Compositions for the modification of the human c9ORF72 gene

Abstract

Provided herein are compositions, systems, and methods comprising effector proteins for treating amyotrophic lateral sclerosis and Frontotemporal dementia. These effector proteins may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification of the C9ORF72 gene.

Description

Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO COMPOSITIONS FOR THE MODIFICATION OF THE HUMAN C9ORF72 GENE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 514,863, filed July 21, 2023; U.S. Provisional Application 63 / 584,260, filed September 21, 2023; U.S. Provisional Application 63 / 602,843, filed November 27, 2023; U.S. Provisional Application 63 / 634,037, filed April 15, 2024; U.S. Provisional Application 63 / 641,579, filed May 2, 2024; and U.S. Provisional Application 63 / 655,166, filed June 3, 2024, the contents of each of which are incorporated herein by reference in their entireties. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (MABI_046_06WO_SeqList_ST26.xml; Size: 9,927,882 bytes; and Date of Creation: June 21, 2024) are herein incorporated by reference in its entirety. BACKGROUND

[0003] Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s Disease, are neurodegenerative diseases characterized by behavioral and motor neuron dysfunction. FTD symptoms include antisocial behavior, unsteadiness, rigidity, slowness, twitching, muscle weakness, and difficulty swallowing. The symptoms of ALS include twitching, cramping, spasticity, muscle weakness, slurred speech and difficulty chewing and swallowing. Both ALS and FTD are characterized by neuropathological aggregates containing the ubiquitinated protein, TDP-43. A mutation of the human gene, chromosome 9 open reading frame 72 (C9ORF72) is associated with familial forms of FTD and ALS, in particular a hexanucleotide (G4C2)n repeat expansion (HRE) in the non-coding region of C9ORF72. In a healthy individual, the HRE contains 2–30 repeats, but patients with C9orf72-associated ALS / FTD, may have several hundreds or thousands of repeats. The C9ORF72 gene can produce three different transcript variants, V1, V2, and V3. V1 produces a 222 amino acid protein. V2 and V3 produce a 481 amino acid protein. In the case of V1 and V3, the HRE is located in intron 1. In the case of V2, the HRE is located in the promoter region. The HRE containing mutant allele results in a loss-of-function effect due to C9ORF72 haploinsufficiency and a gain of function effect, wherein the expanded RNAs accumulate in neuropathological aggregates. Patients with ALS associated with theCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO C9ORF72 repeat expansion may have FTD as well. Suppressing expression of the HRE containing mutant allele may be therapeutic for ALS and FTD.

[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 human C9ORF72 gene. Compositions, systems, kits and methods described herein comprise a CRISPR system or a use thereof. Such compositions, systems, kits and methods may be useful for the treatment of ALS and FTD. In some embodiments, compositions, systems, kits and methods disclosed herein produce a double stranded break in an C9ORF72 gene comprising a mutation, thereby disrupting expression and reducing production of a pathogenic C9orf72 variant. In some embodiments, compositions, systems, kits and methods do not cleave an C9ORF72 gene, but otherwise modify its expression, e.g., through epigenetic silencing.

[0006] In some aspects, the present disclosure provides systems or 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 C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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 C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector protein that binds the proteinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO binding sequence or a nucleic acid encoding the effector protein. 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 C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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 SEQ ID NOS: 1-2849. 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 7. In some embodiments, the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 10,847, 10,854, 10,855, 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: 5424-8272. 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 any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919. 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 SEQ ID NOS: 2850-5423. In some embodiments, the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 8. In some embodiments, the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 10,848-10,853 and 10,920. 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: 8273-10,846. 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 any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918. 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 isCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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. In some embodiments, the systems, compositions, or kits provided herein comprise 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 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. In some embodiments, the systems, compositions, or kits provided herein comprise a lipid or a lipid nanoparticle. 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. In some embodiments, the systems, compositions, or kits provided herein comprise an adenoviral associated viral (AAV) vector, wherein the AAV 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 AAV vector comprises an additional guide nucleic acid, wherein the additional guide nucleic acid comprises the protein binding sequence and an additional spacer sequence that is complementary to an additional target sequence of the C9ORF72 gene.

[0007] In some aspects, the present disclosure provides expression cassettes 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 C9ORF72 gene, wherein the spacer sequence selected from SEQ ID NOs: 1-5423; 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 acidCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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:10,857. In some embodiments, the effector protein comprises the amino acid substitution of L26R relative to SEQ ID NO: 10,857. In some embodiments, the effector protein comprises SEQ ID NO: 10,879. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10,856. In some embodiments, the effector protein comprises the amino acid substitution of D220R relative to SEQ ID NO: 10,856. In some embodiments, the effector protein comprises SEQ ID NO: 10,880. In some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector comprising an expression cassette described herein.

[0008] In some aspects, the present disclosure provides cells comprising or modified by the systems, compositions, kits, or nucleic acid expression vectors 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 cells described herein.

[0009] In some aspects, the present disclosure provides pharmaceutical compositions comprising compositions or nucleic acid expression vectors described herein, and a pharmaceutical acceptable carrier.

[0010] In some aspects, the present disclosure provides methods of modifying a C9ORF72 gene, comprising contacting the C9ORF72 gene with systems, compositions, kits, AAV vectors or pharmaceutical compositions described herein. In some embodiments, the method comprises contacting a cell that comprises the C9ORF72 gene with the system, composition, kit, or AAV vectors or pharmaceutical compositions described herein. In some embodiments, an amount of a C9orf72 protein in the cell is reduced relative to the amount of the C9orf72 protein in the cell prior to contacting the cell with the system, composition, kit, or AAV vectors or pharmaceutical compositions described herein.

[0011] In some aspects, the present disclosure provides methods 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 system, composition, kit, AAV vectors or pharmaceutical compositions described herein. In some embodiments, the subject harbors aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO mutation in the C9ORF72 gene. In some embodiments, the mutation is a hexanucleotide (G4C2)n repeat expansion (HRE) in a non-coding region of C9ORF72. In some embodiments, the non- coding region is selected from a promoter and intron 1. In some embodiments, the hexanucleotide (G4C2)n repeat expansion comprises more than 100, more than 150, more than 200, more than 250, or more than 300 repeats of G4C2.

[0012] In some aspects, the present disclosure provides fusion proteins, or nucleic acids encoding the fusion proteins, wherein the fusion protein comprises an effector protein and a base editing enzyme, wherein the effector protein sequence comprises an amino acid 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: 10,856; 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: 10,881. In some embodiments, the effector protein comprises the amino acid substitutions of D220R and E335Q relative to SEQ ID NO: 10,856. 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: 10,882. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 10,882.

[0013] In some aspects, 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 targeting sequence that is complementary to a target sequence of a C9ORF72 gene and comprising a spacer sequence selected from SEQ ID NOs:1-2849, wherein the first region is located 5’ of the second region; (b) a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

[0014] In some aspects, 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 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: 5424-8272; 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: 10,882.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0015] In some aspects, the present disclosure provides fusion proteins comprising 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: 10,857; 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: 10,881. In some embodiments, effector protein comprises the amino acid substitutions of L26K and E567Q relative to SEQ ID NO: 10,857. 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: 10,883. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 10,883.

[0016] In some aspects, 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 targeting sequence that is complementary to a target sequence of a C9ORF72 gene and comprising a spacer sequence selected from SEQ ID NOs:2850-5423, wherein the first region is located 5’ of the second region; (b) a fusion protein comprising an effector protein and a base editing enzyme, or a nucleic acid encoding the fusion protein.

[0017] In some aspects, the present disclosure provides compositions 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: 8273-10,846; 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: 10,883.

[0018] In some aspects, 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 containing SNP rs78074330 located in the sequence of SEQ ID NO: 10,896 in intron 1 of a C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector proteinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO or a nucleic acid encoding an effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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 sequence selected from SEQ ID NOS: 513, 1063, 1336, 10,901 and 10,902. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5936, 6486, 6759, 10,910, and 10,911. 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: 10,856. 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 sequence selected from SEQ ID NOS: 4221, 4300, 10,899, and 10,900. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 9644, 9723, 10,908, and 10,909. 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: 10,857.

[0019] In some aspects, 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 containing SNP rs112048460 located in the sequence of SEQ ID NO: 10,897 in intron 1 of a C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector protein or a nucleic acid encoding an effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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% identicalCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO to a sequence selected from SEQ ID NOS: 662, 1117, 1660, 10,903, and 10,904. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 6085, 6540, 7083, 10,912, and 10,913. 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: 10,856.

[0020] In some aspects, 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 containing SNP rs2282240 located in the sequence of SEQ ID NO: 10,898 in intron 1 of a C9ORF72 gene, wherein the first region is located 5’ of the second region; and (b) optionally, an effector protein or a nucleic acid encoding an effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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 sequence selected from SEQ ID NOS: 111, 859, 1244, 2059, and 10,905-10,907. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5534, 6282, 6667, 7482, and 10,914-10,916. 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: 10,856.

[0021] In some aspects, the present disclosure provides compositions comprising an upstream guide nucleic acid or a DNA molecule encoding the upstream guide nucleic acid, wherein the upstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence upstream of a HRE in a C9ORF72 gene; a downstream guide nucleic acid or a DNA molecule encoding the downstream guide nucleic acid, wherein the downstream guide nucleic acidCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO comprises a spacer sequence that is complementary to a target sequence downstream of the HRE in the C9ORF72 gene; and an effector protein or a nucleic acid encoding the effector protein, that binds the protein binding sequence or a nucleic acid encoding the effector protein. 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 SEQ ID NO: 10,857. In some embodiments, the effector protein comprises the amino acid substitutions of L26R and I471T relative to SEQ ID NO: 10,857. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403. In some embodiments, the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 4571, 4370, 4710, 4533, and 4449. In some embodiments, the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 9994, 9793, 10,133, 9956, and 9872. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4571. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9994. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4370. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9793. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4710. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 10,133. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4533. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9956. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4449. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9872. 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 SEQ ID NO: 10,856. In some embodiments, the effector protein comprises theCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO amino acid substitution of D220R relative to SEQ ID NO: 10,856. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 35, 415, 479, and 1653. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 5458, 5838, 5902, and 7076. In some embodiments, the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 463, 711, and 1336. In some embodiments, the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 5886, 6134, and 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, wherein the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at leastCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NOs: 6759.

[0022] In some aspects, the present disclosure provides AAV vectors comprising: a) a first nucleotide sequence that is 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% identical to SEQ ID NO:10,918; b) a second nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 8273-10,846; and c) optionally, a third nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 8273-10,846. In some embodiments, the second and third nucleotide sequences are selected from SEQ ID NOS: 8273-10,846. In some embodiments, the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9403, and the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ IDCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO NOs: 9994, 9793, 10,133, 9956, and 9872. In some embodiments, the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9656, and the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 10,207, 9793 and 9678. In some embodiments, the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9673, and the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 10,207 and 9793. In some embodiments, the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9403, and the third nucleotide sequence is at least 90% or 100% identical to SEQ ID NOs: 10,207.

[0023] In some aspects, the present disclosure provides AAV vectors comprising: a) a first nucleotide sequence that is 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% identical to SEQ ID NO: 10,880; b) a second nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-8272; and c) optionally, a third nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-8272. In some embodiments, the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 6627, and the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 5838, 5902, 5458, and 7106. INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 depicts a schematic for selecting spacer sequence placement for editing of the C9ORF72 gene with two guide RNAs.

[0026] FIG.2 shows an exemplary AAV vector comprising a plurality of expression cassettes.

[0027] FIG.3 shows C9ORF72 HRE deletion by CasPhi.12 L264, I471T and guide nucleic acid pairings.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0028] FIG. 4 shows % indel results in the C9ORF72 gene produced by CasM.265466 D220R and C9ORF72 guide nucleic acids.

[0029] FIG.5 depicts in vivo gene editing of PCSK9 in muscle tissues using AAV9-A4 delivery of CasPhi.12 and CasM.265466 variants. DETAILED DESCRIPTION OF THE INVENTION

[0030] 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.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0032] 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

[0033] 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:

[0034] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0035] The terms, “or” and “and / or,” as used herein, include any, and all, combinations of one or more of the associated listed items.

[0036] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0037] 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.

[0038] 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.

[0039] 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 sameCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0040] 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.

[0041] 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.

[0042] 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-occurringCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0043] 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.

[0044] 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 double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0045] 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-strandedCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[0046] 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.

[0047] 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.

[0048] The term, “detectable signal,” as used herein, refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical and other detection methods known in the art.

[0049] 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.

[0050] 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.

[0051] 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 moreCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence selective manner.

[0052] 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.

[0053] 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.

[0054] 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 in aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 hereinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO with the term “guide RNA” (gRNA) however it is understood that guide nucleic acids 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.

[0060] 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 embodiments, the heterologous polypeptide or nucleotide sequence is not found covalently bound to the reference nucleic acid or protein. In some embodiments, the heterologous polypeptide or nucleotide sequence is not found non-covalently interacting with the reference nucleic acid or protein. In some embodiments, 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. The fusion partner protein 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.

[0061] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0062] 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) or a first nucleic acid to a second nucleic acid (e.g., by a phosphodiester bond, or one or more nucleotides).

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.).Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0068] 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-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).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0074] 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 “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

[0075] 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.

[0076] 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, a sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence.

[0077] 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 polypeptidesCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0078] 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.

[0079] 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, 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.

[0080] 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).

[0081] 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.

[0082] 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 fromCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO (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.

[0083] 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).

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 theCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0089] 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.^^ OHQWLYLUXVHV^ DQG^ Ȗ-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A viral vector 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

[0090] The present disclosure describes compositions, systems and methods for modifying the C9ORF72 gene. 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 C9ORF72 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0091] 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.

[0092] In some embodiments, the compositions and systems comprise guide nucleic acids that are capable of hybridizing to a target sequence of the C9ORF72 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 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 C9ORF72 gene). In some embodiments, the present disclosure provides methods for treating a disease (e.g., amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD)) by modifying one or more target genes (e.g., the C9ORF72 gene). In some embodiments, ALS is familial ALS (fALS).

[0093] 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 regionCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0094] 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 order or proximity that is not observed in nature. Therefore, guide nucleic acid compositions described herein are not naturally occurring. 3. Guide Nucleic Acids

[0095] 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.

[0096] In general, guide nucleic acids, also referred to as guide RNA (gRNA), comprise a nucleotide sequence. Such nucleotide sequence may be described as a nucleotide sequence of either DNA or RNA. Due to WIPO Standard ST.26, the Us are being represented as Ts in RNA in the Sequence Listing provided herein. 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 aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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, or a deletion, or an insertion.

[0097] 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.

[0098] 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 to as a protein binding sequence. In some embodiments, the first nucleotide sequence is located 5’ to 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 C9ORF72 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0099] 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.

[0100] 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 C9ORF72 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 acid (e.g., the C9ORF72 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.

[0101] 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 comprisingCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0102] 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.

[0103] 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 C9ORF72 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., an exon of the C9ORF72 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.

[0104] 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 linkedCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0105] 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.

[0106] 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 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.

[0107] 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, intermolecularCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.).

[0108] 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.

[0109] 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.

[0110] 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 may bind to a portion of a gene associated with a genetic disorder, or an amplicon thereof, as described herein.

[0111] 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: 5424-10,846. Spacer Sequences

[0112] Guide nucleic acids described herein generally comprise a spacer region represented by a spacer sequence. The terms, “spacer region,” “targeting sequence” and “spacer sequence” may be used interchangeably. In some embodiments, a spacer region is capable ofCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0113] 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.

[0114] 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 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0115] 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.

[0116] 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 C9ORF72 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 C9ORF72 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.

[0117] 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 C9ORF72 gene. In some embodiments, the target sequence is within an exon region of the C9ORF72 gene. In some embodiments, the target sequence is within an intron region of the C9ORF72 gene. In some embodiments, the target sequence is spans and exon and intron of the C9ORF72 gene. In some embodiments, the target sequence is located in a regulatory element of the C9ORF72 gene. In some embodiments, the regulatory element is a promoter. In some embodiments, the target sequence contains one or more single nucleotide polymorphisms (SNPs) of the C9ORF72 gene.

[0118] 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 C9ORF72 gene. In someCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 C9ORF72 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 C9ORF72 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 C9ORF72 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 C9ORF72 gene.

[0119] In some embodiments, a spacer sequence is complementary to a target sequence that is adjacent to a PAM in the C9ORF72 gene. In some embodiments, a spacer sequence is complementary to a target sequence that is within 0-10 nucleotides of a PAM in the C9ORF72 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.

[0120] In some embodiments, a spacer sequence is complementary to a target sequence that contains one or more SNPs in the C9ORF72 gene. Non-limiting examples of SNPs in the C9ORF72 gene are described in Ben-Dor, et al., PLoS Genet.2021 Mar 29;17(3), the contents of which are incorporated herein by reference in their entirety. 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 containing SNP rs78074330 located in the sequence of TTGCT[A / G]CAGGC (SEQ ID NO: 10,896) in intron 1 of the C9ORF72 gene. 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 SEQ ID NOS: 513, 1063, 1336, 4221, 4300, and 10,899-10,902. 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 containing SNP rs112048460 locatedCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO in the sequence of CAGTA[C / T]CCGAG (SEQ ID NO: 10,897) in intron 1 of the C9ORF72 gene. 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 SEQ ID NOS: 662, 1117, 1660, 10,903, and 10,904. 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 containing SNP rs2282240 located in the sequence of GGCCA[C / T]CCCTC (SEQ ID NO: 10,898) in intron 1 of the C9ORF72 gene. 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 SEQ ID NOS: 111, 859, 1244, 2059, and 10,905- 10,907.

[0121] 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 SEQ ID NOS: 1-5423.

[0122] Guide nucleic acids disclosed herein may target various regions of the C9ORF72 gene. In some embodiments, the spacer sequences hybridize to a target sequence in intron 1 of the C9ORF72 gene. By way of non-limiting example, the spacer sequences that are complementary to intron 1 of the C9ORF72 gene include SEQ ID NOs: 1-2248 and 2850-4857. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of VNTR. By way of non- limiting example, the spacer sequences that are complementary to the promoter of the C9ORF72 gene and are adjacent to a PAM of VNTR include SEQ ID NOs: 2249-2549. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of TNTR. By way of non- limiting example, the spacer sequences that are complementary to the promoter of the C9ORF72 gene and are adjacent to a PAM of TNTR include SEQ ID NOs: 2550-2669. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of TNTY. By way of non- limiting example, the spacer sequences that are complementary to the promoter of the C9ORF72Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO gene and are adjacent to a PAM of TNTY include SEQ ID NOs: 2670-2849. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of NTTN. By way of non- limiting example, the spacer sequences that are complementary to the promoter of the C9ORF72 gene and are adjacent to a PAM of NTTN include SEQ ID NOs: 4858-5423.

[0123] In some embodiments, the spacer sequence comprises one or more nucleobase alterations at one or more positions in any one of the sequences selected from SEQ ID NOS: 1- 5423. 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 selected from SEQ ID NOS: 1-5423. 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 selected from SEQ ID NOS: 1-5423.

[0124] 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: 10,856, 10,878, 10,880, and 10,919; 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 SEQ ID NOS: 1-2849. 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: 10,856, 10,878, 10,880, and 10,919; 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 C9ORF72 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO GCTA, GCTG, GGTA, GGTG, GTTA, GTTG, TATA, TATC, TATG, TATT, TCTA, TCTC, TCTG, TCTT, TGTA, TGTC, TGTG, TGTT, TTTA, TTTC, TTTG, and TTTT.

[0125] 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: 10,856, 10,878, 10,880, and 10,919; and two guide nucleic acids each 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 SEQ ID NOS: 1-2849. In such embodiments, the spacer sequences of the two guide nucleic acids can be selected to excise a portion of the C9ORF72 gene. See for example, Fig.1. In order to excise the disease-causing HRE region, a first spacer sequence targeting the upstream guide RNA region and a second spacer sequence targeting the Exon1b-sparing region can be selected. Such a guide RNA pairing would excise the disease-causing HRE while leaving Exon1b (which drives robust expression of C9ORF72) intact.

[0126] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 SEQ ID NOS: 2850-5423. 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 C9ORF72 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. 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 leastCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; and two guide nucleic acids each 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 SEQ ID NOS: 2850-5423.

[0127] 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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 1-2248. In some embodiments, the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM selected from TNTR, VNTR, and TNTY. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 5424-7671. In some embodiments, the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.

[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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 2249-2549. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of VNTR. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 7672-7972. In some embodiments, the composition or system comprises a gene silencing protein, such as a transcriptional repressor. A non-limiting example of aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO transcriptional repressor is a DNA methyltransferase. In some embodiments, the effector protein is fused to the gene silencing protein.

[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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 2550-2669. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of TNTR. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 7973-8092. In some embodiments, the composition or system comprises a gene silencing protein, such as a transcriptional repressor. A non-limiting example of a transcriptional repressor is a DNA methyltransferase. In some embodiments, the effector protein is fused to the gene silencing protein.

[0130] 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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 2670-2849. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of TNTY. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 8093-8272. In some embodiments, the composition or system comprises a gene silencing protein, such as a transcriptional repressor. A non-limiting example of a transcriptionalCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO repressor is a DNA methyltransferase. In some embodiments, the effector protein is fused to the gene silencing protein.

[0131] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 nucleotide sequence selected from SEQ ID NOS: 2850-3931. In some embodiments, the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of NTTN. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 8273-9354. In some embodiments, the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.

[0132] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 nucleotide sequence selected from SEQ ID NOS: 3932-4857. In some embodiments, the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of NTTN. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 9355-10,280. In some embodiments, the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.

[0133] 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%,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 nucleotide sequence selected from SEQ ID NOS: 4858-5151. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of NTTN. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 10,281-10,574. In some embodiments, the composition or system comprises a gene silencing protein, such as a transcriptional repressor. A non-limiting example of a transcriptional repressor is a DNA methyltransferase. In some embodiments, the effector protein is fused to the gene silencing protein.

[0134] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 nucleotide sequence selected from SEQ ID NOS: 5152-5423. In some embodiments, the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene. In some embodiments, the target sequence is adjacent to a PAM of NTTN. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOs: 10,575-10,846. In some embodiments, the composition or system comprises a gene silencing protein, such as a transcriptional repressor. A non-limiting example of a transcriptional repressor is a DNA methyltransferase. In some embodiments, the effector protein is fused to the gene silencing protein.

[0135] 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: 10,856, 10,878, 10,880,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 513, 1063, 1336, 10,901 and 10,902. In some embodiments, the spacer sequence hybridizes to a target sequence containing SNP rs78074330 located in the sequence of SEQ ID NO: 10,896 in intron 1 of a C9ORF72 gene. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5936, 6486, 6759, 10,910, and 10,911.

[0136] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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 nucleotide sequence selected from SEQ ID NOS: 4221, 4300, 10,899, and 10,900. In some embodiments, the spacer sequence hybridizes to a target sequence containing SNP rs78074330 located in the sequence of SEQ ID NO: 10,896 in intron 1 of a C9ORF72 gene. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 9644, 9723, 10,908, and 10,909.

[0137] 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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 662, 1117, 1660, 10,903, and 10,904. In some embodiments, the spacer sequence hybridizes to a target sequence containing SNP rs112048460 located in the sequence of SEQ ID NO: 10,897 in intron 1 of a C9ORF72 gene. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, atCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100% identical to a sequence selected from SEQ ID NOS: 6085, 6540, 7083, 10,912, and 10,913.

[0138] 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: 10,856, 10,878, 10,880, and 10,919; 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 nucleotide sequence selected from SEQ ID NOS: 111, 859, 1244, 2059, and 10,905-10,907. In some embodiments, the spacer sequence hybridizes to a target sequence containing SNP rs2282240 located in the sequence of SEQ ID NO: 10,898 in intron 1 of a C9ORF72 gene. In some embodiments, the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5534, 6282, 6667, 7482, and 10,914-10,916.

[0139] In some embodiments, compositions and systems provided herein comprise an upstream guide nucleic acid or a DNA molecule encoding the upstream guide nucleic acid, wherein the upstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence upstream of a HRE in a C9ORF72 gene; a downstream guide nucleic acid or a DNA molecule encoding the upstream guide nucleic acid, wherein the downstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence downstream of the HRE in the C9ORF72 gene; and an effector protein or a nucleic acid encoding the effector protein. Such a guide RNA pairing would excise the disease-causing HRE while leaving Exon1b intact.

[0140] 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 SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918. In some embodiments, the effector protein comprises the amino acid substitutions of L26R and I471T relative to SEQ ID NO: 10,857. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403. In someCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO embodiments, the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 4571, 4370, 4710, 4533, and 4449. In some embodiments, the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 9994, 9793, 10133, 9956, and 9872. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4571. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9994. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4370. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9793. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4710. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 10133. In someCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4533. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9956. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4449. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9872.

[0141] 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 SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919. In some embodiments, the effector protein comprises the amino acid substitution of D220R relative to SEQ ID NO: 10,856. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 35, 415, 479, and 1653. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 5458, 5838, 5902, and 7076. In some embodiments, the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 463, 711, and 1336. In some embodiments, the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequenceCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO selected from SEQ ID NOs: 5886, 6134, and 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, wherein the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotideCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and theCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, atCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134. In some embodiments, the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336. In some embodiments, the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NOs: 6759. Nucleic acid linkers

[0142] 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.

[0143] 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: 10,921).

[0144] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO Repeat Sequences

[0145] 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.”

[0146] 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. 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: 10,921.

[0147] 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.).

[0148] TABLE 1 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 1.

[0149] 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 1.

[0150] 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 selected from SEQ ID NOS: 1-2849 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: 10,847. 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 SEQ ID NOS: 2850-5423, 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: 10,848-10,853 and 10,920. The combination of spacer and repeat sequences provided in SEQ ID NOs: 5424- 10,846 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: 10,847- 10,853 and 10,920). For example, in some embodiments, the guide sequence comprises a spacer sequence selected from any one of SEQ ID NOs: 1-5423 with a repeat sequence selected from any one of SEQ ID NOs: 10,847-10,853 and 10,920.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO Intermediary sequences

[0151] 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.

[0152] 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.

[0153] 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 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.

[0154] 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.

[0155] 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 firstCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0156] 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: 10,856, 10,878, 10,880, and 10,919; 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: 10,854). In some embodiments, an intermediary 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: 10,854. Handle sequences

[0157] 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 handleCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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: 10,921). 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.

[0158] In some embodiments, compositions and systems comprise a 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.

[0159] 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.

[0160] 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, which 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 aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO combination of the intermediary RNA and the repeat sequence is capable of being non-covalently bound by an effector protein.

[0161] In some embodiments, the handle sequence of a 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 a 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.

[0162] 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.

[0163] In some embodiments, the length of a handle sequence in a 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 a sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in a 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 a 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 linkedCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 40 to 70 nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides.

[0164] 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: ACAGCUUAUUUGGAAGCUGAAAUGUGAGGUUUAUAACACUCACAAGAAUCCUGA AAAAGGAUGCCAAAC (SEQ ID NO: 10,855). 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: 10,855. 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: 10,856, 10,878, 10,880, and 10,919.

[0165] In some embodiments, the sequences selected from SEQ ID NOS: 1-10,853, 10,854, and 10,855 and 10,920 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-O-N(CH3)-CH2- , -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiesterCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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

[0166] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO crRNA

[0167] 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 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).

[0168] 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.

[0169] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918; 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: 8,273-10,846. 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: 8,273-10,846.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0170] 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 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

[0171] 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, a 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.

[0172] 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 aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO sgRNA. A sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. A 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.

[0173] In some embodiments, a 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, a sgRNA comprises a handle sequence and a spacer sequence; an intermediary sequence and an crRNA; an intermediary sequence, a repeat sequence and a spacer sequence; and the like.

[0174] In some embodiments, a 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, a 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.

[0175] In some embodiments, a 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, a 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.

[0176] In some embodiments, a 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, a 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 byCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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, a 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.

[0177] An exemplary handle sequence in a 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 phosphodiester bond). In some embodiments, the 5’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).

[0178] 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: 10,856, 10,878, 10,880, and 10,919; 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: 5424-8272. 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: 5424-8272. Dual Nucleic Acid Systems

[0179] 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 ofCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0180] 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.

[0181] 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 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

[0182] 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 anCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO engineered mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature.

[0183] 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.

[0184] 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 single-stranded DNA (ssDNA). Effector proteins may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof.

[0185] 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).

[0186] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0187] TABLEs 2-5 provide illustrative amino acid sequences of effector proteins. In some embodiments, the amino acid sequence of an effector protein 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 as set forth in TABLEs 2-5. 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 2-5.

[0188] 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 2-5.

[0189] 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 2- 5. 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);Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO (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).

[0190] In some embodiments, effector proteins are engineered variants of CasM.265466 (SEQ ID NO: 10,856) and CasPhi.12 (SEQ ID NO: 10,857). Engineered variants of CasM.265466 (SEQ ID NO: 10,856) and CasPhi.12 (SEQ ID NO: 10,857) may comprise amino acid substitutions relative to SEQ ID NO: 10,856 and SEQ ID NO: 10,857, respectively. Exemplary amino acid substitutions are described in TABLE 3 and TABLE 4. The amino acid substitutions in TABLE 5 can be combined with any of the substitutions listed in either TABLE 3 or TABLE 4. The amino acid substitutions in TABLE 3 and TABLE 4 may be combined with other amino acid alterations described herein.

[0191] 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: 10,856, and comprises the amino acid substitution of D220R relative to SEQ ID NO: 10,856. 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: 10,856, and comprises the amino acid substitutions of D220R and K250N relative to SEQ ID NO: 10,856. 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: 10,856, and comprises the amino acid substitutions of D220R and any one of A306K, A306H, and A306R, relative to SEQ ID NO: 10,856.

[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: 10,857, and comprises the amino acid substitution selected from L26R, L26K, I471T, and any combination thereof relative to SEQ ID NO: 10,857. In some embodiments, the effector proteinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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: 10,857, and comprises the amino acid substitutions of: L26R or L26K, and A673G relative to SEQ ID NO: 10,857. 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: 10,857, and comprises the amino acid substitutions of: L26R or L26K, and I471T, relative to SEQ ID NO: 10,857.

[0193] 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: 10,856, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,856, 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: 10,856, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,856, 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.

[0194] 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: 10,856, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,856, 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: 10,856, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,856, 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0195] 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: 10,856, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,856, 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: 10,856, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,856, 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 3 provides the exemplary amino acid alterations relative to SEQ ID NO: 10,856 useful in compositions, systems, and methods described herein.

[0196] 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: 10,856, with the exception of two amino acid substitutions at D220 and E335 relative to SEQ ID NO: 10,856. In some embodiments, the amino acid substitutions are D220R and 335Q. In some embodiments, the engineered effector protein comprises or consists of SEQ ID NO: 10,878.

[0197] 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: 10,857, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,857, 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 someCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 10,857, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,857, 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.

[0198] 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: 10,857, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,857 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: 10,857, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,857, 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0199] 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: 10,857, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,857 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: 10,857, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,857, 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 / Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[0200] In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 10,857, with the exception of at least two amino acid substitutions relative to SEQ ID NO: 10,857, wherein the amino acid substitutions comprise L26K / E567Q. In some embodiments, the polypeptide comprises or consists of SEQ ID NO: 10,877.

[0201] 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: 10,857, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,857 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: 10,857, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,857, 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0202] 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: 10,857, 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: 10,857, wherein amino acids S478-S505 have been deleted and replaced with SDLYIERGGDPRDVHQQVETKPKGKRKSEIRILKIR (SEQ ID NO: 10,885) or SDYIVDHGGDPEKVFFETKSKKDKTKRYKRR (SEQ ID NO: 10,886). 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: 10875. 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: 10,876.

[0203] 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: 10,857, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 10,857, 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: 10,857, with the exception of at least one amino acid substitution relative to SEQ ID NO: 10,857, 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 4 provides the exemplary amino acid alterations relative to SEQ ID NO: 10,857 useful in compositions, systems, and methods described herein.

[0204] Exemplary engineered effector proteins are provided in TABLE 5.

[0205] 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 2-5, 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 2-5. In some embodiments, anCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 2-5, 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 2-5. 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 2-5, 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 2-5.

[0206] 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 2-5. 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–^^^^^^^^^^^^^^WKDW^LV^WUDQVIRUPHG^VR^WKDW^DQ\^YDOXH^^^^^LV^UHSODFHG^ZLWK^^^^ DQG^DQ\^YDOXH^^^^^LV^UHSODFHG^ZLWK^^^^)RU^H[DPSOH^^DQ^,OH^^,^^WR^ / HX^^ / ^^VXEVWLWXWLRQ^LV^VFRUHG^DW^ +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 andCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 similarity. The % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given WKH^SDUDPHWHUV^PDWUL[^ ^% / 2680^^^DQG^WKUHVKROG^^^^^

[0207] 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.

[0208] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0209] 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); 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.

[0210] 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.

[0211] TABLE 6 provides exemplary nuclear localization sequences and endosomal escape polypeptide sequences. In TABLE 6, 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

[0212] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0213] 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 7. In some embodiments, the effector protein recognizes a PAM sequence as shown in TABLE 7, 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 7. In some embodiments, the PAM is selected from: AATA, AATC, AATG, AATT, ACTA, ACTC, ACTG, ACTT, AGTA, AGTG, AGTC, AGTG, AGTT, ATTA, ATTC, ATTG, 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: 10,856, 10,878, 10,880, and 10,919.

[0214] In some embodiments, the PAM is 5’-NTTN-3’, wherein N = any nucleic acid. Exemplary PAM sequences are disclosed in TABLE 8. In some embodiments, the effector protein recognizes a PAM sequence as shown in TABLE 8. In some embodiments, the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 8. 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918. Nuclease-dead effector proteins

[0215] 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 anCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 2. 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 2.

[0216] 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 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.

[0217] 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: 10,856, 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: 10,856, and wherein the effectorCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO protein further comprises one or more alterations selected from D237A, D418A, D418N, E335A, and E335Q.

[0218] 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: 10,857, 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: 10,857, and wherein the effector protein further comprises one or more alterations selected from D369A, D369N, E567A, E567Q, D658A and D658N. Fusion Proteins

[0219] 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 proteinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0220] 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 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.

[0221] 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.

[0222] 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: 10,856, 10,878, 10,880, and 10,919 and aCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO guide RNA comprising a repeat sequence that is at least 90% identical to SEQ ID NO: 10,847, and a spacer sequence that is at least 90% identical a sequence selected from SEQ ID NOS: 1- 2849.

[0223] 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: 10,856, 10,878, 10,880, and 10,919 and a guide RNA comprising a repeat sequence that is at least 95% identical to SEQ ID NO: 10,847, and a spacer sequence that is at least 95% identical a sequence selected from SEQ ID NOS: 1- 2849.

[0224] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919 and a guide RNA comprising a repeat sequence comprising SEQ ID NO: 10,847, and a spacer sequence comprising any one of SEQ ID NOS: 1-2849.

[0225] 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: 10,856, 10,878, 10,880, and 10,919 and a guide RNA sequence that is at least 90% identical to any one of SEQ ID NOs: 5424-8272.

[0226] 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: 10, 10,856, 10,878, 10,880, and 10,919 and a guide RNA sequence that is at least 95% identical to any one of SEQ ID NOs: 5424-8272.

[0227] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919 and a guide RNA sequence comprising any one of SEQ ID NOs: 5424-8272.

[0228] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA comprising a repeat sequence that is at least 90% identical to any one ofCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO SEQ ID NOs: 10,848-10853 and 10,920, and a spacer sequence that is at least 90% identical a sequence selected from SEQ ID NOS: 2850-5423.

[0229] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA comprising a repeat sequence that is at least 95% identical to any one of SEQ ID NOs: 10,848-10853 and 10,920, and a spacer sequence that is at least 95% identical a sequence selected from SEQ ID NOS: 2850-5423.

[0230] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA comprising a repeat sequence comprising any one of SEQ ID NOs: 10,848-10853 and 10,920, and a spacer sequence comprising any one of SEQ ID NOS: 2850-5423.

[0231] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA sequence that is at least 90% identical to any one of SEQ ID NOs: 8273- 10,846.

[0232] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA sequence that is at least 95% identical to any one of SEQ ID NOs: 8273- 10,846.

[0233] In some embodiments of the above, the systems and compositions provided herein comprise a fusion protein comprising an effector protein comprising any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 and a guide RNA sequence comprising any one of SEQ ID NOs: 8273-10,846.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO Nucleic Acid Modification Activity

[0234] 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), DNA methyltransferase 3L (DNMT3L), 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.

[0235] 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.

[0236] 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0237] 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.

[0238] 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

[0239] 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 embodiments, the aminoCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0240] 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.

[0241] 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.

[0242] 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 herebyCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al., Nature, 533, 420-424 (2016); 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.

[0243] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0244] 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 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.

[0245] 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.

[0246] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0247] 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, saBE4, and saBE4-Gam as described in WO2021163587, WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.

[0248] In some embodiments, a base editor is a cytosine to guanine base editor (CGBE). A CGBE may convert a cytosine to a guanine.

[0249] 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0250] 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: 10,881. In some embodiments, the ABE is ABE8e and comprises or consists of SEQ ID NO: 10,881.

[0251] 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.

[0252] 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: 10,856, 10,878, 10,880, and 10,919 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: 10,881. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919 and a base editing enzyme comprising or consisting of SEQ ID NO: 10,881. In some embodiments, the fusion protein comprises a linker sequence comprising SEQ ID NO: 10,884. 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: 10,882. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 10,882.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0253] 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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918 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: 10,881. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918and a base editing enzyme comprising or consisting of SEQ ID NO: 10,881. In some embodiments, the fusion protein comprises a linker sequence comprising SEQ ID NO: 10,884. 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: 10,883. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 10,883. Exemplary base editing enzyme and base editing enzyme fusion proteins are provided in TABLE 9.

[0254] 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.

[0255] 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 comprisesCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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).

[0256] 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.

[0257] 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.

[0258] 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 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

[0259] 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.

[0260] 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 (retRNA) or nucleic acid encoding the retRNA, wherein the retRNA comprises (i) a primer binding sequence (PBS), and (ii) a template sequence that hybridizes to the target sequence of a second strand of the dsDNACooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO target nucleic acid. In some embodiments, the RDDP comprises a reverse transcriptase. In some embodiments, the guide RNA is linked to the retRNA. 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 retRNA is circularized. In some embodiments, the retRNA and / or guide RNA comprises a protein localization sequence that can localize a protein to the retRNA. 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 retRNA sequence comprises a difference of at least one nucleotide relative to an equal length portion of the target sequence.

[0261] 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 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.

[0262] 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.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO

[0263] 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

[0264] 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, 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

[0265] 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, fusionCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.

[0266] 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.

[0267] 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: 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).

[0268] 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 expressionCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0269] In some embodiments, the guide nucleic acids disclosed herein can be used in combination with a fusion protein for epigenetic modification of the C9ORF72 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 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: 5424-8272. 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: 8273-10,846.

[0270] 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,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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)).

[0271] 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.

[0272] 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-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, particularlyCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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-[^VSOLFLQJ^LVRIRUPV^LV^UHJXODWHG^E\^PXOWLSOH^F^-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

[0273] 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.

[0274] 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 the recombinase to the Cas-CRISPR domain of the effector protein. In some embodiments, the linker is The-Ser.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 5. Exemplary Systems

[0275] 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 C9ORF72 gene; and (2) an effector protein or fusion protein thereof or a polynucleotide encoding the same.

[0276] 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 2, 3 and 5, 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 of SEQ ID NO: 10,847 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: 1-2849. 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: 10,854 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: 10,855. 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: 5424-8272.

[0277] In some embodiments, the effector protein comprises any one of the sequences recited in TABLEs 2, 3 and 5, and the guide RNA comprises (a) a repeat sequence that comprises a sequence of SEQ ID NO: 10,847 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 1-2849. In some embodiments, the system further comprises an (c) intermediary sequence that comprises SEQ ID NO: 10,854 or (d) a handle sequence that comprises SEQ ID NO: 10,855. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 5424-8272.

[0278] In some embodiments, the effector protein consists of any one of the sequences recited in TABLEs 2, 3 and 5, and the guide RNA comprises (a) a repeat sequence that consists of a sequence of SEQ ID NO: 10,847 and (b) a spacer sequence that consists of a sequence selectedCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO from to any one of SEQ ID NOs: 1-2849. In some embodiments, the system further comprises an (c) intermediary sequence that consists of SEQ ID NO: 10,854 or (d) a handle sequence that consists of SEQ ID NO: 10,855. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 5424-8272.

[0279] 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: 10,856, 10,878, 10,880, and 10,919, 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: 10,847 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: 1-2849. 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: 10,854 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: 10,855. In some embodiments, the guide RNA sequence comprises a 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: 5424-8272.

[0280] In some embodiments, the effector protein comprises any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919, and the guide RNA comprises (a) a repeat sequence that comprises SEQ ID NO: 10,847 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 1-2849. In some embodiments, the system further comprises an (c) intermediary sequence that comprises SEQ ID NO: 10,854 or (d) a handle sequence that comprises SEQ ID NO: 10,855. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 5424-8272.

[0281] In some embodiments, the effector protein consists of any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919, and the guide RNA comprises (a) a repeat sequence that consists of SEQ ID NO: 10,847 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 1-2849. In some embodiments, the system further comprises anCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO (c) intermediary sequence that consists of SEQ ID NO: 10,854 or (d) a handle sequence that consists of SEQ ID NO: 10,855. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 5424-8272.

[0282] 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 2, 4 and 5, 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: 10,848-10853 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: 2850-5423. 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: 8273-10,846.

[0283] In some embodiments, the effector protein comprises any one of the sequences recited in TABLEs 2, 4 and 5, and the guide RNA comprises (a) a repeat sequence that comprises a sequence selected from any one of SEQ ID NOs: 10,848-10853 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 2850-5423. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 8273-10,846.

[0284] In some embodiments, the effector protein consists of any one of the sequences recited in TABLEs 2, 4 and 5, and the guide RNA comprises (a) a repeat sequence that consists of a sequence selected from any one of SEQ ID NOs: 10,848-10853 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 2850-5423. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 8273-10,846.

[0285] 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: 10,857, 10,875- 10,877, 10,879, 10,917 and 10,918, 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 IDCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO NOs: 10,848-10853 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: 2850-5423. In some embodiments, the guide RNA sequence comprises a 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: 8273-10,846.

[0286] In some embodiments, the effector protein comprises any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918, and the guide RNA comprises (a) a repeat sequence that comprises a sequence selected from any one of SEQ ID NOs: 10,848-10853 and (b) a spacer sequence that comprises a sequence selected from to any one of SEQ ID NOs: 2850- 5423. In some embodiments, the guide RNA sequence comprises any one of SEQ ID NOs: 8273- 10,846.

[0287] In some embodiments, the effector protein consists of any one of SEQ ID NOs: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918, and the guide RNA comprises (a) a repeat sequence that consists of a sequence selected from any one of SEQ ID NOs: 10,848-10853 and (b) a spacer sequence that consists of a sequence selected from to any one of SEQ ID NOs: 2850- 5423. In some embodiments, the guide RNA sequence consists of any one of SEQ ID NOs: 8273- 10,846. 6. Target Nucleic Acids

[0288] Disclosed herein are compositions, systems and methods for modifying the C9ORF72 gene. Exemplary reference C9ORF72 nucleotide sequences are provided in TABLE 10. In general, the target nucleic acid is the C9ORF72 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 C9ORF72 gene. In some embodiments, the target sequence is within an exon region of the C9ORF72 gene. In some embodiments, the target sequence is within an intron region of the C9ORF72 gene. In some embodiments, the target sequences spans an exon and intron of the C9ORF72 gene. In some embodiments, the target sequence is located in a regulatory region of the C9ORF72 gene. In some embodiments, the regulatory region is a promoter. Exemplary reference functional C9orf72 proteins are listed in TABLE 11. While exemplary sequences are provided in TABLE 10 and TABLE 11 may be considered wildtype sequences that do not necessarily contain a hexanucleotide (G4C2)nrepeat expansion, the compositions, systems, andCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO methods disclosed herein are capable of modifying similar or otherwise identical sequences that do contain a hexanucleotide (G4C2)nrepeat expansion. 7. Subject and Environmental Samples

[0289] Systems, compositions, and methods described herein may be useful for detecting a mutated C9ORF72 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 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

[0290] Compositions, systems, and methods described herein comprise an expression vector or a use thereof. In some embodiments, the nucleic acid of interest (e.g., a C9ORF72 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.

[0291] 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 proteinsCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0292] 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. 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.

[0293] 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.

[0294] In some embodiments, a vector may comprise or encode one or more regulatory elements. Regulatory elements may refer to transcriptional and translational control sequences,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0295] 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 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.

[0296] 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, 500Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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.

[0297] 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, 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.

[0298] 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.

[0299] 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 eukaryoticCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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. I...

Claims

Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 C9ORF72 gene, wherein the first region is located 5’ of the second region; and b) optionally, an 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 SEQ ID NOS: 1-2849.

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 7.

4. The composition of any one of claims 1-3, wherein the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 10,847, 10,854, 10,855, 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: 5424-8272.

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: 10,856, 10,878, 10,880, and 10,919.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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 SEQ ID NOS: 2850-5423.

8. The composition of claim 1 or 7, wherein the target sequence is adjacent to a PAM of 5’- NTTN-3’, optionally wherein the PAM is selected from TABLE 8.

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: 10,848-10,853 and 10,920.

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: 8273-10,846.

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: 10,857, 10,875-10,877, 10,879, 10,917 and 10,918.

12. The composition of any one of claims 1-11, wherein the effector protein is a dCas protein, optionally wherein the dCas protein is covalently linked to a heterologous polypeptide.

13. The composition of claim 12, 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.

14. An adenoviral associated viral (AAV) vector encoding the composition of any one of claims 1-13.

15. The AAV vector of claim 14, wherein the AAV vector encodes an additional guide nucleic acid, wherein the additional guide nucleic acid comprises the protein binding sequence and an additional spacer sequence that is complementary to an additional target sequence of the C9ORF72 gene.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 16. 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 C9ORF72 gene, wherein the spacer sequence selected from SEQ ID NOs: 1-5423; 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; optionally wherein the expression cassette further comprises a WPRE sequence located between the nucleic acid sequence encoding an effector protein and the poly(A) signal; optionally wherein the first promoter is a U6 promoter; optionally wherein the second promoter is a CK8E promoter or a SPC5 promoter; or optionally wherein the poly(A) signal is a bGH or an hGH poly(A) signal.

17. The expression cassette of claim 16, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10,856 or SEQ ID NO: 10,857.

18. An adeno-associated virus (AAV) vector comprising the expression cassette of claim 16 or 17.

19. A cell comprising or modified by the composition of any one of claims 1-15, the expression cassette of claim 16 or 17, or the AAV vector of claim 18.

20. The cell of claim 19, wherein the cell is a human cell.

21. The cell of claim 19 or 20, wherein the cell is a myeloid or a neuron, optionally wherein the neuron is a motor neuron.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 22. A pharmaceutical composition comprising the composition of any one of claims 1-15, the expression cassette of any one of claims 16-17, or the AAV vector of claim 18, and a pharmaceutical acceptable carrier.

23. A method of modifying a C9ORF72 gene, comprising contacting the C9ORF72 gene with the composition of any one of claims 1-15, the expression cassette of any one of claims 16- 17, or the AAV vector of claim 18, or the pharmaceutical composition of claim 22.

24. The method of claim 23, comprising contacting a cell that comprises the C9ORF72 gene with the composition of any one of claims 1-15, the expression cassette of any one of claims 16- 17, or the AAV vector of claim 18, or the pharmaceutical composition of claim 22.

25. The method of claim 23 or 24, wherein an amount of a C9orf72 protein in the cell is reduced relative to the amount of the C9orf72 protein in the cell prior to contacting the cell with the composition of any one of claims 1-15, the expression cassette of any one of claims 16-17, or the AAV vector of claim 18, or the pharmaceutical composition of claim 22.

26. A method of treating or preventing amyotrophic lateral sclerosis in a human subject in need thereof, the method comprising administering to the human subject the composition of any one of claims 1-15, the expression cassette of any one of claims 16-17, the AAV vector of claim 18, or the pharmaceutical composition of claim 22.

27. The method of claim 26, wherein the subject harbors a mutation in the C9ORF72 gene.

28. The method of claim 27, wherein the mutation is a hexanucleotide (G4C2)nrepeat expansion (HRE) in a non-coding region of the C9ORF72 gene.

29. The method of claim 28, wherein the non-coding region is selected from a promoter and intron 1.

30. The method of claim 27 or 28, wherein the hexanucleotide (G4C2)nrepeat expansion comprises more than 100, more than 150, more than 200, more than 250, or more than 300 repeats of G4C2.

31. A fusion protein comprising an effector protein and a base editing enzyme, whereinCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 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: 10,856 or 10,857; 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: 10,881; optionally, wherein the fusion protein comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10,882 or 10,883.

32. 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 targeting sequence that is complementary to a target sequence of a C9ORF72 gene and comprising a spacer sequence selected from SEQ ID NOs:1-5423; wherein the first region is located 5’ of the second region; and b) the fusion protein of claim 31.

33. 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 containing SNP rs78074330 located in the sequence of SEQ ID NO: 10,896 in intron 1 of a C9ORF72 gene, wherein the first region is located 5’ of the second region; and b) optionally, an effector protein or a nucleic acid encoding an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

34. The composition of claim 33, wherein 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%,Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100% identical to a sequence selected from SEQ ID NOS: 513, 1063, 1336, 10,901 and 10,902.

35. The composition of claim 33 or 34, wherein the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5936, 6486, 6759, 10,910, and 10,911.

36. The composition of any one of claims 33-35, 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: 10,856.

37. The composition of claim 33, wherein 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 sequence selected from SEQ ID NOS: 4221, 4300, 10,899, and 10,900.

38. The composition of claim 33 or 34, wherein the guide nucleic acid 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 sequence selected from SEQ ID NOS: 9644, 9723, 10,908, and 10,909.

39. The composition of any one of claims 33, 37-38, 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: 10,857.

40. A composition comprising: a) a guide nucleic acid or a DNA molecule encoding the guide nucleic acid, wherein the guide nucleic acid comprises:Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO (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 containing SNP rs112048460 located in the sequence of SEQ ID NO: 10,897 in intron 1 of a C9ORF72 gene; wherein the first region is located 5’ of the second region; and b) optionally, an effector protein or a nucleic acid encoding an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

41. The composition of claim 40, wherein 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 sequence selected from SEQ ID NOS: 662, 1117, 1660, 10,903, and 10,904.

42. The composition of claim 40 or 41, wherein the guide nucleic acid 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 sequence selected from SEQ ID NOS: 6085, 6540, 7083, 10,912, and 10,913.

43. The composition of any one of claims 40-42, 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: 10,856.

44. 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 containing SNP rs2282240 located in the sequence of SEQ ID NO: 10,898 in intron 1 of a C9ORF72 gene; wherein the first region is located 5’ of the second region; andCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO b) optionally, an effector protein or a nucleic acid encoding an effector protein that binds the protein binding sequence or a nucleic acid encoding the effector protein.

45. The composition of claim 44, wherein 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 sequence selected from SEQ ID NOS: 111, 859, 1244, 2059, and 10,905-10,907.

46. The composition of claim 44 or 45, wherein the guide nucleic acid 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 sequence selected from SEQ ID NOS: 5534, 6282, 6667, 7482, and 10,914- 10,916.

47. The composition of any one of claims 44-46, 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: 10,856.

48. A composition comprising: a) an upstream guide nucleic acid or a DNA molecule encoding the upstream guide nucleic acid, wherein the upstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence upstream of a HRE in a C9ORF72 gene; b) a downstream guide nucleic acid or a DNA molecule encoding the upstream guide nucleic acid, wherein the downstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence downstream of the HRE in the C9ORF72 gene; and c) an effector protein or a nucleic acid encoding the effector protein.

49. The composition of claim 48, 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: 10,857;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO optionally wherein the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NO: 3980 and 9403; or optionally wherein the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 4571, 4370, 4710, 4533, 4449, 9994, 9793, 10,133, 9956, and 9872.

50. The composition of claim 48 or 49, wherein a) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4571; b) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9994; c) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4370; d) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9793; e) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ IDCooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4710; f) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 10,133; g) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4533; h) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9956; i) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 3980, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 4449; or j) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9403, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 9872.Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO 51. The composition of claim 48, 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: 10,856; optionally wherein the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 35, 415, 479, 1653, 5458, 5838, 5902, and 7076; or optionally wherein the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 463, 711, 1336, 5886, 6134, and 6759.

52. The composition of claim 48 or 51, wherein a) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463; b) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886; c) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711; d) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO e) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 35, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336; f) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5458, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759; g) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463; h) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886; i) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711; j) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO k) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 415, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336; l) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5838, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759; m) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463; n) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NOs: 5886; o) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711; p) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO q) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 479, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336; r) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5902, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759; s) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 463; t) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 5886; u) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 711; v) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO w) the upstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1653, and the downstream guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 1336; or x) the upstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 7076, and the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6759.

53. An AAV vector comprising: a) a first nucleotide sequence that is 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% identical to SEQ ID NO:10,918; b) a second nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 8273-10,846; and c) optionally, a third nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 8273-10,846.

54. The AAV vector of claim 53, wherein the second and third nucleotide sequences are selected from SEQ ID NOs: 8273-10,846.

55. The AAV vector of claim 53, wherein a) the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9403, and wherein the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 9994, 9793, 10,133, 9956, and 9872; b) the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9656, and wherein the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 10,207, 9793 and 9678;Cooley Ref: MABI-046 / 06WO 344183-2321 Mammoth Ref: MB0125WO c) the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9673, and wherein the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 10,207 and 9793; or d) the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9403, and wherein the third nucleotide sequence is at least 90% or 100% identical to SEQ ID NOs: 10,207.

56. An AAV vector comprising: a) a first nucleotide sequence that is 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% identical to SEQ ID NO: 10,880; b) a second nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-8272; and c) optionally, a third nucleotide sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-8272.

57. The AAV vector of claim 56, wherein the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 6627, and wherein the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 5838, 5902, 5458, and 7106.

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