ^Cooley Ref: MABI-044 / 03WO 344183-2319 editor and a guide nucleic acid, wherein the guide nucleic acid directs the base editor to a sequence in a target gene. Precision Editing Systems

[0249] In some embodiments, the fusion partner comprises a polymerase. In some embodiments, the fusion partner is an RNA-directed DNA polymerase (RDDP). In some embodiments, the RDDP is a reverse transcriptase.

[0250] In some embodiments, the present disclosure provides system comprising: (a) an effector protein or a nucleic acid encoding the effector protein; (b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; (c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises (i) a first region comprising a protein binding sequence, and (ii) a second region comprising a spacer sequence that hybridizes to a target sequence of a first strand of a double stranded DNA (dsDNA) target nucleic acid, wherein the first region is located 5’ of the second region; and (d) a template RNA (rttRNA) or nucleic acid encoding the rttRNA, wherein the rttRNA comprises (i) a primer binding sequence (PBS), and (ii) a template sequence that hybridizes to the target sequence of a second strand of the dsDNA target nucleic acid. In some embodiments, the RDDP comprises a reverse transcriptase. In some embodiments, the guide RNA is linked to the rttRNA. In some embodiments, the template sequence is located 5’ of the PBS, optionally wherein the 3’ end of the PBS is linked to the 5’ end of the template sequence. In some embodiments, the rttRNA is circularized. In some embodiments, the rttRNA and / or guide RNA comprises a protein localization sequence that can localize a protein to the rttRNA. By way of non-limiting example, the protein localization sequence comprises an MS2 coat protein localization sequence and the RDDP is fused to an MS2 coat protein. In some embodiments, the RDDP is fused to the effector protein. In some embodiments, the rttRNA sequence comprises a difference of at least one nucleotide relative to an equal length portion of the target sequence.

[0251] In some embodiments, the RDDP that is capable of catalyzing the modification of the target nucleic acid forms a complex with an extended guide RNA. In some embodiments, the extended guide RNA comprises (not necessarily in this order): a first region (also referred to as a protein binding region or protein binding sequence) that interacts with an effector protein; a second region comprising a spacer sequence that is complementary to a target sequence of a first strand of a target dsDNA molecule; a third region comprising a template sequence that is complementary to at least a portion of the target sequence on the non-target strand of the target dsDNA molecule with the exception of at least one nucleotide; and a fourth region comprising ^Cooley Ref: MABI-044 / 03WO 344183-2319 a primer binding sequence that hybridizes to a primer sequence of the target dsDNA molecule that is formed when target nucleic acid is cleaved. The third region or template sequence may comprise a nucleotide having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when the template sequence and the target sequence are aligned for maximum identity. In some embodiments, there is a linker between any one of the first, second, third and fourth regions. In some embodiments, the linker comprises a nucleotide. In some embodiments, the linker comprises multiple nucleotides.

[0252] In some embodiments, the third and fourth regions are 5’ of the first and second regions. In some embodiments, the order of the regions of the extended guide RNA from 5’ to 3’ is: third region, fourth region, first region, and second region. In some embodiments, there is a linker between any one of the first, second, third and fourth regions. In some embodiments, there is a linker between the first and fourth regions. In some embodiments, the effector protein is linked to an RDDP. In some embodiments, the RDDP comprises a reverse transcriptase.

[0253] In some embodiments, the third and fourth regions are 3’ of the first and second regions. In some embodiments, the order of the regions of the extended guide RNA from 5’ to 3’ is: first region, second region, third region, and fourth region. In some embodiments, there is a linker between the second and third regions. Protein Modification Activity

[0254] In some embodiments, a fusion partner provides enzymatic activity that modifies a protein associated with a target nucleic acid. The protein may be a histone, an RNA binding protein, or a DNA binding protein. Examples of such protein modification activities include: methyltransferase activity, such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1); demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU- 1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3); acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK); deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, 70 ^Cooley Ref: MABI-044 / 03WO 344183-2319 HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11); kinase activity; phosphatase activity; ubiquitin ligase activity; deubiquitinating activity; adenylation activity; deadenylation activity; SUMOylating activity; deSUMOylating activity; ribosylation activity; deribosylation activity; myristoylation activity; and demyristoylation activity. CRISPRa Fusions and CRISPRi fusions

[0255] In some embodiments, fusion partners include, but are not limited to, a protein that directly and / or indirectly provides for increased or decreased transcription and / or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription and / or translation regulator, a translation-regulating protein, etc.). In some embodiments, fusion partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.

[0256] In some embodiments, fusion partners activate or increase expression of a target nucleic acid. Such fusion proteins comprising the described fusion partners and an effector protein may be referred to as CRISPRa fusions. In some embodiments, fusion partners increase expression of the target nucleic acid relative to its expression in the absence of the fusion effector protein. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR. In some embodiments, fusion partners comprise a transcriptional activator. In some embodiments, the transcriptional activators may promote transcription by: recruitment of other transcription factor proteins; modification of target DNA such as demethylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones; or a combination thereof. In some embodiments, the fusion partner is a reverse transcriptase.

[0257] Non-limiting examples of fusion partners that promote or increase transcription include: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1; and functional domains thereof. Other non-limiting examples of suitable fusion partners include: ^Cooley Ref: MABI-044 / 03WO 344183-2319 proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for stimulation of RNA splicing (e.g., Serine / Arginine-rich (SR) domains); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat).

[0258] In some embodiments, fusions partners inhibit or reduce expression of a target nucleic acid. Such fusion proteins comprising described fusion partners and an effector protein may be referred to as CRISPRi fusions. In some embodiments, fusion partners reduce expression of the target nucleic acid relative to its expression in the absence of the fusion effector protein. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR. In some embodiments, fusion partners may comprise a transcriptional repressor. In some embodiments, the transcriptional repressors may inhibit transcription by: recruitment of other transcription factor proteins; modification of target DNA such as methylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones; or a combination thereof.

[0259] In some embodiments, the guide nucleic acids disclosed herein can be used in combination with a fusion protein for epigenetic modification of the SOD1 gene. In some embodiments, the fusion protein comprises an effector protein and a methyltransferase. In some embodiments, the fusion protein further comprises a KRAB domain. In some embodiments, the methyltransferase is selected from M.HhaI, DNMT1, DNMT3A, DNMT3B, DNMT3L, and a combination thereof. In some embodiments, the methyltransferase is selected from DNMT3A, DNMT3L, and a combination thereof. In some embodiments, the methyltransferase is DNMT3L. In some embodiments, the fusion protein does not comprise DNMT3A. In some embodiments, the effector protein is CasPhi.12 or a variant thereof, and the guide nucleic acid comprises a sequence that is at least at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100% identical to any one of the sequences of SEQ ID NOs: 797-1038. In some embodiments, the effector protein is CasM.265466 or a variant thereof, and the guide nucleic acid comprises a sequence that is at least at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100% identical to any one of the sequences of SEQ ID NOs: 534-796. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0260] Non-limiting examples of fusion partners that decrease or inhibit transcription include: transcriptional repressors such as the Krüppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11; DNA methylases such as HhaI DNA m5c- methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and periphery recruitment elements such as Lamin A, and Lamin B; and functional domains thereof. Other non-limiting examples of suitable fusion partners include: proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for repression of RNA splicing (e.g., PTB, Sam68, and hnRNP A1); proteins and protein domains responsible for reducing the efficiency of transcription (e.g., FUS (TLS)).

[0261] In some embodiments, fusion proteins are targeted by a guide nucleic acid (e.g., guide RNA) to a specific location in a target nucleic acid and exert locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and / or changes a local chromatin status (e.g., when a fusion sequence is used that edits the target nucleic acid or modifies a protein associated with the target nucleic acid). In some embodiments, the modifications are transient (e.g., transcription repression or activation). In some embodiments, the modifications are inheritable. For example, epigenetic modifications made to a target nucleic acid, or to proteins associated with the target nucleic acid, e.g., nucleosomal histones, in a cell, can be observed in a successive generation.

[0262] In some embodiments, fusion partner comprises an RNA splicing factor. The RNA splicing factor may be used (in whole or as fragments thereof) for modular organization, with separate sequence-specific RNA binding modules and splicing effector domains. In some embodiments, the RNA splicing factors comprise members of the Serine / Arginine-rich (SR) protein family containing N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. In some embodiments, a hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal Glycine- ^Cooley Ref: MABI-044 / 03WO 344183-2319 rich domain. In some embodiments, the RNA splicing factors may regulate alternative use of splice site (ss) by binding to regulatory sequences between two alternative sites. For example, in some embodiments, ASF / SF2 may recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al may bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. Long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. Short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). A ratio of the two Bcl-x splicing isoforms is regulated by multiple c^-elements that are located in either core exon region or exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety. Recombinases

[0263] In some embodiments, fusion partners comprise a recombinase. In some embodiments, effector proteins described herein are linked with the recombinase. In some embodiments, the effector proteins have reduced nuclease activity or no nuclease activity. In some embodiments, the recombinase is a site-specific recombinase.

[0264] In some embodiments, a catalytically inactive effector protein is linked with a recombinase, wherein the recombinase can be a site-specific recombinase. Such polypeptides can be used for site-directed transgene insertion. Non-limiting examples of site-specific recombinases include a tyrosine recombinase (e.g., Cre, Flp or lambda integrase), a serine recombinase (e.g., gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase and integrase), or mutants or variants thereof. In some embodiments, the recombinase is a serine recombinase. Non-limiting examples of serine recombinases include gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase, and IS607 integrase. In some embodiments, the site-specific recombinase is an integrase. Non-limiting examples of integrases include:Bxb1, wBeta, BL3, phiR4, A118, TG1, MR11, phi370, SPBc, TP901-1, phiRV, FC1, K38, phiBT1, and phiC31. Further discussion and examples of suitable recombinase fusion partners are described in US 10,975,392, which is incorporated herein by reference in its entirety. In some embodiments, the fusion protein comprises a linker that links ^Cooley Ref: MABI-044 / 03WO 344183-2319 the recombinase to the Cas-CRISPR domain of the effector protein. In some embodiments, the linker is The-Ser. 5. Exemplary systems

[0265] In some embodiments, the present disclosure provides a system comprising (1) a guide RNA or a polynucleotide encoding the same, wherein the guide RNA comprises a spacer sequence that is capable of hybridizing to a target nucleic acid sequence in the SOD1 gene; and (2) an effector protein or fusion protein thereof or a polynucleotide encoding the same.

[0266] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences recited in TABLEs 4 and 6-7, and the guide RNA comprises (a) a repeat sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 797-1038.

[0267] In some embodiments, the effector protein comprises any one of the sequences recited in TABLEs 4 and 6-7, and the guide RNA comprises (a) a repeat sequence that comprises a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 797-1038.

[0268] In some embodiments, the effector protein consists of any one of the sequences recited in TABLEs 4 and 6-7, and the guide RNA comprises (a) a repeat sequence that consists of a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 797-1038.

[0269] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046, and the guide RNA comprises (a) a repeat sequence that is at least 90%, ^Cooley Ref: MABI-044 / 03WO 344183-2319 at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 797-1038.

[0270] In some embodiments, the effector protein comprises any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046, and the guide RNA comprises (a) a repeat sequence that comprises a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 797-1038.

[0271] In some embodiments, the effector protein consists of any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046, and the guide RNA comprises (a) a repeat sequence that consists of a sequence selected from any one of SEQ ID NOs: 507-512 and 1064 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 264-505. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 797-1038.

[0272] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences recited in TABLEs 4-5 and 7, and the guide RNA comprises (a) a repeat sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:506 and (b) a spacer sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a sequence selected from to any one of SEQ ID NOs: 1-263. In some embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 534-796. In some embodiments, the system further comprises an (c) intermediary sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 513 or (d) a handle sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 514. In some ^Cooley Ref: MABI-044 / 03WO 344183-2319 embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 534-796.

[0273] In some embodiments, the effector protein comprises any one of the sequences recited in TABLEs 4-5 and 7, and the guide RNA comprises (a) a repeat sequence that comprises SEQ ID NO: 506 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 1-263. In some embodiments, the system further comprises an (c) intermediary sequence that comprises SEQ ID NO: 513 or (d) a handle sequence that comprises SEQ ID NO: 514. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 534-796.

[0274] In some embodiments, the effector protein consists of any one of the sequences recited in TABLEs 4-5 and 7, and the guide RNA comprises (a) a repeat sequence that consists of SEQ ID NO: 506 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 1-263. In some embodiments, the system further comprises an (c) intermediary sequence that consists of SEQ ID NO: 513 or (d) a handle sequence that consists of SEQ ID NO: 514. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 534-796.

[0275] In some embodiments, the effector protein comprises an amino acid sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:^515, 1045, or 1047, and the guide RNA comprises (a) a repeat sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:506 and (b) a spacer sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a sequence selected from to any one of SEQ ID NOs: 1- 263. In some embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 534-796. In some embodiments, the system further comprises an (c) intermediary sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 513 or (d) a handle sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 514. In some embodiments, the guide RNA sequence is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, ^Cooley Ref: MABI-044 / 03WO 344183-2319 at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 534-796.

[0276] In some embodiments, the effector protein comprises any one of SEQ ID NOs:^515, 1045, or 1047, and the guide RNA comprises (a) a repeat sequence that comprises SEQ ID NO: 506 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 1-263. In some embodiments, the system further comprises an (c) intermediary sequence that comprises SEQ ID NO: 513 or (d) a handle sequence that comprises SEQ ID NO: 514. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 534-796.

[0277] In some embodiments, the effector protein consists of any one of SEQ ID NOs:^515, 1045, or 1047, and the guide RNA comprises (a) a repeat sequence that consists of SEQ ID NO: 506 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 1-263. In some embodiments, the system further comprises an (c) intermediary sequence that consists of SEQ ID NO: 513 or (d) a handle sequence that consists of SEQ ID NO: 514. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 534-796. 6. Target Nucleic Acids

[0278] Disclosed herein are compositions, systems, and methods for modifying the SOD1 gene. Exemplary reference SOD1 gene sequences are provided in TABLE 12 In general, the target nucleic acid is the SOD1 gene or a portion thereof. In general, guide nucleic acids described herein comprise a sequence that is complementary to and / or hybridizes to a target sequence of the SOD1 gene. In some embodiments, the target sequence is within an exon region of the SOD1 gene. In some embodiments, the target sequence is within an intron region of the SOD1 gene. In some embodiments, the target sequence spans an exon and intron of the SOD1 gene. In some embodiments, the target sequence is located in a regulatory region of the SOD1 gene. In some embodiments, the regulatory region is a promoter. Exemplary reference functional SOD1 proteins are listed in TABLE 13. 7. Subject and Environmental Samples

[0279] Systems, compositions, and methods described herein may be useful for detecting a mutated SOD1 gene in a sample. In some embodiments, the sample is a biological sample, an environmental sample, or a combination thereof. Non-limiting examples of biological samples are blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, and a tissue sample (e.g., a biopsy sample). A tissue sample ^Cooley Ref: MABI-044 / 03WO 344183-2319 from a subject may be dissociated or liquified prior to application to detection system of the present disclosure. Non-limiting examples of environmental samples are soil, air, or water. In some embodiments, an environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. 8. Expression Vectors

[0280] Compositions, systems, and methods described herein comprise an expression vector or a use thereof. In some embodiments, the nucleic acid of interest (e.g., an SOD1-targeting guide nucleic acid) comprises one or more components of a composition or system described herein. In some embodiments, the nucleic acid of interest comprises a nucleotide sequence that encodes one or more components of the composition or system described herein. In some embodiments, one or more components comprises polypeptide(s), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s). In some embodiments, the component comprises a nucleic acid encoding an effector protein and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid. The vector may be part of a vector system, wherein a vector system comprises a library of vectors each encoding one or more component of a composition or system described herein. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and / or a target nucleic acid) are encoded by the same vector. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and / or a target nucleic acid) are each encoded by different vectors of the system.

[0281] In some embodiments, a vector comprises a nucleotide sequence encoding one or more effector proteins as described herein. In some embodiments, the one or more effector proteins comprise at least two effector proteins. In some embodiments, the at least two effector protein are the same. In some embodiments, the at least two effector proteins are different from each other. In some embodiments, the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the vector comprises the nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more effector proteins.

[0282] In some embodiments, a vector may encode one or more of any system components, including but not limited to effector proteins, guide nucleic acids, donor nucleic acids, and target nucleic acids as described herein. In some embodiments, a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector may encode 1, 2, 3, 4 or more of any system components. ^Cooley Ref: MABI-044 / 03WO 344183-2319 For example, a vector may encode two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence. A vector may comprise the nucleic acid encoding an effector protein and a guide nucleic acid. A vector may encode an effector protein, a guide nucleic acid, and a donor nucleic acid.

[0283] In some embodiments, a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein. In some embodiments, the one or more guide nucleic acids comprise at least two guide nucleic acids. In some embodiments, the at least two guide nucleic acids are the same. In some embodiments, the at least two guide nucleic acids are different from each other. In some embodiments, the guide nucleic acid or the nucleotide sequence encoding the guide nucleic acid is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids. In some embodiments, the vector comprises a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids.^

[0284] In some embodiments, a vector may comprise or encode one or more regulatory elements. Regulatory elements may refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence or a coding sequence and / or regulate translation of an encoded polypeptide. In some embodiments, a vector may comprise or encode for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like. In some embodiments, a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites.

[0285] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3’ direction) coding or non-coding sequence. A promoter can be linked at its 3’ terminus to a nucleic acid, the expression or transcription of which is desired, and extends upstream (5’ direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. The promoter sequence can include a transcription initiation ^Cooley Ref: MABI-044 / 03WO 344183-2319 site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest. Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.

[0286] Promotors may be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and / or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 2 fold, 5 fold, 10 fold, 50 fold, by 100 fold, 500 fold, or by 1000 fold, or more. In addition, vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and / or a nucleic acid that encodes an effector protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and / or the effector protein.

[0287] In general, vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the vector comprises a nucleotide sequence of a promoter. In some embodiments, the vector comprises two promoters. In some embodiments, the vector comprises three promoters. In some embodiments, a length of the promoter is less than about 500, less than about 400, less than about 300, or less than about 200 linked nucleotides. In some embodiments, a length of the promoter is at least 100, at least 200, at least 300, at least 400, or at least 500 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK, EF1a, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1-10, H1, TEF1, GDS, ADH1, HSV TK, Ubi, U6, MNDU3, ^Cooley Ref: MABI-044 / 03WO 344183-2319 MSCV, MND and CAG. In some embodiments, the promoter allows for expression in a motor neuron. In some embodiments, the motor neuron specific promoter is Hb9.

[0288] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter only drives expression of its corresponding coding sequence (e.g., polypeptide or guide nucleic acid) when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline- inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter. In some embodiments, the promoter for expressing effector protein is a motor neuron-specific promoter, such as a Hb9 promoter. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence.

[0289] In some embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). In some embodiments, the promoter is EF1a. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.

[0290] In some embodiments, a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA.

[0291] In some embodiments, the expression vector comprises the DNA molecule encoding a guide nucleic acid. In some embodiments, the expression vector further comprises the nucleic acid encoding an effector protein. In some embodiments, the expression vector further comprises or encodes a donor nucleic acid. In some embodiments, the expression vector encoding a guide nucleic acid, wherein the guide nucleic acid comprises a first region comprising a repeat sequence; and a second region comprising a spacer sequence that is complementary to a target sequence of an SOD1 gene. In some embodiments, wherein the first region is located 5’ of the second region. In some embodiments, the expression vector further comprises an effector protein that binds the repeat sequence or a nucleic acid encoding the ^Cooley Ref: MABI-044 / 03WO 344183-2319 effector protein. In some embodiments, the spacer comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 564-505 the repeat sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 507-512 and 1064; the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence listed in TABLEs 4 and 6-7; or a combination thereof. In some embodiments, the spacer comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 1-263; the repeat sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 506; the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence listed in TABLES 4- 5 and 7; or a combination thereof.

[0292] In some embodiments, a vector described herein is a delivery vector. In some embodiments, the delivery vector is a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the delivery vector is a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some embodiments, the plasmid comprises circular double-stranded DNA. In some embodiments, the plasmid is linear. In some embodiments, the plasmid comprises one or more coding sequences of interest and one or more regulatory elements. In some embodiments, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some embodiments, the plasmid is a minicircle plasmid. In some embodiments, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmids are engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements are assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which is then be readily ligated to another genetic sequence. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0293] In some embodiments, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and / or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV- I.

[0294] In some embodiments, disclosed herein comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. In some embodiments, of the above, the nucleic acid expression vector comprises a polynucleotide encoding an effector protein that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences recited in TABLEs 4-7.

[0295] The one or more nucleic acids may comprise a plasmid. The one or more nucleic acids may comprise a nucleic acid expression vector. The one or more nucleic acids may comprise a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, compositions, including pharmaceutical compositions, comprise a viral vector encoding a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion effector protein. In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. Administration of a non-viral vector

[0296] In some embodiments, an administration of a non-viral vector comprises contacting a cell, such as a host cell, with the non-viral vector. In some embodiments, a physical method or a chemical method is employed for delivering the vector into the cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide by liposomes such as, cationic lipids or neutral lipids; lipofection; dendrimers; lipid nanoparticle (LNP); or cell-penetrating peptides. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0297] In some embodiments, a vector is administered as part of a method of nucleic acid detection, editing, and / or treatment as described herein. In some embodiments, a vector is administered in a single vehicle, such as a single expression vector. In some embodiments, at least two of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid, are provided in the single expression vector. In some embodiments, components, such as a guide nucleic acid and an effector protein, are encoded by the same vector. In some embodiments, an effector protein (or a nucleic acid encoding same) and / or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle. In some embodiments, an effector protein (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and / or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.

[0298] In some embodiments, a vector may be part of a vector system. In some embodiments, the vector system comprises a library of vectors each encoding one or more components of a composition or system described herein. In some embodiments, a vector system is administered as part of a method of nucleic acid detection, editing, and / or treatment as described herein, wherein at least two vectors are co-administered. In some embodiments, the at least two vectors comprise different components. In some embodiments, the at least two vectors comprise the same component having different sequences. In some embodiments, at least one of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof is provided in a different vector. In some embodiments, the nucleic acid encoding the effector protein, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors. In some embodiments, the donor nucleic acid is encoded by a different vector than the vector encoding the effector protein and the guide nucleic acid. Lipid Particles and Non-viral Vectors

[0299] In some embodiments, compositions and systems provided herein comprise a lipid particle. In some embodiments, a lipid particle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector as described herein. LNPs are a non-viral delivery system for delivery of the composition and / or system ^Cooley Ref: MABI-044 / 03WO 344183-2319 components described herein. LNPs are particularly effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi- dosing capabilities and flexibility of design (Kulkarni et al., (2018) Nucleic Acid Therapeutics, 28(3):146-157). In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce one or more effector proteins, one or more guide nucleic acids, one or more donor nucleic acids, or any combinations thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio-responsive polymers. In some embodiments, the ionizable lipids exploits chemical-physical properties of the endosomal environment (e.g., pH) offering improved delivery of nucleic acids. In some embodiments, the ionizable lipids are neutral at physiological pH. In some embodiments, the ionizable lipids are protonated under acidic pH. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.

[0300] In some embodiments, a LNP comprises an outer shell and an inner core. In some embodiments, the outer shell comprises lipids. In some embodiments, the lipids comprise modified lipids. In some embodiments, the modified lipids comprise pegylated lipids. In some embodiments, the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids. In some embodiments, the LNP comprises one or more of N1,N3,N5- tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), 2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2- dimyristoyl-sn-glycerol, and methoxypolyethylene glycol (DMG-PEChooo), derivatives, analogs, or variants thereof. In some embodiments, the LNP has a negative net overall charge prior to complexation with one or more of a guide nucleic acid, a nucleic acid encoding the one or more guide nucleic acid, a nucleic acid encoding the effector protein, and / or a donor nucleic acid. In some embodiments, the inner core is a hydrophobic core. In some embodiments, the one or more of a guide nucleic acid, the nucleic acid encoding the one or more guide nucleic acid, the nucleic acid encoding the effector protein, and / or the donor nucleic acid forms a complex with one or more of the cationic lipids and the ionizable lipids. In some embodiments, the nucleic acid encoding the effector protein or the nucleic acid encoding the guide nucleic acid is self-replicating. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0301] In some embodiments, a LNP comprises one or more of cationic lipids, ionizable lipids, and modified versions thereof. In some embodiments, the ionizable lipid comprises TT3 or a derivative thereof. Accordingly, in some embodiments, the LNP comprises one or more of TT3 and pegylated TT3. The publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and representative methods of delivering LNP formulations in Example 7.

[0302] In some embodiments, a LNP comprises a lipid composition targeting to a specific organ. In some embodiments, the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes). Delivery of Viral Vectors

[0303] In some embodiments, a vector described herein comprises a viral vector. In some embodiments, the viral vector comprises a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. In some embodiments, the vector is an adeno- associated viral vector. There are a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and Ȗ-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a recombinant viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector comprises gamma-retroviral vector. A viral vector provided herein may be derived from or based on any such virus. For example, in some embodiments, the gamma-retroviral vector is derived from a Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or a Murine Stem cell Virus (MSCV) genome. In some embodiments, the lentiviral vector is derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the chimeric viral vector comprises ^Cooley Ref: MABI-044 / 03WO 344183-2319 viral portions from two or more viruses. In some embodiments, the viral vector corresponds to a virus of a specific serotype.

[0304] In some embodiments, a viral vector is an adeno-associated viral vector (AAV vector). In some embodiments, a viral particle that delivers a viral vector described herein is an AAV. In some embodiments, the AAV comprises any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific AAV serotype. In some embodiments, the AAV serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, an AAV-rh10 serotype, and any combination, derivative, or variant thereof. In some embodiments, the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self- complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.

[0305] In some embodiments, an AAV vector described herein is a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[0306] In some embodiments, AAV vector described herein comprises two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. A nucleotide sequence between the ITRs of an AAV vector provided herein comprises a sequence encoding genome editing tools. In some embodiments, the genome editing tools comprise a nucleic acid encoding one or more effector proteins, a nucleic acid encoding one or more fusion proteins (e.g., a nuclear localization signal (NLS), polyA tail), one or more guide nucleic acids, a nucleic acid encoding the one or more guide nucleic acids, respective promoter(s), one or more donor nucleic acid, or any combinations thereof. In some embodiments, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, a coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating the AAV vector that is a self-complementary AAV (scAAV) vector. In some embodiments, the scAAV vector comprises the sequence encoding genome editing tools that has a length of about 2 kb to about 3 kb. In some embodiments, the AAV vector provided herein is a self-inactivating ^Cooley Ref: MABI-044 / 03WO 344183-2319 AAV vector. In some embodiments, the AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild- type AAV vector. Producing AAV Delivery Vectors

[0307] In some embodiments, methods of producing AAV delivery vectors herein comprise packaging a nucleic acid encoding an effector protein and a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging an effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector. In some examples, the AAV vector may package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[0308] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes may be not the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2 / 9. In some examples, the hybrid AAV delivery vector comprises an AAV2 / 1, AAV2 / 2, AAV 2 / 4, AAV2 / 5, AAV2 / 8, or AAV2 / 9 vector.

[0309] In some embodiments, the AAV vector comprises a recombinant AAV expression cassette comprising sequences encoding: a) a first inverted terminal repeat (ITR) and a first promoter; b) an effector protein disclosed herein; c) optionally a second promoter; d) a second polynucleotide encoding a guide nucleic acid disclosed here; and e) a second ITR. In some embodiments,the AAV expression cassette is a self-complementary AAV vector. Producing AAV Particles

[0310] In some embodiments, AAV particles described herein are recombinant AAV (rAAV). In some embodiments, rAAV particles are generated by transfecting AAV producing ^Cooley Ref: MABI-044 / 03WO 344183-2319 cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing cells are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian cells. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a variant thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell comprises transfecting vectors that express the rep protein, the capsid protein, and the gene-of- interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31(4):317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties.

[0311] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, the insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells may comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells may comprise infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Ther., 1;13(16):1935-43; and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety. ^Cooley Ref: MABI-044 / 03WO 344183-2319 9. Cells and a Population of Cells

[0312] Disclosed herein are one or more cells comprising or modified by the compositions, systems, or nucleic acid expression vectors disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is a neuron. In some embodiments, the human cell is a motor neuron.

[0313] Also disclosed herein are a population of cells comprising at least one cell disclosed herein.

[0314] The cells or a population of cells disclosed herein can be used as a component of a pharmaceutical composition disclosed herein. The cells or a population of cells disclosed herein can be used in a method disclosed herein. In some embodiments, the cells or a population of cells disclosed herein can be used to treat a disease such as amyotrophic lateral sclerosis (ALS). 10. Pharmaceutical Compositions and Modes of Administration

[0315] Disclosed herein are compositions comprising one or more effector proteins described herein or nucleic acids encoding the one or more effector proteins, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or combinations thereof. Also disclosed herein are compositions comprising the nucleic acid expression vector, the cell, or the population of cells disclosed herein. In some embodiments, a repeat sequence of the one or more guide nucleic acids are capable of interacting with the one or more of the effector proteins. In some embodiments, spacer sequences of the one or more guide nucleic acids hybridizes with a target sequence of a target nucleic acid. In some embodiments, the compositions are capable of editing a target nucleic acid in a cell or a subject. In some embodiments, the compositions are capable of editing a target nucleic acid or the expression thereof in a cell, in a tissue, in an organ, in vitro, in vivo, or ex vivo. In some embodiments, the compositions are capable of editing a target nucleic acid in a sample comprising the target nucleic.

[0316] In some embodiments, compositions described herein comprise plasmids described herein, viral vectors described herein, non-viral vectors described herein, or combinations thereof. In some embodiments, compositions described herein comprise the viral vectors. In some embodiments, compositions described herein comprise an AAV. In some embodiments, compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell-penetrating peptides. In some embodiments, compositions described herein comprise an LNP. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0317] In some embodiments, compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein and a pharmaceutically acceptable carrier or diluent. Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives. In some embodiments, the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method. In some embodiments, the pharmaceutical compositions comprise a virus vector or a non-viral vector.

[0318] Pharmaceutical compositions described herein comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In someembodiments, the salt is KNO3. In some embodiments, the salt is Mg2+SO42í.

[0319] Pharmaceutical compositions described herein are in the form of a solution (e.g., a liquid). In some embodiments, the solution is formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7.

[0320] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising the nucleic acid expression vector, the cell, or the population of cells disclosed herein. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. In some embodiments, the pharmaceutical composition disclosed herein also ^Cooley Ref: MABI-044 / 03WO 344183-2319 comprise a pharmaceutical acceptable carrier. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo. In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. 11. Methods of Nucleic Acid Modification

[0321] Provided herein are methods of editing and modifying the expression of the human SOD1 gene. In general, editing refers to modifying the nucleobase sequence of a target nucleic acid. However, compositions and systems disclosed herein may also be capable of making epigenetic modifications of target nucleic acids. Effector proteins, multimeric complexes thereof and systems described herein may be used for editing or modifying a target nucleic acid. Editing a target nucleic acid may comprise one or more of: cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or modifying (e.g., methylating, demethylating, deaminating, or oxidizing) of one or more nucleotides of the target nucleic acid.

[0322] Methods of modifying may comprise contacting the SOD1 gene with a composition, system, or nucleic acid expression vector disclosed herein, wherein the system or nucleic acid comprises an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in TABLEs 4-7. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in TABLEs 4-7. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 515, 1045, 1047. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, 1046. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0323] Editing may introduce a mutation (e.g., point mutations, deletions) in a target nucleic acid relative to a corresponding wildtype nucleobase sequence. Editing may remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleobase sequence. Editing may remove / correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. Editing may be used to generate gene knock-out, gene knock-in, gene editing, gene tagging, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a cell.

[0324] Editing may comprise single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof. In some embodiments, cleavage (single-stranded or double-stranded) is site-specific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer region. In some embodiments, the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g., homology directed repair (HDR)) or non-homologous end joining (NHEJ). In some cases, a double-stranded break in the target nucleic acid may be repaired (e.g., by NHEJ or HDR) without insertion of a donor template, such that the repair results in an indel in the target nucleic acid at or near the site of the double-stranded break.

[0325] In some embodiments, an indel, sometimes referred to as an insertion-deletion or indel mutation, is a type of genetic mutation that results from the insertion and / or deletion of nucleotides in a target nucleic acid. An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including sequencing. If the number of nucleotides in the insertion / deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation.

[0326] In some embodiments, wherein the compositions, systems, and methods of the present disclosure comprise an additional guide nucleic acid or a use thereof, the dual-guided compositions, systems, and methods described herein can modify the target nucleic acid in two locations. In some cases, dual-guided editing can comprise cleavage of the target nucleic acid in the two locations targeted by the guide RNAs. In some embodiments, upon removal of the sequence between the guide nucleic acids, the wild-type reading frame is restored. A wild-type reading frame can be a reading frame that produces at least a partially, or fully, functional protein. A non-wild-type reading frame can be a reading frame that produces a non-functional or partially non-functional protein. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0327] Accordingly, in some embodiments, compositions, systems, and methods described herein can edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid. In some embodiments, 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides can be edited by the compositions, systems, and methods described herein. In some embodiments, 10, 20, 30, 40, 50, 60, 70, 80 90, 100 or more nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein. In some embodiments, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein.

[0328] In some cases, methods comprise editing a target nucleic acid with two or more effector proteins. Editing a target nucleic acid may comprise introducing a two or more single- stranded breaks in a target nucleic acid. In some embodiments, a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid. The guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting the effector protein to the region of the target nucleic acid. Binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid. In some embodiments, modifying a target nucleic acid may comprise introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid. For example, modifying a target nucleic acid may comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second programmable nickase and hybridizes to a second region of the target nucleic acid. The first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid, and the second effector protein may introduce a second break in a second strand at the second region of the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be removed, thereby modifying the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be replaced (e.g., with donor nucleic acid), thereby modifying the target nucleic acid.

[0329] Methods, systems, and compositions described herein can edit or modify a target nucleic acid wherein such editing or modification can be measured by indel activity. Indel ^Cooley Ref: MABI-044 / 03WO 344183-2319 activity measures the amount of change in a target nucleic acid (e.g., nucleotide deletion(s) and / or insertion(s)) compared to a target nucleic acid that has not been contacted by a polypeptide described in compositions, systems, and methods described herein. For example, indel activity can be detected by next generation sequencing of one or more target loci of a target nucleic acid where indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence. In some embodiments, methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein can exhibit about 0.0001% to about 65% or more indel activity upon contact to a target nucleic acid compared to a target nucleic acid non-contacted with compositions, systems, or by methods described herein. For example, methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein can exhibit about 0.0001%, about 0.001%, about 0.01%, about 0.1%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or more indel activity.

[0330] In some embodiments, sequence deletion is a modification where one or more sequences in a target nucleic acid are deleted relative to a target nucleic acid without the sequence deletion. In some embodiments, a sequence deletion can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, a sequence deletion result in or effect a splicing disruption.

[0331] In some embodiments, a modification is a deletion of an entire exon. In some embodiments, the exon is associated with a disease. In some embodiments, compositions, systems, and methods described herein comprise a combination of a first gRNA, a second gRNA, a first effector protein, and a second effector protein, wherein the combination can be used for deleting the entire exon or a portion thereof. In some embodiments, the first effector protein and the second effector protein are the same. In some embodiments, the first effector protein and the second effector protein are not the same.

[0332] In some embodiments, sequence skipping is a modification where one or more sequences in a target nucleic acid are skipped upon transcription or translation of the target nucleic acid relative to a target nucleic acid without the sequence skipping. In some embodiments, sequence skipping can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, sequence skipping can result in or effect a splicing disruption.

[0333] In some embodiments, sequence reframing is a modification where one or more bases in a target are modified so that the reading frame of the sequence is reframed relative to a target nucleic acid without the sequence reframing. In some embodiments, sequence reframing can ^Cooley Ref: MABI-044 / 03WO 344183-2319 result in or effect a splicing disruption or a frameshift mutation. In some embodiments, sequence reframing can result in or effect a frameshift mutation.

[0334] In some embodiments, sequence knock-in is a modification where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in. In some embodiments, sequence knock-in can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, sequence knock-in can result in or effect a splicing disruption.

[0335] In some embodiments, editing or modification of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit or modify a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising splicing disruption mutations, frameshift mutations, sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. For example, editing or modification of a specific locus can affect any one of a splicing disruption, frameshift (e.g., 1+ or 2+ frameshift), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. In some embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both. In some embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise an effector protein described herein and a guide nucleic acid described herein.

[0336] Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vitro. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed ex vivo. Editing methods include, but are not limited to, introduction of double stranded breaks (DSB), which can result in deleting some nucleotides and disrupting the translation of a functional protein, base editing, and splice acceptor disruption (SA).

[0337] In some embodiments the method of editing by the effector proteins can be promotor silencing, frameshift mutation, base editing, or splice disruption.

[0338] In some embodiments, the editing by the effector protein targets an exon of the SOD1 gene. In some embodiments, the editing by the effector protein targets an intron of the SOD1 gene. In some embodiments, the editing by the effector protein decreases transcription of the DNA sequence of the SOD1 gene. In some embodiments, the editing by the effector protein decreases translation of the RNA sequence of the SOD1 gene. In some embodiments, the effector protein targets exon 1 of the SOD1 gene. In some embodiments, the effector protein ^Cooley Ref: MABI-044 / 03WO 344183-2319 targets exon 2 of the SOD1 gene. In some embodiments, the effector protein targets exon 3 of the SOD1 gene. In some embodiments, the effector protein targets exon 4 of the SOD1 gene. In some embodiments, the effector protein targets exon 5 of the SOD1 gene.

[0339] In some embodiments the gene regulation is regulated by effector protein repressing a promoter. In some embodiments the repression is temporary or transient. In some embodiments the repression is permanent. In some embodiments the effector protein is linked to a KRAB sequence. In some embodiments the effector protein is linked to an acetylase sequence. In some embodiments the effector protein is linked to a methyltransferase. In some embodiments the effector protein is linked to a Ezh2 sequence.

[0340] In some embodiments the effector protein causes a frameshift mutation. In some embodiments the effector protein causes the addition of one or more nucleotides causing a shift in the reading frame. In some embodiments the effector protein causes a deletion of one or more nucleotides causing a shift in the reading frame. In some embodiments the effector protein causes the deletion or addition of 1, 2, or 4 nucleotides. In some embodiments the effector protein causes an alternation in the amino acid sequence at protein translation. In some embodiments the alteration is a missense mutation. In some embodiments the alteration is a premature stop codon. In some embodiments the effector protein causes a change in the ribosome reading frame and cause premature termination of translation at a new nonsense or chain termination codon (TAA, TAG, and TGA).

[0341] In some embodiments the effector protein causes a base to be edited. In some embodiments the effector protein is linked to an adenine base editing enzyme (ABE). In some embodiments the effector protein is linked to a cytosine base editing enzyme (CBE). In some embodiments the fusion protein causes a cytodine to thymidine transition. In some embodiments the fusion protein causes a cytodine to uracil transition. In some embodiments the fusion protein causes a thymidine to cytodine transition. In some embodiments the fusion protein causes an adenosine to guanosine transition. In some embodiments the fusion protein causes a guanosine to adenosine conversion. In some embodiments the alteration results in a missense mutation. In some embodiments the alteration is a premature stop codon. In some embodiments the fusion protein causes a premature termination of translation at a new nonsense or chain termination codon (TAA, TAG, and TGA). 12. Methods of Detecting a Target Nucleic Acid

[0342] Provided herein are methods of detecting target nucleic acids. Methods may comprise detecting target nucleic acids with compositions or systems described herein. Methods may ^Cooley Ref: MABI-044 / 03WO 344183-2319 comprise detecting a target nucleic acid in a sample, e.g., a cell lysate, a biological fluid, or environmental sample. Methods may comprise detecting a target nucleic acid in a cell. In some embodiments, methods of detecting a target nucleic acid in a sample or cell comprises contacting the sample or cell with an effector protein or a multimeric complex thereof, a guide nucleic acid, wherein at least a portion of the guide nucleic acid is complementary to at least a portion of the target nucleic acid, and a reporter nucleic acid that is cleaved in the presence of the effector protein, the guide nucleic acid, and the target nucleic acid, and detecting a signal produced by cleavage of the reporter nucleic acid, thereby detecting the target nucleic acid in the sample. In some embodiments, methods result in trans cleavage of the reporter nucleic acid. In some embodiments, methods result in cis cleavage of the reporter nucleic acid. 13. Methods of Treating a Disorder

[0343] Described herein are methods for treating or preventing a disease in a subject by modifying a target nucleic acid associated with a gene or expression of a gene related to the disease. In some embodiments, the disease or disorder is caused by a misfolded SOD1 protein in a subject. In some embodiments, the disease or disorder is caused by a mutation in the SOD1 gene in a subject. In some embodiments, the gene is set forth in TABLE 12. In some embodiments the disease or disorder is amyotrophic lateral sclerosis (ALS). In some instances, the ALS is sporadic ALS (sALS). In some instances, the ALS is familial ALS (fALS).

[0344] In some embodiments, methods comprise administering a guide RNA comprising one or more sequences that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from the sequences in TABLES 1-3 or a DNA molecule encoding the same. In some embodiments, methods comprise administering an effector protein described herein or a nucleic acid encoding the same. In some embodiments, the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLES 4-7. Error! Reference source not found..

[0345] The effector protein or nucleic acid encoding the same, and the guide RNA or nucleic acid encoding the same may be administered in a single composition. The effector protein or nucleic acid encoding the same, and the guide RNA or nucleic acid encoding the same may be administered separately (formulaically or chronologically). In some embodiments, methods comprise administering: an effector protein or a messenger RNA encoding an effector protein and a lipid nanoparticle; and a viral vector encoding a guide RNA. In some embodiments, ^Cooley Ref: MABI-044 / 03WO 344183-2319 methods comprise administering: an effector protein or a messenger RNA encoding an effector protein and a lipid nanoparticle; and a guide RNA, optionally wherein the synthetic guide RNA comprises at least one chemical modification. In some embodiments, methods comprise administering a viral vector encoding the effector protein and the guide RNA. In some embodiments, methods comprise administering an effector protein and a lipid nanoparticle. In some embodiments, methods comprise administering a messenger RNA encoding an effector protein. In some embodiments, methods decrease an amount of SOD1 protein in a cell. The SOD1 protein may be a misfolded SOD1 protein and / or an SOD1 protein variant associated with ALS. SOD1 protein variants associated with ALS are known in the art. In some embodiments, the cell is a motor neuron. A non-limiting example of an SOD1 protein variant is an SOD1 protein with a G93A substitution that is encoded in exon 4 of SOD1.

[0346] In some embodiments, methods comprise administering premedication prior to administering the guide RNA. In some embodiments, the premedication includes a corticosteroid. In some embodiments, the premedication includes a histamine antagonist or inverse agonist. In some embodiments, the premedication includes dexamethasone. In some embodiments, the premedication includes famotidine. In some embodiments, the premedication includes diphenhydramine. NUMBERED EMBODIMENTS

[0347] The present invention is also described, for example and without limitation, in the following numbered embodiments which are not to be construed as limiting the scope thereof in any manner.

[0348] Embodiment 1: A system comprising: a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: a. a first region comprising a protein binding sequence; and b. a second region comprising a spacer sequence that is complementary to a target sequence of a superoxide dismutase 1 (SOD1) gene, wherein the first region is located 5’ of the second region; and b) optionally, an effector protein or nucleic acid encoding the effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0349] Embodiment 2: The system of embodiment 1, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1.

[0350] Embodiment 3: The system of embodiment 1, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 9.

[0351] Embodiment 4: The system of any one of embodiments 1-3, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 506, 513, 514, 1063 and combinations thereof.

[0352] Embodiment 5: The system of embodiment 1, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534- 796.

[0353] Embodiment 6: The system of any one of embodiments 1-5, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515.

[0354] Embodiment 7: The system of embodiment 1, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2.

[0355] Embodiment 8: The system of embodiment 1, wherein the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 10.

[0356] Embodiment 9: The system of any one of embodiments 1, 7, and 8, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064.

[0357] Embodiment 10: The system of embodiment 1, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 797- 1038.

[0358] Embodiment 11: The system of any one of embodiments 1 and 7-10, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0359] Embodiment 12: The system of any one of embodiments 1-11, wherein the effector protein is a dCas protein.

[0360] Embodiment 13: The system of embodiment 12, comprising a heterologous polypeptide.

[0361] Embodiment 14: The system of embodiment 13, wherein the dCas protein is covalently linked to the heterologous polypeptide.

[0362] Embodiment 15: The system of embodiment 13 or 14, wherein the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor.

[0363] Embodiment 16: The system of any one of embodiments 1-15, wherein the effector protein is linked to a nuclear localization signal.

[0364] Embodiment 17: The system of any one of embodiments 1-16, comprising the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in a single composition.

[0365] Embodiment 18: The system of any one of embodiments 1-16, comprising the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in separate compositions, separate solutions, separate containers, or a combination thereof.

[0366] Embodiment 19: The system of any one of embodiments 1-18, wherein the nucleic acid encoding the effector protein is a messenger RNA.

[0367] Embodiment 20: The system of any one of embodiments 1-19, comprising a lipid or a lipid nanoparticle.

[0368] Embodiment 21: The system of any one of embodiments 1-20, wherein the guide nucleic acid comprises at least one chemical modification, optionally wherein the at least one chemical modification is selected from a 2’-O-Methyl sugar modification, a 2’-fluoro sugar modification, a phosphorothioate internucleoside linkage, and a combination thereof.

[0369] Embodiment 22: The system of any one of embodiments 1-18, comprising a nucleic acid expression vector, wherein the nucleic acid expression vector comprises the nucleic acid encoding the effector protein, the DNA molecule encoding the guide nucleic acid, or a combination thereof. ^Cooley Ref: MABI-044 / 03WO 344183-2319

[0370] Embodiment 23: The system of embodiment 22, wherein the nucleic acid expression vector is an adenoviral associated viral (AAV) vector.

[0371] Embodiment 24: The system of embodiment 22 or 23, wherein the nucleic acid expression vector comprises or encodes a donor nucleic acid.

[0372] Embodiment 25: The system of any one of embodiments 1-23, comprising a donor nucleic acid.

[0373] Embodiment 26: A nucleic acid expression vector encoding a) a guide nucleic acid, wherein the guide nucleic acid comprises: a. a first region comprising a protein binding sequence; and b. a second region comprising a spacer sequence that is complementary to a target sequence of an SOD1 gene, wherein the first region is located 5’ of the second region; and optionally, an effector protein that binds the protein binding sequence of the guide nucleic acid.

[0374] Embodiment 27: The nucleic acid expression vector of embodiment 26, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1.

[0375] Embodiment 28: The nucleic acid expression vector of any one of embodiments 26- 27, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN- 3’, optionally wherein the PAM is selected from TABLE 9.

[0376] Embodiment 29: The nucleic acid expression vector of any one of embodiments 26- 28, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 506, 513, 514, 1063 and combinations thereof.

[0377] Embodiment 30: The nucleic acid expression vector of any one of embodiments 26- 29, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796.

[0378] Embodiment 31: The nucleic acid expression vector of any one of embodiments 26- 30, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515.

[0379] Embodiment 32: The nucleic acid expression vector of embodiment 26, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least ^Cooley Ref: MABI-044 / 03WO 344183-2319 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2.

[0380] Embodiment 33: The nucleic acid expression vector of any one of embodiments 26 or 32, wherein the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 10.

[0381] Embodiment 34: The nucleic acid expression vector of any one of embodiments 26, 32, and 33, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064.

[0382] Embodiment 35: The nucleic acid expression vector of any one of embodiments 26 and 32-34, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 797-1038.

[0383] Embodiment 36: The nucleic acid expression vector of any one of embodiments 26 and 32-35, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516.

[0384] Embodiment 37: The nucleic acid expression vector of any one of embodiments 26- 36, wherein the effector protein is a dCas protein.

[0385] Embodiment 38: The nucleic acid expression vector of embodiment 37, comprising a third region encoding a heterologous polypeptide.

[0386] Embodiment 39: The nucleic acid expression vector of embodiment 38, wherein the dCas protein is covalently linked to the heterologous polypeptide upon expression of the nucleic acid expression vector.

[0387] Embodiment 40: The nucleic acid expression vector of embodiment 38 or 39, wherein the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor.

[0388] Embodiment 41: The nucleic acid expression vector of any one of embodiments 26- 40, wherein the expression vector is an adenoviral associated viral (AAV) vector.

[0389] Embodiment 42: An expression cassette comprising, from 5’ to 3’: a) a first inverted terminal repeat (ITR); b) a first promoter sequence operably linked to a nucleic acid sequence encoding a guide RNA wherein the guide RNA comprises: a. a first region comprising a protein binding sequence; and ^Cooley Ref: MABI-044 / 03WO 344183-2319 b. a second region comprising a spacer sequence that is complementary to a target sequence of a SOD1 gene, wherein the spacer sequence selected from SEQ ID NOs: 1-505; c) a second promoter sequence operably linked to a nucleic acid sequence encoding an effector protein; d) a poly(A) signal; and e) a second ITR.

[0390] Embodiment 43: The expression cassette of embodiment 42, wherein the expression cassette further comprises a WPRE sequence located between ...

Claims

Cooley Ref: MABI-044 / 03WO 344183-2319 CLAIMS 1. A composition comprising: a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of a superoxide dismutase 1 (SOD1) gene, wherein the first region is located 5’ of the second region; and b) optionally, an effector protein or nucleic acid encoding the effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein.

2. The composition of claim 1, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1.

3. The composition of claim 1 or 2, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 9.

4. The composition of any one of claims 1-3, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 506, 513, 514, 1063 and combinations thereof.

5. The composition of claim 1, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796.

6. The composition of any one of claims 1-5, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047.

7. The composition of claim 1, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2. ^Cooley Ref: MABI-044 / 03WO 344183-2319 8. The composition of claim 1, wherein the target sequence is adjacent to a PAM of 5’- NTTN-3’, optionally wherein the PAM is selected from TABLE 10.

9. The composition of any one of claims 1, 7, and 8, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507-512 and 1064.

10. The composition of claim 1, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 797-1038.

11. The composition of any one of claims 1 and 7-10, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046.

12. The composition of any one of claims 1-11, wherein the effector protein is a dCas protein.

13. The composition of claim 12, comprising a heterologous polypeptide.

14. The composition of claim 13, wherein the dCas protein is covalently linked to the heterologous polypeptide.

15. The composition of claim 13 or 14, wherein the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor.

16. The composition of any one of claims 1-15, wherein the effector protein is linked to a nuclear localization signal.

17. The composition of any one of claims 1-16, comprising the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in a single composition.

18. The composition of any one of claims 1-16, comprising the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein or the nucleic acid ^Cooley Ref: MABI-044 / 03WO 344183-2319 encoding the effector protein, and the guide nucleic acid or the DNA molecule encoding the guide nucleic acid, are provided in separate compositions, separate solutions, separate containers, or a combination thereof.

19. The composition of any one of claims 1-18, wherein the nucleic acid encoding the effector protein is a messenger RNA.

20. The composition of any one of claims 1-19, comprising a lipid or a lipid nanoparticle.

21. The composition of any one of claims 1-20, wherein the guide nucleic acid comprises at least one chemical modification, optionally wherein the at least one chemical modification is selected from a 2’-O-Methyl sugar modification, a 2’-fluoro sugar modification, a phosphorothioate internucleoside linkage, and a combination thereof.

22. The composition of any one of claims 1-18, comprising a nucleic acid expression vector, wherein the nucleic acid expression vector comprises the nucleic acid encoding the effector protein, the DNA molecule encoding the guide nucleic acid, or a combination thereof.

23. The composition of claim 22, wherein the nucleic acid expression vector is an adenoviral associated viral (AAV) vector.

24. The composition of claim 22 or 23, wherein the nucleic acid expression vector comprises or encodes a donor nucleic acid.

25. The composition of any one of claims 1-23, comprising a donor nucleic acid.

26. A nucleic acid expression vector encoding a) a guide nucleic acid, wherein the guide nucleic acid comprises: (i) a first region comprising a protein binding sequence; and (ii) a second region comprising a spacer sequence that is complementary to a target sequence of an SOD1 gene, wherein the first region is located 5’ of the second region; and b) optionally, an effector protein that binds the protein binding sequence of the guide nucleic acid. ^Cooley Ref: MABI-044 / 03WO 344183-2319 27. The nucleic acid expression vector of claim 26, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1.

28. The nucleic acid expression vector of any one of claims 26-27, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 9.

29. The nucleic acid expression vector of any one of claims 26-28, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 506, 513, 514, 1063 and combinations thereof.

30. The nucleic acid expression vector of any one of claims 26-29, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796.

31. The nucleic acid expression vector of any one of claims 26-30, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 515, 1045, or 1047.

32. The nucleic acid expression vector of claim 26, wherein the spacer sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2.

33. The nucleic acid expression vector of any one of claims 26 or 32, wherein the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 10.

34. The nucleic acid expression vector of any one of claims 26, 32, and 33, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 507- 512 and 1064.

35. The nucleic acid expression vector of any one of claims 26 and 32-34, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, ^Cooley Ref: MABI-044 / 03WO 344183-2319 at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 797-1038.

36. The nucleic acid expression vector of any one of claims 26 and 32-35, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 516, 1041, 1042, 1044, or 1046.

37. The nucleic acid expression vector of any one of claims 26-36, wherein the effector protein is a dCas protein.

38. The nucleic acid expression vector of claim 37, comprising a third region encoding a heterologous polypeptide.

39. The nucleic acid expression vector of claim 38, wherein the dCas protein is covalently linked to the heterologous polypeptide upon expression of the nucleic acid expression vector.

40. The nucleic acid expression vector of claim 38 or 39, wherein the heterologous polypeptide is selected from the group consisting of a polymerase, a reverse transcriptase, a deaminase, an integrase, a recombinase, a transposase, a methyltransferase, an acetyltransferase, a transcriptional activator, and a transcriptional repressor.

41. The nucleic acid expression vector of any one of claims 26-40, wherein the expression vector is an adenoviral associated viral (AAV) vector.

42. An expression cassette comprising, from 5’ to 3’: a. a first inverted terminal repeat (ITR); b. a first promoter sequence operably linked to a nucleic acid sequence encoding a guide RNA wherein the guide RNA comprises: i. a first region comprising a protein binding sequence; and ii. a second region comprising a spacer sequence that is complementary to a target sequence of a SOD1 gene, wherein the spacer sequence selected from SEQ ID NOs: 1-505; c. a second promoter sequence operably linked to a nucleic acid sequence encoding an effector protein; d. a poly(A) signal; and ^Cooley Ref: MABI-044 / 03WO 344183-2319 e. a second ITR.

43. The expression cassette of claim 42, wherein the expression cassette further comprises a WPRE sequence located between the nucleic acid sequence encoding an effector protein and the poly(A) signal.

44. The expression cassette of claim 42 or 43, wherein the first promoter is a U6 promoter.

45. The expression cassette of any one of claims 42-44, wherein the second promoter is a CK8E promoter or a SPC5 promoter.

46. The expression cassette of any one of claims 42-45, wherein the poly(A) signal is a bGH or an hGH poly(A) signal.

47. The expression cassette of any one of claims 42-46, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

516.

48. The expression cassette of claim 47, wherein the effector protein comprises the amino acid substitution of L26R relative to SEQ ID NO:

516.

49. The expression cassette of claim 48, wherein the effector protein comprises SEQ ID NO: 1046.

50. The expression cassette of any one of claims 42-46, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

515.

51. The expression cassette of claim 50, wherein the effector protein comprises the amino acid substitution of D220R relative to SEQ ID NO:

515.

52. The expression cassette of claim 51, wherein the effector protein comprises SEQ ID NO: 1047.

53. An adeno-associated virus (AAV) vector comprising the expression cassette of any one of claims 42-52.

54. A cell comprising or modified by the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, or the AAV vector of claim 53.

55. The cell of claim 54, wherein the cell is a human cell. ^Cooley Ref: MABI-044 / 03WO 344183-2319 56. The cell of claim 54 or 55, wherein the cell is a motor neuron.

57. A population of cells comprising at least one cell of any one of claims 54-56.

58. A pharmaceutical composition comprising the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, or the AAV vector of claim 53, and a pharmaceutical acceptable carrier.

59. A method of modifying an SOD1 gene, comprising contacting the SOD1 gene with the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, the AAV vector of claim 53, or the pharmaceutical composition of claim 58.

60. The method of claim 59, comprising contacting a cell that comprises the SOD1 gene with the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, the AAV vector of claim 53, or the pharmaceutical composition of claim 58.

61. The method of claim 60, wherein an amount of a superoxide dismutase protein in the cell is reduced relative to the amount of the superoxide dismutase protein in the cell prior to contacting the cell with the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, the AAV vector of claim 53, or the pharmaceutical composition of claim 58.

62. A method of treating or preventing amyotrophic lateral sclerosis in a human subject in need thereof, the method comprising administering to the human subject components of the composition of any one of claims 1-25, the nucleic acid expression vector of any one of claims 26-41, the AAV vector of claim 53, or the pharmaceutical composition of claim 58.

63. The method of claim 62, wherein the human subject harbors a mutation in the SOD1 gene.

64. A fusion protein comprising an effector protein and a base editing enzyme, wherein a. the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 515; and b. the base editing enzyme comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1062. ^Cooley Ref: MABI-044 / 03WO 344183-2319 65. The fusion protein of claim 64, wherein the effector protein comprises the amino acid substitutions of D220R and E335Q relative to SEQ ID NO:

515.

66. The fusion protein of claim 64 or 65, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1048.

67. The fusion protein of claim 64 or 65, wherein the fusion protein comprises or consists of SEQ ID NO: 1048.

68. A system comprising a. a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: i. a first region comprising a protein binding sequence; and ii. a second region comprising a targeting sequence that is complementary to a target sequence of an SOD1 gene, wherein the targeting sequence comprises a spacer sequence comprising a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 1, wherein the first region is located 5’ of the second region; and b. a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

69. A system comprising a. a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 534-796; and b. a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1048.

70. A fusion protein comprising an effector protein and a base editing enzyme, wherein a. the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 516; and ^Cooley Ref: MABI-044 / 03WO 344183-2319 b. the base editing enzyme comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1062.

71. The fusion protein of claim 70, wherein the effector protein comprises the amino acid substitutions of L26K and E567Q relative to SEQ ID NO:

516.

72. The fusion protein of claim 70 or 71, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 1049.

73. The fusion protein of claim 70 or 71, wherein the fusion protein comprises or consists of SEQ ID NO: 1049.

74. A system comprising a. a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises: i. a first region comprising a protein binding sequence; and ii. a second region comprising a targeting sequence that is complementary to a target sequence of an SOD1 gene, wherein the targeting sequence comprises a spacer sequence comprising a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2, wherein the first region is located 5’ of the second region; and b. a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

75. A system comprising a. a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 797-1038; and b. a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO:^

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