US20230034581A1 - Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof - Google Patents

Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof Download PDF

Info

Publication number
US20230034581A1
US20230034581A1 US17/832,114 US202217832114A US2023034581A1 US 20230034581 A1 US20230034581 A1 US 20230034581A1 US 202217832114 A US202217832114 A US 202217832114A US 2023034581 A1 US2023034581 A1 US 2023034581A1
Authority
US
United States
Prior art keywords
ldha
seq
sequence
nucleotide
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/832,114
Inventor
Quinton Norman WESSELLS
Jeffrey Raymond HASWELL
Tia Marie Ditommaso
Noah Michael Jakimo
Sejuti SENGUPTA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arbor Biotechnologies Inc
Original Assignee
Arbor Biotechnologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arbor Biotechnologies Inc filed Critical Arbor Biotechnologies Inc
Priority to US17/832,114 priority Critical patent/US20230034581A1/en
Priority to US18/052,791 priority patent/US11939607B2/en
Assigned to Arbor Biotechnologies, Inc. reassignment Arbor Biotechnologies, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HASWELL, Jeffrey Raymond, JAKIMO, NOAH MICHAEL, SENGUPTA, Sejuti, WESSELLS, Quinton Norman, DITOMMASO, Tia Marie
Assigned to Arbor Biotechnologies, Inc. reassignment Arbor Biotechnologies, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HASWELL, Jeffrey Raymond, JAKIMO, NOAH MICHAEL, SENGUPTA, Sejuti, WESSELLS, Quinton Norman, DITOMMASO, Tia Marie
Publication of US20230034581A1 publication Critical patent/US20230034581A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/1137—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 against enzymes
    • 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 RNAses, DNAses
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)

Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • the present disclosure is based, at least in part, on the development of a system for genetic editing of a lactate dehydrogenase A (LDHA) gene.
  • the system involves a Cas12i polypeptide such as a Cas12i2 polypeptide and an RNA guide mediating cleavage at a genetic site within the LDHA gene by the CRISPR nuclease polypeptide.
  • the gene editing system disclosed herein has achieved successful editing of LDHA gene with high editing efficiency and accuracy.
  • the gene editing system disclosed herein may exhibit one or more of the following advantageous features.
  • Cas12i effectors are smaller (1033 to 1093aa) which, in conjunction with their short mature crRNA (40-43 nt), is preferable in terms of delivery and cost of synthesis.
  • Cas12i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage.
  • Cas12i PAM sequences also differ from those of Cas9. Therefore, larger and different portions of genetic sites of interest can be disrupted with a Cas12i polypeptide and RNA guide compared to Cas9.
  • Cas12i such as Cas12i2 may be more specific than Cas9.
  • gene editing systems for editing LDHA gene pharmaceutical compositions or kits comprising such, methods of using the gene editing systems to produce genetically modified cells, and the resultant cells thus produced. Also provided herein are uses of the gene editing systems disclosed herein, the pharmaceutical compositions and kits comprising such, and/or the genetically modified cells thus produced for treating primary hyperoxaluria (PH) in a subject.
  • PH primary hyperoxaluria
  • the present disclosure features system for genetic editing of a hydroxyacid oxidase 1 (LDHA) gene, comprising (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, and (ii) an RNA guide or a second nucleic acid encoding the RNA guide.
  • the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
  • PAM protospacer adjacent motif
  • the Cas12i is a Cas12i2 polypeptide. In other embodiments, the Cas12i is a Cas12i4 polypeptide.
  • the Cas12i polypeptide is a Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1166.
  • the Cas12i2 polypeptide may comprise one or more mutations relative to SEQ ID NO: 1166.
  • the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO: 1166.
  • the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • the Cas12i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G).
  • the Cas12i2 polypeptide comprises mutations at positions D581, I926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G).
  • the Cas12i2 polypeptide comprises mutations at positions D581, I926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G).
  • the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G).
  • the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).
  • Exemplary Cas12i2 polypeptides for use in any of the gene editing systems disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 1167-1171.
  • the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1168.
  • the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1171.
  • the gene editing system may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide).
  • the first nucleic acid is located in a first vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector).
  • the first nucleic acid is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the coding sequence for the Cas12i polypeptide is codon optimized.
  • the target sequence may be within exon 1 or exon 2 of the LDHA gene.
  • the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249).
  • the target sequence may comprise SEQ ID NO: 1248.
  • the spacer sequence may be 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length. In some examples, the spacer sequence comprises 5′-UAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1273). In some examples, the spacer sequence may comprise SEQ ID NO: 1272.
  • the RNA guide comprises the spacer and a direct repeat sequence.
  • the direct repeat sequence is 23-36-nucleotide in length.
  • the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length.
  • the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length.
  • the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
  • the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225).
  • the RNA guide may comprise SEQ ID NO: 1224.
  • the system may comprise the second nucleic acid encoding the RNA guide.
  • the nucleic acid encoding the RNA guide may be located in a viral vector.
  • the viral vector comprises the both the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) and the second nucleic acid encoding the RNA guide.
  • any of the systems described herein may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide), which is located in a first vector, and the second nucleic acid encoding the RNA guide, which is located on a second vector.
  • the first and/or second vector is a viral vector. In some specific examples, the first and second vectors are the same vector.
  • any of the systems described herein may comprise one or more lipid nanoparticles (LNPs), which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, the RNA guide or the second nucleic acid encoding the RNA guide, or both.
  • LNPs lipid nanoparticles
  • the system described herein may comprise a LNP, which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, and a viral vector comprising the second nucleic acid encoding the RNA guide.
  • the viral vector is an AAV vector.
  • the system described herein may comprise a LNP, which encompass the RNA guide or the second nucleic acid encoding the RNA guide, and a viral vector comprising the first nucleic acid encoding the Cas12i polypeptide.
  • the viral vector is an AAV vector.
  • the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein, and a kit comprising the components of the gene editing system.
  • the present disclosure also features a method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with any of the systems disclosed herein to genetically edit the LDHA gene in the host cell.
  • the host cell is cultured in vitro.
  • the contacting step is performed by administering the system for editing the LDHA gene to a subject comprising the host cell.
  • a cell comprising a disrupted a lactate dehydrogenase A (LDHA) gene, which can be produced by contacting a host cell with the system disclosed herein genetically edit the LDHA gene in the host cell.
  • LDHA lactate dehydrogenase A
  • the present disclosure provides a method for treating primary hyperoxaluria (PH) in a subject.
  • the method may comprise administering to a subject in need thereof any of the systems for editing a lactate dehydrogenase A (LDHA) gene or any of the cells disclosed herein.
  • LDHA lactate dehydrogenase A
  • the subject may be a human patient having the PH.
  • the PH is PH1, PH2, or PH3.
  • the PH is PH1.
  • RNA guide comprising (i) a spacer sequence as disclosed herein that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence.
  • LDHA lactate dehydrogenase A
  • PAM protospacer adjacent motif
  • the spacer may be 20-30-nucleotide in length. In some examples, the spacer is 20-nucleotide in length.
  • the direct repeat sequence may be 23-36-nucleotide in length. In some examples, the direct repeat sequence is 23-nucleotide in length.
  • the target sequence may be within exon 3 or exon 5 of the LDHA gene.
  • the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249).
  • the target sequence may comprise SEQ ID NO: 1248.
  • the spacer sequence may comprise 5′-AGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 1273).
  • the spacer sequence may comprise SEQ ID NO: 1272.
  • the direct repeat sequence may be at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. In some examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. By way of non-limiting example, the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
  • the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225).
  • the RNA guide may comprise SEQ ID NO: 1224.
  • compositions or kits comprising such, or genetically modified cells generated by the gene editing system for use in treating PH in a subject, as well as uses of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such, or genetically modified cells generated by the gene editing system for manufacturing a medicament for treatment of PH in a subject.
  • FIG. 1 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit the LDHA gene in HEK293 cells.
  • the darker grey bars represent target sequences with perfect homology to both rhesus macaque ( Macaca mulatta ) and crab-eating macaque ( Macaca fascicularis ) sequences.
  • FIG. 2 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in HepG2 cells.
  • FIG. 3 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in primary hepatocytes.
  • FIG. 4 is a graph showing knockdown of LDHA mRNA in primary human hepatocytes with a Cas12i2 polypeptide and an LDHA-targeting crRNA, E3T1 (SEQ ID NO: 1214).
  • FIG. 5 A is a graph showing % indels induced by LDHA-targeting crRNAs and the variant Cas12i2 polypeptide of SEQ ID NO: 1168 or SEQ ID NO: 1171 in HepG2 cells.
  • FIG. 5 B shows the size (left) and start position (right) of indels induced in HepG2 cells by the variant Cas12i2 of SEQ ID NO: 1168 and the LDHA-targeting RNA guide of E5T9 (SEQ ID NO: 1224).
  • FIG. 6 is a graph showing % indels induced by chemically modified LDHA-targeting crRNAs of SEQ ID NO: 1267 and SEQ ID NO: 1268 and the variant Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266.
  • FIG. 7 A shows plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E3T1 (SEQ ID NO: 1214), E5T10 (SEQ ID NO: 1225), and EST1 (SEQ ID NO: 1221).
  • the black wedge and centered number represent the fraction of on-target TTISS reads.
  • Each gray wedge represents a unique off-target site identified by TTISS.
  • the size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target.
  • FIG. 7 B shows plots depicting two replicates of TTISS reads for variant Cas12i2 of SEQ ID NO: 1171 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E5T10 (SEQ ID NO: 1225), and E3T1 (SEQ ID NO: 1214).
  • the black wedge and centered number represent the fraction of on-target TTISS reads.
  • Each gray wedge represents a unique off-target site identified by TTISS.
  • the size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target.
  • FIG. 8 is a Western Blot showing knockdown of LDHA protein following electroporation of primary human hepatoyctes with variant Cas12i2 of SEQ ID NO: 1168 and RNA guides E3T1 (SEQ ID NO: 1214), E5T9 (SEQ ID NO: 1224), E5T1 (SEQ ID NO: 1221), or E5T10 (SEQ ID NO: 1225).
  • the present disclosure relates to a system for genetic editing of a lactate dehydrogenase A (LDHA) gene, which comprises (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
  • PAM protospacer adjacent motif
  • a pharmaceutical composition or a kit comprising such system as well as uses thereof.
  • RNA guide that comprises (i) a spacer that is specific to a target sequence in a LDHA gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence as well as uses thereof.
  • PAM protospacer adjacent motif
  • the Cas12i polypeptide for use in the gene editing system disclosed herein may be a Cas12i2 polypeptide, e.g., a wild-type Cas12i polypeptide or a variant thereof as those disclosed herein.
  • the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 922 and comprises one or more mutations relative to SEQ ID NO: 922.
  • the Cas12i polypeptide may be a Cas12i4 polypeptide, which is also disclosed herein.
  • activity refers to a biological activity.
  • activity includes enzymatic activity, e.g., catalytic ability of a Cas12i polypeptide.
  • activity can include nuclease activity.
  • LDHA refers to “lactate dehydrogenase A.”
  • LDHA is an enzyme that catalyzes the inter-conversion of pyruvate and L-lactate with concomitant inter-conversion of NADH and NAD+.
  • LDHA plays roles in development, as well as invasion and metastasis of cancer. Many cancers are characterized by higher LDHA levels than normal tissues.
  • SEQ ID NO: 1172 as set forth herein provides an example of an LDHA gene sequence.
  • Cas12i polypeptide refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Cas12i polypeptide.
  • the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Pat. No. 10,808,245, which is incorporated by reference for the subject matter and purpose referenced herein.
  • a Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with any one of SEQ ID NOs: 8, 2, 11, and 9 of the present application.
  • a Cas12i polypeptide of the disclosure is a Cas12i2 polypeptide as described in WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.
  • the Cas12i polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).
  • the term “complex” refers to a grouping of two or more molecules.
  • the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another.
  • the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Cas12i polypeptide).
  • the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and the complementary region of a target sequence.
  • the term “complex” can refer to a grouping of an LDHA-targeting RNA guide and a Cas12i polypeptide.
  • the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., an LDHA target sequence) to which a complex comprising an RNA guide (e.g., an LDHA-targeting RNA guide) and a Cas12i polypeptide binds.
  • a target sequence e.g., an LDHA target sequence
  • a complex comprising an RNA guide (e.g., an LDHA-targeting RNA guide) and a Cas12i polypeptide binds.
  • the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.”
  • the RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein.
  • the PAM strand is a coding (e.g., sense) strand.
  • the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand.
  • target sequence refers to a DNA fragment adjacent to a PAM motif (on the PAM strand).
  • the complementary region of the target sequence is on the non-PAM strand.
  • a target sequence may be immediately adjacent to the PAM motif.
  • the target sequence and the PAM may be separately by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide).
  • a target sequence may be located at the 3′ end of the PAM motif or at the 5′ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art.
  • a target sequence is located at the 3′ end of a PAM motif for a Cas12i polypeptide (e.g., a Cas12i2 polypeptide such as those disclosed herein).
  • the target sequence is a sequence within an LDHA gene sequence, including, but not limited, to the sequence set forth in SEQ ID NO: 1172.
  • a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides).
  • a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides.
  • a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.
  • the term “spacer” or “spacer sequence” is a portion in an RNA guide that is the RNA equivalent of the target sequence (a DNA sequence).
  • the spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand).
  • Such a spacer is also known as specific to the target sequence.
  • the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference.
  • the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.
  • RNA guide refers to any RNA molecule or a modified RNA molecule that facilitates the targeting of a polypeptide (e.g., a Cas12i polypeptide) described herein to a target sequence (e.g., a sequence of an LDHA gene).
  • a target sequence e.g., a sequence of an LDHA gene.
  • an RNA guide can be a molecule that is designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., an LDHA nucleic acid sequence).
  • An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence.
  • DR direct repeat
  • the RNA guide can be a modified RNA molecule comprising one or more deoxyribonucleotides, for example, in a DNA-binding sequence contained in the RNA guide, which binds a sequence complementary to the target sequence.
  • the DNA-binding sequence may contain a DNA sequence or a DNA/RNA hybrid sequence.
  • CRISPR RNA (crRNA), pre-crRNA and mature crRNA are also used herein to refer to an RNA guide.
  • the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide.
  • first polynucleotide e.g., a spacer sequence of an RNA guide
  • a second polynucleotide e.g., the complementary sequence of a target sequence
  • the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide.
  • the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide.
  • the term “edit” refers to one or more modifications introduced into a target nucleic acid, e.g., within the LDHA gene.
  • the edit can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof.
  • substitution refers to a replacement of a nucleotide or nucleotides with a different nucleotide or nucleotides, relative to a reference sequence.
  • the term “insertion” refers to a gain of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence.
  • the term “deletion” refers to a loss of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence.
  • a sequence comprising a deletion can be synthesized directly from individual nucleotides.
  • a deletion is made by providing and then altering a reference sequence.
  • the nucleic acid sequence can be in a genome of an organism.
  • the nucleic acid sequence can be in a cell.
  • the nucleic acid sequence can be a DNA sequence.
  • the deletion can be a frameshift mutation or a non-frameshift mutation.
  • a deletion described herein refers to a deletion of up to several kilobases.
  • upstream and downstream refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5′ to 3′ direction, respectively, in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3′ end of the first sequence occurs before the 5′ end of the second sequence. A first sequence is downstream of a second sequence when the 5′ end of the first sequence occurs after the 3′ end of the second sequence.
  • the 5′-NTTN-3′ or 5′-TTN-3′ sequence is upstream of an indel described herein, and a Cas12i-induced indel is downstream of the 5′-NTTN-3′ or 5′-TTN-3′ sequence.
  • the present disclosure provides gene editing systems comprising an RNA guide targeting an LDHA gene or a portion of the LDHA gene.
  • a gene editing system can be used to edit the LDHA target gene, e.g., to disrupt the LDHA gene.
  • Lactate dehydrogenase is an enzyme found in nearly every cell that regulates both the homeostasis of lactate and pyruvate, and of glyoxylate and oxalate metabolism.
  • LDH is comprised of 4 polypeptides that form a tetramer. Five isozymes of LDH differing in their subunit composition and tissue distribution have been identified. The two most common forms of LDH are the muscle (M) form encoded by the LDHA gene, and the heart (H) form encoded by LDHB gene.
  • M muscle
  • H heart
  • LDH is the key enzyme responsible for converting glyoxalate to oxalate which is then secreted into the plasma and excreted by the kidneys.
  • reduction of LDHA can reduce hepatic LDH and prevent calcium oxalate crystal deposition.
  • the RNA guide is comprised of a direct repeat component and a spacer component.
  • the RNA guide binds a Cas12i polypeptide.
  • the spacer component is specific to an LDHA target sequence, wherein the LDHA target sequence is adjacent to a 5′-NTTN-3′ or 5′-TTN-3′ PAM sequence as described herein.
  • the RNA guide binds to a first strand of the target (i.e., the non-PAM strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the PAM strand).
  • the present disclosure described herein comprises compositions comprising a complex, wherein the complex comprises an RNA guide targeting LDHA.
  • the present disclosure comprises a complex comprising an RNA guide and a Cas12i polypeptide.
  • the RNA guide and the Cas12i polypeptide bind to each other in a molar ratio of about 1:1.
  • a complex comprising an RNA guide and a Cas12i polypeptide binds to an LDHA target sequence.
  • a complex comprising an RNA guide targeting LDHA and a Cas12i polypeptide binds to an LDHA target sequence at a molar ratio of about 1:1.
  • the complex comprises enzymatic activity, such as nuclease activity, that can cleave the LDHA target sequence.
  • the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229.
  • the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • the present disclosure described herein comprises compositions comprising an RNA guide as described herein and/or an RNA encoding a Cas12i polypeptide as described herein.
  • the RNA guide and the RNA encoding a Cas12i polypeptide are comprised together within the same composition.
  • the RNA guide and the RNA encoding a Cas12i polypeptide are comprised within separate compositions.
  • the RNA guide comprises a direct repeat and/or a spacer sequence described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • Cas12i polypeptides are smaller than other nucleases.
  • Cas12i2 is 1,054 amino acids in length
  • S. pyogenes Cas9 (SpCas9) is 1,368 amino acids in length
  • S. thermophilus Cas9 (StCas9) is 1,128 amino acids in length
  • FnCpf1 is 1,300 amino acids in length
  • AsCpf1 is 1,307 amino acids in length
  • LbCpf1 is 1,246 amino acids in length.
  • Cas12i RNA guides which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides.
  • the smaller Cas12i polypeptide and RNA guide sizes are beneficial for delivery.
  • Compositions comprising a Cas12i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.
  • indels induced by compositions comprising a Cas12i polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide.
  • SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length.
  • Cas12i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of a gene such as LDHA.
  • a system for genetic editing of an LDHA gene which comprises (i) a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a first nucleic acid encoding the Cas12i polypeptide (e.g., a Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166, which may comprise one or more mutations relative to SEQ ID NO: 1166); and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene (e.g., within exon 3 or exon 5 of the LDHA gene), the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′ (5′-NTTN-3′), which is located 5′ to the target sequence.
  • PAM protospacer adjacent motif
  • the gene editing system described herein comprises an RNA guide targeting an LDHA gene, e.g., targeting exon 3 or exon 5 of the LDHA gene. In some embodiments, the gene editing system described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting LDHA.
  • the RNA guide may direct the Cas12i polypeptide contained in the gene editing system as described herein to an LDHA target sequence.
  • Two or more RNA guides may direct two or more separate Cas12i polypeptides (e.g., Cas12i polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) LDHA target sequences.
  • an RNA guide is LDHA target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more LDHA target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).
  • the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.
  • the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule.
  • the spacer and direct repeat sequences are linked directly to one another.
  • a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length.
  • the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.
  • RNA guides Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.
  • the RNA guide comprises a direct repeat sequence.
  • the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).
  • the direct repeat sequence is a sequence of Table 1 or a portion of a sequence of Table 1.
  • the direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 12 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence is set forth in SEQ ID NO: 10.
  • the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.
  • the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.
  • compositions comprising a Cas12i2 polypeptide and an RNA guide comprising the direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides are capable of introducing indels into an LDHA target sequence.
  • Example 1 where indels were measured at seventeen LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HEK293T cells by RNP;
  • Example 2 where indels were measured at four LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HepG2 cells by RNP;
  • Example 3 where indels were measured at three LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 primary hepatocytes by RNP.
  • the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1-10.
  • the direct repeat sequence is a sequence of Table 2 or a portion of a sequence of Table 2.
  • the direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs:
  • the direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 95% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • the direct repeat sequence is at least 90% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is at least 95% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is 100% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200.
  • the direct repeat sequence is a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is the reverse complement
  • the direct repeat sequence is a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1208-1210.
  • a direct repeat sequence described herein comprises a uracil (U). In some embodiments, a direct repeat sequence described herein comprises a thymine (T). In some embodiments, a direct repeat sequence according to Tables 1-4 comprises a sequence comprising a thymine in one or more places indicated as uracil in Tables 1-4.
  • the RNA guide comprises a DNA targeting or spacer sequence.
  • the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence).
  • the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.
  • the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence.
  • the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence.
  • the percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
  • the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
  • the spacer sequence is a sequence of Table 5 or a portion of a sequence of Table 5. It should be understood that an indication of SEQ ID NOs: 588-1164 should be considered as equivalent to a listing of SEQ ID NOs: 588-1164, with each of the intervening numbers present in the listing, i.e., 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 6
  • the spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 5 or a portion of a sequence of Table 5.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of 588-1164.
  • the present disclosure includes all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein.
  • a spacer sequence described herein comprises a uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5.
  • RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g., as set forth in Table 5, above).
  • the sequence of an RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229.
  • an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • exemplary RNA guides provided herein may comprise a spacer sequence of any one of SEQ ID NOs: 1269-1273.
  • the RNA guide may comprise a spacer of SEQ ID NO: 1272.
  • the RNA guide may comprise a spacer of SEQ ID NO: 1269.
  • the RNA guide may comprise a spacer of SEQ ID NO: 1270.
  • the RNA guide may comprise a spacer of SEQ ID NO: 1271.
  • the RNA guide may comprise a spacer of SEQ ID NO: 1273.
  • any of the exemplary RNA guides disclosed herein may comprise a direct sequence of any one of SEQ ID NOs:1-10 or a fragment thereof that is at least 23-nucleotide in length.
  • the direct sequence may comprise SEQ ID NO: 10.
  • the RNA guides provide herein may comprise the nucleotide sequence of SEQ ID NOs: 1214, 1235, 1221, 1224 or 1225.
  • the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1224.
  • the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1214.
  • the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1235.
  • the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1221.
  • the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1225.
  • the RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this invention.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof.
  • Some of the exemplary modifications provided herein are described in detail below.
  • the RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the modification may include a chemical or cellular induced modification.
  • RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to RNA guide-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
  • nucleotide modifications may exist at various positions in the sequence.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased.
  • the sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%,
  • sugar modifications e.g., at the 2′ position or 4′ position
  • replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages.
  • Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone.
  • Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.
  • Various salts, mixed salts and free acid forms are also included.
  • the sequence may be negatively or positively charged.
  • the modified nucleotides which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone).
  • internucleoside linkage e.g., phosphate backbone
  • the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the ⁇ -thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-p
  • internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.
  • the sequence may include one or more cytotoxic nucleosides.
  • cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification.
  • Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4′-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine,
  • Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5′-elaidic acid ester).
  • the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc).
  • the one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).
  • the first isolated nucleic acid comprises messenger RNA (mRNA).
  • the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-p
  • the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-
  • the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoy
  • mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • nucleoside selected from the group consisting of ino
  • the sequence may or may not be uniformly modified along the entire length of the molecule.
  • nucleotides e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the sequence includes a pseudouridine.
  • the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
  • one or more of the nucleotides of an RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.
  • each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification
  • each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification
  • nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.
  • composition or system of the present disclosure includes a Cas12i polypeptide as described in WO/2019/178427, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.
  • the composition of the present disclosure includes a Cas12i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1166 and/or encoded by SEQ ID NO: 1165).
  • the Cas12i2 polypeptide comprises at least one RuvC domain.
  • a nucleic acid sequence encoding the Cas12i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165.
  • the Cas12i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1165.
  • the percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency). See, e.g., Tijssen, “Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation” (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).
  • the Cas12i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165.
  • the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1166.
  • the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1166.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1166 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171.
  • the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1168 or SEQ ID NO: 1171.
  • the Cas12i2 polypeptide may contain one or more mutations relative to SEQ ID NO: 1166, for example, at position D581, G624, F626, P868, I926, V1030, E1035, S1046, or any combination thereof.
  • the one or more mutations are amino acid substitutions, for example, D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • the Cas12i2 polypeptide contains mutations at positions D581, D911, 1926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, D911R, I926R, and V1030G (e.g., SEQ ID NO: 1167). In some examples, the Cas12i2 polypeptide contains mutations at positions D581, I926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, and V1030G (e.g., SEQ ID NO: 1168).
  • the Cas12i2 polypeptide may contain mutations at positions D581, I926, V1030, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, V1030G, and S1046G (e.g., SEQ ID NO: 1169). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046.
  • Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1170).
  • the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046.
  • Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1171).
  • the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171.
  • the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • enzymatic activity e.g., nuclease or endonuclease activity
  • the composition of the present disclosure includes a Cas12i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1202 and/or encoded by SEQ ID NO: 1201).
  • the Cas12i4 polypeptide comprises at least one RuvC domain.
  • a nucleic acid sequence encoding the Cas12i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201.
  • the Cas12i4 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1201.
  • the percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency).
  • the Cas12i4 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201.
  • the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1202.
  • the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1202.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1202 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • the Cas12i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.
  • the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204.
  • a Cas12i4 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate it from its respective parent/reference sequence.
  • the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1203 or SEQ ID NO: 1204 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • composition of the present disclosure includes a Cas12i1 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1211).
  • the Cas12i4 polypeptide comprises at least one RuvC domain.
  • the Cas12i1 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1211.
  • the present disclosure describes a Cas12i1 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1211.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i1 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1211 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • composition of the present disclosure includes a Cas12i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1212).
  • the Cas12i4 polypeptide comprises at least one RuvC domain.
  • the Cas12i3 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1212.
  • the present disclosure describes a Cas12i3 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1212.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Cas12i3 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1212 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • changes to the Cas12i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions.
  • the Cas12i polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a polyhistidine tag (His-tag), Myc, and FLAG.
  • the Cas12i polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • the Cas12i polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.
  • the nucleotide sequence encoding the Cas12i polypeptide described herein can be codon-optimized for use in a particular host cell or organism.
  • the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety.
  • nucleic acid encoding the Cas12i polypeptides such as Cas12i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized.
  • the gene editing system disclosed herein may comprise a Cas12i polypeptide as disclosed herein.
  • the gene editing system may comprise a nucleic acid encoding the Cas12i polypeptide.
  • the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11 and AAV12) encoding the Cas12i polypeptide.
  • the gene editing system may comprise a mRNA molecule encoding the Cas12i polypeptide. In some instances, the mRNA molecule may be codon-optimized.
  • the present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the RNA guide, methods for production of the Cas12i polypeptide, and methods for complexing the RNA guide and Cas12i polypeptide.
  • the RNA guide is made by in vitro transcription of a DNA template.
  • the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence).
  • the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide.
  • the RNA guide is made using chemical synthetic methods.
  • the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide.
  • the plasmid encodes multiple different RNA guides.
  • multiple different plasmids, each encoding a different RNA guide are transfected into the cells.
  • the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Cas12i polypeptide.
  • the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Cas12i polypeptide.
  • the RNA guide is purchased from a commercial vendor.
  • the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.
  • the Cas12i polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Cas12i polypeptide of the present disclosure, isolating the Cas12i polypeptide, optionally, purifying the Cas12i polypeptide, and complexing the Cas12i polypeptide with an RNA guide.
  • the Cas12i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Cas12i polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell.
  • the Cas12i polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.
  • a host cell is used to express the Cas12i polypeptide.
  • the host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli , yeasts (budding yeast, Saccharomyces cerevisiae , and fission yeast, Schizosaccharomyces pombe ), nematodes ( Caenorhabditis elegans ), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells).
  • the method for transferring the expression vector described above into host cells i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.
  • the host cells After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Cas12i polypeptide. After expression of the Cas12i polypeptide, the host cells can be collected and Cas12i polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).
  • the methods for Cas12i polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Cas12i polypeptide.
  • the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Cas12i polypeptide.
  • a variety of methods can be used to determine the level of production of a Cas12i polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Cas12i polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).
  • the present disclosure provides methods of in vivo expression of the Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide, expressing the Cas12i polypeptide in the cell, and obtaining the Cas12i polypeptide from the cell.
  • the present disclosure further provides methods of in vivo expression of a Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide and expressing the Cas12i polypeptide in the cell.
  • the polyribonucleotide encoding the Cas12i polypeptide is delivered to the cell with an RNA guide and, once expressed in the cell, the Cas12i polypeptide and the RNA guide form a complex.
  • the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are delivered to the cell within a single composition. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are comprised within separate compositions. In some embodiments, the host cell is present in a subject, e.g., a human patient.
  • an RNA guide targeting LDHA is complexed with a Cas12i polypeptide to form a ribonucleoprotein.
  • complexation of the RNA guide and Cas12i polypeptide occurs at a temperature lower than about any one of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C.
  • the RNA guide does not dissociate from the Cas12i polypeptide at about 37° C. over an incubation period of at least about any one of 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hr, 2 hr, 3 hr, 4 hr, or more hours.
  • the RNA guide and Cas12i polypeptide are complexed in a complexation buffer.
  • the Cas12i polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide.
  • the Cas12i polypeptide is stored in a complexation buffer.
  • the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.
  • the Cas12i polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein.
  • mRNA or DNA encoding the Cas12i polypeptide is introduced into a cell so that the Cas12i polypeptide is expressed in the cell.
  • the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.
  • the disclosure also provides methods of modifying a target site within the LDHA gene.
  • the methods comprise introducing an LDHA-targeting RNA guide and a Cas12i polypeptide into a cell.
  • the LDHA-targeting RNA guide and Cas12i polypeptide can be introduced as a ribonucleoprotein complex into a cell.
  • the LDHA-targeting RNA guide and Cas12i polypeptide can be introduced on a nucleic acid vector.
  • the Cas12i polypeptide can be introduced as an mRNA.
  • the RNA guide can be introduced directly into the cell.
  • the composition described herein is delivered to a cell/tissue/liver/person to reduce LDHA in the cell/tissue/liver/person.
  • the composition described herein is delivered to a cell/tissue/liver/person to reduce oxalate production in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to correct calcium oxalate crystal deposition in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a person with primary hyperoxaluria.
  • the gene editing system may comprise a RNA guide and a Cas12i2 polypeptide.
  • the RNA guide comprises a spacer sequence specific to a target sequence in the LDHA gene, e.g., specific to a region in exon 3 or exon 5 of the LDHA gene.
  • an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5′-TTN-3′ PAM sequence or 5′-NTTN-3′ PAM sequence.
  • the target sequence is within an LDHA gene or a locus of an LDHA gene (e.g., exon 3 or exon 5), to which the RNA guide can bind via base pairing.
  • a cell has only one copy of the target sequence.
  • a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.
  • the LDHA gene is a mammalian gene. In some embodiments, the LDHA gene is a human gene.
  • the target sequence is within the sequence of SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within an exon of the LDHA gene set forth in SEQ ID NO: 1172, e.g., within a sequence of SEQ ID NO: 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, or 1181 (or a reverse complement thereof).
  • Target sequences within an exon region of the LDHA gene of SEQ ID NO: 1172 are set forth in Table 5.
  • the target sequence is within an intron of the LDHA gene set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the LDHA gene sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). For examples, in some embodiments, the LDHA gene sequence is a non-human LDHA sequence. In some embodiments, the LDHA gene sequence is a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof).
  • the target sequence is adjacent to a 5′-NTTN-3′ PAM sequence or 5′-TTN-3′ PAM sequence, wherein N is any nucleotide.
  • the 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence.
  • the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G.
  • the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • the PAM sequence may be 5′ to the target sequence.
  • the 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence.
  • the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G.
  • the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • the RNA guide is designed to bind to a first strand of a double-stranded target nucleic acid (i.e., the non-PAM strand), and the 5′-NTTN-3′ PAM sequence is present in the second, complementary strand (i.e., the PAM strand).
  • the RNA guide binds to a region on the non-PAM strand that is complementary to a target sequence on the PAM strand, which is adjacent to a 5′-NAAN-3′ sequence.
  • the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5′ or 3′ untranslated region, etc.
  • the target sequence is present in a readily accessible region of the target sequence. In some embodiments, the target sequence is in an exon of a target gene. In some embodiments, the target sequence is across an exon-intron junction of a target gene. In some embodiments, the target sequence is present in a non-coding region, such as a regulatory region of a gene.
  • the Cas12i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Cas12i polypeptide induces one or more DNA double-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA single-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).
  • an RNA guide disclosed herein forms a complex with the Cas12i polypeptide and directs the Cas12i polypeptide to a target sequence adjacent to a 5′-NTTN-3′ sequence.
  • the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5′-NTTN-3′ sequence.
  • the complex induces a deletion adjacent to a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • the complex induces a deletion adjacent to a T/C-rich sequence.
  • the deletion is downstream of a 5′-NTTN-3′ sequence. In some embodiments, the deletion is downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.
  • the deletion alters expression of the LDHA gene. In some embodiments, the deletion alters function of the LDHA gene. In some embodiments, the deletion inactivates the LDHA gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5,
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8,
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion
  • the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g.,
  • the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g
  • the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g
  • the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleo
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleo
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends within about 20 to about 30 nucleotides e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TT
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends within about 25 to about 30 nucleotides e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends e.g., about 22, 23, 24,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TT
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion is up to about 40 nucleotides in length (e.g., about 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 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, or 45 nucleotides).
  • the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 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, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).
  • the methods described herein are used to engineer a cell comprising a deletion as described herein in an LDHA gene.
  • the methods are carried out using a complex comprising a Cas12i enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229.
  • an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is synthetic or purified RNA. In some embodiments, the Cas12i polypeptide is encoded in a plasmid. In some embodiments, the Cas12i polypeptide is encoded by an RNA that is synthetic or purified.
  • Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.).
  • a carrier such as a carrier and/or a polymeric carrier, e.g., a liposome
  • transfection e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers
  • electroporation or other methods of membrane disruption e.g., nucleofection
  • viral delivery e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus,
  • the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Cas12i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed.
  • nucleic acids e.g., nucleic acids encoding the Cas12i polypeptide, RNA guide, donor DNA, etc.
  • a pre-formed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed.
  • an RNA guide and an RNA encoding a Cas12i polypeptide are delivered together in a single composition.
  • an RNA guide and an RNA encoding a Cas12i polypeptide are delivered in separate compositions.
  • an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using different delivery technologies.
  • Exemplary intracellular delivery methods include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection.
  • viruses such as AAV, or virus-like agents
  • chemical-based transfection methods such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethyleni
  • a lipid nanoparticle comprises an mRNA encoding a Cas12i polypeptide, an RNA guide, or an mRNA encoding a Cas12i polypeptide and an RNA guide.
  • the mRNA encoding the Cas12i polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 1165 or SEQ ID NO: 1201 or a variant thereof.
  • the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
  • the cell is an isolated cell.
  • the cell is in cell culture or a co-culture of two or more cell types.
  • the cell is ex vivo.
  • the cell is obtained from a living organism and maintained in a cell culture.
  • the cell is a single-cellular organism.
  • the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.
  • the cell is derived from a cell line.
  • a wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).
  • the cell is an immortal or immortalized cell.
  • the cell is a primary cell.
  • the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell.
  • the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC.
  • the cell is a differentiated cell.
  • the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex
  • the cell is a terminally differentiated cell.
  • the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell.
  • the cell is an immune cell.
  • the immune cell is a T cell.
  • the immune cell is a B cell.
  • the immune cell is a Natural Killer (NK) cell.
  • the immune cell is a Tumor Infiltrating Lymphocyte (TIL).
  • the cell is a mammalian cell, e.g., a human cell or a murine cell.
  • the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model.
  • the cell is a cell within a living tissue, organ, or organism.
  • modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure.
  • modified cells may comprise a disrupted LDHA gene.
  • compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy.
  • Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject.
  • the disease or condition is primary hyperoxaluria (PH).
  • the PH is PH1, PH2, or PH3.
  • Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing an LDHA sequence as disclosed herein.
  • a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.
  • any of the gene editing systems or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease that is associated with the LDHA gene, for example, primary hyperoxaluria (PH).
  • PH primary hyperoxaluria
  • the PH is PH1, PH2, or PH3.
  • the target disease is PH1.
  • PH is a rare genetic disorder effecting subjects of all ages from infants to elderly. PH includes three subtypes involving genetic defects that alter the expression of three distinct proteins. PH1 involves alanine-glyoxylate aminotransferase, or AGT/AGT1. PH2 involves glyoxylate/hydroxypyruvate reductase, or GR/HPR, and PH3 involves 4-hydroxy-2-oxoglutarate aldolase, or HOGA.
  • excess oxalate can also combine with calcium to form calcium oxalate in the kidney and other organs.
  • Deposits of calcium oxalate can produce widespread deposition of calcium oxalate (nephrocalcinosis) or formation of kidney and bladder stones (urolithiasis) and lead to kidney damage.
  • Common kidney complications in PH1 include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PH1 may begin to fail, and levels of oxalate may rise in the blood.
  • oxalate in tissues throughout the body may occur due to high blood levels of oxalate and can lead to complications in bone, skin, and eye.
  • Patients with PH1 normally have kidney failure at an early age, with renal dialysis or dual kidney/liver organ transplant as the only treatment options.
  • a method for treating a target disease as disclosed herein comprising administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein.
  • the gene editing system may be delivered to a specific tissue or specific type of cells where the gene edit is needed.
  • the gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof.
  • Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers.
  • modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of the treatment.
  • the modified cells may comprise a substitution, insertion, and/or deletion described herein.
  • the modified cells may include a cell line modified by a CRISPR nuclease, reverse transcriptase polypeptide, and editing template RNA (e.g., RNA guide and RT donor RNA).
  • the modified cells may be a heterogenous population comprising cells with different types of gene edits.
  • the modified cells may comprise a substantially homogenous cell population (e.g., at least 80% of the cells in the whole population) comprising one particular gene edit in the LDHA gene.
  • the cells can be suspended in a suitable media.
  • compositions comprising the gene editing system or components thereof.
  • a composition can be a pharmaceutical composition.
  • a pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • a pharmaceutical composition comprising the gene editing system or components thereof as described herein may be administered to a subject in need thereof, e.g., one who suffers from a liver disease associated with the LDHA gene.
  • the gene editing system or components thereof may be delivered to specific cells or tissue (e.g., to liver cells), where the gene editing system could function to genetically modify the LDHA gene in such cells.
  • a formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • kits that can be used, for example, to carry out a method described herein for genetical modification of the LDHA gene.
  • the kits include an RNA guide and a Cas12i polypeptide.
  • the kits include a polynucleotide that encodes such a Cas12i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein.
  • the Cas12i polypeptide and the RNA guide e.g., as a ribonucleoprotein
  • the kits can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Cas12i polypeptide.
  • the kit may be useful for research purposes.
  • the kit may be useful to study gene function.
  • Embodiment 1 A composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or complete complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.
  • RNA guide comprises (i) a spacer sequence that is substantially complementary or complete complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.
  • PAM protospacer adjacent motif
  • the target sequence may be within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene.
  • the LDHA gene comprises the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of
  • the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one
  • the direct repeat sequence may comprise: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleo
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 118
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205;
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 12
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleot
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • the PAM may comprise the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • the target sequence is immediately adjacent to the PAM sequence.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229.
  • the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 2 The composition of Embodiment 1 may further comprise a Cas12i polypeptide or a polyribonucleotide encoding a Cas12i polypeptide, which can be one of the following: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO
  • the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
  • the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex.
  • the ribonucleoprotein complex binds a target nucleic acid.
  • the composition is present within a cell.
  • the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector.
  • the RNA guide and the Cas12i polypeptide are encoded in a single vector.
  • the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.
  • Embodiment 3 A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide.
  • the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide.
  • the vectors may be expression vectors.
  • Embodiment 4 A composition comprising an RNA guide and a Cas12i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.
  • the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence
  • the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence that is at
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleo
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 118
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205;
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 12
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleot
  • the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.
  • PAM protospacer adjacent motif
  • the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • the target sequence is immediately adjacent to the PAM sequence. In some examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.
  • the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
  • the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex.
  • the ribonucleoprotein complex binds a target nucleic acid.
  • the composition may be present within a cell.
  • the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector.
  • the RNA guide and the Cas12i polypeptide are encoded in a single vector.
  • the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.
  • Embodiment 5 A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide.
  • the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide.
  • the vectors are expression vectors.
  • Embodiment 6 An RNA guide comprising (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.
  • the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence
  • the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence that is at
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleo
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 118
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205;
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 12
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleot
  • the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • the target sequence may be adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′, wherein N is any nucleotide.
  • PAM protospacer adjacent motif
  • the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • the target sequence is immediately adjacent to the PAM sequence. In other examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 7 A nucleic acid encoding an RNA guide as described herein.
  • Embodiment 8 A vector comprising such an RNA guide as described herein.
  • Embodiment 9 A cell comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.
  • the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • Embodiment 10 A kit comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.
  • Embodiment 11 A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with a composition or an RNA guide as described herein. In some examples, the method is carried out in vitro. In other examples, the method is carried out ex vivo.
  • the LDHA sequence is in a cell.
  • the composition or the RNA guide induces a deletion in the LDHA sequence.
  • the deletion is adjacent to a 5′-NTTN-3′ sequence, wherein N is any nucleotide.
  • the deletion is downstream of the 5′-NTTN-3′ sequence.
  • the deletion is up to about 40 nucleotides in length. In some instances, the deletion is from about 4 nucleotides to 40 nucleotides, about 4 nucleotides to 25 nucleotides, about 10 nucleotides to 25 nucleotides, or about 10 nucleotides to 15 nucleotides in length.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, or about 25 nucleotides to about 30 nucleotides of the 5′-NTTN-3′ sequence.
  • the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • the 5′-NTTN-3′ sequence is 5′-CTTT-3′, 5′-CTTC-3′, 5′-GTTT-3′, 5′-GTTC-3′, 5′-TTTC-3′, 5′-GTTA-3′, or 5′-GTTG-3′.
  • the deletion overlaps with a mutation in the LDHA sequence. In some instances, the deletion overlaps with an insertion in the LDHA sequence. In some instances, the deletion removes a repeat expansion of the LDHA sequence or a portion thereof. In some instances, the deletion disrupts one or both alleles of the LDHA sequence.
  • the RNA guide may comprise the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 12 A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering a composition, an RNA guide, or a cell described herein to the subject.
  • PH primary hyperoxaluria
  • the RNA guide and/or the polyribonucleotide encoding the Cas12i polypeptide are comprised within a lipid nanoparticle. In some examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within the same lipid nanoparticle. In other examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within separate lipid nanoparticles.
  • Embodiment 13 An RNA guide comprising (i) a spacer sequence that is complementary to a target site within an LDHA gene (the target site being on the non-PAM strand and complementary to a target sequence), and (ii) a direct repeat sequence, wherein the target sequence is any one of SEQ ID NOs: 1237, 1239, 1248, 1245, or 1249, or the reverse complement thereof.
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence that is at
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleo
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 118
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205;
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 12
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through
  • the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleot
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225.
  • each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1254-1263. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1254-1263.
  • an LDHA-targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprises any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248.
  • the LDHA-targeting RNA guide of SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245.
  • the LDHA-targeting RNA guide of SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249.
  • Embodiment 14 A nucleic acid encoding an RNA guide as described herein.
  • Embodiment 15 A vector comprising the nucleic acid as described herein.
  • Embodiment 16 A vector system comprising one or more vectors encoding (i) the RNA guide of Embodiment 13 as described herein and (ii) a Cas12i polypeptide.
  • the vector system comprises a first vector encoding the RNA guide and a second vector encoding the Cas12i polypeptide.
  • Embodiment 17 A cell comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein.
  • the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • Embodiment 18 A kit comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein.
  • Embodiment 19 A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with an RNA guide of Embodiment 13 as described herein.
  • the LDHA sequence is in a cell.
  • the RNA guide induces an indel (e.g., an insertion or deletion) in the LDHA sequence.
  • Embodiment 20 A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering the RNA guide of Embodiment 13 as described herein to the subject.
  • PH primary hyperoxaluria
  • This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HEK293T cells.
  • Cas12i2 RNA guides were designed and ordered from Integrated DNA Technologies (IDT). For initial guide screening in HEK293T cells, target sequences were designed by tiling the coding exons of LDHA for 5′-NTTN-3′ PAM sequences, and then spacer sequences were designed for the 20-bp target sequences downstream of the PAM sequence.
  • the LDHA-targeting RNA guide sequences are shown in Table 7. TS stands for “top strand” of the LDHA gene, and BS stands for “bottom strand” of the LDHA gene. In the figures, “E #T #” can also be represented as “exon #target #.”
  • Cas12i2 RNP complexation reactions were made by mixing purified Cas12i2 polypeptide (400 ⁇ M) with crRNA (1 mM in 250 mM NaCl) at a 1:1 (Cas12i2:crRNA) volume ratio (2.5:1 crRNA:Cas12i2 molar ratio). Complexations were incubated on ice for 30-60 min.
  • HEK293T cells were harvested using TRYPLETM (recombinant cell-dissociation enzymes; Thermo Fisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTORTM X KIT S; Lonza #V4XC-2032) at a concentration of 16,480 cells/ ⁇ L. Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips.
  • TRYPLETM recombinant cell-dissociation enzymes
  • the strips were electroporated using an electroporation device (program CM-130, Lonza 4D-NUCLEOFECTORTM). Immediately following electroporation, 80 ⁇ L of pre-warmed DMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 ⁇ L (30,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 ⁇ L DMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO 2 .
  • NGS Next Generation Sequencing
  • the indel mapping function used a sample's fastq file, the amplicon reference sequence, and the forward primer sequence.
  • a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence.
  • the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well.
  • Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion.
  • the % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000).
  • the QC standard for the minimum number of reads passing filters was 10,000.
  • FIG. 1 shows LDHA indels in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Delivery of E3T1 (SEQ ID NO: 1214), E3T9 (SEQ ID NO: 1216), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), and E5T10 (SEQ ID NO: 1225) resulted in indels in over 70% of the NGS reads. Therefore, LDHA-targeting RNA guides induced indels in exon 2, exon 3, and exon 5 in HEK293T cells.
  • E3T1 SEQ ID NO: 1214
  • E3T9 SEQ ID NO: 1216
  • EST1 SEQ ID NO: 1221
  • E5T9 SEQ ID NO: 1224
  • E5T10 SEQ ID NO: 1225
  • This Example thus shows that LDHA can be individually targeted by Cas12i2 RNPs in mammalian cells such as HEK293T cells.
  • This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HepG2 cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 7.
  • HepG2 cells were harvested using TRYPLETM (recombinant cell-dissociation enzymes; ThermoFisher) and counted.
  • Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTORTM X KIT S; Lonza #V4XC-2032) at a concentration of 13,889 cells/ ⁇ L. Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips.
  • the strips were electroporated using an electroporation device (program DJ-100, Lonza 4D-NUCLEOFECTORTM). Immediately following electroporation, 80 ⁇ L of pre-warmed EMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 ⁇ L (25,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 ⁇ L EMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO 2 .
  • FIG. 2 shows LDHA indels in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Therefore, LDHA-targeting RNA guides induced indels in exon 3 and exon 5 in HepG2 cells.
  • This Example describes the genomic editing of the LDHA using Cas12i2 introduced into primary hepatocytes cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with RNA guides of Table 7.
  • Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath.
  • the cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged at 100 g for 10 minutes.
  • the cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermofisher CM3000).
  • the cells were subjected to trypan blue viability count with an INCUCYTE® disposable hemocytometer (Fisher scientific, 22-600-100). The cells were then washed in PBS and resuspended in P3 buffer+supplement (P3 PRIMARY CELL 4D-NUCLEOFECTORTM X Kit; Lonza, VXP-3032) at a concentration of ⁇ 7,500 cells/ ⁇ L. Resuspended cells were dispensed at 150,000 cells/reaction into the 16 well Lonza NUCLEOCUVETTE strips or 500,000 cells/reaction into the single Lonza NUCLEOCUVETTES® for the mRNA readout.
  • P3 buffer+supplement P3 PRIMARY CELL 4D-NUCLEOFECTORTM X Kit
  • Resuspended cells were dispensed at 150,000 cells/reaction into the 16 well Lonza NUCLEOCUVETTE strips or 500,000 cells/reaction into the single Lonza NUCLEOCUVETTES® for the mRNA
  • Complexed Cas12i2 RNP was added to each reaction at a final concentration of 20 ⁇ M (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 ⁇ M.
  • the final volume of each electroporated reaction was either 20 ⁇ L in the 16 well nucleocuvette strip format or 100 ⁇ L in the single nucleocuvette format. Non-targeting guides were used as negative controls.
  • the strips were electroporated using DS-150 program, while the single nucleocuvettes were electroporated using CA137 program (Lonza 4D-NUCLEOFECTORTM).
  • pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting.
  • the media was changed to hepatocyte maintenance media (Williams' Medium E, Thermofisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. Fresh hepatocyte maintenance media was replaced after 2 days.
  • RNA readout For the mRNA readout, cell pellets were frozen at ⁇ 80° C. and subsequently resuspended in lysis buffer and DNA/RNA extracted with the RNeasy kit (Qiagen) following manufacturer's instructions. The DNA extracted from the samples were analyzed by NGS. The RNA isolated was checked for quantity and purity using nanodrop, and subsequently used for cDNA synthesis using 5 ⁇ iScript reverse transcription reaction mix (Bio-Rad laboratories), following manufacturer's recommendations. cDNA templated was appropriately diluted to be in linear range of the subsequent analysis.
  • Diluted cDNA was used to set up a 20 ⁇ L Digital Droplet PCR (ddPCR-BioRad laboratories) reaction using target-specific primer and probe for LDHA, TTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAAC TCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTC TTAATTTGGTCSEQ ID NO: 1264), and 2 ⁇ ddPCR Supermix for Probes No dUTP (BioRad laboratories) following manufacturer's instructions.
  • the reaction was used to generate droplets using Automated Droplet Generator (BioRad Laboratories), following manufacture's recommendations.
  • the plate was sealed using PX1 PCR Plate Sealer (BioRad Laboratories) generated droplets were subjected to PCR amplification using C1000 Touch Thermal Cycler (BioRad Laboratories) using conditions recommended by the manufacturer.
  • the PCR amplified droplets were read on QX200 Droplet Reader (BioRad Laboratories) and the acquired data was analyzed using QX Manager version 1.2 (BioRad Laboratories) to determine presence of absolute copy number of mRNA present in each reaction for the appropriate targets.
  • each RNA guide tested induced indels within and/or adjacent to the LDHA target sites. Indels were not induced with the non-targeting control. Therefore, LDHA-targeting RNA guides induced indels in primary hepatocytes. Indels for RNA guide E3T1 were then correlated with mRNA levels to determine whether indels led to mRNA knockdown and subsequent protein knockdown.
  • FIG. 4 shows % mRNA knockdown of LDHA in edited cells compared to unedited control cells. RNA guide E3T1 resulted in knockdown of LDHA mRNA.
  • LDHA can be targeted by Cas12i2 RNPs in mammalian cells such as primary human hepatocytes.
  • This Example describes indel assessment on LDHA target sites using variants introduced into HepG2 cells by transient transfection.
  • the Cas12i2 variants of SEQ ID NO: 1168 and SEQ ID NO: 1171 were individually cloned into a pcda3.1 backbone (Invitrogen). Nucleic acids encoding RNA guides E3T1, E3T3, EST1, E5T9, and E5T10 (Table 7) were cloned into a pUC19 backbone (New England Biolabs). The plasmids were then maxi-prepped and diluted.
  • HepG2 cells were harvested using TRYPLETM (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTORTM X KIT S; Lonza #V4XC-2032).
  • TRYPLETM recombinant cell-dissociation enzymes
  • FIG. 5 A shows the indel size frequency (left) and indel start position relative to the PAM for E5T9 and the variant Cas12i2 of SEQ ID NO: 1168 in HepG2 cells. As shown on the left, deletions ranged in size from 1 nucleotide to about 40 nucleotides.
  • the majority of the deletions were about 8 nucleotides to about 23 nucleotides in length. As shown on the right, the target sequence is represented as starting at position 0 and ending at position 20. Indels started within about 5 nucleotides and about 35 nucleotides downstream of the PAM sequence. The majority of indels started about 10 nucleotides to about 30 nucleotides downstream of the PAM sequence.
  • this Example shows that LDHA is capable of being targeted by multiple Cas12i2 polypeptides.
  • This Example describes indel assessment on LDHA target sites via delivery of Cas12i2 mRNA and chemically modified LDHA-targeting RNA guides.
  • mRNA sequences corresponding to the variant Cas12i2 sequence of SEQ ID NO: 1168 and the variant Cas12i2 sequence of SEQ ID NO: 1171 were synthesized by Aldeveron with 1-pseudo-U modified nucleotides and using CleanCap® Reagent AG (TriLink Biotechnologies).
  • the Cas12i2 mRNA sequences, shown in Table 8, further comprised a C-terminal NLS.
  • Cas12i2 RNA guides were designed and ordered from Integrated DNA Technologies (IDT) as having 3′ end modified phosphorothioated 2′ O-methyl bases or 5′ end and 3′ end modified phosphorothioated 2′ O-methyl bases guides, as specified in Table 9.
  • IDTT Integrated DNA Technologies
  • Each variant Cas12i2 mRNA was mixed with a crRNA at a 1:1 (Cas12i2:crRNA) volume ratio (1050:1 crRNA:Cas12i2 molar ratio). The mRNA and crRNA were mixed immediately before electroporation.
  • the primary human hepatocyte cells were cultured and electroporated as described in Example 3.
  • RNA Guide Sequence 3′ end modified AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA*mA*mC* E3T1 mU (SEQ ID NO: 1267) 5′ and 3′ end mA*mG*mA*AAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA* modified E3T1 mA*mC*mU (SEQ ID NO: 1268)
  • FIG. 6 shows editing of an LDHA target site by a variant Cas12i2 mRNA and 3′ end modified E3T1 (SEQ ID NO: 1267) or 5′ and 3′ end modified E3T1 (SEQ ID NO: 1268) RNA guide.
  • Indels in the LDHA target site were introduced following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266 and either the RNA guide of SEQ ID NO: 1267 or SEQ ID NO: 1268.
  • NGS reads A higher percentage of NGS reads exhibited indels for RNA guide E3T1 with 5′ and 3′ end modifications (SEQ ID NO: 1268) compared to NGS reads for RNA guide with 3′ end modifications only (SEQ ID NO: 1267). Approximately 50% of NGS reads comprised indels following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1266 and the RNA guide of SEQ ID NO: 1268.
  • This Example thus shows that LDHA can be targeted by Cas12i2 mRNA constructs and chemically modified RNA guides in mammalian cells.
  • This Example describes on-target versus off-target assessment of a Cas12i2 variant and an LDHA-targeting RNA guide.
  • HEK293T cells were transfected with a plasmid encoding the variant Cas12i2 of SEQ ID NO: 1168 or the variant Cas12i2 of SEQ ID NO: 1171 and a plasmid encoding E3T1 (SEQ ID NO: 1214), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), or E5T10 (SEQ ID NO: 1225) according the method described in Example 16 of PCT/US21/25257.
  • the tagmentation-based tag integration site sequencing (TTISS) method described in Example 16 of PCT/US21/25257 was then carried out.
  • FIG. 7 A and FIG. 7 B show plots depicting on-target and off-target TTISS reads.
  • the black wedge and centered number represent the fraction of on-target TTISS reads.
  • Each grey wedge represents a unique off-target site identified by TTISS.
  • the size of each grey wedge represents the fraction of TTISS reads mapping to a given off-target site.
  • FIG. 7 A shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1168
  • FIG. 7 B shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1171.
  • variant Cas12i2 of SEQ ID NO: 1168 paired with E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on-target. No TTISS reads mapped to potential off-target sites.
  • E3T1 and E5T10 also showed a low likelihood of off-target editing.
  • E3T1 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads.
  • E5T10 97% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 3% of TTISS reads.
  • E5T1 demonstrated a higher likelihood of off-target editing using the TTISS method.
  • variant Cas12i2 of SEQ ID NO: 1171 paired with the E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads in replicate 1 and 93% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 7% of TTISS reads in replicate 2.
  • E5T10 also showed a low likelihood of off-target editing; 92% of TTISS reads in replicate 1 and 100% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 8% of TTISS reads in replicate 1.
  • Variant Cas12i2 of SEQ ID NO: 1171 paired with the E3T1 demonstrated a higher likelihood of off-target editing. 86% and 93% of TTISS reads mapping to the on-target in replicate 1 and replicate 2, respectively. 5 potential off-target sites represented the remaining 14% of TTISS reads in replicate 1, and 2 potential off-target sites represented the remaining 7% off TTISS reads in replicate 2 for E3T1.
  • compositions comprising Cas12i2 and LDHA-targeting RNA guides comprise different off-target activity profiles.
  • This Example describes use of a Western Blot to identify knockdown of LDHA protein using variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides.
  • hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath.
  • the cells were added to pre-warmed hepatocyte recovery media (Thermo Fisher, CM7000) and centrifuged at 100 g for 10 minutes.
  • the cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermo Fisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermo Fisher CM3000).
  • the cells were subjected to trypan blue viability count with an Inucyte disposable hemocytometer (Fisher scientific, 22-600-100).
  • the cells were then washed in PBS and resuspended in P3 buffer+supplement (Lonza, VXP-3032) at a concentration of ⁇ 5000 cells/ ⁇ L. Resuspended cells were dispensed at 500,000 cells/reaction into Lonza electroporation cuvettes
  • E3T1 SEQ ID NO: 1214
  • E5T9 SEQ ID NO: 1224
  • E5T1 SEQ ID NO: 1221
  • E5T10 SEQ ID NO: 1225
  • RNPs were added to each reaction at a final concentration of 20 ⁇ M (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 Unelectroporated cells and cells electroporated without cargo were used as negative controls.
  • the strips were electroporated using an electroporation device (program CA137, Lonza 4D-nucleofector). Immediately following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 500,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 24-well plate (Thermo Fisher) with wells containing Hepatocyte plating medium. The cells were then incubated at 37° C. The media was changed to hepatocyte maintenance media (Williams' Medium E, Thermo Fisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermo Fisher CM 4000) after the cells attached after 24 hours. Fresh hepatocyte maintenance media was replaced every 48 hours.
  • hepatocyte maintenance media Woodiams' Medium E, Thermo Fisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermo Fisher CM 4000
  • Samples were run on a 4-15% TGX gel (BioRad 5671084) at 200V for 45 minutes. Samples were transferred to a 0.2 um nitrocellulose membrane (BioRad 1704159) using the Trans Blot Turbo System. The membrane was blocked in Intercept TBS Blocking Buffer (Li-cor 927-60001) for 30 minutes at room temperature. The blot was then incubated in a 1:1000 dilution of primary anti-LDHA antibody (Abcam ab52488) and 1:2500 dilution of primary anti-vinculin antibody (Sigma V9131) in blocking buffer at 4 C overnight.
  • the blot was washed three times with TBST (Thermo Fisher 28360) for 5 minutes each, then incubated with a 1:12500 dilution of IR680 anti-mouse (Thermo Fisher PI35518) and IR800 anti-rabbit secondary antibodies (Thermo Fisher PISA535571) in TBST for 1 hour at room temperature. The blot was then washed three times with TBST for 5 minutes each and visualized on the Li-cor Odyssey CLX.
  • LDHA protein (monomer and dimer) was observed in primary human hepatocytes at Day 7 post editing by Cas12i2 RNPs targeting the LDHA gene ( FIG. 8 ). This knockdown was seen across each of the four RNA guides, E3T1, E5T9, EST1, and E5T10 (lanes 1-8). LDHA knockdown was not observed for the buffer only (lanes 9 and 10) or unelectroporated controls (lanes 11 and 12).
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Abstract

Provided herein are gene editing systems and/or compositions comprising RNA guides targeting LDHA for use in genetic editing of the LDHA gene. Also provide herein are methods of using the gene editing system for introducing edits to the LDHA gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/197,067, filed Jun. 4, 2021, U.S. Provisional Application No. 63/225,214, filed Jul. 23, 2021, U.S. Provisional Application No. 63/292,912, filed Dec. 22, 2021, and U.S. Provisional Application No. 63/300,743, filed Jan. 19, 2022, the contents of each of which are incorporated by reference herein in their entirety.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 3, 2022, is named 116928-0036-0003US00_SEQ.txt and is 381,734 bytes in size.
    • BACKGROUND
  • Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements.
    • SUMMARY OF THE INVENTION
  • The present disclosure is based, at least in part, on the development of a system for genetic editing of a lactate dehydrogenase A (LDHA) gene. The system involves a Cas12i polypeptide such as a Cas12i2 polypeptide and an RNA guide mediating cleavage at a genetic site within the LDHA gene by the CRISPR nuclease polypeptide. As reported herein, the gene editing system disclosed herein has achieved successful editing of LDHA gene with high editing efficiency and accuracy.
  • Without being bound by theory, the gene editing system disclosed herein may exhibit one or more of the following advantageous features. Compared to SpCas9 and Cas12a, Cas12i effectors are smaller (1033 to 1093aa) which, in conjunction with their short mature crRNA (40-43 nt), is preferable in terms of delivery and cost of synthesis. Cas12i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage. Cas12i PAM sequences also differ from those of Cas9. Therefore, larger and different portions of genetic sites of interest can be disrupted with a Cas12i polypeptide and RNA guide compared to Cas9. Using an unbiased approach of tagmentation-based tag integration site sequencing (TTISS), more potential off-target sites with a higher number of unique integration events were identified for SpCas9 compared to Cas12i2. See WO/2021/202800. Therefore, Cas12i such as Cas12i2 may be more specific than Cas9.
  • Accordingly, provided herein are gene editing systems for editing LDHA gene, pharmaceutical compositions or kits comprising such, methods of using the gene editing systems to produce genetically modified cells, and the resultant cells thus produced. Also provided herein are uses of the gene editing systems disclosed herein, the pharmaceutical compositions and kits comprising such, and/or the genetically modified cells thus produced for treating primary hyperoxaluria (PH) in a subject.
  • In some aspects, the present disclosure features system for genetic editing of a hydroxyacid oxidase 1 (LDHA) gene, comprising (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, and (ii) an RNA guide or a second nucleic acid encoding the RNA guide. The RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
  • In some embodiments, the Cas12i is a Cas12i2 polypeptide. In other embodiments, the Cas12i is a Cas12i4 polypeptide.
  • In some embodiments, the Cas12i polypeptide is a Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1166. In some instances, the Cas12i2 polypeptide may comprise one or more mutations relative to SEQ ID NO: 1166. In some examples, the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO: 1166. In some examples, the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • In one example, the Cas12i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In yet another example, the Cas12i2 polypeptide comprises mutations at positions D581, I926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G). In still another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G). In another example, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).
  • Exemplary Cas12i2 polypeptides for use in any of the gene editing systems disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 1167-1171. In one example, the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1168. In another example, the exemplary Cas12i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1171.
  • In some embodiments, the gene editing system may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide). In some instances, the first nucleic acid is located in a first vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector). In some instances, the first nucleic acid is a messenger RNA (mRNA). In some instances, the coding sequence for the Cas12i polypeptide is codon optimized.
  • In some embodiments, the target sequence may be within exon 1 or exon 2 of the LDHA gene. In some examples, the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249). In some examples, the target sequence may comprise SEQ ID NO: 1248.
  • In some embodiments, the spacer sequence may be 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length. In some examples, the spacer sequence comprises 5′-UAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1273). In some examples, the spacer sequence may comprise SEQ ID NO: 1272.
  • In some embodiments, the RNA guide comprises the spacer and a direct repeat sequence. In some examples, the direct repeat sequence is 23-36-nucleotide in length. In one example, the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. In some specific examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. By way of non-limiting example, the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
  • In specific examples, the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225). In one example, the RNA guide may comprise SEQ ID NO: 1224.
  • In some embodiments, the system may comprise the second nucleic acid encoding the RNA guide. In some examples, the nucleic acid encoding the RNA guide may be located in a viral vector. In some examples, the viral vector comprises the both the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) and the second nucleic acid encoding the RNA guide.
  • In some embodiments, any of the systems described herein may comprise the first nucleic acid encoding the Cas12i polypeptide (e.g., the Cas12i2 polypeptide), which is located in a first vector, and the second nucleic acid encoding the RNA guide, which is located on a second vector. In some examples, the first and/or second vector is a viral vector. In some specific examples, the first and second vectors are the same vector.
  • In some embodiments, any of the systems described herein may comprise one or more lipid nanoparticles (LNPs), which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, the RNA guide or the second nucleic acid encoding the RNA guide, or both.
  • In some embodiments, the system described herein may comprise a LNP, which encompass the Cas12i polypeptide (e.g., the Cas12i2 polypeptide) or the first nucleic acid encoding the Cas12i polypeptide, and a viral vector comprising the second nucleic acid encoding the RNA guide. In some examples, the viral vector is an AAV vector. In other embodiments, the system described herein may comprise a LNP, which encompass the RNA guide or the second nucleic acid encoding the RNA guide, and a viral vector comprising the first nucleic acid encoding the Cas12i polypeptide. In some examples, the viral vector is an AAV vector.
  • In some aspects, the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein, and a kit comprising the components of the gene editing system.
  • In other aspects, the present disclosure also features a method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with any of the systems disclosed herein to genetically edit the LDHA gene in the host cell. In some examples, the host cell is cultured in vitro. In other examples, the contacting step is performed by administering the system for editing the LDHA gene to a subject comprising the host cell.
  • Also within the scope of the present disclosure is a cell comprising a disrupted a lactate dehydrogenase A (LDHA) gene, which can be produced by contacting a host cell with the system disclosed herein genetically edit the LDHA gene in the host cell.
  • Still in other aspects, the present disclosure provides a method for treating primary hyperoxaluria (PH) in a subject. The method may comprise administering to a subject in need thereof any of the systems for editing a lactate dehydrogenase A (LDHA) gene or any of the cells disclosed herein.
  • In some embodiments, the subject may be a human patient having the PH. In some examples, the PH is PH1, PH2, or PH3. In a specific example, the PH is PH1.
  • Also provided herein is an RNA guide, comprising (i) a spacer sequence as disclosed herein that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence.
  • In some embodiments, the spacer may be 20-30-nucleotide in length. In some examples, the spacer is 20-nucleotide in length.
  • In some embodiments, the direct repeat sequence may be 23-36-nucleotide in length. In some examples, the direct repeat sequence is 23-nucleotide in length.
  • In some embodiments, the target sequence may be within exon 3 or exon 5 of the LDHA gene. In some examples, the target sequence comprises 5′-TAGGACTTGGCAGATGAACT-3′ (SEQ ID NO: 1237), 5′-GATGACATCAACAAGAGCAA-3′ (SEQ ID NO: 1239), 5′-TTCATAGTGGATATCTTGAC-3′ (SEQ ID NO: 1245), 5′-TCATAGTGGATATCTTGACC-3′ (SEQ ID NO: 1248), or 5′-CATAGTGGATATCTTGACCT-3′ (SEQ ID NO: 1249). In some examples, the target sequence may comprise SEQ ID NO: 1248.
  • In some embodiments, the spacer sequence may comprise 5′-AGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1269); 5′-GAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1270); 5′-UUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1271); 5′-UCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1272); or 5′-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 1273). In some examples, the spacer sequence may comprise SEQ ID NO: 1272.
  • In some embodiments, the direct repeat sequence may be at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length. In some examples, the direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. By way of non-limiting example, the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
  • In some embodiments, the RNA guide may comprise the nucleotide sequence of 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′ (SEQ ID NO: 1214), 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′ (SEQ ID NO: 1235), 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′ (SEQ ID NO: 1221), 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′ (SEQ ID NO: 1224), or 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′ (SEQ ID NO: 1225). In some examples, the RNA guide may comprise SEQ ID NO: 1224.
  • Also provided herein are any of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such, or genetically modified cells generated by the gene editing system for use in treating PH in a subject, as well as uses of the gene editing systems disclosed herein, pharmaceutical compositions or kits comprising such, or genetically modified cells generated by the gene editing system for manufacturing a medicament for treatment of PH in a subject.
  • The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit the LDHA gene in HEK293 cells. The darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaca mulatta) and crab-eating macaque (Macaca fascicularis) sequences.
  • FIG. 2 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in HepG2 cells.
  • FIG. 3 is a graph showing the ability of RNPs prepared with a Cas12i2 polypeptide and a crRNA to edit LDHA target sequences in primary hepatocytes.
  • FIG. 4 is a graph showing knockdown of LDHA mRNA in primary human hepatocytes with a Cas12i2 polypeptide and an LDHA-targeting crRNA, E3T1 (SEQ ID NO: 1214).
  • FIG. 5A is a graph showing % indels induced by LDHA-targeting crRNAs and the variant Cas12i2 polypeptide of SEQ ID NO: 1168 or SEQ ID NO: 1171 in HepG2 cells. FIG. 5B shows the size (left) and start position (right) of indels induced in HepG2 cells by the variant Cas12i2 of SEQ ID NO: 1168 and the LDHA-targeting RNA guide of E5T9 (SEQ ID NO: 1224).
  • FIG. 6 is a graph showing % indels induced by chemically modified LDHA-targeting crRNAs of SEQ ID NO: 1267 and SEQ ID NO: 1268 and the variant Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266.
  • FIG. 7A shows plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E3T1 (SEQ ID NO: 1214), E5T10 (SEQ ID NO: 1225), and EST1 (SEQ ID NO: 1221). The black wedge and centered number represent the fraction of on-target TTISS reads. Each gray wedge represents a unique off-target site identified by TTISS. The size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target. FIG. 7B shows plots depicting two replicates of TTISS reads for variant Cas12i2 of SEQ ID NO: 1171 and LDHA-targeting RNA guides E5T9 (SEQ ID NO: 1224), E5T10 (SEQ ID NO: 1225), and E3T1 (SEQ ID NO: 1214). The black wedge and centered number represent the fraction of on-target TTISS reads. Each gray wedge represents a unique off-target site identified by TTISS. The size of each gray wedge represents the fraction of TTISS reads mapping to a given off-target.
  • FIG. 8 is a Western Blot showing knockdown of LDHA protein following electroporation of primary human hepatoyctes with variant Cas12i2 of SEQ ID NO: 1168 and RNA guides E3T1 (SEQ ID NO: 1214), E5T9 (SEQ ID NO: 1224), E5T1 (SEQ ID NO: 1221), or E5T10 (SEQ ID NO: 1225).
    •  DETAILED DESCRIPTION
  • The present disclosure relates to a system for genetic editing of a lactate dehydrogenase A (LDHA) gene, which comprises (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence. Also provided in the present disclosure are a pharmaceutical composition or a kit comprising such system as well as uses thereof. Further disclosed herein are a method for editing a LDHA gene in a cell, a cell so produced that comprises a disrupted a LDHA gene, a method of treating primary hyperoxaluria (PH) in a subject, and an RNA guide that comprises (i) a spacer that is specific to a target sequence in a LDHA gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence as well as uses thereof.
  • The Cas12i polypeptide for use in the gene editing system disclosed herein may be a Cas12i2 polypeptide, e.g., a wild-type Cas12i polypeptide or a variant thereof as those disclosed herein. In some examples, the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 922 and comprises one or more mutations relative to SEQ ID NO: 922. In other examples, the Cas12i polypeptide may be a Cas12i4 polypeptide, which is also disclosed herein.
    • Definitions
  • The present disclosure will be described with respect to particular embodiments and with reference to certain Figures, but the disclosure is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.
  • As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of a Cas12i polypeptide. For example, activity can include nuclease activity.
  • As used herein the term “LDHA” refers to “lactate dehydrogenase A.” LDHA is an enzyme that catalyzes the inter-conversion of pyruvate and L-lactate with concomitant inter-conversion of NADH and NAD+. LDHA plays roles in development, as well as invasion and metastasis of cancer. Many cancers are characterized by higher LDHA levels than normal tissues. SEQ ID NO: 1172 as set forth herein provides an example of an LDHA gene sequence.
  • As used herein, the term “Cas12i polypeptide” (also referred to herein as Cas12i) refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Cas12i polypeptide. In some embodiments, the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Pat. No. 10,808,245, which is incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, a Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with any one of SEQ ID NOs: 8, 2, 11, and 9 of the present application. In some embodiments, a Cas12i polypeptide of the disclosure is a Cas12i2 polypeptide as described in WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, the Cas12i polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).
  • As used herein, the term “complex” refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. For example, the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Cas12i polypeptide). Alternatively, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and the complementary region of a target sequence. In another example, the term “complex” can refer to a grouping of an LDHA-targeting RNA guide and a Cas12i polypeptide.
  • As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., an LDHA target sequence) to which a complex comprising an RNA guide (e.g., an LDHA-targeting RNA guide) and a Cas12i polypeptide binds. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein. In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand.
  • As used herein, the term “target sequence” refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separately by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3′ end of the PAM motif or at the 5′ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art. For example, a target sequence is located at the 3′ end of a PAM motif for a Cas12i polypeptide (e.g., a Cas12i2 polypeptide such as those disclosed herein). In some embodiments, the target sequence is a sequence within an LDHA gene sequence, including, but not limited, to the sequence set forth in SEQ ID NO: 1172.
  • As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.
  • As used herein, the term “spacer” or “spacer sequence” is a portion in an RNA guide that is the RNA equivalent of the target sequence (a DNA sequence). The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.
  • As used herein, the term “RNA guide” or “RNA guide sequence” refers to any RNA molecule or a modified RNA molecule that facilitates the targeting of a polypeptide (e.g., a Cas12i polypeptide) described herein to a target sequence (e.g., a sequence of an LDHA gene). For example, an RNA guide can be a molecule that is designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., an LDHA nucleic acid sequence). An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence. In some instances, the RNA guide can be a modified RNA molecule comprising one or more deoxyribonucleotides, for example, in a DNA-binding sequence contained in the RNA guide, which binds a sequence complementary to the target sequence. In some examples, the DNA-binding sequence may contain a DNA sequence or a DNA/RNA hybrid sequence. The terms CRISPR RNA (crRNA), pre-crRNA and mature crRNA are also used herein to refer to an RNA guide.
  • As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide.
  • The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • As used herein, the term “edit” refers to one or more modifications introduced into a target nucleic acid, e.g., within the LDHA gene. The edit can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof. As used herein, the term “substitution” refers to a replacement of a nucleotide or nucleotides with a different nucleotide or nucleotides, relative to a reference sequence. As used herein, the term “insertion” refers to a gain of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence. As used herein, the term “deletion” refers to a loss of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence.
  • No particular process is implied in how to make a sequence comprising a deletion. For instance, a sequence comprising a deletion can be synthesized directly from individual nucleotides. In other embodiments, a deletion is made by providing and then altering a reference sequence. The nucleic acid sequence can be in a genome of an organism. The nucleic acid sequence can be in a cell. The nucleic acid sequence can be a DNA sequence. The deletion can be a frameshift mutation or a non-frameshift mutation. A deletion described herein refers to a deletion of up to several kilobases.
  • As used herein, the terms “upstream” and “downstream” refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5′ to 3′ direction, respectively, in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3′ end of the first sequence occurs before the 5′ end of the second sequence. A first sequence is downstream of a second sequence when the 5′ end of the first sequence occurs after the 3′ end of the second sequence. In some embodiments, the 5′-NTTN-3′ or 5′-TTN-3′ sequence is upstream of an indel described herein, and a Cas12i-induced indel is downstream of the 5′-NTTN-3′ or 5′-TTN-3′ sequence.
    • I. Gene Editing Systems
  • In some aspects, the present disclosure provides gene editing systems comprising an RNA guide targeting an LDHA gene or a portion of the LDHA gene. Such a gene editing system can be used to edit the LDHA target gene, e.g., to disrupt the LDHA gene.
  • Lactate dehydrogenase (LDH) is an enzyme found in nearly every cell that regulates both the homeostasis of lactate and pyruvate, and of glyoxylate and oxalate metabolism. LDH is comprised of 4 polypeptides that form a tetramer. Five isozymes of LDH differing in their subunit composition and tissue distribution have been identified. The two most common forms of LDH are the muscle (M) form encoded by the LDHA gene, and the heart (H) form encoded by LDHB gene. In the perioxisome of liver cells, LDH is the key enzyme responsible for converting glyoxalate to oxalate which is then secreted into the plasma and excreted by the kidneys. As LDH is key in the final step of oxalate production, reduction of LDHA can reduce hepatic LDH and prevent calcium oxalate crystal deposition.
  • In some embodiments, the RNA guide is comprised of a direct repeat component and a spacer component. In some embodiments, the RNA guide binds a Cas12i polypeptide. In some embodiments, the spacer component is specific to an LDHA target sequence, wherein the LDHA target sequence is adjacent to a 5′-NTTN-3′ or 5′-TTN-3′ PAM sequence as described herein. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the non-PAM strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the PAM strand).
  • In some embodiments, the present disclosure described herein comprises compositions comprising a complex, wherein the complex comprises an RNA guide targeting LDHA. In some embodiments, the present disclosure comprises a complex comprising an RNA guide and a Cas12i polypeptide. In some embodiments, the RNA guide and the Cas12i polypeptide bind to each other in a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide and a Cas12i polypeptide binds to an LDHA target sequence. In some embodiments, a complex comprising an RNA guide targeting LDHA and a Cas12i polypeptide binds to an LDHA target sequence at a molar ratio of about 1:1. In some embodiments, the complex comprises enzymatic activity, such as nuclease activity, that can cleave the LDHA target sequence. The RNA guide, the Cas12i polypeptide, and the LDHA target sequence, either alone or together, do not naturally occur. In some embodiments, the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • In some embodiments, the present disclosure described herein comprises compositions comprising an RNA guide as described herein and/or an RNA encoding a Cas12i polypeptide as described herein. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are comprised together within the same composition. In some embodiments, the RNA guide and the RNA encoding a Cas12i polypeptide are comprised within separate compositions. In some embodiments, the RNA guide comprises a direct repeat and/or a spacer sequence described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • Use of the gene editing systems disclosed herein has advantages over those of other known nuclease systems. Cas12i polypeptides are smaller than other nucleases. For example, Cas12i2 is 1,054 amino acids in length, whereas S. pyogenes Cas9 (SpCas9) is 1,368 amino acids in length, S. thermophilus Cas9 (StCas9) is 1,128 amino acids in length, FnCpf1 is 1,300 amino acids in length, AsCpf1 is 1,307 amino acids in length, and LbCpf1 is 1,246 amino acids in length. Cas12i RNA guides, which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides. The smaller Cas12i polypeptide and RNA guide sizes are beneficial for delivery. Compositions comprising a Cas12i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See WO/2021/202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Furthermore, indels induced by compositions comprising a Cas12i polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide. For example, SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length. However, Cas12i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of a gene such as LDHA.
  • Also provided herein is a system for genetic editing of an LDHA gene, which comprises (i) a Cas12i polypeptide (e.g., a Cas12i2 polypeptide) or a first nucleic acid encoding the Cas12i polypeptide (e.g., a Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166, which may comprise one or more mutations relative to SEQ ID NO: 1166); and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene (e.g., within exon 3 or exon 5 of the LDHA gene), the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′ (5′-NTTN-3′), which is located 5′ to the target sequence.
    • A. RNA Guides
  • In some embodiments, the gene editing system described herein comprises an RNA guide targeting an LDHA gene, e.g., targeting exon 3 or exon 5 of the LDHA gene. In some embodiments, the gene editing system described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting LDHA.
  • The RNA guide may direct the Cas12i polypeptide contained in the gene editing system as described herein to an LDHA target sequence. Two or more RNA guides may direct two or more separate Cas12i polypeptides (e.g., Cas12i polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) LDHA target sequences.
  • Those skilled in the art reading the below examples of particular kinds of RNA guides will understand that, in some embodiments, an RNA guide is LDHA target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more LDHA target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).
  • In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.
  • In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. In some embodiments, the spacer and direct repeat sequences are linked directly to one another. In some embodiments, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.
  • Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.
    • (i). Direct Repeat
  • In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).
  • In some embodiments, the direct repeat sequence is a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.
  • In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.
  • In some embodiments, compositions comprising a Cas12i2 polypeptide and an RNA guide comprising the direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides are capable of introducing indels into an LDHA target sequence. See, e.g., Example 1, where indels were measured at seventeen LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HEK293T cells by RNP; Example 2, where indels were measured at four LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 to HepG2 cells by RNP; and Example 3, where indels were measured at three LDHA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1168 primary hepatocytes by RNP.
  • In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1-10.
  • TABLE 1
    Cas12i2 Direct Repeat Sequences
    Sequence
    Identifier Direct Repeat Sequence
    SEQ ID NO: 1 GUUGCAAAACCCAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 2 AAUAGCGGCCCUAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 3 AUUGGAACUGGCGAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 4 CCAGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 5 CGGCGCUCGAAUAGGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 6 GUGGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 7 GUUGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 8 GUUGCAAUGCCUAAGAAAUCCGUCUUUCAUUGACGG
    SEQ ID NO: 9 GCAACACCUAAGAAAUCCGUCUUUCAUUGACGGG
    SEQ ID NO: 10 AGAAAUCCGUCUUUCAUUGACGG
  • In some embodiments, the direct repeat sequence is a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs:
  • 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 95% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 95% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, or 1199.
  • In some embodiments, the direct repeat sequence is at least 90% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is at least 95% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200. In some embodiments, the direct repeat sequence is 100% identical to SEQ ID NO: 1200 or a portion of SEQ ID NO: 1200.
  • TABLE 2
    Cas12i4 Direct Repeat Sequences
    Sequence
    Identifier Direct Repeat Sequence
    SEQ ID NO: UCUCAACGAUAGUCAGACAUGUGUCCUCAGUGACAC
    1182
    SEQ ID NO: UUUUAACAACACUCAGGCAUGUGUCCACAGUGACAC
    1183
    SEQ ID NO: UUGAACGGAUACUCAGACAUGUGUUUCCAGUGACAC
    1184
    SEQ ID NO: UGCCCUCAAUAGUCAGAUGUGUGUCCACAGUGACAC
    1185
    SEQ ID NO: UCUCAAUGAUACUUAGAUACGUGUCCUCAGUGACAC
    1186
    SEQ ID NO: UCUCAAUGAUACUCAGACAUGUGUCCCCAGUGACAC
    1187
    SEQ ID NO: UCUCAAUGAUACUAAGACAUGUGUCCUCAGUGACAC
    1188
    SEQ ID NO: UCUCAACUAUACUCAGACAUGUGUCCUCAGUGACAC
    1189
    SEQ ID NO: UCUCAACGAUACUCAGACAUGUGUCCUCAGUGACAC
    1190
    SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCUCAGCGACAC
    1191
    SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCCCAGUGACAC
    1192
    SEQ ID NO: UCUCAACGAUACUAAGAUAUGUGUCCACAGUGACAC
    1193
    SEQ ID NO: UCUCAACAAUACUCAGACAUGUGUCCCCAGUGACAC
    1194
    SEQ ID NO: UCUCAACAAUACUAAGGCAUGUGUCCCCAGUGACCC
    1195
    SEQ ID NO: UCUCAAAGAUACUCAGACACGUGUCCCCAGUGACAC
    1196
    SEQ ID NO: UCUCAAAAAUACUCAGACAUGUGUCCUCAGUGACAC
    1197
    SEQ ID NO: GCGAAACAACAGUCAGACAUGUGUCCCCAGUGACAC
    1198
    SEQ ID NO: CCUCAACGAUAUUAAGACAUGUGUCCGCAGUGACAC
    1199
    SEQ ID NO: AGACAUGUGUCCUCAGUGACAC
    1200
  • In some embodiments, the direct repeat sequence is a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1205-1207. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1205-1207.
  • TABLE 3
    Cas12il Direct Repeat Sequences
    Sequence Direct Repeat
    Identifier Sequence
    SEQ ID NO: 1205 GUUGGAAUGACUAAUUUUUGUGC
    CCACCGUUGGCAC
    SEQ ID NO: 1206 AAUUUUUGUGCCCAUCGUUGGCAC
    SEQ ID NO: 1207 AUUUUUGUGCCCAUCGUUGGCAC
  • In some embodiments, the direct repeat sequence is a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1208-1210. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1208-1210.
  • TABLE 4
    Cas12i3 Direct Repeat Sequences
    Sequence Direct Repeat
    Identifier Sequence
    SEQ ID NO: 1208 CUAGCAAUGACCUAAUAGUGUG
    UCCUUAGUUGACAU
    SEQ ID NO: 1209 CCUACAAUACCUAAGAAAUCCG
    UCCUAAGUUGACGG
    SEQ ID NO: 1210 AUAGUGUGUCCUUAGUUGACAU
  • In some embodiments, a direct repeat sequence described herein comprises a uracil (U). In some embodiments, a direct repeat sequence described herein comprises a thymine (T). In some embodiments, a direct repeat sequence according to Tables 1-4 comprises a sequence comprising a thymine in one or more places indicated as uracil in Tables 1-4.
    • (ii). Spacer Sequence
  • In some embodiments, the RNA guide comprises a DNA targeting or spacer sequence. In some embodiments, the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence). In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.
  • In some embodiments, the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence. In some embodiments, the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • In some embodiments, the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence. In some embodiments, the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a region on the non-PAM strand that is complementary to the target. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
  • In some embodiments, the spacer sequence is a sequence of Table 5 or a portion of a sequence of Table 5. It should be understood that an indication of SEQ ID NOs: 588-1164 should be considered as equivalent to a listing of SEQ ID NOs: 588-1164, with each of the intervening numbers present in the listing, i.e., 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, and 1164.
  • The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.
  • In some embodiments, the spacer sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 5 or a portion of a sequence of Table 5. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of 588-1164.
  • TABLE 5
    Target and Spacer Sequences
    SEQ SEQ
    ID ID
    LDHA Strand PAM* NO Target Sequence NO Spacer Sequence
    LDHA_exon1 + ATTC 11 CGGATCTCATTGCCAC 588 CGGAUCUCAUUGCCACG
    GCGCCCCCGACGAC CGCCCCCGACGAC
    LDHA_exon1 + ATTG 12 CCACGCGCCCCCGACG 589 CCACGCGCCCCCGACGA
    ACCGCCCGACGTGC CCGCCCGACGUGC
    LDHA_exon1 + ATTC 13 CCGGTACGGTAGGGCC 590 CCGGUACGGUAGGGCCC
    CTGCGCGCACGGCG UGCGCGCACGGCG
    LDHA_exon2 + CTTG 14 CTGTAGGAGCCGGAGT 591 CUGUAGGAGCCGGAGUA
    AGCTCAGAGTGATC GCUCAGAGUGAUC
    LDHA_exon2 + OTTA 15 CACCCAAACGTCGATA 592 CACCCAAACGUCGAUAU
    TTCCTTTTCCACGC UCCUUUUCCACGC
    LDHA_exon2 + GTTA 16 ATAAACCGCGATGGGT 593 AUAAACCGCGAUGGGUG
    GAACCCTCAGGAGG AACCCUCAGGAGG
    LDHA_exon2 + CTTG 17 GGGTTAATAAACCGCG 594 GGGUUAAUAAACCGCGA
    ATGGGTGAACCCTC UGGGUGAACCCUC
    LDHA_exon2 + TTTA 18 CTTGAGAAGCCTGGCT 595 CUUGAGAAGCCUGGCUG
    GTGTCCTTGCTGTA UGUCCUUGCUGUA
    LDHA_exon2 + GTTT 19 ACTTGAGAAGCCTGGC 596 ACUUGAGAAGCCUGGCU
    TGTGTCCTTGCTGT GUGUCCUUGCUGU
    LDHA_exon2 + TTTC 20 TGCACGTATCTCTGGT 597 UGCACGUAUCUCUGGUG
    GTTTACTTGAGAAG UUUACUUGAGAAG
    LDHA_exon2 + TTTT 21 CTGCACGTATCTCTGG 598 CUGCACGUAUCUCUGGU
    TGTTTACTTGAGAA GUUUACUUGAGAA
    LDHA_exon2 + GTTA 22 ATGGCTTTTCTGCACG 599 AUGGCUUUUCUGCACGU
    TATCTCTGGTGTTT AUCUCUGGUGUUU
    LDHA_exon2 + ATTC 23 CTTTTCCACGCTAAGG 600 CUUUUCCACGCUAAGGU
    TATGGGCCTTCACT AUGGGCCUUCACU
    LDHA_exon2 + TTTG 24 TGGCAGTTAATGGCTT 601 UGGCAGUUAAUGGCUUU
    TTCTGCACGTATCT UCUGCACGUAUCU
    LDHA_exon2 + GTTT 25 GTGGCAGTTAATGGCT 602 GUGGCAGUUAAUGGCUU
    TTTCTGCACGTATC UUCUGCACGUAUC
    LDHA_exon2 + CTTG 26 AGCTTTGTGGCAGTTA 603 AGCUUUGUGGCAGUUAA
    ATGGCTTTTCTGCA UGGCUUUUCUGCA
    LDHA_exon2 + CTTG 27 GGCTTGAGCTTTGTGG 604 GGCUUGAGCUUUGUGGC
    CAGTTAATGGCTTT AGUUAAUGGCUUU
    LDHA_exon2 + TTTC 28 CGAGCGGGAAGGAGAG 605 CGAGCGGGAAGGAGAGC
    CCACAAAGCGCGCA CACAAAGCGCGCA
    LDHA_exon2 + CTTG 29 AGAAGCCTGGCTGTGT 606 AGAAGCCUGGCUGUGUC
    CCTTGCTGTAGGAG CUUGCUGUAGGAG
    LDHA_exon2 + CTTT 30 TCTGCACGTATCTCTG 607 UCUGCACGUAUCUCUGG
    GTGTTTACTTGAGA UGUUUACUUGAGA
    LDHA_exon2 + CTTG 31 TCTGAGGAAAGGCCAG 608 UCUGAGGAAAGGCCAGC
    CCCCACTTGGGGTT CCCACUUGGGGUU
    LDHA_exon2 + TTTT 32 CCACGCTAAGGTATGG 609 CCACGCUAAGGUAUGGG
    GCCTTCACTCTTCA CCUUCACUCUUCA
    LDHA_exon2 + TTTC 33 CGCCCACCTTTCCGAG 610 CGCCCACCUUUCCGAGC
    CGGGAAGGAGAGCC GGGAAGGAGAGCC
    LDHA_exon2 − CTTC 34 CCGCTCGGAAAGGTGG 611 CCGCUCGGAAAGGUGGG
    GCGGAAATCAGACT CGGAAAUCAGACU
    LDHA_exon2 − TTTG 35 TGGCTCTCCTTCCCGC 612 UGGCUCUCCUUCCCGCU
    TCGGAAAGGTGGGC CGGAAAGGUGGGC
    LDHA_exon2 − CTTT 36 GTGGCTCTCCTTCCCG 613 GUGGCUCUCCUUCCCGC
    CTCGGAAAGGTGGG UCGGAAAGGUGGG
    LDHA_exon2 − ATTA 37 ACTGCCACAAAGCTCA 614 ACUGCCACAAAGCUCAA
    AGCCCAAGGCACAG GCCCAAGGCACAG
    LDHA_exon2 − CTTC 38 TCAAGTAAACACCAGA 615 UCAAGUAAACACCAGAG
    GATACGTGCAGAAA AUACGUGCAGAAA
    LDHA_exon2 − TTTC 39 CTCAGACAAGATCACT 616 CUCAGACAAGAUCACUC
    CTGAGCTACTCCGG UGAGCUACUCCGG
    LDHA_exon2 − CTTT 40 CCTCAGACAAGATCAC 617 CCUCAGACAAGAUCACU
    TCTGAGCTACTCCG CUGAGCUACUCCG
    LDHA_exon2 − ATTA 41 ACCCCAAGTGGGGCTG 618 ACCCCAAGUGGGGCUGG
    GCCTTTCCTCAGAC CCUUUCCUCAGAC
    LDHA_exon2 − TTTA 42 TTAACCCCAAGTGGGG 619 UUAACCCCAAGUGGGGC
    CTGGCCTTTCCTCA UGGCCUUUCCUCA
    LDHA_exon2 − GTTT 43 ATTAACCCCAAGTGGG 620 AUUAACCCCAAGUGGGG
    GCTGGCCTTTCCTC CUGGCCUUUCCUC
    LDHA_exon2 − GTTC 44 ACCCATCGCGGTTTAT 621 ACCCAUCGCGGUUUAUU
    TAACCCCAAGTGGG AACCCCAAGUGGG
    LDHA_exon2 − TTTG 45 GGTGTAAGTATAGCCT 622 GGUGUAAGUAUAGCCUC
    CCTGAGGGTTCACC CUGAGGGUUCACC
    LDHA_exon2 − GTTT 46 GGGTGTAAGTATAGCC 623 GGGUGUAAGUAUAGCCU
    TCCTGAGGGTTCAC CCUGAGGGUUCAC
    LDHA_exon2 − CTTA 47 GCGTGGAAAAGGAATA 624 GCGUGGAAAAGGAAUAU
    TCGACGTTTGGGTG CGACGUUUGGGUG
    LDHA_exon2 + TTTC 48 CACGCTAAGGTATGGG 625 CACGCUAAGGUAUGGGC
    CCTTCACTCTTCAC CUUCACUCUUCAC
    LDHA_exon2 + GTTT 49 TCCACGCTAAGGTATG 626 UCCACGCUAAGGUAUGG
    GGCCTTCACTCTTC GCCUUCACUCUUC
    LDHA_exon2 + ATTT 50 CCGCCCACCTTTCCGA 627 CCGCCCACCUUUCCGAG
    GCGGGAAGGAGAGC CGGGAAGGAGAGC
    LDHA_exon2 + GTTT 51 CCGAGCGGGAAGGAGA 628 CCGAGCGGGAAGGAGAG
    GCCACAAAGCGCGC CCACAAAGCGCGC
    LDHA_exon2 + ATTA 52 GTCTGATTTCCGCCCA 629 GUCUGAUUUCCGCCCAC
    CCTTTCCGAGCGGG CUUUCCGAGCGGG
    LDHA_exon2 + CTTC 53 ACAGACCCTGTCATTA 630 ACAGACCCUGUCAUUAG
    GGCCT GCCU
    LDHA_exon2 + CTTC 54 ACTCTTCACAGACCCT 631 ACUCUUCACAGACCCUG
    GTCATTAGGCCT UCAUUAGGCCU
    LDHA_exon3 − ATTT 55 AGTGTCACTACAGCTT 632 AGUGUCACUACAGCUUC
    CTTTAATGTTTATT UUUAAUGUUUAUU
    LDHA_exon3 + GTTG 56 TTGGGGTTGGTGCTGT 633 UUGGGGUUGGUGCUGUU
    TGGCATGGCCTGTG GGCAUGGCCUGUG
    LDHA_exon3 + CTTC 57 TAAAGGAAGAACAGAC 634 UAAAGGAAGAACAGACC
    CCCCCAGAATAAGA CCCCAGAAUAAGA
    LDHA_exon3 + TTTA 58 TAATCTTCTAAAGGAA 635 UAAUCUUCUAAAGGAAG
    GAACAGACCCCCCA AACAGACCCCCCA
    LDHA_exon3 + ATTT 59 ATAATCTTCTAAAGGA 636 AUAAUCUUCUAAAGGAA
    AGAACAGACCCCCC GAACAGACCCCCC
    LDHA_exon3 + GTTC 60 CAAGTCCAATATGGCA 637 CAAGUCCAAUAUGGCAA
    ACTCTAAAGGATCA CUCUAAAGGAUCA
    LDHA_exon3 + TTTG 61 GTTCCAAGTCCAATAT 638 GUUCCAAGUCCAAUAUG
    GGCAACTCTAAAGG GCAACUCUAAAGG
    LDHA_exon3 + TTTT 62 GGTTCCAAGTCCAATA 639 GGUUCCAAGUCCAAUAU
    TGGCAACTCTAAAG GGCAACUCUAAAG
    LDHA_exon3 + CTTT 63 TGGTTCCAAGTCCAAT 640 UGGUUCCAAGUCCAAUA
    ATGGCAACTCTAAA UGGCAACUCUAAA
    LDHA_exon3 + ATTC 64 CTTTTGGTTCCAAGTC 641 CUUUUGGUUCCAAGUCC
    CAATATGGCAACTC AAUAUGGCAACUC
    LDHA_exon3 + TTTC 65 CTCCTATAGATTCCTT 642 CUCCUAUAGAUUCCUUU
    TTGGTTCCAAGTCC UGGUUCCAAGUCC
    LDHA_exon3 + TTTT 66 CCTCCTATAGATTCCT 643 CCUCCUAUAGAUUCCUU
    TTTGGTTCCAAGTC UUGGUUCCAAGUC
    LDHA_exon3 + TTTT 67 TCCTCCTATAGATTCC 644 UCCUCCUAUAGAUUCCU
    TTTTGGTTCCAAGT UUUGGUUCCAAGU
    LDHA_exon3 + GTTT 68 TTCCTCCTATAGATTC 645 UUCCUCCUAUAGAUUCC
    CTTTTGGTTCCAAG UUUUGGUUCCAAG
    LDHA_exon3 + ATTA 69 AAGAAGCTGTAGTGAC 646 AAGAAGCUGUAGUGACA
    ACTAAATGTTTTTC CUAAAUGUUUUUC
    LDHA_exon3 + GTTG 70 GGGTTGGTGCTGTTGG 647 GGGUUGGUGCUGUUGGC
    CATGGCCTGTGCCA AUGGCCUGUGCCA
    LDHA_exon3 + GTTG 71 GTGCTGTTGGCATGGC 648 GUGCUGUUGGCAUGGCC
    CTGTGCCATCAGTA UGUGCCAUCAGUA
    LDHA_exon3 + ATTA 72 CAGTTGTTGGGGTTGG 649 CAGUUGUUGGGGUUGGU
    TGCTGTTGGCATGG GCUGUUGGCAUGG
    LDHA_exon3 + CTTA 73 ATGAAGGTAAGTGAGA 650 AUGAAGGUAAGUGAGAG
    GTCTACCACACTGG UCUACCACACUGG
    LDHA_exon3 − GTTG 74 GAACCAAAAGGAATCT 651 GAACCAAAAGGAAUCUA
    ATAGGAGGAAAAAC UAGGAGGAAAAAC
    LDHA_exon3 − ATTG 75 GACTTGGAACCAAAAG 652 GACUUGGAACCAAAAGG
    GAATCTATAGGAGG AAUCUAUAGGAGG
    LDHA_exon3 − GTTG 76 CCATATTGGACTTGGA 653 CCAUAUUGGACUUGGAA
    ACCAAAAGGAATCT CCAAAAGGAAUCU
    LDHA_exon3 + GTTG 77 GCATGGCCTGTGCCAT 654 GCAUGGCCUGUGCCAUC
    CAGTATCTTAATGA AGUAUCUUAAUGA
    LDHA_exon3 − CTTT 78 AGAGTTGCCATATTGG 655 AGAGUUGCCAUAUUGGA
    ACTTGGAACCAAAA CUUGGAACCAAAA
    LDHA_exon3 − ATTA 79 TAAATCAGCTGATCCT 656 UAAAUCAGCUGAUCCUU
    TTAGAGTTGCCATA UAGAGUUGCCAUA
    LDHA_exon3 − TTTA 80 GAAGATTATAAATCAG 657 GAAGAUUAUAAAUCAGC
    CTGATCCTTTAGAG UGAUCCUUUAGAG
    LDHA_exon3 − CTTT 81 AGAAGATTATAAATCA 658 AGAAGAUUAUAAAUCAG
    GCTGATCCTTTAGA CUGAUCCUUUAGA
    LDHA_exon3 − TTTA 82 GAGTTGCCATATTGGA 659 GAGUUGCCAUAUUGGAC
    CTTGGAACCAAAAG UUGGAACCAAAAG
    LDHA_exon3 − GTTC 83 TTCCTTTAGAAGATTA 660 UUCCUUUAGAAGAUUAU
    TAAATCAGCTGATC AAAUCAGCUGAUC
    LDHA_exon3 − ATTO 84 TGGGGGGTCTGTTCTT 661 UGGGGGGUCUGUUCUUC
    CCTTTAGAAGATTA CUUUAGAAGAUUA
    LDHA_exon3 − CTTA 85 TTCTGGGGGGTCTGTT 662 UUCUGGGGGGUCUGUUC
    CTTCCTTTAGAAGA UUCCUUUAGAAGA
    LDHA_exon3 − ATTA 86 AGATACTGATGGCACA 663 AGAUACUGAUGGCACAG
    GGCCATGCCAACAG GCCAUGCCAACAG
    LDHA_exon3 − CTTC 87 ATTAAGATACTGATGG 664 AUUAAGAUACUGAUGGC
    CACAGGCCATGCCA ACAGGCCAUGCCA
    LDHA_exon3 − CTTA 88 CCTTCATTAAGATACT 665 CCUUCAUUAAGAUACUG
    GATGGCACAGGCCA AUGGCACAGGCCA
    LDHA_exon3 − CTTC 89 CAGTGTGGTAGACTCT 666 CAGUGUGGUAGACUCUC
    CACTTACCTTCATT ACUUACCUUCAUU
    LDHA_exon3 − CTTC 90 CTTTAGAAGATTATAA 667 CUUUAGAAGAUUAUAAA
    ATCAGCTGATCCTT UCAGCUGAUCCUU
    LDHA_exon3 − TTTA 91 GTGTCACTACAGCTTC 668 GUGUCACUACAGCUUCU
    TTTAATGTTTATT UUAAUGUUUAUU
    LDHA_exon4 − GTTC 92 TAAGGAAAAGGCTGCC 669 UAAGGAAAAGGCUGCCA
    ATGTTGGAGATCCA UGUUGGAGAUCCA
    LDHA_exon4 − GTTG 93 GAGATCCATCATCTCT 670 GAGAUCCAUCAUCUCUC
    CCCTTCAATTTGTC CCUUCAAUUUGUC
    LDHA_exon4 − CTTC 94 AATTTGTCTTCGATGA 671 AAUUUGUCUUCGAUGAC
    CATCAACAAGAGCA AUCAACAAGAGCA
    LDHA_exon4 − GTTC 95 ATCTGCCAAGTCCTAA 672 AUCUGCCAAGUCCUAAA
    AAGACATCAAATCT AGACAUCAAAUCU
    LDHA_exon4 − TTTG 96 TCTTCGATGACATCAA 673 UCUUCGAUGACAUCAAC
    CAAGAGCAAGTTCA AAGAGCAAGUUCA
    LDHA_exon4 − CTTC 97 GATGACATCAACAAGA 674 GAUGACAUCAACAAGAG
    GCAAGTTCATCTGC CAAGUUCAUCUGC
    LDHA_exon4 − CTTT 98 AGTTAAATGGAAAATT 675 AGUUAAAUGGAAAAUUG
    GCCACTTCTAGATT CCACUUCUAGAUU
    LDHA_exon4 − ATTT 99 GTCTTCGATGACATCA 676 GUCUUCGAUGACAUCAA
    ACAAGAGCAAGTTC CAAGAGCAAGUUC
    LDHA_exon4 − CTTT 100 GGTGTTCTAAGGAAAA 677 GGUGUUCUAAGGAAAAG
    GGCTGCCATGTTGG GCUGCCAUGUUGG
    LDHA_exon4 − TTTG 101 GTGTTCTAAGGAAAAG 678 GUGUUCUAAGGAAAAGG
    GCTGCCATGTTGGA CUGCCAUGUUGGA
    LDHA_exon4 − CTTT 102 GCCAGAGACAATCTTT 679 GCCAGAGACAAUCUUUG
    GGTGTTCTAAGGAA GUGUUCUAAGGAA
    LDHA_exon4 + ATTT 103 TCCATTTAACTAAAGA 680 UCCAUUUAACUAAAGAU
    TTTGATGTCTTTTA UUGAUGUCUUUUA
    LDHA_exon4 + TTTT 104 CCATTTAACTAAAGAT 681 CCAUUUAACUAAAGAUU
    TTGATGTCTTTTAG UGAUGUCUUUUAG
    LDHA_exon4 + TTTC 105 CATTTAACTAAAGATT 682 CAUUUAACUAAAGAUUU
    TGATGTCTTTTAGG GAUGUCUUUUAGG
    LDHA_exon4 + ATTT 106 AACTAAAGATTTGATG 683 AACUAAAGAUUUGAUGU
    TCTTTTAGGACTTG CUUUUAGGACUUG
    LDHA_exon4 + ATTT 107 GATGTCTTTTAGGACT 684 GAUGUCUUUUAGGACUU
    TGGCAGATGAACTT GGCAGAUGAACUU
    LDHA_exon4 + TTTG 108 ATGTCTTTTAGGACTT 685 AUGUCUUUUAGGACUUG
    GGCAGATGAACTTG GCAGAUGAACUUG
    LDHA_exon4 + CTTT 109 TAGGACTTGGCAGATG 686 UAGGACUUGGCAGAUGA
    AACTTGCTCTTGTT ACUUGCUCUUGUU
    LDHA_exon4 + TTTT 110 AGGACTTGGCAGATGA 687 AGGACUUGGCAGAUGAA
    ACTTGCTCTTGTTG CUUGCUCUUGUUG
    LDHA_exon4 + TTTA 111 GGACTTGGCAGATGAA 688 GGACUUGGCAGAUGAAC
    CTTGCTCTTGTTGA UUGCUCUUGUUGA
    LDHA_exon4 + CTTG 112 GCAGATGAACTTGCTC 689 GCAGAUGAACUUGCUCU
    TTGTTGATGTCATC UGUUGAUGUCAUC
    LDHA_exon4 + CTTG 113 CTCTTGTTGATGTCAT 690 CUCUUGUUGAUGUCAUC
    CGAAGACAAATTGA GAAGACAAAUUGA
    LDHA_exon4 + CTTG 114 TTGATGTCATCGAAGA 691 UUGAUGUCAUCGAAGAC
    CAAATTGAAGGGAG AAAUUGAAGGGAG
    LDHA_exon4 + GTTG 115 ATGTCATCGAAGACAA 692 AUGUCAUCGAAGACAAA
    ATTGAAGGGAGAGA UUGAAGGGAGAGA
    LDHA_exon4 + ATTG 116 AAGGGAGAGATGATGG 693 AAGGGAGAGAUGAUGGA
    ATCTCCAACATGGC UCUCCAACAUGGC
    LDHA_exon4 + CTTT 117 TCCTTAGAACACCAAA 694 UCCUUAGAACACCAAAG
    GATTGTCTCTGGCA AUUGUCUCUGGCA
    LDHA_exon4 + TTTT 118 CCTTAGAACACCAAAG 695 CCUUAGAACACCAAAGA
    ATTGTCTCTGGCAA UUGUCUCUGGCAA
    LDHA_exon4 + TTTC 119 CTTAGAACACCAAAGA 696 CUUAGAACACCAAAGAU
    TTGTCTCTGGCAAA UGUCUCUGGCAAA
    LDHA_exon4 + CTTA 120 GAACACCAAAGATTGT 697 GAACACCAAAGAUUGUC
    CTCTGGCAAAGGTT UCUGGCAAAGGUU
    LDHA_exon4 + ATTG 121 TCTCTGGCAAAGGTTG 698 UCUCUGGCAAAGGUUGA
    ATTTCAACAAGTTT UUUCAACAAGUUU
    LDHA_exon4 + GTTG 122 ATTTCAACAAGTTTAT 699 AUUUCAACAAGUUUAUA
    ATTATAATCCATGC UUAUAAUCCAUGC
    LDHA_exon4 + ATTT 123 CAACAAGTTTATATTA 700 CAACAAGUUUAUAUUAU
    TAATCCATGCTTGA AAUCCAUGCUUGA
    LDHA_exon4 + TTTC 124 AACAAGTTTATATTAT 701 AACAAGUUUAUAUUAUA
    AATCCATGCTTGAC AUCCAUGCUUGAC
    LDHA_exon4 + GTTT 125 ATATTATAATCCATGC 702 AUAUUAUAAUCCAUGCU
    TTGACTTAAATTCT UGACUUAAAUUCU
    LDHA_exon4 + TTTA 126 TATTATAATCCATGCT 703 UAUUAUAAUCCAUGCUU
    TGACTTAAATTCTT GACUUAAAUUCUU
    LDHA_exon4 − ATTT 127 AAGTCAAGCATGGATT 704 AAGUCAAGCAUGGAUUA
    ATAATATAAACTTG UAAUAUAAACUUG
    LDHA_exon4 − TTTA 128 AGTCAAGCATGGATTA 705 AGUCAAGCAUGGAUUAU
    TAATATAAACTTGT AAUAUAAACUUGU
    LDHA_exon4 − ATTA 129 TAATATAAACTTGTTG 706 UAAUAUAAACUUGUUGA
    AAATCAACCTTTGC AAUCAACCUUUGC
    LDHA_exon4 − CTTG 130 TTGAAATCAACCTTTG 707 UUGAAAUCAACCUUUGC
    CCAGAGACAATCTT CAGAGACAAUCUU
    LDHA_exon4 − GTTG 131 AAATCAACCTTTGCCA 708 AAAUCAACCUUUGCCAG
    GAGACAATCTTTGG AGACAAUCUUUGG
    LDHA_exon4 − TTTG 132 CCAGAGACAATCTTTG 709 CCAGAGACAAUCUUUGG
    GTGTTCTAAGGAAA UGUUCUAAGGAAA
    LDHA_exon4 + TTTA 133 ACTAAAGATTTGATGT 710 ACUAAAGAUUUGAUGUC
    CTTTTAGGACTTGG UUUUAGGACUUGG
    LDHA_exon4 + ATTA 134 TAATCCATGCTTGACT 711 UAAUCCAUGCUUGACUU
    TAAATTCTTT AAAUUCUUU
    LDHA_exon4 − TTTA 135 GTTAAATGGAAAATTG 712 GUUAAAUGGAAAAUUGC
    CCACTTCTAGATT CACUUCUAGAUU
    LDHA_exon4 − GTTA 136 AATGGAAAATTGCCAC 713 AAUGGAAAAUUGCCACU
    TTCTAGATT UCUAGAUU
    LDHA_exon5 + ATTT 137 ATTCTAAAGGCCTTAA 714 AUUCUAAAGGCCUUAAU
    TCTGGTCATTATTC CUGGUCAUUAUUC
    LDHA_exon5 − ATTA 138 TAGTCTAGAGAAAAGG 715 UAGUCUAGAGAAAAGGG
    GGAATAATGACCAG GAAUAAUGACCAG
    LDHA_exon5 + TTTT 139 GACTGCATAAAAATTG 716 GACUGCAUAAAAAUUGA
    ACAAGCTATAGTAA CAAGCUAUAGUAA
    LDHA_exon5 + GTTT 140 TGACTGCATAAAAATT 717 UGACUGCAUAAAAAUUG
    GACAAGGTATAGTA ACAAGCUAUAGUA
    LDHA_exon5 + TTTG 141 AAATCCAGGTGAGGCT 718 AAAUCCAGGUGAGGCUU
    TTTGACTGCATAAA UUGACUGCAUAAA
    LDHA_exon5 + GTTT 142 CAAATCCAGGTGAGGC 719 CAAAUCCAGGUGAGGCU
    TTTTGACTGCATAA UUUGACUGCAUAA
    LDHA_exon5 + ATTG 143 TTTCAAATCCAGGTGA 720 UUUCAAAUCCAGGUGAG
    GGCTTTTGACTGCA GCUUUUGACUGCA
    LDHA_exon5 + GTTA 144 TTGTTTCAAATCCAGG 721 UUGUUUCAAAUCCAGGU
    TGAGGCTTTTGACT GAGGCUUUUGACU
    LDHA_exon5 + GTTG 145 CTTATTGTTTCAAATC 722 CUUAUUGUUUCAAAUCC
    CAGGTGAGGCTTTT AGGUGAGGCUUUU
    LDHA_exon5 + GTTG 146 TAAAATACAGCCCGAA 723 UAAAAUACAGCCCGAAC
    CTGCAAGTTGCTTA UGCAAGUUGCUUA
    LDHA_exon5 + ATTC 147 CTAATGTTGTAAAATA 724 CUAAUGUUGUAAAAUAC
    CAGCCCGAACTGCA AGCCCGAACUGCA
    LDHA_exon5 + ATTC 148 ATCATTCCTAATGTTG 725 AUCAUUCCUAAUGUUGU
    TAAAATACAGCCCG AAAAUACAGCCCG
    LDHA_exon5 + TTTA 149 AATTCATCATTCCTAA 726 AAUUCAUCAUUCCUAAU
    TGTTGTAAAATACA GUUGUAAAAUACA
    LDHA_exon5 + TTTG 150 ACTGCATAAAAATTGA 727 ACUGCAUAAAAAUUGAC
    CAAGCTATAGTAAA AAGCUAUAGUAAA
    LDHA_exon5 + GTTT 151 AAATTCATCATTCCTA 728 AAAUUCAUCAUUCCUAA
    ATGTTGTAAAATAC UGUUGUAAAAUAC
    LDHA_exon5 + ATTT 152 GGTCCAGCGTAACGTG 729 GGUCCAGCGUAACGUGA
    AACATCTTTAAATT ACAUCUUUAAAUU
    LDHA_exon5 + CTTA 153 ATTTGGTCCAGCGTAA 730 AUUUGGUCCAGCGUAAC
    CGTGAACATCTTTA GUGAACAUCUUUA
    LDHA_exon5 + ATTA 154 TCACGGCTGGGGCACG 731 UCACGGCUGGGGCACGU
    TCAGCAAGAGGGAG CAGCAAGAGGGAG
    LDHA_exon5 + TTTC 155 TCTAGACTATAATGTA 732 UCUAGACUAUAAUGUAA
    ACTGCAAACTCCAA CUGCAAACUCCAA
    LDHA_exon5 + TTTT 156 CTCTAGACTATAATGT 733 CUCUAGACUAUAAUGUA
    AACTGCAAACTCCA ACUGCAAACUCCA
    LDHA_exon5 + CTTT 157 TCTCTAGACTATAATG 734 UCUCUAGACUAUAAUGU
    TAACTGCAAACTCC AACUGCAAACUCC
    LDHA_exon5 + ATTC 158 CCCTTTTCTCTAGACT 735 CCCUUUUCUCUAGACUA
    ATAATGTAACTGCA UAAUGUAACUGCA
    LDHA_exon5 + ATTA 159 TTCCCCTTTTCTCTAG 736 UUCCCCUUUUCUCUAGA
    ACTATAATGTAACT CUAUAAUGUAACU
    LDHA_exon5 + CTTA 160 ATCTGGTCATTATTCC 737 AUCUGGUCAUUAUUCCC
    CCTTTTCTCTAGAC CUUUUCUCUAGAC
    LDHA_exon5 + ATTC 161 TAAAGGCCTTAATCTG 738 UAAAGGCCUUAAUCUGG
    GTCATTATTCCCCT UCAUUAUUCCCCU
    LDHA_exon5 + TTTA 162 TTCTAAAGGCCTTAAT 739 UUCUAAAGGCCUUAAUC
    CTGGTCATTATTCC UGGUCAUUAUUCC
    LDHA_exon5 + TTTG 163 GTCCAGCGTAACGTGA 740 GUCCAGCGUAACGUGAA
    ACATCTTTAAATTC CAUCUUUAAAUUC
    LDHA_exon5 − TTTT 164 ACTATAGCTTGTCAAT 741 ACUAUAGCUUGUCAAUU
    TTTTATGCAGTCAA UUUAUGCAGUCAA
    LDHA_exon5 − GTTT 165 TACTATAGCTTGTCAA 742 UACUAUAGCUUGUCAAU
    TTTTTATGCAGTCA UUUUAUGCAGUCA
    LDHA_exon5 − ATTT 166 AAAGATGTTCACGTTA 743 AAAGAUGUUCACGUUAC
    CGCTGGACCAAATT GCUGGACCAAAUU
    LDHA_exon5 − GTTT 167 GCAGTTACATTATAGT 744 GCAGUUACAUUAUAGUC
    CTAGAGAAAAGGGG UAGAGAAAAGGGG
    LDHA_exon5 − CTTG 168 GAGTTTGCAGTTACAT 745 GAGUUUGCAGUUACAUU
    TATAGTCTAGAGAA AUAGUCUAGAGAA
    LDHA_exon5 − CTTG 169 CTGACGTGCCCCAGCC 746 CUGACGUGCCCCAGCCG
    GTGATAATGACCAG UGAUAAUGACCAG
    LDHA_exon5 − TTTC 170 TCCCTCTTGCTGACGT 747 UCCCUCUUGCUGACGUG
    GCCCCAGCCGTGAT CCCCAGCCGUGAU
    LDHA_exon5 − CTTT 171 CTCCCTCTTGCTGACG 748 CUCCCUCUUGCUGACGU
    TGCCCCAGCCGTGA GCCCCAGCCGUGA
    LDHA_exon5 − ATTA 172 AGACGGCTTTCTCCCT 749 AGACGGCUUUCUCCCUC
    CTTGCTGACGTGCC UUGCUGACGUGCC
    LDHA_exon5 − GTTA 173 CGCTGGACCAAATTAA 750 CGCUGGACCAAAUUAAG
    GACGGCTTTCTCCC ACGGCUUUCUCCC
    LDHA_exon5 − GTTC 174 ACGTTACGCTGGACCA 751 ACGUUACGCUGGACCAA
    AATTAAGACGGCTT AUUAAGACGGCUU
    LDHA_exon5 − TTTA 175 AAGATGTTCACGTTAC 752 AAGAUGUUCACGUUACG
    GCTGGACCAAATTA CUGGACCAAAUUA
    LDHA_exon5 − TTTA 176 CTATAGCTTGTCAATT 753 CUAUAGCUUGUCAAUUU
    TTTATGCAGTCAAA UUAUGCAGUCAAA
    LDHA_exon5 − ATTA 177 GGAATGATGAATTTAA 754 GGAAUGAUGAAUUUAAA
    AGATGTTCACGTTA GAUGUUCACGUUA
    LDHA_exon5 − TTTA 178 CAACATTAGGAATGAT 755 CAACAUUAGGAAUGAUG
    GAATTTAAAGATGT AAUUUAAAGAUGU
    LDHA_exon5 − TTTT 179 ACAACATTAGGAATGA 756 ACAACAUUAGGAAUGAU
    TGAATTTAAAGATG GAAUUUAAAGAUG
    LDHA_exon5 − ATTT 180 TACAACATTAGGAATG 757 UACAACAUUAGGAAUGA
    ATGAATTTAAAGAT UGAAUUUAAAGAU
    LDHA_exon5 − GTTC 181 GGGCTGTATTTTACAA 758 GGGCUGUAUUUUACAAC
    CATTAGGAATGATG AUUAGGAAUGAUG
    LDHA_exon5 − CTTG 182 CAGTTCGGGCTGTATT 759 CAGUUCGGGCUGUAUUU
    TTACAACATTAGGA UACAACAUUAGGA
    LDHA_exon5 − TTTG 183 AAACAATAAGCAACTT 760 AAACAAUAAGCAACUUG
    GCAGTTCGGGCTGT CAGUUCGGGCUGU
    LDHA_exon5 − ATTT 184 GAAACAATAAGCAACT 761 GAAACAAUAAGCAACUU
    TGCAGTTCGGGCTG GCAGUUCGGGCUG
    LDHA_exon5 − TTTA 185 TGCAGTCAAAAGCCTC 762 UGCAGUCAAAAGCCUCA
    ACCTGGATTTGAAA CCUGGAUUUGAAA
    LDHA_exon5 − TTTT 186 ATGCAGTCAAAAGCCT 763 AUGCAGUCAAAAGCCUC
    CACCTGGATTTGAA ACCUGGAUUUGAA
    LDHA_exon5 − TTTT 187 TATGCAGTCAAAAGCC 764 UAUGCAGUCAAAAGCCU
    TCACCTGGATTTGA CACCUGGAUUUGA
    LDHA_exon5 − ATTT 188 TTATGCAGTCAAAAGC 765 UUAUGCAGUCAAAAGCC
    CTCACCTGGATTTG UCACCUGGAUUUG
    LDHA_exon5 − CTTG 189 TCAATTTTTATGCAGT 766 UCAAUUUUUAUGCAGUC
    CAAAAGCCTCACCT AAAAGCCUCACCU
    LDHA_exon5 − TTTG 190 CAGTTACATTATAGTC 767 CAGUUACAUUAUAGUCU
    TAGAGAAAAGGGGA AGAGAAAAGGGGA
    LDHA_exon5 − GTTA 191 CATTATAGTCTAGAGA 768 CAUUAUAGUCUAGAGAA
    AAAGGGGAATAATG AAGGGGAAUAAUG
    LDHA_exon5 + ATTG 192 ACAAGCTATAGTAAAA 769 ACAAGCUAUAGUAAAAC
    CTGATAG UGAUAG
    LDHA_exon5 − ATTA 193 AGGCCTTTAGAATAAA 770 AGGCCUUUAGAAUAAAU
    TTTT UUU
    LDHA_exon6 − GTTA 194 TCTTCCAAGCCACGTA 771 UCUUCCAAGCCACGUAG
    GGTCAAGATATCCA GUCAAGAUAUCCA
    LDHA_exon6 − CTTG 195 CAAGCCACGTAGGTCA 772 CAAGCCACGUAGGUCAA
    AGATATCCACTATG GAUAUCCACUAUG
    LDHA_exon6 − TTTG 196 GGAAAACCACTTATCT 773 GGAAAACCACUUAUCUU
    TCCAAGCCACGTAG CCAAGCCACGUAG
    LDHA_exon6 + CTTG 197 ACCTACGTGGCTTGGA 774 ACCUACGUGGCUUGGAA
    AGATAAGTGGTTTT GAUAAGUGGUUUU
    LDHA_exon6 − TTTT 198 TGGGAAAACCACTTAT 775 UGGGAAAACCACUUAUC
    CTTCCAAGCCACGT UUCCAAGCCACGU
    LDHA_exon6 + GTTA 199 CCTAATGGGGGAAAGG 776 CCUAAUGGGGGAAAGGC
    CTGGGAGTTCACCC UGGGAGUUCACCC
    LDHA_exon6 + ATTC 200 CGTTACCTAATGGGGG 777 CGUUACCUAAUGGGGGA
    AAAGGCTGGGAGTT AAGGCUGGGAGUU
    LDHA_exon6 + ATTC 201 AGCCCGATTCCGTTAC 778 AGCCCGAUUCCGUUACC
    CTAATGGGGGAAAG UAAUGGGGGAAAG
    LDHA_exon6 + GTTG 202 CAATCTGGATTCAGCC 779 CAAUCUGGAUUCAGCCC
    CGATTCCGTTACCT GAUUCCGUUACCU
    LDHA_exon6 + ATTG 203 GAAGCGGTTGCAATCT 780 GAAGCGGUUGCAAUCUG
    GGATTCAGCCCGAT GAUUCAGCCCGAU
    LDHA_exon6 + TTTC 204 CCAAAAACCGTGTTAT 781 CCAAAAACCGUGUUAUU
    TGGAAGCGGTTGCA GGAAGCGGUUGCA
    LDHA_exon6 + TTTT 205 CCCAAAAACCGTGTTA 782 CCCAAAAACCGUGUUAU
    TTGGAAGCGGTTGC UGGAAGCGGUUGC
    LDHA_exon6 + GTTT 206 TCCCAAAAACCGTGTT 783 UCCCAAAAACCGUGUUA
    ATTGGAAGCGGTTG UUGGAAGCGGUUG
    LDHA_exon6 + CTTG 207 GAAGATAAGTGGTTTT 784 GAAGAUAAGUGGUUUUC
    CCCAAAAACCGTGT CCAAAAACCGUGU
    LDHA_exon6 + TTTC 208 ATAGTGGATATCTTGA 785 AUAGUGGAUAUCUUGAC
    CCTACGTGGCTTGG CUACGUGGCUUGG
    LDHA_exon6 + TTTT 209 CATAGTGGATATCTTG 786 CAUAGUGGAUAUCUUGA
    ACCTACGTGGCTTG CCUACGUGGCUUG
    LDHA_exon6 + TTTT 210 TCATAGTGGATATCTT 787 UCAUAGUGGAUAUCUUG
    GACCTACGTGGCTT ACCUACGUGGCUU
    LDHA_exon6 + GTTT 211 TTCATAGTGGATATCT 788 UUCAUAGUGGAUAUCUU
    TGACCTACGTGGCT GACCUACGUGGCU
    LDHA_exon6 + TTTC 212 TCCTTTTTCATAGTGG 789 UCCUUUUUCAUAGUGGA
    ATATCTTGACCTAC UAUCUUGACCUAC
    LDHA_exon6 + TTTT 213 CTCCTTTTTCATAGTG 790 CUCCUUUUUCAUAGUGG
    GATATCTTGACCTA AUAUCUUGACCUA
    LDHA_exon6 + ATTT 214 TCTCCTTTTTCATAGT 791 UCUCCUUUUUCAUAGUG
    GGATATCTTGACCT GAUAUCUUGACCU
    LDHA_exon6 + TTTA 215 TTTTCTCCTTTTTCAT 792 UUUUCUCCUUUUUCAUA
    AGTGGATATCTTGA GUGGAUAUCUUGA
    LDHA_exon6 + TTTT 216 ATTTTCTCCTTTTTCA 793 AUUUUCUCCUUUUUCAU
    TAGTGGATATCTTG AGUGGAUAUCUUG
    LDHA_exon6 + TTTT 217 TATTTTCTCCTTTTTC 794 UAUUUUCUCCUUUUUCA
    ATAGTGGATATCTT UAGUGGAUAUCUU
    LDHA_exon6 + ATTT 218 TTATTTTCTCCTTTTT 795 UUAUUUUCUCCUUUUUC
    CATAGTGGATATCT AUAGUGGAUAUCU
    LDHA_exon6 − TTTT 219 GGGAAAACCACTTATC 796 GGGAAAACCACUUAUCU
    TTCCAAGCCACGTA UCCAAGCCACGUA
    LDHA_exon6 + GTTC 220 ACCCATTAAGCTGTCA 797 ACCCAUUAAGCUGUCAU
    TGGGTGGGTCCTTG GGGUGGGUCCUUG
    LDHA_exon6 + ATTA 221 AGCTGTCATGGGTGGG 798 AGCUGUCAUGGGUGGGU
    TCCTTGGGGAACAT CCUUGGGGAACAU
    LDHA_exon6 + GTTA 222 TTGGAAGCGGTTGCAA 799 UUGGAAGCGGUUGCAAU
    TCTGGATTCAGCCC CUGGAUUCAGCCC
    LDHA_exon6 + ATTO 223 CAGTGGTAAGCATAAG 800 CAGUGGUAAGCAUAAGU
    TTATTTTCTTTTTG UAUUUUCUUUUUG
    LDHA_exon6 − GTTT 224 TTGGGAAAACCACTTA 801 UUGGGAAAACCACUUAU
    TCTTCCAAGCCACG CUUCCAAGCCACG
    LDHA_exon6 − CTTC 225 CAATAACACGGTTTTT 802 CAAUAACACGGUUUUUG
    GGGAAAACCACTTA GGAAAACCACUUA
    LDHA_exon6 − ATTG 226 CAACCGCTTCCAATAA 803 CAACCGCUUCCAAUAAC
    CACGGTTTTTGGGA ACGGUUUUUGGGA
    LDHA_exon6 − ATTA 227 GGTAACGGAATCGGGC 804 GGUAACGGAAUCGGGCU
    TGAATCCAGATTGC GAAUCCAGAUUGC
    LDHA_exon6 + CTTG 228 GGGAACATGGAGATTC 805 GGGAACAUGGAGAUUCC
    CAGTGGTAAGCATA AGUGGUAAGCAUA
    LDHA_exon6 − CTTT 229 CCCCCATTAGGTAACG 806 CCCCCAUUAGGUAACGG
    GAATCGGGCTGAAT AAUCGGGCUGAAU
    LDHA_exon6 − CTTA 230 ATGGGTGAACTCCCAG 807 AUGGGUGAACUCCCAGC
    CCTTTCCCCCATTA CUUUCCCCCAUUA
    LDHA_exon6 − GTTC 231 CCCAAGGACCCACCCA 808 CCCAAGGACCCACCCAU
    TGACAGCTTAATGG GACAGCUUAAUGG
    LDHA_exon6 − CTTA 232 CCACTGGAATCTCCAT 809 CCACUGGAAUCUCCAUG
    GTTCCCCAAGGACC UUCCCCAAGGACC
    LDHA_exon6 − CTTA 233 TGCTTACCACTGGAAT 810 UGCUUACCACUGGAAUC
    CTCCATGTTCCCCA UCCAUGUUCCCCA
    LDHA_exon6 − TTTC 234 AAAAACAAAAAGAAAA 811 AAAAACAAAAAGAAAAU
    TAACTTATGCTTAC AACUUAUGCUUAC
    LDHA_exon6 − TTTC 235 CCCCATTAGGTAACGG 812 CCCCAUUAGGUAACGGA
    AATCGGGCTGAATC AUCGGGCUGAAUC
    LDHA_exon6 + TTTT 236 CTTTTTGTTTTTGAAA 813 CUUUUUGUUUUUGAAAA
    AGATTATATAAAAA GAUUAUAUAAAAA
    LDHA_exon6 − CTTT 237 TCAAAAACAAAAAGAA 814 UCAAAAACAAAAAGAAA
    AATAACTTATGCTT AUAACUUAUGCUU
    LDHA_exon6 − TTTA 238 TATAATCTTTTCAAAA 815 UAUAAUCUUUUCAAAAA
    ACAAAAAGAAAATA CAAAAAGAAAAUA
    LDHA_exon6 − TTTT 239 ATATAATCTTTTCAAA 816 AUAUAAUCUUUUCAAAA
    AACAAAAAGAAAAT ACAAAAAGAAAAU
    LDHA_exon6 − TTTT 240 TATATAATCTTTTCAA 817 UAUAUAAUCUUUUCAAA
    AAACAAAAAGAAAA AACAAAAAGAAAA
    LDHA_exon6 − CTTT 241 TTATATAATCTTTTCA 818 UUAUAUAAUCUUUUCAA
    AAAACAAAAAGAAA AAACAAAAAGAAA
    LDHA_exon6 + TTTC 242 TTTTTGTTTTTGAAAA 819 UUUUUGUUUUUGAAAAG
    GATTATATAAAAAG AUUAUAUAAAAAG
    LDHA_exon6 + GTTA 243 TTTTCTTTTTGTTTTT 820 UUUUCUUUUUGUUUUUG
    GAAAAGATTATATA AAAAGAUUAUAUA
    LDHA_exon6 + ATTT 244 TCTTTTTGTTTTTGAA 821 UCUUUUUGUUUUUGAAA
    AAGATTATATAAAA AGAUUAUAUAAAA
    LDHA_exon6 − TTTT 245 CAAAAACAAAAAGAAA 822 CAAAAACAAAAAGAAAA
    ATAACTTATGCTTA UAACUUAUGCUUA
    LDHA_exon6 + TTTT 246 GAAAAGATTATATAAA 823 GAAAAGAUUAUAUAAAA
    AAGT AGU
    LDHA_exon6 + TTTT 247 TGAAAAGATTATATAA 824 UGAAAAGAUUAUAUAAA
    AAAGT AAGU
    LDHA_exon6 + GTTT 248 TTGAAAAGATTATATA 825 UUGAAAAGAUUAUAUAA
    AAAAGT AAAGU
    LDHA_exon6 + TTTT 249 GTTTTTGAAAAGATTA 826 GUUUUUGAAAAGAUUAU
    TATAAAAAGT AUAAAAAGU
    LDHA_exon6 + TTTT 250 TGTTTTTGAAAAGATT 827 UGUUUUUGAAAAGAUUA
    ATATAAAAAGT UAUAAAAAGU
    LDHA_exon6 + CTTT 251 TTGTTTTTGAAAAGAT 828 UUGUUUUUGAAAAGAUU
    TATATAAAAAGT AUAUAAAAAGU
    LDHA_exon6 + TTTG 252 TTTTTGAAAAGATTAT 829 UUUUUGAAAAGAUUAUA
    ATAAAAAGT UAAAAAGU
    LDHA_exon7 + GTTG 253 AGAGGTAATAAATCTT 830 AGAGGUAAUAAAUCUUU
    TCAATTTGGCAACA CAAUUUGGCAACA
    LDHA_exon7 + GTTG 254 GTACATGAAAATAAAT 831 GUACAUGAAAAUAAAUG
    GTAGTCTGTACTAT UAGUCUGUACUAU
    LDHA_exon7 + TTTC 255 AATTTGGCAACACAGA 832 AAUUUGGCAACACAGAA
    ATATTAACATTTAC UAUUAACAUUUAC
    LDHA_exon7 + GTTC 256 ACAAGCAGGTGGTTGA 833 ACAAGCAGGUGGUUGAG
    GAGGTAATAAATCT AGGUAAUAAAUCU
    LDHA_exon7 + ATTT 257 GGCAACACAGAATATT 834 GGCAACACAGAAUAUUA
    AACATTTACTATTT ACAUUUACUAUUU
    LDHA_exon7 + CTTT 258 CAATTTGGCAACACAG 835 CAAUUUGGCAACACAGA
    AATATTAACATTTA AUAUUAACAUUUA
    LDHA_exon7 + TTTA 259 GGGACTGATAAAGATA 836 GGGACUGAUAAAGAUAA
    AGGAACAGTGGAAA GGAACAGUGGAAA
    LDHA_exon7 + CTTT 260 TAGTGCCTGTATGGAG 837 UAGUGCCUGUAUGGAGU
    TGGAATGAATGTTG GGAAUGAAUGUUG
    LDHA_exon7 + GTTG 261 CTGGTGTCTCTCTGAA 838 CUGGUGUCUCUCUGAAG
    GACTCTGCACCCAG ACUCUGCACCCAG
    LDHA_exon7 + TTTA 262 GTGCCTGTATGGAGTG 839 GUGCCUGUAUGGAGUGG
    GAATGAATGTTGCT AAUGAAUGUUGCU
    LDHA_exon7 + TTTT 263 AGTGCCTGTATGGAGT 840 AGUGCCUGUAUGGAGUG
    GGAATGAATGTTGC GAAUGAAUGUUGC
    LDHA_exon7 + TTTC 264 TTTTAGTGCCTGTATG 841 UUUUAGUGCCUGUAUGG
    GAGTGGAATGAATG AGUGGAAUGAAUG
    LDHA_exon7 + ATTT 265 CTTTTAGTGCCTGTAT 842 CUUUUAGUGCCUGUAUG
    GGAGTGGAATGAAT GAGUGGAAUGAAU
    LDHA_exon7 + TTTG 266 GCAACACAGAATATTA 843 GCAACACAGAAUAUUAA
    ACATTTACTATTTT CAUUUACUAUUUU
    LDHA_exon7 + ATTT 267 AGGGACTGATAAAGAT 844 AGGGACUGAUAAAGAUA
    AAGGAACAGTGGAA AGGAACAGUGGAA
    LDHA_exon7 − GTTA 268 ATATTCTGTGTTGCCA 845 AUAUUCUGUGUUGCCAA
    AATTGAAAGATTTA AUUGAAAGAUUUA
    LDHA_exon7 − TTTA 269 TCAGTCCCTAAATCTG 846 UCAGUCCCUAAAUCUGG
    GGTGCAGAGTCTTC GUGCAGAGUCUUC
    LDHA_exon7 − GTTG 270 CCAAATTGAAAGATTT 847 CCAAAUUGAAAGAUUUA
    ATTACCTCTCAACC UUACCUCUCAACC
    LDHA_exon7 − ATTC 271 TGTGTTGCCAAATTGA 848 UGUGUUGCCAAAUUGAA
    AAGATTTATTACCT AGAUUUAUUACCU
    LDHA_exon7 − ATTC 272 CACTCCATACAGGCAC 849 CACUCCAUACAGGCACU
    TAAAAGAAATAGTA AAAAGAAAUAGUA
    LDHA_exon7 − OTTO 273 AGAGAGACACCAGCAA 850 AGAGAGACACCAGCAAC
    CATTCATTCCACTC AUUCAUUCCACUC
    LDHA_exon7 − CTTT 274 ATCAGTCCCTAAATCT 851 AUCAGUCCCUAAAUCUG
    GGGTGCAGAGTCTT GGUGCAGAGUCUU
    LDHA_exon7 − CTTA 275 TCTTTATCAGTCCCTA 852 UCUUUAUCAGUCCCUAA
    AATCTGGGTGCAGA AUCUGGGUGCAGA
    LDHA_exon7 − GTTC 276 CTTATCTTTATCAGTC 853 CUUAUCUUUAUCAGUCC
    CCTAAATCTGGGTG CUAAAUCUGGGUG
    LDHA_exon7 − ATTC 277 ATTCCACTCCATACAG 854 AUUCCACUCCAUACAGG
    GCACTAAAAGAAAT CACUAAAAGAAAU
    LDHA_exon7 − CTTT 278 CCACTGTTCCTTATCT 855 CCACUGUUCCUUAUCUU
    TTATCAGTCCCTAA UAUCAGUCCCUAA
    LDHA_exon7 − CTTG 279 TGAACCTCTTTCCACT 856 UGAACCUCUUUCCACUG
    GTTCCTTATCTTTA UUCCUUAUCUUUA
    LDHA_exon7 − ATTA 280 CCTCTCAACCACCTGC 857 CCUCUCAACCACCUGCU
    TTGTGAACCTCTTT UGUGAACCUCUUU
    LDHA_exon7 − TTTA 281 TTACCTCTCAACCACC 858 UUACCUCUCAACCACCU
    TGCTTGTGAACCTC GCUUGUGAACCUC
    LDHA_exon7 − ATTT 282 ATTACCTCTCAACCAC 859 AUUACCUCUCAACCACC
    CTGCTTGTGAACCT UGCUUGUGAACCU
    LDHA_exon7 − TTTC 283 CACTGTTCCTTATCTT 860 CACUGUUCCUUAUCUUU
    TATCAGTCCCTAAA AUCAGUCCCUAAA
    LDHA_exon7 − ATTG 284 AAAGATTTATTACCTC 861 AAAGAUUUAUUACCUCU
    TCAACCACCTGCTT CAACCACCUGCUU
    LDHA_exon7 − TTTA 285 TTTTCATGTACCAACA 862 UUUUCAUGUACCAACAG
    GATTAG AUUAG
    LDHA_exon7 − ATTT 286 ATTTTCATGTACCAAC 863 AUUUUCAUGUACCAACA
    AGATTAG GAUUAG
    LDHA_exon8 + ATTG 287 GACTCTCTGTAGCAGA 864 GACUCUCUGUAGCAGAU
    TTTGGCAGAGAGTA UUGGCAGAGAGUA
    LDHA_exon8 + CTTA 288 TGAGGTGATCAAACTC 865 UGAGGUGAUCAAACUCA
    AAAGGCTACACATC AAGGCUACACAUC
    LDHA_exon8 + TTTC 289 CTATCATACAGTGCTT 866 CUAUCAUACAGUGCUUA
    ATGAGGTGATCAAA UGAGGUGAUCAAA
    LDHA_exon8 + GTTT 290 CCTATCATACAGTGCT 867 CCUAUCAUACAGUGCUU
    TATGAGGTGATCAA AUGAGGUGAUCAA
    LDHA_exon8 + CTTT 291 ACCTATGGTTTCCTAT 868 ACCUAUGGUUUCCUAUC
    CATACAGTGCTTAT AUACAGUGCUUAU
    LDHA_exon8 + TTTC 292 TGCCTTTACCTATGGT 869 UGCCUUUACCUAUGGUU
    TTCCTATCATACAG UCCUAUCAUACAG
    LDHA_exon8 + TTTT 293 CTGCCTTTACCTATGG 870 CUGCCUUUACCUAUGGU
    TTTCCTATCATACA UUCCUAUCAUACA
    LDHA_exon8 + ATTT 294 GGCAGAGAGTATAATG 871 GGCAGAGAGUAUAAUGA
    AAGAATCTTAGGCG AGAAUCUUAGGCG
    LDHA_exon8 + TTTA 295 CCTATGGTTTCCTATC 872 CCUAUGGUUUCCUAUCA
    ATACAGTGCTTATG UACAGUGCUUAUG
    LDHA_exon8 + TTTG 296 GCAGAGAGTATAATGA 873 GCAGAGAGUAUAAUGAA
    AGAATCTTAGGCGG GAAUCUUAGGCGG
    LDHA_exon8 − CTTC 297 ATTATACTCTCTGCCA 874 AUUAUACUCUCUGCCAA
    AATCTGCTACAGAG AUCUGCUACAGAG
    LDHA_exon8 + GTTT 298 CCACCATGATTAAGGT 875 CCACCAUGAUUAAGGUA
    AGGTCTATGTAGTG GGUCUAUGUAGUG
    LDHA_exon8 + TTTC 299 CACCATGATTAAGGTA 876 CACCAUGAUUAAGGUAG
    GGTCTATGTAGTGA GUCUAUGUAGUGA
    LDHA_exon8 + ATTA 300 AGGTAGGTCTATGTAG 877 AGGUAGGUCUAUGUAGU
    TGATACGCTGCATT GAUACGCUGCAUU
    LDHA_exon8 − ATTC 301 AAATGCAGCGTATCAC 878 AAAUGCAGCGUAUCACU
    TACATAGACCTACC ACAUAGACCUACC
    LDHA_exon8 − CTTA 302 ATCATGGTGGAAACTG 879 AUCAUGGUGGAAACUGG
    GGTGCACCCGCCTA GUGCACCCGCCUA
    LDHA_exon8 − ATTC 303 TTCATTATACTCTCTG 880 UUCAUUAUACUCUCUGC
    CCAAATCTGCTACA CAAAUCUGCUACA
    LDHA_exon8 − ATTA 304 TACTCTCTGCCAAATC 881 UACUCUCUGCCAAAUCU
    TGCTACAGAGAGTC GCUACAGAGAGUC
    LDHA_exon8 − CTTT 305 GAGTTTGATCACCTCA 882 GAGUUUGAUCACCUCAU
    TAAGCACTGTATGA AAGCACUGUAUGA
    LDHA_exon8 − TTTG 306 AGTTTGATCACCTCAT 883 AGUUUGAUCACCUCAUA
    AAGCACTGTATGAT AGCACUGUAUGAU
    LDHA_exon8 + TTTT 307 TCTGCCTTTACCTATG 884 UCUGCCUUUACCUAUGG
    GTTTCCTATCATAC UUUCCUAUCAUAC
    LDHA_exon8 + CTTA 308 GGCGGGTGCACCCAGT 885 GGCGGGUGCACCCAGUU
    TTCCACCATGATTA UCCACCAUGAUUA
    LDHA_exon8 + CTTT 309 TTCTGCCTTTACCTAT 886 UUCUGCCUUUACCUAUG
    GGTTTCCTATCATA GUUUCCUAUCAUA
    LDHA_exon8 − TTTG 310 ATCACCTCATAAGCAC 887 AUCACCUCAUAAGCACU
    TGTATGATAGGAAA GUAUGAUAGGAAA
    LDHA_exon8 − GTTT 311 GATCACCTCATAAGCA 888 GAUCACCUCAUAAGCAC
    CTGTATGATAGGAA UGUAUGAUAGGAA
    LDHA_exon8 + ATTT 312 GAATGCTTTTTGCTGG 889 GAAUGCUUUUUGCUGGC
    CTTTT UUUU
    LDHA_exon8 + TTTG 313 AATGCTTTTTGCTGGC 890 AAUGCUUUUUGCUGGCU
    TTTT UUU
    LDHA_exon8 + CTTC 314 TGAGGAAGAGGCCCGT 891 UGAGGAAGAGGCCCGUU
    TTGAAGAAGAGTGC UGAAGAAGAGUGC
    LDHA_exon8 − TTTC 315 CAAATTAATATAATAA 892 CAAAUUAAUAUAAUAAC
    CTAGCAGCTTTATG UAGCAGCUUUAUG
    LDHA_exon9 − ATTA 316 ATATAATAACTAGCAG 893 AUAUAAUAACUAGCAGC
    CTTTATGACTTTAT UUUAUGACUUUAU
    LDHA_exon9 − CTTT 317 ATGACTTTATATCTTA 894 AUGACUUUAUAUCUUAA
    ATATAATGAATTAA UAUAAUGAAUUAA
    LDHA_exon9 − TTTA 318 TGACTTTATATCTTAA 895 UGACUUUAUAUCUUAAU
    TATAATGAATTAAC AUAAUGAAUUAAC
    LDHA_exon9 − CTTT 319 ATATCTTAATATAATG 896 AUAUCUUAAUAUAAUGA
    AATTAACCAAAGTA AUUAACCAAAGUA
    LDHA_exon9 − TTTA 320 TATCTTAATATAATGA 897 UAUCUUAAUAUAAUGAA
    ATTAACCAAAGTAG UUAACCAAAGUAG
    LDHA_exon9 − CTTA 321 ATATAATGAATTAACC 898 AUAUAAUGAAUUAACCA
    AAAGTAGTCACTGT AAGUAGUCACUGU
    LDHA_exon9 − ATTA 322 ACCAAAGTAGTCACTG 899 ACCAAAGUAGUCACUGU
    TTCAAGGTTTATTG UCAAGGUUUAUUG
    LDHA_exon9 − GTTC 323 AAGGTTTATTGGGGGT 900 AAGGUUUAUUGGGGGUU
    TTTAGTTGGTATAA UUAGUUGGUAUAA
    LDHA_exon9 − GTTT 324 ATTGGGGGTTTTAGTT 901 AUUGGGGGUUUUAGUUG
    GGTATAACACTTGG GUAUAACACUUGG
    LDHA_exon9 − TTTA 325 TTGGGGGTTTTAGTTG 902 UUGGGGGUUUUAGUUGG
    GTATAACACTTGGA UAUAACACUUGGA
    LDHA_exon9 − ATTG 326 GGGGTTTTAGTTGGTA 903 GGGGUUUUAGUUGGUAU
    TAACACTTGGATAG AACACUUGGAUAG
    LDHA_exon9 − GTTT 327 TAGTTGGTATAACACT 904 UAGUUGGUAUAACACUU
    TGGATAGTTGGTTG GGAUAGUUGGUUG
    LDHA_exon9 − ATTT 328 CCAAATTAATATAATA 905 CCAAAUUAAUAUAAUAA
    ACTAGCAGCTTTAT CUAGCAGCUUUAU
    LDHA_exon9 − TTTT 329 AGTTGGTATAACACTT 906 AGUUGGUAUAACACUUG
    GGATAGTTGGTTGC GAUAGUUGGUUGC
    LDHA_exon9 − GTTG 330 GTATAACACTTGGATA 907 GUAUAACACUUGGAUAG
    GTTGGTTGCATTGT UUGGUUGCAUUGU
    LDHA_exon9 − CTTG 331 GATAGTTGGTTGCATT 908 GAUAGUUGGUUGCAUUG
    GTTTGTATGTAGAT UUUGUAUGUAGAU
    LDHA_exon9 − GTTG 332 GTTGCATTGTTTGTAT 909 GUUGCAUUGUUUGUAUG
    GTAGATCTTTTTAC UAGAUCUUUUUAC
    LDHA_exon9 − GTTG 333 CATTGTTTGTATGTAG 910 CAUUGUUUGUAUGUAGA
    ATCTTTTTACATTA UCUUUUUACAUUA
    LDHA_exon9 − ATTG 334 TTTGTATGTAGATCTT 911 UUUGUAUGUAGAUCUUU
    TTTACATTATATGG UUACAUUAUAUGG
    LDHA_exon9 − GTTT 335 GTATGTAGATCTTTTT 912 GUAUGUAGAUCUUUUUA
    ACATTATATGGTAA CAUUAUAUGGUAA
    LDHA_exon9 − TTTG 336 TATGTAGATCTTTTTA 913 UAUGUAGAUCUUUUUAC
    CATTATATGGTAAT AUUAUAUGGUAAU
    LDHA_exon9 − CTTT 337 TTACATTATATGGTAA 914 UUACAUUAUAUGGUAAU
    TGTACACTACTGAT GUACACUACUGAU
    LDHA_exon9 − TTTT 338 TACATTATATGGTAAT 915 UACAUUAUAUGGUAAUG
    GTACACTACTGATA UACACUACUGAUA
    LDHA_exon9 − TTTT 339 ACATTATATGGTAATG 916 ACAUUAUAUGGUAAUGU
    TACACTACTGATAT ACACUACUGAUAU
    LDHA_exon9 − TTTA 340 CATTATATGGTAATGT CAUUAUAUGGUAAUGUA
    ACACTACTGATATA CACUACUGAUAUA
    LDHA_exon9 − ATTA 341 TATGGTAATGTACACT 918 UAUGGUAAUGUACACUA
    ACTGATATAGTTCA CUGAUAUAGUUCA
    LDHA_exon9 − GTTC 342 ACAAAATAAGATCCTT 919 ACAAAAUAAGAUCCUUU
    TGGAAGAATTATGC GGAAGAAUUAUGC
    LDHA_exon9 − CTTT 343 GGAAGAATTATGCACA 920 GGAAGAAUUAUGCACAA
    AGACATGATATTGG GACAUGAUAUUGG
    LDHA_exon9 − TTTA 344 GTTGGTATAACACTTG 921 GUUGGUAUAACACUUGG
    GATAGTTGGTTGCA AUAGUUGGUUGCA
    LDHA_exon9 − GTTG 345 CCCAAGAATAGCCTAA 922 CCCAAGAAUAGCCUAAU
    TATTTCCAAATTAA AUUUCCAAAUUAA
    LDHA_exon9 − GTTG 346 CAGGGTTGCCCAAGAA 923 CAGGGUUGCCCAAGAAU
    TAGCCTAATATTTC AGCCUAAUAUUUC
    LDHA_exon9 − GTTA 347 GAAAAAATCGTTGCAG 924 GAAAAAAUCGUUGCAGG
    GGTTGCCCAAGAAT GUUGCCCAAGAAU
    LDHA_exon9 − ATTG 348 TTTTTAATTGTTACCA 925 UUUUUAAUUGUUACCAG
    GCTTCCAGAGGACA CUUCCAGAGGACA
    LDHA_exon9 − GTTT 349 TTAATTGTTACCAGCT 926 UUAAUUGUUACCAGCUU
    TCCAGAGGACAAGA CCAGAGGACAAGA
    LDHA_exon9 − TTTT 350 TAATTGTTACCAGCTT 927 UAAUUGUUACCAGCUUC
    CCAGAGGACAAGAT CAGAGGACAAGAU
    LDHA_exon9 − TTTT 351 AATTGTTACCAGCTTC 928 AAUUGUUACCAGCUUCC
    CAGAGGACAAGATC AGAGGACAAGAUC
    LDHA_exon9 − TTTA 352 ATTGTTACCAGCTTCC 929 AUUGUUACCAGCUUCCA
    AGAGGACAAGATCT GAGGACAAGAUCU
    LDHA_exon9 − ATTG 353 TTACCAGCTTCCAGAG 930 UUACCAGCUUCCAGAGG
    GACAAGATCTCAAA ACAAGAUCUCAAA
    LDHA_exon9 − GTTA 354 CCAGCTTCCAGAGGAC 931 CCAGCUUCCAGAGGACA
    AAGATCTCAAAAAT AGAUCUCAAAAAU
    LDHA_exon9 − GTTG 355 CAGAGGACAAGATCTC 932 CAGAGGACAAGAUCUCA
    AAAAATCTGTGTTC AAAAUCUGUGUUC
    LDHA_exon9 − GTTG 356 CCTATAGTGACACACT 933 CCUAUAGUGACACACUA
    ATCATTGCCTATAT UCAUUGCCUAUAU
    LDHA_exon9 − ATTG 357 CCTATATTCAGTTGGC 934 CCUAUAUUCAGUUGGCA
    AAATAAATTTTACA AAUAAAUUUUACA
    LDHA_exon9 − ATTG 358 AGTTGGCAAATAAATT 935 AGUUGGCAAAUAAAUUU
    TTACATTTACATAT UACAUUUACAUAU
    LDHA_exon9 − GTTG 359 GCAAATAAATTTTACA 936 GCAAAUAAAUUUUACAU
    TTTACATATAGAAT UUACAUAUAGAAU
    LDHA_exon9 − ATTT 360 TACATTTACATATAGA 937 UACAUUUACAUAUAGAA
    ATGTTACTTTCCAA UGUUACUUUCCAA
    LDHA_exon9 − TTTT 361 ACATTTACATATAGAA 938 ACAUUUACAUAUAGAAU
    TGTTACTTTCCAAT GUUACUUUCCAAU
    LDHA_exon9 − TTTG 362 GAAGAATTATGCACAA 939 GAAGAAUUAUGCACAAG
    GACATGATATTGGA ACAUGAUAUUGGA
    LDHA_exon9 − TTTA 363 CATTTACATATAGAAT 940 CAUUUACAUAUAGAAUG
    GTTACTTTCCAATT UUACUUUCCAAUU
    LDHA_exon9 − TTTA 364 CATATAGAATGTTACT 941 CAUAUAGAAUGUUACUU
    TTCCAATTATGATT UCCAAUUAUGAUU
    LDHA_exon9 − GTTA 365 CTTTCCAATTATGATT 942 CUUUCCAAUUAUGAUUA
    AGCATTATTATCAA GCAUUAUUAUCAA
    LDHA_exon9 − CTTT 366 CCAATTATGATTAGCA 943 CCAAUUAUGAUUAGCAU
    TTATTATCAAATAT UAUUAUCAAAUAU
    LDHA_exon9 − TTTC 367 CAATTATGATTAGCAT 944 CAAUUAUGAUUAGCAUU
    TATTATCAAATATA AUUAUCAAAUAUA
    LDHA_exon9 − ATTA 368 TGATTAGCATTATTAT 945 UGAUUAGCAUUAUUAUC
    CAAATATATAATAC AAAUAUAUAAUAC
    LDHA_exon9 − ATTA 369 GCATTATTATCAAATA 946 GCAUUAUUAUCAAAUAU
    TATAATACTTTGGG AUAAUACUUUGGG
    LDHA_exon9 − ATTA 370 TTATCAAATATATAAT 947 UUAUCAAAUAUAUAAUA
    ACTTTGGGACTTAC CUUUGGGACUUAC
    LDHA_exon9 − ATTA 371 TCAAATATATAATACT 948 UCAAAUAUAUAAUACUU
    TTGGGACTTACAAT UGGGACUUACAAU
    LDHA_exon9 − CTTT 372 GGGACTTACAATGGAA 949 GGGACUUACAAUGGAAG
    GTGGTACCAATACA UGGUACCAAUACA
    LDHA_exon9 − TTTG 373 GGACTTACAATGGAAG 950 GGACUUACAAUGGAAGU
    TGGTACCAATACAA GGUACCAAUACAA
    LDHA_exon9 − CTTA 374 CAATGGAAGTGGTACC 951 CAAUGGAAGUGGUACCA
    AATACAACTCAGTT AUACAACUCAGUU
    LDHA_exon9 − GTTG 375 ACTATTACATCCTCTG 952 ACUAUUACAUCCUCUGC
    CTATTAGTCAATAA UAUUAGUCAAUAA
    LDHA_exon9 − ATTA 376 CATCCTCTGCTATTAG 953 CAUCCUCUGCUAUUAGU
    TCAATAATATCCCT CAAUAAUAUCCCU
    LDHA_exon9 − ATTA 377 GTCAATAATATCCCTG 954 GUCAAUAAUAUCCCUGU
    TTAGAAAAAATCGT UAGAAAAAAUCGU
    LDHA_exon9 − ATTT 378 ACATATAGAATGTTAC 955 ACAUAUAGAAUGUUACU
    TTTCCAATTATGAT UUCCAAUUAUGAU
    LDHA_exon9 − ATTA 379 TGCACAAGACATGATA 956 UGCACAAGACAUGAUAU
    TTGGATTTATACAC UGGAUUUAUACAC
    LDHA_exon9 − ATTG 380 GATTTATACACTGGAT 957 GAUUUAUACACUGGAUC
    CCCAGGATGTGACT CCAGGAUGUGACU
    LDHA_exon9 − ATTT 381 ATACACTGGATCCCAG 958 AUACACUGGAUCCCAGG
    GATGTGACTCACTG AUGUGACUCACUG
    LDHA_exon9 − CTTC 382 AAACGGGCCTCTTCCT 959 AAACGGGCCUCUUCCUC
    CAGAAGTCAGAGTC AGAAGUCAGAGUC
    LDHA_exon9 − CTTC 383 CTCAGAAGTCAGAGTC 960 CUCAGAAGUCAGAGUCA
    ACCTTCACAAGGTC CCUUCACAAGGUC
    LDHA_exon9 − CTTC 384 ACAAGGTCTGAGATTC 961 ACAAGGUCUGAGAUUCC
    CATTCTGTCCCAAA AUUCUGUCCCAAA
    LDHA_exon9 − ATTC 385 CATTCTGTCCCAAAAT 962 CAUUCUGUCCCAAAAUG
    GCAAGGAACACTAA CAAGGAACACUAA
    LDHA_exon9 − ATTC 386 TGTCCCAAAATGCAAG 963 UGUCCCAAAAUGCAAGG
    GAACACTAAGGAAG AACACUAAGGAAG
    LDHA_exon9 − CTTT 387 ATTCCGTAAAGACCCT 964 AUUCCGUAAAGACCCUG
    GAAGATGAAATGAA AAGAUGAAAUGAA
    LDHA_exon9 − TTTA 388 TTCCGTAAAGACCCTG 965 UUCCGUAAAGACCCUGA
    AAGATGAAATGAAA AGAUGAAAUGAAA
    LDHA_exon9 − ATTC 389 CGTAAAGACCCTGAAG 966 CGUAAAGACCCUGAAGA
    ATGAAATGAAAAAA UGAAAUGAAAAAA
    LDHA_exon9 + TTTG 390 GGACAGAATGGAATCT 967 GGACAGAAUGGAAUCUC
    CAGACCTTGTGAAG AGACCUUGUGAAG
    LDHA_exon9 + TTTT 391 GGGACAGAATGGAATC 968 GGGACAGAAUGGAAUCU
    TCAGACCTTGTGAA CAGACCUUGUGAA
    LDHA_exon9 + ATTT 392 TGGGACAGAATGGAAT 969 UGGGACAGAAUGGAAUC
    CTCAGACCTTGTGA UCAGACCUUGUGA
    LDHA_exon9 + CTTG 393 CATTTTGGGACAGAAT 970 CAUUUUGGGACAGAAUG
    GGAATCTCAGACCT GAAUCUCAGACCU
    LDHA_exon9 + GTTC 394 CTTGCATTTTGGGACA 971 CUUGCAUUUUGGGACAG
    GAATGGAATCTCAG AAUGGAAUCUCAG
    LDHA_exon9 + CTTC 395 CTTAGTGTTCCTTGCA 972 CUUAGUGUUCCUUGCAU
    TTTTGGGACAGAAT UUUGGGACAGAAU
    LDHA_exon9 − CTTC 396 TTCAAACGGGCCTCTT 973 UUCAAACGGGCCUCUUC
    CCTCAGAAGTCAGA CUCAGAAGUCAGA
    LDHA_exon9 + TTTA 397 CGGAATAAAGGATGAT 974 CGGAAUAAAGGAUGAUG
    GTCTTCCTTAGTGT UCUUCCUUAGUGU
    LDHA_exon9 + CTTC 398 AGGGTCTTTACGGAAT 975 AGGGUCUUUACGGAAUA
    AAAGGATGATGTCT AAGGAUGAUGUCU
    LDHA_exon9 + TTTC 399 ATCTTCAGGGTCTTTA 976 AUCUUCAGGGUCUUUAC
    CGGAATAAAGGATG GGAAUAAAGGAUG
    LDHA_exon9 + ATTT 400 CATCTTCAGGGTCTTT 977 CAUCUUCAGGGUCUUUA
    ACGGAATAAAGGAT CGGAAUAAAGGAU
    LDHA_exon9 + TTTC 401 ATTTCATCTTCAGGGT 978 AUUUCAUCUUCAGGGUC
    CTTTACGGAATAAA UUUACGGAAUAAA
    LDHA_exon9 + TTTT 402 CATTTCATCTTCAGGG 979 CAUUUCAUCUUCAGGGU
    TCTTTACGGAATAA CUUUACGGAAUAA
    LDHA_exon9 + TTTT 403 TCATTTCATCTTCAGG 980 UCAUUUCAUCUUCAGGG
    GTCTTTACGGAATA UCUUUACGGAAUA
    LDHA_exon9 + TTTT 404 TTCATTTCATCTTCAG 981 UUCAUUUCAUCUUCAGG
    GGTCTTTACGGAAT GUCUUUACGGAAU
    LDHA_exon9 + TTTT 405 TTTCATTTCATCTTCA 982 UUUCAUUUCAUCUUCAG
    GGGTCTTTACGGAA GGUCUUUACGGAA
    LDHA_exon9 + TTTT 406 TTTTCATTTCATCTTC 983 UUUUCAUUUCAUCUUCA
    AGGGTCTTTACGGA GGGUCUUUACGGA
    LDHA_exon9 + TTTT 407 TTTTTCATTTCATCTT 984 UUUUUCAUUUCAUCUUC
    CAGGGTCTTTACGG AGGGUCUUUACGG
    LDHA_exon9 + TTTT 408 TTTTTTCATTTCATCT 985 UUUUUUCAUUUCAUCUU
    TCAGGGTCTTTACG CAGGGUCUUUACG
    LDHA_exon9 + TTTT 409 TTTTTTTCATTTCATC 986 UUUUUUUCAUUUCAUCU
    TTCAGGGTCTTTAC UCAGGGUCUUUAC
    LDHA_exon9 + TTTT 410 TTTTTTTTCATTTCAT 987 UUUUUUUUCAUUUCAUC
    CTTCAGGGTCTTTA UUCAGGGUCUUUA
    LDHA_exon9 + ATTT 411 TTTTTTTTTCATTTCA 988 UUUUUUUUUCAUUUCAU
    TCTTCAGGGTCTTT CUUCAGGGUCUUU
    LDHA_exon9 + CTTT 412 ACGGAATAAAGGATGA 989 ACGGAAUAAAGGAUGAU
    TGTCTTCCTTAGTG GUCUUCCUUAGUG
    LDHA_exon9 − CTTA 413 AGATTGTTTTTAATTG 990 AGAUUGUUUUUAAUUGU
    TTACCAGCTTCCAG UACCAGCUUCCAG
    LDHA_exon9 − TTTG 414 GATCCCCCAAAGTGTA 991 GAUCCCCCAAAGUGUAU
    TCTGCACTCTTCTT CUGCACUCUUCUU
    LDHA_exon9 − CTTT 415 TGGATCCCCCAAAGTG 992 UGGAUCCCCCAAAGUGU
    TATCTGCACTCTTC AUCUGCACUCUUC
    LDHA_exon9 − TTTA 416 TACACTGGATCCCAGG 993 UACACUGGAUCCCAGGA
    ATGTGACTCACTGG UGUGACUCACUGG
    LDHA_exon9 − GTTG 417 GACTAGGCATGTTCAG 994 GACUAGGCAUGUUCAGU
    TGAAGGAGCCAGGA GAAGGAGCCAGGA
    LDHA_exon9 − GTTC 418 AGTGAAGGAGCCAGGA 995 AGUGAAGGAGCCAGGAA
    AGTTATATAACACA GUUAUAUAACACA
    LDHA_exon9 − GTTA 419 TATAACACACGGTAAA 996 UAUAACACACGGUAAAC
    CATCCACCTGGCTC AUCCACCUGGCUC
    LDHA_exon9 − ATTG 420 GCAGTGGTGCGTCAGA 997 GCAGUGGUGCGUCAGAG
    GGTGGCAGAACTAT GUGGCAGAACUAU
    LDHA_exon9 − ATTT 421 CACACTAACCAGTTGA 998 CACACUAACCAGUUGAA
    AGACTACACAAGAT GACUACACAAGAU
    LDHA_exon9 − TTTC 422 ACACTAACCAGTTGAA 999 ACACUAACCAGUUGAAG
    GACTACACAAGATT ACUACACAAGAUU
    LDHA_exon9 − GTTG 423 AAGACTACACAAGATT 1000 AAGACUACACAAGAUUA
    AATACCATCCAGCA AUACCAUCCAGCA
    LDHA_exon9 − ATTA 424 ATACCATCCAGCATCA 1001 AUACCAUCCAGCAUCAG
    GGATATAGCTGTGG GAUAUAGCUGUGG
    LDHA_exon9 − ATTT 425 TACAAACCATTCTTAT 1002 UACAAACCAUUCUUAUU
    TTCTAACTTCAGGA UCUAACUUCAGGA
    LDHA_exon9 − TTTT 426 ACAAACCATTCTTATT 1003 ACAAACCAUUCUUAUUU
    TCTAACTTCAGGAG CUAACUUCAGGAG
    LDHA_exon9 − TTTA 427 CAAACCATTCTTATTT 1004 CAAACCAUUCUUAUUUC
    CTAACTTCAGGAGT UAACUUCAGGAGU
    LDHA_exon9 − ATTC 428 TTATTTCTAACTTCAG 1005 UUAUUUCUAACUUCAGG
    GAGTTGATGTTTTT AGUUGAUGUUUUU
    LDHA_exon9 − CTTA 429 TTTCTAACTTCAGGAG 1006 UUUCUAACUUCAGGAGU
    TTGATGTTTTTCCC UGAUGUUUUUCCC
    LDHA_exon9 − TTTT 430 GGATCCCCCAAAGTGT 1007 GGAUCCCCCAAAGUGUA
    ATCTGCACTCTTCT UCUGCACUCUUCU
    LDHA_exon9 − ATTT 431 CTAACTTCAGGAGTTG 1008 CUAACUUCAGGAGUUGA
    ATGTTTTTCCCAGT UGUUUUUCCCAGU
    LDHA_exon9 − CTTC 432 AGGAGTTGATGTTTTT 1009 AGGAGUUGAUGUUUUUC
    CCCAGTCCATCTTA CCAGUCCAUCUUA
    LDHA_exon9 − GTTG 433 ATGTTTTTCCCAGTCC 1010 AUGUUUUUCCCAGUCCA
    ATCTTAAAATATTA UCUUAAAAUAUUA
    LDHA_exon9 − GTTT 434 TTCCCAGTCCATCTTA 1011 UUCCCAGUCCAUCUUAA
    AAATATTACTGCTT AAUAUUACUGCUU
    LDHA_exon9 − TTTT 435 TCCCAGTCCATCTTAA 1012 UCCCAGUCCAUCUUAAA
    AATATTACTGCTTT AUAUUACUGCUUU
    LDHA_exon9 − TTTT 436 CCCAGTCCATCTTAAA 1013 CCCAGUCCAUCUUAAAA
    ATATTACTGCTTTA UAUUACUGCUUUA
    LDHA_exon9 − TTTC 437 CCAGTCCATCTTAAAA 1014 CCAGUCCAUCUUAAAAU
    TATTACTGCTTTAA AUUACUGCUUUAA
    LDHA_exon9 − CTTA 438 AAATATTACTGCTTTA 1015 AAAUAUUACUGCUUUAA
    ATCACAGATCAGAT UCACAGAUCAGAU
    LDHA_exon9 − ATTA 439 CTGCTTTAATCACAGA 1016 CUGCUUUAAUCACAGAU
    TCAGATAAAAAGGA CAGAUAAAAAGGA
    LDHA_exon9 − CTTT 440 AATCACAGATCAGATA 1017 AAUCACAGAUCAGAUAA
    AAAAGGACAACATG AAAGGACAACAUG
    LDHA_exon9 − TTTA 441 ATCACAGATCAGATAA 1018 AUCACAGAUCAGAUAAA
    AAAGGACAACATGC AAGGACAACAUGC
    LDHA_exon9 − GTTG 442 TAGCCTAGACAGTGAA 1019 UAGCCUAGACAGUGAAA
    ATGATATGACATCA UGAUAUGACAUCA
    LDHA_exon9 − CTTT 443 AAAATTGCAGCTCCTT 1020 AAAAUUGCAGCUCCUUU
    TTGGATCCCCCAAA UGGAUCCCCCAAA
    LDHA_exon9 − TTTA 444 AAATTGCAGCTCCTTT 1021 AAAUUGCAGCUCCUUUU
    TGGATCCCCCAAAG GGAUCCCCCAAAG
    LDHA_exon9 − ATTG 445 CAGCTCCTTTTGGATC 1022 CAGCUCCUUUUGGAUCC
    CCCCAAAGTGTATC CCCAAAGUGUAUC
    LDHA_exon9 − TTTC 446 TAACTTCAGGAGTTGA 1023 UAACUUCAGGAGUUGAU
    TGTTTTTCCCAGTC GUUUUUCCCAGUC
    LDHA_exon9 + CTTG 447 TGAAGGTGACTCTGAC 1024 UGAAGGUGACUCUGACU
    TTCTGAGGAAGAGG UCUGAGGAAGAGG
    LDHA_exon9 − ATTA 448 TAGGCATGAGCCACTG 1025 UAGGCAUGAGCCACUGC
    CACCCTGCCTTAAG ACCCUGCCUUAAG
    LDHA_exon9 − ATTC 449 CTGGCCTCCAGTGATC 1026 CUGGCCUCCAGUGAUCA
    AGCCCACCTGGGCT GCCCACCUGGGCU
    LDHA_exon9 + GTTA 450 TATAACTTCCTGGCTC 1027 UAUAACUUCCUGGCUCC
    CTTCACTGAACATG UUCACUGAACAUG
    LDHA_exon9 + CTTC 451 CTGGCTCCTTCACTGA 1028 CUGGCUCCUUCACUGAA
    ACATGCCTAGTCCA CAUGCCUAGUCCA
    LDHA_exon9 + CTTC 452 ACTGAACATGCCTAGT 1029 ACUGAACAUGCCUAGUC
    CCAACATTTTTTCC CAACAUUUUUUCC
    LDHA_exon9 + ATTT 453 TTTCCCAGTGAGTCAC 1030 UUUCCCAGUGAGUCACA
    ATCCTGGGATCCAG UCCUGGGAUCCAG
    LDHA_exon9 + TTTT 454 TTCCCAGTGAGTCACA 1031 UUCCCAGUGAGUCACAU
    TCCTGGGATCCAGT CCUGGGAUCCAGU
    LDHA_exon9 + TTTT 455 TCCCAGTGAGTCACAT 1032 UCCCAGUGAGUCACAUC
    CCTGGGATCCAGTG CUGGGAUCCAGUG
    LDHA_exon9 + TTTT 456 CCCAGTGAGTCACATC 1033 CCCAGUGAGUCACAUCC
    CTGGGATCCAGTGT UGGGAUCCAGUGU
    LDHA_exon9 + TTTC 457 CCAGTGAGTCACATCC 1034 CCAGUGAGUCACAUCCU
    TGGGATCCAGTGTA GGGAUCCAGUGUA
    LDHA_exon9 + CTTG 458 TGCATAATTCTTCCAA 1035 UGCAUAAUUCUUCCAAA
    AGGATCTTATTTTG GGAUCUUAUUUUG
    LDHA_exon9 + ATTC 459 TTCCAAAGGATCTTAT 1036 UUCCAAAGGAUCUUAUU
    TTTGTGAACTATAT UUGUGAACUAUAU
    LDHA_exon9 + CTTC 460 CAAAGGATCTTATTTT 1037 CAAAGGAUCUUAUUUUG
    GTGAACTATATCAG UGAACUAUAUCAG
    LDHA_exon9 + CTTA 461 TTTTGTGAACTATATC 1038 UUUUGUGAACUAUAUCA
    AGTAGTGTACATTA GUAGUGUACAUUA
    LDHA_exon9 + ATTT 462 TGTGAACTATATCAGT 1039 UGUGAACUAUAUCAGUA
    AGTGTACATTACCA GUGUACAUUACCA
    LDHA_exon9 + TTTT 463 GTGAACTATATCAGTA 1040 GUGAACUAUAUCAGUAG
    GTGTACATTACCAT UGUACAUUACCAU
    LDHA_exon9 + TTTA 464 CCGTGTGTTATATAAC 1041 CCGUGUGUUAUAUAACU
    TTCCTGGCTCCTTC UCCUGGCUCCUUC
    LDHA_exon9 + TTTG 465 TGAACTATATCAGTAG 1042 UGAACUAUAUCAGUAGU
    TGTACATTACCATA GUACAUUACCAUA
    LDHA_exon9 + GTTA 466 TACCAACTAAAACCCC 1043 UACCAACUAAAACCCCC
    CAATAAACCTTGAA AAUAAACCUUGAA
    LDHA_exon9 + CTTG 467 AACAGTGACTACTTTG 1044 AACAGUGACUACUUUGG
    GTTAATTCATTATA UUAAUUCAUUAUA
    LDHA_exon9 + CTTT 468 GGTTAATTCATTATAT 1045 GGUUAAUUCAUUAUAUU
    TAAGATATAAAGTC AAGAUAUAAAGUC
    LDHA_exon9 + TTTG 469 GTTAATTCATTATATT 1046 GUUAAUUCAUUAUAUUA
    AAGATATAAAGTCA AGAUAUAAAGUCA
    LDHA_exon9 + GTTA 470 ATTCATTATATTAAGA 1047 AUUCAUUAUAUUAAGAU
    TATAAAGTCATAAA AUAAAGUCAUAAA
    LDHA_exon9 + ATTC 471 ATTATATTAAGATATA 1048 AUUAUAUUAAGAUAUAA
    AAGTCATAAAGCTG AGUCAUAAAGCUG
    LDHA_exon9 + ATTA 472 TATTAAGATATAAAGT 1049 UAUUAAGAUAUAAAGUC
    CATAAAGCTGCTAG AUAAAGCUGCUAG
    LDHA_exon9 + ATTA 473 AGATATAAAGTCATAA 1050 AGAUAUAAAGUCAUAAA
    AGCTGCTAGTTATT GCUGCUAGUUAUU
    LDHA_exon9 + GTTA 474 TTATATTAATTTGGAA 1051 UUAUAUUAAUUUGGAAA
    ATATTAGGCTATTC UAUUAGGCUAUUC
    LDHA_exon9 + ATTA 475 TATTAATTTGGAAATA 1052 UAUUAAUUUGGAAAUAU
    TTAGGCTATTCTTG UAGGCUAUUCUUG
    LDHA_exon9 + ATTA 476 ATTTGGAAATATTAGG 1053 AUUUGGAAAUAUUAGGC
    CTATTCTTGGGCAA UAUUCUUGGGCAA
    LDHA_exon9 + ATTT 477 GGAAATATTAGGCTAT 1054 GGAAAUAUUAGGCUAUU
    TCTTGGGCAACCCT CUUGGGCAACCCU
    LDHA_exon9 + TTTG 478 GAAATATTAGGCTATT 1055 GAAAUAUUAGGCUAUUC
    CTTGGGCAACCCTG UUGGGCAACCCUG
    LDHA_exon9 + ATTA 479 GGCTATTCTTGGGCAA 1056 GGCUAUUCUUGGGCAAC
    CCCTGCAACGATTT CCUGCAACGAUUU
    LDHA_exon9 + ATTA 480 CCATATAATGTAAAAA 1057 CCAUAUAAUGUAAAAAG
    GATCTACATACAAA AUCUACAUACAAA
    LDHA_exon9 + ATTC 481 TTGGGCAACCCTGCAA 1058 UUGGGCAACCCUGCAAC
    CGATTTTTTCTAAC GAUUUUUUCUAAC
    LDHA_exon9 + GTTT 482 ACCGTGTGTTATATAA 1059 ACCGUGUGUUAUAUAAC
    CTTCCTGGCTCCTT UUCCUGGCUCCUU
    LDHA_exon9 + TTTG 483 CCCCTTGAGCCAGGTG 1060 CCCCUUGAGCCAGGUGG
    GATGTTTACCGTGT AUGUUUACCGUGU
    LDHA_exon9 + GTTT 484 GAAGAAGAGTGCAGAT 1061 GAAGAAGAGUGCAGAUA
    ACACTTTGGGGGAT CACUUUGGGGGAU
    LDHA_exon9 + TTTG 485 AAGAAGAGTGCAGATA 1062 AAGAAGAGUGCAGAUAC
    CACTTTGGGGGATC ACUUUGGGGGAUC
    LDHA_exon9 + GTTT 486 GGGGGATCCAAAAGGA 1063 GGGGGAUCCAAAAGGAG
    GCTGCAATTTTAAA CUGCAAUUUUAAA
    LDHA_exon9 + TTTG 487 GGGGATCCAAAAGGAG 1064 GGGGAUCCAAAAGGAGC
    CTGCAATTTTAAAG UGCAAUUUUAAAG
    LDHA_exon9 + ATTT 488 TAAAGTCTTCTGATGT 1065 UAAAGUCUUCUGAUGUC
    CATATCATTTCACT AUAUCAUUUCACU
    LDHA_exon9 + TTTT 489 AAAGTCTTCTGATGTC 1066 AAAGUCUUCUGAUGUCA
    ATATCATTTCAGTG UAUCAUUUCACUG
    LDHA_exon9 + TTTA 490 AAGTCTTCTGATGTCA 1067 AAGUCUUCUGAUGUCAU
    TATCATTTCACTGT AUCAUUUCACUGU
    LDHA_exon9 + CTTC 491 TGATGTCATATCATTT 1068 UGAUGUCAUAUCAUUUC
    CACTGTCTAGGCTA ACUGUCUAGGCUA
    LDHA_exon9 + ATTT 492 CACTGTCTAGGCTACA 1069 CACUGUCUAGGCUACAA
    ACAGGATTCTAGGT CAGGAUUCUAGGU
    LDHA_exon9 + TTTG 493 ACTGTCTAGGCTACAA 1070 ACUGUCUAGGCUACAAC
    CAGGATTCTAGGTG AGGAUUCUAGGUG
    LDHA_exon9 + ATTC 494 TAGGTGGAGGTTGTGC 1071 UAGGUGGAGGUUGUGCA
    ATGTTGTCCTTTTT UGUUGUCCUUUUU
    LDHA_exon9 + GTTG 495 TGCATGTTGTCCTTTT 1072 UGCAUGUUGUCCUUUUU
    TATCTGATCTGTGA AUCUGAUCUGUGA
    LDHA_exon9 + GTTG 496 TCCTTTTTATCTGATC 1073 UCCUUUUUAUCUGAUCU
    TGTGATTAAAGCAG GUGAUUAAAGCAG
    LDHA_exon9 + GTTT 497 TTATCTGATCTGTGAT 1074 UUAUCUGAUCUGUGAUU
    TAAAGCAGTAATAT AAAGCAGUAAUAU
    LDHA_exon9 + GTTG 498 AGCCAGGTGGATGTTT 1075 AGCCAGGUGGAUGUUUA
    ACCGTGTGTTATAT CCGUGUGUUAUAU
    LDHA_exon9 + TTTT 499 TATCTGATCTGTGATT 1076 UAUCUGAUCUGUGAUUA
    AAAGCAGTAATATT AAGCAGUAAUAUU
    LDHA_exon9 + TTTA 500 TCTGATCTGTGATTAA 1077 UCUGAUCUGUGAUUAAA
    AGCAGTAATATTTT GCAGUAAUAUUUU
    LDHA_exon9 + ATTA 501 AAGCAGTAATATTTTA 1078 AAGCAGUAAUAUUUUAA
    AGATGGACTGGGAA GAUGGACUGGGAA
    LDHA_exon9 + ATTT 502 TAAGATGGACTGGGAA 1079 UAAGAUGGACUGGGAAA
    AAACATCAACTCCT AACAUCAACUCCU
    LDHA_exon9 + TTTT 503 AAGATGGACTGGGAAA 1080 AAGAUGGACUGGGAAAA
    AACATCAACTCCTG ACAUCAACUCCUG
    LDHA_exon9 + TTTA 504 AGATGGACTGGGAAAA 1081 AGAUGGACUGGGAAAAA
    ACATCAACTCCTGA CAUCAACUCCUGA
    LDHA_exon9 + GTTA 505 GAAATAAGAATGGTTT 1082 GAAAUAAGAAUGGUUUG
    GTAAAATCCACAGC UAAAAUCCACAGC
    LDHA_exon9 + GTTT 506 GTAAAATCCACAGCTA 1083 GUAAAAUCCACAGCUAU
    TATCCTGATGCTGG AUCCUGAUGCUGG
    LDHA_exon9 + TTTG 507 TAAAATCCACAGCTAT 1084 UAAAAUCCACAGCUAUA
    ATCCTGATGCTGGA UCCUGAUGCUGGA
    LDHA_exon9 + ATTA 508 ATCTTGTGTAGTCTTC 1085 AUCUUGUGUAGUCUUCA
    AACTGGTTAGTGTG ACUGGUUAGUGUG
    LDHA_exon9 + CTTG 509 TGTAGTCTTCAACTGG 1086 UGUAGUCUUCAACUGGU
    TTAGTGTGAAATAG UAGUGUGAAAUAG
    LDHA_exon9 + CTTC 510 AACTGGTTAGTGTGAA 1087 AACUGGUUAGUGUGAAA
    ATAGTTCTGCCACC UAGUUCUGCCACC
    LDHA_exon9 + GTTA 511 GTGTGAAATAGTTCTG 1088 GUGUGAAAUAGUUCUGC
    CCACCTCTGACGCA CACCUCUGACGCA
    LDHA_exon9 + GTTC 512 TGCCACCTCTGACGCA 1089 UGCCACCUCUGACGCAC
    CCACTGCCAATGCT CACUGCCAAUGCU
    LDHA_exon9 + ATTT 513 GCCCCTTGAGCCAGGT 1090 GCCCCUUGAGCCAGGUG
    GGATGTTTACCGTG GAUGUUUACCGUG
    LDHA_exon9 + TTTT 514 ATCTGATCTGTGATTA 1091 AUCUGAUCUGUGAUUAA
    AAGCAGTAATATTT AGCAGUAAUAUUU
    LDHA_exon9 − CTTC 515 CCAAAGTGCTGGGATT 1092 CCAAAGUGCUGGGAUUA
    ATAGGCATGAGCCA UAGGCAUGAGCCA
    LDHA_exon9 + CTTG 516 GGCAACCCTGCAACGA 1093 GGCAACCCUGCAACGAU
    TTTTTTCTAACAGG UUUUUCUAACAGG
    LDHA_exon9 + TTTT 517 TTCTAACAGGGATATT 1094 UUCUAACAGGGAUAUUA
    ATTGACTAATAGCA UUGACUAAUAGCA
    LDHA_exon9 − ATTT 518 TCAGAAAAATGTGCAG 1095 UCAGAAAAAUGUGCAGA
    AAAACTTGAGTAGA AAACUUGAGUAGA
    LDHA_exon9 − TTTT 519 CAGAAAAATGTGCAGA 1096 CAGAAAAAUGUGCAGAA
    AAACTTGAGTAGAC AACUUGAGUAGAC
    LDHA_exon9 − TTTC 520 AGAAAAATGTGCAGAA 1097 AGAAAAAUGUGCAGAAA
    AACTTGAGTAGACA ACUUGAGUAGACA
    LDHA_exon9 − CTTG 521 AGTAGACATCCACCAA 1098 AGUAGACAUCCACCAAG
    GGTTACTTGTTTTT GUUACUUGUUUUU
    LDHA_exon9 − GTTA 522 CTTGTTTTTTTTGGTT 1099 CUUGUUUUUUUUGGUUU
    TTGTTTTGTTTTTT UGUUUUGUUUUUU
    LDHA_exon9 − CTTG 523 TTTTTTTTGGTTTTGT 1100 UUUUUUUUGGUUUUGUU
    TTTGTTTTTTTAAC UUGUUUUUUUAAC
    LDHA_exon9 − GTTT 524 TTTTTGGTTTTGTTTT 1101 UUUUUGGUUUUGUUUUG
    GTTTTTTTAACAGA UUUUUUUAACAGA
    LDHA_exon9 − TTTT 525 TTTTGGTTTTGTTTTG 1102 UUUUGGUUUUGUUUUGU
    TTTTTTTAACAGAT UUUUUUAACAGAU
    LDHA_exon9 − TTTT 526 TTTGGTTTTGTTTTGT 1103 UUUGGUUUUGUUUUGUU
    TTTTTTAACAGATG UUUUUAACAGAUG
    LDHA_exon9 − TTTT 527 TTGGTTTTGTTTTGTT 1104 UUGGUUUUGUUUUGUUU
    TTTTTAACAGATGG UUUUAACAGAUGG
    LDHA_exon9 − TTTT 528 TGGTTTTGTTTTGTTT 1105 UGGUUUUGUUUUGUUUU
    TTTTAACAGATGGG UUUAACAGAUGGG
    LDHA_exon9 − TTTT 529 GGTTTTGTTTTGTTTT 1106 GGUUUUGUUUUGUUUUU
    TTTAACAGATGGGG UUAACAGAUGGGG
    LDHA_exon9 − TTTG 530 GTTTTGTTTTGTTTTT 1107 GUUUUGUUUUGUUUUUU
    TTAACAGATGGGGT UAACAGAUGGGGU
    LDHA_exon9 − GTTT 531 TGTTTTGTTTTTTTAA 1108 UGUUUUGUUUUUUUAAC
    CAGATGGGGTTTTG AGAUGGGGUUUUG
    LDHA_exon9 − GTTG 532 TATTTTCAGAAAAATG 1109 UAUUUUCAGAAAAAUGU
    TGCAGAAAACTTGA GCAGAAAACUUGA
    LDHA_exon9 − TTTT 533 GTTTTGTTTTTTTAAC 1110 GUUUUGUUUUUUUAACA
    AGATGGGGTTTTGT GAUGGGGUUUUGU
    LDHA_exon9 − GTTT 534 TGTTTTTTTAACAGAT 1111 UGUUUUUUUAACAGAUG
    GGGGTTTTGTTGTG GGGUUUUGUUGUG
    LDHA_exon9 − TTTT 535 GTTTTTTTAACAGATG 1112 GUUUUUUUAACAGAUGG
    GGGTTTTGTTGTGT GGUUUUGUUGUGU
    LDHA_exon9 − TTTG 536 TTTTTTTAACAGATGG 1113 UUUUUUUAACAGAUGGG
    GGTTTTGTTGTGTT GUUUUGUUGUGUU
    LDHA_exon9 − GTTT 537 TTTTAACAGATGGGGT 1114 UUUUAACAGAUGGGGUU
    TTTGTTGTGTTGGC UUGUUGUGUUGGC
    LDHA_exon9 − TTTT 538 TTTAACAGATGGGGTT 1115 UUUAACAGAUGGGGUUU
    TTGTTGTGTTGGCC UGUUGUGUUGGCC
    LDHA_exon9 − TTTT 539 TTAACAGATGGGGTTT 1116 UUAACAGAUGGGGUUUU
    TGTTGTGTTGGCCA GUUGUGUUGGCCA
    LDHA_exon9 − TTTT 540 TAACAGATGGGGTTTT 1117 UAACAGAUGGGGUUUUG
    GTTGTGTTGGCCAG UUGUGUUGGCCAG
    LDHA_exon9 − TTTT 541 AACAGATGGGGTTTTG 1118 AACAGAUGGGGUUUUGU
    TTGTGTTGGCCAGG UGUGUUGGCCAGG
    LDHA_exon9 − TTTA 542 ACAGATGGGGTTTTGT 1119 ACAGAUGGGGUUUUGUU
    TGTGTTGGCCAGGC GUGUUGGCCAGGC
    LDHA_exon9 − GTTT 543 TGTTGTGTTGGCCAGG 1120 UGUUGUGUUGGCCAGGC
    CTGGTCCCCAATTC UGGUCCCCAAUUC
    LDHA_exon9 − TTTT 544 GTTGTGTTGGCCAGGC 1121 GUUGUGUUGGCCAGGCU
    TGGTCCCCAATTCC GGUCCCCAAUUCC
    LDHA_exon9 − TTTG 545 TTGTGTTGGCCAGGCT 1122 UUGUGUUGGCCAGGCUG
    GGTCCCCAATTCCT GUCCCCAAUUCCU
    LDHA_exon9 − GTTG 546 TGTTGGCCAGGCTGGT 1123 UGUUGGCCAGGCUGGUC
    CCCCAATTCCTGGC CCCAAUUCCUGGC
    LDHA_exon9 − GTTG 547 GCCAGGCTGGTCCCCA 1124 GCCAGGCUGGUCCCCAA
    ATTCCTGGCCTCCA UUCCUGGCCUCCA
    LDHA_exon9 − TTTG 548 TTTTGTTTTTTTAACA 1125 UUUUGUUUUUUUAACAG
    GATGGGGTTTTGTT AUGGGGUUUUGUU
    LDHA_exon9 + ATTT 549 TTTCTAACAGGGATAT 1126 UUUCUAACAGGGAUAUU
    TATTGACTAATAGC AUUGACUAAUAGC
    LDHA_exon9 + TTTG 550 TGCACATTTTTCTGAA 1127 UGCACAUUUUUCUGAAA
    AATACAACTGTGAC AUACAACUGUGAC
    LDHA_exon9 + GTTT 551 TCTGCACATTTTTCTG 1128 UCUGCACAUUUUUCUGA
    AAAATACAACTGTG AAAUACAACUGUG
    LDHA_exon9 + TTTT 552 TCTAACAGGGATATTA 1129 UCUAACAGGGAUAUUAU
    TTGACTAATAGCAG UGACUAAUAGCAG
    LDHA_exon9 + TTTT 553 CTAACAGGGATATTAT 1130 CUAACAGGGAUAUUAUU
    TGACTAATAGCAGA GACUAAUAGCAGA
    LDHA_exon9 + TTTG 554 TAACAGGGATATTATT 1131 UAACAGGGAUAUUAUUG
    GACTAATAGCAGAG ACUAAUAGCAGAG
    LDHA_exon9 + ATTA 555 TTGACTAATAGCAGAG 1132 UUGACUAAUAGCAGAGG
    GATGTAATAGTCAA AUGUAAUAGUCAA
    LDHA_exon9 + ATTG 556 ACTAATAGCAGAGGAT 1133 ACUAAUAGCAGAGGAUG
    GTAATAGTCAACTG UAAUAGUCAACUG
    LDHA_exon9 + GTTG 557 TATTGGTACCACTTCC 1134 UAUUGGUACCACUUCCA
    ATTGTAAGTCCCAA UUGUAAGUCCCAA
    LDHA_exon9 + ATTG 558 GTACCACTTCCATTGT 1135 GUACCACUUCCAUUGUA
    AAGTCCCAAAGTAT AGUCCCAAAGUAU
    LDHA_exon9 + CTTC 559 CATTGTAAGTCCCAAA 1136 CAUUGUAAGUCCCAAAG
    GTATTATATATTTG UAUUAUAUAUUUG
    LDHA_exon9 + ATTG 560 TAAGTCCCAAAGTATT 1137 UAAGUCCCAAAGUAUUA
    ATATATTTGATAAT UAUAUUUGAUAAU
    LDHA_exon9 + ATTA 561 TATATTTGATAATAAT 1138 UAUAUUUGAUAAUAAUG
    GCTAATCATAATTG CUAAUCAUAAUUG
    LDHA_exon9 + ATTT 562 GATAATAATGCTAATC 1139 GAUAAUAAUGCUAAUCA
    ATAATTGGAAAGTA UAAUUGGAAAGUA
    LDHA_exon9 + TTTG 563 ATAATAATGCTAATCA 1140 AUAAUAAUGCUAAUCAU
    TAATTGGAAAGTAA AAUUGGAAAGUAA
    LDHA_exon9 + ATTG 564 GAAAGTAACATTCTAT 1141 GAAAGUAACAUUCUAUA
    ATGTAAATGTAAAA UGUAAAUGUAAAA
    LDHA_exon9 + ATTG 565 TATATGTAAATGTAAA 1142 UAUAUGUAAAUGUAAAA
    ATTTATTTGCCAAC UUUAUUUGCCAAC
    LDHA_exon9 + TTTT 566 CTGCACATTTTTCTGA 1143 CUGCACAUUUUUCUGAA
    AAATACAACTGTGA AAUACAACUGUGA
    LDHA_exon9 + ATTT 567 ATTTGCCAACTGAATA 1144 AUUUGCCAACUGAAUAU
    TAGGCAATGATAGT AGGCAAUGAUAGU
    LDHA_exon9 + ATTT 568 GCCAACTGAATATAGG 1145 GCCAACUGAAUAUAGGC
    CAATGATAGTGTGT AAUGAUAGUGUGU
    LDHA_exon9 + TTTG 569 CCAACTGAATATAGGC 1146 CCAACUGAAUAUAGGCA
    AATGATAGTGTGTC AUGAUAGUGUGUC
    LDHA_exon9 + ATTT 570 TTGAGATCTTGTCCTC 1147 UUGAGAUCUUGUCCUCU
    TGGAAGCTGGTAAC GGAAGCUGGUAAC
    LDHA_exon9 + TTTT 571 TGAGATCTTGTCCTCT 1148 UGAGAUCUUGUCCUCUG
    GGAAGCTGGTAACA GAAGCUGGUAACA
    LDHA_exon9 + TTTT 572 GAGATCTTGTCCTCTG 1149 GAGAUCUUGUCCUCUGG
    GAAGCTGGTAACAA AAGCUGGUAACAA
    LDHA_exon9 + TTTG 573 AGATCTTGTCCTCTGG 1150 AGAUCUUGUCCUCUGGA
    AAGCTGGTAACAAT AGCUGGUAACAAU
    LDHA_exon9 + GTTG 574 TCCTCTGGAAGCTGGT 1151 UCCUCUGGAAGCUGGUA
    AACAATTAAAAACA ACAAUUAAAAACA
    LDHA_exon9 + ATTA 575 AAAACAATCTTAAGGC 1152 AAAACAAUCUUAAGGCA
    AGGGTGCAGTGGCT GGGUGCAGUGGCU
    LDHA_exon9 + CTTA 576 AGGCAGGGTGCAGTGG 1153 AGGCAGGGUGCAGUGGC
    CTCATGCCTATAAT UCAUGCCUAUAAU
    LDHA_exon9 + CTTT 577 GGGAAGCCCAGGTGGG 1154 GGGAAGCCCAGGUGGGC
    CTGATCACTGGAGG UGAUCACUGGAGG
    LDHA_exon9 + TTTG 578 GGAAGCCCAGGTGGGC 1155 GGAAGCCCAGGUGGGCU
    TGATCACTGGAGGC GAUCACUGGAGGC
    LDHA_exon9 + ATTG 579 GGGACCAGCCTGGCCA 1156 GGGACCAGCCUGGCCAA
    ACACAACAAAACCC CACAACAAAACCC
    LDHA_exon9 + GTTA 580 AAAAAACAAAACAAAA 1157 AAAAAACAAAACAAAAC
    CCAAAAAAAACAAG CAAAAAAAACAAG
    LDHA_exon9 + GTTG 581 GTGGATGTCTACTCAA 1158 GUGGAUGUCUACUCAAG
    GTTTTCTGCACATT UUUUCUGCACAUU
    LDHA_exon9 + TTTA 582 TTTGCCAACTGAATAT 1159 UUUGCCAACUGAAUAUA
    AGGCAATGATAGTG GGCAAUGAUAGUG
    LDHA_exon9 + CTTA 583 GTGTTCCTTGCATTTT 1160 GUGUUCCUUGCAUUUUG
    GGGACAGAATGGAA GGACAGAAUGGAA
    LDHA_exon9 + ATTT 584 TTCTGAAAATACAACT 1161 UUCUGAAAAUACAACUG
    GTGACCCTTA UGACCCUUA
    LDHA_exon9 + TTTT 585 TCTGAAAATACAACTG 1162 UCUGAAAAUACAACUGU
    TGACCCTTA GACCCUUA
    LDHA_exon9 + TTTT 586 CTGAAAATACAACTGT 1163 CUGAAAAUACAACUGUG
    GACCCTTA ACCCUUA
    LDHA_exon9 + TTTC 587 TGAAAATACAACTGTG 1164 UGAAAAUACAACUGUGA
    ACCCTTA CCCUUA
    *The 3′ three nucleotides represent the 5′-TTN-3′ motif.
  • The present disclosure includes all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein.
  • In some embodiments, a spacer sequence described herein comprises a uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5.
    • (iii). Exemplary RNA Guides
  • The present disclosure includes RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g., as set forth in Table 5, above). In some embodiments, the sequence of an RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229.
  • In some embodiments, exemplary RNA guides provided herein may comprise a spacer sequence of any one of SEQ ID NOs: 1269-1273. In one example, the RNA guide may comprise a spacer of SEQ ID NO: 1272. In another example, the RNA guide may comprise a spacer of SEQ ID NO: 1269. In still another example, the RNA guide may comprise a spacer of SEQ ID NO: 1270. In still another example, the RNA guide may comprise a spacer of SEQ ID NO: 1271. In yet another example, the RNA guide may comprise a spacer of SEQ ID NO: 1273.
  • Any of the exemplary RNA guides disclosed herein may comprise a direct sequence of any one of SEQ ID NOs:1-10 or a fragment thereof that is at least 23-nucleotide in length. In one example, the direct sequence may comprise SEQ ID NO: 10.
  • In specific examples, the RNA guides provide herein may comprise the nucleotide sequence of SEQ ID NOs: 1214, 1235, 1221, 1224 or 1225. In one example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1224. In another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1214. In still another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1235. In still another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1221. In yet another example, the RNA guide provided herein comprise the nucleotide sequence of SEQ ID NO: 1225.
    • (iv). Modifications
  • The RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this invention.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.
  • The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.
  • In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to RNA guide-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
  • Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
  • In some embodiments, sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone.
  • Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.
  • The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
  • Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.
  • In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4′-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5′-elaidic acid ester).
  • In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
  • In some embodiments, one or more of the nucleotides of an RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification. In some embodiments, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • When a gene editing system disclosed herein comprises nucleic acids encoding the Cas12i polypeptide disclosed herein, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.
    • B. Cas12i Polypeptide
  • In some embodiments, the composition or system of the present disclosure includes a Cas12i polypeptide as described in WO/2019/178427, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.
  • In some embodiments, the composition of the present disclosure includes a Cas12i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1166 and/or encoded by SEQ ID NO: 1165). In some embodiments, the Cas12i2 polypeptide comprises at least one RuvC domain.
  • A nucleic acid sequence encoding the Cas12i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165. In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1165. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency). See, e.g., Tijssen, “Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation” (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).
  • In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165.
  • In some embodiments, the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1166.
  • In some embodiments, the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1166. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1166 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • In some embodiments, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. In specific examples, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1168 or SEQ ID NO: 1171.
  • In some examples, the Cas12i2 polypeptide may contain one or more mutations relative to SEQ ID NO: 1166, for example, at position D581, G624, F626, P868, I926, V1030, E1035, S1046, or any combination thereof. In some instances, the one or more mutations are amino acid substitutions, for example, D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • In some examples, the Cas12i2 polypeptide contains mutations at positions D581, D911, 1926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, D911R, I926R, and V1030G (e.g., SEQ ID NO: 1167). In some examples, the Cas12i2 polypeptide contains mutations at positions D581, I926, and V1030. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, and V1030G (e.g., SEQ ID NO: 1168). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, I926, V1030, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, I926R, V1030G, and S1046G (e.g., SEQ ID NO: 1169). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1170). In some examples, the Cas12i2 polypeptide may contain mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046. Such a Cas12i2 polypeptide may contain amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G (e.g., SEQ ID NO: 1171).
  • In some embodiments, the Cas12i2 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. In some embodiments, a Cas12i2 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate the polypeptide from its respective parent/reference sequence.
  • In some embodiments, the present disclosure describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • In some embodiments, the composition of the present disclosure includes a Cas12i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1202 and/or encoded by SEQ ID NO: 1201). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.
  • A nucleic acid sequence encoding the Cas12i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201. In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1201. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency).
  • In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201.
  • In some embodiments, the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1202.
  • In some embodiments, the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1202. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1202 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • In some embodiments, the Cas12i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.
  • In some embodiments, the Cas12i4 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204. In some embodiments, a Cas12i4 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1203 or SEQ ID NO: 1204 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate it from its respective parent/reference sequence.
  • In some embodiments, the present disclosure describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i4 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1203 or SEQ ID NO: 1204 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • In some embodiments, the composition of the present disclosure includes a Cas12i1 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1211). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.
  • In some embodiments, the Cas12i1 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1211.
  • In some embodiments, the present disclosure describes a Cas12i1 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1211. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i1 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1211 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • In some embodiments, the composition of the present disclosure includes a Cas12i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1212). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.
  • In some embodiments, the Cas12i3 polypeptide of the present disclosure comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1212.
  • In some embodiments, the present disclosure describes a Cas12i3 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, 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%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1212. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Also provided is a Cas12i3 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1212 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • Although the changes described herein may be one or more amino acid changes, changes to the Cas12i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions. For example, the Cas12i polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a polyhistidine tag (His-tag), Myc, and FLAG. In some embodiments, the Cas12i polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).
  • In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • In some embodiments, the Cas12i polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.
  • In some embodiments, the nucleotide sequence encoding the Cas12i polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.). In some examples, the nucleic acid encoding the Cas12i polypeptides such as Cas12i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized.
  • Exemplary Cas12i polypeptide sequences and corresponding nucleotide sequences are listed in Table 6.
  • TABLE 6
    Cas12i and LDHA Sequences
    SEQ ID Description
    NO Sequence
    1165 ATGAGCAGCGCGATCAAAAGCTACAAGAGCGTTCTGCGTCCGAACGAGCGTAAGAA Nucleotide
    CCAACTGCTGAAAAGCACCATTCAGTGCCTGGAAGACGGTAGCGCGTTCTTTTTCA sequence
    AGATGCTGCAAGGCCTGTTTGGTGGCATCACCCCGGAGATTGTTCGTTTCAGCACC encoding
    GAACAGGAGAAACAGCAACAGGATATCGCGCTGTGGTGCGCGGTTAACTGGTTCCG parent
    TCCGGTGAGCCAAGACAGCCTGACCCACACCATTGCGAGCGATAACCTGGTGGAGA Cas12i2
    AGTTTGAGGAATACTATGGTGGCACCGCGAGCGACGCGATCAAACAGTACTTCAGC
    GCGAGCATTGGCGAAAGCTACTATTGGAACGACTGCCGTCAACAGTACTATGATCT
    GTGCCGTGAGCTGGGTGTTGAGGTGAGCGACCTGACCCATGATCTGGAGATCCTGT
    GCCGTGAAAAGTGCCTGGCGGTTGCGACCGAGAGCAACCAGAACAACAGCATCATT
    AGCGTTCTGTTTGGCACCGGCGAAAAAGAGGACCGTAGCGTGAAACTGCGTATCAC
    CAAGAAAATTCTGGAGGCGATCAGCAACCTGAAAGAAATCCCGAAGAACGTTGCGC
    CGATTCAAGAGATCATTCTGAACGTGGCGAAAGCGACCAAGGAAACCTTCCGTCAG
    GTGTATGCGGGTAACCTGGGTGCGCCGAGCACCCTGGAGAAATTTATCGCGAAGGA
    CGGCCAAAAAGAGTTCGATCTGAAGAAACTGCAGACCGACCTGAAGAAAGTTATTC
    GTGGTAAAAGCAAGGAGCGTGATTGGTGCTGCCAGGAAGAGCTGCGTAGCTACGTG
    GAGCAAAACACCATCCAGTATGACCTGTGGGCGTGGGGCGAAATGTTCAACAAAGC
    GCACACCGCGCTGAAAATCAAGAGCACCCGTAACTACAACTTTGCGAAGCAACGTC
    TGGAACAGTTCAAAGAGATTCAGAGCCTGAACAACCTGCTGGTTGTGAAGAAGCTG
    AACGACTTTTTCGATAGCGAATTTTTCAGCGGCGAGGAAACCTACACCATCTGCGT
    TCACCATCTGGGTGGCAAGGACCTGAGCAAACTGTATAAGGCGTGGGAGGATGATC
    CGGCGGACCCGGAAAACGCGATTGTGGTTCTGTGCGACGATCTGAAAAACAACTTT
    AAGAAAGAGCCGATCCGTAACATTCTGCGTTACATCTTCACCATTCGTCAAGAATG
    CAGCGCGCAGGACATCCTGGCGGCGGCGAAGTACAACCAACAGCTGGATCGTTATA
    AAAGCCAAAAGGCGAACCCGAGCGTTCTGGGTAACCAGGGCTTTACCTGGACCAAC
    GCGGTGATCCTGCCGGAGAAGGCGCAGCGTAACGACCGTCCGAACAGCCTGGATCT
    GCGTATTTGGCTGTACCTGAAACTGCGTCACCCGGACGGTCGTTGGAAGAAACACC
    ATATCCCGTTCTACGATACCCGTTTCTTCCAAGAAATTTATGCGGCGGGCAACAGC
    CCGGTTGACACCTGCCAGTTTCGTACCCCGCGTTTCGGTTATCACCTGCCGAAACT
    GACCGATCAGACCGCGATCCGTGTTAACAAGAAACATGTGAAAGCGGCGAAGACCG
    AGGCGCGTATTCGTCTGGCGATCCAACAGGGCACCCTGCCGGTGAGCAACCTGAAG
    ATCACCGAAATTAGCGCGACCATCAACAGCAAAGGTCAAGTGCGTATTCCGGTTAA
    GTTTGACGTGGGTCGTCAAAAAGGCACCCTGCAGATCGGTGACCGTTTCTGCGGCT
    ACGATCAAAACCAGACCGCGAGCCACGCGTATAGCCTGTGGGAAGTGGTTAAAGAG
    GGTCAATACCATAAAGAGCTGGGCTGCTTTGTTCGTTTCATCAGCAGCGGTGACAT
    CGTGAGCATTACCGAGAACCGTGGCAACCAATTTGATCAGCTGAGCTATGAAGGTC
    TGGCGTACCCGCAATATGCGGACTGGCGTAAGAAAGCGAGCAAGTTCGTGAGCCTG
    TGGCAGATCACCAAGAAAAACAAGAAAAAGGAAATCGTGACCGTTGAAGCGAAAGA
    GAAGTTTGACGCGATCTGCAAGTACCAGCCGCGTCTGTATAAATTCAACAAGGAGT
    ACGCGTATCTGCTGCGTGATATTGTTCGTGGCAAAAGCCTGGTGGAACTGCAACAG
    ATTCGTCAAGAGATCTTTCGTTTCATTGAACAGGACTGCGGTGTTACCCGTCTGGG
    CAGCCTGAGCCTGAGCACCCTGGAAACCGTGAAAGCGGTTAAGGGTATCATTTACA
    GCTATTTTAGCACCGCGCTGAACGCGAGCAAGAACAACCCGATCAGCGACGAACAG
    CGTAAAGAGTTTGATCCGGAACTGTTCGCGCTGCTGGAAAAGCTGGAGCTGATTCG
    TACCCGTAAAAAGAAACAAAAAGTGGAACGTATCGCGAACAGCCTGATTCAGACCT
    GCCTGGAGAACAACATCAAGTTCATTCGTGGTGAAGGCGACCTGAGCACCACCAAC
    AACGCGACCAAGAAAAAGGCGAACAGCCGTAGCATGGATTGGTTGGCGCGTGGTGT
    TTTTAACAAAATCCGTCAACTGGCGCCGATGCACAACATTACCCTGTTCGGTTGCG
    GCAGCCTGTACACCAGCCACCAGGACCCGCTGGTGCATCGTAACCCGGATAAAGCG
    ATGAAGTGCCGTTGGGCGGCGATCCCGGTTAAGGACATTGGCGATTGGGTGCTGCG
    TAAGCTGAGCCAAAACCTGCGTGCGAAAAACATCGGCACCGGCGAGTACTATCACC
    AAGGTGTTAAAGAGTTCCTGAGCCATTATGAACTGCAGGACCTGGAGGAAGAGCTG
    CTGAAGTGGCGTAGCGATCGTAAAAGCAACATTCCGTGCTGGGTGCTGCAGAACCG
    TCTGGCGGAGAAGCTGGGCAACAAAGAAGCGGTGGTTTACATCCCGGTTCGTGGTG
    GCCGTATTTATTTTGCGACCCACAAGGTGGCGACCGGTGCGGTGAGCATCGTTTTC
    GACCAAAAACAAGTGTGGGTTTGCAACGCGGATCATGTTGCGGCGGCGAACATCGC
    GCTGACCGTGAAGGGTATTGGCGAACAAAGCAGCGACGAAGAGAACCCGGATGGTA
    GCCGTATCAAACTGCAGCTGACCAGC
    1166 MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFST Parent
    EQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFS Cas12i2
    ASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSII amino acid
    SVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQ sequence
    VYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYV
    EQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKL
    NDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNF
    KKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTN
    AVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNS
    PVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLK
    ITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKE
    GQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSL
    WQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQ
    IRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQ
    RKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTN
    NATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKA
    MKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEEL
    LKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVF
    DQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
    1167 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant
    IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of
    GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID
    REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 3 of
    PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20
    KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257
    TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET
    YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI
    LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV
    ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA
    AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ
    QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ
    NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS
    YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ
    PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS
    LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE
    LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR
    SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR
    WAAIPVKDIG RWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE
    ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA
    TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGSRIKL
    QLTS
    1168 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant
    IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of
    GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID
    REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 4 of
    PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20
    KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257
    TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET
    YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI
    LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV
    ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA
    AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ
    QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ
    NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS
    YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ
    PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS
    LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE
    LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR
    SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR
    WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE
    ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA
    TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGSRIKL
    QLTS
    1169 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant
    IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of
    GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID
    REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 5 of
    PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20
    KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257
    TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET
    YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI
    LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV
    ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA
    AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ
    QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ
    NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS
    YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ
    PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS
    LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE
    LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR
    SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR
    WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE
    ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA
    TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGEQSSDE ENPDGGRIKL
    QLTS
    1170 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant
    IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of
    GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID
    REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 495 of
    PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20
    KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257
    TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET
    YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI
    LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV
    ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA
    AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ
    QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ
    NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS
    YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ
    PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS
    LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE
    LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR
    SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR
    WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE
    ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA
    TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGRQSSDE ENPDGGRIKL
    QLTS
    1171 MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE Variant
    IVRFSTEQEK QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG Cas12i2 of
    GTASDAIKQY FSASIGESYY WNDCRQQYYD LCRELGVEVS DLTHDLEILC SEQ ID
    REKCLAVATE SNQNNSIISV LFGTGEKEDR SVKLRITKKI LEAISNLKEI NO: 496 of
    PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI AKDGQKEFDL PCT/US20
    KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH 21/025257
    TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET
    YTICVHHLGG KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI
    LRYIFTIRQE CSAQDILAAA KYNQQLDRYK SQKANPSVLG NQGFTWTNAV
    ILPEKAQRND RPNSLDLRIW LYLKLRHPDG RWKKHHIPFY DTRFFQEIYA
    AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK TEARIRLAIQ
    QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ
    NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS
    YEGLAYPQYA DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ
    PRLYKFNKEY AYLLRDIVRG KSLVELQQIR QEIFRFIEQD CGVTRLGSLS
    LSTLETVKAV KGIIYSYFST ALNASKNNPI SDEQRKEFDP ELFALLEKLE
    LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN NATKKKANSR
    SMDWLARGVF NKIRQLATMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR
    WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE
    ELLKWRSDRK SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA
    TGAVSIVFDQ KQVWVCNADH VAAANIALTG KGIGRQSSDE ENPDGGRIKL
    QLTS
    1201 ATGGCTTCCATCTCTAGGCCATACGGCACCAAGCTGCGACCGGACGCACGGAAGAA Nucleotide
    GGAGATGCTCGATAAGTTCTTTAATACACTGACTAAGGGTCAGCGCGTGTTCGCAG sequence
    ACCTGGCCCTGTGCATCTATGGCTCCCTGACCCTGGAGATGGCCAAGTCTCTGGAG encoding
    CCAGAAAGTGATTCAGAACTGGTGTGCGCTATTGGGTGGTTTCGGCTGGTGGACAA parent
    GACCATCTGGTCCAAGGATGGCATCAAGCAGGAGAATCTGGTGAAACAGTACGAAG Cas12i4
    CCTATTCCGGAAAGGAGGCTTCTGAAGTGGTCAAAACATACCTGAACAGCCCCAGC
    TCCGACAAGTACGTGTGGATCGATTGCAGGCAGAAATTCCTGAGGTTTCAGCGCGA
    GCTCGGCACTCGCAACCTGTCCGAGGACTTCGAATGTATGCTCTTTGAACAGTACA
    TTAGACTGACCAAGGGCGAGATCGAAGGGTATGCCGCTATTTCAAATATGTTCGGA
    AACGGCGAGAAGGAAGACCGGAGCAAGAAAAGAATGTACGCTACACGGATGAAAGA
    TTGGCTGGAGGCAAACGAAAATATCACTTGGGAGCAGTATAGAGAGGCCCTGAAGA
    ACCAGCTGAATGCTAAAAACCTGGAGCAGGTTGTGGCCAATTACAAGGGGAACGCT
    GGCGGGGCAGACCCCTTCTTTAAGTATAGCTTCTCCAAAGAGGGAATGGTGAGCAA
    GAAAGAACATGCACAGCAGCTCGACAAGTTCAAAACCGTCCTGAAGAACAAAGCCC
    GGGACCTGAATTTTCCAAACAAGGAGAAGCTGAAGCAGTACCTGGAGGCCGAAATC
    GGCATTCCGGTCGACGCTAACGTGTACTCCCAGATGTTCTCTAACGGGGTGAGTGA
    GGTCCAGCCTAAGACCACACGGAATATGTCTTTTAGTAACGAGAAACTGGATCTGC
    TCACTGAACTGAAGGACCTGAACAAGGGCGATGGGTTCGAGTACGCCAGAGAAGTG
    CTGAACGGGTTCTTTGACTCCGAGCTCCACACTACCGAGGATAAGTTTAATATCAC
    CTCTAGGTACCTGGGAGGCGACAAATCAAACCGCCTGAGCAAACTCTATAAGATCT
    GGAAGAAAGAGGGTGTGGACTGCGAGGAAGGCATTCAGCAGTTCTGTGAAGCCGTC
    AAAGATAAGATGGGCCAGATCCCCATTCGAAATGTGCTGAAGTACCTGTGGCAGTT
    CCGGGAGACAGTCAGTGCCGAGGATTTTGAAGCAGCCGCTAAGGCTAACCATCTGG
    AGGAAAAGATCAGCCGGGTGAAAGCCCACCCAATCGTGATTAGCAATAGGTACTGG
    GCTTTTGGGACTTCCGCACTGGTGGGAAACATTATGCCCGCAGACAAGAGGCATCA
    GGGAGAGTATGCCGGTCAGAATTTCAAAATGTGGCTGGAGGCTGAACTGCACTACG
    ATGGCAAGAAAGCAAAGCACCATCTGCCTTTTTATAACGCCCGCTTCTTTGAGGAA
    GTGTACTGCTATCACCCCTCTGTCGCCGAGATCACTCCTTTCAAAACCAAGCAGTT
    TGGCTGTGAAATCGGGAAGGACATTCCAGATTACGTGAGCGTCGCTCTGAAGGACA
    ATCCGTATAAGAAAGCAACCAAACGAATCCTGCGTGCAATCTACAATCCCGTCGCC
    AACACAACTGGCGTTGATAAGACCACAAACTGCAGCTTCATGATCAAACGCGAGAA
    TGACGAATATAAGCTGGTCATCAACCGAAAAATTTCCGTGGATCGGCCTAAGAGAA
    TCGAAGTGGGCAGGACAATTATGGGGTACGACCGCAATCAGACAGCTAGCGATACT
    TATTGGATTGGCCGGCTGGTGCCACCTGGAACCCGGGGCGCATACCGCATCGGAGA
    GTGGAGCGTCCAGTATATTAAGTCCGGGCCTGTCCTGTCTAGTACTCAGGGAGTTA
    ACAATTCCACTACCGACCAGCTGGTGTACAACGGCATGCCATCAAGCTCCGAGCGG
    TTCAAGGCCTGGAAGAAAGCCAGAATGGCTTTTATCCGAAAACTCATTCGTCAGCT
    GAATGACGAGGGACTGGAATCTAAGGGTCAGGATTATATCCCCGAGAACCCTTCTA
    GTTTCGATGTGCGGGGCGAAACCCTGTACGTCTTTAACAGTAATTATCTGAAGGCC
    CTGGTGAGCAAACACAGAAAGGCCAAGAAACCTGTTGAGGGGATCCTGGACGAGAT
    TGAAGCCTGGACATCTAAAGACAAGGATTCATGCAGCCTGATGCGGCTGAGCAGCC
    TGAGCGATGCTTCCATGCAGGGAATCGCCAGCCTGAAGAGTCTGATTAACAGCTAC
    TTCAACAAGAATGGCTGTAAAACCATCGAGGACAAAGAAAAGTTTAATCCCGTGCT
    GTATGCCAAGCTGGTTGAGGTGGAACAGCGGAGAACAAACAAGCGGTCTGAGAAAG
    TGGGAAGAATCGCAGGTAGTCTGGAGCAGCTGGCCCTGCTGAACGGGGTTGAGGTG
    GTCATCGGCGAAGCTGACCTGGGGGAGGTCGAAAAAGGAAAGAGTAAGAAACAGAA
    TTCACGGAACATGGATTGGTGCGCAAAGCAGGTGGCACAGCGGCTGGAGTACAAAC
    TGGCCTTCCATGGAATCGGTTACTTTGGAGTGAACCCCATGTATACCAGCCACCAG
    GACCCTTTCGAACATAGGCGCGTGGCTGATCACATCGTCATGCGAGCACGTTTTGA
    GGAAGTCAACGTGGAGAACATTGCCGAATGGCACGTGCGAAATTTCTCAAACTACC
    TGCGTGCAGACAGCGGCACTGGGCTGTACTATAAGCAGGCCACCATGGACTTCCTG
    AAACATTACGGTCTGGAGGAACACGCTGAGGGCCTGGAAAATAAGAAAATCAAGTT
    CTATGACTTTAGAAAGATCCTGGAGGATAAAAACCTGACAAGCGTGATCATTCCAA
    AGAGGGGCGGGCGCATCTACATGGCCACCAACCCAGTGACATCCGACTCTACCCCG
    ATTACATACGCCGGCAAGACTTATAATAGGTGTAACGCTGATGAGGTGGCAGCCGC
    TAATATCGTTATTTCTGTGCTGGCTCCCCGCAGTAAGAAAAACGAGGAACAGGACG
    ATATCCCTCTGATTACCAAGAAAGCCGAGAGTAAGTCACCACCGAAAGACCGGAAG
    AGATCAAAAACAAGCCAGCTGCCTCAGAAA
    1202 MASISRPYGTKLRPDARKKEMLDKFFNTLTKGQRVFADLALCIYGSLTLEMAKSLE Parent
    PESDSELVCAIGWFRLVDKTIWSKDGIKQENLVKQYEAYSGKEASEVVKTYLNSPS Cas12i4
    SDKYVWIDCRQKFLRFQRELGTRNLSEDFECMLFEQYIRLTKGEIEGYAAISNMFG
    NGEKEDRSKKRMYATRMKDWLEANENITWEQYREALKNQLNAKNLEQVVANYKGNA
    GGADPFFKYSFSKEGMVSKKEHAQQLDKFKTVLKNKARDLNFPNKEKLKQYLEAEI
    GIPVDANVYSQMFSNGVSEVQPKTTRNMSFSNEKLDLLTELKDLNKGDGFEYAREV
    LNGFFDSELHTTEDKFNITSRYLGGDKSNRLSKLYKIWKKEGVDCEEGIQQFCEAV
    KDKMGQIPIRNVLKYLWQFRETVSAEDFEAAAKANHLEEKISRVKAHPIVISNRYW amino acid
    AFGTSALVGNIMPADKRHQGEYAGQNFKMWLEAELHYDGKKAKHHLPFYNARFFEE sequence
    VYCYHPSVAEITPFKTKQFGCEIGKDIPDYVSVALKDNPYKKATKRILRAIYNPVA
    NTTGVDKTTNCSFMIKRENDEYKLVINRKISVDRPKRIEVGRTIMGYDRNQTASDT
    YWIGRLVPPGTRGAYRIGEWSVQYIKSGPVLSSTQGVNNSTTDQLVYNGMPSSSER
    FKAWKKARMAFIRKLIRQLNDEGLESKGQDYIPENPSSFDVRGETLYVFNSNYLKA
    LVSKHRKAKKPVEGILDEIEAWTSKDKDSCSLMRLSSLSDASMQGIASLKSLINSY
    FNKNGCKTIEDKEKFNPVLYAKLVEVEQRRTNKRSEKVGRIAGSLEQLALLNGVEV
    VIGEADLGEVEKGKSKKQNSRNMDWCAKQVAQRLEYKLAFHGIGYFGVNPMYTSHQ
    DPFEHRRVADHIVMRARFEEVNVENIAEWHVRNFSNYLRADSGTGLYYKQATMDFL
    KHYGLEEHAEGLENKKIKFYDFRKILEDKNLTSVIIPKRGGRIYMATNPVTSDSTP
    ITYAGKTYNRCNADEVAAANIVISVLAPRSKKNEEQDDIPLITKKAESKSPPKDRK
    RSKTSQLPQK
    1203 MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE Variant
    MAKSLEPESD SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA Cas12i4 A
    SEVVKTYLNS PSSDKYVWID CRQKFLRFQR ELGTRNLSED FECMLFEQYI
    RLTKGEIEGY AAISNMFGNG EKEDRSKKRM YATRMKDWLE ANENITWEQY
    REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM VSKKEHAQQL
    DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV
    QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED
    KFNITSRYLG GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP
    IRNVLKYLWQ FRETVSAEDF EAAAKANHLE EKISRVKAHP IVISNRYWAF
    GTSALVGNIM PADKRHQGEY AGQNFKMWLE AELHYDGKKA KHHLPFYNAR
    FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK DNPYKKATKR
    ILRAIYNPVA NTTGVDKTTN CSFMIKREND EYKLVINRKI SRDRPKRIEV
    GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST
    QGVNNSTTDQ LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG
    QDYIPENPSS FDVRGETLYV FNSNYLKALV SKHRKAKKPV EGILDEIEAW
    TSKDKDSCSL MRLSSLSDAS MQGIASLKSL INSYFNKNGC KTIEDKEKFN
    PVLYAKLVEV EQRRTNKRSE KVGRIAGSLE QLALLNGVEV VIGEADLGEV
    EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE
    HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD
    FLKHYGLEEH AEGLENKKIK FYDFRKILED KNLTSVIIPK RGGRIYMATN
    PVTSDSTPIT YAGKTYNRCN ADEVAAANIV ISVLAPRSKK NREQDDIPLI
    TKKAESKSPP KDRKRSKTSQ LPQK
    1204 MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE Variant
    MAKSLEPESD SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA Cas12i4 B
    SEVVKTYLNS PSSDKYVWID CRQKFLRFQR ELGTRNLSED FECMLFEQYI
    RLTKGEIEGY AAISNMFGNG EKEDRSKKRM YATRMKDWLE ANENITWEQY
    REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM VSKKEHAQQL
    DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV
    QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED
    KFNITSRYLG GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP
    IRNVLKYLWQ FRETVSAEDF EAAAKANHLE EKISRVKAHP IVISNRYWAF
    GTSALVGNIM PADKRHQGEY AGQNFKMWLR AELHYDGKKA KHHLPFYNAR
    FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK DNPYKKATKR
    ILRAIYNPVA NTTRVDKTTN CSFMIKREND EYKLVINRKI SRDRPKRIEV
    GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST
    QGVNNSTTDQ LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG
    QDYIPENPSS FDVRGETLYV FNSNYLKALV SKHRKAKKPV EGILDEIEAW
    TSKDKDSCSL MRLSSLSDAS MQGIASLKSL INSYFNKNGC KTIEDKEKFN
    PVLYAKLVEV EQRRTNKRSE KVGRIAGSLE QLALLNGVEV VIGEADLGEV
    EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE
    HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD
    FLKHYGLEEH AEGLENKKIK FYDFRKILED KNLTSVIIPK RGGRIYMATN
    PVTSDSTPIT YAGKTYNRCN ADEVAAANIV ISVLAPRSKK NREQDDIPLI
    TKKAESKSPP KDRKRSKTSQ LPQK
    1172 GTGCTGCAGCCGCTGCCGCCGATTCCGGATCTCATTGCCACGCGCCCCCGACGACC LDHA
    GCCCGACGTGCATTCCCGGTACGGTAGGGCCCTGCGCGCACGGCGCCAGAGGGATG
    GGCGGGTAGAGCCAACTGCCTCTGGTTCTGCTGGCCTCCGCTGCTCGCGAAGGGAT
    TCCTGCTCCCGGGAGGTGTAGGAGCCGCTTTCCAGAAGCACAGCCCAGAGACGTCT
    GGGCGGCGGCCCACACAACGCATGTGTTCGGAGCTCGCCGCGCTCTGCTTTTGCTC
    TAAGCGGGAACCATGGCTTCTGGCCACGCTGGGGAACCGAGGAGGTGGCCGCACCC
    AAGCAGGGGTCGAAAGCCCGGGTGGATGCGGAACAAGGATATGATAGGCCTTAAGG
    GTGGGGGATACCTCTGGGCTCGAAATCGGCGGGCGGTGCAAAACTCGAGGTCCAGT
    TCTCGGAGCCCATAGAGCCAAAAAAGCCTCAGCTTGTCCGGGGCGGGTTCTTGAAA
    GACGGAAAGCGGCTGAGTACCACGCGGCTTGCATTTTTCTCTTGGGACGCTCGAGA
    GGTGGGCTCCGTGAGGGCAGCTGCTGCCTGCAGATTATAGGGAGCCCTTTGCGCAT
    TTATTAAGAAGCTACTGGTGTATCTCGGGCTGCGCTAGGCACGGCGCATGCAAAGA
    TGAAGCAGGCAGCATCCCAGCCCTTCCGCACCTCAGACGGTCAGTTGAGTAGGATC
    CGCCGGTACCAACTCCTCCTTTTAACAAATAGGGAGACCGAAAGCTAGGAGACAGT
    CAGGGATCTCTAAGTTCCCAGTGAGTAGGAGGCAGAGGTGAGGTGTAGAACTCGTT
    TTTGCATGTCTCTCGCCTCTAGACGCACCCTTCCCTCATCCCATGCCCTCCCACCT
    CCGCCCCTACATTAAAGGTAGCATTGGATCCCGGGGCCGTTCAGTGAAGCTAGCAG
    GTGTCCGCAGGAACTCCCTTCCCCCTGCCAGGCTAGAAACCTTACAAGGCTGTCTA
    GAAATAGCAGTGATTTGTAAGGAGAGACCCGGCTCCAGCTTGGTGACTCTGGGCTG
    ACTGCCTGCCTAGAGGTCCTCTCGGATTTTTGCCCTTTGGAGTGGTGTCAAAACTA
    GACGTGATACTTTGGGGATGCAGCCTGTGATATTTCCTCCAGCGAATGCAGTGCAG
    GGTTGGATTAACAAGGTGGAAAGAATTCGAGGGTTCCACCAAGTAGCTATTAACTC
    TAGGGCTGCAGGCCTCAGGCCTTCTGCAGCTATTTCTACACTCCCTGTACTGAAAC
    TATTTCTTCATACTGGGCCTGACAGGCCTTTGCAACAAGGATCACGGCCGAAGCCA
    CACCGTGCGCCTCCCTCCCGGTTGGTTAACAGGCCCTGGTTTCTAGTATTGCGATT
    TAAAGTCTGGCGCTGGCTGCGCGCCAGACCTGGGAGGCTGCCAGCTAGGCTTCACG
    TTGCTGGCGTCTGCTTCGGGGCATTCATTAGGTCTGAAGTCTGAATCCCAGCTCCC
    TCCCTCTCACCCACTGAGCTGCATAGCTCCAGATTGCCTCTGCTTACGGGCGGGGC
    TTCTCAGCCTTCTGCCTTCTGGCCCGATGCCCGCTTCCCAACGGCCGGAGGCCGCT
    AGACTAATCGGCTTCGCCCTGCGCGCTGTAATGCGCATGCGCACGCGCACAAGTTC
    CTGGGCCCGCCCATCTTCCGGACTTGGGCGGGGCGTAAAAGCCGGGCGTTCGGAGG
    ACCCAGCAATTAGTCTGATTTCCGCCCACCTTTCCGAGCGGGAAGGAGAGCCACAA
    AGCGCGCATGCGCGCGGATCACCGCAGGCTCCTGTGCCTTGGGCTTGAGCTTTGTG
    GCAGTTAATGGCTTTTCTGCACGTATCTCTGGTGTTTACTTGAGAAGCCTGGCTGT
    GTCCTTGCTGTAGGAGCCGGAGTAGCTCAGAGTGATCTTGTCTGAGGAAAGGCCAG
    CCCCACTTGGGGTTAATAAACCGCGATGGGTGAACCCTCAGGAGGCTATACTTACA
    CCCAAACGTCGATATTCCTTTTCCACGCTAAGGTATGGGCCTTCACTCTTCACAGA
    CCCTGTCATTAGGCCTTTCAACTCTCTTTTGGCAACCATTAGGTTTTTTCCCCTCC
    CTTTTTAGTCATCTCTAGTGATTTATAGTGGCAAATACCCCCAAAGGAAGTAAAAT
    AGCTTAAAAAAATCTCTTGGTTAATAAACATTAAAGAAGCTGTAGTGACACTAAAT
    GTTTTTCCTCCTATAGATTCCTTTTGGTTCCAAGTCCAATATGGCAACTCTAAAGG
    ATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCCCCAGAATAAGATTACA
    GTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCAGTATCTTAATGAAGGT
    AAGTGAGAGTCTACCACACTGGAAGCCCATACCTTGACCCCATCCTCTACCCCCAC
    TCCTACCCCTAGAACTGTATTATTACATTTCATGTAACAGTATTTAGATTTATGCA
    CTCATTCGGATAACTTTCTGTGAAACAAACTTTTGAAATATGATAATACACCAAAA
    GTGTATCTGAAATTAAAAAGAATCAAAGGTTGTCAGGCTGGAGACCCAGTTCCTAA
    AATTCATTATTCTGTATTAACATGCATGGATTGACTACCAATGAAAAGGAAGGGTC
    CATGATTTTAAATGAGCCAAAATTCTTTTAAAGTGATTTTTGAATTGAAAATGACA
    ATTCAAAAATTGTCATTTATTGGTAAAATTATATGGGAAATCATAAGTTCTCCCAC
    TCAAATCTCATTGCCCCTGTGCCTTGGATAGCAATTTTGTTATCAATTATGGAGCT
    AAAATTTAATTAGAAAAAAGAAATTGTGAGTAAAGCACTCCTTATTACACTATTGA
    AAGCTGATTTATATTTAAAAGAAATTGAGGCAGCTTACAACATTAAAATGTCTGAG
    GCGGGGCACAGTGGCTCATGCTTGTAATGCCAGCACTTTAGGAGGCTGAGGTGGGT
    GGATCACGAGGTCAGGAGATGGAGACCATCCTGGCTAACACGATGAAACCCCATCT
    TTACTAGAAATACAAAAAATTAGCCGGGCGTGGTGGCATACGCCTATAGTCCCAGC
    TACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAGGAGGTGGAGGTGGCAGT
    GACCCGAGATAGCACCACTCCACTCCAGCCTGGGCGACAGTGAGACTCCATCTCAA
    AAAAAAAAATCTGAAGTTAAGATGTGGAGTGTCTAATAAAAGTAAAATGATGAATT
    CTGGGTTCTAAATAGAAATGGATTCAAGTGAGAAGGGACTAAAGACAGAAATGAGC
    TATGAAAAGGCCTCGTAACAACACAGGTGACTCTACATATGTTCTTAGGAAAGGCC
    ACATAATACACCAACTTTTATTCCTTACCCACTAGATGAGAAATTGATGCTGTTTT
    CCCCACACCTACAAACCGCCTATGTTTTTTCTCTGTGATGGCCTCTGGCTCAGGTG
    TGGGTAAGAAGAGTAACTGACACTCATTATATTGTGGATGATTTAGGGATAGATCT
    GCAGCTTGAATAACTTTTGGTAACGATAGACCACATCCAGTTGTATTAAAGCTGTT
    ATTGGTGCTCCTGGCCTGAAATGGACCTATGAACTTTGAGTTGCAACTATAAGGAT
    ATTTTTTGCCAGTATTATACACTGCACAAACCTATTTATCCATAACTGTTAGTATT
    GGTTCATATATGGAATCAACCAGGGAATAGTTCAGATTCCATCTCTGAAAGATGGG
    CGGAAATCAGACTTTTTAACTTTTTAAGTTTTTTTTTTTTGAGACGGAATCTCGCT
    TTGTTGCCCTGGCTGGAGTGCAGTGGCACGATCTTGGCTCACTTGACCTCCTGGGT
    TCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCACCCACCG
    CCACGCCTGGCTGATTTTTGTATTTTTAGTAGAGACAGGCCTTCACCATATTGGCC
    AGGCTGGTCTTTTTTTTTTTTTTTTTTTTTTTTTTCTGAGAAGGAGTCTCGCCGTG
    TCGCCCAGGCTAGAGTGCAGTGGCGTGAACTCCGCTCACTGCTAGCTCTGCCTCCC
    GGGTTCATACCATTCTCCTGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCC
    ACCACCACGCCTGGCTAAATGTTTGTATTTTTTAGTAGAGACGGGGTTTCACCATG
    TTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCTACCTTGGCCTCCCA
    AAGTGCTGGGATTACAAGCGTGAGCCACCGTGCCTGGCCTGGCCAGGCTGTCTTGA
    ACTCCTGACCTCAAGTGATGTGCCCGCCTCGGCCTCCCAAAGTGTTGGGATTACAG
    ATGTGAGTCACTATGCCCGGCCAGAACATTTCTTACTAATTTCAAGTCTTGATGCT
    GGTCAATATCACCTAGTTAAATGAATAACAACCTAAAATTGGTGTGTAGGATGGAA
    TTTGAGAGAGTAGACAGAGCAGTTTTATATAATTGGAAGTTATTCTAGCAACTGCC
    AGTCCAGTGTTCTGCTTCCACATCTGCAGTGGTGGAACTCCTATAGAGCTCGCTTC
    AGTGGGGAGACAGGGCTGGAGAGAGGGTCAGTGCTATCTATGTAGGGTGTAATCTG
    TAAGTCAGCTTTTGAAATGGGGTGCCCTCTACTTTGAATATCTCGATACTGTACTA
    ATAAAGTAACAGAACTCTCCTATGCCAGAAATATAGAAATTTTTCATGCTCTTCTA
    AAAATCTAGAAGTGGCAATTTTCCATTTAACTAAAGATTTGATGTCTTTTAGGACT
    TGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATG
    ATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAA
    AGGTTGATTTCAACAAGTTTATATTATAATCCATGCTTGACTTAAATTCTTTTTCC
    AGATGGTCTCCATTTGTTGCTTAGGGTAGAGTGCAGTTGCACAATTATGGCTCACC
    ACAGCCTCGAACCCTGGGCTCAAGCAATCCTCCTTCCACTTCATTACCCCCTCCCC
    CTCACAAAGAAACTGGGACTATAGGGTATGCTACCATGCCCGGCTAATTTTTTTAC
    TTTTTGTAGAGATGGGGACCCACTGTGTTGCCCAGGCCTGTCTTGAACCACTGGGC
    TCAAGTGATCCTCCCTCCTTAGCCTTCCGAAGTACTGGGATTGCAGGTGTGAACCA
    CTGTGCCCGGCTTTAGACTTAAATGTTTTATCAGGCTTGAAATCCTAGCTCTTTAA
    AGATTTTGTTTTAAATGCCGGGTGCAAGAGCCTGGGAACAATTTCACTTAGGTGCC
    TGTGAATATCAAAGTTTCAATTTCTGGCAAATGGTTTAAAATAGAAATCCAATTTG
    TCCATGCTATGCAAACCATCTGAATTAGAATGTAATGAGTAAAGCTTAAACCTTAG
    GTCTGTATTTAACCACATTGTGTTACTTACTTGCCCCCACATCCTTTCACACACGA
    AGTTGAGAATAGGGTAAATAAATGAGCCTGTTCAGCTAATACTCTTGGCTTGACCC
    TTTCACACTTAACAGCACCAGCCAAGAAACCTGAATGTGAGCCCAAATAGTGTCTA
    TTTTGATACCTGAAAATCACTGGCCACCTTGCTGATGGGCAACTCCCTTCATCACT
    GGTTTAACTCTCTTGTGCCATAGGGTATCTAGAAGCAAAATATGTTTGTTAAGTGT
    AAAGCTGTCTCTGCTTAAAAACAAGTCCCCCTACCACCACCACCACACACACACAC
    ACACACACACACACACACACACACACACACACACACACACGAAATTGCCTGTTCCT
    GGGCTGATAGGACACCAGTTAAGTAGAAACAGGAGTATGGAAGAGTGTGAACGTTG
    AGCTTGGGGATCAAAAATTTGAGGATATGTAAGAAATTAATAGGAGAATCAAATAA
    TAAACTTGATTTCCTCCAGCTCTCCCTAATTGTAGTTACATAAAGTTACAACTTGA
    CTAAAACTACAAGGAAGATGTTGACATGCTCTTCCTCCATTTAAGAAGCCATAATG
    ATAAAACTCTAAGAACAAGAAAGGTTTGTGGAGCATTTATGGAACAAATTTTTGCT
    GCCTAGGTAAAATTTATTCTAAAGGCCTTAATCTGGTCATTATTCCCCTTTTCTCT
    AGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTC
    AGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAA
    TTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTC
    AAATCCAGGTGAGGCTTTTGACTGCATAAAAATTGACAAGCTATAGTAAAACTGAT
    AGTATATGATATATATATTATATATATTTTAAATATTTTGAAATATTTTAAAAAAT
    ACATTTTTAAAAATATTTTCGAATATTATTTTAAAATATATATATATATTTTGAGG
    CGGAGTTTTGCTCTTGTCGCCCAGGTTGGAGTGCAGTGGCGCAATCTGGGCTCACT
    GCAACCTCTGCCTCATGGGTTCAAGCGATTCTTTTGCCTCAGCCTCTCAAGTAGCT
    GGGATTATAAGCGCCTGCCACCACACATGGCTAATTTTTTATATTTTTAGTAGAGA
    CAGGGTTTCACCATGTTGGCCAGGCTGGTTTTGAACTCCTGGCCTCAAGCAGTCCA
    TCTGCCTCCCAAAGTGCTAGGATTACAGGCGTGAGCCACCGTGCCCAGCCACGCAT
    ATTTATTGATTCATTTATTTTTCTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTC
    TTGCTCTGTCACCCTGGCTGGAGTACAGTGGCTTGATCTTGGCTCACTGCAAGCTC
    CGCCTCCCGGGTTCATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTAC
    AGGTGCCCACCACGACGCCTGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTT
    CATCAGGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCCCGCCTTGG
    CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGTGATTCATTTAT
    TTTTCATGTTTCATTTCCCTTCTAAGGAGATTTGTGTGTGTGTGTTTTTTGTTTTT
    TAATAATTTTAAAACATTAAAGGGAATACAATGCCTTTAAATGTAGTTGGAGCTTA
    AAATTACCTGCCCAAGATCTTGGATAAGGGATAAGTTTGTGAATAATTGTTATTCT
    CTTTTTTTTTTTTTTTTTTTTTGAGACAGTCTCACTTTGTAGCTCAGGCTGGAGTG
    CAGTGGTTCGATCTTGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGCAATTCTC
    CTGCCTCAGCCTCCCAAGAGCTGGGATTACAGGCACGTGCCACCATGCTCGGCTAA
    TTTTTGAAGTTTTAGTAGAAAGGGGTTTCACCATGTTGCCCAGGCTGGTCTCAAAT
    TCCTGAGCTCAGGTGATCCATCTGCCTCAGCCTCCCAAAGTATTAGGATTACAGGC
    GTGAGCCACCGTGCCCGGGCCCATAATTGTCTCTTAGTTGATAAACAGTTTATTTT
    CATAAAACTGTTACTATACTTTTTTTTTGAGAGCATGTCTCACTCTGTCGCCCAAG
    CTGGAGGGCAATGGGATGATCATGGCAGCTTTGACCTACTAGGCTCAGGTGATCCT
    TCTTCCTCAGCCTCTTAAGTAGCTAGGACTACAGGCGTGCACCAATATGCCTGGCT
    AGTTTGTTAAAAGTTTTTTTGTAGAGATGGGGTTTTGCTATGTTGCCCAGGCTGGT
    CTTGAACTGCTGGCCTCAGGCAGTCCTCCCACCTCAGCCTCCCAAAGTGTTGGGAT
    AACAGGTGTGAGTTGTCATGCCCAGCCAAAACTACTTTTTGAATAATTAATGGACT
    TGATATACATAGTGTAGAGGCTTAAAAATATTAACAAAATTATTGGTTAGCCATGA
    TCAATATCAAGATCCTGAAAAGCCATATATCTGGAGTAGCCTATTATTATCTAATG
    ATCACCTAGTATCTGGTTAAGTGTTTTCTTCATAGTAGGTATATCTTTTTTGTGTG
    TAGGGAGAGGATAATGGGTGATTTTTATTTTCTCCTTTTTCATAGTGGATATCTTG
    ACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGG
    TTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTC
    ACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGATTCCAGTGGTAAG
    CATAAGTTATTTTCTTTTTGTTTTTGAAAAGATTATATAAAAAGTCGATGGGCATT
    ATATTATTCAATTAGAGCCTAATCAAATATCCATTCAGTAGGATGGAATGGTTTCC
    CGAAATCTAGCATTTTGTATAATTATATGTTAAGAATTGTTAAGATTGTTGCCATT
    TTATATGGCATTTTATGGCGAGGGGGACGGGAAATGAAATTTCTCTTCTTACCATG
    GATATCTTAAGACTGTAGTTCTTAGGATGTCTTCAGTCATTTAATATCACAGCTGT
    TTATACCTGACTTGTACTGCCTGGCCCTGAAAAGATGAGCAAATCCAAATGCACAA
    AAGTTATATTATCACAGTTGAAAAATGTTATGATTAGGTTCTGTATGCTAAGAAAA
    CCCCCCTTATGTTCTCATACTATCTTTATATTTCAAATATACATGGGTTAAACATT
    TCAATTGGCTAGAGAAACAGGTTAGAATACAGTTAAAATTCTTAGTTTTACATAAT
    GTAAGTAAATGAAAATCTAATCTAAAAGTGAGTAATGACTACATTAGTAGTCTTGA
    CCATCTACCAAAATTGAGTATTCTTCCTCCGAAGATAAGAGAATTAGGAAAATGAA
    TCACAATTACTAATCTGTTGGTACATGAAAATAAATGTAGTCTGTACTATTTCTTT
    TAGTGCCTGTATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCTGCAC
    CCAGATTTAGGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGCAGGT
    GGTTGAGAGGTAATAAATCTTTCAATTTGGCAACACAGAATATTAACATTTACTAT
    TTTTATTTAAAAGGTTAAAATTGTAATAGTATTTGCATTTGAGAACTTTTTGTTAG
    AAAACTTGTGTGGTTTTTTTGTTTTGTTTTGTTTGAGACAGAATCTTGCCCTTTCG
    CACAGGCTGCAGTGCAGTGGCGCAATCTTGGCTCACTGCAACCTCTGCCTCCCGGG
    TTCAGGCGATTCTCCTGCCTCAGCCTCCTGGGTACCTGGGACTACAGGCATATGCC
    ATGACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTAG
    CCAAAAAAAAAAGAATGTGCCTCACCTTGCAAGGCCCAGGCCCTAGGATCACTTGA
    GCTCAGGAGTTCAAGGCCAGCCTGGGCAACAGGGCAAAACCCTGTCTCTACAATAA
    ATACACAAATTAGCCAGGCATGGTGGTGAGCACCTGTGGTCCTAGCTACTTGAGAG
    GCTGAGGCAGGAGGATCGCATGAGCCTGGGAGGTCAAGGCTGCAGTGAAGCGAGAT
    CCTGCCACTGCACTCCAGAGCCTGCTAGCCTGGGTGACAGAGTAAGAGCCTGTCTC
    AAAGGAAAAAAAAAATTATTGAAATAGGGAAGCTTTCAACTTGGTGGCATTATTTA
    CCTTTGTGGTCCTGTGTGGACCTCAGGTCTATAGAATTAAAAAATGAATCATAGCC
    GGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGA
    TCACGAGGTCAGGAGATGGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTA
    CTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTAC
    TCGGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGTAGTTGGAGCTTGCAGTGAG
    CCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAA
    AAAAAAAGAATCATAATCTTTAGTTCATAACATATTCTTGTGATTGGTCAAGCAAG
    GCCCTCTTGTTTGTATTTGTTTAATTAAATAAAACCTGTGAACCCACCACCCAGCT
    CAAGAAAGAAACACAATATCTGTCAAATAACATTGTTGAATCAGAATTTAGTATTC
    TGCTGGTGTTTGGAAATAAGTGGATTCTGTGCTCTTTCCCCCAGCTATCCCTCTGT
    CCCCCTCACGCTCCCACTTGAGATAATCCTGAGTTAAGGATGCTATGTTATCTTGG
    ATTTCTTTTTAAAATTCAATATTATATTTTTAAGAATTATCCAATTTTTTTTACAA
    GTAGCTATAGTTTATTTTTTGATAGCTGTGTAATATTCCATTGTATCAGTATACCA
    TGATTTATCCATTCTTCTGTTGGAGGACATTGGAAAGATTGTCATGTTTTTGCTGT
    TACTAACAGTACTGTTAATGAATATCCCTGTACATAATATCCTAGCATACATGTGT
    GCAAGGGTTATTCTTGGTATAATGCAACATTGTGGCATTATTTACTGTAAAATGTG
    TATTAATGAAAACTTTGTTTTTCTTTCTTTCTCCCACCCTGCTTTTTCTGCCTTTA
    CCTATGGTTTCCTATCATACAGTGCTTATGAGGTGATCAAACTCAAAGGCTACACA
    TCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAATGAAGAATCT
    TAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGTAGGTCTATGTAGTGATACGC
    TGCATTTGAATGCTTTTTGCTGGCTTTTTAAAAAAGATTCTTCTGAGAAAGATTAA
    TACAAGTCTTCCATTACTGACTTAAGTGAAATAAATTAATGTACCCACAGCTTACC
    TTTTTTGAAAGAAATGGTTGAGCTTTAGGATTAATGTCCATTAGGCCTGTTCAACA
    CATAGATACTTGATAATTTGACTACAAAAAAGTCTTGTTCAATTATGCTGAGGTAG
    GTGGAAGACTATAAAAGAAATAAACTATTTCTCCATTGGGGAAAATAGAAATTATA
    TTCAAGTTAGCATTATGTTACTATTTTTAATGACTTTCTTTTATACTATTAATTAA
    ATCATAACTGAACACCTGGAAAGGAATTTCTACTTATCAAAGTTTTTTATTTTTTT
    GAGACAGTCTCCCTCTGTCACCCAGGCTGCAGTGCAGTGGCCGATCTCGGCTCACC
    GCAACCTCTGCCTCCCAGGTTTAAGCGATTCTTCTGCCTCAGCCTCCTAAGTAGCT
    GGGACTACAGGTGCGTGCCACCACGCCCGGCTACTTTTTGTATTTTTAGTAGAGAT
    GGAGTTTCACCATATTGGCTAGGCTGGTCTCGAACTCCTGACCTTGTGATCCACCC
    GCCTCGGCCTCCCCGAATGCTGGGATTGCAGGTGTGAGCCACCGCACCTGGCCTCA
    AGTTGTATTTTAAAATCTTCATAATTAGGCCACACACAGTGACTGACAGCTGTAAT
    GCCAGCACTTTGGAAGGCCAAGGGCAGGAGAATTGCTTGAGCCCAGGTGTTTGAGA
    CCACCCTAGGCAGTATAGTGAGATCTTGCCTCTGTTAAAAAAAAAAAAAAAAAAAA
    AGGCCATGTGCGGGCAGCTGATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGGG
    TGGATCACCTGAGGTCAGTAGTTCAAGACCAGCCTGACCAACATGGTGAAACCCTG
    TCTCTACTAAAAATACAGAATTAGCCAGGTGTGGTGGCAGGCGCCTGTAATCCCAG
    CTACTTGGGAGACTGAGGCAGAAGAATCACTTGAACCCAGGAGGTGGAGGTTGCAG
    TGAGCTGAGATCGCACCATTGCACTCCAGCCTGGGCAACAAGAGTGAAACTCCATC
    TCAAAAGAAAAAAAAAAGCGGCTGGGCTCTGTGGCTCATGCTTGTAACCCCAGCAC
    TTTGGGAGGCCAAGAGGTGGATCACCTGAGGTCAAGAATTTGAGACCAACCTGGCC
    AACATGGTGAAACCCCATCTGTACTAAACATACAAAAATTAGCCAAGTGTGGTGGC
    GCACGCCTGTAGTCCCAGAAGGCTGAAGCAGGAGAATTACTTGAACCCTGGAGGTG
    GAGGTTGCGGTGAGCTGAGATCGTGCCACTGCACTCCAGCCTGGGCGACAGAGCGA
    GACTCTGCCTCAAAAAAAAATTAAAAAAAAAAAGCTTTATAATTATAGAGACTGTA
    AGTCTTGGGAAACCTGGGAATGCATAGACAAAATGTGAGATTTTTTTTTTTTCATT
    TCATCTTCAGGGTCTTTACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCA
    TTTTGGGACAGAATGGAATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAA
    GAGGCCCGTTTGAAGAAGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCA
    ATTTTAAAGTCTTCTGATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCT
    AGGTGGAGGTTGTGCATGTTGTCCTTTTTATCTGATCTGTGATTAAAGCAGTAATA
    TTTTAAGATGGACTGGGAAAAACATCAACTCCTGAAGTTAGAAATAAGAATGGTTT
    GTAAAATCCACAGCTATATCCTGATGCTGGATGGTATTAATCTTGTGTAGTCTTCA
    ACTGGTTAGTGTGAAATAGTTCTGCCACCTCTGACGCACCACTGCCAATGCTGTAC
    GTACTGCATTTGCCCCTTGAGCCAGGTGGATGTTTACCGTGTGTTATATAACTTCC
    TGGCTCCTTCACTGAACATGCCTAGTCCAACATTTTTTCCCAGTGAGTCACATCCT
    GGGATCCAGTGTATAAATCCAATATCATGTCTTGTGCATAATTCTTCCAAAGGATC
    TTATTTTGTGAACTATATCAGTAGTGTACATTACCATATAATGTAAAAAGATCTAC
    ATACAAACAATGCAACCAACTATCCAAGTGTTATACCAACTAAAACCCCCAATAAA
    CCTTGAACAGTGACTACTTTGGTTAATTCATTATATTAAGATATAAAGTCATAAAG
    CTGCTAGTTATTATATTAATTTGGAAATATTAGGCTATTCTTGGGCAACCCTGCAA
    CGATTTTTTCTAACAGGGATATTATTGACTAATAGCAGAGGATGTAATAGTCAACT
    GAGTTGTATTGGTACCACTTCCATTGTAAGTCCCAAAGTATTATATATTTGATAAT
    AATGCTAATCATAATTGGAAAGTAACATTCTATATGTAAATGTAAAATTTATTTGC
    CAACTGAATATAGGCAATGATAGTGTGTCACTATAGGGAACACAGATTTTTGAGAT
    CTTGTCCTCTGGAAGCTGGTAACAATTAAAAACAATCTTAAGGCAGGGTGCAGTGG
    CTCATGCCTATAATCCCAGCACTTTGGGAAGCCCAGGTGGGCTGATCACTGGAGGC
    CAGGAATTGGGGACCAGCCTGGCCAACACAACAAAACCCCATCTGTTAAAAAAACA
    AAACAAAACCAAAAAAAACAAGTAACCTTGGTGGATGTCTACTCAAGTTTTCTGCA
    CATTTTTCTGAAAATACAACTGTGACCCTTA
    1173 GTGCTGCAGCCGCTGCCGCCGATTCCGGATCTCATTGCCACGCGCCCCCGACGACC LDHA
    GCCCGACGTGCATTCCCGGTACGGTAGGGCCCTGCGCGCACGGCGCCAGAGGGATG exon 1
    GGGGGGTAGAGC
    1174 CCAGCAATTAGTCTGATTTCCGCCCACCTTTCCGAGCGGGAAGGAGAGCCACAAAG LDHA
    CGCGCATGCGCGCGGATCACCGCAGGCTCCTGTGCCTTGGGCTTGAGCTTTGTGGC exon 2
    AGTTAATGGCTTTTCTGCACGTATCTCTGGTGTTTACTTGAGAAGCCTGGCTGTGT
    CCTTGCTGTAGGAGCCGGAGTAGCTCAGAGTGATCTTGTCTGAGGAAAGGCCAGCC
    CCACTTGGGGTTAATAAACCGCGATGGGTGAACCCTCAGGAGGCTATACTTACACC
    CAAACGTCGATATTCCTTTTCCACGCTAAGGTATGGGCCTTCACTCTTCACAGACC
    CTGTCATTAGGCCT
    1175 AATAAACATTAAAGAAGCTGTAGTGACACTAAATGTTTTTCCTCCTATAGATTCCT LDHA
    TTTGGTTCCAAGTCCAATATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCT exon 3
    AAAGGAAGAACAGACCCCCCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTG
    GCATGGCCTGTGCCATCAGTATCTTAATGAAGGTAAGTGAGAGTCTACCACACTGG
    AAGCCCATACCTTGACCCCATCCTCT
    1176 AATCTAGAAGTGGCAATTTTCCATTTAACTAAAGATTTGATGTCTTTTAGGACTTG LDHA
    GCAGATGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATGAT exon 4
    GGATCTCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAG
    GTTGATTTCAACAAGTTTATATTATAATCCATGCTTGACTTAAATTCTTT
    1177 AAAATTTATTCTAAAGGCCTTAATCTGGTCATTATTCCCCTTTTCTCTAGACTATA LDHA
    ATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAG exon 5
    GGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAATTCATCAT
    TCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTCAAATCCAG
    GTGAGGCTTTTGACTGCATAAAAATTGACAAGCTATAGTAAAACTGATAG
    1178 GTGTGTAGGGAGAGGATAATGGGTGATTTTTATTTTCTCCTTTTTCATAGTGGATA LDHA
    TCTTGACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGTTATTGGA exon 6
    AGCGGTTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAAGGCTGGG
    AGTTCACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGATTCCAGTG
    GTAAGCATAAGTTATTTTCTTTTTGTTTTTGAAAAGATTATATAAAAAGT
    1179 CTAATCTGTTGGTACATGAAAATAAATGTAGTCTGTACTATTTCTTTTAGTGCCTG LDHA
    TATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTA exon 7
    GGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAG
    GTAATAAATCTTTCAATTTGGCAACACAGAATATTAACATTTACTATTTT
    1180 TTCTCCCACCCTGCTTTTTCTGCCTTTACCTATGGTTTCCTATCATACAGTGCTTA LDHA
    TGAGGTGATCAAACTCAAAGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAG exon 8
    ATTTGGCAGAGAGTATAATGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATG
    ATTAAGGTAGGTCTATGTAGTGATACGCTGCATTTGAATGCTTTTTGCTGGCTTTT
    1181 GGAATGCATAGACAAAATGTGAGATTTTTTTTTTTTCATTTCATCTTCAGGGTCTT LDHA
    TACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGG exon 9
    AATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGA
    AGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAAAGTCTTCTG
    ATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCTAGGTGGAGGTTGTGCA
    TGTTGTCCTTTTTATCTGATCTGTGATTAAAGCAGTAATATTTTAAGATGGACTGG
    GAAAAACATCAACTCCTGAAGTTAGAAATAAGAATGGTTTGTAAAATCCACAGCTA
    TATCCTGATGCTGGATGGTATTAATCTTGTGTAGTCTTCAACTGGTTAGTGTGAAA
    TAGTTCTGCCACCTCTGACGCACCACTGCCAATGCTGTACGTACTGCATTTGCCCC
    TTGAGCCAGGTGGATGTTTACCGTGTGTTATATAACTTCCTGGCTCCTTCACTGAA
    CATGCCTAGTCCAACATTTTTTCCCAGTGAGTCACATCCTGGGATCCAGTGTATAA
    ATCCAATATCATGTCTTGTGCATAATTCTTCCAAAGGATCTTATTTTGTGAACTAT
    ATCAGTAGTGTACATTACCATATAATGTAAAAAGATCTACATACAAACAATGCAAC
    CAACTATCCAAGTGTTATACCAACTAAAACCCCCAATAAACCTTGAACAGTGACTA
    CTTTGGTTAATTCATTATATTAAGATATAAAGTCATAAAGCTGCTAGTTATTATAT
    TAATTTGGAAATATTAGGCTATTCTTGGGCAACCCTGCAACGATTTTTTCTAACAG
    GGATATTATTGACTAATAGCAGAGGATGTAATAGTCAACTGAGTTGTATTGGTACC
    ACTTCCATTGTAAGTCCCAAAGTATTATATATTTGATAATAATGCTAATCATAATT
    GGAAAGTAACATTCTATATGTAAATGTAAAATTTATTTGCCAACTGAATATAGGCA
    ATGATAGTGTGTCACTATAGGGAACACAGATTTTTGAGATCTTGTCCTCTGGAAGC
    TGGTAACAATTAAAAACAATCTTAAGGCAGGGTGCAGTGGCTCATGCCTATAATCC
    CAGCACTTTGGGAAGCCCAGGTGGGCTGATCACTGGAGGCCAGGAATTGGGGACCA
    GCCTGGCCAACACAACAAAACCCCATCTGTTAAAAAAACAAAACAAAACCAAAAAA
    AACAAGTAACCTTGGTGGATGTCTACTCAAGTTTTCTGCACATTTTTCTGAAAATA
    CAACTGTGACCCTTA
    1211 MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGID (Cas12i1 of
    RDIISGTANKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQ SEQ ID
    AQEYFASNFDTEKHQWKDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLST NO: 3 of
    AHYITSVMFGTGAKNNRQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSY U.S. Pat.
    RKLRIRCSRKGATPSILKIVQDYELGTNHDDEVNVPSLIANLKEKLGRFEYECEWK No.
    CMEKIKAFLASKVGPYYLGSYSAMLENALSPIKGMTTKNCKFVLKQIDAKNDIKYE 10,808,245)
    NEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWEDLNAIHSKYEEDIA
    SLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKI
    IDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRN
    QVDNYIWIEIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFRTK
    TNGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNIN
    ICKRGNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYN
    DLPVKPIESGFVTVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMK
    DNRGNNIQIDFMKDFEAIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFE
    LLRDGKLSVLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDE
    DKQKADPEMFALRLALEEKRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENI
    SDTTKKGKKSSTNSFLMDWLARGVANKVKEMVMMHQGLEFVEVNPNFTSHQDPFVH
    KNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKRPTNAYYNEGAMAFLATYG
    LKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKLDADATSVNWN
    GKQFWVCNADLVAAYNVGLVDIQKDFKKK
    1212 MSISNNNILPYNPKLLPDDRKHKMLVDTFNQLDLIRNNLHDMIIALYGALKYDNIK (Cas12i3 of
    QFASKEKPHISADALCSINWFRLVKTNERKPAIESNQIISKFIQYSGHTPDKYALS SEQ ID
    HITGNHEPSHKWIDCREYAINYARIMHLSFSQFQDLATACLNCKILILNGTLTSSW NO: 14 of
    AWGANSALFGGSDKENFSVKAKILNSFIENLKDEMNTTKFQVVEKVCQQIGSSDAA U.S. Pat.
    DLFDLYRSTVKDGNRGPATGRNPKVMNLFSQDGEISSEQREDFIESFQKVMQEKNS No.
    KQIIPHLDKLKYHLVKQSGLYDIYSWAAAIKNANSTIVASNSSNLNTILNKTEKQQ 10,808,245)
    TFEELRKDEKIVACSKILLSVNDTLPEDLHYNPSTSNLGKNLDVFFDLLNENSVHT
    IENKEEKNKIVKECVNQYMEECKGLNKPPMPVLLTFISDYAHKHQAQDFLSAAKMN
    FIDLKIKSIKVVPTVHGSSPYTWISNLSKKNKDGKMIRTPNSSLIGWIIPPEEIHD
    QKFAGQNPIIWAVLRVYCNNKWEMHHFPFSDSRFFTEVYAYKPNLPYLPGGENRSK
    RFGYRHSTNLSNESRQILLDKSKYAKANKSVLRCMENMTHNVVFDPKTSLNIRIKT
    DKNNSPVLDDKGRITFVMQINHRILEKYNNTKIEIGDRILAYDQNQSENHTYAILQ
    RTEEGSHAHQFNGWYVRVLETGKVTSIVQGLSGPIDQLNYDGMPVTSHKFNCWQAD
    RSAFVSQFASLKISETETFDEAYQAINAQGAYTWNLFYLRILRKALRVCHMENINQ
    FREEILAISKNRLSPMSLGSLSQNSLKMIRAFKSIINCYMSRMSFVDELQKKEGDL
    ELHTIMRLTDNKLNDKRVEKINRASSFLTNKAHSMGCKMIVGESDLPVADSKTSKK
    QNVDRMDWCARALSHKVEYACKLMGLAYRGIPAYMSSHQDPLVHLVESKRSVLRPR
    FVVADKSDVKQHHLDNLRRMLNSKTKVGTAVYYREAVELMCEELGIHKTDMAKGKV
    SLSDFVDKFIGEKAIFPQRGGRFYMSTKRLTTGAKLICYSGSDVWLSDADEIAAIN
    IGMFVVCDQTGAFKKKKKEKLDDEECDILPFRPM
    1230 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA
    CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 1
    GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA
    AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC
    AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA
    TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT
    AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG
    CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA
    TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC
    CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG
    GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG
    TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG
    GAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGTGCTTATGAGGTGATCAA
    ACTCAAAGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGA
    GTATAATGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGGTCTT
    TACGGAATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGG
    AATCTCAGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGA
    AGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAA
    1231 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA
    CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 2
    GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA
    AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC
    AAAGATTGTCTCTGGCAAAGTGGATATCTTGACCTACGTGGCTTGGAAGATAAGTG
    GTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCCCGATTC
    CGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGGGTGGGT
    CCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATGTTGCTG
    GTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAGGAACAG
    TGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGTGCTTATGAGGTGATCAAACTCAA
    AGGCTACACATCCTGGGCTATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAA
    TGAAGAATCTTAGGCGGGTGCACCCAGTTTCCACCATGATTAAGGGTCTTTACGGA
    ATAAAGGATGATGTCTTCCTTAGTGTTCCTTGCATTTTGGGACAGAATGGAATCTC
    AGACCTTGTGAAGGTGACTCTGACTTCTGAGGAAGAGGCCCGTTTGAAGAAGAGTG
    CAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTTAA
    1232 ATGGGTGAACCCTCAGGAGGCTATACTTACACCCAAACGTCGATATTCCTTTTCCA LDHA
    CGCTAAGATTCCTTTTGGTTCCAAGTCCAATATGGCAACTCTAAAGGATCAGCTGA isoform 3
    TTTATAATCTTCTAAAGGAAGAACAGACCCCCCAGAATAAGATTACAGTTGTTGGG cDNA
    GTTGGTGCTGTTGGCATGGCCTGTGCCATCAGTATCTTAATGAAGGACTTGGCAGA
    TGAACTTGCTCTTGTTGATGTCATCGAAGACAAATTGAAGGGAGAGATGATGGATC
    TCCAACATGGCAGCCTTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTAT
    AATGTAACTGCAAACTCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGA
    GGGAGAAAGCCGTCTTAATTTGGTCCAGCGTAACGTGAACATCTTTAAATTCATCA
    TTCCTAATGTTGTAAAATACAGCCCGAACTGCAAGTTGCTTATTGTTTCAAATCCA
    GTGGATATCTTGACCTACGTGGCTTGGAAGATAAGTGGTTTTCCCAAAAACCGTGT
    TATTGGAAGCGGTTGCAATCTGGATTCAGCCCGATTCCGTTACCTAATGGGGGAAA
    GGCTGGGAGTTCACCCATTAAGCTGTCATGGGTGGGTCCTTGGGGAACATGGAGAT
    TCCAGTGTGCCTGTATGGAGTGGAATGAATGTTGCTGGTGTCTCTCTGAAGACTCT
    GCACCCAGATTTAGGGACTGATAAAGATAAGGAACAGTGGAAAGAGGTTCACAAGC
    AGGTGGTTGAGAGTGCTTATGAGGTGATCAAACTCAAAGGCTACACATCCTGGGCT
    ATTGGACTCTCTGTAGCAGATTTGGCAGAGAGTATAATGAAGAATCTTAGGCGGGT
    GCACCCAGTTTCCACCATGATTAAGGGTCTTTACGGAATAAAGGATGATGTCTTCC
    TTAGTGTTCCTTGCATTTTGGGACAGAATGGAATCTCAGACCTTGTGAAGGTGACT
    CTGACTTCTGAGGAAGAGGCCCGTTTGAAGAAGAGTGCAGATACACTTTGGGGGAT
    CCAAAAGGAGCTGCAATTTTAA
    1233 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA
    CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 4
    GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA
    AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC
    AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA
    TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT
    AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG
    CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA
    TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC
    CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG
    GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG
    TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG
    GAACAGTGGAAAGAGTGCAGATACACTTTGGGGGATCCAAAAGGAGCTGCAATTTT
    AAAGTCTTCTGATGTCATATCATTTCACTGTCTAGGCTACAACAGGATTCTAGGTG
    GAGGTTGTGCATGTTGTCCTTTTTATCTGATCTGTGATTAA
    1234 ATGGCAACTCTAAAGGATCAGCTGATTTATAATCTTCTAAAGGAAGAACAGACCCC LDHA
    CCAGAATAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCCTGTGCCATCA isoform 4
    GTATCTTAATGAAGGACTTGGCAGATGAACTTGCTCTTGTTGATGTCATCGAAGAC cDNA
    AAATTGAAGGGAGAGATGATGGATCTCCAACATGGCAGCCTTTTCCTTAGAACACC
    AAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCCAAGCTGGTCATTA
    TCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTTGGTCCAGCGT
    AACGTGAACATCTTTAAATTCATCATTCCTAATGTTGTAAAATACAGCCCGAACTG
    CAAGTTGCTTATTGTTTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAGA
    TAAGTGGTTTTCCCAAAAACCGTGTTATTGGAAGCGGTTGCAATCTGGATTCAGCC
    CGATTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCATTAAGCTGTCATGG
    GTGGGTCCTTGGGGAACATGGAGATTCCAGTGTGCCTGTATGGAGTGGAATGAATG
    TTGCTGGTGTCTCTCTGAAGACTCTGCACCCAGATTTAGGGACTGATAAAGATAAG
    GAACAGTGGAAAGAGGTTCACAAGCAGGTGGTTGAGAGGGTCTTTACGGAATAA
    1254 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrArGrGrA 3′ end
    rCrUrUrGrGrCrArGrArUrGmA*mA*mC*rU modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1237
    1255 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrArGr 5′ and 3′
    GrArCrUrUrGrGrCrArGrArUrGmA*mA*mC*rU end
    modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1237
    1256 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrGrArUrGrA 3′ end
    rCrArUrCrArArCrArArGrAmG*mC*mA*rA modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1239
    1257 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrGrArUr 5′ and 3′
    GrArCrArUrCrArArCrArArGrAmG*mC*mA*rA end
    modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1239
    1258 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrCrArUrA 3′ end
    rGrUrGrGrArUrArUrCrUrUmG*mA*mC*rC modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1248
    1259 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrCrAr 5′ and 3′
    UrArGrUrGrGrArUrArUrCrUrUmG*mA*mC*rC end
    modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1248
    1260 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrUrCrArU 3′ end
    rArGrUrGrGrArUrArUrCrUmU*mG*mA*rC modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1245
    1261 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrUrUrCr 5′ and 3′
    ArUrArGrUrGrGrArUrArUrCrUmU*mG*mA*rC end
    modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1245
    1262 rArGrArArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrCrArUrArG 3′ end
    rUrGrGrArUrArUrCrUrUrGmA*mC*mC*rU modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1249
    1263 mA*mG*mA*rArArUrCrCrGrUrCrUrUrUrCrArUrUrGrArCrGrGrCrArUr 5′ and 3′
    ArGrUrGrGrArUrArUrCrUrUrGmA*mC*mC*rU end
    modified
    RNA guide
    targeting
    LDHA
    sequence
    of SEQ ID
    NO: 1249
  • In some embodiments, the gene editing system disclosed herein may comprise a Cas12i polypeptide as disclosed herein. In other embodiments, the gene editing system may comprise a nucleic acid encoding the Cas12i polypeptide. For example, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11 and AAV12) encoding the Cas12i polypeptide. Alternatively, the gene editing system may comprise a mRNA molecule encoding the Cas12i polypeptide. In some instances, the mRNA molecule may be codon-optimized.
    • II. Preparation of Gene Editing System Components
  • The present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the RNA guide, methods for production of the Cas12i polypeptide, and methods for complexing the RNA guide and Cas12i polypeptide.
    • A. RNA Guide
  • In some embodiments, the RNA guide is made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence).
  • In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Cas12i polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Cas12i polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.
    • B. Cas12i Polypeptide
  • In some embodiments, the Cas12i polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Cas12i polypeptide of the present disclosure, isolating the Cas12i polypeptide, optionally, purifying the Cas12i polypeptide, and complexing the Cas12i polypeptide with an RNA guide. The Cas12i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Cas12i polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell. Alternatively, the Cas12i polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.
  • In some embodiments, a host cell is used to express the Cas12i polypeptide. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.
  • After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Cas12i polypeptide. After expression of the Cas12i polypeptide, the host cells can be collected and Cas12i polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).
  • In some embodiments, the methods for Cas12i polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Cas12i polypeptide. In some embodiments, the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Cas12i polypeptide.
  • A variety of methods can be used to determine the level of production of a Cas12i polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Cas12i polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).
  • The present disclosure provides methods of in vivo expression of the Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide, expressing the Cas12i polypeptide in the cell, and obtaining the Cas12i polypeptide from the cell.
  • The present disclosure further provides methods of in vivo expression of a Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide and expressing the Cas12i polypeptide in the cell. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide is delivered to the cell with an RNA guide and, once expressed in the cell, the Cas12i polypeptide and the RNA guide form a complex. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are delivered to the cell within a single composition. In some embodiments, the polyribonucleotide encoding the Cas12i polypeptide and the RNA guide are comprised within separate compositions. In some embodiments, the host cell is present in a subject, e.g., a human patient.
    • C. Complexing
  • In some embodiments, an RNA guide targeting LDHA is complexed with a Cas12i polypeptide to form a ribonucleoprotein. In some embodiments, complexation of the RNA guide and Cas12i polypeptide occurs at a temperature lower than about any one of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C. In some embodiments, the RNA guide does not dissociate from the Cas12i polypeptide at about 37° C. over an incubation period of at least about any one of 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hr, 2 hr, 3 hr, 4 hr, or more hours.
  • In some embodiments, the RNA guide and Cas12i polypeptide are complexed in a complexation buffer. In some embodiments, the Cas12i polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the Cas12i polypeptide is stored in a complexation buffer.
  • In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.
  • In some embodiments, the Cas12i polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In some embodiments, mRNA or DNA encoding the Cas12i polypeptide is introduced into a cell so that the Cas12i polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.
    • III. Genetic Editing Methods
  • The disclosure also provides methods of modifying a target site within the LDHA gene. In some embodiments, the methods comprise introducing an LDHA-targeting RNA guide and a Cas12i polypeptide into a cell. The LDHA-targeting RNA guide and Cas12i polypeptide can be introduced as a ribonucleoprotein complex into a cell. The LDHA-targeting RNA guide and Cas12i polypeptide can be introduced on a nucleic acid vector. The Cas12i polypeptide can be introduced as an mRNA. The RNA guide can be introduced directly into the cell. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to reduce LDHA in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to reduce oxalate production in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a cell/tissue/liver/person to correct calcium oxalate crystal deposition in the cell/tissue/liver/person. In some embodiments, the composition described herein is delivered to a person with primary hyperoxaluria.
  • Any of the gene editing systems disclosed herein may be used to genetically engineered an LDHA gene. The gene editing system may comprise a RNA guide and a Cas12i2 polypeptide. The RNA guide comprises a spacer sequence specific to a target sequence in the LDHA gene, e.g., specific to a region in exon 3 or exon 5 of the LDHA gene.
    • A. Target Sequence
  • In some embodiments, an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5′-TTN-3′ PAM sequence or 5′-NTTN-3′ PAM sequence.
  • In some embodiments, the target sequence is within an LDHA gene or a locus of an LDHA gene (e.g., exon 3 or exon 5), to which the RNA guide can bind via base pairing. In some embodiments, a cell has only one copy of the target sequence. In some embodiments, a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.
  • In some embodiments, the LDHA gene is a mammalian gene. In some embodiments, the LDHA gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within an exon of the LDHA gene set forth in SEQ ID NO: 1172, e.g., within a sequence of SEQ ID NO: 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, or 1181 (or a reverse complement thereof). Target sequences within an exon region of the LDHA gene of SEQ ID NO: 1172 are set forth in Table 5. In some embodiments, the target sequence is within an intron of the LDHA gene set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the LDHA gene sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). For examples, in some embodiments, the LDHA gene sequence is a non-human LDHA sequence. In some embodiments, the LDHA gene sequence is a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof). In some embodiments, the LDHA gene sequence is a homolog of a coding sequence set forth in any one of SEQ ID NOs: 1230-1234 (or the reverse complement thereof).
  • In some embodiments, the target sequence is adjacent to a 5′-NTTN-3′ PAM sequence or 5′-TTN-3′ PAM sequence, wherein N is any nucleotide. The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′. The PAM sequence may be 5′ to the target sequence.
  • The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5′-DTTR-3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′. In some embodiments, the RNA guide is designed to bind to a first strand of a double-stranded target nucleic acid (i.e., the non-PAM strand), and the 5′-NTTN-3′ PAM sequence is present in the second, complementary strand (i.e., the PAM strand). In some embodiments, the RNA guide binds to a region on the non-PAM strand that is complementary to a target sequence on the PAM strand, which is adjacent to a 5′-NAAN-3′ sequence.
  • In some embodiments, the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5′ or 3′ untranslated region, etc.
  • In some embodiments, the target sequence is present in a readily accessible region of the target sequence. In some embodiments, the target sequence is in an exon of a target gene. In some embodiments, the target sequence is across an exon-intron junction of a target gene. In some embodiments, the target sequence is present in a non-coding region, such as a regulatory region of a gene.
    • B. Gene Editing
  • In some embodiments, the Cas12i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Cas12i polypeptide induces one or more DNA double-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA single-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).
  • In some embodiments, an RNA guide disclosed herein forms a complex with the Cas12i polypeptide and directs the Cas12i polypeptide to a target sequence adjacent to a 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a T/C-rich sequence.
  • In some embodiments, the deletion is downstream of a 5′-NTTN-3′ sequence. In some embodiments, the deletion is downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.
  • In some embodiments, the deletion alters expression of the LDHA gene. In some embodiments, the deletion alters function of the LDHA gene. In some embodiments, the deletion inactivates the LDHA gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • In some embodiments, the deletion is up to about 40 nucleotides in length (e.g., about 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 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, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).
  • In some embodiments, the methods described herein are used to engineer a cell comprising a deletion as described herein in an LDHA gene. In some embodiments, the methods are carried out using a complex comprising a Cas12i enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein. In some embodiments, the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1213-1229. In some embodiments, the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is synthetic or purified RNA. In some embodiments, the Cas12i polypeptide is encoded in a plasmid. In some embodiments, the Cas12i polypeptide is encoded by an RNA that is synthetic or purified.
    • C. Delivery
  • Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.
  • In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Cas12i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered together in a single composition. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered in separate compositions. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding a Cas12i polypeptide, an RNA guide, or an mRNA encoding a Cas12i polypeptide and an RNA guide. In some embodiments, the mRNA encoding the Cas12i polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 1165 or SEQ ID NO: 1201 or a variant thereof. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
    • D. Genetically Modified Cells
  • Any of the gene editing systems disclosed herein can be delivered to a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.
  • In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.
  • In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.
  • In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.
  • In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.
  • Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure. Such modified cells may comprise a disrupted LDHA gene.
  • Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy. Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject. In some embodiments, the disease or condition is primary hyperoxaluria (PH). In some embodiments, the PH is PH1, PH2, or PH3. Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing an LDHA sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.
    • IV. Therapeutic Applications
  • Any of the gene editing systems or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease that is associated with the LDHA gene, for example, primary hyperoxaluria (PH). In some embodiments, the PH is PH1, PH2, or PH3. In specific examples, the target disease is PH1.
  • PH is a rare genetic disorder effecting subjects of all ages from infants to elderly. PH includes three subtypes involving genetic defects that alter the expression of three distinct proteins. PH1 involves alanine-glyoxylate aminotransferase, or AGT/AGT1. PH2 involves glyoxylate/hydroxypyruvate reductase, or GR/HPR, and PH3 involves 4-hydroxy-2-oxoglutarate aldolase, or HOGA.
  • In PH1, excess oxalate can also combine with calcium to form calcium oxalate in the kidney and other organs. Deposits of calcium oxalate can produce widespread deposition of calcium oxalate (nephrocalcinosis) or formation of kidney and bladder stones (urolithiasis) and lead to kidney damage. Common kidney complications in PH1 include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PH1 may begin to fail, and levels of oxalate may rise in the blood. Deposition of oxalate in tissues throughout the body, e.g., systemic oxalosis, may occur due to high blood levels of oxalate and can lead to complications in bone, skin, and eye. Patients with PH1 normally have kidney failure at an early age, with renal dialysis or dual kidney/liver organ transplant as the only treatment options.
  • In some embodiments, provided herein is a method for treating a target disease as disclosed herein (e.g., PH such as PH1) comprising administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue or specific type of cells where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers.
  • In some embodiments, modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of the treatment. The modified cells may comprise a substitution, insertion, and/or deletion described herein. In some examples, the modified cells may include a cell line modified by a CRISPR nuclease, reverse transcriptase polypeptide, and editing template RNA (e.g., RNA guide and RT donor RNA). In some instances, the modified cells may be a heterogenous population comprising cells with different types of gene edits. Alternatively, the modified cells may comprise a substantially homogenous cell population (e.g., at least 80% of the cells in the whole population) comprising one particular gene edit in the LDHA gene. In some examples, the cells can be suspended in a suitable media.
  • In some embodiments, provided herein is a composition comprising the gene editing system or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • In some embodiments, a pharmaceutical composition comprising the gene editing system or components thereof as described herein may be administered to a subject in need thereof, e.g., one who suffers from a liver disease associated with the LDHA gene. In some instances, the gene editing system or components thereof may be delivered to specific cells or tissue (e.g., to liver cells), where the gene editing system could function to genetically modify the LDHA gene in such cells.
  • A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
    • V. Kits and Uses Thereof
  • The present disclosure also provides kits that can be used, for example, to carry out a method described herein for genetical modification of the LDHA gene. In some embodiments, the kits include an RNA guide and a Cas12i polypeptide. In some embodiments, the kits include a polynucleotide that encodes such a Cas12i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. The Cas12i polypeptide and the RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Cas12i polypeptide.
  • In some embodiments, the kit may be useful for research purposes. For example, in some embodiments, the kit may be useful to study gene function.
  • All references and publications cited herein are hereby incorporated by reference.
    • Additional Embodiments
  • Provided below are additional embodiments, which are also within the scope of the present disclosure.
  • Embodiment 1: A composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or complete complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.
  • In Embodiment 1, the target sequence may be within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene. In some examples, the LDHA gene comprises the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • In Embodiment 1, the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.
  • In any of the compositions of Embodiment 1, the spacer sequence may comprise: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.
  • In any of the compositions of Embodiment 1, the direct repeat sequence may comprise: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • In any of the composition of Embodiment 1, the PAM may comprise the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • In some examples, the target sequence is immediately adjacent to the PAM sequence.
  • In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229.
  • In some examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 2: The composition of Embodiment 1 may further comprise a Cas12i polypeptide or a polyribonucleotide encoding a Cas12i polypeptide, which can be one of the following: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.
  • In specific examples, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
  • In any of the compositions of Embodiment 2, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some examples, the ribonucleoprotein complex binds a target nucleic acid. In some examples, the composition is present within a cell.
  • In any of the compositions of Embodiment 2, the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.
  • Embodiment 3: A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide. The vectors may be expression vectors.
  • Embodiment 4: A composition comprising an RNA guide and a Cas12i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.
  • In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.
  • In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.
  • In any of the compositions of Embodiment 4, the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • In some examples, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′. In some examples, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • In some examples, the target sequence is immediately adjacent to the PAM sequence. In some examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • In any of the compositions of Embodiment 4, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.
  • In some examples, the Cas12i polypeptide is: (a) a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1166, SEQ ID NO: 1167, SEQ ID NO: 1168, SEQ ID NO: 1169, SEQ ID NO: 1170, or SEQ ID NO: 1171; (b) a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; (c) a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 1211; or (d) a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
  • In any of the composition of Embodiment 4, the RNA guide and the Cas12i polypeptide may form a ribonucleoprotein complex. In some examples, the ribonucleoprotein complex binds a target nucleic acid.
  • In any of the composition of Embodiment 4, the composition may be present within a cell.
  • In any of the composition of Embodiment 4, the RNA guide and the Cas12i polypeptide may be encoded in a vector, e.g., expression vector. In some examples, the RNA guide and the Cas12i polypeptide are encoded in a single vector. In other examples, the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.
  • Embodiment 5: A vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide. In some examples, the vectors are expression vectors.
  • Embodiment 6: An RNA guide comprising (i) a spacer sequence that is substantially complementary or completely complementary to a region on a non-PAM strand (the complementary sequence of a target sequence) within an LDHA gene, and (ii) a direct repeat sequence.
  • In some examples, the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or exon 9 of the LDHA gene, which may comprise the sequence of SEQ ID NO: 1172, the reverse complement of SEQ ID NO: 1172, a variant of the sequence of SEQ ID NO: 1172, or the reverse complement of a variant of SEQ ID NO: 1172.
  • In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164.
  • In some examples, the spacer sequence comprises: (a) nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 588-1164; (b) nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 588-1164; (c) nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 588-1164; (d) nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 588-1164; (e) nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 588-1164; (f) nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 588-1164; (g) nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 588-1164; (h) nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 588-1164; (i) nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 588-1164; (j) nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 588-1164; (k) nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 588-1164; (l) nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 588-1164; (m) nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 588-1164; (n) nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 588-1164; or (o) nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 588-1164.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; (or o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.
  • In any of the RNA guide of Embodiment 6, the spacer sequence may be substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587.
  • In any of the RNA guide of Embodiment 6, the target sequence may be adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′, wherein N is any nucleotide. In some examples, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.
  • In some examples, the target sequence is immediately adjacent to the PAM sequence. In other examples, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1213-1229. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 7: A nucleic acid encoding an RNA guide as described herein.
  • Embodiment 8: A vector comprising such an RNA guide as described herein.
  • Embodiment 9: A cell comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • Embodiment 10: A kit comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.
  • Embodiment 11: A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with a composition or an RNA guide as described herein. In some examples, the method is carried out in vitro. In other examples, the method is carried out ex vivo.
  • In some examples, the LDHA sequence is in a cell.
  • In some examples, the composition or the RNA guide induces a deletion in the LDHA sequence. In some examples, the deletion is adjacent to a 5′-NTTN-3′ sequence, wherein N is any nucleotide. In some specific examples, the deletion is downstream of the 5′-NTTN-3′ sequence. In some specific examples, the deletion is up to about 40 nucleotides in length. In some instances, the deletion is from about 4 nucleotides to 40 nucleotides, about 4 nucleotides to 25 nucleotides, about 10 nucleotides to 25 nucleotides, or about 10 nucleotides to 15 nucleotides in length.
  • In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, or about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, or about 25 nucleotides to about 30 nucleotides of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion ends within about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.
  • In some examples, the 5′-NTTN-3′ sequence is 5′-CTTT-3′, 5′-CTTC-3′, 5′-GTTT-3′, 5′-GTTC-3′, 5′-TTTC-3′, 5′-GTTA-3′, or 5′-GTTG-3′.
  • In some examples, the deletion overlaps with a mutation in the LDHA sequence. In some instances, the deletion overlaps with an insertion in the LDHA sequence. In some instances, the deletion removes a repeat expansion of the LDHA sequence or a portion thereof. In some instances, the deletion disrupts one or both alleles of the LDHA sequence.
  • In any of the composition, RNA guide, nucleic acid, vector, cell, kit, or method of Embodiments 1-11 described herein, the RNA guide may comprise the sequence of any one of SEQ ID NOs: 1213-1229.
  • Embodiment 12: A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering a composition, an RNA guide, or a cell described herein to the subject.
  • In any of the compositions, RNA guides, cells, kits, or methods described herein, the RNA guide and/or the polyribonucleotide encoding the Cas12i polypeptide are comprised within a lipid nanoparticle. In some examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within the same lipid nanoparticle. In other examples, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within separate lipid nanoparticles.
  • Embodiment 13: An RNA guide comprising (i) a spacer sequence that is complementary to a target site within an LDHA gene (the target site being on the non-PAM strand and complementary to a target sequence), and (ii) a direct repeat sequence, wherein the target sequence is any one of SEQ ID NOs: 1237, 1239, 1248, 1245, or 1249, or the reverse complement thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or (aa) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (1) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; (o) nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; (p) nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; (q) nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; (r) nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; (s) nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; (t) nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; (u) nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; (v) nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; (w) nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; (x) nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; (y) nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; (z) nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or (aa) SEQ ID NO: 10 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1182-1199; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (b) nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (c) nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (d) nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (e) nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (f) nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (g) nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (h) nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (i) nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (j) nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (k) nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (l) nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (m) nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; (n) nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1182-1199; or (o) SEQ ID NO: 1200 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1205; or (o) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1205; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1205; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1205; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1205; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1205; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1205; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1205; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1205; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1205; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1205; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1205; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1205; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1205; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1205; or (o) SEQ ID NO: 1206 or SEQ ID NO: 1207 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the direct repeat sequence comprises: (a) nucleotide 1 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (b) nucleotide 2 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (c) nucleotide 3 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (d) nucleotide 4 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (e) nucleotide 5 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (f) nucleotide 6 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (g) nucleotide 7 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (h) nucleotide 8 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (i) nucleotide 9 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (j) nucleotide 10 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (k) nucleotide 11 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (l) nucleotide 12 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (m) nucleotide 13 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (n) nucleotide 14 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; (o) nucleotide 15 through nucleotide 36 of SEQ ID NO: 1208 or SEQ ID NO: 1209; or (p) SEQ ID NO: 1210 or a portion thereof.
  • In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1214, 1235, 1224, 1221, or 1225.
  • In some examples, each of the first three nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • In some examples, each of the last four nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification.
  • In some examples, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2′-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.
  • In some examples, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1254-1263. In specific examples, the RNA guide has the sequence of any one of SEQ ID NOs: 1254-1263.
  • In some embodiments, an LDHA-targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprises any one of SEQ ID NOs: 1254-1263. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1254 or SEQ ID NO: 1255 binds the complementary region of LDHA target sequence of SEQ ID NO: 1237. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1256 or SEQ ID NO: 1257 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1239. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1258 or SEQ ID NO: 1259 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1248. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1260 or SEQ ID NO: 1261 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1245. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 1262 or SEQ ID NO: 1263 binds the complementary region of the LDHA target sequence of SEQ ID NO: 1249.
  • Embodiment 14: A nucleic acid encoding an RNA guide as described herein.
  • Embodiment 15: A vector comprising the nucleic acid as described herein.
  • Embodiment 16: A vector system comprising one or more vectors encoding (i) the RNA guide of Embodiment 13 as described herein and (ii) a Cas12i polypeptide. In some examples, the vector system comprises a first vector encoding the RNA guide and a second vector encoding the Cas12i polypeptide.
  • Embodiment 17: A cell comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein. In some examples, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • Embodiment 18: A kit comprising the RNA guide, the nucleic acid, the vector, or the vector system of Embodiments 13-16 as described herein.
  • Embodiment 19: A method of editing an LDHA sequence, the method comprising contacting an LDHA sequence with an RNA guide of Embodiment 13 as described herein. In some examples, the LDHA sequence is in a cell.
  • In some examples, the RNA guide induces an indel (e.g., an insertion or deletion) in the LDHA sequence.
  • Embodiment 20: A method of treating primary hyperoxaluria (PH), which optionally is PH1, PH2, or PH3, in a subject, the method comprising administering the RNA guide of Embodiment 13 as described herein to the subject.
    • General Techniques
  • The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985»; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984»; Animal Cell Culture (R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (1RL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).
  • Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
    • EXAMPLES
  • The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the present disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
    • Example 1—Cas12i2-Mediated Editing of LDHA Target Sites in HEK293T Cells
  • This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HEK293T cells.
  • Cas12i2 RNA guides (crRNAs) were designed and ordered from Integrated DNA Technologies (IDT). For initial guide screening in HEK293T cells, target sequences were designed by tiling the coding exons of LDHA for 5′-NTTN-3′ PAM sequences, and then spacer sequences were designed for the 20-bp target sequences downstream of the PAM sequence. The LDHA-targeting RNA guide sequences are shown in Table 7. TS stands for “top strand” of the LDHA gene, and BS stands for “bottom strand” of the LDHA gene. In the figures, “E #T #” can also be represented as “exon #target #.”
  • TABLE 7
    crRNA sequences for LDHA
    Target
    strand
    (non-
    PAM
    guide name PAM* strand) crRNA sequence target sequence
    LDHA_E2T23 CTTA TS AGAAAUCCGUCUUUCAUUG CCTTCATTAAGATA
    ACGGCCUUCAUUAAGAUAC CTGATG (SEQ ID
    UGAUG (SEQ ID NO: 1213) NO: 1236)
    LDHA_E3T1 CTTT BS AGAAAUCCGUCUUUCAUUG TAGGACTTGGCAG
    ACGGUAGGACUUGGCAGAU ATGAACT (SEQ ID
    GAACU (SEQ ID NO: 1214) NO: 1237)
    LDHA_E3T2 GTTC TS AGAAAUCCGUCUUUCAUUG ATCTGCCAAGTCCT
    ACGGAUCUGCCAAGUCCUA AAAAGA (SEQ ID
    AAAGA (SEQ ID NO: 1215) NO: 1238)
    LDHA_E3T3 CTTC TS AGAAAUCCGUCUUUCAUUG GATGACATCAACA
    ACGGGAUGACAUCAACAAG AGAGCAA (SEQ ID
    AGCAA (SEQ ID NO: 1235) NO: 1239)
    LDHA_E3T9 ATTT BS AGAAAUCCGUCUUUCAUUG GATGTCTTTTAGGA
    ACGGGAUGUCUUUUAGGAC CTTGGC (SEQ ID
    UUGGC (SEQ ID NO: 1216) NO: 1240)
    LDHA_E3T10 TTTG BS AGAAAUCCGUCUUUCAUUG ATGTCTTTTAGGAC
    ACGGAUGUCUUUUAGGACU TTGGCA (SEQ ID
    UGGCA (SEQ ID NO: 1217) NO: 1241)
    LDHA_E3T12 TTTA BS AGAAAUCCGUCUUUCAUUG GGACTTGGCAGAT
    ACGGGGACUUGGCAGAUGA GAACTTG (SEQ ID
    ACUUG (SEQ ID NO: 1218) NO: 1242)
    LDHA_E3T26 GTTG TS AGAAAUCCGUCUUUCAUUG AAATCAACCTTTGC
    ACGGAAAUCAACCUUUGCC CAGAGA (SEQ ID
    AGAGA (SEQ ID NO: 1219) NO: 1243)
    LDHA_E3T27 CTTG TS AGAAAUCCGUCUUUCAUUG TTGAAATCAACCTT
    ACGGUUGAAAUCAACCUUU TGCCAG (SEQ ID
    GCCAG (SEQ ID NO: 1220) NO: 1244)
    LDHA_E5T1 CTTT BS AGAAAUCCGUCUUUCAUUG TTCATAGTGGATAT
    ACGGUUCAUAGUGGAUAUC CTTGAC (SEQ ID
    UUGAC (SEQ ID NO: 1221) NO: 1245)
    LDHA_E5T7 TTTT BS AGAAAUCCGUCUUUCAUUG CTCCTTTTTCATAG
    ACGGCUCCUUUUUCAUAGU TGGATA (SEQ ID
    GGAUA (SEQ ID NO: 1222) NO: 1246)
    LDHA_E5T8 TTTC BS AGAAAUCCGUCUUUCAUUG TCCTTTTTCATAGT
    ACGGUCCUUUUUCAUAGUG GGATAT (SEQ ID
    GAU AU (SEQ ID NO: 1223) NO: 1247)
    LDHA_E5T9 TTTT BS AGAAAUCCGUCUUUCAUUG TCATAGTGGATATC
    ACGGUCAUAGUGGAUAUCU TTGACC (SEQ ID
    UGACC (SEQ ID NO: 1224) NO: 1248)
    LDHA_E5T10 TTTT BS AGAAAUCCGUCUUUCAUUG CATAGTGGATATCT
    ACGGCAUAGUGGAUAUCUU TGACCT (SEQ ID
    GACCU (SEQ ID NO: 1225) NO: 1249)
    LDHA_E5T11 TTTC BS AGAAAUCCGUCUUUCAUUG ATAGTGGATATCTT
    ACGGAUAGUGGAUAUCUUG GACCTA (SEQ ID
    ACCUA (SEQ ID NO: 1226) NO: 1250)
    LDHA_E5T28 ATTA TS AGAAAUCCGUCUUUCAUUG GGTAACGGAATCG
    ACGGGGUAACGGAAUCGGG GGCTGAA (SEQ ID
    CUGAA (SEQ ID NO: 1227) NO: 1251)
    LDHA_E5T32 CTTA TS AGAAAUCCGUCUUUCAUUG CCACTGGAATCTCC
    ACGGCCACUGGAAUCUCCA ATGTTC (SEQ ID
    UGUUC (SEQ ID NO: 1228) NO: 1252)
    LDHA_E5T33 CTTA TS AGAAAUCCGUCUUUCAUUG TGCTTACCACTGGA
    ACGGUGCUUACCACUGGAA ATCTCC (SEQ ID
    UCUCC (SEQ ID NO: 1229) NO: 1253)
    *The 3′ three nucleotides represent the 5′-TTN-3′ motif.
  • Cas12i2 RNP complexation reactions were made by mixing purified Cas12i2 polypeptide (400 μM) with crRNA (1 mM in 250 mM NaCl) at a 1:1 (Cas12i2:crRNA) volume ratio (2.5:1 crRNA:Cas12i2 molar ratio). Complexations were incubated on ice for 30-60 min.
  • HEK293T cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; Thermo Fisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032) at a concentration of 16,480 cells/μL. Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 10 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 μM. The final volume of each electroporated reaction was 20 μL. Non-targeting guides were used as negative controls.
  • The strips were electroporated using an electroporation device (program CM-130, Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, 80 μL of pre-warmed DMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 μL (30,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 μL DMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO2.
  • After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; Thermo Fisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C.
  • Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing runs were done using a 150 cycle NGS instrument (NEXTSEQ™ v2.5) mid or high output kit (Illumina) and run on an NGS instrument (NEXTSEQ™ 550; Illumina).
  • For NGS analysis, the indel mapping function used a sample's fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000.
  • FIG. 1 shows LDHA indels in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Delivery of E3T1 (SEQ ID NO: 1214), E3T9 (SEQ ID NO: 1216), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), and E5T10 (SEQ ID NO: 1225) resulted in indels in over 70% of the NGS reads. Therefore, LDHA-targeting RNA guides induced indels in exon 2, exon 3, and exon 5 in HEK293T cells.
  • This Example thus shows that LDHA can be individually targeted by Cas12i2 RNPs in mammalian cells such as HEK293T cells.
    • Example 2—Cas12i2-Mediated Editing of LDHA Target Sites in Hepg2 Cells
  • This Example describes the genomic editing of the LDHA gene using Cas12i2 introduced into HepG2 cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 7. HepG2 cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032) at a concentration of 13,889 cells/μL. Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 20 μM (Cas12i2), with no transfection enhancer oligo. The final volume of each electroporated reaction was 20 Non-targeting guides were used as negative controls.
  • The strips were electroporated using an electroporation device (program DJ-100, Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, 80 μL of pre-warmed EMEM+10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 μL (25,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 μL EMEM+10% FBS. Editing plates were incubated for 3 days at 37° C. with 5% CO2.
  • After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C. Samples were analyzed by NGS as described in Example 1.
  • FIG. 2 shows LDHA indels in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the LDHA target sites with each of the RNA guides. Therefore, LDHA-targeting RNA guides induced indels in exon 3 and exon 5 in HepG2 cells.
    • Example 3—Cas12i2-Mediated Editing of LDHA Target Sites in Primary Hepatocytes
  • This Example describes the genomic editing of the LDHA using Cas12i2 introduced into primary hepatocytes cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with RNA guides of Table 7. Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged at 100 g for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermofisher CM3000). The cells were subjected to trypan blue viability count with an INCUCYTE® disposable hemocytometer (Fisher scientific, 22-600-100). The cells were then washed in PBS and resuspended in P3 buffer+supplement (P3 PRIMARY CELL 4D-NUCLEOFECTOR™ X Kit; Lonza, VXP-3032) at a concentration of ˜7,500 cells/μL. Resuspended cells were dispensed at 150,000 cells/reaction into the 16 well Lonza NUCLEOCUVETTE strips or 500,000 cells/reaction into the single Lonza NUCLEOCUVETTES® for the mRNA readout. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 20 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 μM. The final volume of each electroporated reaction was either 20 μL in the 16 well nucleocuvette strip format or 100 μL in the single nucleocuvette format. Non-targeting guides were used as negative controls.
  • The strips were electroporated using DS-150 program, while the single nucleocuvettes were electroporated using CA137 program (Lonza 4D-NUCLEOFECTOR™). Immediately following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, plated all the cell suspension of diluted nucleofected cells into a pre-warmed collagen-coated 96-well plate or 24-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37° C. incubator. The media was changed to hepatocyte maintenance media (Williams' Medium E, Thermofisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. Fresh hepatocyte maintenance media was replaced after 2 days.
  • After 4-5 days post RNP electroporation, media was aspirated and the cells were harvested by shaking (500 rpm) in a 37° C. incubator with 2 mg/ml collagenase IV (Thermofisher, 17104019) dissolved in PBS containing Ca/Mg (Thermofisher). After cells were dissociated from the plate, they were transferred to 96-well TWIN.TEC® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cell pellets for the NGS readout were resuspended in 20 μL. QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min and analyzed by NGS as described in Example 1.
  • For the mRNA readout, cell pellets were frozen at −80° C. and subsequently resuspended in lysis buffer and DNA/RNA extracted with the RNeasy kit (Qiagen) following manufacturer's instructions. The DNA extracted from the samples were analyzed by NGS. The RNA isolated was checked for quantity and purity using nanodrop, and subsequently used for cDNA synthesis using 5× iScript reverse transcription reaction mix (Bio-Rad laboratories), following manufacturer's recommendations. cDNA templated was appropriately diluted to be in linear range of the subsequent analysis. Diluted cDNA was used to set up a 20 μL Digital Droplet PCR (ddPCR-BioRad laboratories) reaction using target-specific primer and probe for LDHA, TTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAAC TCCAAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTC TTAATTTGGTCSEQ ID NO: 1264), and 2×ddPCR Supermix for Probes No dUTP (BioRad laboratories) following manufacturer's instructions. The reaction was used to generate droplets using Automated Droplet Generator (BioRad Laboratories), following manufacture's recommendations. The plate was sealed using PX1 PCR Plate Sealer (BioRad Laboratories) generated droplets were subjected to PCR amplification using C1000 Touch Thermal Cycler (BioRad Laboratories) using conditions recommended by the manufacturer. The PCR amplified droplets were read on QX200 Droplet Reader (BioRad Laboratories) and the acquired data was analyzed using QX Manager version 1.2 (BioRad Laboratories) to determine presence of absolute copy number of mRNA present in each reaction for the appropriate targets.
  • As shown in FIG. 3 , each RNA guide tested induced indels within and/or adjacent to the LDHA target sites. Indels were not induced with the non-targeting control. Therefore, LDHA-targeting RNA guides induced indels in primary hepatocytes. Indels for RNA guide E3T1 were then correlated with mRNA levels to determine whether indels led to mRNA knockdown and subsequent protein knockdown. FIG. 4 shows % mRNA knockdown of LDHA in edited cells compared to unedited control cells. RNA guide E3T1 resulted in knockdown of LDHA mRNA.
  • This Example thus shows that LDHA can be targeted by Cas12i2 RNPs in mammalian cells such as primary human hepatocytes.
    • Example 4—Editing of LDHA Target Sites in HepG2 Cells with Cas12i2 Variants
  • This Example describes indel assessment on LDHA target sites using variants introduced into HepG2 cells by transient transfection.
  • The Cas12i2 variants of SEQ ID NO: 1168 and SEQ ID NO: 1171 were individually cloned into a pcda3.1 backbone (Invitrogen). Nucleic acids encoding RNA guides E3T1, E3T3, EST1, E5T9, and E5T10 (Table 7) were cloned into a pUC19 backbone (New England Biolabs). The plasmids were then maxi-prepped and diluted.
  • HepG2 cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer+supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032).
  • Approximately 16 hours prior to transfection, 25,000 HepG2 cells in EMEM/10% FBS were plated into each well of a 96-well plate. On the day of transfection, the cells were 70-90% confluent. For each well to be transfected, a mixture of Lipofectamine™ 3000 and Opti-MEM® was prepared and then incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine™:OptiMEM® mixture was added to a separate mixture containing nuclease plasmid and RNA guide plasmid and P3000 reagent (Solution 2). In the case of negative controls, the crRNA was not included in Solution 2. The Solution 1 and Solution 2 were mixed by pipetting up and down and then incubated at room temperature for 15 minutes. Following incubation, the Solution 1 and Solution 2 mixture was added dropwise to each well of a 96 well plate containing the cells.
  • After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 μL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C. and analyzed by NGS as described in Example 1.
  • As shown in FIG. 5A, two guides, E3T3 and E5T1, demonstrated significantly higher activity with variant Cas12i2 of SEQ ID NO: 1171 compared to variant Cas12i2 of SEQ ID NO: 1168. Comparable indel activity with the two Cas12i2 variants was observed for E3T1, E5T9, and E5T10. FIG. 5B shows the indel size frequency (left) and indel start position relative to the PAM for E5T9 and the variant Cas12i2 of SEQ ID NO: 1168 in HepG2 cells. As shown on the left, deletions ranged in size from 1 nucleotide to about 40 nucleotides. The majority of the deletions were about 8 nucleotides to about 23 nucleotides in length. As shown on the right, the target sequence is represented as starting at position 0 and ending at position 20. Indels started within about 5 nucleotides and about 35 nucleotides downstream of the PAM sequence. The majority of indels started about 10 nucleotides to about 30 nucleotides downstream of the PAM sequence.
  • Thus, this Example shows that LDHA is capable of being targeted by multiple Cas12i2 polypeptides.
    • Example 5—Editing of LDHA in Primary Human Hepatocytes Using Cas12i2 mRNA Constructs
  • This Example describes indel assessment on LDHA target sites via delivery of Cas12i2 mRNA and chemically modified LDHA-targeting RNA guides. mRNA sequences corresponding to the variant Cas12i2 sequence of SEQ ID NO: 1168 and the variant Cas12i2 sequence of SEQ ID NO: 1171 were synthesized by Aldeveron with 1-pseudo-U modified nucleotides and using CleanCap® Reagent AG (TriLink Biotechnologies). The Cas12i2 mRNA sequences, shown in Table 8, further comprised a C-terminal NLS.
  • TABLE 8
    Cas12i2 mRNA Sequences
    Description mRNA sequence
    mRNA AUGAGCUCCGCCAUCAAGUCCUACAAGUCUGUGCUGCGGCCAAACGAGAGAAAGAAUCAGC
    corresponding to UGCUGAAGUCCACCAUCCAGUGCCUGGAGGACGGCUCCGCCUUCUUUUUCAAGAUGCUGCA
    variant Cas12i2 GGGCCUGUUUGGCGGCAUCACCCCCGAGAUCGUGAGAUUCAGCACAGAGCAGGAGAAGCAG
    of SEQ ID NO: CAGCAGGAUAUCGCCCUGUGGUGUGCCGUGAAUUGGUUCAGGCCUGUGAGCCAGGACUCCC
    1168 UGACCCACACAAUCGCCUCCGAUAACCUGGUGGAGAAGUUUGAGGAGUACUAUGGCGGCAC
    AGCCAGCGACGCCAUCAAGCAGUACUUCAGCGCCUCCAUCGGCGAGUCCUACUAUUGGAAU
    GACUGCCGCCAGCAGUACUAUGAUCUGUGUCGGGAGCUGGGCGUGGAGGUGUCUGACCUGA
    CCCACGAUCUGGAGAUCCUGUGCCGGGAGAAGUGUCUGGCCGUGGCCACAGAGAGCAACCA
    GAACAAUUCUAUCAUCAGCGUGCUGUUUGGCACCGGCGAGAAGGAGGAUAGGUCUGUGAAG
    CUGCGCAUCACAAAGAAGAUCCUGGAGGCCAUCAGCAACCUGAAGGAGAUCCCAAAGAAUG
    UGGCCCCCAUCCAGGAGAUCAUCCUGAAUGUGGCCAAGGCCACCAAGGAGACAUUCAGACA
    GGUGUACGCAGGAAACCUGGGAGCACCAUCCACCCUGGAGAAGUUUAUCGCCAAGGACGGC
    CAGAAGGAGUUCGAUCUGAAGAAGCUGCAGACAGACCUGAAGAAAGUGAUCCGGGGCAAGU
    CUAAGGAGAGAGAUUGGUGCUGUCAGGAGGAGCUGAGGAGCUACGUGGAGCAGAAUACCAU
    CCAGUAUGACCUGUGGGCCUGGGGCGAGAUGUUCAACAAGGCCCACACCGCCCUGAAGAUC
    AAGUCCACAAGAAACUACAAUUUUGCCAAGCAGAGGCUGGAGCAGUUCAAGGAGAUCCAGU
    CUCUGAACAAUCUGCUGGUGGUGAAGAAGCUGAACGACUUUUUCGAUAGCGAGUUUUUCUC
    CGGCGAGGAGACCUACACAAUCUGCGUGCACCACCUGGGCGGCAAGGACCUGUCCAAGCUG
    UAUAAGGCCUGGGAGGACGAUCCCGCCGAUCCUGAGAAUGCCAUCGUGGUGCUGUGCGACG
    AUCUGAAGAACAAUUUUAAGAAGGAGCCUAUCAGGAACAUCCUGCGCUACAUCUUCACCAU
    CCGCCAGGAGUGUAGCGCACAGGACAUCCUGGCAGCAGCAAAGUACAAUCAGCAGCUGGAU
    CGGUAUAAGAGCCAGAAGGCCAACCCAUCCGUGCUGGGCAAUCAGGGCUUUACCUGGACAA
    ACGCCGUGAUCCUGCCAGAGAAGGCCCAGCGGAACGACAGACCCAAUUCUCUGGAUCUGCG
    CAUCUGGCUGUACCUGAAGCUGCGGCACCCUGACGGCAGAUGGAAGAAGCACCACAUCCCA
    UUCUACGAUACCCGGUUUUUCCAGGAGAUCUAUGCCGCCGGCAAUAGCCCUGUGGACACCU
    GUCAGUUUAGGACACCCCGCUUCGGCUAUCACCUGCCUAAGCUGACCGAUCAGACAGCCAU
    CCGCGUGAACAAGAAGCACGUGAAGGCAGCAAAGACCGAGGCACGGAUCAGACUGGCCAUC
    CAGCAGGGCACACUGCCAGUGUCCAAUCUGAAGAUCACCGAGAUCUCCGCCACAAUCAACU
    CUAAGGGCCAGGUGCGCAUCCCCGUGAAGUUUCGGGUGGGAAGGCAGAAGGGAACCCUGCA
    GAUCGGCGACCGGUUCUGCGGCUACGAUCAGAACCAGACAGCCUCUCACGCCUAUAGCCUG
    UGGGAGGUGGUGAAGGAGGGCCAGUACCACAAGGAGCUGGGCUGUUUUGUGCGCUUCAUCU
    CUAGCGGCGACAUCGUGUCCAUCACCGAGAACCGGGGCAAUCAGUUUGAUCAGCUGUCUUA
    UGAGGGCCUGGCCUACCCCCAGUAUGCCGACUGGAGAAAGAAGGCCUCCAAGUUCGUGUCU
    CUGUGGCAGAUCACCAAGAAGAACAAGAAGAAGGAGAUCGUGACAGUGGAGGCCAAGGAGA
    AGUUUGACGCCAUCUGCAAGUACCAGCCUAGGCUGUAUAAGUUCAACAAGGAGUACGCCUA
    UCUGCUGCGGGAUAUCGUGAGAGGCAAGAGCCUGGUGGAGCUGCAGCAGAUCAGGCAGGAG
    AUCUUUCGCUUCAUCGAGCAGGACUGUGGAGUGACCCGCCUGGGAUCUCUGAGCCUGUCCA
    CCCUGGAGACAGUGAAGGCCGUGAAGGGCAUCAUCUACUCCUAUUUUUCUACAGCCCUGAA
    UGCCUCUAAGAACAAUCCCAUCAGCGACGAGCAGCGGAAGGAGUUUGAUCCUGAGCUGUUC
    GCCCUGCUGGAGAAGCUGGAGCUGAUCAGGACUCGGAAGAAGAAGCAGAAGGUGGAGAGAA
    UCGCCAAUAGCCUGAUCCAGACAUGCCUGGAGAACAAUAUCAAGUUCAUCAGGGGCGAGGG
    CGACCUGUCCACCACAAACAAUGCCACCAAGAAGAAGGCCAACUCUAGGAGCAUGGAUUGG
    CUGGCCAGAGGCGUGUUUAAUAAGAUCCGGCAGCUGGCCCCAAUGCACAACAUCACCCUGU
    UCGGCUGCGGCAGCCUGUACACAUCCCACCAGGACCCUCUGGUGCACAGAAACCCAGAUAA
    GGCCAUGAAGUGUAGAUGGGCAGCAAUCCCAGUGAAGGACAUCGGCGAUUGGGUGCUGAGA
    AAGCUGUCCCAGAACCUGAGGGCCAAGAAUCGGGGCACCGGCGAGUACUAUCACCAGGGCG
    UGAAGGAGUUCCUGUCUCACUAUGAGCUGCAGGACCUGGAGGAGGAGCUGCUGAAGUGGCG
    GUCUGAUAGAAAGAGCAACAUCCCUUGCUGGGUGCUGCAGAAUAGACUGGCCGAGAAGCUG
    GGCAACAAGGAGGCCGUGGUGUACAUCCCAGUGAGGGGCGGCCGCAUCUAUUUUGCAACCC
    ACAAGGUGGCAACAGGAGCCGUGAGCAUCGUGUUCGACCAGAAGCAAGUGUGGGUGUGUAA
    UGCAGAUCACGUGGCAGCAGCAAACAUCGCACUGACCGGCAAGGGCAUCGGCGAGCAGUCC
    UCUGACGAGGAGAACCCCGAUGGCUCCAGGAUCAAGCUGCAGCUGACAUCUAAAAGGCCGG
    CGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGUAA (SEQ ID NO: 1265)
    mRNA AUGAGCUCCGCCAUCAAGUCCUACAAGUCUGUGCUGCGGCCAAACGAGAGAAAGAAUCAGC
    corresponding to UGCUGAAGUCCACCAUCCAGUGCCUGGAGGACGGCUCCGCCUUCUUUUUCAAGAUGCUGCA
    variant Cas12i2 GGGCCUGUUUGGCGGCAUCACCCCCGAGAUCGUGAGAUUCAGCACAGAGCAGGAGAAGCAG
    of SEQ ID NO: CAGCAGGAUAUCGCCCUGUGGUGUGCCGUGAAUUGGUUCAGGCCUGUGAGCCAGGACUCCC
    1171 UGACCCACACAAUCGCCUCCGAUAACCUGGUGGAGAAGUUUGAGGAGUACUAUGGCGGCAC
    AGCCAGCGACGCCAUCAAGCAGUACUUCAGCGCCUCCAUCGGCGAGUCCUACUAUUGGAAU
    GACUGCCGCCAGCAGUACUAUGAUCUGUGUCGGGAGCUGGGCGUGGAGGUGUCUGACCUGA
    CCCACGAUCUGGAGAUCCUGUGCCGGGAGAAGUGUCUGGCCGUGGCCACAGAGAGCAACCA
    GAACAAUUCUAUCAUCAGCGUGCUGUUUGGCACCGGCGAGAAGGAGGAUAGGUCUGUGAAG
    CUGCGCAUCACAAAGAAGAUCCUGGAGGCCAUCAGCAACCUGAAGGAGAUCCCAAAGAAUG
    UGGCCCCCAUCCAGGAGAUCAUCCUGAAUGUGGCCAAGGCCACCAAGGAGACAUUCAGACA
    GGUGUACGCAGGAAACCUGGGAGCACCAUCCACCCUGGAGAAGUUUAUCGCCAAGGACGGC
    CAGAAGGAGUUCGAUCUGAAGAAGCUGCAGACAGACCUGAAGAAAGUGAUCCGGGGCAAGU
    CUAAGGAGAGAGAUUGGUGCUGUCAGGAGGAGCUGAGGAGCUACGUGGAGCAGAAUACCAU
    CCAGUAUGACCUGUGGGCCUGGGGCGAGAUGUUCAACAAGGCCCACACCGCCCUGAAGAUC
    AAGUCCACAAGAAACUACAAUUUUGCCAAGCAGAGGCUGGAGCAGUUCAAGGAGAUCCAGU
    CUCUGAACAAUCUGCUGGUGGUGAAGAAGCUGAACGACUUUUUCGAUAGCGAGUUUUUCUC
    CGGCGAGGAGACCUACACAAUCUGCGUGCACCACCUGGGCGGCAAGGACCUGUCCAAGCUG
    UAUAAGGCCUGGGAGGACGAUCCCGCCGAUCCUGAGAAUGCCAUCGUGGUGCUGUGCGACG
    AUCUGAAGAACAAUUUUAAGAAGGAGCCUAUCAGGAACAUCCUGCGCUACAUCUUCACCAU
    CCGCCAGGAGUGUAGCGCACAGGACAUCCUGGCAGCAGCAAAGUACAAUCAGCAGCUGGAU
    CGGUAUAAGAGCCAGAAGGCCAACCCAUCCGUGCUGGGCAAUCAGGGCUUUACCUGGACAA
    ACGCCGUGAUCCUGCCAGAGAAGGCCCAGCGGAACGACAGACCCAAUUCUCUGGAUCUGCG
    CAUCUGGCUGUACCUGAAGCUGCGGCACCCUGACGGCAGAUGGAAGAAGCACCACAUCCCA
    UUCUACGAUACCCGGUUUUUCCAGGAGAUCUAUGCCGCCGGCAAUAGCCCUGUGGACACCU
    GUCAGUUUAGGACACCCCGCUUCGGCUAUCACCUGCCUAAGCUGACCGAUCAGACAGCCAU
    CCGCGUGAACAAGAAGCACGUGAAGGCAGCAAAGACCGAGGCACGGAUCAGACUGGCCAUC
    CAGCAGGGCACACUGCCAGUGUCCAAUCUGAAGAUCACCGAGAUCUCCGCCACAAUCAACU
    CUAAGGGCCAGGUGCGCAUCCCCGUGAAGUUUCGGGUGGGAAGGCAGAAGGGAACCCUGCA
    GAUCGGCGACCGGUUCUGCGGCUACGAUCAGAACCAGACAGCCUCUCACGCCUAUAGCCUG
    UGGGAGGUGGUGAAGGAGGGCCAGUACCACAAGGAGCUGCGGUGUCGGGUGCGCUUCAUCU
    CUAGCGGCGACAUCGUGUCCAUCACCGAGAACCGGGGCAAUCAGUUUGAUCAGCUGUCUUA
    UGAGGGCCUGGCCUACCCCCAGUAUGCCGACUGGAGAAAGAAGGCCUCCAAGUUCGUGUCU
    CUGUGGCAGAUCACCAAGAAGAACAAGAAGAAGGAGAUCGUGACAGUGGAGGCCAAGGAGA
    AGUUUGACGCCAUCUGCAAGUACCAGCCUAGGCUGUAUAAGUUCAACAAGGAGUACGCCUA
    UCUGCUGCGGGAUAUCGUGAGAGGCAAGAGCCUGGUGGAGCUGCAGCAGAUCAGGCAGGAG
    AUCUUUCGCUUCAUCGAGCAGGACUGUGGAGUGACCCGCCUGGGAUCUCUGAGCCUGUCCA
    CCCUGGAGACAGUGAAGGCCGUGAAGGGCAUCAUCUACUCCUAUUUUUCUACAGCCCUGAA
    UGCCUCUAAGAACAAUCCCAUCAGCGACGAGCAGCGGAAGGAGUUUGAUCCUGAGCUGUUC
    GCCCUGCUGGAGAAGCUGGAGCUGAUCAGGACUCGGAAGAAGAAGCAGAAGGUGGAGAGAA
    UCGCCAAUAGCCUGAUCCAGACAUGCCUGGAGAACAAUAUCAAGUUCAUCAGGGGCGAGGG
    CGACCUGUCCACCACAAACAAUGCCACCAAGAAGAAGGCCAACUCUAGGAGCAUGGAUUGG
    CUGGCCAGAGGCGUGUUUAAUAAGAUCCGGCAGCUGGCCACCAUGCACAACAUCACCCUGU
    UCGGCUGCGGCAGCCUGUACACAUCCCACCAGGACCCUCUGGUGCACAGAAACCCAGAUAA
    GGCCAUGAAGUGUAGAUGGGCAGCAAUCCCAGUGAAGGACAUCGGCGAUUGGGUGCUGAGA
    AAGCUGUCCCAGAACCUGAGGGCCAAGAAUCGGGGCACCGGCGAGUACUAUCACCAGGGCG
    UGAAGGAGUUCCUGUCUCACUAUGAGCUGCAGGACCUGGAGGAGGAGCUGCUGAAGUGGCG
    GUCUGAUAGAAAGAGCAACAUCCCUUGCUGGGUGCUGCAGAAUAGACUGGCCGAGAAGCUG
    GGCAACAAGGAGGCCGUGGUGUACAUCCCAGUGAGGGGCGGCCGCAUCUAUUUUGCAACCC
    ACAAGGUGGCAACAGGAGCCGUGAGCAUCGUGUUCGACCAGAAGCAAGUGUGGGUGUGUAA
    UGCAGAUCACGUGGCAGCAGCAAACAUCGCACUGACCGGCAAGGGCAUCGGCCGGCAGUCC
    UCUGACGAGGAGAACCCCGAUGGCGGCAGGAUCAAGCUGCAGCUGACAUCUAAAAGGCCGG
    CGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGUAA (SEQ ID NO: 1266)
  • Cas12i2 RNA guides were designed and ordered from Integrated DNA Technologies (IDT) as having 3′ end modified phosphorothioated 2′ O-methyl bases or 5′ end and 3′ end modified phosphorothioated 2′ O-methyl bases guides, as specified in Table 9. Each variant Cas12i2 mRNA was mixed with a crRNA at a 1:1 (Cas12i2:crRNA) volume ratio (1050:1 crRNA:Cas12i2 molar ratio). The mRNA and crRNA were mixed immediately before electroporation. The primary human hepatocyte cells were cultured and electroporated as described in Example 3.
  • TABLE 9
    Chemically Modified RNA Guide Sequences
    RNA Guide Sequence
    3′ end modified AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA*mA*mC*
    E3T1 mU (SEQ ID NO: 1267)
    5′ and 3′ end mA*mG*mA*AAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGA*
    modified E3T1 mA*mC*mU (SEQ ID NO: 1268)
  • FIG. 6 shows editing of an LDHA target site by a variant Cas12i2 mRNA and 3′ end modified E3T1 (SEQ ID NO: 1267) or 5′ and 3′ end modified E3T1 (SEQ ID NO: 1268) RNA guide. Indels in the LDHA target site were introduced following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1265 or SEQ ID NO: 1266 and either the RNA guide of SEQ ID NO: 1267 or SEQ ID NO: 1268. A higher percentage of NGS reads exhibited indels for RNA guide E3T1 with 5′ and 3′ end modifications (SEQ ID NO: 1268) compared to NGS reads for RNA guide with 3′ end modifications only (SEQ ID NO: 1267). Approximately 50% of NGS reads comprised indels following electroporation of the Cas12i2 mRNA of SEQ ID NO: 1266 and the RNA guide of SEQ ID NO: 1268.
  • This Example thus shows that LDHA can be targeted by Cas12i2 mRNA constructs and chemically modified RNA guides in mammalian cells.
    • Example 6—Off-Target Analysis of Cas12i2 and LDHA-Targeting RNA Guides
  • This Example describes on-target versus off-target assessment of a Cas12i2 variant and an LDHA-targeting RNA guide.
  • HEK293T cells were transfected with a plasmid encoding the variant Cas12i2 of SEQ ID NO: 1168 or the variant Cas12i2 of SEQ ID NO: 1171 and a plasmid encoding E3T1 (SEQ ID NO: 1214), EST1 (SEQ ID NO: 1221), E5T9 (SEQ ID NO: 1224), or E5T10 (SEQ ID NO: 1225) according the method described in Example 16 of PCT/US21/25257. The tagmentation-based tag integration site sequencing (TTISS) method described in Example 16 of PCT/US21/25257 was then carried out.
  • FIG. 7A and FIG. 7B show plots depicting on-target and off-target TTISS reads. The black wedge and centered number represent the fraction of on-target TTISS reads. Each grey wedge represents a unique off-target site identified by TTISS. The size of each grey wedge represents the fraction of TTISS reads mapping to a given off-target site. FIG. 7A shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1168, and FIG. 7B shows TTISS reads for variant Cas12i2 of SEQ ID NO: 1171.
  • As shown in FIG. 7A, variant Cas12i2 of SEQ ID NO: 1168 paired with E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on-target. No TTISS reads mapped to potential off-target sites. E3T1 and E5T10 also showed a low likelihood of off-target editing. For E3T1, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For E5T10, 97% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 3% of TTISS reads. E5T1 demonstrated a higher likelihood of off-target editing using the TTISS method.
  • As shown in FIG. 7B, variant Cas12i2 of SEQ ID NO: 1171 paired with the E5T9 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads in replicate 1 and 93% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 7% of TTISS reads in replicate 2. E5T10 also showed a low likelihood of off-target editing; 92% of TTISS reads in replicate 1 and 100% of TTISS reads in replicate 2 mapped to the on-target, and two potential off-target sites represented the remaining 8% of TTISS reads in replicate 1. Variant Cas12i2 of SEQ ID NO: 1171 paired with the E3T1 demonstrated a higher likelihood of off-target editing. 86% and 93% of TTISS reads mapping to the on-target in replicate 1 and replicate 2, respectively. 5 potential off-target sites represented the remaining 14% of TTISS reads in replicate 1, and 2 potential off-target sites represented the remaining 7% off TTISS reads in replicate 2 for E3T1.
  • Therefore, this Example shows that compositions comprising Cas12i2 and LDHA-targeting RNA guides comprise different off-target activity profiles.
    • Example 7—LDHA Protein Knockdown with Cas12i2 and LDHA-Targeting RNA Guides
  • This Example describes use of a Western Blot to identify knockdown of LDHA protein using variant Cas12i2 of SEQ ID NO: 1168 and LDHA-targeting RNA guides.
  • Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37° C. water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermo Fisher, CM7000) and centrifuged at 100 g for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams' Medium E, Thermo Fisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermo Fisher CM3000). The cells were subjected to trypan blue viability count with an Inucyte disposable hemocytometer (Fisher scientific, 22-600-100). The cells were then washed in PBS and resuspended in P3 buffer+supplement (Lonza, VXP-3032) at a concentration of ˜5000 cells/μL. Resuspended cells were dispensed at 500,000 cells/reaction into Lonza electroporation cuvettes
  • For the RNP reactions, E3T1 (SEQ ID NO: 1214), E5T9 (SEQ ID NO: 1224), E5T1 (SEQ ID NO: 1221), and E5T10 (SEQ ID NO: 1225) were used as the LDHA-targeting RNA guides. RNPs were added to each reaction at a final concentration of 20 μM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 Unelectroporated cells and cells electroporated without cargo were used as negative controls.
  • The strips were electroporated using an electroporation device (program CA137, Lonza 4D-nucleofector). Immediately following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 500,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 24-well plate (Thermo Fisher) with wells containing Hepatocyte plating medium. The cells were then incubated at 37° C. The media was changed to hepatocyte maintenance media (Williams' Medium E, Thermo Fisher A1217601 supplemented with William's E medium Cell Maintenance Cocktail, Thermo Fisher CM 4000) after the cells attached after 24 hours. Fresh hepatocyte maintenance media was replaced every 48 hours.
  • 7 days post RNP electroporation, the media was aspirated, and the cells were washed gently with PBS. Cells were then lysed with RIPA Lysis and Extraction buffer (Thermo Fisher 89901)+1× protease inhibitors (Thermo Fisher 78440) for 30 minutes on ice, mixing the samples every 5 minutes. Cell lysate was quantified via Pierce BCA Protein Assay Kit (Thermo Fisher 23227). 15 μg of total protein per sample was prepared for SDS-PAGE in 1× Laemmlli Sample buffer (BioRad 1610747) and 100 mM DTT, then heated at 95° C. for 10 minutes. Samples were run on a 4-15% TGX gel (BioRad 5671084) at 200V for 45 minutes. Samples were transferred to a 0.2 um nitrocellulose membrane (BioRad 1704159) using the Trans Blot Turbo System. The membrane was blocked in Intercept TBS Blocking Buffer (Li-cor 927-60001) for 30 minutes at room temperature. The blot was then incubated in a 1:1000 dilution of primary anti-LDHA antibody (Abcam ab52488) and 1:2500 dilution of primary anti-vinculin antibody (Sigma V9131) in blocking buffer at 4 C overnight. The blot was washed three times with TBST (Thermo Fisher 28360) for 5 minutes each, then incubated with a 1:12500 dilution of IR680 anti-mouse (Thermo Fisher PI35518) and IR800 anti-rabbit secondary antibodies (Thermo Fisher PISA535571) in TBST for 1 hour at room temperature. The blot was then washed three times with TBST for 5 minutes each and visualized on the Li-cor Odyssey CLX.
  • Knockdown of LDHA protein (monomer and dimer) was observed in primary human hepatocytes at Day 7 post editing by Cas12i2 RNPs targeting the LDHA gene (FIG. 8 ). This knockdown was seen across each of the four RNA guides, E3T1, E5T9, EST1, and E5T10 (lanes 1-8). LDHA knockdown was not observed for the buffer only (lanes 9 and 10) or unelectroporated controls (lanes 11 and 12).
  • This Example thus shows that LDHA protein levels were decreased following editing with Cas12i2 and LDHA-targeting RNA guides.
    • Other Embodiments
  • All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
    • EQUIVALENTS
  • While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
  • All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
  • All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
  • The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
  • The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
  • As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims (33)

1. A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising
(i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide, wherein the Cas12i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1166 and comprises one or more mutations relative to SEQ ID NO: 1166;
(ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
2. The gene editing system of claim 1, wherein the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, I926, V1030, E1035, and/or S1046 of SEQ ID NO: 1166.
3. The gene editing system of claim 2, wherein the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
4. The gene editing system of claim 3, wherein the Cas12i2 polypeptide comprises:
(i) mutations at positions D581, D911, I926, and V1030, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G;
(ii) mutations at positions D581, I926, and V1030, which optionally are amino acid substitutions of D581R, I926R, and V1030G;
(iii) mutations at positions D581, I926, V1030, and S1046, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G;
(iv) mutations at positions D581, G624, F626, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or
(v) mutations at positions D581, G624, F626, P868, I926, V1030, E1035, and S1046, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G.
5. The gene editing system of claim 1, wherein the Cas12i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 1167, 1168, 1169, 1170, or 1171.
6. The gene editing system of claim 1, which comprises the first nucleic acid encoding the Cas12i2 polypeptide.
7. The gene editing system of claim 6, wherein the first nucleic acid is a messenger RNA (mRNA), and/or is included in a viral vector.
8. (canceled)
9. The gene editing system of claim 1, wherein the target sequence is within exon 3 or exon 5 of the LDHA gene, and/or comprises:
(i) (SEQ ID NO: 1237) 5′-TAGGACTTGGCAGATGAACT-3′; (ii) (SEQ ID NO: 1239) 5′-GATGACATCAACAAGAGCAA-3; (iii) (SEQ ID NO: 1245) 5′-TTCATAGTGGATATCTTGAC-3′; (iv) (SEQ ID NO: 1248) 5′-TCATAGTGGATATCTTGACC-3′; or (v) (SEQ ID NO: 1249) 5′-CATAGTGGATATCTTGACCT-3′.
10. (canceled)
11. The gene editing system of claim 9, wherein the spacer sequence comprises:
(i) (SEQ ID NO: 1269) 5′-UAGGACUUGGCAGAUGAACU-3′; (ii) (SEQ ID NO: 1270) 5′-GAUGACAUCAACAAGAGCAA-3′; (iii) (SEQ ID NO: 1271) 5′-UUCAUAGUGGAUAUCUUGAC-3′; (iv) (SEQ ID NO: 1272) 5′-UCAUAGUGGAUAUCUUGACC-3′; or (v)  (SEQ ID NO: 1273) 5′-CAUAGUGGAUAUCUUGACCU-3.
12. (canceled)
13. The gene editing system of claim 1, wherein the RNA guide comprises the spacer sequence and a direct repeat sequence, which is at least 90% identical to any one of SEQ ID NOs: 1-10 or a fragment thereof that is at least 23-nucleotide in length.
14-16. (canceled)
17. The gene editing system of claim 13, wherein the direct repeat sequence is 5′-AGAAAUCCGUCUUUCAUUGACGG-3′ (SEQ ID NO: 10).
18. The gene editing system of claim 1, wherein the RNA guide comprises the nucleotide sequence of:
(i) (SEQ ID NO: 1214) 5′-AGAAAUCCGUCUUUCAUUGACGGUAGGACUUGGCAGAUGAACU-3′; (ii) (SEQ ID NO: 1235) 5′-AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3′; (iii) (SEQ ID NO: 1221) 5′-AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3′; (iv) (SEQ ID NO: 1224) 5′-AGAAAUCCGUCUUUCAUUGACGGUCAUAGUGGAUAUCUUGACC-3′; or (v) (SEQ ID NO: 1225) 5′-AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-3′.
19. The gene editing system of claim 1, wherein the system comprises the second nucleic acid encoding the RNA guide, or wherein the nucleic acid encoding the RNA guide is located in a viral vector.
20. (canceled)
21. The gene editing system of claim 7, wherein the viral vector comprises both the first nucleic acid encoding the Cas12i2 polypeptide and the second nucleic acid encoding the RNA guide.
22. The gene editing system of claim 21, wherein the system comprises the first nucleic acid encoding the Cas12i2 polypeptide, which is located on a first vector, and wherein the system comprises the second nucleic acid encoding the RNA guide, which is located on a second vector; or
wherein the system comprises one or more lipid nanoparticles (LNPs), which encompass (i), (ii), or both.
23-24. (canceled)
25. The gene editing system of claim 22, wherein the system comprises the LNP, which encompass (i), and wherein the system comprises a viral vector comprising the second nucleic acid encoding the RNA guide; or
wherein the system comprises the LNP, which encompass (ii), and wherein the system comprises a viral vector comprising the first nucleic acid encoding Cas12i2 polypeptide.
26. The gene editing system of claim 25, wherein the viral vector is an AAV vector.
27. A gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene, comprising
(i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, optionally wherein the Cas12i polypeptide is a Cas12i2 polypeptide; and
(ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within exon 3 or exon 5 of an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
28-46. (canceled)
47. A pharmaceutical composition comprising the gene editing system set forth in claim 1.
48. A kit comprising the elements (i) and (ii) of the gene editing system set forth in claim 1.
49. A method for editing a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with the gene editing system for editing the LDHA gene set forth in claim 1 to genetically edit the LDHA gene in the host cell.
50-52. (canceled)
53. A method for treating primary hyperoxaluria (PH) in a subject, comprising administering to a subject in need thereof a gene editing system for editing a lactate dehydrogenase A (LDHA) gene set forth in claim 1.
54-55. (canceled)
56. An RNA guide, comprising (i) a spacer sequence that is specific to a target sequence in a lactate dehydrogenase A (LDHA) gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence; and (ii) a direct repeat sequence.
57-65. (canceled)
US17/832,114 2021-06-04 2022-06-03 Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof Pending US20230034581A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/832,114 US20230034581A1 (en) 2021-06-04 2022-06-03 Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof
US18/052,791 US11939607B2 (en) 2021-06-04 2022-11-04 Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US202163197067P 2021-06-04 2021-06-04
US202163225214P 2021-07-23 2021-07-23
US202163292912P 2021-12-22 2021-12-22
US202263300743P 2022-01-19 2022-01-19
US17/832,114 US20230034581A1 (en) 2021-06-04 2022-06-03 Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/052,791 Continuation US11939607B2 (en) 2021-06-04 2022-11-04 Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof

Publications (1)

Publication Number Publication Date
US20230034581A1 true US20230034581A1 (en) 2023-02-02

Family

ID=82492872

Family Applications (2)

Application Number Title Priority Date Filing Date
US17/832,114 Pending US20230034581A1 (en) 2021-06-04 2022-06-03 Gene editing systems comprising an rna guide targeting lactate dehydrogenase a (ldha) and uses thereof
US18/052,791 Active US11939607B2 (en) 2021-06-04 2022-11-04 Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
US18/052,791 Active US11939607B2 (en) 2021-06-04 2022-11-04 Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof

Country Status (3)

Country Link
US (2) US20230034581A1 (en)
TW (1) TW202313968A (en)
WO (1) WO2022256655A2 (en)

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2015330670B2 (en) * 2014-10-10 2022-01-06 Novo Nordisk Health Care Ag Therapeutic inhibition of lactate dehydrogenase and agents therefor
JP7256739B2 (en) 2016-09-07 2023-04-12 サンガモ セラピューティクス， インコーポレイテッド Modulation of liver genes
AU2019236210A1 (en) 2018-03-14 2020-09-10 Arbor Biotechnologies, Inc. Novel CRISPR DNA targeting enzymes and systems
MX2021001070A (en) 2018-07-31 2021-05-27 Intellia Therapeutics Inc COMPOSITIONS AND METHODS FOR HYDROXYACID OXIDASE 1 ( <i>HAO1</i>) GENE EDITING FOR TREATING PRIMARY HYPEROXALURIA TYPE 1 (PH1).
SG11202102660RA (en) * 2018-09-28 2021-04-29 Intellia Therapeutics Inc Compositions and methods for lactate dehydrogenase (ldha) gene editing
EP3898661A1 (en) 2018-12-21 2021-10-27 Precision BioSciences, Inc. Genetic modification of the hydroxyacid oxidase 1 gene for treatment of primary hyperoxaluria
JP2023520504A (en) 2020-03-31 2023-05-17 アーバー バイオテクノロジーズ， インコーポレイテッド Compositions Comprising Cas12i2 Mutant Polypeptides and Uses Thereof
WO2021257730A2 (en) * 2020-06-16 2021-12-23 Arbor Biotechnologies, Inc. Cells modified by a cas12i polypeptide
CN112195164B (en) * 2020-12-07 2021-04-23 中国科学院动物研究所 Engineered Cas effector proteins and methods of use thereof

Also Published As

Publication number Publication date
TW202313968A (en) 2023-04-01
WO2022256655A2 (en) 2022-12-08
US11939607B2 (en) 2024-03-26
US20230212540A1 (en) 2023-07-06
WO2022256655A3 (en) 2023-01-12

Similar Documents

Publication Publication Date Title
US20220396782A1 (en) Gene editing systems comprising an rna guide targeting transthyretin (ttr) and uses thereof
US20230203539A1 (en) Gene editing systems comprising an rna guide targeting stathmin 2 (stmn2) and uses thereof
US20230407343A1 (en) Compositions comprising an rna guide targeting pdcd1 and uses thereof
US11939607B2 (en) Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof
US11821012B2 (en) Gene editing systems comprising an RNA guide targeting hydroxyacid oxidase 1 (HAO1) and uses thereof
WO2023122433A1 (en) Gene editing systems targeting hydroxyacid oxidase 1 (hao1) and lactate dehydrogenase a (ldha)
US20240026351A1 (en) Compositions comprising an rna guide targeting trac and uses thereof
US20230416732A1 (en) Compositions comprising an rna guide targeting bcl11a and uses thereof
CN117897485A (en) RNA-guided gene editing system comprising a target hydroxy acid oxidase 1 (HAO 1) and uses thereof
US20230399639A1 (en) Compositions comprising an rna guide targeting b2m and uses thereof
US20230193243A1 (en) Compositions comprising a cas12i2 polypeptide and uses thereof
WO2023018856A1 (en) Gene editing systems comprising an rna guide targeting polypyrimidine tract binding protein 1 (ptbp1) and uses thereof
WO2023081377A2 (en) Compositions comprising an rna guide targeting ciita and uses thereof
WO2023137451A1 (en) Compositions comprising an rna guide targeting cd38 and uses thereof
CN117813382A (en) Gene editing system including RNA guide targeting STATHMIN 2 (STMN 2) and uses thereof
KR20240052763A (en) Gene editing system comprising an RNA guide targeting Stasmin 2 (STMN2) and uses thereof

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: ARBOR BIOTECHNOLOGIES, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WESSELLS, QUINTON NORMAN;HASWELL, JEFFREY RAYMOND;DITOMMASO, TIA MARIE;AND OTHERS;SIGNING DATES FROM 20221003 TO 20221006;REEL/FRAME:061714/0206

AS Assignment

Owner name: ARBOR BIOTECHNOLOGIES, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WESSELLS, QUINTON NORMAN;HASWELL, JEFFREY RAYMOND;DITOMMASO, TIA MARIE;AND OTHERS;SIGNING DATES FROM 20221003 TO 20221006;REEL/FRAME:062534/0235