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WO2023122433A1 - Gene editing systems targeting hydroxyacid oxidase 1 (hao1) and lactate dehydrogenase a (ldha) - Google Patents

Gene editing systems targeting hydroxyacid oxidase 1 (hao1) and lactate dehydrogenase a (ldha) Download PDF

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Publication number
WO2023122433A1
WO2023122433A1 PCT/US2022/081082 US2022081082W WO2023122433A1 WO 2023122433 A1 WO2023122433 A1 WO 2023122433A1 US 2022081082 W US2022081082 W US 2022081082W WO 2023122433 A1 WO2023122433 A1 WO 2023122433A1
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seq
sequence
nucleotide
gene
editing system
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Inventor
Quinton Norman WESSELLS
Jeffrey Raymond HASWELL
Tia Marie DITOMMASO
Noah Michael Jakimo
Sejuti SENGUPTA
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Arbor Biotechnologies Inc
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Arbor Biotechnologies Inc
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • 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
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    • 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)
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    • 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)
    • 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)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

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 gene editing system for genetic editing of both a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase (LDHA) gene.
  • the system involves a Casl2i CRISPR nuclease polypeptide (e.g. , a Casl2i2 polypeptide), an RNA guide mediating cleavage at a genetic site within the HAO1 gene by the Casl2i polypeptide, and an RNA guide mediating cleavage at a genetic site within the LDHA gene by the Casl2i polypeptide.
  • the gene editing system disclosed herein has achieved successful editing of HAO1 and LDHA genes with high editing efficiency and accuracy.
  • the gene editing system disclosed herein may further exhibit one or more of the following advantageous features.
  • Casl2i 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.
  • Casl2i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage.
  • Casl2i PAM sequences also differ from those of Cas9. Therefore, larger and different portions of genetic sites of interest can be disrupted with a Casl2i polypeptide and RNA guide compared to Cas9.
  • Casl2i such as Casl2i2 may be more specific than Cas9.
  • gene editing systems for editing HA01 and LDHA genes are provided herein, 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
  • a gene editing system for genetic editing of a hydroxy acid oxidase 1 (HA01) gene and a lactate dehydrogenase A (LDHA) gene comprising: (i) a first RNA guide or a first nucleic acid encoding the RNA guide, wherein the first RNA guide comprises a first spacer sequence specific to a first target sequence within an HA01 gene, the first 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; (ii) a second RNA guide or a second nucleic acid encoding the RNA guide, wherein the second RNA guide comprises a second spacer sequence specific to a second target sequence within an LDHA gene, the second 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; and (iii) a Casl2
  • the Casl2i polypeptide is a Casl2i2 polypeptide, which 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.
  • the one or more mutations in the Casl2i2 polypeptide are at positions D581, G624, F626, P868, 1926, 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 Casl2i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, 1926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, 1926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).
  • Exemplary Casl2i2 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 Casl2i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1168.
  • the exemplary Casl2i2 polypeptide for use in any of the gene editing systems disclosed herein comprises the amino acid sequence of SEQ ID NO: 1171.
  • the first target sequence within the HA01 gene is within exon 1 or exon 2 of the HAO1 gene.
  • the first target sequence may comprise:
  • the first target sequence may comprise 5’- CGGAGCATCCTTGGATACAG-3’ (SEQ ID NO: 2234). In some specific examples, the first target sequence may comprise 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212).
  • the second target sequence in the LDHA gene may be within exon 3 or exon 5 of the LDHA gene.
  • the second target sequence may comprise:
  • the second target sequence may comprise: 5’- TTCATAGTGGATATCTTGAC-3’ (SEQ ID NO: 2278). In specific examples, the second target sequence may comprise: 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281).
  • the first spacer sequence specific to the HAO1 gene and/or the second spacer sequence specific to the LDHA gene is 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length.
  • the first spacer sequence targeting the HAO1 gene may comprise the nucleotide sequence of:
  • the first spacer sequence may comprise 5’- CGGAGCAUCCUUGGAUACAG-3’ (SEQ ID NO: 2314). In specific examples, the first spacer sequence may comprise 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2311).
  • the second spacer sequence specific to the LDHA gene may comprise the nucleotide sequence of:
  • the second spacer sequence may comprise 5’- UUCAUAGUGGAUAUCUUGAC-3’ (SEQ ID NO: 2319). In specific examples, the second spacer sequence may comprise 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316).
  • any of the first RNA guides specific to the HAO1 gene may comprise the first spacer sequence as disclosed herein and a first direct repeat sequence.
  • any of the second RNA guides specific to the LDHA gene may comprise the second spacer sequence disclosed herein and a second direct repeat sequence.
  • the first direct repeat sequence and/or the second direct repeat sequence is 23-36-nucleotide in length.
  • the first direct repeat sequence and/or the second 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 first direct repeat sequence and/or the second 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 first direct repeat sequence and/or the second direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 10).
  • Exemplary first RNA guides specific to the HAO1 gene includes:
  • the first RNA guide is (a-i). In another specific example, the first RNA guide is (a-iv).
  • Exemplary second RNA guides specific to the LDHA gene includes:
  • the first RNA guide is (b-i). In another specific example, the second RNA guide is (b-iv).
  • the gene editing system may comprise the third nucleic acid encoding the Casl2i polypeptide (e.g., the Casl2i2 polypeptide as disclosed herein).
  • the third nucleic acid is located in a third vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector).
  • the third nucleic acid is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the nucleic acid encoding the Casl2i polypeptide is codon-optimized.
  • any of the gene editing systems disclosed herein may comprise the first nucleic acid encoding the first RNA guide.
  • the gene editing system may comprise the second nucleic acid encoding the second RNA guide.
  • the first nucleic acid encoding the first RNA guide is located in a first vector; and/or the second nucleic acid encoding the second RNA guide is located in a second vector.
  • the first vector and the second vector are the same vector.
  • the gene editing system comprises one or more vectors, which collectively comprise the first nucleic acid encoding the first RNA guide in a first vector, the second nucleic acid encoding the second RNA guide in a second vector, and/or the third nucleic acid encoding the Casl2i polypeptide such as the Casl2i2 polypeptide.
  • the first, second and/or third vector is a viral vector, for example, an AAV vector.
  • the gene editing system comprises one or more lipid nanoparticles (LNPs), which are associated (e.g., encompass) (i), (ii), (iii), or a combination thereof.
  • the system comprises a lipid nanoparticle, which encompass one or two of (i)-(iii), and a viral vector, which comprising the nucleic acid(s) encoding the remaining.
  • the viral vector is an AAV vector.
  • the LNPs are associated with (i) the first RNA guide, (ii) the second RNA guide, and (iii) an mRNA molecule encoding the Casl2i2 polypeptide. In some examples, at least a portion of (i), (ii), and/or (iii) is encapsulated by the LNPs.
  • the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein, or a kit comprising the components of the gene editing system.
  • the present disclosure also features a method for editing a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with any of the gene editing systems disclosed herein to genetically edit the HAO1 gene and 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 HA01 gene and the LDHA gene to a subject comprising the host cell.
  • a cell comprising a disrupted an HA01 gene and an LDHA gene, which can be produced by contacting a host cell with the system disclosed herein genetically edit the HA01 gene and the LDHA gene in the host cell.
  • 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 an HA01 gene and an LDHA gene or any of the modified cells disclosed herein.
  • the subject may be a human patient having the PH.
  • the PH is PHI, PH2, or PH3.
  • the PH is PHI.
  • 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 % of NGS reads comprising indels in HEK293T cells following delivery of a variant Casl2i2 and HAO 1 -targeting RNA guide RNP.
  • the darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaco mulatto) and crab-eating macaque (Macaco fascicularis) sequences.
  • FIG. 2 is a graph showing % of NGS reads comprising indels in HEK293T cells following delivery of a variant Casl2i2 and LDHA-targeting RNA guide RNP.
  • the darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaco, mulato) and crab-eating macaque (Macaco fascicularis) sequences.
  • FIG. 3 is a graph showing HAO1 indels in HepG2 cells following RNP delivery.
  • FIG. 4 is a graph showing LDHA indels in HepG2 cells following RNP delivery.
  • FIG. 5 is a graph showing HAO1 and LDHA indels in primary hepatocytes.
  • FIG. 6 is a graph showing dual RNA guide editing with HAO 1 -targeting RNA guides and LDHA-targeting RNA guides in primary hepatocytes.
  • FIG. 7 is a graph showing knockdown of HAO1 mRNA in primary human hepatocytes with a variant Casl2i2 polypeptide and an HAO 1 -targeting crRNA or a variant Casl2i2 polypeptide, an HAO 1 -targeting crRNA, and an LDHA-targeting crRNA.
  • FIG. 8 is a graph showing knockdown of LDHA mRNA in primary human hepatocytes with a variant Casl2i2 polypeptide and an LDHA-targeting crRNA or a variant Casl2i2 polypeptide, an LDHA-targeting crRNA, and an HAO 1 -targeting crRNA.
  • FIG. 9 A is a graph showing % of NGS reads comprising indels following delivery of HAO 1 -targeting crRNAs or LDHA-targeting crRNAs and the variant Casl2i2 polypeptide of SEQ ID NO: 1168 or SEQ ID NO: 1171.
  • FIG. 9B shows the size (left) and start position (right) of indels induced in HepG2 cells by a variant Casl2i2 and HAO 1 -targeting RNA guide E1T3.
  • FIG. 9C shows the size (left) and start position (right) of indels induced in HepG2 cells by a variant Casl2i2 and LDHA-targeting RNA guide E5T9.
  • FIG. 10 is a graph showing % of NGS reads comprising indels induced by chemically modified HAO 1 -targeting crRNAs or LDHA-targeting crRNAs and variant Casl2i2 mRNA.
  • FIGS. 11A-11B show plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for HAO 1 -targeting RNA guides.
  • 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. 11A shows plots for HAO 1 -targeting RNA guides E2T5, E1T2, E1T3, and E2T10 and the variant Casl2i2 of SEQ ID NO: 1168.
  • FIG. 11B shows plots for HAO 1 -targeting RNA guides E2T5, E1T2, and E1T3 and the variant Casl2i2 of SEQ ID NO: 1171.
  • FIGS. 12A-12B show plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for LDHA-targeting RNA guides.
  • 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. 12A shows plots for LDHA-targeting RNA guides E5T9, E3T1, E5T10, and E5T1 and the variant Casl2i2 of SEQ ID NO: 1168.
  • FIG. 12B shows plots for LDHA-targeting RNA guides E5T9, E5T10, and E1T3 and the variant Casl2i2 of SEQ ID NO: 1171.
  • FIGs. 13A-13B include diagrams showing knockdown of HAO 1 and LDHA genes as examined by protein expression via Western Blot.
  • FIG. 13A is a Western Blot showing knockdown of HAO1 protein following electroporation of primary human hepatocytes variant Casl2i2 and HAO 1 -targeting RNA guide E2T5.
  • FIG. 13B is a Western Blot showing knockdown of LDHA protein following electroporation of primary human hepatocytes with variant Casl2i2 and LDHA-targeting RNA guides E3T1, E5T9, E5T1, or E5T10.
  • the present disclosure relates to a gene editing system for genetic editing of a hydroxy acid oxidase 1 (HAO1) gene (a.k.a., glycolate oxidase gene) and a lactate dehydrate A (LDHA) gene.
  • the gene editing system may comprise: (i) an RNA guide specific to the HAO1 gene or a first nucleic acid encoding the RNA guide, (ii) an RNA guide specific to the LDHA gene or a second nucleic acid encoding the RNA guide, and (iii) a Casl2i polypeptide or a first nucleic acid encoding the Casl2i polypeptide.
  • the RNA guide specific to the HAO1 gene may comprise a first spacer sequence specific to a first target sequence within an HAO1 gene, the first target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-TTN-3’, which is located 5’ to the first target sequence.
  • the RNA guide specific to the LDHA gene may comprise a second spacer sequence specific to a second target sequence within an LDHA gene, the second target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-TTN-3’, which is located 5’ to the second target sequence.
  • the Casl2i polypeptide for use in the gene editing system disclosed herein may be a Casl2i2 polypeptide, e.g., a wild- type Casl2i polypeptide or a variant thereof as those disclosed herein.
  • the Casl2i2 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.
  • the Casl2i polypeptide may be a Casl2i4 polypeptide, which is also disclosed herein.
  • compositions or kits comprising any of the gene editing systems disclosed herein, as well as uses thereof.
  • a method for editing a HA01 gene and an LDHA gene in a cell a cell so produced that comprises a disrupted the HAO1 and LDHA genes, a method of treating primary hyperoxaluria (PH) in a subject.
  • PH primary hyperoxaluria
  • activity refers to a biological activity.
  • activity includes enzymatic activity, e.g., catalytic ability of a Casl2i 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 interconversion 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.
  • HAO1 refers to “glycolate oxidase 1,” which is also known as “hydroxyacid oxidase.” HAO1 is a peroxisome protein expressed primarily in the liver and pancreas, and its activities include oxidation of glycolate and 2-hydroxy fatty acids. SEQ ID NO: 2123 as set forth herein provides an example of an HAO1 gene sequence.
  • 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 Casl2i polypeptide).
  • the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence.
  • the term “complex” can refer to a grouping of an LDHA-targeting RNA guide and a Casl2i polypeptide or a grouping of an HAO 1 -targeting RNA guide and a Casl2i polypeptide.
  • the term “protospacer adjacent motif’ or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., an LDHA target sequence or an HAO1 target sequence) to which a complex comprising an RNA guide (e.g. , an LDHA-targeting RNA guide or an HAO 1 -targeting RNA guide) and a Casl2i polypeptide binds.
  • a target sequence e.g., an LDHA-targeting RNA guide or an HAO 1 -targeting RNA guide
  • a Casl2i 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 Casl2i polypeptide (e.g. , a Casl2i2 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.
  • the target sequence is a sequence within an HAO1 gene sequence, including, but not limited to, the sequence set forth in SEQ ID NO: 2123.
  • 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.
  • 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 sequence 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. In some instances, 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 Casl2i 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 Casl2i-induced indel is downstream of the 5’-NTTN-3’ or 5’- TTN-3’ sequence.
  • the present disclosure described herein comprises gene editing systems or compositions comprising RNA guides targeting an LDHA gene and an HAO1 gene.
  • Such gene editing systems or compositions can be used to edit both HAO1 and LDHA genes in cells, for example, to disrupt both genes.
  • HAO1 Hydroxyacid oxidase 1
  • GOX glycolate oxidase
  • 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 Casl2i polypeptide.
  • the spacer component is specific to a target sequence (e.g., an LDHA target sequence or an HAO1 target sequence), wherein the 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 the PAM strand).
  • the present described herein comprises compositions comprising complexes, wherein a first complex comprises an RNA guide targeting HAO1 and a second complex comprises an RNA guide targeting LDHA.
  • the present disclosure provides complexes comprising an RNA guide and a Casl2i polypeptide.
  • the RNA guides and the Casl2i polypeptides bind to each other in molar ratios of about 1:1.
  • a complex comprising an RNA guide and a Casl2i polypeptide binds to an HAO1 target sequence.
  • a complex comprising an RNA guide and a Casl2i polypeptide binds to an LDHA target sequence.
  • a complex comprising an RNA guide targeting LDHA and a Casl2i polypeptide binds to the complementary region of an HAO1 target sequence at a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide targeting LDHA and a Casl2i polypeptide binds to the complementary region of 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 HAO1 target sequence and/or the complementary sequence thereof.
  • the complex comprises enzymatic activity, such as nuclease activity, that can cleave the LDHA target sequence and/or the complementary sequence thereof.
  • enzymatic activity such as nuclease activity
  • the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein.
  • the sequence of the RNA guide targeting HAO1 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: 2131-2187.
  • the RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187.
  • the sequence of the RNA guide targeting LDHA 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: 2188-2204. In some embodiments, the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
  • the present disclosure provides gene editing systems and compositions comprising one or more RNA guides as described herein and/or an RNA encoding a Casl2i polypeptide as described herein.
  • an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide are comprised together within the same composition, while an RNA guide targeting LDHA is comprised within a different composition.
  • an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide are comprised within the same composition, while an RNA guide targeting HAO1 is comprised within a different composition.
  • an RNA guide targeting HAO1, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide are comprised within the same composition. In some embodiments, an RNA guide targeting HAO1 and an RNA guide targeting LDHA are comprised within the same composition, while an RNA encoding a Casl2i polypeptide is comprised within a different composition. In some embodiments, an RNA guide targeting HAO1, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide are each comprised within separate compositions.
  • an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide are comprised together within the same composition, while an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide are comprised within a different composition.
  • an 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: 2131-2204.
  • the RNA guide has a sequence of any one of SEQ ID NOs: 2131-2204.
  • Casl2i polypeptides are smaller than other nucleases.
  • Casl2i2 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
  • FnCpfl is 1,300 amino acids in length
  • AsCpfl is 1,307 amino acids in length
  • LbCpfl is 1,246 amino acids in length.
  • Casl2i RNA guides which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides.
  • compositions comprising a Casl2i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See PCT/US2021/025257, which is incorporated by reference in its entirety.
  • indels induced by compositions comprising a Casl2i polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide.
  • SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length.
  • Casl2i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of genes such as HAO1 and LDHA.
  • the gene editing system described herein comprises an RNA guide targeting HAO1 and an RNA guide targeting LDHA.
  • 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 HAO1 and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting LDHA.
  • the HAO 1 -targeting RNA guide may direct the Casl2i polypeptide as described herein to an HAO1 target sequence.
  • Two or more RNA guides may target two or more separate Casl2i polypeptides (e.g., Casl2i polypeptides having the same or different sequence) as described herein to two or more (e.g.
  • the LDHA-targeting RNA guide may direct the Casl2i polypeptide as described herein to an LDHA target sequence.
  • Two or more RNA guides may target two or more separate Casl2i polypeptides (e.g., Casl2i 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 or is HAO1 target- specific. That is, in some embodiments, an RNA guide binds specifically to one or more LDHA target sequences or one or more HAO1 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. 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 Casl2i2 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 or an HA01 target sequence.
  • Example 1 where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 polypeptide of SEQ ID NO: 1168 to HEK293T cells by RNP;
  • Example 2 where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 polypeptide of SEQ ID NO: 1168 to HepG2 cells by RNP;
  • Example 3 where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 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: 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,
  • 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, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192,
  • 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,
  • 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,
  • 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,
  • 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,
  • 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,
  • 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,
  • 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,
  • 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,
  • 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. Table 2. Casl2i4 Direct Repeat Sequences
  • 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. (ii) Spacer Sequences
  • 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 a specific target 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 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 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 spacer sequence is a sequence of Table 5 or a portion of a sequence of Table 5.
  • 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,
  • 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 spacer sequence is a sequence of Table 6 or a portion of a sequence of Table 6. It should be understood that an indication of SEQ ID NOs: 1668-2122 should be considered as equivalent to a listing of SEQ ID NOs: 1668-2122, with each of the intervening numbers present in the listing, consistent with the listing of SEQ ID NOs: 588- 1164, above.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 1668- 2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 1668-2122.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 1668-2122.
  • 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 6 or a portion of a sequence of Table 6.
  • 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: 1668- 2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • 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: 1668-2122.
  • the spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of 1668-2122.
  • 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 an uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 or Table 6 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5 or Table 6. (iii) Exemplary RNA Guides
  • RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g. , as set forth in Tables 5 and 6, above).
  • the sequence of the RNA guide targeting HAO1 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: 2131-2187.
  • the RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187.
  • the sequence of the RNA guide targeting LDHA 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: 2188-2204. In some embodiments, the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
  • RNA guide targeting HAO1 may be E1T2 disclosed in Table 8 below (e.g., the modified version provided in Table 8) and the RNA guide targeting LDHA may be E5T9 also provided in Table 8 (e.g., the modified version provided in Table 8).
  • 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 the present disclosure.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the intemucleoside 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
  • 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.
  • 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.
  • 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).
  • phosphate backbone e.g., phosphate backbone
  • 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 intemucleoside 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).
  • a-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-( l-thiophosphate)-adenosine, 5'-O-( l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(l-thiophosphate)-guanosine, 5'-O-( l-thiophosphate)-uridine, or 5’-O-(l- thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5 , -O-( l-thiophosphate)-adenosine, 5'-O-( l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(l-thiophosphate)-guanosine, 5'-O-( l-thiophosphate)-uridine, or 5’-O
  • 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, l-(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-fhioro-l-(tetrahydrofuran-2-yl)pyrimidine- 2,4(lH,3H)-dione), t
  • Additional examples include fludarabine phosphate, N4-behenoyl-l- beta-D-arabinofuranosylcytosine, N4-octadecyl-l-beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(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 -carboxymethylpseudouridine, 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,
  • 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-l -methyl-pseudoisocytidine, 4-thio-l - methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-
  • 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 carbam
  • 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, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • nucleoside selected from
  • 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 AD ARI 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
  • an HAO 1 -targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 2287-2292. In some embodiments, an HAO 1 -targeting RNA guide comprises any one of SEQ ID NOs: 2287-2292. In some embodiments, an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2288 or SEQ ID NO: 2289 recognizes the HAO1 target sequence of SEQ ID NO: 2234. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2288 or SEQ ID NO: 2289 recognizes the HAO1 target sequence of SEQ ID NO: 2234.
  • an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2290 or SEQ ID NO: 2291 recognizes the HAO1 target sequence of SEQ ID NO: 2213. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2290 or SEQ ID NO: 2291 recognizes the HAO1 target sequence of SEQ ID NO: 2213. In some embodiments, an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2292 or SEQ ID NO: 2293 recognizes the HAO1 target sequence of SEQ ID NO: 2212. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2292 or SEQ ID NO: 2293 recognizes the HAO1 target sequence of SEQ ID NO: 2212.
  • an LDHA-targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 2293-2302. In some embodiments, an LDHA-targeting RNA guide comprises any one of SEQ ID NOs: 2293-2302. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2293 or SEQ ID NO: 2294 recognizes the LDHA target sequence of SEQ ID NO: 2270. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2293 or SEQ ID NO: 2294 recognizes the LDHA target sequence of SEQ ID NO: 2270.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2295 or SEQ ID NO: 2296 recognizes the LDHA target sequence of SEQ ID NO: 2272. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2295 or SEQ ID NO: 2296 recognizes the LDHA target sequence of SEQ ID NO: 2272. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2297 or SEQ ID NO: 2298 recognizes the LDHA target sequence of SEQ ID NO: 2281.
  • the LDHA-targeting RNA guide of SEQ ID NO: 2297 or SEQ ID NO: 2298 recognizes the LDHA target sequence of SEQ ID NO: 2281.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2299 or SEQ ID NO: 2300 recognizes the LDHA target sequence of SEQ ID NO: 2278.
  • the LDHA-targeting RNA guide of SEQ ID NO: 2299 or SEQ ID NO: 2300 recognizes the LDHA target sequence of SEQ ID NO: 2278.
  • an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2301 or SEQ ID NO: 2302 recognizes the LDHA target sequence of SEQ ID NO: 2282. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2301 or SEQ ID NO: 2302 recognizes the LDHA target sequence of SEQ ID NO: 2282.
  • nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.
  • composition of the present disclosure includes a Casl2i 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 Casl2i2 polypeptide described herein (e.g. , a polypeptide comprising SEQ ID NO: 1166 and/or encoded by SEQ ID NO: 1165).
  • the Casl2i2 polypeptide comprises at least one RuvC domain.
  • a nucleic acid sequence encoding the Casl2i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165.
  • the Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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.
  • Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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.
  • Casl2i2 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 Casl2i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1202 and/or encoded by SEQ ID NO: 1201).
  • the Casl2i4 polypeptide comprises at least one RuvC domain.
  • a nucleic acid sequence encoding the Casl2i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201.
  • the Casl2i4 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.
  • nucleic acid sequences 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 Casl2i4 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 Casl2i4 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 Casl2i4 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.
  • Casl2i4 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 Casl2i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.
  • the Casl2i4 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 Casl2i4 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 Casl2i4 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.
  • Casl2i4 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.
  • the composition of the present disclosure includes a Casl2il polypeptide described herein (e.g. , a polypeptide comprising SEQ ID NO: 1211).
  • the Casl2i4 polypeptide comprises at least one RuvC domain.
  • the Casl2il 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 Casl2il 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.
  • a Casl2il 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.
  • the composition of the present disclosure includes a Casl2i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1212).
  • the Casl2i4 polypeptide comprises at least one RuvC domain.
  • the Casl2i3 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 Casl2i3 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.
  • Casl2i3 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 Casl2i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions.
  • the Casl2i 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 Casl2i 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 Casl2i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Casl2i polypeptide comprises at least one (e.g. , two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Casl2i 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 Casl2i polypeptide described herein can be selfinactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Then, 24 (2016): S50, which is incorporated by reference in its entirety.
  • the nucleotide sequence encoding the Casl2i 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 Casl2i polypeptides such as Casl2i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized.
  • Exemplary Casl2i polypeptide sequences and corresponding nucleotide sequences are listed in Table 7.
  • the gene editing system disclosed herein may comprise a Casl2i polypeptide as disclosed herein.
  • the gene editing system may comprise a nucleic acid encoding the Casl2i 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, AAVrhlO, AAV11 and AAV12) encoding the Casl2i polypeptide.
  • the gene editing system may comprise a mRNA molecule encoding the Casl2i 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 guides, methods for production of the Casl2i polypeptide, and methods for complexing the RNA guide and Casl2i 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 may encode 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 Casl2i polypeptide.
  • the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Casl2i 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 Casl2i polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Casl2i polypeptide of the present disclosure, isolating the Casl2i polypeptide, optionally, purifying the Casl2i polypeptide, and complexing the Casl2i polypeptide with an RNA guide.
  • the Casl2i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Casl2i 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 Casl2i 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 Casl2i 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 Casl2i polypeptide. After expression of the Casl2i polypeptide, the host cells can be collected and Casl2i 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 Casl2i 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 Casl2i 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 Casl2i polypeptide.
  • a variety of methods can be used to determine the level of production of a Casl2i 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 Casl2i 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 Casl2i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Casl2i polypeptide to a host cell wherein the polyribonucleotide encodes the Casl2i polypeptide, expressing the Casl2i polypeptide in the cell, and obtaining the Casl2i polypeptide from the cell.
  • the present disclosure further provides methods of in vivo expression of Casl2i polypeptides in a cell, comprising providing a polyribonucleotide encoding the Casl2i polypeptides to a host cell wherein the polyribonucleotide encodes the Casl2i polypeptides and expressing the Casl2i polypeptides in the cell.
  • the polyribonucleotide encoding the Casl2i polypeptides is delivered to the cell with RNA guides and, once expressed in the cell, the Casl2i polypeptides and the RNA guides form complexes.
  • the polyribonucleotide encoding the Casl2i polypeptides and the RNA guides are delivered to the cell within a single composition (i.e., a composition comprising an RNA guide targeting HA01, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide).
  • the polyribonucleotide encoding the Casl2i polypeptides and the RNA guides are comprised within separate compositions (e.g., (i) a composition comprising an RNA guide targeting HAO1 and an RNA guide targeting LDHA, and a separate composition comprising an RNA encoding a Casl2i polypeptide, (ii) a composition comprising an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide, and a separate composition comprising an RNA guide targeting LDHA, (iii) a composition comprising an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide, and a separate composition comprising an RNA guide targeting HAO1, (iv) a composition comprising an RNA guide targeting HAO1, a separate composition comprising an RNA guide targeting LDHA, and a separate composition comprising a Casl2i, and (v) a composition comprising an RNA guide targeting H
  • an RNA guide targeting LDHA or HAO1 is complexed with a Casl2i polypeptide to form a ribonucleoprotein.
  • complexation of the RNA guide and Casl2i polypeptide occurs at a temperature lower than about any one of
  • the RNA guide does not dissociate from the Casl2i polypeptide at about 37°C over an incubation period of at least about any one of lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Jackpot, 2hr, 3hr, 4hr, or more hours.
  • the RNA guide and Casl2i polypeptide are complexed in a complexation buffer.
  • the Casl2i polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide.
  • the Casl2i 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 Casl2i 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 Casl2i polypeptide is introduced into a cell so that the Casl2i 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 HAO1 gene and a target site with the LDHA gene.
  • the methods comprise introducing an HAO 1 -targeting RNA guide, an LDHA-targeting RNA guide, and a Casl2i polypeptide into a cell.
  • the HAO 1 -targeting RNA guide and/or the LDHA-targeting RNA guide, and the Casl2i polypeptide can be introduced as a ribonucleoprotein complex into a cell.
  • the HAO 1 -targeting RNA guide, the LDHA-targeting guide, and/or the Casl2i polypeptide can be introduced on a nucleic acid vector.
  • the Casl2i polypeptide can be introduced as an mRNA.
  • the RNA guides can be introduced directly into the cell.
  • the gene editing system described herein is delivered to a cell/tissue/liver/person to reduce HAO1 and LDHA in the cell/tissue/liver/person.
  • the gene editing system described herein is delivered to a cell/tissue/liver/person to reduce oxalate production in the cell/tissue/liver/person.
  • the composition described herein is delivered to a cell/tissue/liver/person to correct calcium oxalate crystal deposition in the cell/tissue/liver/person.
  • the composition described herein is delivered to a person with primary hyperoxaluria.
  • the gene editing system may comprise RNA guides and a Casl2i2 polypeptide.
  • the RNA guides each comprise a spacer sequence specific to a target sequence in the HAO1 gene, e.g. , specific to a region in exonl or exon 2 of the HAO1 gene, or 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.
  • the LDHA gene is a mammalian gene.
  • the LDHA gene is a human gene.
  • the sequence of the LDHA gene is set forth in SEQ ID NO: 1172 (or is the reverse complement thereof).
  • the target sequence is in an exon of an LDHA gene, such as an exon having a sequence set forth in any one of SEQ ID NO: 1173, SEQ ID NO: 1174, SEQ ID NO: 1175, SEQ ID NO: 1176, SEQ ID NO: 1177, SEQ ID NO: 1178, SEQ ID NO: 1179, SEQ ID NO: 1180, or SEQ ID NO: 1181 (or a reverse complement thereof).
  • the target sequence is in an intron of an LDHA gene (e.g. , an intron of the sequence set forth in SEQ ID NO: 1172 or the reverse complement thereof).
  • the sequence of the LDHA gene is a variant of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof).
  • the target sequence is polymorphic variant of the LDHA sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof) or a non-human form of the LDHA gene.
  • the target sequence is within the sequence of SEQ ID NO: 1172 (or the reverse complement thereof).
  • 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).
  • 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).
  • the LDHA gene sequence is a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof).
  • the LDHA gene sequence is a non-human LDHA sequence.
  • the LDHA gene sequence is a coding sequence set forth in any one of SEQ ID NOs: 2205-2209 (or a reverse complement thereof).
  • the LDHA gene sequence is a homolog of a coding sequence set forth in any one of SEQ ID NOs: 2205-2209 (or a reverse complement thereof).
  • the sequence of the HAO1 gene is set forth in SEQ ID NO: 2123 (or is the reverse complement thereof).
  • the target sequence is in an exon of an HAO1 gene, such as an exon having a sequence set forth in any one of SEQ ID NO: 2124, SEQ ID NO: 2125, SEQ ID NO: 2126, SEQ ID NO: 2127, SEQ ID NO: 2128, SEQ ID NO: 2129, and SEQ ID NO: 2130 (or a reverse complement of any thereof).
  • the target sequence is in an intron of an HAO1 gene (e.g. , an intron of the sequence set forth in SEQ ID NO: 2123 or the reverse complement thereof).
  • the sequence of the HAO1 gene is a variant of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof).
  • the target sequence is polymorphic variant of the HAO1 sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof) or a non-human form of the HAO1 gene.
  • the target sequence is within an HAO1 gene or a locus of an HAO1 gene.
  • the HAO1 gene is a mammalian gene.
  • the HAO1 gene is a human gene.
  • the target sequence is within the sequence of SEQ ID NO: 2123 (or the reverse complement thereof).
  • the target sequence is within an exon of the HAO1 gene set forth in SEQ ID NO: 2123, e.g., within a sequence of SEQ ID NO: 2124, 2125, 2126, 2127, 2128, 2129, or 2130 (or a reverse complement thereof).
  • Target sequences within an exon region of the HAO1 gene of SEQ ID NO: 2123 are set forth in Table 6.
  • the target sequence is within an intron of the HAO1 gene set forth in SEQ ID NO: 2123 (or the reverse complement thereof).
  • the target sequence is within a variant (e.g.
  • the HAO1 gene sequence is a homolog of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof).
  • the HAO1 gene sequence is a non-human HAO1 sequence.
  • the HAO1 gene sequence is a coding sequence set forth in SEQ ID NO: 2210 (or the reverse complement thereof).
  • the HAO1 gene sequence is a homolog of a coding sequence set forth in SEQ ID NO: 2210 (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 Casl2i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Casl2i polypeptide induces one or more DNA doublestranded breaks in the cell. In some embodiments, the Casl2i polypeptide induces one or more DNA single- stranded breaks in the cell. In some embodiments, the Casl2i 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 Casl2i polypeptide and directs the Casl2i 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 alters expression of the HAO1 gene. In some embodiments, the deletion alters function of the HAO1 gene. In some embodiments, the deletion inactivates the HAO1 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 nucleot
  • 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.
  • 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.
  • 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 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 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
  • 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,
  • 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.
  • 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.
  • 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.
  • 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’-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
  • 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
  • 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 nucleot
  • 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 nu
  • 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
  • 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’,
  • 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
  • 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. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10,
  • 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,
  • 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. 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 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.
  • 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,
  • 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. 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.
  • 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,
  • 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,
  • 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’
  • 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.
  • 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
  • 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,
  • 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 ’-ATTIS’, 5’-ATTG-3’, 5’-ATTC-3’, 5
  • 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,
  • 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,
  • 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 and in an HAO1 gene.
  • the methods are carried out using complexes comprising a Casl2i enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein.
  • the sequence of the RNA guide targeting HAO1 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: 2131-2187.
  • an RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187.
  • the sequence of the RNA guide targeting LDHA 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: 2188-2204.
  • an RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
  • the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is a synthetic or purified RNA. In some embodiments, the Casl2i polypeptide is encoded in a plasmid. In some embodiments, the Casl2i 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 Casl2i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Casl2i polypeptide complex to a cell, where a ternary complex is formed.
  • nucleic acids e.g., nucleic acids encoding the Casl2i polypeptide, RNA guide, donor DNA, etc.
  • a pre-formed RNA guide/Casl2i polypeptide complex to a cell, where a ternary complex is formed.
  • an RNA guide and an RNA encoding a Casl2i polypeptide are delivered together in a single composition, e.g. , as described herein.
  • an RNA guide and an RNA encoding a Casl2i polypeptide are delivered in separate compositions, e.g., as described herein.
  • an RNA guide and an RNA encoding a Casl2i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Casl2i polypeptide delivered in separate compositions are delivered using different delivery technologies.
  • Exemplary intracellular delivery methods include, but are not limited to: viruses 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 or virus-like agents include, but are not limited to: viruses 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 polye
  • a lipid nanoparticle comprises an mRNA encoding a Casl2i polypeptide, an RNA guide, or an mRNA encoding a Casl2i polypeptide and an RNA guide.
  • the mRNA encoding the Casl2i 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 gene editing system comprises a nucleic acid such as an mRNA encoding any of the Casl2i polypeptides disclosed herein (e.g., any of the Casl2i2 polypeptides disclosed herein), an RNA guide targeting the HAO1 gene (e.g., those disclosed herein, e.g., in Table 8), and an RNA guide targeting the LDHA gene (e.g., those disclosed herein, e.g., in Table 8) and LNPs associated with one or all of the just noted components.
  • a nucleic acid such as an mRNA encoding any of the Casl2i polypeptides disclosed herein (e.g., any of the Casl2i2 polypeptides disclosed herein), an RNA guide targeting the HAO1 gene (e.g., those disclosed herein, e.g., in Table 8), and an RNA guide targeting the LDHA gene (e.g., those disclosed herein, e.g., in Table 8)
  • 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 cell.
  • an immune cell e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage
  • fibroblast e a
  • 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 and/or disrupted HAO1 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 PHI, 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 PHI, PH2, or PH3.
  • the target disease is PHI.
  • PH Primary hyperoxaluria
  • AGT/AGT1 alanine-glyoxylate aminotransferase
  • PH2 glyoxylate/hydroxypyruvate reductase
  • PH3 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 PHI include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PHI 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 PHI normally have kidney failure at an early age, with renal dialysis or dual kidney /liver organ transplant as the only treatment options.
  • 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. 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.
  • HAO1 Hydroxyacid oxidase 1
  • GOX glycolate oxidase
  • 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 associated with (e.g., encompassing) one or more of the components, one or more nucleic acids (e.g., vectors such as viral vectors or mRNA molecules) 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 and/or one particular gene edit in the HAO1 gene.
  • the cells can be suspended in a suitable media.
  • an RNA guide targeting HAO1 is contacted with a cell before an RNA guide targeting LDHA. In some embodiments, an RNA guide targeting HAO1 is contacted with a cell at the same time as an RNA guide targeting LDHA. In some embodiments, an RNA guide targeting HAO1 is contacted with a cell after the cell is contacted with an RNA guide targeting LDHA. In some embodiments, a composition comprising an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell before an RNA guide targeting LDHA and a second Casl2i polypeptide.
  • an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell at the same time as an RNA guide targeting LDHA and a second Casl2i polypeptide.
  • an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell after the cell is contacted with an RNA guide targeting LDHA and a second Casl2i polypeptide.
  • the RNA guide targeting HAO1 is encoded in a plasmid.
  • the RNA guide targeting LDHA is encoded in a plasmid.
  • the RNA guide targeting HAO1 is synthetic or purified RNA.
  • the RNA guide targeting LDHA is synthetic or purified RNA.
  • the first Casl2i polypeptide is encoded in a plasmid.
  • the second Casl2i polypeptide is encoded in a plasmid.
  • the first Casl2i polypeptide and the second Casl2i polypeptide are identical in sequence.
  • the first Casl2i polypeptide and the second Casl2i polypeptide are different sequences.
  • an RNP targeting HAO1 e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1
  • an RNP targeting LDHA e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA
  • an RNP targeting HAO1 e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1
  • a cell at the same time as an RNP targeting LDHA e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA.
  • an RNP targeting HAO1 e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1
  • an RNP targeting LDHA e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA
  • the Casl2i polypeptide of the RNP targeting HAO1 and the Casl2i polypeptide of the RNP targeting LDHA are identical in sequence.
  • the Casl2i polypeptide of the RNP targeting HAO1 and the Casl2i polypeptide of the RNP targeting LDHA are different sequences.
  • compositions comprising any of the gene editing systems described above 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 and/or HAO1 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 and/or HAO1 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 Casl2i polypeptide.
  • the kits include a polynucleotide that encodes such a Casl2i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g. , as described herein.
  • the Casl2i 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 Casl2i polypeptide.
  • the kit may be useful for research purposes.
  • the kit may be useful to study gene function.
  • the target sequence within the LDHA gene 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
  • the target sequence within the HAO1 gene is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the HAO1 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 HAO1 gene comprises the sequence of SEQ ID NO: 2123, the reverse complement of SEQ ID NO: 2123, a variant of SEQ ID NO: 2123, or the reverse complement of a variant of SEQ ID NO: 2123.
  • the spacer sequence that is substantially complementary to a target sequence within an LDHA gene 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 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 588-1164; and/or (ii) the spacer sequence that is substantially complementary to a target sequence within an HA01 gene 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: 1668-2122; 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: 1668-2122; c.
  • the spacer sequence that is substantially complementary to a target sequence within an LDHA gene 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 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; and/or (ii) the spacer sequence that is substantially complementary to a target sequence within an HA01 gene comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 1668-2122; b.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1.
  • 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1.
  • 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1. 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1.
  • 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.
  • the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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.
  • the spacer sequence that is substantially complementary to a target sequence within an LDHA gene is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587, and/or the spacer sequence that is substantially complementary to a target sequence within an HAO1 gene is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 1213-1667.
  • 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 sequences are each immediately adjacent to the PAM sequence.
  • the RNA guide targeting HAO1 has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2131-2187 and/or wherein the RNA guide targeting LDHA has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2188-2204.
  • the RNA guide targeting HAO1 has the sequence of any one of SEQ ID NOs: 2131-2187 and/or wherein the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
  • the composition further comprises Casl2i polypeptides or a polynucleotide encoding Casl2i polynucleotides.
  • the Casl2i polypeptides are selected from: a. a Casl2i2 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 Casl2i4 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 Casl2il polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or d. a Casl2i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.
  • the Casl2i polypeptides are selected from: a. a Casl2i2 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 Casl2i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; c. a Casl2il polypeptide comprising a sequence of SEQ ID NO: 1211; or d. a Casl2i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
  • the RNA guides and the Casl2i polypeptides form ribonucleoprotein complexes.
  • the ribonucleoprotein complexes bind target nucleic acids.
  • the composition is present within a cell.
  • the RNA guides and the Casl2i polypeptides are encoded in one or more vectors, e.g., expression vectors.
  • the RNA guides and the Casl2i polypeptides are encoded in a single vector, the RNA guides are encoded in a first vector and the Casl2i polypeptides are encoded in a second vector, or the RNA guides are separately encoded in first and second vectors and the Casl2i polypeptides are encoded in a third vector.
  • the present disclosure yet further provides a nucleic acid encoding an RNA guide targeting HAO1 and an RNA guide targeting LDHA, wherein the RNA guides are of a composition described herein.
  • the present disclosure yet further provides a vector comprising the nucleic acid.
  • the present disclosure yet further provides a vector system comprising one or more vectors encoding (i) an RNA guide targeting HAO1 and an RNA guide targeting LDHA, as described herein, and (ii) a Casl2i polypeptide, optionally wherein the vector system comprises a first vector encoding one or more of the RNA guides and a second vector encoding the Casl2i polypeptide.
  • a cell comprising a composition described herein, a nucleic acid described herein, a vector described herein, or a vector system 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 hepatocyte.
  • kits comprising a composition described herein, a nucleic acid described herein, a vector described herein, or a vector system described herein.
  • the present disclosure yet further provides a method of editing an LDHA sequence and an HAO1 sequence, the method comprising contacting an LDHA sequence and an HAO1 sequence with a composition of any described herein.
  • the LDHA sequence and the HAO1 sequence are in a cell.
  • composition or the RNA guides induce a deletion in the LDHA sequence and/or a deletion in the HAO1 sequence.
  • At least one deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide.
  • At least one deletion is downstream of the 5’-NTTN- 3’ sequence.
  • At least one deletion is up to about 50 nucleotides in length.
  • At least one deletion is up to about 40 nucleotides in length.
  • At least one deletion is from about 4 nucleotides to 40 nucleotides in length.
  • At least one deletion is from about 4 nucleotides to 25 nucleotides in length.
  • At least one deletion is from about 10 nucleotides to 25 nucleotides in length.
  • At least one deletion is from about 10 nucleotides to 15 nucleotides in length.
  • At least one deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence. In another aspect of the method, at least one deletion starts within about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence.
  • At least one deletion starts within about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
  • At least one deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • At least one 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.
  • At least one 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.
  • At least one 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.
  • At least one 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 another aspect of the method, at least one 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.
  • At least one 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.
  • At least one 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.
  • At least one 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.
  • At least one 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 in the LDHA sequence overlaps with a mutation in the LDHA sequence, and/or the deletion in the HA01 sequence overlaps with a mutation in the HA01 sequence.
  • the deletion in the LDHA sequence overlaps with an insertion in the LDHA sequence, and/or the deletion in the HA01 sequence overlaps with an insertion in the HA01 sequence.
  • the deletion in the LDHA sequence removes a repeat expansion of the LDHA sequence or a portion thereof, and/or the deletion in the HA01 sequence removes a repeat expansion of the LDHA sequence or a portion thereof.
  • the deletion in the LDHA sequence disrupts one or both alleles of the LDHA sequence, and/or the deletion in the HA01 sequence disrupts one or both alleles of the HA01 sequence.
  • the RNA guide comprises the sequence of any one of SEQ ID NOs: 2131- 2204.
  • the present disclosure yet further provides a method of treating primary hyperoxaluria (PH), which optionally is PHI, PH2, or PH3, in a subject, the method comprising administering a composition or a cell described herein to the subject.
  • PH primary hyperoxaluria
  • the RNA guides and/or the polyribonucleotide encoding the Casl2i polypeptide are comprised within a lipid nanoparticle.
  • the RNA guides and the polyribonucleotide encoding the Casl2i polypeptide are comprised within the same lipid nanoparticle.
  • the RNA guides and the polyribonucleotide encoding the Casl2i polypeptide are comprised within separate lipid nanoparticles.
  • composition comprising: a. a first RNA guide comprising (i) a spacer sequence that is complementary to a first target sequence and (ii) a direct repeat sequence, wherein the first target sequence is an LDHA gene sequence of any one of SEQ ID NOs: 2270, 2272, 2281, 2278, or 2282 or the reverse complement thereof; and b. a second RNA guide comprising (i) a spacer sequence that is complementary to a second target sequence and (ii) a direct repeat sequence, wherein the second target sequence is an HAO1 gene sequence of any one of SEQ ID NOs: 2234, 2213, or 2212 or the reverse complement thereof.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1.
  • 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1.
  • 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1.
  • 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1.
  • 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.
  • the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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.
  • the first RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2189, 2211, 2199, 2196, or 2200, and wherein the second RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2153, 2132, or 2131.
  • the first RNA guide has the sequence of any one of SEQ ID NOs: 2189, 2211, 2199, 2196, or 2200, and wherein the second RNA guide has a sequence of any one of SEQ ID NOs: 2153, 2132, or 2131.
  • each of the first three nucleotides of the first RNA guide and/or the second RNA guide comprises a 2’-O-methyl phosphorothioate modification.
  • each of the last four nucleotides of the first RNA guide and/or the second 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 first RNA guide and/or the second RNA guide comprises a 2’-O- methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.
  • the first RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2293-2302, and wherein the second RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2287-2292.
  • the first RNA guide has the sequence of any one of SEQ ID NOs: 2293-2302, and wherein the second RNA guide has a sequence of any one of SEQ ID NOs: 2287-2292.
  • the present disclosure further provides a nucleic acid encoding the first RNA guide of the composition described herein.
  • the present disclosure further provides a nucleic acid encoding the second RNA guide of the composition described herein.
  • the present disclosure further provides a vector comprising the nucleic acid described herein.
  • the present disclosure further provides a vector system comprising one or more vectors encoding (i) the first RNA guide of the composition described herein, (ii) the second RNA guide of the composition described herein, and (iii) a Casl2i polypeptide, optionally wherein the vector system comprises a first vector encoding the first RNA guide, a second vector encoding the second RNA guide, and a third vector encoding the Casl2i polypeptide.
  • the present disclosure further provides a cell comprising the composition, the nucleic acid, the vector, or the vector system 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.
  • the present disclosure further provides a kit comprising the composition, the nucleic acid, the vector, or the vector system described herein.
  • the present disclosure further provides a method of editing an LDHA sequence and an HAO1 sequence, the method comprising contacting an LDHA sequence with the composition described herein.
  • the LDHA sequence and the HAO1 sequence are in a cell.
  • the first RNA guide induces an indel in the LDHA sequence and the second RNA guide induces an indel in the HAO1 sequence.
  • the present disclosure further provides a method of treating primary hyperoxaluria (PH), which optionally is PHI, PH2, or PH3, in a subject, the method comprising administering the composition or the cell described herein.
  • PH primary hyperoxaluria
  • This Example describes the genomic editing of the HA01 gene or the LDHA gene using Casl2i2 introduced into HEK293T cells.
  • Casl2i2 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 HAO 1 or 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 HAO 1 -targeting RNA guide sequences are shown in Table 8.
  • the LDHA-targeting RNA guide sequences are shown in Table 9.
  • TS stands for “top strand” of the HA01 or LDHA gene
  • BS stands for “bottom strand” of the HAO1 or LDHA gene.
  • E#T# can also be represented as “exon # target #.”
  • Casl2i2 RNP complexation reactions were made by mixing purified Casl2i2 polypeptide (400 pM) with an HAO 1 -targeting crRNA or an LDHA-targeting crRNA (1 mM in 250 mM NaCl) at a 1:1 (Casl2i2:crRNA) volume ratio (2.5:1 crRNA:Casl2i2 molar ratio). Complexations were incubated on ice for 30-60 min.
  • HEK293T 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 CEEE FINE 4D-NUCEEOFECTORTM X KIT S; Eonza #V4XC- 2032) at a concentration of 16,480 cells/pE. Resuspended cells were dispensed at 3e5 cells/reaction into Eonza 16-well NUCLEOCUVETTE® strips.
  • the strips were electroporated using an electroporation device (program CM- 130, Lonza 4D-NUCLEOFECTORTM). Immediately following electroporation, 80 pL of prewarmed DMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 pL (30,000 cells) of diluted nucleofected cells into prewarmed 96-well plate with wells containing 100 pL DMEM + 10% FBS. Editing plates were incubated for 3 days at 37°C with 5% CO2.
  • 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 indels at HA01 target sites in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the HA01 target sites with each of the RNA guides. Delivery of E1T2, E1T3, E1T6, E1T7, E1T13, T1T17, E2T4, E2T5, E2T9, E2T10, E3T6, E3T19, E3T22, and E3T28 resulted in indels in over 70% of the NGS reads. Therefore, HAO 1 -targeting RNA guides induced indels in exon 1, exon 2, and exon 3 of the HAO1 gene in HEK293T cells.
  • FIG. 2 shows indels at LDHA target sites in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the LDHA target sites with each of the RNA guides. Delivery of E3T1, E3T9, E5T1, E5T9, and E5T10 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 of the LDHA gene in HEK293T cells. This Example thus shows that HAO1 and LDHA can be individually targeted by Casl2i2 RNPs in mammalian cells such as HEK293T cells.
  • This Example describes the genomic editing of the HAO1 gene or the LDHA gene using Casl2i2 introduced into HepG2 cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 8 or Table 9.
  • 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/pL. 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 pL of prewarmed EMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 pL (25,000 cells) of diluted nucleofected cells into prewarmed 96-well plate with wells containing 100 pL EMEM + 10% FBS. Editing plates were incubated for 3 days at 37°C with 5% CO2.
  • FIG. 3 shows indels at HAO1 target sites in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the HAO1 target sites with each of the RNA guides. Therefore, HAO 1 -targeting RNA guides induced indels in exon 1, exon 2, and exon 3 of the HAO1 gene in HepG2 cells.
  • FIG. 4 shows indels at LDHA target sites in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in 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 of the LDHA gene in HepG2 cells.
  • This Example thus shows that HAO1 and LDHA can be individually targeted by Casl2i2 RNPs in mammalian cells such as HepG2 cells.
  • This Example describes the genomic editing of the HAO1 using Casl2i2 introduced into primary hepatocytes cells by RNP.
  • RNP complexation reactions were performed as described in Example 1 with RNA guides for HAO1 of Table 8 and RNA guides for LDHA of Table 9. Additionally, for multiplexing experiments, shown in FIG. 6, separate HAO 1 -targeting RNPs and LDHA- targeting RNPs were mixed together at a 1 : 1 volume ratio prior to electroporation.
  • 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 100g 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/pL.
  • 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 Casl2i2 RNP was added to each reaction at a final concentration of 20 pM (Casl2i2), and transfection enhancer oligos were then added at a final concentration of 4 pM.
  • the final volume of each electroporated reaction was either 20 pL in the 16 well nucleocuvette strip format or 100 pL 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 CAI 37 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 5x 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 pL Digital Droplet PCR (ddPCR- BioRad laboratories) reaction using target specific primer and probes for HA01 and LDHA, and 2x 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.
  • 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.
  • ddPCR primers :
  • HAO1 ATTGTGCACTGTCAGATCTTGGAAACGGCCAAAGGATTTTTCCTCACCAATGTCTTGTCG ATGACTTTCACATTCTGGCACCCACTCAGAGCCATGGCCAACCGGAATTCTTCCTTTAGT AT (SEQ ID NO: 2303)
  • each HAO 1 -targeting RNA guide induced indels within and/or adjacent to the HAO1 target sites and each LDHA-targeting RNA guide induced indels within and/or adjacent to the LDHA target sites. Indels were not induced with the nontargeting control. Therefore, HAO 1 -targeting RNA guides and LDHA-targeting RNA guides individually induced indels in primary hepatocytes.
  • FIG. 6 shows dual RNA guide editing with HAO 1 -targeting RNA guides (E2T10, E1T3, E1T2, E2T4, and E2T5 from Table 8) and LDHA-targeting RNA guides (E3T1, E3T3, E5T9, and E5T10 from Table 9) in primary human hepatocytes.
  • HAO 1 -targeting RNA guides E2T10, E1T3, E1T2, E2T4, and E2T5 from Table 8
  • LDHA-targeting RNA guides E3T1, E3T3, E5T9, and E5T10 from Table 9
  • the top performing dual RNA guide pairings (HAO1 E1T2 and LDHA E3T1; HAO1 E1T3 and LDHA E3T1; HAO1 E2T5 and LDHA E3T1) induced HAO1 indels and LDHA indels in over about 90% of the NGS reads.
  • FIG. 7 and FIG. 8 show % knockdown of HAO1 mRNA or LDHA mRNA, respectively, in edited cells compared to unedited control cells.
  • HAO1 E2T4 and HAO 1 E2T5 reduced HAO1 mRNA when delivered with or without LDHA E3T1.
  • HAO1 E2T4 resulted in a greater knockdown of HAO1 mRNA.
  • LDHA E3T1 reduced LDHA mRNA when delivered with or without HAO1 E2T4 or HAO1 E2T5.
  • HAO1 and LDHA can be individually targeted or simultaneously targeted by Casl2i2 RNPs in mammalian cells such as primary human hepatocytes, leading to indels in HAO1 and LDHA target sites and knockdown of HAO1 mRNA and LDHA mRNA.
  • This Example describes indel assessment on HAO1 target sites and LDHA target sites using Casl2i2 variants introduced into HepG2 cells by transient transfection.
  • the Casl2i2 variants of SEQ ID NO: 1168 and SEQ ID NO: 1171 were individually cloned into a pcda3.1 backbone (Invitrogen).
  • HAO1 E1T2, HAO1 E1T3, HAO1 E2T4, HAO1 E2T5, and HAO1 E2T10) (Table 8) and LDHA-targeting RNA guides (LDHA E3T1, LDHA E3T3, LDHA E5T1, LDHA E5T9, and LDHA E5T10) (Table 9) were individually 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
  • two LDHA-targeting guides demonstrated significantly higher activity with variant Casl2i2 of SEQ ID NO: 1171 compared to variant Casl2i2 of SEQ ID NO: 1168. Comparable indel activity with the two Casl2i2 variants was observed for LDHA-targeting guides LDHA E3T1, LDHA E5T9, and LDHA E5T10 as well as each of the HAO 1 -targeting guides (HAO1 E1T2, HAO1 E1T3, HAO1 E2T4, HAO1 E2T5, and HAOl E2T10).
  • FIG. 9B shows the indel size frequency (left) and indel start position relative to the PAM for HAO1 E1T3 and the variant Casl2i2 of SEQ ID NO: 1168 in HepG2 cells.
  • deletions ranged in size from 1 nucleotide to about 40 nucleotides. The majority of the deletions were about 6 nucleotides to about 27 nucleotides in length.
  • the target sequence is represented as starting at position 0 and ending at position 20. Indels started within about 10 nucleotides and about 35 nucleotides downstream of the PAM sequence.
  • FIG. 9C shows the indel size frequency (left) and indel start position relative to the PAM for LDHA E5T9 and the variant Casl2i2 of SEQ ID NO: 1168 in HepG2 cells.
  • deletions ranged in size from 1 nucleotide to about 40 nucleotides; a small percentage of NGS reads comprised a 1 nucleotide insertion.
  • the majority of the deletions were about 8 nucleotides to about 23 nucleotides in length.
  • 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 describes indel assessment on HA01 and LDHA target sites via delivery of Casl2i2 mRNA and chemically modified HAO 1 -targeting and LDHA-targeting RNA guides.
  • mRNA sequences corresponding to the variant Casl2i2 sequence of SEQ ID NO: 1168 and the variant Casl2i2 sequence of SEQ ID NO: 1171 were synthesized with 1- pseudo-U modified nucleotides and using CleanCap® Reagent AG (TriLink Biotechnologies).
  • the Casl2i2 mRNA sequences, shown in Table 10, further comprised a C- terminal NLS.
  • Casl2i2 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 11.
  • TS stands for “top strand” of the HA01 gene
  • BS stands for “bottom strand” of the LDHA gene.
  • Each variant Casl2i2 mRNA was mixed with a crRNA at a 1:1 (Casl2i2:crRNA) volume ratio (1050: 1 crRNA:Casl2i2 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.
  • FIG. 10 shows editing of an HA01 target site by a variant Casl2i2 mRNA and a 3’ end modified HA01 E2T5 guide or a 5’ and 3’ end modified HAO1 E2T5 RNA guide and editing of an LDHA target site by a variant Casl2i2 mRNA and a 3’ end modified LDHA E3T1 RNA guide or a 5’ and 3’ end modified LDHA E3T1 RNA guide.
  • Indels in the HAO1 target site or the LDHA target site were introduced following electroporation of the Casl2i2 mRNA and each of the RNA guides.
  • NGS reads comprised an indel following electroporation of the Casl2i2 mRNA of SEQ ID NO: 2306 and the RNA guide of SEQ ID NO: 2307 or SEQ ID NO: 2308.
  • Statistically significant higher % indels were observed using variant Casl2i2 mRNA of SEQ ID NO: 2306 compared to variant Casl2i2 mRNA of SEQ ID NO: 2305. No statistical difference was observed using 5’ and 3’ modifications versus 3’ only modifications to RNA guide HAO1 E2T5.
  • NGS reads For LDHA, a higher percentage of NGS reads exhibited indels for RNA guide LDHA E3T1 with 5’ and 3’ end modifications compared to percentage of NGS reads for RNA guide LDHA E3T1 with 3’ end modifications only. Approximately 50% of NGS reads comprised indels following electroporation of the Casl2i2 mRNA of SEQ ID NO: 2306 and the LDHA RNA guide of SEQ ID NO: 2310.
  • HAO1 and LDHA can be targeted by Casl2i2 mRNA constructs and chemically modified HAO 1 -targeting and LDHA-targeting RNA guides in mammalian cells.
  • This Example describes on-target versus off-target assessment of a Casl2i2 variant and an HAO 1 -targeting RNA guide or an LDHA-targeting guide.
  • HEK293T cells were transfected with a plasmid encoding the variant Casl2i2 of SEQ ID NO: 1168 or the variant Casl2i2 of SEQ ID NO: 1171 and a plasmid encoding HAO1 E3T1, HAO1 E5T1, HAO1 E5T9, HAO1 E5T10, LDHA E3T1, LDHA E5T1, LDHA E5T9, or LDHA E5T10 according to 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.
  • FIGS. 11A/B and 12A/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. 11A and FIG. 12A show TTISS reads for variant Casl2i2 of SEQ ID NO: 1168
  • FIG. 11B and FIG. 12B show TTISS reads for variant Casl2i2 of SEQ ID NO: 1171. As shown in FIG.
  • variant Casl2i2 of SEQ ID NO: 1168 paired with HAO1 E2T5 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. HAO1 E1T2 also showed a low likelihood of off-target editing. For HAO1 E1T2, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For HAO1 E5T10, 95% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 5% of TTISS reads.
  • HA01 E2T10 demonstrated a higher likelihood of off-target editing using the TTISS method. For HA01 E2T10, only 65% of TTISS reads mapped to the on-target and 4 potential off-target sites represented the remaining combined 35% of TTISS reads. One potential off-target represented the majority of potential off-target TTISS reads for HA01 E2T10.
  • variant Casl2i2 of SEQ ID NO: 1171 paired with HA01 E2T5 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.
  • Variant Casl2i2 of SEQ ID NO: 1171 paired with the HA01 E1T2 or HA01 E1T3 also demonstrated a low likelihood of off-target editing.
  • variant Casl2i2 of SEQ ID NO: 1168 paired with EDHA 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.
  • EDHA E3T1 and LDHA E5T10 also showed a low likelihood of off-target editing.
  • LDHA E3T1 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads.
  • LDHA E5T10 97% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 3% of TTISS reads.
  • LDHA E5T1 demonstrated a higher likelihood of off-target editing using the TTISS method.
  • variant Casl2i2 of SEQ ID NO: 1171 paired with the LDHA 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.
  • LDHA 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.
  • compositions comprising Casl2i2 and HA01- targeting and LDHA-targeting RNA guides comprise different off-target activity profiles.
  • This Example describes use of a Western Blot to identify knockdown of HA01 or LDHA protein using variant Casl2i2 of SEQ ID NO: 1168 and HAO 1 -targeting or 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 100g 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 (serumcontaining), 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/pL. Resuspended cells were dispensed at 500,000 cells/reaction into Lonza electroporation cuvettes
  • HA01 E2T5 was used as the HAO 1 -targeting RNA guide
  • LDHA E3T1, LDHA E5T9, LDHA E5T1, and LDHA E5T10 were used as the LDHA- targeting RNA guides.
  • RNPs were added to each reaction at a final concentration of 20 pM (Casl2i2), and transfection enhancer oligos were then added at a final concentration of 4 pM.
  • Unelectroporated cells and cells electroporated without cargo were used as negative controls.
  • the strips were electroporated using an electroporation device (program CAI 37, 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 prewarmed 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.
  • electroporation device program CAI 37, Lonza 4D-nucleofector
  • the blot was washed three times with TBST (ThermoFisher 28360) for 5 minutes each, then incubated with a 1:12500 dilution of IR680 anti-mouse (ThermoFisher PI35518) and IR800 anti -rabbit secondary antibodies (ThermoFisher 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 HAO1 protein was observed in primary human hepatocytes at day 16 post delivery of Casl2i2 RNPs targeting the HAO1 gene with HAO1 E2T5 (lanes 1-3 of FIG. 13A). HAO1 knockdown was not observed for the buffer only controls (lanes 4-7). Knockdown of LDHA protein (monomer and dimer) was observed in primary human hepatocytes at Day 7 post delivery of Casl2i2 RNPs targeting the LDHA gene (FIG. 13B). This knockdown was seen across each of the four RNA guides, LDHA E3T1, LDHA E5T9, LDHA E5T1, and LDHA E5T10 (lanes 1-8). LDHA knockdown was not observed for the buffer only (lanes 9 and 10) or electroporated 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.

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Abstract

The present disclosure relates to gene editing systems and/or compositions comprising RNA guides targeting HAO1 and RNA guides targeting LDHA. Also provide herein are methods of using the gene editing system for introducing edits to the HAO1 gene, introducing edits to the LDHA gene, and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

Description

GENE EDITING SYSTEMS TARGETING HYDROXYACID OXIDASE 1 (HAO1) AND LACTATE DEHYDROGENASE A (LDHA)
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/292,938, filed December 22, 2021, U.S. Provisional Application No. 63/300,841, filed January 19, 2022, and U.S. Provisional Application No. 63/348,830, filed June 3, 2022, the contents 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 XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 7, 2022, is named 116928-0056-006WO00_SEQ.xml and is 2,181,422 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 gene editing system for genetic editing of both a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase (LDHA) gene. The system involves a Casl2i CRISPR nuclease polypeptide (e.g. , a Casl2i2 polypeptide), an RNA guide mediating cleavage at a genetic site within the HAO1 gene by the Casl2i polypeptide, and an RNA guide mediating cleavage at a genetic site within the LDHA gene by the Casl2i polypeptide. As reported herein, the gene editing system disclosed herein has achieved successful editing of HAO1 and LDHA genes with high editing efficiency and accuracy.
Without being bound by theory, the gene editing system disclosed herein may further exhibit one or more of the following advantageous features. Compared to SpCas9 and Casl2a, Casl2i 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. Casl2i cleavage results in larger deletions compared to the small deletions and +1 insertions induced by Cas9 cleavage. Casl2i PAM sequences also differ from those of Cas9. Therefore, larger and different portions of genetic sites of interest can be disrupted with a Casl2i 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 Casl2i2. See WO/2021/202800. Therefore, Casl2i such as Casl2i2 may be more specific than Cas9.
Accordingly, provided herein are gene editing systems for editing HA01 and LDHA genes, 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, provided herein is a gene editing system for genetic editing of a hydroxy acid oxidase 1 (HA01) gene and a lactate dehydrogenase A (LDHA) gene, comprising: (i) a first RNA guide or a first nucleic acid encoding the RNA guide, wherein the first RNA guide comprises a first spacer sequence specific to a first target sequence within an HA01 gene, the first 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; (ii) a second RNA guide or a second nucleic acid encoding the RNA guide, wherein the second RNA guide comprises a second spacer sequence specific to a second target sequence within an LDHA gene, the second 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; and (iii) a Casl2i polypeptide or a third nucleic acid encoding the Casl2i. polypeptide. In some embodiments, the Casl2i polypeptide can be a Casl2i2 polypeptide. In other embodiments, the Casl2i polypeptide can be a Casl2i4 polypeptide.
In some embodiments, the Casl2i polypeptide is a Casl2i2 polypeptide, which 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. In some embodiments, the one or more mutations in the Casl2i2 polypeptide are at positions D581, G624, F626, P868, 1926, 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 Casl2i2 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 Casl2i2 polypeptide comprises mutations at positions D581, 1926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In yet another example, the Casl2i2 polypeptide comprises mutations at positions D581, 1926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G). In still another example, the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G). In another example, the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).
Exemplary Casl2i2 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 Casl2i2 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 Casl2i2 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 first target sequence within the HA01 gene is within exon 1 or exon 2 of the HAO1 gene. In some examples, the first target sequence may comprise:
(a-i) 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212);
(a-ii) 5’-GGAAGTACTGATTTAGCATG-3’ (SEQ ID NO: 2213);
(a-iii) 5’-TAGATGGAAGCTGTATCCAA-3’ (SEQ ID NO: 2233);
(a-iv) 5’-CGGAGCATCCTTGGATACAG-3’ (SEQ ID NO: 2234); or (a-v) 5’-AGGACAGAGGGTCAGCATGC-3 (SEQ ID NO: 2239).
In some specific examples, the first target sequence may comprise 5’- CGGAGCATCCTTGGATACAG-3’ (SEQ ID NO: 2234). In some specific examples, the first target sequence may comprise 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212).
Alternatively or in addition, the second target sequence in the LDHA gene may be within exon 3 or exon 5 of the LDHA gene. In some examples, the second target sequence may comprise:
(b-i) 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281);
(b-ii) 5’-TAGGACTTGGCAGATGAACT-3’ (SEQ ID NO: 2270); (b-iii) 5’-GATGACATCAACAAGAGCAA-3’ (SEQ ID NO: 2272);
(b-iv) 5’-TTCATAGTGGATATCTTGAC-3’ (SEQ ID NO: 2278); or
(b-v) 5 ’-CATAGTGGATATCTTG ACCT-3 (SEQ ID NO: 2282).
In specific examples, the second target sequence may comprise: 5’- TTCATAGTGGATATCTTGAC-3’ (SEQ ID NO: 2278). In specific examples, the second target sequence may comprise: 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281).
In some embodiments, the first spacer sequence specific to the HAO1 gene and/or the second spacer sequence specific to the LDHA gene is 20-30-nucleotide in length. In some examples, the spacer sequence is 20-nucleotide in length.
In some embodiments, the first spacer sequence targeting the HAO1 gene may comprise the nucleotide sequence of:
(a-i) 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2311);
(a-ii) 5’-GGAAGUACUGAUUUAGCAUG-3’ (SEQ ID NO: 2312);
(a-iii) 5’-UAGAUGGAAGCUGUAUCCAA-3’ (SEQ ID NO: 2313);
(a-iv) 5’-CGGAGCAUCCUUGGAUACAG-3’ (SEQ ID NO: 2314); or
(a-v) 5’-AGGACAGAGGGUCAGCAUGC-3 (SEQ ID NO: 2315).
In specific examples, the first spacer sequence may comprise 5’- CGGAGCAUCCUUGGAUACAG-3’ (SEQ ID NO: 2314). In specific examples, the first spacer sequence may comprise 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2311).
In some embodiments, the second spacer sequence specific to the LDHA gene may comprise the nucleotide sequence of:
(b-i) 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316);
(b-ii) 5’-UAGGACUUGGCAGAUGAACU-3’ (SEQ ID NO: 2317);
(b-iii) 5’-GAUGACAUCAACAAGAGCAA-3’ (SEQ ID NO: 2318);
(b-iv) 5’-UUCAUAGUGGAUAUCUUGAC-3’ (SEQ ID NO: 2319); or
(b-v) 5’-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 2320).
In specific examples, the second spacer sequence may comprise 5’- UUCAUAGUGGAUAUCUUGAC-3’ (SEQ ID NO: 2319). In specific examples, the second spacer sequence may comprise 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316).
Any of the first RNA guides specific to the HAO1 gene may comprise the first spacer sequence as disclosed herein and a first direct repeat sequence. Alternatively or in addition, any of the second RNA guides specific to the LDHA gene may comprise the second spacer sequence disclosed herein and a second direct repeat sequence. In some embodiments, the first direct repeat sequence and/or the second direct repeat sequence is 23-36-nucleotide in length. In some examples, the first direct repeat sequence and/or the second 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. For example, the first direct repeat sequence and/or the second direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length. In one example, the first direct repeat sequence and/or the second direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 10).
Exemplary first RNA guides specific to the HAO1 gene includes:
(a-i) 5’ AGAAAUCCGUCUUUC AUUGACGGCAAAGUCUAU AU AUGACUAU- 3’ (SEQ ID NO: 2131);
(a-ii) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGGGAAGUACUGAUUUAGCAUG- 3’ (SEQ ID NO: 2132);
(a-iii) 5 ’ AGAAAUCCGUCUUUC AUUGACGGUAGAUGGAAGCUGUAUCCAA- 3’ (SEQ ID NO: 2152);
(a-iv) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGCGGAGC AUCCUUGGAU AC AG- 3’ (SEQ ID NO: 2153); or
(a-v) 5 ’ - AGAAAUCCGUCUUUC AUUGACGG AGGAC AGAGGGUC AGC AUGC- 3’ (SEQ ID NO: 2158).
In one specific example, the first RNA guide is (a-i). In another specific example, the first RNA guide is (a-iv).
Exemplary second RNA guides specific to the LDHA gene includes:
(b-i) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUC AUAGUGG AU AUCUUGACC- 3’ (SEQ ID NO: 2199);
(b-ii) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUAGG ACUUGGCAGAUG AACU- 3’ (SEQ ID NO: 2189);
(b-iii) 5 ’ -AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGC AA-3’ (SEQ ID NO: 2211);
(b-iv) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUUCAUAGUGGAUAUCUUG AC-3’ (SEQ ID NO: 2196); or
(b-v) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU-
3’ (SEQ ID NO: 2200). In one specific example, the first RNA guide is (b-i). In another specific example, the second RNA guide is (b-iv).
In some embodiments, the gene editing system may comprise the third nucleic acid encoding the Casl2i polypeptide (e.g., the Casl2i2 polypeptide as disclosed herein). In some instances, the third nucleic acid is located in a third vector (e.g., a viral vector such as an adeno-associated viral vector or AAV vector). In some instances, the third nucleic acid is a messenger RNA (mRNA). In some instances, the nucleic acid encoding the Casl2i polypeptide (e.g., the Casl2i2 polypeptide as disclosed herein) is codon-optimized.
Any of the gene editing systems disclosed herein may comprise the first nucleic acid encoding the first RNA guide. Alternatively or in addition, the gene editing system may comprise the second nucleic acid encoding the second RNA guide. In some embodiments, the first nucleic acid encoding the first RNA guide is located in a first vector; and/or the second nucleic acid encoding the second RNA guide is located in a second vector. In some examples, the first vector and the second vector are the same vector.
In some instances, the gene editing system comprises one or more vectors, which collectively comprise the first nucleic acid encoding the first RNA guide in a first vector, the second nucleic acid encoding the second RNA guide in a second vector, and/or the third nucleic acid encoding the Casl2i polypeptide such as the Casl2i2 polypeptide. In some examples, the first, second and/or third vector is a viral vector, for example, an AAV vector.
In some instances, the gene editing system comprises one or more lipid nanoparticles (LNPs), which are associated (e.g., encompass) (i), (ii), (iii), or a combination thereof. In some instances, the system comprises a lipid nanoparticle, which encompass one or two of (i)-(iii), and a viral vector, which comprising the nucleic acid(s) encoding the remaining. In some examples, the viral vector is an AAV vector. In other instances, the LNPs are associated with (i) the first RNA guide, (ii) the second RNA guide, and (iii) an mRNA molecule encoding the Casl2i2 polypeptide. In some examples, at least a portion of (i), (ii), and/or (iii) is encapsulated by the LNPs.
In some aspects, the present disclosure also provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein, or a kit comprising the components of the gene editing system.
In other aspects, the present disclosure also features a method for editing a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with any of the gene editing systems disclosed herein to genetically edit the HAO1 gene and 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 HA01 gene and the LDHA gene to a subject comprising the host cell.
Also within the scope of the present disclosure is a cell comprising a disrupted an HA01 gene and an LDHA gene, which can be produced by contacting a host cell with the system disclosed herein genetically edit the HA01 gene and 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 an HA01 gene and an LDHA gene or any of the modified cells disclosed herein. In some embodiments, the subject may be a human patient having the PH. In some examples, the PH is PHI, PH2, or PH3. In a specific example, the PH is PHI.
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
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.
FIG. 1 is a graph showing % of NGS reads comprising indels in HEK293T cells following delivery of a variant Casl2i2 and HAO 1 -targeting RNA guide RNP. The darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaco mulatto) and crab-eating macaque (Macaco fascicularis) sequences.
FIG. 2 is a graph showing % of NGS reads comprising indels in HEK293T cells following delivery of a variant Casl2i2 and LDHA-targeting RNA guide RNP. The darker grey bars represent target sequences with perfect homology to both rhesus macaque (Macaco, mulato) and crab-eating macaque (Macaco fascicularis) sequences.
FIG. 3 is a graph showing HAO1 indels in HepG2 cells following RNP delivery.
FIG. 4 is a graph showing LDHA indels in HepG2 cells following RNP delivery.
FIG. 5 is a graph showing HAO1 and LDHA indels in primary hepatocytes.
FIG. 6 is a graph showing dual RNA guide editing with HAO 1 -targeting RNA guides and LDHA-targeting RNA guides in primary hepatocytes.
FIG. 7 is a graph showing knockdown of HAO1 mRNA in primary human hepatocytes with a variant Casl2i2 polypeptide and an HAO 1 -targeting crRNA or a variant Casl2i2 polypeptide, an HAO 1 -targeting crRNA, and an LDHA-targeting crRNA.
FIG. 8 is a graph showing knockdown of LDHA mRNA in primary human hepatocytes with a variant Casl2i2 polypeptide and an LDHA-targeting crRNA or a variant Casl2i2 polypeptide, an LDHA-targeting crRNA, and an HAO 1 -targeting crRNA.
FIG. 9 A is a graph showing % of NGS reads comprising indels following delivery of HAO 1 -targeting crRNAs or LDHA-targeting crRNAs and the variant Casl2i2 polypeptide of SEQ ID NO: 1168 or SEQ ID NO: 1171. FIG. 9B shows the size (left) and start position (right) of indels induced in HepG2 cells by a variant Casl2i2 and HAO 1 -targeting RNA guide E1T3. FIG. 9C shows the size (left) and start position (right) of indels induced in HepG2 cells by a variant Casl2i2 and LDHA-targeting RNA guide E5T9.
FIG. 10 is a graph showing % of NGS reads comprising indels induced by chemically modified HAO 1 -targeting crRNAs or LDHA-targeting crRNAs and variant Casl2i2 mRNA.
FIGS. 11A-11B show plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for HAO 1 -targeting RNA guides. 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. 11A shows plots for HAO 1 -targeting RNA guides E2T5, E1T2, E1T3, and E2T10 and the variant Casl2i2 of SEQ ID NO: 1168. FIG. 11B shows plots for HAO 1 -targeting RNA guides E2T5, E1T2, and E1T3 and the variant Casl2i2 of SEQ ID NO: 1171.
FIGS. 12A-12B show plots depicting tagmentation-based tag integration site sequencing (TTISS) reads for LDHA-targeting RNA guides. 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. 12A shows plots for LDHA-targeting RNA guides E5T9, E3T1, E5T10, and E5T1 and the variant Casl2i2 of SEQ ID NO: 1168. FIG. 12B shows plots for LDHA-targeting RNA guides E5T9, E5T10, and E1T3 and the variant Casl2i2 of SEQ ID NO: 1171.
FIGs. 13A-13B include diagrams showing knockdown of HAO 1 and LDHA genes as examined by protein expression via Western Blot. FIG. 13A is a Western Blot showing knockdown of HAO1 protein following electroporation of primary human hepatocytes variant Casl2i2 and HAO 1 -targeting RNA guide E2T5. FIG. 13B is a Western Blot showing knockdown of LDHA protein following electroporation of primary human hepatocytes with variant Casl2i2 and LDHA-targeting RNA guides E3T1, E5T9, E5T1, or E5T10.
DETAILED DESCRIPTION
The present disclosure relates to a gene editing system for genetic editing of a hydroxy acid oxidase 1 (HAO1) gene (a.k.a., glycolate oxidase gene) and a lactate dehydrate A (LDHA) gene. The gene editing system may comprise: (i) an RNA guide specific to the HAO1 gene or a first nucleic acid encoding the RNA guide, (ii) an RNA guide specific to the LDHA gene or a second nucleic acid encoding the RNA guide, and (iii) a Casl2i polypeptide or a first nucleic acid encoding the Casl2i polypeptide. The RNA guide specific to the HAO1 gene may comprise a first spacer sequence specific to a first target sequence within an HAO1 gene, the first target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-TTN-3’, which is located 5’ to the first target sequence. The RNA guide specific to the LDHA gene may comprise a second spacer sequence specific to a second target sequence within an LDHA gene, the second target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-TTN-3’, which is located 5’ to the second target sequence.
The Casl2i polypeptide for use in the gene editing system disclosed herein may be a Casl2i2 polypeptide, e.g., a wild- type Casl2i polypeptide or a variant thereof as those disclosed herein. In some examples, the Casl2i2 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. In other examples, the Casl2i polypeptide may be a Casl2i4 polypeptide, which is also disclosed herein.
Also provided in the present disclosure are a pharmaceutical composition or a kit comprising any of the gene editing systems disclosed herein, as well as uses thereof. Further disclosed herein are a method for editing a HA01 gene and an LDHA gene in a cell, a cell so produced that comprises a disrupted the HAO1 and LDHA genes, a method of treating primary hyperoxaluria (PH) in a subject.
Definitions
The present invention will be described with respect to particular embodiments, but the invention 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 Casl2i 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 interconversion 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 “HAO1” refers to “glycolate oxidase 1,” which is also known as “hydroxyacid oxidase.” HAO1 is a peroxisome protein expressed primarily in the liver and pancreas, and its activities include oxidation of glycolate and 2-hydroxy fatty acids. SEQ ID NO: 2123 as set forth herein provides an example of an HAO1 gene sequence.
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 Casl2i polypeptide). In some instances, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence. For example, the term “complex” can refer to a grouping of an LDHA-targeting RNA guide and a Casl2i polypeptide or a grouping of an HAO 1 -targeting RNA guide and a Casl2i 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 or an HAO1 target sequence) to which a complex comprising an RNA guide (e.g. , an LDHA-targeting RNA guide or an HAO 1 -targeting RNA guide) and a Casl2i polypeptide binds. In a doublestranded 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 Casl2i polypeptide (e.g. , a Casl2i2 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. In some embodiments, the target sequence is a sequence within an HAO1 gene sequence, including, but not limited to, the sequence set forth in SEQ ID NO: 2123.
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 sequence 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 Casl2i 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 Casl2i-induced indel is downstream of the 5’-NTTN-3’ or 5’- TTN-3’ sequence.
I. Gene Editing Systems
In some aspects, the present disclosure described herein comprises gene editing systems or compositions comprising RNA guides targeting an LDHA gene and an HAO1 gene. Such gene editing systems or compositions can be used to edit both HAO1 and LDHA genes in cells, for example, to disrupt both genes.
Hydroxyacid oxidase 1 (HAO1, also known as glycolate oxidase [GOX or GO]), converts glycolate into glyoxylate. It has been proposed that inhibition of HAO1 in individuals with PHI would block formation of glyoxylate, and excess glycolate would be excreted through the urine. The idea of treating PHI by inhibition of HAO1 is further supported that some individuals with abnormal splice variants of HAO1 are asymptomatic for glycolic aciduria, whereby there was increased urinary glycolic acid excretion without apparent kidney pathology. Thus, inhibition of HAO1 expression would block production of glyoxylate, and in turn block production of its metabolite, oxalate. Accordingly, the gene editing systems disclosed here, targeting the HAO1 gene, could be used to treat primary hyperoxaluria (PH) in a subject in need of the treatment.
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 Casl2i polypeptide. In some embodiments, the spacer component is specific to a target sequence (e.g., an LDHA target sequence or an HAO1 target sequence), wherein the 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
Figure imgf000017_0001
the PAM strand).
In some embodiments, the present described herein comprises compositions comprising complexes, wherein a first complex comprises an RNA guide targeting HAO1 and a second complex comprises an RNA guide targeting LDHA. In some embodiments, the present disclosure provides complexes comprising an RNA guide and a Casl2i polypeptide. In some embodiments, the RNA guides and the Casl2i polypeptides bind to each other in molar ratios of about 1:1. In some embodiments, a complex comprising an RNA guide and a Casl2i polypeptide binds to an HAO1 target sequence. In some embodiments, a complex comprising an RNA guide and a Casl2i polypeptide binds to an LDHA target sequence. In some embodiments, a complex comprising an RNA guide targeting LDHA and a Casl2i polypeptide binds to the complementary region of an HAO1 target sequence at a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide targeting LDHA and a Casl2i polypeptide binds to the complementary region of 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 HAO1 target sequence and/or the complementary sequence thereof. In some embodiments, the complex comprises enzymatic activity, such as nuclease activity, that can cleave the LDHA target sequence and/or the complementary sequence thereof. The RNA guide, the Casl2i polypeptide, and the complementary region of the HAO1 target sequence, either alone or together, do not naturally occur. The RNA guide, the Casl2i polypeptide, and the complementary region of 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 targeting HAO1 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: 2131-2187. In some embodiments, the RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187. In some embodiments, the sequence of the RNA guide targeting LDHA 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: 2188-2204. In some embodiments, the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
In some embodiments, the present disclosure provides gene editing systems and compositions comprising one or more RNA guides as described herein and/or an RNA encoding a Casl2i polypeptide as described herein. In some embodiments, an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide are comprised together within the same composition, while an RNA guide targeting LDHA is comprised within a different composition. In some embodiments, an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide are comprised within the same composition, while an RNA guide targeting HAO1 is comprised within a different composition. In some embodiments, an RNA guide targeting HAO1, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide are comprised within the same composition. In some embodiments, an RNA guide targeting HAO1 and an RNA guide targeting LDHA are comprised within the same composition, while an RNA encoding a Casl2i polypeptide is comprised within a different composition. In some embodiments, an RNA guide targeting HAO1, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide are each comprised within separate compositions. In some embodiments, an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide are comprised together within the same composition, while an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide are comprised within a different composition. In some embodiments, an 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: 2131-2204. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 2131-2204.
Use of the compositions disclosed herein has advantages over those of other known nuclease systems. Casl2i polypeptides are smaller than other nucleases. For example, Casl2i2 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, FnCpfl is 1,300 amino acids in length, AsCpfl is 1,307 amino acids in length, and LbCpfl is 1,246 amino acids in length. Casl2i RNA guides, which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides. The smaller Casl2i polypeptide and RNA guide sizes are beneficial for delivery. Compositions comprising a Casl2i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See PCT/US2021/025257, which is incorporated by reference in its entirety. Furthermore, indels induced by compositions comprising a Casl2i 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, Casl2i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of genes such as HAO1 and LDHA. A. RNA Guides
In some embodiments, the gene editing system described herein comprises an RNA guide targeting HAO1 and an RNA guide targeting LDHA. 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 HAO1 and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting LDHA. The HAO 1 -targeting RNA guide may direct the Casl2i polypeptide as described herein to an HAO1 target sequence. Two or more RNA guides may target two or more separate Casl2i polypeptides (e.g., Casl2i 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) HAO1 target sequences. The LDHA-targeting RNA guide may direct the Casl2i polypeptide as described herein to an LDHA target sequence. Two or more RNA guides may target two or more separate Casl2i polypeptides (e.g., Casl2i 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 or is HAO1 target- specific. That is, in some embodiments, an RNA guide binds specifically to one or more LDHA target sequences or one or more HAO1 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 Casl2i2 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 or an HA01 target sequence. See, e.g., Example 1, where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 polypeptide of SEQ ID NO: 1168 to HEK293T cells by RNP; Example 2, where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 polypeptide of SEQ ID NO: 1168 to HepG2 cells by RNP; and Example 3, where indels were measured at HA01 and LDHA target sequences following delivery of an RNA guide and a Casl2i2 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. Casl2i2 Direct Repeat Sequences
Figure imgf000023_0001
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. Casl2i4 Direct Repeat Sequences
Figure imgf000027_0001
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. Casl2il Direct Repeat Sequences
Figure imgf000028_0001
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. Casl2i3 Direct Repeat Sequences
Figure imgf000028_0002
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 Sequences
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 a specific target 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 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 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 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.
In some embodiments, the spacer sequence is a sequence of Table 6 or a portion of a sequence of Table 6. It should be understood that an indication of SEQ ID NOs: 1668-2122 should be considered as equivalent to a listing of SEQ ID NOs: 1668-2122, with each of the intervening numbers present in the listing, consistent with the listing of SEQ ID NOs: 588- 1164, above.
The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 1668- 2122. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 1668-2122. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 1668-2122.
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 6 or a portion of a sequence of Table 6. 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: 1668- 2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. 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: 1668-2122. The spacer sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of 1668-2122.
Table 5. Target and Spacer Sequences - LDHA
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Table 6. Target and Spacer Sequences - HAO1
Figure imgf000053_0002
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
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 an uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 or Table 6 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5 or Table 6. (iii) Exemplary RNA Guides
The present disclosure provides RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g. , as set forth in Tables 5 and 6, above). In some embodiments, the sequence of the RNA guide targeting HAO1 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: 2131-2187. In some embodiments, the RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187. In some embodiments, the sequence of the RNA guide targeting LDHA 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: 2188-2204. In some embodiments, the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
In specific examples, the RNA guide targeting HAO1 may be E1T2 disclosed in Table 8 below (e.g., the modified version provided in Table 8) and the RNA guide targeting LDHA may be E5T9 also provided in Table 8 (e.g., the modified version provided in Table 8).
(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 the present disclosure.
Exemplary modifications can include any modification to the sugar, the nucleobase, the intemucleoside 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 intemucleoside 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 intemucleoside 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 a-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-( l-thiophosphate)-adenosine, 5'-O-( l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(l-thiophosphate)-guanosine, 5'-O-( l-thiophosphate)-uridine, or 5’-O-(l- 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, l-(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-fhioro-l-(tetrahydrofuran-2-yl)pyrimidine- 2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2 ’-deoxy-2’ -methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l- beta-D-arabinofuranosylcytosine, N4-octadecyl-l-beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(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 -carboxymethylpseudouridine, 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-l -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-l -methyl-pseudoisocytidine, 4-thio-l - methyl-l-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, l-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 AD ARI 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.
In some embodiments, an HAO 1 -targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 2287-2292. In some embodiments, an HAO 1 -targeting RNA guide comprises any one of SEQ ID NOs: 2287-2292. In some embodiments, an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2288 or SEQ ID NO: 2289 recognizes the HAO1 target sequence of SEQ ID NO: 2234. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2288 or SEQ ID NO: 2289 recognizes the HAO1 target sequence of SEQ ID NO: 2234. In some embodiments, an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2290 or SEQ ID NO: 2291 recognizes the HAO1 target sequence of SEQ ID NO: 2213. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2290 or SEQ ID NO: 2291 recognizes the HAO1 target sequence of SEQ ID NO: 2213. In some embodiments, an HAO 1 -targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2292 or SEQ ID NO: 2293 recognizes the HAO1 target sequence of SEQ ID NO: 2212. In some embodiments, the HAO 1 -targeting RNA guide of SEQ ID NO: 2292 or SEQ ID NO: 2293 recognizes the HAO1 target sequence of SEQ ID NO: 2212.
In some embodiments, an LDHA-targeting RNA guide comprises at least 90% identity to any one of SEQ ID NOs: 2293-2302. In some embodiments, an LDHA-targeting RNA guide comprises any one of SEQ ID NOs: 2293-2302. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2293 or SEQ ID NO: 2294 recognizes the LDHA target sequence of SEQ ID NO: 2270. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2293 or SEQ ID NO: 2294 recognizes the LDHA target sequence of SEQ ID NO: 2270. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2295 or SEQ ID NO: 2296 recognizes the LDHA target sequence of SEQ ID NO: 2272. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2295 or SEQ ID NO: 2296 recognizes the LDHA target sequence of SEQ ID NO: 2272. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2297 or SEQ ID NO: 2298 recognizes the LDHA target sequence of SEQ ID NO: 2281. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2297 or SEQ ID NO: 2298 recognizes the LDHA target sequence of SEQ ID NO: 2281. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2299 or SEQ ID NO: 2300 recognizes the LDHA target sequence of SEQ ID NO: 2278. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2299 or SEQ ID NO: 2300 recognizes the LDHA target sequence of SEQ ID NO: 2278. In some embodiments, an LDHA-targeting RNA guide comprising at least 90% identity to SEQ ID NO: 2301 or SEQ ID NO: 2302 recognizes the LDHA target sequence of SEQ ID NO: 2282. In some embodiments, the LDHA-targeting RNA guide of SEQ ID NO: 2301 or SEQ ID NO: 2302 recognizes the LDHA target sequence of SEQ ID NO: 2282.
When a gene editing system disclosed herein comprises nucleic acids encoding the Casl2i polypeptide disclosed herein, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.
B. CasI2i Polypeptide
In some embodiments, the composition of the present disclosure includes a Casl2i 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 Casl2i2 polypeptide described herein (e.g. , a polypeptide comprising SEQ ID NO: 1166 and/or encoded by SEQ ID NO: 1165). In some embodiments, the Casl2i2 polypeptide comprises at least one RuvC domain.
A nucleic acid sequence encoding the Casl2i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1165. In some embodiments, the Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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 some embodiments, the Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i2 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 Casl2i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1202 and/or encoded by SEQ ID NO: 1201). In some embodiments, the Casl2i4 polypeptide comprises at least one RuvC domain.
A nucleic acid sequence encoding the Casl2i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1201. In some embodiments, the Casl2i4 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 Casl2i4 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 Casl2i4 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 Casl2i4 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 Casl2i4 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 Casl2i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1203 or SEQ ID NO: 1204.
In some embodiments, the Casl2i4 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 Casl2i4 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 Casl2i4 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 Casl2i4 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 Casl2il polypeptide described herein (e.g. , a polypeptide comprising SEQ ID NO: 1211). In some embodiments, the Casl2i4 polypeptide comprises at least one RuvC domain.
In some embodiments, the Casl2il 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 Casl2il 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 Casl2il 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 Casl2i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1212). In some embodiments, the Casl2i4 polypeptide comprises at least one RuvC domain.
In some embodiments, the Casl2i3 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 Casl2i3 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 Casl2i3 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 Casl2i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions. For example, the Casl2i 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 Casl2i 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 Casl2i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Casl2i polypeptide comprises at least one (e.g. , two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Casl2i 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 Casl2i polypeptide described herein can be selfinactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Then, 24 (2016): S50, which is incorporated by reference in its entirety.
In some embodiments, the nucleotide sequence encoding the Casl2i 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 Casl2i polypeptides such as Casl2i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized. Exemplary Casl2i polypeptide sequences and corresponding nucleotide sequences are listed in Table 7.
Table 7. Casl2i, LDHA, HAO1, and RNA Guide Sequences
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
In some embodiments, the gene editing system disclosed herein may comprise a Casl2i polypeptide as disclosed herein. In other embodiments, the gene editing system may comprise a nucleic acid encoding the Casl2i 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, AAVrhlO, AAV11 and AAV12) encoding the Casl2i polypeptide. Alternatively, the gene editing system may comprise a mRNA molecule encoding the Casl2i 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 guides, methods for production of the Casl2i polypeptide, and methods for complexing the RNA guide and Casl2i 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 may encode 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 Casl2i polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Casl2i 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. Casl2i Polypeptide
In some embodiments, the Casl2i polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Casl2i polypeptide of the present disclosure, isolating the Casl2i polypeptide, optionally, purifying the Casl2i polypeptide, and complexing the Casl2i polypeptide with an RNA guide. The Casl2i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Casl2i 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 Casl2i 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 Casl2i 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 Casl2i polypeptide. After expression of the Casl2i polypeptide, the host cells can be collected and Casl2i 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 Casl2i 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 Casl2i 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 Casl2i polypeptide.
A variety of methods can be used to determine the level of production of a Casl2i 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 Casl2i 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 Casl2i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Casl2i polypeptide to a host cell wherein the polyribonucleotide encodes the Casl2i polypeptide, expressing the Casl2i polypeptide in the cell, and obtaining the Casl2i polypeptide from the cell.
The present disclosure further provides methods of in vivo expression of Casl2i polypeptides in a cell, comprising providing a polyribonucleotide encoding the Casl2i polypeptides to a host cell wherein the polyribonucleotide encodes the Casl2i polypeptides and expressing the Casl2i polypeptides in the cell. In some embodiments, the polyribonucleotide encoding the Casl2i polypeptides is delivered to the cell with RNA guides and, once expressed in the cell, the Casl2i polypeptides and the RNA guides form complexes. In some embodiments, the polyribonucleotide encoding the Casl2i polypeptides and the RNA guides are delivered to the cell within a single composition (i.e., a composition comprising an RNA guide targeting HA01, an RNA guide targeting LDHA, and an RNA encoding a Casl2i polypeptide). In some embodiments, the polyribonucleotide encoding the Casl2i polypeptides and the RNA guides are comprised within separate compositions (e.g., (i) a composition comprising an RNA guide targeting HAO1 and an RNA guide targeting LDHA, and a separate composition comprising an RNA encoding a Casl2i polypeptide, (ii) a composition comprising an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide, and a separate composition comprising an RNA guide targeting LDHA, (iii) a composition comprising an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide, and a separate composition comprising an RNA guide targeting HAO1, (iv) a composition comprising an RNA guide targeting HAO1, a separate composition comprising an RNA guide targeting LDHA, and a separate composition comprising a Casl2i, and (v) a composition comprising an RNA guide targeting HAO1 and an RNA encoding a Casl2i polypeptide, and a separate composition comprising an RNA guide targeting LDHA and an RNA encoding a Casl2i polypeptide). 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 or HAO1 is complexed with a Casl2i polypeptide to form a ribonucleoprotein. In some embodiments, complexation of the RNA guide and Casl2i 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 Casl2i polypeptide at about 37°C over an incubation period of at least about any one of lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Ihr, 2hr, 3hr, 4hr, or more hours.
In some embodiments, the RNA guide and Casl2i polypeptide are complexed in a complexation buffer. In some embodiments, the Casl2i 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 Casl2i 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 Casl2i 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 Casl2i polypeptide is introduced into a cell so that the Casl2i 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 HAO1 gene and a target site with the LDHA gene. In some embodiments, the methods comprise introducing an HAO 1 -targeting RNA guide, an LDHA-targeting RNA guide, and a Casl2i polypeptide into a cell. The HAO 1 -targeting RNA guide and/or the LDHA-targeting RNA guide, and the Casl2i polypeptide can be introduced as a ribonucleoprotein complex into a cell. The HAO 1 -targeting RNA guide, the LDHA-targeting guide, and/or the Casl2i polypeptide can be introduced on a nucleic acid vector. The Casl2i polypeptide can be introduced as an mRNA. The RNA guides can be introduced directly into the cell. In some embodiments, the gene editing system described herein is delivered to a cell/tissue/liver/person to reduce HAO1 and LDHA in the cell/tissue/liver/person. In some embodiments, the gene editing system 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 HAO1 gene and an LDHA gene. The gene editing system may comprise RNA guides and a Casl2i2 polypeptide. The RNA guides each comprise a spacer sequence specific to a target sequence in the HAO1 gene, e.g. , specific to a region in exonl or exon 2 of the HAO1 gene, or 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. In some embodiments, the LDHA gene is a mammalian gene. In some embodiments, the LDHA gene is a human gene. In some embodiments, the sequence of the LDHA gene is set forth in SEQ ID NO: 1172 (or is the reverse complement thereof). In some embodiments, the target sequence is in an exon of an LDHA gene, such as an exon having a sequence set forth in any one of SEQ ID NO: 1173, SEQ ID NO: 1174, SEQ ID NO: 1175, SEQ ID NO: 1176, SEQ ID NO: 1177, SEQ ID NO: 1178, SEQ ID NO: 1179, SEQ ID NO: 1180, or SEQ ID NO: 1181 (or a reverse complement thereof). In some embodiments, the target sequence is in an intron of an LDHA gene (e.g. , an intron of the sequence set forth in SEQ ID NO: 1172 or the reverse complement thereof). In other embodiments, the sequence of the LDHA gene is a variant of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof). For example, in some embodiments, the target sequence is polymorphic variant of the LDHA sequence set forth in SEQ ID NO: 1172 (or the reverse complement thereof) or a non-human form of the LDHA 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: 2205-2209 (or a 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: 2205-2209 (or a reverse complement thereof).
In some embodiments, the sequence of the HAO1 gene is set forth in SEQ ID NO: 2123 (or is the reverse complement thereof). In some embodiments, the target sequence is in an exon of an HAO1 gene, such as an exon having a sequence set forth in any one of SEQ ID NO: 2124, SEQ ID NO: 2125, SEQ ID NO: 2126, SEQ ID NO: 2127, SEQ ID NO: 2128, SEQ ID NO: 2129, and SEQ ID NO: 2130 (or a reverse complement of any thereof). In some embodiments, the target sequence is in an intron of an HAO1 gene (e.g. , an intron of the sequence set forth in SEQ ID NO: 2123 or the reverse complement thereof). In other embodiments, the sequence of the HAO1 gene is a variant of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof). For example, in some embodiments, the target sequence is polymorphic variant of the HAO1 sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof) or a non-human form of the HAO1 gene. In some embodiments, the target sequence is within an HAO1 gene or a locus of an HAO1 gene. In some embodiments, the HAO1 gene is a mammalian gene. In some embodiments, the HAO1 gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of SEQ ID NO: 2123 (or the reverse complement thereof). In some embodiments, the target sequence is within an exon of the HAO1 gene set forth in SEQ ID NO: 2123, e.g., within a sequence of SEQ ID NO: 2124, 2125, 2126, 2127, 2128, 2129, or 2130 (or a reverse complement thereof). Target sequences within an exon region of the HAO1 gene of SEQ ID NO: 2123 are set forth in Table 6. In some embodiments, the target sequence is within an intron of the HAO1 gene set forth in SEQ ID NO: 2123 (or the reverse complement thereof). In some embodiments, the target sequence is within a variant (e.g. , a polymorphic variant) of the HAO1 gene sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof). In some embodiments, the HAO1 gene sequence is a homolog of the sequence set forth in SEQ ID NO: 2123 (or the reverse complement thereof). For examples, in some embodiments, the HAO1 gene sequence is a non-human HAO1 sequence. In some embodiments, the HAO1 gene sequence is a coding sequence set forth in SEQ ID NO: 2210 (or the reverse complement thereof). In some embodiments, the HAO1 gene sequence is a homolog of a coding sequence set forth in SEQ ID NO: 2210 (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 Casl2i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Casl2i polypeptide induces one or more DNA doublestranded breaks in the cell. In some embodiments, the Casl2i polypeptide induces one or more DNA single- stranded breaks in the cell. In some embodiments, the Casl2i 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 Casl2i polypeptide and directs the Casl2i 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 alters expression of the HAO1 gene. In some embodiments, the deletion alters function of the HAO1 gene. In some embodiments, the deletion inactivates the HAO1 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 ’-ATTIS’, 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 and in an HAO1 gene. In some embodiments, the methods are carried out using complexes comprising a Casl2i 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 targeting HAO1 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: 2131-2187. In some embodiments, an RNA guide targeting HAO1 has a sequence of any one of SEQ ID NOs: 2131-2187. In some embodiments, the sequence of the RNA guide targeting LDHA 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: 2188-2204. In some embodiments, an RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
In some embodiments, the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is a synthetic or purified RNA. In some embodiments, the Casl2i polypeptide is encoded in a plasmid. In some embodiments, the Casl2i 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 Casl2i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Casl2i polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and an RNA encoding a Casl2i polypeptide are delivered together in a single composition, e.g. , as described herein. In some embodiments, an RNA guide and an RNA encoding a Casl2i polypeptide are delivered in separate compositions, e.g., as described herein. In some embodiments, an RNA guide and an RNA encoding a Casl2i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Casl2i polypeptide delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses 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 Casl2i polypeptide, an RNA guide, or an mRNA encoding a Casl2i polypeptide and an RNA guide. In some embodiments, the mRNA encoding the Casl2i 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.
In some embodiments, the gene editing system comprises a nucleic acid such as an mRNA encoding any of the Casl2i polypeptides disclosed herein (e.g., any of the Casl2i2 polypeptides disclosed herein), an RNA guide targeting the HAO1 gene (e.g., those disclosed herein, e.g., in Table 8), and an RNA guide targeting the LDHA gene (e.g., those disclosed herein, e.g., in Table 8) and LNPs associated with one or all of the just noted components. In some instances, at least a portion of the mRNAs, the RNA guide targeting HAO1, and the RNA guide targeting LDHA are encapsulated by the LNPs.
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 and/or disrupted HAO1 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 PHI, 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 PHI, PH2, or PH3. In specific examples, the target disease is PHI.
Primary hyperoxaluria (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. PHI 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 PHI, 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 PHI include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PHI 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 PHI normally have kidney failure at an early age, with renal dialysis or dual kidney /liver organ transplant as the only treatment options.
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. Hydroxyacid oxidase 1 (HAO1, also known as glycolate oxidase [GOX or GO]) converts glycolate into glyoxylate. It has been proposed that inhibition of HAO1 in individuals with PHI would block formation of glyoxylate, and excess glycolate would be excreted through the urine. The idea of treating PHI by inhibition of HAO1 is further supported that some individuals with abnormal splice variants of HAO1 are asymptomatic for glycolic aciduria, whereby there was increased urinary glycolic acid excretion without apparent kidney pathology. Thus, inhibition of HAO1 expression would block production of glyoxylate, and in turn block production of its metabolite, oxalate.
In some embodiments, provided herein is a method for treating a target disease as disclosed herein (e.g., PH such as PHI) 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 associated with (e.g., encompassing) one or more of the components, one or more nucleic acids (e.g., vectors such as viral vectors or mRNA molecules) 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 and/or one particular gene edit in the HAO1 gene. In some examples, the cells can be suspended in a suitable media.
In some embodiments, an RNA guide targeting HAO1 is contacted with a cell before an RNA guide targeting LDHA. In some embodiments, an RNA guide targeting HAO1 is contacted with a cell at the same time as an RNA guide targeting LDHA. In some embodiments, an RNA guide targeting HAO1 is contacted with a cell after the cell is contacted with an RNA guide targeting LDHA. In some embodiments, a composition comprising an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell before an RNA guide targeting LDHA and a second Casl2i polypeptide. In some embodiments, an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell at the same time as an RNA guide targeting LDHA and a second Casl2i polypeptide. In some embodiments, an RNA guide targeting HAO1 and a first Casl2i polypeptide is contacted with a cell after the cell is contacted with an RNA guide targeting LDHA and a second Casl2i polypeptide. In some embodiments, the RNA guide targeting HAO1 is encoded in a plasmid. In some embodiments, the RNA guide targeting LDHA is encoded in a plasmid. In some embodiments, the RNA guide targeting HAO1 is synthetic or purified RNA. In some embodiments, the RNA guide targeting LDHA is synthetic or purified RNA. In some embodiments, the first Casl2i polypeptide is encoded in a plasmid. In some embodiments, the second Casl2i polypeptide is encoded in a plasmid. In some embodiments, the first Casl2i polypeptide and the second Casl2i polypeptide are identical in sequence. In some embodiments, the first Casl2i polypeptide and the second Casl2i polypeptide are different sequences.
In some embodiments, an RNP targeting HAO1 (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1) is contacted with a cell before an RNP targeting LDHA (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA). In some embodiments, an RNP targeting HAO1 (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1) is contacted with a cell at the same time as an RNP targeting LDHA (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA). In some embodiments, an RNP targeting HAO1 (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting HAO1) is contacted with a cell after the cell is contacted with an RNP targeting LDHA (e.g., an RNP comprising a Casl2i polypeptide and an RNA guide targeting LDHA). In some embodiments, the Casl2i polypeptide of the RNP targeting HAO1 and the Casl2i polypeptide of the RNP targeting LDHA are identical in sequence. In some embodiments, the Casl2i polypeptide of the RNP targeting HAO1 and the Casl2i polypeptide of the RNP targeting LDHA are different sequences.
In some embodiments, provided herein is a composition comprising any of the gene editing systems described above 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 and/or HAO1 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 and/or HAO1 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 Casl2i polypeptide. In some embodiments, the kits include a polynucleotide that encodes such a Casl2i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g. , as described herein. The Casl2i 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 Casl2i 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.
In one aspect of the composition, the target sequence within the LDHA gene 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, and/or the target sequence within the HAO1 gene is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the HAO1 gene.
In another aspect of the composition, 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, and/or the HAO1 gene comprises the sequence of SEQ ID NO: 2123, the reverse complement of SEQ ID NO: 2123, a variant of SEQ ID NO: 2123, or the reverse complement of a variant of SEQ ID NO: 2123.
In another aspect of the composition, (i) the spacer sequence that is substantially complementary to a target sequence within an LDHA gene 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; 1. 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; and/or (ii) the spacer sequence that is substantially complementary to a target sequence within an HA01 gene 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1668-2122; 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: 1668-2122; 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: 1668-2122; 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: 1668-2122.
In another aspect of the composition (i) the spacer sequence that is substantially complementary to a target sequence within an LDHA gene 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; 1. 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; and/or (ii) the spacer sequence that is substantially complementary to a target sequence within an HA01 gene comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 1668-2122; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 1668-2122; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 1668-2122; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 1668-2122; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 1668-2122; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 1668-2122; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 1668-2122; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 1668-2122; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 1668-2122; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 1668-2122; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 1668-2122; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 1668-2122; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 1668-2122; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 1668-2122; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 1668-2122.
In another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HAO1 gene comprise(s): 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; 1. 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 another aspect of the composition, the direct repeat sequence of the RNA guide targeting the LDHA gene and/or the RNA guide targeting the HA01 gene comprise(s): 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; 1. 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 another aspect of the composition the spacer sequence that is substantially complementary to a target sequence within an LDHA gene is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-587, and/or the spacer sequence that is substantially complementary to a target sequence within an HAO1 gene is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 1213-1667.
In another aspect of the composition, 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 another aspect of the composition, the target sequences are each immediately adjacent to the PAM sequence.
In another aspect of the composition, the RNA guide targeting HAO1 has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2131-2187 and/or wherein the RNA guide targeting LDHA has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2188-2204.
In another aspect of the composition, the RNA guide targeting HAO1 has the sequence of any one of SEQ ID NOs: 2131-2187 and/or wherein the RNA guide targeting LDHA has a sequence of any one of SEQ ID NOs: 2188-2204.
In another aspect of the composition, the composition further comprises Casl2i polypeptides or a polynucleotide encoding Casl2i polynucleotides.
In another aspect of the composition, the Casl2i polypeptides are selected from: a. a Casl2i2 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 Casl2i4 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 Casl2il polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1211; or d. a Casl2i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1212.
In another aspect of the composition, the Casl2i polypeptides are selected from: a. a Casl2i2 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 Casl2i4 polypeptide comprising a sequence of SEQ ID NO: 1202, SEQ ID NO: 1203, or SEQ ID NO: 1204; c. a Casl2il polypeptide comprising a sequence of SEQ ID NO: 1211; or d. a Casl2i3 polypeptide comprising a sequence of SEQ ID NO: 1212.
In another aspect of the composition, the RNA guides and the Casl2i polypeptides form ribonucleoprotein complexes.
In another aspect of the composition, the ribonucleoprotein complexes bind target nucleic acids.
In another aspect of the composition, the composition is present within a cell.
In another aspect of the composition, the RNA guides and the Casl2i polypeptides are encoded in one or more vectors, e.g., expression vectors.
In another aspect of the composition, the RNA guides and the Casl2i polypeptides are encoded in a single vector, the RNA guides are encoded in a first vector and the Casl2i polypeptides are encoded in a second vector, or the RNA guides are separately encoded in first and second vectors and the Casl2i polypeptides are encoded in a third vector.
The present disclosure yet further provides a nucleic acid encoding an RNA guide targeting HAO1 and an RNA guide targeting LDHA, wherein the RNA guides are of a composition described herein.
The present disclosure yet further provides a vector comprising the nucleic acid.
The present disclosure yet further provides a vector system comprising one or more vectors encoding (i) an RNA guide targeting HAO1 and an RNA guide targeting LDHA, as described herein, and (ii) a Casl2i polypeptide, optionally wherein the vector system comprises a first vector encoding one or more of the RNA guides and a second vector encoding the Casl2i polypeptide. The present disclosure yet further provides a cell comprising a composition described herein, a nucleic acid described herein, a vector described herein, or a vector system described herein.
In another aspect of the cell, 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 hepatocyte.
The present disclosure yet further provides a kit comprising a composition described herein, a nucleic acid described herein, a vector described herein, or a vector system described herein.
The present disclosure yet further provides a method of editing an LDHA sequence and an HAO1 sequence, the method comprising contacting an LDHA sequence and an HAO1 sequence with a composition of any described herein.
In one aspect of the method, the LDHA sequence and the HAO1 sequence are in a cell.
In another aspect of the method, the composition or the RNA guides induce a deletion in the LDHA sequence and/or a deletion in the HAO1 sequence.
In another aspect of the method, at least one deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide.
In another aspect of the method, at least one deletion is downstream of the 5’-NTTN- 3’ sequence.
In another aspect of the method, at least one deletion is up to about 50 nucleotides in length.
In another aspect of the method, at least one deletion is up to about 40 nucleotides in length.
In another aspect of the method, at least one deletion is from about 4 nucleotides to 40 nucleotides in length.
In another aspect of the method, at least one deletion is from about 4 nucleotides to 25 nucleotides in length.
In another aspect of the method, at least one deletion is from about 10 nucleotides to 25 nucleotides in length.
In another aspect of the method, at least one deletion is from about 10 nucleotides to 15 nucleotides in length.
In another aspect of the method, at least one deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence. In another aspect of the method, at least one deletion starts within about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion starts within about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
In another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, at least one 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 another aspect of the method, 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 another aspect of the method, the deletion in the LDHA sequence overlaps with a mutation in the LDHA sequence, and/or the deletion in the HA01 sequence overlaps with a mutation in the HA01 sequence.
In another aspect of the method, the deletion in the LDHA sequence overlaps with an insertion in the LDHA sequence, and/or the deletion in the HA01 sequence overlaps with an insertion in the HA01 sequence.
In another aspect of the method, the deletion in the LDHA sequence removes a repeat expansion of the LDHA sequence or a portion thereof, and/or the deletion in the HA01 sequence removes a repeat expansion of the LDHA sequence or a portion thereof.
In another aspect of the method, the deletion in the LDHA sequence disrupts one or both alleles of the LDHA sequence, and/or the deletion in the HA01 sequence disrupts one or both alleles of the HA01 sequence.
In another aspect of the composition, nucleic acid, vector, cell, kit or method of described herein, the RNA guide comprises the sequence of any one of SEQ ID NOs: 2131- 2204. The present disclosure yet further provides a method of treating primary hyperoxaluria (PH), which optionally is PHI, PH2, or PH3, in a subject, the method comprising administering a composition or a cell described herein to the subject.
In another aspect of the composition, the cell, the kit, or the method described herein, the RNA guides and/or the polyribonucleotide encoding the Casl2i polypeptide are comprised within a lipid nanoparticle.
In another aspect of the composition, the cell, the kit, or the method described herein, the RNA guides and the polyribonucleotide encoding the Casl2i polypeptide are comprised within the same lipid nanoparticle.
In another aspect of the composition, the cell, the kit, or the method described herein, the RNA guides and the polyribonucleotide encoding the Casl2i polypeptide are comprised within separate lipid nanoparticles.
The present disclosure yet furthers provides a composition comprising: a. a first RNA guide comprising (i) a spacer sequence that is complementary to a first target sequence and (ii) a direct repeat sequence, wherein the first target sequence is an LDHA gene sequence of any one of SEQ ID NOs: 2270, 2272, 2281, 2278, or 2282 or the reverse complement thereof; and b. a second RNA guide comprising (i) a spacer sequence that is complementary to a second target sequence and (ii) a direct repeat sequence, wherein the second target sequence is an HAO1 gene sequence of any one of SEQ ID NOs: 2234, 2213, or 2212 or the reverse complement thereof.
In another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the direct repeat sequence of the first RNA guide and/or the second RNA guide 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; 1. 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 another aspect of the composition, the first RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2189, 2211, 2199, 2196, or 2200, and wherein the second RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2153, 2132, or 2131.
In another aspect of the composition, the first RNA guide has the sequence of any one of SEQ ID NOs: 2189, 2211, 2199, 2196, or 2200, and wherein the second RNA guide has a sequence of any one of SEQ ID NOs: 2153, 2132, or 2131.
In another aspect of the composition, each of the first three nucleotides of the first RNA guide and/or the second RNA guide comprises a 2’-O-methyl phosphorothioate modification.
In another aspect of the composition, each of the last four nucleotides of the first RNA guide and/or the second RNA guide comprises a 2’-O-methyl phosphorothioate modification. In another aspect of the composition, each of the first to last, second to last, and third to last nucleotides of the first RNA guide and/or the second RNA guide comprises a 2’-O- methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified.
In another aspect of the composition, the first RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2293-2302, and wherein the second RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 2287-2292.
In another aspect of the composition, the first RNA guide has the sequence of any one of SEQ ID NOs: 2293-2302, and wherein the second RNA guide has a sequence of any one of SEQ ID NOs: 2287-2292.
The present disclosure further provides a nucleic acid encoding the first RNA guide of the composition described herein.
The present disclosure further provides a nucleic acid encoding the second RNA guide of the composition described herein.
The present disclosure further provides a vector comprising the nucleic acid described herein.
The present disclosure further provides a vector system comprising one or more vectors encoding (i) the first RNA guide of the composition described herein, (ii) the second RNA guide of the composition described herein, and (iii) a Casl2i polypeptide, optionally wherein the vector system comprises a first vector encoding the first RNA guide, a second vector encoding the second RNA guide, and a third vector encoding the Casl2i polypeptide.
The present disclosure further provides a cell comprising the composition, the nucleic acid, the vector, or the vector system described herein.
In yet another aspect of the cell, 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.
The present disclosure further provides a kit comprising the composition, the nucleic acid, the vector, or the vector system described herein.
The present disclosure further provides a method of editing an LDHA sequence and an HAO1 sequence, the method comprising contacting an LDHA sequence with the composition described herein.
In yet another aspect of the method, the LDHA sequence and the HAO1 sequence are in a cell. In yet another aspect of the method, the first RNA guide induces an indel in the LDHA sequence and the second RNA guide induces an indel in the HAO1 sequence.
The present disclosure further provides a method of treating primary hyperoxaluria (PH), which optionally is PHI, PH2, or PH3, in a subject, the method comprising administering the composition or the cell described herein.
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 (IRL 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 invention but are not intended to limit the scope of the invention; 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 - Casl2i2-Mediated Editing of HAO1 and LDHA Target Sites in HEK293T cells
This Example describes the genomic editing of the HA01 gene or the LDHA gene using Casl2i2 introduced into HEK293T cells.
Casl2i2 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 HAO 1 or 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 HAO 1 -targeting RNA guide sequences are shown in Table 8. The LDHA-targeting RNA guide sequences are shown in Table 9. TS stands for “top strand” of the HA01 or LDHA gene, and BS stands for “bottom strand” of the HAO1 or LDHA gene. In the figures, “E#T#” can also be represented as “exon # target #.”
Table 8. crRNA Sequence for HAO1
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Table 9. crRNA Sequences for LDHA
Figure imgf000161_0002
Figure imgf000162_0001
Figure imgf000163_0001
Casl2i2 RNP complexation reactions were made by mixing purified Casl2i2 polypeptide (400 pM) with an HAO 1 -targeting crRNA or an LDHA-targeting crRNA (1 mM in 250 mM NaCl) at a 1:1 (Casl2i2:crRNA) volume ratio (2.5:1 crRNA:Casl2i2 molar ratio). Complexations were incubated on ice for 30-60 min.
HEK293T 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 CEEE FINE 4D-NUCEEOFECTOR™ X KIT S; Eonza #V4XC- 2032) at a concentration of 16,480 cells/pE. Resuspended cells were dispensed at 3e5 cells/reaction into Eonza 16-well NUCLEOCUVETTE® strips. Complexed Casl2i2 RNP was added to each reaction at a final concentration of 10 pM (Casl2i2), and transfection enhancer oligos were then added at a final concentration of 4 pM. The final volume of each electroporated reaction was 20 pL. 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 pL of prewarmed DMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 pL (30,000 cells) of diluted nucleofected cells into prewarmed 96-well plate with wells containing 100 pL 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; ThermoFisher) and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 pL 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 indels at HA01 target sites in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the HA01 target sites with each of the RNA guides. Delivery of E1T2, E1T3, E1T6, E1T7, E1T13, T1T17, E2T4, E2T5, E2T9, E2T10, E3T6, E3T19, E3T22, and E3T28 resulted in indels in over 70% of the NGS reads. Therefore, HAO 1 -targeting RNA guides induced indels in exon 1, exon 2, and exon 3 of the HAO1 gene in HEK293T cells.
FIG. 2 shows indels at LDHA target sites in HEK293T cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the LDHA target sites with each of the RNA guides. Delivery of E3T1, E3T9, E5T1, E5T9, and E5T10 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 of the LDHA gene in HEK293T cells. This Example thus shows that HAO1 and LDHA can be individually targeted by Casl2i2 RNPs in mammalian cells such as HEK293T cells.
Example 2 - Casl2i2-Mediated Editing of HAO1 and LDHA Target Sites in HepG2 Cells
This Example describes the genomic editing of the HAO1 gene or the LDHA gene using Casl2i2 introduced into HepG2 cells by RNP.
RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 8 or Table 9. 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/pL. Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Casl2i2 RNP was added to each reaction at a final concentration of 20 pM (Casl2i2), with no transfection enhancer oligo. The final volume of each electroporated reaction was 20 pL. 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 pL of prewarmed EMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 pL (25,000 cells) of diluted nucleofected cells into prewarmed 96-well plate with wells containing 100 pL 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 pL 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. 3 shows indels at HAO1 target sites in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in each of the HAO1 target sites with each of the RNA guides. Therefore, HAO 1 -targeting RNA guides induced indels in exon 1, exon 2, and exon 3 of the HAO1 gene in HepG2 cells.
FIG. 4 shows indels at LDHA target sites in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected in 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 of the LDHA gene in HepG2 cells.
This Example thus shows that HAO1 and LDHA can be individually targeted by Casl2i2 RNPs in mammalian cells such as HepG2 cells.
Example 3 - Casl2i2-Mediated Editing of HAO1 and LDHA Target Sites in Primary Hepatocytes
This Example describes the genomic editing of the HAO1 using Casl2i2 introduced into primary hepatocytes cells by RNP.
RNP complexation reactions were performed as described in Example 1 with RNA guides for HAO1 of Table 8 and RNA guides for LDHA of Table 9. Additionally, for multiplexing experiments, shown in FIG. 6, separate HAO 1 -targeting RNPs and LDHA- targeting RNPs were mixed together at a 1 : 1 volume ratio prior to electroporation. 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 100g 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/pL. 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 Casl2i2 RNP was added to each reaction at a final concentration of 20 pM (Casl2i2), and transfection enhancer oligos were then added at a final concentration of 4 pM. The final volume of each electroporated reaction was either 20 pL in the 16 well nucleocuvette strip format or 100 pL 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 CAI 37 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 (500rpm) in a 37°C incubator with 2mg/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 pL 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 5x 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 pL Digital Droplet PCR (ddPCR- BioRad laboratories) reaction using target specific primer and probes for HA01 and LDHA, and 2x 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. ddPCR primers:
HAO1: ATTGTGCACTGTCAGATCTTGGAAACGGCCAAAGGATTTTTCCTCACCAATGTCTTGTCG ATGACTTTCACATTCTGGCACCCACTCAGAGCCATGGCCAACCGGAATTCTTCCTTTAGT AT (SEQ ID NO: 2303)
LDHA:
TTTTCCTTAGAACACCAAAGATTGTCTCTGGCAAAGACTATAATGTAACTGCAAACTCC AAGCTGGTCATTATCACGGCTGGGGCACGTCAGCAAGAGGGAGAAAGCCGTCTTAATTT GGTC (SEQ ID NO: 2304)
As shown in FIG. 5, each HAO 1 -targeting RNA guide induced indels within and/or adjacent to the HAO1 target sites and each LDHA-targeting RNA guide induced indels within and/or adjacent to the LDHA target sites. Indels were not induced with the nontargeting control. Therefore, HAO 1 -targeting RNA guides and LDHA-targeting RNA guides individually induced indels in primary hepatocytes.
FIG. 6 shows dual RNA guide editing with HAO 1 -targeting RNA guides (E2T10, E1T3, E1T2, E2T4, and E2T5 from Table 8) and LDHA-targeting RNA guides (E3T1, E3T3, E5T9, and E5T10 from Table 9) in primary human hepatocytes. The data shown is from 3 technical replicates. Indels at the HAO1 and LDHA target loci were observed for each dual RNA guide pairing. The top performing dual RNA guide pairings (HAO1 E1T2 and LDHA E3T1; HAO1 E1T3 and LDHA E3T1; HAO1 E2T5 and LDHA E3T1) induced HAO1 indels and LDHA indels in over about 90% of the NGS reads.
Indels were then correlated with mRNA levels for each target to determine whether indels led to mRNA knockdown and subsequent protein knockdown. FIG. 7 and FIG. 8 show % knockdown of HAO1 mRNA or LDHA mRNA, respectively, in edited cells compared to unedited control cells. As shown in FIG. 7, HAO1 E2T4 and HAO 1 E2T5 reduced HAO1 mRNA when delivered with or without LDHA E3T1. Although a higher percentage of NGS reads comprised indels using HAO1 E2T5 compared to HAO1 E2T4, HAO1 E2T4 resulted in a greater knockdown of HAO1 mRNA. As shown in FIG. 8, LDHA E3T1 reduced LDHA mRNA when delivered with or without HAO1 E2T4 or HAO1 E2T5.
This Example thus shows that HAO1 and LDHA can be individually targeted or simultaneously targeted by Casl2i2 RNPs in mammalian cells such as primary human hepatocytes, leading to indels in HAO1 and LDHA target sites and knockdown of HAO1 mRNA and LDHA mRNA.
Example 4 - Editing of HAO1 and LDHA Target Sites in HepG2 Cells with Casl2i2 Variants
This Example describes indel assessment on HAO1 target sites and LDHA target sites using Casl2i2 variants introduced into HepG2 cells by transient transfection. The Casl2i2 variants of SEQ ID NO: 1168 and SEQ ID NO: 1171 were individually cloned into a pcda3.1 backbone (Invitrogen). Nucleic acids encoding HAO 1 -targeting RNA guides (HAO1 E1T2, HAO1 E1T3, HAO1 E2T4, HAO1 E2T5, and HAO1 E2T10) (Table 8) and LDHA-targeting RNA guides (LDHA E3T1, LDHA E3T3, LDHA E5T1, LDHA E5T9, and LDHA E5T10) (Table 9) were individually 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™: Op tiMEM® 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 pL 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. 9A, two LDHA-targeting guides, E3T3 and E5T1, demonstrated significantly higher activity with variant Casl2i2 of SEQ ID NO: 1171 compared to variant Casl2i2 of SEQ ID NO: 1168. Comparable indel activity with the two Casl2i2 variants was observed for LDHA-targeting guides LDHA E3T1, LDHA E5T9, and LDHA E5T10 as well as each of the HAO 1 -targeting guides (HAO1 E1T2, HAO1 E1T3, HAO1 E2T4, HAO1 E2T5, and HAOl E2T10).
FIG. 9B shows the indel size frequency (left) and indel start position relative to the PAM for HAO1 E1T3 and the variant Casl2i2 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 6 nucleotides to about 27 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 10 nucleotides and about 35 nucleotides downstream of the PAM sequence. The majority of indels started near the end of the target sequence, e.g., about 18 nucleotides to about 25 nucleotides downstream of the PAM sequence. FIG. 9C shows the indel size frequency (left) and indel start position relative to the PAM for LDHA E5T9 and the variant Casl2i2 of SEQ ID NO: 1168 in HepG2 cells. As shown on the left, deletions ranged in size from 1 nucleotide to about 40 nucleotides; a small percentage of NGS reads comprised a 1 nucleotide insertion. 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 therefore shows that HA01 and LDHA are capable of being targeted by multiple Casl2i2 polypeptides.
Example 5 - Editing of HAO1 and LDHA in Primary Human Hepatocytes using Casl2i2 mRNA Constructs
This Example describes indel assessment on HA01 and LDHA target sites via delivery of Casl2i2 mRNA and chemically modified HAO 1 -targeting and LDHA-targeting RNA guides. mRNA sequences corresponding to the variant Casl2i2 sequence of SEQ ID NO: 1168 and the variant Casl2i2 sequence of SEQ ID NO: 1171 were synthesized with 1- pseudo-U modified nucleotides and using CleanCap® Reagent AG (TriLink Biotechnologies). The Casl2i2 mRNA sequences, shown in Table 10, further comprised a C- terminal NLS.
Table 10. Casl2i2 mRNA Sequences
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Casl2i2 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 11. TS stands for “top strand” of the HA01 gene, and BS stands for “bottom strand” of the LDHA gene. Each variant Casl2i2 mRNA was mixed with a crRNA at a 1:1 (Casl2i2:crRNA) volume ratio (1050: 1 crRNA:Casl2i2 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 11. Chemically Modified RNA Guide Sequences
Figure imgf000173_0002
* The 3’ three nucleotides represent the 5’-TTN-3’ motif.
FIG. 10 shows editing of an HA01 target site by a variant Casl2i2 mRNA and a 3’ end modified HA01 E2T5 guide or a 5’ and 3’ end modified HAO1 E2T5 RNA guide and editing of an LDHA target site by a variant Casl2i2 mRNA and a 3’ end modified LDHA E3T1 RNA guide or a 5’ and 3’ end modified LDHA E3T1 RNA guide. Indels in the HAO1 target site or the LDHA target site were introduced following electroporation of the Casl2i2 mRNA and each of the RNA guides.
For HAO1, approximately 50% of NGS reads comprised an indel following electroporation of the Casl2i2 mRNA of SEQ ID NO: 2306 and the RNA guide of SEQ ID NO: 2307 or SEQ ID NO: 2308. Statistically significant higher % indels were observed using variant Casl2i2 mRNA of SEQ ID NO: 2306 compared to variant Casl2i2 mRNA of SEQ ID NO: 2305. No statistical difference was observed using 5’ and 3’ modifications versus 3’ only modifications to RNA guide HAO1 E2T5.
For LDHA, a higher percentage of NGS reads exhibited indels for RNA guide LDHA E3T1 with 5’ and 3’ end modifications compared to percentage of NGS reads for RNA guide LDHA E3T1 with 3’ end modifications only. Approximately 50% of NGS reads comprised indels following electroporation of the Casl2i2 mRNA of SEQ ID NO: 2306 and the LDHA RNA guide of SEQ ID NO: 2310.
This Example thus shows that HAO1 and LDHA can be targeted by Casl2i2 mRNA constructs and chemically modified HAO 1 -targeting and LDHA-targeting RNA guides in mammalian cells.
Example 6 - Off-Target Analysis of Casl2i2 and HAO 1 -Targeting RNA Guides
This Example describes on-target versus off-target assessment of a Casl2i2 variant and an HAO 1 -targeting RNA guide or an LDHA-targeting guide.
HEK293T cells were transfected with a plasmid encoding the variant Casl2i2 of SEQ ID NO: 1168 or the variant Casl2i2 of SEQ ID NO: 1171 and a plasmid encoding HAO1 E3T1, HAO1 E5T1, HAO1 E5T9, HAO1 E5T10, LDHA E3T1, LDHA E5T1, LDHA E5T9, or LDHA E5T10 according to 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.
FIGS. 11A/B and 12A/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. 11A and FIG. 12A show TTISS reads for variant Casl2i2 of SEQ ID NO: 1168, and FIG. 11B and FIG. 12B show TTISS reads for variant Casl2i2 of SEQ ID NO: 1171. As shown in FIG. 11A, variant Casl2i2 of SEQ ID NO: 1168 paired with HAO1 E2T5 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. HAO1 E1T2 also showed a low likelihood of off-target editing. For HAO1 E1T2, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For HAO1 E5T10, 95% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 5% of TTISS reads. HA01 E2T10 demonstrated a higher likelihood of off-target editing using the TTISS method. For HA01 E2T10, only 65% of TTISS reads mapped to the on-target and 4 potential off-target sites represented the remaining combined 35% of TTISS reads. One potential off-target represented the majority of potential off-target TTISS reads for HA01 E2T10.
As shown in FIG. 11B, variant Casl2i2 of SEQ ID NO: 1171 paired with HA01 E2T5 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. Variant Casl2i2 of SEQ ID NO: 1171 paired with the HA01 E1T2 or HA01 E1T3 also demonstrated a low likelihood of off-target editing. For HA01 E1T2, 100% of TTISS reads in replicate 1 and 96% of TTISS reads in replicate 2 mapped to the on-target; two potential off-target sites represented the remaining 4% of TTISS reads in replicate 2. For HA01 E1T3, 100% of TTISS reads in replicate 1 and 92% of TTISS reads in replicate 2 mapped to the on-target; two potential off-target sites represented the remaining 8% of TTISS reads in replicate 2.
As shown in FIG. 12A, variant Casl2i2 of SEQ ID NO: 1168 paired with EDHA 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. EDHA E3T1 and LDHA E5T10 also showed a low likelihood of off-target editing. For LDHA E3T1, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For LDHA E5T10, 97% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 3% of TTISS reads. LDHA E5T1 demonstrated a higher likelihood of off-target editing using the TTISS method.
As shown in FIG. 12B, variant Casl2i2 of SEQ ID NO: 1171 paired with the LDHA 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. LDHA 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 Casl2i2 of SEQ ID NO: 1171 paired with the LDHA 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 LDHA E3T1.
Therefore, this Example shows that compositions comprising Casl2i2 and HA01- targeting and LDHA-targeting RNA guides comprise different off-target activity profiles.
Example 7 - HAO1 and LDHA Protein Knockdown with Casl2i2 and HAOl-Targeting and LDHA -Targeting RNA Guides
This Example describes use of a Western Blot to identify knockdown of HA01 or LDHA protein using variant Casl2i2 of SEQ ID NO: 1168 and HAO 1 -targeting or 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 100g 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 (serumcontaining), 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/pL. Resuspended cells were dispensed at 500,000 cells/reaction into Lonza electroporation cuvettes
For the RNP reactions, HA01 E2T5 was used as the HAO 1 -targeting RNA guide, and LDHA E3T1, LDHA E5T9, LDHA E5T1, and LDHA E5T10 were used as the LDHA- targeting RNA guides. RNPs were added to each reaction at a final concentration of 20 pM (Casl2i2), and transfection enhancer oligos were then added at a final concentration of 4 pM. Unelectroporated cells and cells electroporated without cargo were used as negative controls.
The strips were electroporated using an electroporation device (program CAI 37, 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 prewarmed 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.
Following electroporation (16 days for HAO1 and 7 days for EDHA), the media was aspirated, and the cells were washed gently with PBS. Cells were then lysed with RIPA Eysis and Extraction buffer (Thermo Fisher 89901) + IX 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 pg of total protein per sample was prepared for SDS-PAGE in IX Laemmlli Sample buffer (BioRad 1610747) and lOOmM DTT, then heated at 95C for 10 minutes. Samples were run on a 4-15% TGX gel (BioRad 5671084) at 200V for 45 minutes. Samples were transferred to a 0.2um 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-HAOl antibody (Genetex GTX81144) or 1:1000 dilution of primary anti-LDHA antibody (Abeam ab52488) and 1:2500 dilution of primary anti-vinculin antibody (Sigma V9131) in blocking buffer at 4C overnight. The blot was washed three times with TBST (ThermoFisher 28360) for 5 minutes each, then incubated with a 1:12500 dilution of IR680 anti-mouse (ThermoFisher PI35518) and IR800 anti -rabbit secondary antibodies (ThermoFisher 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 HAO1 protein was observed in primary human hepatocytes at day 16 post delivery of Casl2i2 RNPs targeting the HAO1 gene with HAO1 E2T5 (lanes 1-3 of FIG. 13A). HAO1 knockdown was not observed for the buffer only controls (lanes 4-7). Knockdown of LDHA protein (monomer and dimer) was observed in primary human hepatocytes at Day 7 post delivery of Casl2i2 RNPs targeting the LDHA gene (FIG. 13B). This knockdown was seen across each of the four RNA guides, LDHA E3T1, LDHA E5T9, LDHA E5T1, and LDHA E5T10 (lanes 1-8). LDHA knockdown was not observed for the buffer only (lanes 9 and 10) or electroporated controls (lanes 11 and 12).
This Example thus shows that HAO1 protein levels were decreased following editing with Casl2i2 and HAO 1 -targeting RNA guides and that LDHA protein levels were decreased following editing with Casl2i2 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

WHAT IS CLAIMED IS:
1. A gene editing system for genetic editing of a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase A (LDHA) gene, comprising:
(i) a first RNA guide or a first nucleic acid encoding the RNA guide, wherein the first RNA guide comprises a first spacer sequence specific to a first target sequence within an HAO1 gene, the first 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;
(ii) a second RNA guide or a second nucleic acid encoding the RNA guide, wherein the second RNA guide comprises a second spacer sequence specific to a second target sequence within an LDHA gene, the second 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; and
(iii) a Casl2i2 polypeptide or a third nucleic acid encoding the Casl2i2 polypeptide, wherein the Casl2i2 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.
2. The gene editing system of claim 1 , wherein the one or more mutations in the Casl2i2 polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S 1046 of SEQ ID NO: 1166.
3. The gene editing system of claim 1 or 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 gene editing system of claim 3, wherein the Casl2i2 polypeptide comprises:
(i) mutations at positions D581, D911, 1926, and V1030, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G;
(ii) mutations at positions D581, 1926, and V1030, which optionally are amino acid substitutions of D581R, I926R, and V1030G;
(iii) mutations at positions D581, 1926, V1030, and S1046, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) mutations at positions D581, G624, F626, 1926, 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, 1926, 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 Casl2i2 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1167-1171, optionally wherein the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 1168 or 1171.
6. The gene editing system of any one of claims 1-5, which comprises the third nucleic acid encoding the Casl2i2 polypeptide; optionally wherein the third nucleic acid is codon-optimized.
7. The gene editing system of claim 6, wherein the third nucleic acid is a messenger RNA (mRNA).
8. The gene editing system of claim 6, wherein the third nucleic acid is included in a viral vector, which optionally is an adeno-associated viral (AAV) vector.
9. The gene editing system of any one of claims 1-8, wherein the first target sequence is within exon 1 or exon 2 of the HAO1 gene.
10. The gene editing system of claim 9, wherein the first target sequence comprises:
(a-i) 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212);
(a-ii) 5’-GGAAGTACTGATTTAGCATG-3’ (SEQ ID NO: 2213);
(a-iii) 5’-TAGATGGAAGCTGTATCCAA-3’ (SEQ ID NO: 2233);
(a-iv) 5’-CGGAGCATCCTTGGATACAG-3’ (SEQ ID NO: 2234); or (a-v) 5’-AGGACAGAGGGTCAGCATGC-3 (SEQ ID NO: 2239).
11. The gene editing system of claim 10, wherein the first spacer sequence is set forth as:
(a-i) 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2311);
(a-ii) 5’-GGAAGUACUGAUUUAGCAUG-3’ (SEQ ID NO: 2312);
(a-iii) 5’-UAGAUGGAAGCUGUAUCCAA-3’ (SEQ ID NO: 2313);
(a-iv) 5’-CGGAGCAUCCUUGGAUACAG-3’ (SEQ ID NO: 2314); or (a-v) 5’-AGGACAGAGGGUCAGCAUGC-3 (SEQ ID NO: 2315).
12. The gene editing system of any one of claims 1-11, wherein the second target sequence is within exon 3 or exon 5 of the LDHA gene.
13. The system of claim 12, wherein the second target sequence comprises: (b-i) 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281); (b-ii) 5’-TAGGACTTGGCAGATGAACT-3’ (SEQ ID NO: 2270); (b-iii) 5’-GATGACATCAACAAGAGCAA-3’ (SEQ ID NO: 2272); (b-iv) 5’-TTCATAGTGGATATCTTGAC-3’ (SEQ ID NO: 2278); or (b-v) 5 ’-CATAGTGGATATCTTG ACCT-3 (SEQ ID NO: 2282).
14. The gene editing system of claim 13, wherein the second spacer sequence is set forth as:
(b-i) 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316);
(b-ii) 5’-UAGGACUUGGCAGAUGAACU-3’ (SEQ ID NO: 2317);
(b-iii) 5’-GAUGACAUCAACAAGAGCAA-3’ (SEQ ID NO: 2318);
(b-iv) 5’-UUCAUAGUGGAUAUCUUGAC-3’ (SEQ ID NO: 2319); or (b-v) 5’-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 2320).
15. The gene editing system of any one of claims 1-8, wherein the first target sequence comprises (a-i) 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212); and wherein the second target sequence comprises (b-i) 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281).
16. The gene editing system of claim 15, wherein the first spacer sequence is set forth as (a-i) 5 ’ -CAAAGUCUAUAUAUGACUAU-3 ’ (SEQ ID NO: 2311); and wherein the
181 second spacer sequence is set forth as (b-i) 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316).
17. The gene editing system of any one of claims 1-16, wherein the first spacer sequence and/or the second spacer sequence is 20-30-nucleotide in length, optionally wherein the spacer sequence is 20-nucleotide in length.
18. The gene editing system of any one of claims 1-17, wherein the first RNA guide comprises the first spacer sequence and a first direct repeat sequence; and/or wherein the second RNA guide comprises the second spacer sequence and a second direct repeat sequence.
19. The gene editing system of claim 18, wherein the first direct repeat sequence and/or the second direct repeat sequence is 23-36-nucleotide in length.
20. The gene editing system of claim 19, wherein the first direct repeat sequence and/or the second 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.
21. The gene editing system of claim 20, wherein the first direct repeat sequence and/or the second direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length.
22. The gene editing system of claim 21, wherein the first direct repeat sequence and/or the second direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 10).
23. The gene editing system of claim 1, wherein the first RNA guide comprises the nucleotide sequence of:
(a-i) 5 ’ -AGAAAUCCGUCUUUCAUUGACGGCAAAGUCUAUAUAUGACUAU- 3’ (SEQ ID NO: 2131);
(a-ii) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGGGAAGUACUGAUUUAGCAUG- 3’ (SEQ ID NO: 2132);
182 (a-iii) 5’-
AGAAAUCCGUCUUUCAUUGACGGUAGAUGGAAGCUGUAUCCAA-3 ’ (SEQ ID NO: 2152);
(a-iv) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGCGGAGCAUCCUUGGAUAC AG- 3’ (SEQ ID NO: 2153); or
(a- v) 5 ’ - AGAAAUCCGUCUUUC AUUGACGG AGGAC AGAGGGUC AGO AUGC- 3’ (SEQ ID NO: 2158).
24. The gene editing system of claim 1 or claim 23, wherein the second RNA guide comprises the nucleotide sequence of:
(b-i) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUC AUAGUGG AU AUCUUGACC- 3’ (SEQ ID NO: 2199);
(b-ii) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUAGG ACUUGGCAGAUG AACU- 3’ (SEQ ID NO: 2189);
(b-iii) 5’-
AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3 ’ (SEQ
ID NO: 2211);
(b-iv) 5’-
AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3 ’ (SEQ ID NO: 2196); or
(b-v) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU- 3’ (SEQ ID NO: 2200).
25. The gene editing system of claim 1, wherein the first the first RNA guide comprises the nucleotide sequence of (a-i) 5’-AGAAAUCCGUCUUUCAUUGACGGC AAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2131), optionally a modified version thereof set forth as SEQ ID NO: 2321; and wherein the second RNA guide comprises the nucleotide sequence of (b-i) 5 ’-AGAAAUCCGUCUUUC AUUGACGGUC AUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2199), optionally a modified version thereof set forth as SEQ ID NO: 2322.
26. The gene editing system of any one of claims 1-25, wherein the system comprises the first nucleic acid encoding the first RNA guide; and/or wherein the system comprises the second nucleic acid encoding the second RNA guide.
183
27. The gene editing system of claim 26, wherein the first nucleic acid encoding the first RNA guide is located in a first vector; and/or wherein the second nucleic acid encoding the second RNA guide is located in a second vector.
28. The gene editing system of claim 27, wherein the first vector and the second vector are the same vector.
29. The gene editing system of any one of claims 1-25, wherein the gene editing system comprises one or more vectors, which collectively comprise the first nucleic acid encoding the first RNA guide in a first vector, the second nucleic acid encoding the second RNA guide in a second vector, and/or the third nucleic acid encoding the Casl2i2 polypeptide.
30. The gene editing system of claim 29, wherein the first, second and/or third vector is a viral vector, optionally wherein the viral vector is an AAV vector.
31. The gene editing system of any one of claims 1-25, wherein the gene editing system comprises one or more lipid nanoparticles (LNPs), which are associated with (i), (ii), (iii), or a combination thereof.
32. The gene editing system of claim 31, which comprises (i) the first RNA guide, (ii) the second RNA guide, and (iii) an mRNA molecule encoding the Casl2i2 polypeptide; and wherein the LNPs are associated with (i)-(iii).
33. The gene editing system of claim 32, wherein at least a portion of (i), (ii), and/or (iii) is encapsulated by the LNPs.
34. The gene editing system of claim 31, wherein the gene editing system comprises a lipid nanoparticle, which encompass one or two of (i)-(iii), and a viral vector, which comprising the nucleic acid(s) encoding the remaining; optionally wherein the viral vector is an AAV vector.
184
35. A gene editing system for genetic editing of a hydroxyacid oxidase 1 (HAO1) gene and a lactate dehydrogenase A (LDHA) gene, comprising:
(i) a first RNA guide or a first nucleic acid encoding the first RNA guide, wherein the first RNA guide comprises a first spacer sequence specific to a first target sequence within exon 1 or exon 2 of an HAO1 gene, the first 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;
(ii) a second RNA guide or a second nucleic acid encoding the second RNA guide, wherein the second RNA guide comprises a second spacer sequence specific to a second target sequence within exon 3 or exon 5 of an LDHA gene, the second target sequence being adjacent to a protospacer sequence adjacent motif (PAM) comprising the motif of 5’-TTN-3’, which is located 5’ to the target sequence; and
(iii) a Casl2i polypeptide or a third nucleic acid encoding the Casl2i polypeptide; optionally wherein the Casl2i polypeptide is a Casl2i2 polypeptide.
36. The gene editing system of claim 35, wherein the first target sequence comprises:
(a-i) 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212);
(a-ii) 5’-GGAAGTACTGATTTAGCATG-3’ (SEQ ID NO: 2213);
(a-iii) 5’-TAGATGGAAGCTGTATCCAA-3’ (SEQ ID NO: 2233);
(a-iv) 5’-CGGAGCATCCTTGGATACAG-3’ (SEQ ID NO: 2234); or (a-v) 5’-AGGACAGAGGGTCAGCATGC-3 (SEQ ID NO: 2239).
37. The gene editing system of claim 36, wherein the first spacer sequence is set forth as:
(a-i) 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2311);
(a-ii) 5’-GGAAGUACUGAUUUAGCAUG-3’ (SEQ ID NO: 2312);
(a-iii) 5’-UAGAUGGAAGCUGUAUCCAA-3’ (SEQ ID NO: 2313);
(a-iv) 5’-CGGAGCAUCCUUGGAUACAG-3’ (SEQ ID NO: 2314); or (a-v) 5’-AGGACAGAGGGUCAGCAUGC-3 (SEQ ID NO: 2315).
38. The gene editing system of any one of claims 35-37, wherein the second target sequence comprises:
(b-i) 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281);
185 (b-ii) 5’-TAGGACTTGGCAGATGAACT-3’ (SEQ ID NO: 2270);
(b-iii) 5’-GATGACATCAACAAGAGCAA-3’ (SEQ ID NO: 2272);
(b-iv) 5’-TTCATAGTGGATATCTTGAC-3’ (SEQ ID NO: 2278); or (b-v) 5 ’-CATAGTGGATATCTTG ACCT-3 (SEQ ID NO: 2282).
39. The gene editing system of claim 38, wherein the second spacer sequence is set forth as:
(b-i) 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316);
(b-ii) 5’-UAGGACUUGGCAGAUGAACU-3’ (SEQ ID NO: 2317);
(b-iii) 5’-GAUGACAUCAACAAGAGCAA-3’ (SEQ ID NO: 2318);
(b-iv) 5’-UUCAUAGUGGAUAUCUUGAC-3’ (SEQ ID NO: 2319); or (b-v) 5’-CAUAGUGGAUAUCUUGACCU-3 (SEQ ID NO: 2320).
40. The gene editing system of claim 35, wherein the first target sequence comprises (a-i) 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 2212); and wherein the second target sequence comprises (b-i) 5’-TCATAGTGGATATCTTGACC-3’ (SEQ ID NO: 2281).
41. The gene editing system of claim 40, wherein the first spacer sequence is set forth as (a-i) 5 ’ -CAAAGUCUAUAUAUGACUAU-3 ’ (SEQ ID NO: 2311); and wherein the second spacer sequence is set forth as (b-i) 5’-UCAUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2316).
42. The gene editing system of any one of claims 35-41, which comprises the third nucleic acid encoding the Casl2i polypeptide.
43. The gene editing system of claim 42, wherein the third nucleic acid is a messenger RNA (mRNA).
44. The gene editing system of claim 43, wherein the third nucleic acid is included in a viral vector, which optionally is an adeno-associated viral (AAV) vector.
45. The gene editing system of any one of claims 35-44, wherein the first spacer sequence and/or the second spacer sequence is 20-30-nucleotide in length, optionally wherein the first spacer sequence and/or the second spacer sequence is 20-nucleotide in length.
46. The gene editing system of any one of claims 35-45, wherein the first RNA guide comprises the first spacer sequence and a first direct repeat sequence; and/or wherein the second RNA guide comprises the second spacer sequence and a second direct repeat sequence.
47. The gene editing system of claim 46, wherein the first direct repeat sequence and/or the second direct repeat sequence is 23-36-nucleotide in length.
48. The gene editing system of claim 47, wherein the first direct repeat sequence and/or the second 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.
49. The gene editing system of claim 48, wherein the first direct repeat sequence and/or the second direct repeat sequence is any one of SEQ ID NOs: 1-10, or a fragment thereof that is at least 23-nucleotide in length.
50. The gene editing system of claim 49, wherein the first direct repeat sequence and/or the second direct sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 10).
51. The gene editing system of claim 35, wherein the first RNA guide comprises the nucleotide sequence of:
(a-i) 5 ’ -AGAAAUCCGUCUUUCAUUGACGGCAAAGUCUAUAUAUGACUAU- 3’ (SEQ ID NO: 2131);
(a-ii) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGGGAAGUACUGAUUUAGCAUG- 3’ (SEQ ID NO: 2132);
(a-iii) 5’-
AGAAAUCCGUCUUUCAUUGACGGUAGAUGGAAGCUGUAUCCAA-3 ’ (SEQ ID NO: 2152); (a-iv) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGCGGAGCAUCCUUGGAUAC AG- 3’ (SEQ ID NO: 2153); or
(a- v) 5 ’ - AGAAAUCCGUCUUUC AUUGACGG AGGAC AGAGGGUC AGO AUGC- 3’ (SEQ ID NO: 2158).
52. The gene editing system of claim 35 or claim 51 , wherein the second RNA guide comprises the nucleotide sequence of:
(b-i) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUC AUAGUGG AU AUCUUGACC- 3’ (SEQ ID NO: 2199);
(b-ii) 5 ’ - AGAAAUCCGUCUUUC AUUGACGGUAGG ACUUGGCAGAUG AACU- 3’ (SEQ ID NO: 2189);
(b-iii) 5’-
AGAAAUCCGUCUUUCAUUGACGGGAUGACAUCAACAAGAGCAA-3 ’ (SEQ
ID NO: 2211);
(b-iv) 5’-
AGAAAUCCGUCUUUCAUUGACGGUUCAUAGUGGAUAUCUUGAC-3 ’ (SEQ ID NO: 2196); or
(b-v) 5 ’ - AGAAAUCCGUCUUUCAUUGACGGCAUAGUGGAUAUCUUGACCU- 3’ (SEQ ID NO: 2200).
53. The gene editing system of claim 35, wherein the first the first RNA guide comprises the nucleotide sequence of (a-i) 5’-AGAAAUCCGUCUUUCAUUGACGGC AAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 2131), optionally a modified version thereof set forth as SEQ ID NO: 2321; and wherein the second RNA guide comprises the nucleotide sequence of (b-i) 5 ’-AGAAAUCCGUCUUUC AUUGACGGUC AUAGUGGAUAUCUUGACC-3’ (SEQ ID NO: 2199), optionally a modified version thereof set forth as SEQ ID NO: 2322.
54. The gene editing system of any one of claims 35-53, wherein the system comprises the first nucleic acid encoding the first RNA guide; and/or wherein the system comprises the second nucleic acid encoding the second RNA guide.
55. The gene editing system of claim 54, wherein the first nucleic acid encoding the first RNA guide is located in a first viral vector; and/or wherein the second nucleic acid
188 encoding the second RNA guide is located in a second viral vector; optionally wherein the first viral vector and the second viral vector are the same vector.
56. The gene editing system of any one of claims 35-55, wherein the system comprises one or more vectors, which collectively comprise the first nucleic acid encoding the first RNA guide in a first vector, the second nucleic acid encoding the second RNA guide in a second vector, and/or the third nucleic acid encoding the Casl2i2 polypeptide.
57. The gene editing system of any one of claims 35-56, wherein the system comprises one or more lipid nanoparticles (LNPs), which are associated with (i), (ii), (iii), or a combination thereof.
58. The gene editing system of claim 57, which comprises (i) the first RNA guide, (ii) the second RNA guide, and (iii) an mRNA molecule encoding the Casl2i polypeptide, and wherein the LNPs are associated with (i)-(iii).
59. The gene editing system of claim 58, wherein at least a portion of (i), (ii), and/or (iii) is encapsulated by the LNPs.
60. The gene editing system of claim 59, wherein the system comprises a lipid nanoparticle, which encompass one or two of (i)-(iii), and a viral vector, which comprising the nucleic acid(s) encoding the remaining; optionally wherein the viral vector is an AAV vector.
61. A pharmaceutical composition comprising the gene editing system set forth in any one of claims 1-60.
62. A kit comprising the elements (i)-(iii) of the gene editing system set forth in any one of claims 1-60.
63. A method for editing a hydroxy acid oxidase 1 (HAO1) gene and a lactate dehydrogenase A (LDHA) gene in a cell, the method comprising contacting a host cell with the gene editing system set forth in any one of claims 1-60 to genetically edit the HAO1 gene and the LDHA gene in the host cell.
189
64. The method of claim 63, wherein the host cell is cultured in vitro.
65. The method of claim 64, wherein contacting step is performed by administering the gene editing system for editing the HA01 gene and the LDHA gene to a subject comprising the host cell.
66. A cell comprising a disrupted hydroxy acid oxidase 1 (HA01) gene and a disrupted lactate dehydrogenase A (LDHA) gene, wherein the cell optionally is produced by contacting a host cell with the gene editing system of any one of claims 1-60 to genetically edit the HA01 gene and the LDHA gene in the host cell, thereby disrupting the HA01 gene and the LDHA gene.
67. 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 hydroxyacid oxidase 1 (HA01) gene and a lactate dehydrogenase A (LDHA) gene set forth in any one of claims 1-60 or the cell of claim 66.
68. The method of claim 67, wherein the subject is a human patient having the PH, which optionally is PHI, PH2, or PH3.
69. The method of claim 68, wherein the PH is PHI.
190
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178427A1 (en) 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
WO2020069296A1 (en) * 2018-09-28 2020-04-02 Intellia Therapeutics, Inc. Compositions and methods for lactate dehydrogenase (ldha) gene editing
WO2020132659A1 (en) * 2018-12-21 2020-06-25 Precision Biosciences, Inc. Genetic modification of the hydroxyacid oxidase 1 gene for treatment of primary hyperoxaluria
WO2021202800A1 (en) 2020-03-31 2021-10-07 Arbor Biotechnologies, Inc. Compositions comprising a cas12i2 variant polypeptide and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178427A1 (en) 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
WO2020069296A1 (en) * 2018-09-28 2020-04-02 Intellia Therapeutics, Inc. Compositions and methods for lactate dehydrogenase (ldha) gene editing
WO2020132659A1 (en) * 2018-12-21 2020-06-25 Precision Biosciences, Inc. Genetic modification of the hydroxyacid oxidase 1 gene for treatment of primary hyperoxaluria
WO2021202800A1 (en) 2020-03-31 2021-10-07 Arbor Biotechnologies, Inc. Compositions comprising a cas12i2 variant polypeptide and uses thereof

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Antibodies: a practice approach", 1988, IRL PRESS
"Cell and Tissue Culture: Laboratory Procedures", 1993, J. WILEY AND SONS
"Current Protocols in Immunology", 1991
"DNA Cloning: A practical Approach", vol. 1, 2, 1985
"Gene Transfer Vectors for Mammalian Cells", 1987, HUMANA PRESS
"Handbook of Experimental Immunology", 1994, ACADEMIC PRESS, INC
"Immobilized Cells and Enzymes", 1986, LRL PRESS
"Monoclonal antibodies: a practical approach", 2000, OXFORD UNIVERSITY PRESS
"The Antibodies", 1995, HARWOOD ACADEMIC PUBLISHERS
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, no. 17, 1997, pages 3389 - 3402
B. PERBAL: "A practical Guide To Molecular Cloning", 1984
C. A. JANEWAY, P. TRAVERS, IMMUNOBIOLOGY, 1997
E. HARLOWD. LANE: "Using antibodies: a laboratory manual", 1999, COLD SPRING HARBOR LABORATORY PRESS
EPSTEIN ET AL.: "Engineering a Self-Inactivating CRISPR System for AAV Vectors", MOL. THER., vol. 24, 2016, pages S50, XP002777646, DOI: 10.1016/S1525-0016(16)32928-8
J. P. MATHEP. E. ROBERTS: "Introuction to Cell and Tissue Culture", 1998, PLENUM PRESS
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 77
KARLINALTSCHUL, PROC. NATL. ACAD., vol. 87, 1990, pages 2264 - 68
LEWISPAN: "RNA modifications and structures cooperate to RNA guide-protein interactions", NAT REVIEWS MOL CELL BIOL, vol. 18, 2017, pages 202 - 210, XP055451248
MADDOX ET AL., J. EXP. MED., vol. 158, 1983, pages 1211
NAKAMURA ET AL., NUCL. ACIDS RES., vol. 28, 2000, pages 292
NEREA ZABALETA ET AL: "CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I", NATURE COMMUNICATIONS, vol. 9, no. 1, 1 December 2018 (2018-12-01), XP055633086, DOI: 10.1038/s41467-018-07827-1 *
P. FINCH, ANTIBODIES, 1997
ROZENSKI, JCRAIN, PMCCLOSKEY, J: "The RNA Modification Database: 1999 update", NUCL ACIDS RES, vol. 27, 1999, pages 196 - 197
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
TIJSSEN: "Laboratory Techniques in Biochemistry and Molecular Biology", vol. 24, article "Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation"
YU, Z ET AL.: "RNA editing by ADAR1 marks dsRNA as ''self", CELL RES, vol. 25, 2015, pages 1283 - 1284
ZHENG RUI ET AL: "Knockdown of lactate dehydrogenase by adeno-associated virus-delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1", CLINICAL AND TRANSLATIONAL MEDICINE, vol. 10, no. 8, 1 December 2020 (2020-12-01), SE, pages 1 - 12, XP093037380, ISSN: 2001-1326, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1002/ctm2.261> DOI: 10.1002/ctm2.261 *

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