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WO2025024285A1 - Compositions pour la modification du gène c9orf72 humain - Google Patents

Compositions pour la modification du gène c9orf72 humain Download PDF

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Publication number
WO2025024285A1
WO2025024285A1 PCT/US2024/038776 US2024038776W WO2025024285A1 WO 2025024285 A1 WO2025024285 A1 WO 2025024285A1 US 2024038776 W US2024038776 W US 2024038776W WO 2025024285 A1 WO2025024285 A1 WO 2025024285A1
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sequence
nucleic acid
seq
nucleotide sequence
guide nucleic
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Inventor
Renan B. SPER
Julia Karolina NUSSBACHER
Jeffrey Yi-Wen HUANG
Maggie Lindsey BOBBIN
Ning CHAI
Wiputra Jaya HARTONO
Benjamin Julius RAUCH
Aaron DELOUGHERY
Stepan TYMOSHENKO
William Douglass WRIGHT
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Mammoth Biosciences Inc
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Mammoth Biosciences Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Cooley Ref MABI-046/06WO 344183-2321
  • Mammoth Ref MB0125WO COMPOSITIONS FOR THE MODIFICATION OF THE HUMAN C9ORF72 GENE CROSS-REFERENCE TO RELATED APPLICATIONS
  • 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.
  • 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.
  • 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.
  • the HRE is located in intron 1.
  • 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.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas proteins associated proteins
  • 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 [005] 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.
  • compositions, systems, kits and methods may be useful for the treatment of ALS and FTD.
  • 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.
  • compositions, systems, kits and methods do not cleave an C9ORF72 gene, but otherwise modify its expression, e.g., through epigenetic silencing.
  • 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.
  • 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 Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO binding sequence or a nucleic acid encoding the effector protein.
  • 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.
  • 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.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5’-NNTN-3’, optionally wherein the PAM is selected from TABLE 7.
  • the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 10,847, 10,854, 10,855, and combinations thereof.
  • 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.
  • 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.
  • 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.
  • the target sequence is adjacent to a PAM of 5’-NTTN-3’, optionally wherein the PAM is selected from TABLE 8.
  • the protein binding sequence comprises a nucleotide sequence selected from SEQ ID NOs: 10,848-10,853 and 10,920.
  • 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.
  • 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.
  • the effector protein is a dCas protein.
  • the dCas protein comprises a heterologous polypeptide.
  • the dCas protein is Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO covalently linked to the heterologous polypeptide.
  • 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.
  • the effector protein is linked to a nuclear localization signal.
  • 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.
  • 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.
  • the nucleic acid encoding the effector protein is a messenger RNA.
  • the systems, compositions, or kits provided herein comprise a lipid or a lipid nanoparticle.
  • 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.
  • 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.
  • AAV adenoviral associated viral
  • 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.
  • 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.
  • ITR inverted terminal repeat
  • the expression cassette further comprises a WPRE sequence located between the nucleic acid Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO sequence encoding an effector protein and the poly(A) signal.
  • the first promoter is a U6 promoter.
  • the second promoter is a CK8E promoter or a SPC5 promoter.
  • the poly(A) signal is a bGH or an hGH poly(A) signal.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10,857.
  • 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.
  • AAV adeno-associated virus
  • the present disclosure provides cells comprising or modified by the systems, compositions, kits, or nucleic acid expression vectors described herein.
  • the cell is a human cell.
  • the cell is a motor neuron.
  • the present disclosure provides a population of cells described herein.
  • the present disclosure provides pharmaceutical compositions comprising compositions or nucleic acid expression vectors described herein, and a pharmaceutical acceptable carrier.
  • 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.
  • the method comprises contacting a cell that comprises the C9ORF72 gene with the system, composition, kit, or AAV vectors or pharmaceutical compositions described herein.
  • 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.
  • 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.
  • the subject harbors a Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO mutation in the C9ORF72 gene.
  • the mutation is a hexanucleotide (G4C2)n repeat expansion (HRE) in a non-coding region of C9ORF72.
  • the non- coding region is selected from a promoter and intron 1.
  • 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.
  • 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.
  • the effector protein comprises the amino acid substitutions of D220R and E335Q relative to SEQ ID NO: 10,856.
  • 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.
  • 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.
  • 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.
  • 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.
  • effector protein comprises the amino acid substitutions of L26K and E567Q relative to SEQ ID NO: 10,857.
  • the fusion protein comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 10,883.
  • the fusion protein comprises or consists of SEQ ID NO: 10,883.
  • 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.
  • 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.
  • 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 protein Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 acid Cooley 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.
  • the upstream guide nucleic acid comprises a spacer sequence that is complementary to a target sequence upstream of a HRE in a C9ORF72 gene
  • 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.
  • the effector protein comprises the amino acid substitutions of L26R and I471T relative to SEQ ID NO: 10,857.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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 Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO nucleotide sequence of SEQ ID NO: 9403
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • the effector protein comprises the Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO amino acid substitution of D220R relative to SEQ ID NO: 10,856.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • the downstream guide nucleic acid comprises a spacer sequence that is at least Cooley 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • the second and third nucleotide sequences are selected from SEQ ID NOS: 8273-10,846.
  • the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 9403
  • the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO NOs: 9994, 9793, 10,133, 9956, and 9872.
  • 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.
  • 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.
  • the second nucleotide sequence is at least 90% or 100% identical to SEQ ID NO: 6627
  • the third nucleotide sequence is at least 90% or 100% identical to a sequence selected from: SEQ ID NOs: 5838, 5902, 5458, and 7106.
  • FIG.2 shows an exemplary AAV vector comprising a plurality of expression cassettes.
  • 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
  • FIG. 4 shows % indel results in the C9ORF72 gene produced by CasM.265466 D220R and C9ORF72 guide nucleic acids.
  • FIG.5 depicts in vivo gene editing of PCSK9 in muscle tissues using AAV9-A4 delivery of CasPhi.12 and CasM.265466 variants.
  • % identical refers to the extent to which two sequences (nucleotide or amino acid) have the same Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO residue at the same positions in an alignment.
  • 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.
  • 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.
  • base editing enzyme 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.
  • 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.
  • base editor 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.
  • the effector protein may comprise a catalytically inactive effector protein.
  • the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.
  • dCas protein refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring Cooley 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.
  • the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.
  • cleavage 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.
  • nucleic acid molecule or nucleotide sequence refers 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.
  • 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.
  • 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.
  • cleave refers to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond.
  • the result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded Cooley 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.
  • MB0125WO e.g., ssDNA or ssRNA
  • double-stranded e.g., dsDNA
  • CRISPR clustered regularly interspaced short palindromic repeats
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene regulatory element is a splicing- regulatory element.
  • the original function of the gene regulatory element is repressing exonic splicing.
  • donor nucleic acid refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.
  • dual nucleic acid system refers to a system that uses a transactivated or transactivating tracrRNA-crRNA duplex complexed with one or more Cooley 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.
  • effector protein 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.
  • the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid.
  • 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.
  • engineered modification 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).
  • 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.
  • 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.
  • 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 a Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO relationship permitting them to function in their intended manner.
  • 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.
  • fusion protein refers to a protein comprising at least two heterologous polypeptides. The fusion protein may comprise one or more effector proteins and fusion partners.
  • an effector protein and fusion partner are not found connected to one another as a native protein or complex that occurs together in nature.
  • 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.
  • 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.
  • a fusion partner directly interacts with (e.g., binds to/by) an effector protein.
  • 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.
  • 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.
  • guide nucleic acid 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.
  • 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.
  • guide nucleic acid may be used interchangeably herein Cooley 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.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • a combination thereof e.g., RNA with a thymine base
  • biochemically or chemically modified nucleobases e.g., one or more engineered modifications described here
  • 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.
  • the heterologous polypeptide or nucleotide sequence is not found covalently bound to the reference nucleic acid or protein.
  • the heterologous polypeptide or nucleotide sequence is not found non-covalently interacting with the reference nucleic acid or protein.
  • the heterologous polypeptide or nucleotide sequence is not expressed by the same species as that which expresses the reference nucleic acid or protein.
  • 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.
  • a heterologous protein is not encoded by a species that encodes the effector protein.
  • the heterologous protein exhibits an activity (e.g., enzymatic activity) when it is linked to the effector protein.
  • 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.
  • 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.
  • fused and “linked” are used interchangeably herein.
  • linker 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).
  • modified target nucleic acid refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein.
  • the modification is an alteration in the sequence of the target nucleic acid.
  • the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target nucleic acid.
  • 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.
  • 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.
  • a composition may include an effector protein and a guide nucleic acid that do not naturally occur together.
  • 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.
  • nucleic acid expression vector refers to a nucleic acid that can be used to express a nucleic acid of interest.
  • nuclear localization signal 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.
  • nuclease activity 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.).
  • composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • 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.
  • 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).
  • PAM protospacer adjacent motif
  • 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.
  • the complex does not require a PAM to edit the target nucleic acid.
  • 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.
  • regulatory element 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.
  • a non-coding sequence e.g., a guide nucleic acid
  • a coding sequence e.g., effector proteins, fusion proteins, and the like
  • peat sequence refers to a sequence of nucleotides in a guide nucleic acid that is capable of, at least partially, interacting with an effector protein.
  • ribonucleotide protein complex and “RNP,” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes “ribonucleotides” it is understood that the one or more nucleic acid may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a Cooley 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.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • a combination thereof e.g., RNA with a Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO th
  • RuvC and RuvC domain 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.
  • 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.
  • the sample is a biological sample, such as a biological fluid or tissue sample.
  • the sample is an environmental sample.
  • the sample may be a biological sample or environmental sample that is modified or manipulated.
  • samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.
  • single guide nucleic acid 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).
  • 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).
  • a sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence.
  • single nucleic acid system refers to a system that uses a guide nucleic acid complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence specific manner, and wherein the guide nucleic acid is capable of non-covalently interacting with the one or more polypeptides Cooley 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.
  • spacer sequence 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.
  • subject 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.
  • target nucleic acid 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).
  • 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.
  • trans cleavage in the context of cleavage (e.g., hydrolysis of a phosphodiester bond) of one or more target nucleic acids or non-target nucleic acids, or both, by an effector protein that is complexed with a guide nucleic acid and the target nucleic acid.
  • Trans cleavage activity may be triggered by the hybridization of a guide nucleic acid to a target nucleic acid.
  • the effector may cleave a target strand as well as non-target strand, wherein the target nucleic is a double stranded nucleic acid.
  • Trans cleavage of the target nucleic acid may occur away from Cooley 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.
  • trans-activating RNA refers 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).
  • transactivating 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.
  • transcriptional activator refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule.
  • transcriptional repressor refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid.
  • transgene 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.
  • 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.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the Cooley 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.
  • viral vector 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.
  • viruses or viral particles that can deliver a viral vector include retroviruses (e.g. ⁇ OHQWLYLUXVHV ⁇ DQG ⁇ ⁇ -retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • retroviruses e.g. ⁇ OHQWLYLUXVHV ⁇ DQG ⁇ ⁇ -retroviruses
  • adenoviruses e.g. ⁇ OHQWLYLUXVHV ⁇ DQG ⁇ ⁇ -retroviruses
  • AAVs adeno-associated viruses
  • baculoviruses 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.
  • 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.
  • the first region is located 5’ of the second region.
  • the protein binding sequence comprises a repeat sequence.
  • 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.
  • 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.
  • the compositions and systems comprise guide nucleic acids that are capable of hybridizing to a target sequence of the C9ORF72 gene.
  • compositions and systems comprise an effector protein that is capable of binding a portion of the guide nucleic acid.
  • the effector protein is a CRISPR-associated (Cas) protein.
  • 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).
  • 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).
  • a disease e.g., amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD)
  • target genes e.g., the C9ORF72 gene.
  • ALS is familial ALS (fALS).
  • Compositions and systems disclosed herein are not naturally occurring.
  • 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.
  • systems and compositions herein comprise at least one non-naturally occurring component.
  • 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.
  • compositions and systems comprise at least two components that do not naturally occur together.
  • compositions and systems may comprise a guide nucleic acid comprising a repeat sequence and a spacer sequence which do not naturally occur together.
  • a guide nucleic acid may comprise a sequence of a naturally-occurring repeat region and a spacer region that is complementary to a naturally-occurring eukaryotic sequence.
  • the guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region Cooley 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.
  • composition and systems may comprise a guide nucleic acid and an effector protein that do not naturally occur together.
  • 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.
  • a guide nucleic acid may comprise a repeat sequence, an intermediary sequence, and a spacer sequence coupled by one or more linker sequences.
  • the guide nucleic acid comprises two heterologous sequences arranged in an order or proximity that is not observed in nature.
  • compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof.
  • 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.
  • compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid.
  • guide nucleic acids comprise two heterologous sequences arranged in an order or proximity that is not observed in nature.
  • guide nucleic acids also referred to as guide RNA (gRNA)
  • gRNA guide RNA
  • 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.
  • 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.
  • 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.
  • 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.
  • the first nucleotide sequence is located 5’ to second nucleotide sequence.
  • the second nucleotide sequence is located 5’ to the first nucleotide sequence.
  • 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.
  • the first nucleotide sequence comprises a repeat sequence.
  • the first nucleotide sequence comprises an intermediary sequence and a repeat sequence.
  • the first nucleotide sequence comprises a handle sequence.
  • an effector protein binds to at least a portion of the first nucleotide sequence.
  • 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).
  • a target nucleic acid e.g., the C9ORF72 gene.
  • a gRNA may comprise one or more deoxyribonucleotides and/or a deoxyribonucleotide nucleobase (e.g., thymine).
  • the majority of the nucleotides in a guide nucleic acid are ribonucleotides.
  • 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.
  • nucleic acids described herein e.g., engineered guide nucleic acids
  • modifications of a nucleic acid include a nucleobase base modification, a backbone modification, a sugar modification, a phosphorothioate internucleotide linkage, or combinations thereof.
  • the modifications can be of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.
  • 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.
  • 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.
  • a guide nucleic acid-effector protein complex may be described herein as an RNP.
  • 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).
  • a target nucleic acid e.g., a target sequence in the C9ORF72 gene.
  • at least a portion of the guide nucleic acid hybridizes to a target sequence in a target nucleic acid (e.g., the C9ORF72 gene).
  • 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.
  • a guide nucleic acid may comprise or form intramolecular secondary structure (e.g., hairpins, stem-loops, etc.).
  • a guide nucleic acid comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • the guide nucleic acid comprises a pseudoknot (e.g., a secondary structure comprising a stem, at least partially, hybridized to a second stem or half-stem secondary structure).
  • An effector protein may recognize a guide nucleic acid comprising Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the guide nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • the wild-type reading frame may be restored, e.g., by a polymerase, resulting in at least a partially functional protein.
  • 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.
  • 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.
  • 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.
  • a guide nucleic acid comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO nucleotides.
  • the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides.
  • 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.
  • a guide nucleic acid comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides.
  • a guide nucleic acid comprises at least 25 linked nucleotides.
  • a guide nucleic acid may comprise 10 to 50 linked nucleotides.
  • 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.
  • the guide nucleic acid comprises about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides.
  • a length of a guide nucleic acid is about 30 to about 120 linked nucleotides.
  • 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.
  • 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.
  • guide nucleic acids comprise elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. Such elements may be one or more nucleotide alterations, nucleotide sequences, intermolecular Cooley 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.).
  • 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.
  • Guide nucleic acids may comprise deoxyribonucleotides, ribonucleotides or a combination thereof.
  • 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).
  • gRNA guide RNA
  • 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.
  • 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.
  • a guide nucleic acid e.g., a guide RNA
  • 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.
  • a guide nucleic acid may bind to a portion of a gene associated with a genetic disorder, or an amplicon thereof, as described herein.
  • 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.
  • Guide nucleic acids described herein generally comprise a spacer region represented by a spacer sequence.
  • spacer region The terms, “spacer region,” “targeting sequence” and “spacer sequence” may be used interchangeably.
  • a spacer region is capable of Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO hybridizing to a target sequence of a target nucleic acid.
  • 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.
  • 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.
  • 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.
  • the spacer region comprises 5-50 linked nucleotides in length.
  • the spacer region is 15-28 linked nucleotides in length.
  • 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.
  • 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.
  • 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 [00115] 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.
  • a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction.
  • the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule.
  • the spacer(s) and repeat sequence(s) are linked directly to one another.
  • a linker is present between the spacer(s) and repeat sequences. Linkers may be any suitable linker.
  • 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.
  • 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.
  • 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.
  • 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. [00117] 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.
  • 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. [00118] 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.
  • 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.
  • 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. [00119] 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.
  • N represents any nucleotide
  • V represents A, C, or G
  • Y represents C or T
  • R represents A or G.
  • 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.
  • the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT.
  • a spacer sequence is complementary to a target sequence that contains one or more SNPs in the C9ORF72 gene.
  • 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.
  • 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.
  • 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.
  • 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 located Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Guide nucleic acids disclosed herein may target various regions of the C9ORF72 gene.
  • the spacer sequences hybridize to a target sequence in intron 1 of the C9ORF72 gene.
  • the spacer sequences that are complementary to intron 1 of the C9ORF72 gene include SEQ ID NOs: 1-2248 and 2850-4857.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of VNTR.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of TNTR.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of TNTY.
  • the spacer sequences that are complementary to the promoter of the C9ORF72 Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO gene and are adjacent to a PAM of TNTY include SEQ ID NOs: 2670-2849.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of NTTN.
  • 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.
  • 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.
  • the U is pseudouracil.
  • a guanine nucleobase could be replaced with the nucleobase of any one of a cytosine, adenosine, thymine, and uracil.
  • the spacer sequence comprises only one nucleobase alterations relative to a sequence selected from SEQ ID NOS: 1-5423.
  • 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.
  • 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.
  • the PAM is TNTR.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT.
  • 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 Cooley 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.
  • the effector protein comprises an amino acid sequence that is at least Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO 75%, at least 80%, at least
  • 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.
  • the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM selected from TNTR, VNTR, and TNTY.
  • 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.
  • the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of VNTR.
  • 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.
  • the composition or system comprises a gene silencing protein, such as a transcriptional repressor.
  • a non-limiting example of a Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO transcriptional repressor is a DNA methyltransferase.
  • the effector protein is fused to the gene silencing protein.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of TNTR.
  • 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.
  • the composition or system comprises a gene silencing protein, such as a transcriptional repressor.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of TNTY.
  • 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.
  • the composition or system comprises a gene silencing protein, such as a transcriptional repressor.
  • a non-limiting example of a transcriptional Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO repressor is a DNA methyltransferase.
  • the effector protein is fused to the gene silencing protein.
  • 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.
  • the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of NTTN.
  • 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.
  • the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.
  • 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.
  • the spacer sequence hybridizes to a target sequence in intron 1 of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of NTTN.
  • 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.
  • the guide nucleic acid is useful for deleting or excising one or more HRE repeats from intron 1.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of NTTN.
  • 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.
  • the composition or system comprises a gene silencing protein, such as a transcriptional repressor.
  • 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.
  • the spacer sequence hybridizes to a target sequence in the promoter of a C9ORF72 gene.
  • the target sequence is adjacent to a PAM of NTTN.
  • 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.
  • the composition or system comprises a gene silencing protein, such as a transcriptional repressor.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the effector protein comprises the amino acid substitutions of L26R and I471T relative to SEQ ID NO: 10,857.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • the effector protein comprises the amino acid substitution of D220R relative to SEQ ID NO: 10,856.
  • 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.
  • 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.
  • 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.
  • 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 Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO selected from SEQ ID NOs: 5886, 6134, and 6759.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • the upstream guide nucleic acid comprises a nucleotide Cooley 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • the Cooley 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • the downstream guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO least 95% or 100% identical to a nucleotide sequence of SEQ ID NO: 6134.
  • 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
  • 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.
  • 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
  • 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 [00142]
  • 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.
  • 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.
  • 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.
  • the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides.
  • a linker comprises a nucleotide sequence of 5’-GAAA-3’ (SEQ ID NO: 10,921).
  • 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 [00145] 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.
  • effector proteins are capable of non-covalently binding a repeat sequence.
  • a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid.
  • a repeat sequence comprises a nucleotide sequence that may interact with an effector protein.
  • a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex).
  • the repeat sequence may also be referred to as a “protein- binding segment.” [00146] 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.
  • a repeat sequence is linked to a spacer sequence and/or an intermediary sequence.
  • 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.
  • the linker comprises SEQ ID NO: 10,921.
  • the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex).
  • the two sequences are not directly linked and hybridize to form a stem loop structure.
  • the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp).
  • the duplex forming sequence may include a bulge.
  • the repeat sequence comprises a hairpin or stem-loop structure, optionally at the 5’ portion of the repeat sequence.
  • 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.
  • Mammoth Ref: MB0125WO such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity).
  • 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.).
  • TABLE 1 provides illustrative repeat sequences for use with the compositions and methods of the disclosure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Mammoth Ref MB0125WO Intermediary sequences
  • Guide nucleic acids described herein may comprise an intermediary sequence.
  • the protein binding sequence comprises an intermediary sequence.
  • 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.
  • the intermediary sequence non-covalently binds to an effector protein.
  • the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.
  • intermediary RNA 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.
  • a length of the intermediary sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides.
  • 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.
  • 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.
  • the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO sequence and the second sequence.
  • an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • 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.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • an intermediary sequence comprises 1, 2, 3, 4, 5 or more stem regions.
  • 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).
  • 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 [00157]
  • compositions, systems and methods described herein comprise a guide nucleic acid, wherein the guide nucleic acid comprises a handle sequence.
  • the handle sequence comprises a protein binding sequence.
  • the handle sequence is the protein binding sequence.
  • the handle sequence comprises an intermediary sequence.
  • the handle sequence comprises a repeat sequence.
  • the intermediary sequence is at the 5’-end of the handle sequence.
  • the repeat sequence is at the 3’- end of the handle Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO sequence.
  • the handle sequence comprises a linker sequence.
  • 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).
  • the intermediary sequence is 5’ to the repeat sequence.
  • the intermediary sequence is 5’ to the linker.
  • the intermediary sequence is 3’ to the repeat sequence.
  • the intermediary sequence is 3’ to the linker.
  • compositions and systems comprise a sgRNA.
  • 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.
  • an effector protein may recognize a secondary structure of a handle sequence.
  • at least a portion of the handle sequence interacts with an effector protein described herein.
  • at least a portion of the intermediary sequence interacts with the effector protein described herein.
  • both, at least a portion of the intermediary sequence and at least a portion of the repeat sequence interacts with the effector protein.
  • the handle sequence is capable of interacting (e.g., non-covalent binding) with an effector protein described herein.
  • handle sequence 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.
  • sgRNA single guide RNA
  • the handle sequence comprises an intermediary RNA sequence, which is capable of being non- covalently bound by an effector protein.
  • the handle sequence further comprises a repeat sequence.
  • the intermediary RNA sequence or a Cooley 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.
  • the handle sequence of a sgRNA comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • 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.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the sgRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • a handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • 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.
  • 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.
  • 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 linked Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO nucleotides, or 95 to 105 linked nucleotides.
  • 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.
  • 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).
  • 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. [00165] In some embodiments, the sequences selected from SEQ ID NOS: 1-10,853, 10,854, and 10,855 and 10,920 can be modified.
  • nucleic acids e.g., nucleic acids encoding effector proteins, engineered guide nucleic acids, or nucleic acids encoding engineered guide nucleic acids
  • 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
  • the modification includes at least one phosphorothioate (PS) linkage.
  • the modification includes at least one 2’- O-Methyl oligonucleotide (OMe).
  • the modification includes at least one locked nucleic acid (LNA).
  • the modification includes at least one Phosphorodiamidate morpholino oligonucleotide (PMO).
  • the modification includes at least one or more peptide nucleic acid (PNA).
  • the first 3 and last 3 amino acids are O-Me modified, and the first 3 and last 2 linkages are phosphorothioate linkages.
  • 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.
  • a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein.
  • a second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid.
  • 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 [00167]
  • a single guide nucleic acid comprises a crRNA.
  • the guide nucleic acid is the crRNA.
  • 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.
  • the FR consists of a repeat sequence.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • 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.
  • 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.
  • a guide nucleic acid comprises a single guide RNA (sgRNA).
  • the guide nucleic acid is an sgRNA.
  • 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).
  • an sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence.
  • a spacer sequence e.g., a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid
  • 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.
  • the spacer sequence and handle sequence are linked by a phosphodiester bond.
  • the spacer sequence and handle sequence are linked by one or more linked nucleotides.
  • a guide nucleic acid may comprise a spacer sequence, a repeat sequence, or handle sequence, or a combination thereof.
  • the handle sequence may comprise a portion of, or all of, a repeat sequence.
  • 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.
  • compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g., a single nucleic acid system), wherein the guide RNA is a Cooley 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).
  • 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.
  • 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.
  • a sgRNA comprises an intermediary sequence and an crRNA.
  • an intermediary sequence is 5’ to a crRNA in an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and crRNA.
  • an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises a handle sequence and a spacer sequence.
  • a handle sequence is 5’ to a spacer sequence in an sgRNA.
  • a sgRNA comprises a linked handle sequence and spacer sequence.
  • a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • a handle sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence.
  • an intermediary sequence is 5’ to a repeat sequence in an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and repeat sequence.
  • an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a repeat sequence are linked in an sgRNA by Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO any suitable linker, examples of which are provided herein.
  • a repeat sequence is 5’ to a spacer sequence in an sgRNA.
  • a sgRNA comprises a linked repeat sequence and spacer sequence.
  • a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • An exemplary handle sequence in a sgRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region.
  • the 5’ region may hybridize to the 3’ region.
  • the 5’ region does not hybridize to the 3’ region.
  • the 3’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).
  • the 5’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).
  • 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.
  • 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.
  • compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector protein or a nucleotide sequence encoding the one or more effector protein, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA- crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO the target nucleic acid.
  • a repeat hybridization sequence is at the 3’ end of a tracrRNA sequence.
  • 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.
  • the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.
  • 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.
  • the secondary structure modifies activity of the effector protein on a target nucleic acid.
  • the secondary structure comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • 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.
  • nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the secondary structure comprises at least two, at least three, at least four, or at least five stem regions.
  • the secondary structure comprises one or more loops.
  • the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops.
  • compositions provided herein comprise one or more effector proteins.
  • compositions and systems described herein comprise an effector protein that is similar to a naturally occurring effector protein.
  • the effector protein may lack a portion of the naturally occurring effector protein.
  • the effector protein may comprise an Cooley 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.
  • An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid.
  • an effector protein 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.
  • PAM protospacer adjacent motif
  • the effector protein is a programmable nuclease (e.g., a CRISPR-associated (Cas) protein) that modifies a target sequence in a target nucleic acid.
  • 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.
  • 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.
  • 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).
  • the effector protein is a Type V Cas protein.
  • the effector protein is CasPhi.12 or a variant thereof.
  • 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.
  • Mammoth Ref MB0125WO [00187]
  • TABLEs 2-5 provide illustrative amino acid sequences of effector proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • an amino acid alteration comprises a deletion of an amino acid.
  • an amino acid alteration comprises an insertion of an amino acid.
  • an amino acid alteration comprises a conservative amino acid substitution.
  • an amino acid alteration comprises a non-conservative amino acid substitution.
  • 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.
  • 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 (N), G
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the effector protein Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, Y
  • 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, D220
  • 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.
  • 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.
  • 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.
  • 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.
  • the amino acid substitutions are D220R and 335Q.
  • the engineered effector protein comprises or consists of SEQ ID NO: 10,878.
  • 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
  • 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
  • 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, K28
  • 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.
  • 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.
  • 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/
  • 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/L
  • these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.
  • 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.
  • the polypeptide comprises or consists of SEQ ID NO: 10,877.
  • 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.
  • 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.
  • these engineered effector proteins comprise a nickase activity.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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. [00204] Exemplary engineered effector proteins are provided in TABLE 5.
  • 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.
  • 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.
  • 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.
  • 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.
  • the proteins 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.
  • a multilevel consensus sequence or PROSITE motif sequence
  • 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 effector proteins comprise a RuvC domain.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 [00209] 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 (Sl
  • the effector protein is linked to a nuclear localization signal (NLS).
  • 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.
  • effector proteins are linked to an endosomal escape polypeptide (EEP).
  • EEP endosomal escape polypeptide
  • the nucleotide sequence encoding the effector protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.
  • X is any naturally occurring amino acid
  • ⁇ D/E is any naturally occurring amino acid except Asp or Glu.
  • Protospacer Adjacent Motif (PAM) Sequences 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.
  • 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.
  • 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 [00213]
  • N any nucleic acid
  • R a purine nucleic acid (i.e., A or G).
  • Non-limiting examples of 5’-TNTR-3’ PAM sequences are disclosed in TABLE 7.
  • 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.
  • the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 7.
  • 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, TTTA, TTTC, TTTG, and TTTT.
  • 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.
  • the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 8.
  • the PAM is selected from: ATTA, ATTC, ATTG, ATTT, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, GTTT, TTTA, TTTC, TTTG, and TTTT.
  • 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.
  • 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.
  • d catalytically inactive effector protein and/or “dead”
  • an effector protein normally has nuclease activity, in some embodiments, an effector protein does not have nuclease activity.
  • 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.
  • 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.
  • 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.
  • the catalytically inactive effector protein may also be referred to as a catalytically reduced effector protein.
  • a nuclease domain e.g., RuvC domain
  • 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.
  • 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
  • a dead effector polypeptide may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid.
  • 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.
  • 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 effector Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO protein further comprises one or more alterations selected from D237A, D418A, D418N, E335A, and E335Q.
  • 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.
  • 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 [00219]
  • 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.
  • the fusion partner comprises a polypeptide or peptide that is linked to the effector protein.
  • the fusion partner is not linked to the effector protein, but is brought into proximity of the effector protein by other means.
  • 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.
  • the fusion partner is capable of binding or being bound by an effector protein.
  • 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.
  • the fusion protein is a heterologous peptide or polypeptide as described herein.
  • the amino terminus of the fusion partner is linked to the carboxy terminus of the effector protein.
  • the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker.
  • the fusion partner is not an effector protein as described herein.
  • the fusion partner comprises a second effector protein or a multimeric form thereof.
  • the fusion protein Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO comprises more than one effector protein.
  • the fusion protein can comprise at least two effector proteins that are same.
  • the fusion protein comprises at least two effector proteins that are different.
  • the multimeric form is a homomeric form.
  • 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.
  • a fusion partner imparts some function or activity to a fusion protein that is not provided by an effector protein.
  • 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
  • a fusion partner may provide signaling activity.
  • a fusion partner may inhibit or promote the formation of multimeric complex of an effector protein.
  • 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.
  • 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.
  • a fusion partner may modulate transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid.
  • a fusion partner may directly or indirectly inhibit, reduce, activate or increase expression of a target nucleic acid.
  • 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 Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 of Cooley 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fusion partners have enzymatic activity that modifies a nucleic acid, such as a target nucleic acid.
  • the target nucleic acid may comprise or consist of a ssRNA, dsRNA, ssDNA, or a dsDNA.
  • 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 (DN)
  • fusion partners target a ssRNA, dsRNA, ssDNA, or a dsDNA.
  • fusion partners target ssRNA.
  • splicing factors e.g., RS domains
  • protein translation components e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G
  • RNA methylases e.g., 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.
  • splicing factors e.g., RS domains
  • protein translation components e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G
  • RNA methylases e.g., RNA editing enzymes (
  • a fusion partner may include an entire protein, or in some embodiments, may include a fragment of the protein (e.g., a functional domain).
  • 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.
  • 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.
  • 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
  • an effector protein is a fusion protein, wherein the effector protein is linked to a chromatin-modifying enzyme.
  • 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 [00239]
  • 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.
  • 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.
  • a base editor may be a fusion protein comprising a base editing enzyme linked to an effector protein.
  • the amino Cooley 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.
  • 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.
  • the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein).
  • the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.
  • 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).
  • 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).
  • base editors edit a nucleobase on a ssDNA.
  • base editors edit a nucleobase on both strands of dsDNA. In some embodiments, base editors edit a nucleobase of an RNA.
  • a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase.
  • 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”.
  • DNA bases within the R-loop are edited by the base editor having the deaminase enzyme activity.
  • 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.
  • a base editing enzyme comprises a deaminase enzyme. Exemplary deaminases are described in US20210198330, WO2021041945, WO2021050571A1, and WO2020123887, all of which are incorporated herein by reference in their entirety.
  • Exemplary deaminase domains are described WO2018027078 and WO2017070632, and each are hereby Cooley 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.
  • the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein.
  • base editors comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)).
  • the fusion partner is a deaminase, e.g., ADAR1/2, ADAR-2, AID, or any function variant thereof.
  • a base editor is a cytosine base editor (CBE).
  • the CBE may convert a cytosine to a thymine.
  • 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.
  • 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.
  • the catalytically inactive effector protein generated ssDNA R-loop may enable the CBE to perform efficient and localized cytosine deamination in vitro.
  • deamination activity is exhibited in a window of about 4 to about 10 base pairs.
  • 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.
  • the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo.
  • 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. (2016) 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.
  • CBEs comprise a uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG).
  • UMI uracil glycosylase inhibitor
  • UNG uracil N-glycosylase
  • 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.
  • the UNG may be inhibited by fusion of a UGI.
  • the CBE comprises a UGI.
  • a C-terminus of the CBE comprises the UGI.
  • the UGI is a small protein from bacteriophage PBS.
  • the UGI is a DNA mimic that potently inhibits both human and bacterial UNG.
  • the UGI inhibitor is any protein or polypeptide that inhibits UNG.
  • 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.
  • the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.
  • a CBE nicks a non-edited DNA strand.
  • 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.
  • a APOBEC1– nickase–UGI fusion efficiently edits in mammalian cells, while minimizing frequency of non-target indels.
  • base editors do not comprise a functional fragment of the base editing enzyme.
  • 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.
  • the fusion protein further comprises a non-protein uracil- DNA glycosylase inhibitor (npUGI).
  • the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG.
  • 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.
  • a cytosine base editing enzyme and therefore a cytosine base editor, is a cytidine deaminase.
  • 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.
  • a base editor is a cytosine to guanine base editor (CGBE).
  • a CGBE may convert a cytosine to a guanine.
  • a base editor is an adenine base editor (ABE).
  • An ABE may convert an adenine to a guanine.
  • an ABE converts an A•T base pair to a G•C base pair.
  • the ABE converts a target A•T base pair to G•C in vivo or in vitro.
  • ABEs provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations.
  • ABEs provided herein enable correction of pathogenic SNPs ( ⁇ 47% of disease-associated point mutations).
  • the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences).
  • deamination of adenosine yields inosine.
  • 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, ABE8.13
  • 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.
  • the adenine deaminase is an adenine deaminase described by Koblan et al. (2016) Nature Biotechnology 36:848-846, incorporated herein by reference.
  • the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871.
  • 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. [00251] 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.
  • the base editing enzyme is ABE8e.
  • 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.
  • 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.
  • the fusion protein comprises a linker sequence comprising SEQ ID NO: 10,884.
  • 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.
  • the fusion protein comprises or consists of SEQ ID NO: 10,882.
  • 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.
  • 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.
  • the fusion protein comprises a linker sequence comprising SEQ ID NO: 10,884.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a base editor comprises a deaminase dimer.
  • the base editor further comprising a base editing enzyme and an adenine deaminase (e.g., TadA).
  • the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA*9).
  • 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
  • the base editor comprises Cooley 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).
  • a base editing enzyme is a deaminase dimer comprising an ABE.
  • the deaminase dimer comprises an adenosine deaminase.
  • 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.
  • the adenine base editing enzyme is linked to amino-terminus or the carboxy-terminus of TadA.
  • RNA base editors comprise an adenosine deaminase.
  • ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine.
  • RNA base editors comprise an effector protein that is activated by or binds RNA.
  • base editors are used to treat a subject having or a subject suspected of having a disease related to a gene of interest.
  • base editors are useful for treating a disease or a disorder caused by a point mutation in a gene of interest.
  • 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.
  • the fusion partner comprises a polymerase.
  • the fusion partner is an RNA-directed DNA polymerase (RDDP).
  • the RDDP is a reverse transcriptase.
  • 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
  • the RDDP comprises a reverse transcriptase.
  • the guide RNA is linked to the retRNA.
  • 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.
  • the retRNA is circularized.
  • the retRNA and/or guide RNA comprises a protein localization sequence that can localize a protein to the retRNA.
  • the protein localization sequence comprises an MS2 coat protein localization sequence and the RDDP is fused to an MS2 coat protein.
  • the RDDP is fused to the effector protein.
  • the retRNA sequence comprises a difference of at least one nucleotide relative to an equal length portion of the target sequence.
  • the RDDP that is capable of catalyzing the modification of the target nucleic acid forms a complex with an extended guide RNA.
  • 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.
  • a first region also referred to as a protein binding region or protein binding sequence
  • 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
  • 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.
  • the linker comprises a nucleotide.
  • the linker comprises multiple nucleotides.
  • the third and fourth regions are 5’ of the first and second regions.
  • the order of the regions of the extended guide RNA from 5’ to 3’ is: third region, fourth region, first region, and second region.
  • the effector protein is linked to an RDDP.
  • the RDDP comprises a reverse transcriptase. Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO [00263]
  • the third and fourth regions are 3’ of the first and second regions.
  • the order of the regions of the extended guide RNA from 5’ to 3’ is: first region, second region, third region, and fourth region.
  • 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.
  • 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), Vietnamese 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 De
  • HMT histone methyltransferase
  • 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.).
  • 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.
  • fusion Cooley 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.
  • 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.
  • 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.
  • fusion partners comprise a transcriptional activator.
  • 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.
  • the fusion partner is a reverse transcriptase.
  • 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,
  • 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).
  • fusions partners inhibit or reduce expression of a target nucleic acid.
  • fusion proteins comprising described fusion partners and an effector protein may be referred to as CRISPRi fusions.
  • fusion partners reduce expression Cooley Ref: MABI-046/06WO 344183-2321
  • Relative expression including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR.
  • fusion partners may comprise a transcriptional repressor.
  • 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.
  • the guide nucleic acids disclosed herein can be used in combination with a fusion protein for epigenetic modification of the C9ORF72 gene.
  • the fusion protein comprises an effector protein and a methyltransferase.
  • the fusion protein further comprises a KRAB domain.
  • 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.
  • the effector protein is CasM.265466 or a variant thereof
  • 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.
  • the effector protein is CasPhi.12 or a variant thereof
  • 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.
  • 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 Mamm
  • 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)).
  • 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)
  • 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).
  • the modifications are transient (e.g., transcription repression or activation).
  • the modifications are inheritable.
  • 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.
  • 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.
  • RRMs N-terminal RNA recognition motifs
  • ESEs exonic splicing enhancers
  • 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.
  • the RNA splicing factors may regulate alternative use of splice site (ss) by binding to regulatory sequences between two alternative sites.
  • ASF/SF2 may recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al may bind to ESSs and shift splicing towards the use of intron distal sites.
  • One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO disease associated genes.
  • 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).
  • fusion partners comprise a recombinase.
  • effector proteins described herein are linked with the recombinase.
  • the effector proteins have reduced nuclease activity or no nuclease activity.
  • the recombinase is a site-specific recombinase.
  • 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.
  • 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.
  • 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.
  • the site-specific recombinase is an integrase.
  • integrases include:Bxb1, wBeta, BL3, phiR4, A118, TG1, MR11, phi370, SPBc, TP901-1, phiRV, FC1, K38, phiBT1, and phiC31.
  • the fusion protein comprises a linker that links the recombinase to the Cas-CRISPR domain of the effector protein.
  • the linker is The-Ser. Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO 5.
  • 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.
  • 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.
  • 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.
  • an 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: 10,855.
  • 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.
  • 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.
  • 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.
  • the guide RNA sequence comprises any one of SEQ ID NOs: 5424-8272.
  • 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 selected Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO from to any one of SEQ ID NOs: 1-2849.
  • 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.
  • the guide RNA sequence consists of any one of SEQ ID NOs: 5424-8272.
  • 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
  • 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%
  • 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.
  • an 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: 10,855.
  • 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.
  • the effector protein comprises any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919
  • 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.
  • 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.
  • the guide RNA sequence comprises any one of SEQ ID NOs: 5424-8272.
  • the effector protein consists of any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919
  • 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.
  • the system further comprises an Cooley 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.
  • the guide RNA sequence consists of any one of SEQ ID NOs: 5424-8272.
  • 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: 28
  • 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.
  • 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.
  • the guide RNA sequence comprises any one of SEQ ID NOs: 8273-10,846.
  • 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.
  • the guide RNA sequence consists of any one of SEQ ID NOs: 8273-10,846.
  • 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 ID Cooley 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
  • 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.
  • 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.
  • the guide RNA sequence comprises any one of SEQ ID NOs: 8273- 10,846.
  • 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.
  • the guide RNA sequence consists of any one of SEQ ID NOs: 8273- 10,846. 6. Target Nucleic Acids [00288] Disclosed herein are compositions, systems and methods for modifying the C9ORF72 gene.
  • the target nucleic acid is the C9ORF72 gene or a portion thereof.
  • guide nucleic acids described herein comprise a sequence that is complementary to and/or hybridizes to a target sequence of the C9ORF72 gene.
  • the target sequence is within an exon region of the C9ORF72 gene.
  • the target sequence is within an intron region of the C9ORF72 gene.
  • the target sequences spans an exon and intron of the C9ORF72 gene.
  • the target sequence is located in a regulatory region of the C9ORF72 gene.
  • 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 (G 4 C 2 ) n repeat expansion, the compositions, systems, and Cooley 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 (G 4 C 2 ) n repeat expansion. 7.
  • the sample is a biological sample, an environmental sample, or a combination thereof.
  • 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.
  • compositions, systems, and methods described herein comprise an expression vector or a use thereof.
  • the nucleic acid of interest e.g., a C9ORF72 targeting guide nucleic acid
  • the nucleic acid of interest comprises one or more components of a composition or system described herein.
  • the nucleic acid of interest comprises a nucleotide sequence that encodes one or more components of the composition or system described herein.
  • one or more components comprises polypeptide(s), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s).
  • 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.
  • components described herein e.g., an effector protein, a guide nucleic acid, and/or a target nucleic acid
  • components described herein are each encoded by different vectors of the system.
  • a vector comprises a nucleotide sequence encoding one or more effector proteins as described herein.
  • the one or more effector proteins Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO comprise at least two effector proteins.
  • the at least two effector protein are the same.
  • the at least two effector proteins are different from each other.
  • the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell.
  • 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.
  • 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.
  • a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell.
  • a vector may encode 1, 2, 3, 4 or more of any system components.
  • 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.
  • a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein.
  • the one or more guide nucleic acids comprise at least two guide nucleic acids.
  • the at least two guide nucleic acids are the same.
  • the at least two guide nucleic acids are different from each other.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • SV40 early promoter mouse mammary tumor virus long terminal repeat (LTR) promoter
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • Hl human Hl promoter
  • 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.
  • 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.
  • the vector comprises a nucleotide sequence of a promoter.
  • the vector comprises two promoters.
  • the vector comprises three promoters.
  • 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.
  • 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.
  • the promoter allows for expression in a motor neuron.
  • the motor neuron specific promoter is Hb9.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • 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.
  • a signal 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.
  • the promoter is an activation- inducible promoter, such as a CD69 promoter.
  • the promoter for expressing effector protein is a motor neuron-specific promoter, such as a Hb9 promoter.
  • the promoter for expressing effector protein is a ubiquitous promoter.
  • the ubiquitous promoter comprises MND or CAG promoter sequence.
  • the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell).
  • the promoters are eukaryotic Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO promoters, (e.g., drive expression of a gene in a eukaryotic cell).
  • the promoter is EF1a. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.
  • a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA.
  • the expression vector comprises the DNA molecule encoding a guide nucleic acid.
  • the expression vector further comprises the nucleic acid encoding an effector protein. In some embodiments, the expression vector further comprises or encodes a donor nucleic acid. In some embodiments, the expression vector encoding a guide nucleic acid, wherein the guide nucleic acid comprises a first region comprising a repeat sequence; and a second region comprising a spacer sequence that is complementary to a target sequence of a C9ORF72 gene. In some embodiments, wherein the first region is located 5’ of the second region. In some embodiments, the expression vector further comprises an effector protein that binds the repeat sequence or a nucleic acid encoding the effector protein.
  • the spacer comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 1-2849;
  • the repeat sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence of SEQ ID NO: 10,854;
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence listed in TABLEs 2, 3 and 5; or a combination thereof.
  • the spacer comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 2850-5423;
  • the repeat sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 10,848-10853;
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence listed in TABLES 2, 4 and 5; or a combination thereof.
  • a vector described herein is a delivery vector.
  • the delivery vector is a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof.
  • the delivery vehicle is a non-viral vector.
  • the delivery vector is a plasmid.
  • the plasmid comprises DNA.
  • the plasmid comprises RNA.
  • the plasmid comprises circular double-stranded DNA.
  • the plasmid is linear.
  • the plasmid comprises one or more coding sequences of interest and one or more regulatory elements.
  • the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria.
  • the plasmid is a minicircle plasmid.
  • the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid.
  • the plasmids are engineered through synthetic or other suitable means known in the art.
  • the genetic elements are assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which is then be readily ligated to another genetic sequence.
  • vectors comprise an enhancer.
  • Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo base pairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I. [00304] In some embodiments, disclosed herein comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof.
  • the nucleic acid expression vector comprises a polynucleotide encoding an effector protein that is Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences recited in TABLEs 2-5.
  • the one or more nucleic acids may comprise a plasmid.
  • the one or more nucleic acids may comprise a nucleic acid expression vector.
  • the one or more nucleic acids may comprise a viral vector.
  • the viral vector is a lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • compositions, including pharmaceutical compositions comprise a viral vector encoding a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion effector protein.
  • pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • an administration of a non-viral vector comprises contacting a cell, such as a host cell, with the non-viral vector.
  • a physical method or a chemical method is employed for delivering the vector into the cell.
  • Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery.
  • Exemplary chemical methods include delivery of the recombinant polynucleotide by liposomes such as, cationic lipids or neutral lipids; lipofection; dendrimers; lipid nanoparticle (LNP); or cell- penetrating peptides.
  • a vector is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein.
  • a vector is administered in a single vehicle, such as a single expression vector.
  • at least two of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid are provided in the single expression vector.
  • components, such as a guide nucleic acid and an effector protein are encoded by the same vector.
  • an effector protein (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co- administered with donor nucleic acid in a single vehicle.
  • donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.
  • a vector may be part of a vector system.
  • the vector system comprises a library of vectors each encoding one or more components of a composition or system described herein.
  • a vector system is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein, wherein at least two vectors are co-administered.
  • the at least two vectors comprise different components.
  • the at least two vectors comprise the same component having different sequences.
  • At least one of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof is provided in a different vector.
  • the nucleic acid encoding the effector protein, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors.
  • the donor nucleic acid is encoded by a different vector than the vector encoding the effector protein and the guide nucleic acid.
  • compositions and systems provided herein comprise a lipid particle.
  • a lipid particle is a lipid nanoparticle (LNP).
  • LNPs are a non-viral delivery system for delivery of the composition and/or system components described herein. LNPs are particularly effective for delivery of nucleic acids.
  • compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce one or more effector proteins, one or more guide nucleic acids, one or more donor nucleic acids, or any combinations thereof to a cell.
  • lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio- responsive polymers.
  • the ionizable lipids exploits chemical-physical properties of the endosomal environment (e.g., pH) offering improved delivery of nucleic acids.
  • the ionizable lipids are neutral at physiological pH.
  • Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO the ionizable lipids are protonated under acidic pH.
  • the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.
  • a LNP comprises an outer shell and an inner core.
  • the outer shell comprises lipids.
  • the lipids comprise modified lipids.
  • the modified lipids comprise pegylated lipids.
  • the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids.
  • the LNP comprises one or more of N1,N3,N5-tris(3- (didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), 2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2-dimyristoyl-sn- glycerol, and methoxypolyethylene glycol (DMG-PEChooo), derivatives, analogs, or variants thereof.
  • DOPE 2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • POPE l-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine
  • DSPC 1,2-distearoy
  • the LNP has a negative net overall charge prior to complexation with one or more of a guide nucleic acid, a nucleic acid encoding the one or more guide nucleic acid, a nucleic acid encoding the effector protein, and/or a donor nucleic acid.
  • the inner core is a hydrophobic core.
  • the one or more of a guide nucleic acid, the nucleic acid encoding the one or more guide nucleic acid, the nucleic acid encoding the effector protein, and/or the donor nucleic acid forms a complex with one or more of the cationic lipids and the ionizable lipids.
  • a LNP comprises one or more of cationic lipids, ionizable lipids, and modified versions thereof.
  • the ionizable lipid comprises TT3 or a derivative thereof. Accordingly, in some embodiments, the LNP comprises one or more of TT3 and pegylated TT3.
  • the publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and representative methods of delivering LNP formulations in Example 7.
  • a LNP comprises a lipid composition targeting to a specific organ.
  • the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen).
  • the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain).
  • the lipid composition comprises Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes).
  • Delivery of Viral Vectors [00313]
  • a vector described herein comprises a viral vector.
  • the viral vector comprises a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle.
  • the nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented.
  • the nucleic acid may comprise DNA, RNA, or a combination thereof.
  • the vector is an adeno-associated viral vector.
  • retroviruses e.g. ⁇ OHQWLYLUXVHV ⁇ DQG ⁇ ⁇ -retroviruses
  • adenoviruses e.g., adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • the vector is an adeno-associated viral (AAV) vector.
  • the viral vector is a recombinant viral vector.
  • the vector is a retroviral vector.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector comprises gamma-retroviral vector.
  • a viral vector provided herein may be derived from or based on any such virus.
  • the gamma-retroviral vector is derived from a Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or a Murine Stem cell Virus (MSCV) genome.
  • the lentiviral vector is derived from the human immunodeficiency virus (HIV) genome.
  • the viral vector is a chimeric viral vector.
  • the chimeric viral vector comprises viral portions from two or more viruses.
  • the viral vector corresponds to a virus of a specific serotype.
  • a viral vector is an adeno-associated viral vector (AAV vector).
  • AAV vector adeno-associated viral vector
  • a viral particle that delivers a viral vector described herein is an AAV.
  • the AAV comprises any AAV known in the art.
  • the viral vector corresponds to a virus of a specific AAV serotype.
  • the AAV serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, an AAV-rh10 serotype, and any combination, derivative, or variant thereof.
  • the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self- Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.
  • scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.
  • an AAV vector described herein is a chimeric AAV vector.
  • the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes.
  • a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.
  • AAV vector described herein comprises two inverted terminal repeats (ITRs).
  • the viral vector provided herein comprises two inverted terminal repeats of AAV.
  • a nucleotide sequence between the ITRs of an AAV vector provided herein comprises a sequence encoding genome editing tools.
  • the genome editing tools comprise a nucleic acid encoding one or more effector proteins, a nucleic acid encoding one or more fusion proteins (e.g., a nuclear localization signal (NLS), polyA tail), one or more guide nucleic acids, a nucleic acid encoding the one or more guide nucleic acids, respective promoter(s), one or more donor nucleic acid, or any combinations thereof.
  • viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein.
  • a coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating the AAV vector that is a self- complementary AAV (scAAV) vector.
  • the scAAV vector comprises the sequence encoding genome editing tools that has a length of about 2 kb to about 3 kb.
  • the AAV vector provided herein is a self-inactivating AAV vector.
  • the AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector.
  • methods of producing AAV delivery vectors herein comprise packaging a nucleic acid encoding an effector protein and a guide nucleic acid, or a combination thereof, into an AAV vector.
  • methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging an effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector.
  • promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector.
  • the AAV vector may package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof.
  • the AAV vector comprises inverted terminal repeats, e.g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat.
  • the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.
  • a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same.
  • ITR inverted terminal repeat
  • the Rep gene and ITR from a first AAV serotype e.g., AAV2
  • a second AAV serotype e.g., AAV9
  • a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9.
  • the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.
  • the AAV vector comprises at least one recombinant AAV expression cassette comprising: a) a first inverted terminal repeat (ITR) and a first promoter; b) a first polynucleotide sequence encoding an effector protein disclosed herein; c) optionally a second promoter; d) a second polynucleotide encoding a guide nucleic acid disclosed here; and e) a second ITR.
  • the AAV expression cassette is a self-complementary AAV vector.
  • the AAV vector comprises a plurality of expression cassettes comprising a first gRNA expression cassette comprising a first Pol III RNA promoter, and a first polynucleotide sequence encoding a first gRNA disclosed herein (e.g. sgRNA-1); a second gRNA expression cassette comprising a second Pol III RNA promoter, and a second polynucleotide sequence encoding a second gRNA disclosed herein (e.g. sgRNA-2); and an effector protein expression cassette comprising a promoter (e.g.
  • the first Pol III RNA promoter is selected from U6, H1, and 7SK.
  • the second Pol III RNA promoter is selected from U6, H1, and 7SK.
  • the first Pol III promoter and Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO the second Pol III promoter are the same. In some embodiments, the first Pol III promoter and the second Pol III promoter are different.
  • the promoter in the effector protein expression cassette is a tissue specific promoter.
  • the tissue specific promoter includes, but is not limited to, human synapsin (hSyn), calcium/calmodulin-dependent protein kinase II subunit ⁇ ⁇ &D0.,, ⁇ , and Hb9.
  • the promoter in the effector protein expression cassette is a ubiquitous promoter.
  • the ubiquitous promoter includes, but is not limited to, cytRPHJDORYLUXV ⁇ ⁇ &09 ⁇ &09 ⁇ HDUO ⁇ HQKDQFHU ⁇ FKLFNHQ ⁇ ⁇ DFWLQ ⁇ ⁇ CAG), and phosphoglycerate kinase (PGK).
  • the regulatory element is a woodchuck hepatitis virus regulatory element (WPRE).
  • the poly(A) is a human growth hormone (hGH) poly(A).
  • the plurality of expression cassettes that are combined in a single vector may be arranged in any suitable orientation, such as one cassette located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) another cassette.
  • the two gRNA expression cassettes are arranged in the same direction.
  • the two gRNA expression cassettes are arranged in the opposite direction.
  • the two gRNA expression cassettes are arranged in the same direction at distal ends of the AAV vector.
  • the two gRNA expression cassettes are arranged in the opposition direction at distal ends of the AAV vector. In some embodiments, the two gRNA expression cassettes are arranged in the same direction in tandem. In some embodiments, the two sgRNA expression cassettes are arranged in the opposite direction in tandem. In some embodiments, the effector protein expression cassette and at least one gRNA expression cassette are arranged in the same direction. In some embodiments, the effector protein expression cassette and at least one gRNA expression cassette are arranged in the opposite direction. In some embodiments, the effector protein expression cassette is arranged at a distal end of the AAV vector. In some embodiments, the effector protein expression cassette is arranged between the two gRNA cassettes.
  • AAV particles described herein are recombinant AAV (rAAV).
  • rAAV particles are generated by transfecting AAV producing cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA.
  • the AAV producing cells are mammalian cells.
  • host cells for rAAV viral Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO particle production are mammalian cells.
  • a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a variant thereof, or a combination thereof.
  • rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell.
  • producing rAAV virus particles in a mammalian cell comprises transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31(4):317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties. [00322] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell.
  • the insect cell for producing rAAV viral particles comprises a Sf9 cell.
  • production of rAAV virus particles in insect cells may comprise baculovirus.
  • production of rAAV virus particles in insect cells may comprise infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of- interest expression construct enclosed by an ITR on both the 5’ and 3’ end.
  • rAAV virus particles are produced by the One Bac system.
  • rAAV virus particles can be produced by the Two Bac system.
  • the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome.
  • an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene.
  • the cell is a mammalian cell.
  • the mammalian cell is a human cell.
  • the human cell is a neuron.
  • the human cell is a motor neuron.
  • a population of cells comprising at least one cell disclosed herein.
  • the cells or a population of cells disclosed herein can be used as a component of a pharmaceutical composition disclosed herein.
  • the cells or a population of cells disclosed herein can be used in a method disclosed herein.
  • the cells or a population of cells disclosed herein can be used to treat a disease such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). 10.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • compositions comprising one or more effector proteins described herein or nucleic acids encoding the one or more effector proteins, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or combinations thereof. Also disclosed herein are compositions comprising the nucleic acid expression vector, the cell, or the population of cells disclosed herein. In some embodiments, a repeat sequence of the one or more guide nucleic acids are capable of interacting with the one or more of the effector proteins. In some embodiments, spacer sequences of the one or more guide nucleic acids hybridizes with a target sequence of a target nucleic acid.
  • compositions are capable of editing a target nucleic acid in a cell or a subject. In some embodiments, the compositions are capable of editing a target nucleic acid or the expression thereof in a cell, in a tissue, in an organ, in vitro, in vivo, or ex vivo. In some embodiments, the compositions are capable of editing a target nucleic acid in a sample comprising the target nucleic.
  • compositions described herein comprise plasmids described herein, viral vectors described herein, non-viral vectors described herein, or combinations thereof. In some embodiments, compositions described herein comprise the viral vectors. In some embodiments, compositions described herein comprise an AAV.
  • compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell-penetrating peptides. In some embodiments, compositions described herein comprise an LNP. [00328] In some embodiments, compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein and a pharmaceutically acceptable carrier or diluent.
  • Non-limiting examples of Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.
  • buffers e.g., neutral buffered saline, phosphate buffered saline
  • carbohydrates e.g.,
  • the vector is formulated for delivery through injection by a needle carrying syringe.
  • the composition is formulated for delivery by electroporation.
  • the composition is formulated for delivery by chemical method.
  • the pharmaceutical compositions comprise a virus vector or a non-viral vector.
  • Pharmaceutical compositions described herein comprise a salt.
  • the salt is a sodium salt.
  • the salt is a potassium salt.
  • the salt is a magnesium salt.
  • the salt is NaCl.
  • compositions described herein are in the form of a solution (e.g., a liquid).
  • the solution is formulated for injection, e.g., intravenous or subcutaneous injection.
  • the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.
  • the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7. [00331] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof.
  • compositions comprising the nucleic acid expression vector, the cell, or the population of cells disclosed herein.
  • pharmaceutical compositions comprise a plurality of guide nucleic acids.
  • the pharmaceutical composition disclosed herein also comprise a pharmaceutical acceptable Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO carrier.
  • Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.
  • compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • the effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. 11.
  • Methods of Nucleic Acid Modification Provided herein are methods of editing and modifying the expression of the human C9ORF72 gene. In general, editing refers to modifying the nucleobase sequence of a target nucleic acid. However, compositions and systems disclosed herein may also be capable of making epigenetic modifications of target nucleic acids.
  • Editing a target nucleic acid may comprise one or more of: cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or modifying (e.g., methylating, demethylating, deaminating, or oxidizing) of one or more nucleotides of the target nucleic acid.
  • Methods of modifying may comprise contacting the C9ORF72 gene with a composition, system, or nucleic acid expression vector disclosed herein, wherein the system or nucleic acid comprises an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in TABLE 2-5s.
  • the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in TABLEs 2-5. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 10,856, 10,878, 10,880, and 10,919.
  • 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%, Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO 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.
  • Editing may introduce a mutation (e.g., point mutations, deletions) in a target nucleic acid relative to a corresponding wildtype nucleobase sequence.
  • Editing may remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleobase sequence. Editing may remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. Editing may be used to generate gene knock-out, gene knock-in, gene editing, gene tagging, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a cell.
  • Editing may comprise single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof.
  • cleavage is site-specific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer region.
  • the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g., homology directed repair (HDR)) or non-homologous end joining (NHEJ).
  • a double-stranded break in the target nucleic acid may be repaired (e.g., by NHEJ or HDR) without insertion of a donor template, such that the repair results in an indel in the target nucleic acid at or near the site of the double- stranded break.
  • an indel is a type of genetic mutation that results from the insertion and/or deletion of nucleotides in a target nucleic acid.
  • An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including sequencing. If the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation.
  • the dual-guided compositions, systems, and methods described herein can modify the target nucleic acid in two locations.
  • dual-guided editing can comprise cleavage of the target nucleic acid in the two locations targeted by the guide RNAs.
  • the wild-type reading frame is restored.
  • a wild-type reading frame can be a reading frame that produces at least a partially, or fully, functional protein.
  • compositions, systems, and methods described herein can edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid.
  • 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between can be edited by the compositions, systems, and methods described herein.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides can be edited by the compositions, systems, and methods described herein.
  • 10, 20, 30, 40, 50, 60, 70, 8090, 100 or more nucleotides, or any integer in between can be edited by the compositions, systems, and methods described herein.
  • 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between can be edited by the compositions, systems, and methods described herein.
  • methods comprise editing a target nucleic acid with two or more effector proteins. Editing a target nucleic acid may comprise introducing a two or more single- stranded breaks in a target nucleic acid.
  • a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid.
  • the guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting the effector protein to the region of the target nucleic acid. Binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid.
  • modifying a target nucleic acid may comprise introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid.
  • modifying a target nucleic acid may comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second programmable nickase and hybridizes to a second region of the target nucleic acid.
  • the first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid
  • the second effector protein may introduce a second break in a second strand at the second region of the target nucleic acid.
  • a segment of the target nucleic Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO acid between the first break and the second break may be removed, thereby modifying the target nucleic acid.
  • a segment of the target nucleic acid between the first break and the second break may be replaced (e.g., with donor nucleic acid), thereby modifying the target nucleic acid.
  • Methods, systems and compositions described herein can edit or modify a target nucleic acid wherein such editing or modification can be measured by indel activity.
  • Indel activity measures the amount of change in a target nucleic acid (e.g., nucleotide deletion(s) and/or insertion(s)) compared to a target nucleic acid that has not been contacted by a polypeptide described in compositions, systems, and methods described herein.
  • indel activity can be detected by next generation sequencing of one or more target loci of a target nucleic acid where indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence.
  • methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein can exhibit about 0.0001% to about 65% or more indel activity upon contact to a target nucleic acid compared to a target nucleic acid non-contacted with compositions, systems, or by methods described herein.
  • methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein can exhibit about 0.0001%, about 0.001%, about 0.01%, about 0.1%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or more indel activity.
  • sequence deletion is a modification where one or more sequences in a target nucleic acid are deleted relative to a target nucleic acid without the sequence deletion.
  • a sequence deletion can result in or effect a splicing disruption or a frameshift mutation.
  • a sequence deletion result in or effect a splicing disruption.
  • a modification is a deletion of an entire exon. In some embodiments, the exon is associated with a disease.
  • compositions, systems, and methods described herein comprise a combination of a first gRNA, a second gRNA, a first effector protein, and a second effector protein, wherein the combination can be used for deleting the entire exon or a portion thereof.
  • the first effector protein and the second effector protein are the same. In some embodiments, the first effector protein and the second effector protein are not the same.
  • sequence skipping is a modification where one or more sequences in a target nucleic acid are skipped upon transcription or translation of the target nucleic acid relative to a target nucleic acid without the sequence skipping.
  • sequence skipping can result in or effect a splicing disruption or a frameshift mutation.
  • sequence skipping can result in or effect a splicing disruption.
  • sequence reframing is a modification where one or more bases in a target are modified so that the reading frame of the sequence is reframed relative to a target nucleic acid without the sequence reframing.
  • sequence reframing can result in or effect a splicing disruption or a frameshift mutation.
  • sequence reframing can result in or effect a frameshift mutation.
  • sequence knock-in is a modification where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in.
  • sequence knock-in can result in or effect a splicing disruption or a frameshift mutation.
  • sequence knock-in can result in or effect a splicing disruption.
  • editing or modification of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit or modify a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising splicing disruption mutations, frameshift mutations, sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof.
  • editing or modification of a specific locus can affect any one of a splicing disruption, frameshift (e.g., 1+ or 2+ frameshift), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof.
  • editing or modification of a target nucleic acid can be locus specific, modification specific, or both. In some embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise an effector protein described herein and a guide nucleic acid described herein.
  • Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vitro. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed ex vivo.
  • Editing Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO methods include, but are not limited to, introduction of double stranded breaks (DSB), which can result in deleting some nucleotides and disrupting the translation of a functional protein, base editing, and splice acceptor disruption (SA).
  • DSB double stranded breaks
  • SA splice acceptor disruption
  • the method of editing by the effector proteins can be promotor silencing, frameshift mutation, base editing, or splice disruption.
  • the editing by the effector protein targets an exon of the C9ORF72 gene.
  • the editing by the effector protein targets an intron of the C9ORF72 gene.
  • the editing by the effector protein decreases transcription of the DNA sequence of the C9ORF72 gene. In some embodiments, the editing by the effector protein decreases translation of the RNA sequence of the C9ORF72 gene. In some embodiments, the effector protein targets intron 1 of the C9ORF72 gene. In some embodiments, the effector protein targets the promoter of the C9ORF72 gene. [00350] In some embodiments the gene regulation is regulated by effector protein repressing a promoter. In some embodiments the repression is temporary or transient. In some embodiments the repression is permanent. In some embodiments the effector protein is linked to a KRAB sequence. In some embodiments the effector protein is linked to an acetylase sequence.
  • the effector protein is linked to a methyltransferase. In some embodiments the effector protein is linked to a Ezh2 sequence. [00351] In some embodiments the effector protein causes a frameshift mutation. In some embodiments the effector protein causes the addition of one or more nucleotides causing a shift in the reading frame. In some embodiments the effector protein causes a deletion of one or more nucleotides causing a shift in the reading frame. In some embodiments the effector protein causes the deletion or addition of 1, 2, or 4 nucleotides. In some embodiments the effector protein causes an alternation in the amino acid sequence at protein translation. In some embodiments the alteration is a missense mutation.
  • the alteration is a premature stop codon.
  • the effector protein causes a change in the ribosome reading frame and cause premature termination of translation at a new nonsense or chain termination codon (TAA, TAG, and TGA).
  • the effector protein causes a base to be edited.
  • the effector protein is linked to an adenine base editing enzyme (ABE).
  • the effector protein is linked to a cytosine base editing enzyme (CBE).
  • the fusion protein causes a cytodine to thymidine transition.
  • the fusion protein causes a cytodine to uracil transition. In some embodiments the fusion protein causes a thymidine to cytodine transition. In some embodiments the fusion protein causes an adenosine to guanosine transition. In some embodiments the fusion protein causes a guanosine to adenosine conversion. In some embodiments the alteration results in a missense mutation. In some embodiments the alteration is a premature stop codon. In some embodiments the fusion protein causes a premature termination of translation at a new nonsense or chain termination codon (TAA, TAG, and TGA). 12.
  • Methods of detecting target nucleic acids may comprise detecting target nucleic acids with compositions or systems described herein. Methods may comprise detecting a target nucleic acid in a sample, e.g., a cell lysate, a biological fluid, or environmental sample. Methods may comprise detecting a target nucleic acid in a cell.
  • methods of detecting a target nucleic acid in a sample or cell comprises contacting the sample or cell with an effector protein or a multimeric complex thereof, a guide nucleic acid, wherein at least a portion of the guide nucleic acid is complementary to at least a portion of the target nucleic acid, and a reporter nucleic acid that is cleaved in the presence of the effector protein, the guide nucleic acid, and the target nucleic acid, and detecting a signal produced by cleavage of the reporter nucleic acid, thereby detecting the target nucleic acid in the sample.
  • methods result in trans cleavage of the reporter nucleic acid.
  • methods result in cis cleavage of the reporter nucleic acid.
  • Methods of Treating a Disorder Described herein are methods for treating or preventing a disease in a subject by modifying a target nucleic acid associated with a gene or expression of a gene related to the disease.
  • the disease or disorder is caused by a C9orf72 protein variant in a subject.
  • the C9orf72 protein variant comprises a hexanucleotide (G 4 C 2 ) n repeat expansion (HRE).
  • the HRE is located in intron 1 of the C9ORF72 gene.
  • the HRE is located in the promoter of the C9ORF72 gene.
  • the disease or disorder is caused by HRE repeats in the C9ORF72 gene in a subject.
  • the disease or disorder is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the ALS is familial ALS (fALS).
  • FTD frontotemporal dementia
  • methods comprise administering a guide RNA comprising one or more sequences that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 1-10,853 and 10,920, or a DNA molecule encoding the same.
  • methods comprise administering an effector protein described herein or a nucleic acid encoding the same.
  • the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences recited in TABLEs 2-5.
  • the effector protein or nucleic acid encoding the same, and the guide RNA or nucleic acid encoding the same may be administered in a single composition.
  • the effector protein or nucleic acid encoding the same, and the guide RNA or nucleic acid encoding the same may be administered separately (formulaically or chronologically).
  • methods comprise administering: an effector protein or a messenger RNA encoding an effector protein and a lipid nanoparticle; and a viral vector encoding a guide RNA.
  • methods comprise administering: an effector protein or a messenger RNA encoding an effector protein and a lipid nanoparticle; and a guide RNA, optionally wherein the synthetic guide RNA comprises at least one chemical modification.
  • methods comprise administering a viral vector encoding the effector protein and the guide RNA.
  • methods comprise administering an effector protein and a lipid nanoparticle.
  • methods comprise administering a messenger RNA encoding an effector protein.
  • methods decrease an amount of C9orf72 protein in a cell.
  • Cooley Ref MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO TABLES AND SEQUENCES TABLE 1. Exemplary Repeat Sequences TABLE 2. Exemplary Effector Proteins Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO TABLE 3: Exemplary Amino Acid Alterations Relative to SEQ ID NO: 10,856 TABLE 4.
  • Exemplary PAM Sequences N represents any nucleotide; V represents A, C, or G; Y represents C or T; and R represents A or G TABLE 8.
  • Exemplary PAM Sequences N represents any nucleotide TABLE 9: Exemplary base editing enzyme and base editor fusion proteins Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO TABLE 10.
  • Example 1 Gene Editing of C9ORF72 by CasM.265466
  • Combinations of the effector protein as set forth in SEQ ID NO: 10,856 and guide nucleic acids (SEQ ID NOs: 5424-8272) that target intron 1 or the promoter of the C9ORF72 gene are tested for their ability to produce indels in HEK293T cells.
  • plasmids expressing the effector protein and guide nucleic acids are delivered by lipofection to HEK293T cells.
  • Cells are incubated for 3 days before being lysed and subjected to PCR amplification.
  • Indels are detected by next generation sequencing of PCR amplicons at the targeted loci and indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to the unedited C9ORF72 gene sequence.
  • Example 2 Gene Editing of C9ORF72 by CasPhi.12
  • Combinations of the effector protein (as set forth in SEQ ID NO: 10,857) and guide nucleic acids (SEQ ID NOs: 8273-10,846) that target intron 1 or the promoter of the C9ORF72 gene are tested for their ability to produce indels in HEK293T cells.
  • SEQ ID NOs: 1073-10,846 guide nucleic acids that target intron 1 or the promoter of the C9ORF72 gene are tested for their ability to produce indels in HEK293T cells.
  • 300 ng of plasmids expressing the effector protein and guide nucleic acids are delivered by lipofection to HEK293T cells. Cells are incubated for 3 days before being lysed and subjected to PCR amplification.
  • Indels are detected by next generation sequencing of PCR amplicons at the targeted loci and indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to the unedited C9ORF72 gene sequence.
  • Example 3 Base editing of the C9ORF72 gene [00362] Target sequence editing by the base editor fusion proteins described herein will be assessed. Briefly, HEK293T cells will be transfected with plasmids encoding a base editor fusion protein and guide nucleic acids described herein.
  • CasPhi.12 L26R I471T edits C9ORF72 in human neural progenitor cells
  • Human neural progenitor cells (ReNcell®) were transfected with plasmids encoding CasPhi.12 L26R I471T and a C9ORF72 guide nucleic acid. Cells were harvested and NGS data was analyzed to quantify % indel in the C9ORF72 gene.
  • Guide nucleic acids R20722, R20726, R20730, R20731, and R20735 all target C9ORF72 upstream of the HRE. The remainder of the guides tested target C9ORF72 downstream of the HRE. Results are shown in TABLE 12 below.
  • any one of the upstream guides may be combined with any one of the downstream guides tested to remove at least a portion of C9OR72 containing the HRE.
  • upstream guide R20722 (SEQ ID NO: 9403) with any one of R20745 (SEQ ID NO: 9994), R20738 (SEQ ID NO: 9793), R20753 (SEQ ID NO: 10,133), R20744 (SEQ ID NO: 9956), and R20741 (SEQ ID NO: 9872) to remove a portion of C9ORF72 containing the HRE.
  • R20745 SEQ ID NO: 9994
  • R20738 SEQ ID NO: 9793
  • R20753 SEQ ID NO: 10,133
  • R20744 SEQ ID NO: 9956
  • R20741 SEQ ID NO: 9872
  • CasM.265466 D220R edits C9ORF72 in human neural progenitor cells
  • Human neural progenitor cells (ReNcell®) were transfected with plasmids encoding CasM.265466 D220R and a C9ORF72 guide nucleic acid. Cells were harvested and NGS Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO data was analyzed to quantify % indel in the C9ORF72 gene. Results are shown in TABLE 13 below. The target positions of guide nucleic acids relative to the HRE in the C9ORF72 gene are indicated.
  • any one of the upstream guides may be combined with any one of the downstream guides tested to remove at least a portion of C9OR72 containing the HRE.
  • upstream guide R20765 (SEQ ID NO: 5458), R20777 (SEQ ID NO: 5838), R20781 (SEQ ID NO: 5902), R20844 (SEQ ID NO: 1653), R20849 (SEQ ID NO: 7106) with any one of downstream guide R20768 (SEQ ID NO: 5534), R20769 (SEQ ID NO: 5554), R20770 (SEQ ID NO: 5597), R20772 (SEQ ID NO: 5623), R20775 (SEQ ID NO: 5784), R20776 (SEQ ID NO: 5793), R20804 (SEQ ID NO: 6478), R20829 (SEQ ID NO: 6825), R20833 (SEQ ID NO: 6885), R20842 (SEQ ID NO: 7010), R20847 (SEQ ID NO: 7094), R20855 (SEQ ID NO: 7188), R20863 (SEQ ID NO: 7454), and R20864 (SEQ ID NO:
  • CasPhi.12 L26R, I471T generates HRE deletion with dual guides
  • ReNcell CX neuronal precursor cells were transfected with nucleic acids encoding CasPhi.12 L26R, I471T (SEQ ID NO:10,917) and guide nucleic acid pairs described in Example 4.
  • the DNA sequence encoding CasPhi.12 L26R, I471T with NLSs at both termini is provided in TABLE 14. Cells were harvested 48h post-transfection and HRE deletion was quantified by analyzing NGS data.
  • CasM.265466 D220R generates HRE deletion with dual guides [00373] Model systems of ALS such as ALS patient-derived iPSCs and C9-500 transgenic mice are transduced with AAV encoding CasM.265466 variant D220R and guide nucleic acids as described in TABLE 16. Cells are harvested at various time points (e.g., ⁇ 21 days post- transduction) and tissues are collected at various time points, (e.g., ⁇ 8 weeks post-transduction.
  • HRE deletion is quantified by analyzing NGS data. TABLE 16. AAV DNA sequences encoding CasM.265466 variant D220R with NLSs (italicized) and guide nucleic acid pairs tested in EXAMPLE 7 Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO Example 8. Gene Editing of C9ORF72 by CasM.265466 D220R [00374] Human neural progenitor cells (ReNcell®) were transfected with plasmids encoding CasM.265466 D220R (SEQ ID NO: 10,919) or SaCas9, and a corresponding C9ORF72 guide nucleic acid.
  • ReNcell® Human neural progenitor cells
  • Example 9 In vivo muscle targeting via AAV9-4A delivery of CasPhi.12 and CasM.265466 variants
  • the purpose of the study was to assess the capability of two effector protein variants, CasPhi.12 L26R (a variant of CasPhi.12) and CasM.265466 D220R (a variant of CasM.265466), to edit nucleic acid sequences within muscle tissues in vivo.
  • the study focused on PCSK9 as an exemplary gene target.
  • an AAV9-4A vector was employed as the delivery vehicle for introducing the effector protein and gRNA into the specific target tissues.
  • the DNA encoding the effector protein e.g., SaCas9, CasPhi.12 L26R, or CasM.265466 D220R
  • its corresponding promoter e.g., ck8e or spc5
  • PCSK9 in the plasmid the targeting spacer sequence specific to PCSK9
  • its u6 promoter were cloned into the AAV9-4A plasmid between the AAV inverted terminal repeats (ITRs), creating AAV9 constructs as follows: a.
  • pssAAV-ITR-u6-PCSK9-ck8e-saCas9-bGHpolya-ITR b.
  • pssAAV-ITR-u6-PCSK9-ck8e-L26R-wpre-hGHpolya-ITR PL26297
  • pssAAV-ITR-u6-PCSK9-spc5-12-L26R-wpre-hGHpolya-ITR (PL31718)
  • pssAAV-ITR-u6-PCSK9-ck8e-D220R-wpre-hGHpolya-ITR e.
  • Exemplary gRNA sequences [00378] The sequences of the other AAV components are provided in TABLE 18. TABLE 18: AAV Components Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO Cooley Ref: MABI-046/06WO 344183-2321 Mammoth Ref: MB0125WO [00379] 6-week old male C57BL/6J mice were given a single intravenous (IV) bolus through the tail vein using a 1.0 cc syringe with a 27G- ⁇ ⁇ QHHGOH ⁇ RU ⁇ D ⁇ VLQJOH ⁇ LQWUDPXVFXODU ⁇ ,0 ⁇ bolus into the gastrocnemius.
  • IV intravenous
  • mice were euthanized for assessment. Before collecting the tissues, the mice underwent whole-body perfusion via the left ventricle using 5.0- 10.0 mL of PBS to remove any remaining blood from the tissues. Tissue samples weighing 100 mg were then dissected from the liver, heart, gastrocnemius, diaphragm, pectoral, and masseter, respectively, and placed on a plate for subsequent Next-Generation Sequencing (NGS) analysis.
  • NGS Next-Generation Sequencing
  • IM intramuscular
  • both the left and right gastrocnemius were weighed and harvested.
  • only the left gastrocnemius was weighed and harvested.
  • the genomic DNA isolated from the muscle tissues was subject to NGS and aligned to a reference DNA sequence for the analysis of insertions or deletions (indels).
  • indels insertions or deletions
  • CasPhi.12 L26R delivered in the PL26297 vector via IM administration generated about 5% indel rate in the right gastrocnemius and about 3% indel rate in masseter.
  • CasPhi.12 L26R delivered in the PL31718 vector via IV administration was able to generate about 8% indel rate in the PCSK9 gene in the heart.
  • CasM.265466 D220R delivered in the PL31719 vector via IV administration resulted in an about 25% indel rate in the liver, about 10% in the diaphragm, about 15% in the left gastrocnemius, about 20% in the heart, about 5% in the masseter, and about 13% in the pectoral.
  • CasM.265466 D220R demonstrated an approximately 2-fold greater indel rate in the heart compared to SaCas9 delivered in the PL26295 vector via IV administration.
  • CasM.265466 D220R delivered in the PL31719 vector via IV administration generated about 10% in the left gastrocnemius, about 11% in the heart, and about 3% in the pectoral.
  • the results demonstrate that both CasPhi.12 variant L26R and CasM.265466 variant D220R are highly efficient in inducing indels in vivo at the PCSK9 locus across different muscle tissues. Additional experiments are performed to validate in vivo editing using the guide nucleic acids described herein. [00385] This experiment is repeated with various combinations of: (1) an effector protein described herein; and (2) a guide nucleic acid described herein that comprises a spacer sequence complementary to C9ORF72.
  • the effector protein is CasPhi.12 or a variant thereof described herein.
  • the effector protein is CasM.265466 or a variant thereof described herein.

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Abstract

L'invention concerne des compositions, des systèmes et des méthodes comprenant des protéines effectrices pour traiter la sclérose latérale amyotrophique et la démence frontotemporale. Lesdites protéines effectrices peuvent être caractérisées en tant que protéines associées à CRISPR (Cas). Divers compositions, systèmes et méthodes de la présente divulgation peuvent tirer profit des activités de ces protéines effectrices pour la modification du gène C9ORF72.
PCT/US2024/038776 2023-07-21 2024-07-19 Compositions pour la modification du gène c9orf72 humain Pending WO2025024285A1 (fr)

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WO2017109757A1 (fr) * 2015-12-23 2017-06-29 Crispr Therapeutics Ag Matériaux et procédés de traitement de la sclérose latérale amyotrophique et/ou de la dégénérescence lobaire frontotemporale
WO2021188729A1 (fr) * 2020-03-18 2021-09-23 Scribe Therapeutics Inc. Compositions et procédés pour le ciblage de c9orf72
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WO2023004430A1 (fr) * 2021-07-23 2023-01-26 Mammoth Biosciences, Inc. Vecteurs codant pour des systèmes d'édition génique et leurs utilisations
WO2023107967A2 (fr) * 2021-12-07 2023-06-15 Mammoth Biosciences, Inc. Lymphocytes t à récepteurs antigéniques chimériques générés par des protéines effectrices et procédés associés

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US11142760B2 (en) * 2019-02-13 2021-10-12 Beam Therapeutics Inc. Compositions and methods for treating hemoglobinopathies
WO2021188729A1 (fr) * 2020-03-18 2021-09-23 Scribe Therapeutics Inc. Compositions et procédés pour le ciblage de c9orf72
WO2023004430A1 (fr) * 2021-07-23 2023-01-26 Mammoth Biosciences, Inc. Vecteurs codant pour des systèmes d'édition génique et leurs utilisations
WO2023107967A2 (fr) * 2021-12-07 2023-06-15 Mammoth Biosciences, Inc. Lymphocytes t à récepteurs antigéniques chimériques générés par des protéines effectrices et procédés associés

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