EP4649147A2 - Typ-ii-cas-proteine und anwendungen davon - Google Patents

Typ-ii-cas-proteine und anwendungen davon

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Publication number
EP4649147A2
EP4649147A2 EP24700878.2A EP24700878A EP4649147A2 EP 4649147 A2 EP4649147 A2 EP 4649147A2 EP 24700878 A EP24700878 A EP 24700878A EP 4649147 A2 EP4649147 A2 EP 4649147A2
Authority
EP
European Patent Office
Prior art keywords
seq
type
sequence
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24700878.2A
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English (en)
French (fr)
Inventor
Antonio CASINI
Laura PEZZÈ
Matteo CICIANI
Ambra SARRACINO
Sally BERTOLINI
Maddalena BOSETTI
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Alia Therapeutics Srl
Original Assignee
Alia Therapeutics Srl
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Publication date
Application filed by Alia Therapeutics Srl filed Critical Alia Therapeutics Srl
Publication of EP4649147A2 publication Critical patent/EP4649147A2/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • CRISPR-Cas genome editing with Type II Cas proteins and associated guide RNAs is a powerful tool with the potential to treat a variety of genetic diseases.
  • Adeno-associated viral vectors AAVs are commonly used to deliver Cas proteins, for example Streptococcus pyogenes Cas9 (SpCas9), and their guide RNAs (gRNAs).
  • SpCas9 Streptococcus pyogenes Cas9
  • gRNAs guide RNAs
  • packaging a large Cas protein such as SpCas9 together with a guide RNA into a single AAV vector can be challenging due to the limited packaging capacity of AAVs.
  • Type II Cas nucleases with smaller sizes that can be packaged together with a gRNA in a single AAV.
  • the discovery of novel nucleases with new PAM specificities can broaden the range of targetable sites in the cell genome, making genome editing more flexible and efficient.
  • This disclosure is based, in part, on the discovery of a Type II Cas protein from an unclassified bacterium from the Acidaminococcaceae genus (referred to herein as “wild-type AEQH Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Ruminococcaceae family (referred to herein as “wild-type AAOF Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Clostridia class (referred to herein as “wild-type ACEE Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Ruminococcaceae family (referred to herein as “wild-type AQSL Type II Cas”), a Type II Cas protein from an unclassified Proteobacterium (referred to herein as “wild-type ASWC Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Clostridiaceae family (
  • Wild-type AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins are each approximately 1000 amino acids in length, significantly shorter than SpCas9.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:1 (such proteins referred to herein as “AEQH Type II Cas proteins”).
  • AEQH Type II Cas protein sequences are set forth in SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:7 (such proteins referred to herein as “AAOF Type II Cas proteins”).
  • AAOF Type II Cas protein sequences are set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:13 (such proteins referred to herein as “ACEE Type II Cas proteins”).
  • SEQ ID NO:13 such proteins referred to herein as “ACEE Type II Cas proteins”.
  • Exemplary ACEE Type II Cas protein sequences are set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:19 (such proteins referred to herein as “AQSL Type II Cas proteins”).
  • AQSL Type II Cas protein sequences are set forth in SEQ ID NO:19, SEQ ID NQ:20, and SEQ ID NO:21 .
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:25 (such proteins referred to herein as “ASWC Type II Cas proteins”).
  • ASWC Type II Cas proteins such proteins referred to herein as “ASWC Type II Cas proteins”.
  • Exemplary ASWC Type II Cas protein sequences are set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:31 (such proteins referred to herein as “AVFG Type II Cas proteins”).
  • AVFG Type II Cas proteins such proteins referred to herein as “AVFG Type II Cas proteins”.
  • Exemplary AVFG Type II Cas protein sequences are set forth in SEQ ID NO:31 , SEQ ID NO:32, and SEQ ID NO:33.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:37 (such proteins referred to herein as “AWIT Type II Cas proteins”).
  • AWIT Type II Cas proteins such proteins referred to herein as “AWIT Type II Cas proteins”.
  • Exemplary AWIT Type II Cas protein sequences are set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:43 (such proteins referred to herein as “AWMF Type II Cas proteins”).
  • SEQ ID NO:43 such proteins referred to herein as “AWMF Type II Cas proteins”.
  • Exemplary AWMF Type II Cas protein sequences are set forth in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:49 (such proteins referred to herein as “BUMO Type II Cas proteins”).
  • BUMO Type II Cas protein sequences are set forth in SEQ ID NO:49, SEQ ID NQ:50, and SEQ ID NO:51 .
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:55 (such proteins referred to herein as “COIA Type II Cas proteins”).
  • COIA Type II Cas protein sequences are set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:61 (such proteins referred to herein as “DJQA Type II Cas proteins”).
  • DJQA Type II Cas proteins such proteins referred to herein as “DJQA Type II Cas proteins”.
  • Exemplary DJQA Type II Cas protein sequences are set forth in SEQ ID NO:61 , SEQ ID NO:62, and SEQ ID NO:63.
  • the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:68 (such proteins referred to herein as “DWET Type II Cas proteins”).
  • Exemplary DWET Type II Cas protein sequences are set forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69.
  • Type II Cas proteins comprising an amino acid sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or more) sequence identity to a RuvC-l domain, RuvC-ll domain, RuvC-lll domain, BH domain, REC domain, HNH domain, WED domain, or PID domain of an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET Type II Cas protein.
  • AAOF Type II Cas protein ACEE Type II Cas protein
  • AQSL Type II Cas protein ASWC Type II Cas protein
  • a Type II Cas protein of the disclosure is a chimeric Type II Cas protein, for example, comprising one or more domains from an AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and/or DWET Type II Cas protein(s) and one or more domains from a different Type II Cas protein such as SpCas9.
  • the Type II Cas proteins of the disclosure are in the form of a fusion protein, for example, comprising an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET Type II Cas protein sequence fused to one or more additional amino acid sequences, for example, one or more nuclear localization signals and/or one or more tags.
  • Other exemplary fusion partners can enable base editing ⁇ e.g., where the fusion partner is nucleoside deaminase) or prime editing (e.g., where the fusion partner is a reverse transcriptase).
  • Type II Cas proteins of the disclosure are described in Section 6.2 and specific embodiments 1 to 295 and 1132 to 1138, infra.
  • the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs) and combinations of two or more gRNA molecules (e.g., combinations of sgRNA molecules).
  • gRNAs that can be used with the AEQH Type II Cas proteins of the disclosure, gRNAs that can be used with the AAOF Type II Cas proteins of the disclosure, gRNAs that can be used with the ACEE Type II Cas proteins of the disclosure, gRNAs that can be used with the AQSL Type II Cas proteins of the disclosure, gRNAs that can be used with the ASWC Type II Cas proteins of the disclosure, gRNAs that can be used with the AVFG Type II Cas proteins of the disclosure, gRNAs that can be used with the AWIT Type II Cas proteins of the disclosure, gRNAs that can be used with the AWMF Type II Cas proteins of the disclosure, gRNAs that can be used with the BUMO Type II Cas proteins
  • the disclosure provides systems comprising a Type II Cas protein of the disclosure and one or more gRNAs, e.g., sgRNAs.
  • a system can comprise a ribonucleoprotein (RNP) comprising a Type II Cas protein complexed with a gRNA, e.g., an sgRNA or separate crRNA and tracrRNA.
  • RNP ribonucleoprotein
  • Exemplary features of systems are described in Section 6.4 and specific embodiments 1008 to 1072, infra.
  • the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type II Cas protein of the disclosure and, optionally, a guide RNA, for example a sgRNA.
  • the nucleic acids comprise a Type II Cas protein of the disclosure operably linked to a heterologous promoter, e.g., a mammalian promoter, for example a human promoter.
  • the disclosure provides nucleic acids encoding a gRNA, for example a sgRNA, of the disclosure and, optionally, a Type II Cas protein, for example an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET Type II Cas protein.
  • a Type II Cas protein for example an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II
  • the disclosure provides nucleic acids encoding combinations of gRNAs of the disclosure, for example a combination of two gRNAs, and, optionally, a Type II Cas protein.
  • gRNAs of the disclosure for example a combination of two gRNAs, and, optionally, a Type II Cas protein.
  • Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5 and specific embodiments 1073 to 1131 , infra.
  • the disclosure provides particles comprising the Type II Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6 and specific embodiments 1139 to 1154, infra.
  • the disclosure provides cells and populations of cells containing or contacted with a Type II Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.6 and specific embodiments 1156 to 1165 and 1205, infra.
  • compositions comprising a Type II Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients.
  • exemplary features of pharmaceutical compositions are described in Section 6.7 and specific embodiment 1155, infra.
  • the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type II Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure.
  • Cells altered according to the methods of the disclosure can be used, for example, to treat subjects having a disease or disorder, e.g., genetic disease or disorder, for example retinitis pigmentosa caused by a RHO mutation.
  • a disease or disorder e.g., genetic disease or disorder, for example retinitis pigmentosa caused by a RHO mutation.
  • FIG. 1 shows PAM logos for exemplary Type II Cas proteins of the disclosure.
  • FIGS. 2A-2D schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for AAOF Type II Cas protein (FIG. 2A), ACEE Type II Cas protein (FIG. 2B), AEQH Type II Cas protein (FIG. 2C), and AQSL Type II Cas protein (FIG. 2D).
  • Figures 2A-2D disclose SEQ ID NOS 124, 126, 122, and 128, respectively, in order of appearance.
  • FIGS. 3A-3D schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for ASWC Type II Cas protein (FIG. 3A), for AVFG Type II Cas protein (FIG. 3B), AWIT, AWMF and COIA Type II Cas proteins (FIG. 3C), and BUMO Type II Cas protein (FIG. 3D).
  • Figures 3A-3D disclose SEQ ID NOS 130, 132, 134, and 138, respectively, in order of appearance.
  • FIGS. 4A-4B schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for DJQA Type II Cas protein (FIG. 4A) and DWET Type II Cas protein (FIG. 4B).
  • Figures 4A-4B disclose SEQ ID NOS 142 and 144, respectively, in order of appearance.
  • FIGS. 5A-5F show PAM sequence logos for AAOF Type II Cas (FIG. 5A), ACEE Type II Cas (FIG. 5C), and AEQH Type II Cas (FIG.
  • FIGS. 6A-6F show PAM sequence logos for AQSL Type II Cas (FIG. 6A), ASWC Type II Cas (FIG. 6C), and AVFG Type II Cas (FIG. 6E) from an in vitro PAM discovery assay and PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for AQSL Type II Cas (positions 5,6 and 7,8) (FIG. 6B), ASWC Type II Cas (positions 2,3 and 5,6) (FIG 6D), and AVFG Type II Cas (positions 5,6 and 7,8) (FIG. 6F).
  • FIGS. 7A-7F show PAM sequence logos for AWIT Type II Cas (FIG. 7A), AWMF Type II Cas (FIG. 7C), and COIA Type II Cas (FIG. 7E) from an in vitro PAM discovery assay and PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for AWIT Type II Cas (positions 5,6 and 7,8) (FIG. 7B), AWMF Type II Cas (positions 5,6 and 7,8) (FIG. 7D), and COIA Type II Cas (positions 5,6 and 7,8) (FIG. 7F).
  • FIGS. 8A-8F show PAM sequence logos for BUMO Type II Cas (FIG. 8A), DJQA Type II Cas (FIG. 8C) and DWET Type II Cas (FIG. 8E) from an in vitro PAM discovery assay and PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for BUMO Type II Cas (positions 5,6 and 7,8) (FIG. 8B), DJQA Type II Cas (positions 5,6 and 7,8) (FIG. 8D), and DWET Type II Cas (positions 5,6 and 7,8) (FIG. 8F).
  • FIG. 9 shows the activity of selected Type II Cas proteins evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells stably expressing EGFP.
  • FIG. 10 shows a schematic representation of the rs7984 SNP locus with the position of exemplary sgRNAs for AAOF, ACEE, AEQH, AVFG, BUMO, DJQA and DWET Type II Cas proteins (Example 3).
  • Figure 10 discloses SEQ ID NOS 494, 491 , 491 , 485, 493, 493, 487, 487, 490, 490, 492, 495, 495, 859, 486, 486, 484, 496, 497, 488, and 488, respectively, in order of appearance.
  • FIGS. 13A-13B shows the editing activity and allele specificity of selected gRNAs towards the
  • FIG. 13A shows the activity and allele specificity against the rs7984A SNP allele after transient transfection of HEK293T cells with ACEE, DJQA or AVFG Type II Cas in combination with the indicated guides either perfectly matching the rs7984A locus (on-target activity) or the rs7984G locus (off-target activity - allele specificity).
  • FIGS. 14A-14B (including subparts FIGS. 14A-1 to 14A-7 and FIGS. 14B-1 to 14B-7) show a schematic representation of the RHO intron 1 sequence reporting the position of the evaluated guide RNAs for ACEE Type II Cas (FIG. 14A) and DJQA Type II Cas (FIG. 14B) (Example 3).
  • Figures 14A-14B disclose SEQ ID NOS 521 , 860, 518, 520, 517, 519, 862-863, 865, 866, 868, 532, 528, 533, 869-873, 535-536, and 875, respectively, in order of appearance.
  • FIGS. 15A-15B show the evaluation of the editing activity of guide RNAs for ACEE (FIG. 15A) and DJQA (FIG. 15B) Type II Cas targeting RHO intron 1 after transient transfection in HEK293T cells (Example 3).
  • FIGS. 16A-16B show the evaluation of large editing events at the target RHO locus (Example 3).
  • FIG. 16A shows a representative image of an agarose gel electrophoresis of endpoint PCR products generated with primers spanning the deleted RHO fragment using genomic DNA extracted from HEK293T cells transfected with the indicated Type II Cas protein and relative guides.
  • a high molecular weight band corresponds to undeleted/inverted products while low molecular weight bands correspond to deleted RHO alleles.
  • 16B reports the results of a qPCR assay exploiting specifically designed primers to assess the relative amount of RHO alleles which do not contain large edits (deletions/inversions) in a population of HEK293T cells transiently transfected with expression plasmids for ACEE or DJQA Type II Cas together with the selected sgRNAs, as indicated on the graph.
  • FIG. 17 shows the levels of deletions and inversions, as measured by a specifically designed ddPCR assay, which were generated in HEK293T cells after transient transfection of either ACEE or DJQA Type II Cas in combination with the indicated sgRNAs targeting the rs7984A RHO SNP allele and RHO intron 1 (Example 3).
  • Type II Cas proteins e.g., AEQH Type II Cas proteins, AAOF Type II Cas proteins, ACEE Type II Cas proteins, AQSL Type II Cas proteins, ASWC Type II Cas proteins, AVFG Type II Cas proteins, AWIT Type II Cas proteins, AWMF Type II Cas proteins, BUMO Type II Cas proteins, COIA Type II Cas proteins, DJQA Type II Cas proteins, and DWET Type II Cas proteins.
  • Type II Cas proteins of the disclosure can be in the form of fusion proteins.
  • Type II Cas proteins encompass Type II Cas proteins which are not fusion proteins and Type II Cas proteins which are in the form of fusion proteins (e.g., Type II Cas protein comprising one or more nuclear localization signals and/or one or more tags).
  • a Type II Cas protein of the disclosure comprises an amino acid sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or more) sequence identity to a RuvC-l domain, RuvC-ll domain, RuvC-lll domain, BH domain, REC domain, HNH domain, WED domain, or PID domain of an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET Type II Cas protein.
  • AAOF Type II Cas protein ACEE Type II Cas protein
  • AQSL Type II Cas protein ASWC Type II Cas protein
  • a Type II Cas protein of the disclosure is a chimeric Type II Cas protein, for example, comprising one or more domains from an AEQH Type II Cas protein and/or AAOF Type II Cas protein and/or ACEE Type II Cas protein and/or AQSL Type II Cas protein and/or ASWC Type II Cas protein and/or AVFG Type II Cas protein and/or AWIT Type II Cas protein and/or AWMF Type II Cas protein and/or BUMO Type II Cas protein and/or COIA Type II Cas protein and/or DJQA Type II Cas protein and/or or DWET Type II Cas protein, and one or more domains from a different Type II Cas protein such as SpCas9.
  • the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs) and combinations of guide RNA molecules, for example combinations of two or more sgRNAs.
  • gRNAs can include, for example, a gRNA targeting the RHO rs7984 SNP and a second gRNA targeting RHO intron 1.
  • Combinations of gRNAs targeting the RHO rs7984 SNP and RHO intron 1 can be used to selectively edit RHO alleles having pathogenic mutations. This dual targeting approach is further described Section 6.8 and Example 3. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure are further described in Section 6.3.
  • the disclosure provides systems comprising a Type II Cas protein of the disclosure and one or more gRNAs, e.g., sgRNAs. Exemplary features of systems are described in Section 6.4.
  • the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type II Cas protein of the disclosure and, optionally, a guide RNA, for example a sgRNA, and provides nucleic acids encoding a gRNA, for example a sgRNA, of the disclosure and, optionally, a Type II Cas protein.
  • a guide RNA for example a sgRNA
  • nucleic acids encoding a gRNA for example a sgRNA
  • Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5.
  • the disclosure provides particles comprising the Type II Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6.
  • the disclosure provides cells and populations of cells containing or contacted with a Type II Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.6.
  • compositions comprising a Type II Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients.
  • exemplary features of pharmaceutical compositions are described in Section 6.7.
  • the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type II Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure.
  • methods of altering cells e.g., editing the genome of a cell
  • Type II Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure are described in Section 6.8.
  • an agent includes a plurality of agents, including mixtures thereof.
  • an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected).
  • the term “and/or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.
  • a Type II Cas protein refers to a wild-type or engineered Type II Cas protein. Engineered Type II Cas proteins can also be referred to as Type II Cas variants. For the avoidance of doubt, any disclosure pertaining to a “Type II Cas” or “Type II Cas protein” pertains to wild-type Type II Cas proteins and Type II Cas variants, unless the context dictates otherwise.
  • a Type II Cas protein can have nuclease activity or be catalytically inactive (e.g., as in a dCas).
  • the percentage identity between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100, and dividing by the length of the aligned region. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another, nor does it consider substitutions or deletions as matches. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, by manual alignment or using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for achieving maximum alignment.
  • Guide RNA molecule refers to an RNA capable of forming a complex with a Type II Cas protein and which can direct the Type II Cas protein to a target DNA.
  • gRNAs typically comprise a spacer of 15 to 30 nucleotides in length.
  • gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise a spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold.
  • sgRNAs single guide RNAs
  • 3’ sgRNA scaffolds are described in Section 6.3.
  • An sgRNA can in some embodiments comprise no uracil base at the 3’ end of the sgRNA sequence.
  • a sgRNA can comprise one or more uracil bases at the 3’ end of the sgRNA sequence.
  • a sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence, 2 uracil (UU) at the 3’ end of the sgRNA sequence, 3 uracil (UUU) at the 3’ end of the sgRNA sequence, 4 uracil (UUUU) at the 3’ end of the sgRNA sequence, 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence, 6 uracil (UUUUU) at the 3’ end of the sgRNA sequence, 7 uracil (UUUUUU) at the 3’ end of the sgRNA sequence, or 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence.
  • uracil can be appended at the 3’ end of a sgRNA as terminators.
  • the 3’ sgRNA scaffolds set forth in Section 6.3 can be modified by adding or removing one or more uracils at the end of the sequence.
  • Peptide, protein, and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • the amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications.
  • a polypeptide may be attached to other molecules, for instance molecules required for function.
  • polypeptides examples include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc.
  • polypeptides include peptide fragments, denatured/unstructured polypeptides, polypeptides having quaternary or aggregated structures, etc. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected/unintended purposes, or have unknown function.
  • a polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used.
  • the standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V).
  • polypeptide sequence or “amino acid sequence” are an alphabetical representation of a polypeptide molecule.
  • Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • the following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide sequence is the alphabetical representation of a polynucleotide molecule.
  • nucleotide sequences described herein correspond to IUPAC notation.
  • the letter “N” in a nucleotide sequence represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA sequence
  • the letter “R” in a nucleotide sequence represents a nucleotide which can be A or G
  • the letter “V” in a nucleotide sequence represents a nucleotide which can be “A, C, or G.
  • Protospacer adjacent motif refers to a DNA sequence downstream (e.g., immediately downstream) of a target sequence on the non-target strand recognized by a Type II Cas protein.
  • a PAM sequence is located 3’ of the target sequence on the non-target strand.
  • Spacer refers to a region of a gRNA molecule which is partially or fully complementary to a target sequence found in the + or - strand of genomic DNA.
  • the gRNA directs the Type II Cas to the target sequence in the genomic DNA.
  • a spacer of a Type II Cas gRNA is typically 15 to 30 nucleotides in length (e.g., 20-25 nucleotides).
  • the nucleotide sequence of a spacer can be, but is not necessarily, fully complementary to the target sequence.
  • a spacer can contain one or more mismatches with a target sequence, e.g., the spacer can comprise one, two, or three mismatches with the target sequence.
  • the disclosure provides AEQH Type II Cas proteins.
  • AEQH Type II Cas proteins can be further classified as Type IIA Cas proteins.
  • the AEQH Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1 .
  • the AEQH Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 .
  • an AEQH Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:1 .
  • Exemplary AEQH Type II Cas protein sequences and nucleotide sequences encoding exemplary AEQH Type II Cas proteins are set forth in Table 1A.
  • an AEQH Type II Cas protein comprises an amino acid sequence of SEQ
  • an AEQH Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.
  • the one or more amino acid substitutions providing nickase activity comprise a N627A substitution, wherein the position of the N627A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.
  • the one or more amino acid substitutions providing nickase activity comprise an H604A substitution, wherein the position of the H604A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.
  • an AEQH Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N627A substitution or an H604A substitution.
  • the disclosure provides AAOF Type II Cas proteins.
  • AAOF Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the AAOF Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7.
  • the AAOF Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOT.
  • an AAOF Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NOT.
  • Exemplary AAOF Type II Cas protein sequences and nucleotide sequences encoding exemplary AAOF Type II Cas proteins are set forth in Table 2A.
  • an AAOF Type II Cas protein comprises an amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
  • an AAOF Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
  • the one or more amino acid substitutions providing nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.
  • the one or more amino acid substitutions providing nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.
  • the one or more amino acid substitutions providing nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.
  • an AAOF Type II Cas protein is catalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.
  • the disclosure provides ACEE Type II Cas proteins.
  • ACEE Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the ACEE Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:13.
  • the ACEE Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:13.
  • an ACEE Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:13.
  • ACEE Type II Cas protein sequences and nucleotide sequences encoding exemplary ACEE Type II Cas proteins are set forth in Table 2B.
  • an ACEE Type II Cas protein comprises an amino acid sequence of SEQ
  • an ACEE Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.
  • the one or more amino acid substitutions providing nickase activity comprise a D8A substitution, wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.
  • the one or more amino acid substitutions providing nickase activity comprise a N613A substitution, wherein the position of the N613A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.
  • the one or more amino acid substitutions providing nickase activity comprise a H590A substitution, wherein the position of the H590A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.
  • an ACEE Type II Cas protein is catalytically inactive, for example due a D8A substitution in combination with a N613A substitution or a H590A subsitution.
  • the disclosure provides AQSL Type II Cas proteins.
  • AQSL Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the AQSL Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:19.
  • the AQSL Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19.
  • an AQSL Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:19.
  • AQSL Type II Cas protein sequences and nucleotide sequences encoding exemplary AQSL Type II Cas proteins are set forth in Table 2C.
  • an AQSL Type II Cas protein comprises an amino acid sequence of SEQ
  • an AQSL Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:19, SEQ ID NQ:20, or SEQ ID NO:21 .
  • the one or more amino acid substitutions providing nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • the one or more amino acid substitutions providing nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • the one or more amino acid substitutions providing nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • an AQSL Type II Cas protein is catalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.
  • the disclosure provides ASWC Type II Cas proteins.
  • ASWC Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the ASWC Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:25.
  • the ASWC Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25.
  • an ASWC Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:25.
  • ASWC Type II Cas protein sequences and nucleotide sequences encoding exemplary ASWC Type II Cas proteins are set forth in Table 2D.
  • an ASWC Type II Cas protein comprises an amino acid sequence of SEQ
  • an ASWC Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.
  • the one or more amino acid substitutions providing nickase activity comrpise a D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • the one or more amino acid substitutions providing nickase activity comprise a N633A substitution, wherein the position of the N633A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • the one or more amino acid substitutions providing nickase activity comprise an H610A substitution, wherein the position of the H610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • an ASWC Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with a N633A substitution or an H610A substitution.
  • the disclosure provides AVFG Type II Cas proteins.
  • AVFG Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the AVFG Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:31 .
  • the AVFG Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31 .
  • an AVFG Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:31 .
  • Exemplary AVFG Type II Cas protein sequences and nucleotide sequences encoding exemplary AVFG Type II Cas proteins are set forth in Table 2E.
  • an AVFG Type II Cas protein comprises an amino acid sequence of SEQ
  • an AVFG Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:31 , SEQ ID NO:32, or SEQ ID NO:33.
  • the one or more amino acid substitutions providing nickase activity comprise a D12A substitution, wherein the position of the D12A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • the one or more amino acid substitutions providing nickase activity comprise a N607A substitution, wherein the position of the N607A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • the one or more amino acid substitutions providing nickase activity comprise an H584A substitution, wherein the position of the H584A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • an AVFG Type II Cas protein is catalytically inactive, for example due to a D12A substitution in combination with a N607A substitution or an H584A substitution.
  • the disclosure provides AWIT Type II Cas proteins.
  • AWIT Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the AWIT Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:37.
  • the AWIT Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:37.
  • an AWIT Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:37.
  • Exemplary AWIT Type II Cas protein sequences and nucleotide sequences encoding exemplary AWIT Type I Oas proteins are set forth in Table 2F.
  • an AWIT Type II Cas protein comprises an amino acid sequence of SEQ
  • an AWIT Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.
  • the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.
  • the one or more amino acid substitutions providing nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.
  • the one or more amino acid substitutions providing nickase activity comprise an H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.
  • an AWIT Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution. 6.2.2.7. AWMF Type II Cas Proteins
  • the disclosure provides AWMF Type II Cas proteins.
  • AWMF Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the AWMF Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:43.
  • the AWMF Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:43.
  • an AWMF Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:43.
  • Exemplary AWMF Type II Cas protein sequences and nucleotide sequences encoding exemplary AWMF Type II Cas proteins are set forth in Table 2G.
  • an AWMF Type II Cas protein comprises an amino acid sequence of SEQ
  • an AWMF Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.
  • the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.
  • the one or more amino acid substitutions providing nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.
  • the one or more amino acid substitutions providing nickase activity comprise an H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.
  • an AWMF Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.
  • the disclosure provides BUMO Type II Cas proteins.
  • BUMO Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the BUMO Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:49.
  • the BUMO Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:49.
  • a BUMO Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:49.
  • an BUMO Type II Cas protein comprises an amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51 .
  • a BUMO Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51 .
  • the one or more amino acid substitutions providing nickase activity comrpise a D8A substitution wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • the one or more amino acid substitutions providing nickase activity comrpise a N610A substitution wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • the one or more amino acid substitutions providing nickase activity comrpise an H587A substitution wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • a BUMO Type II Cas protein is catalytically inactive, for example due to a D8A substitution in combination with a N610A substitution or an H587A substitution.
  • the disclosure provides COIA Type II Cas proteins.
  • COIA Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the COIA Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:55.
  • the COIA Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:55.
  • a COIA Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:55.
  • a COIA Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57.
  • the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.
  • the one or more amino acid substitutions providing nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.
  • the one or more amino acid substitutions providing nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.
  • a COIA Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution. 6.2.2.10. DJQA Type II Cas Proteins
  • the disclosure provides DJQA Type II Cas proteins.
  • DJQA Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the DJQA Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:61 .
  • the DJQA Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61 .
  • a DJQA Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:61 .
  • DJQA Type II Cas protein sequences and nucleotide sequences encoding exemplary DJQA Type II Cas proteins are set forth in Table 2J.
  • an DJQA Type II Cas protein comprises an amino acid sequence of SEQ
  • a DJQA Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:61 , SEQ ID NO:62, or SEQ ID NO:63.
  • the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • the one or more amino acid substitutions providing nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • the one or more amino acid substitutions providing nickase activity comprise an H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • a DJQA Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.
  • the disclosure provides DWET Type II Cas proteins.
  • DWET Type II Cas proteins can be further classified as Type IIC Cas proteins.
  • the DWET Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:67.
  • the DWET Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67.
  • a DWET Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:67.
  • Exemplary DWET Type II Cas protein sequences and nucleotide sequences encoding exemplary DWET Type I Cas proteins are set forth in Table 2K.
  • an DWET Type II Cas protein comprises an amino acid sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.
  • a DWET Type II Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.
  • the one or more amino acid substitutions providing nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.
  • the one or more amino acid substitutions providing nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.
  • the one or more amino acid substitutions providing nickase activity comprise an H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.
  • a DWET Type II Cas protein is catalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.
  • Type II Cas proteins e.g., an AEQH Type II Cas protein as described in Section 6.2.1 .1 , an AAOF Type II Cas protein as described in Section 6.2.2.1 , an ACEE Type II Cas protein as described in Section 6.2.2.2, an AQSL Type II Cas protein as described in Section 6.2.2.3, an ASWC Type II Cas protein as described in Section 6.2.2.4, an AVFG Type II Cas protein as described in Section 6.2.2.5, an AWIT Type II Cas protein as described in Section 6.2.2.6, an AWMF Type II Cas protein as described in Section 6.2.2.7, a BUMO Type II Cas protein as described in Section 6.2.2.8, a COIA Type II Cas protein as described in Section 6.2.2.9, a DJQA Type II Cas protein as described in Section 6.2.2.10, or a DWET Type II Cas protein as described in Section 6.2.2.11 ,) which are in the form of fusion proteins comprising a Type II Cas protein comprising
  • Fusion proteins can also comprise an amino acid sequence of, for example, a nucleoside deaminase, a reverse transcriptase, a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., Kruppel associated box (KRAB)), a histone-modifying protein, an integrase, or a recombinase.
  • a transcriptional activator e.g., VP64
  • a transcriptional repressor e.g., Kruppel associated box (KRAB)
  • KRAB Kruppel associated box
  • a fusion protein of the disclosure comprises a means for localizing the Type II Cas protein to the nucleus, for example a nuclear localization signal.
  • Non-limiting examples of nuclear localization signals include KRTADGSEFESPKKKRKV (SEQ ID NO:145), PKKKRKV (SEQ ID NO:146), PKKKRRV (SEQ ID NO:147), KRPAATKKAGQAKKKK (SEQ ID NO:148), YGRKKRRQRRR (SEQ ID NO:149), RKKRRQRRR (SEQ ID NQ:150), PAAKRVKLD (SEQ ID NO:151), RQRRNELKRSP (SEQ ID NO:152), VSRKRPRP (SEQ ID NO:153), PPKKARED (SEQ ID NO:154), PQPKKKPL (SEQ ID NO:155), SALIKKKKKMAP (SEQ ID NO:156), PKQKKRK (SEQ ID NO:157), RKLKKKIKKL (SEQ ID NO:158), REKKKFLKRR (SEQ ID NO:159), KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160), RK
  • Exemplary fusion partners include protein tags (e.g., V5-tag e.g., having the sequence GKPIPNPLLGLDST (SEQ ID NO:164) or IPNPLLGLD (SEQ ID NO:165)), FLAG-tag, myc-tag, HA-tag, GST-tag, polyHis-tag, MBP-tag), protein domains, transcription modulators, enzymes acting on small molecule substrates, DNA, RNA and protein modification enzymes (e.g., adenosine deaminase, cytidine deaminase, guanosyl transferase, DNA methyltransferase, RNA methyltransferases, DNA demethylases, RNA demethylases, dioxygenases, polyadenylate polymerases, pseudouridine synthases, acetyltransferases, deacetylase, ubiquitin-ligases, deubiquitinases, kinases, phosphatases
  • a fusion partner is an adenosine deaminase.
  • An exemplary adenosine deaminase is the tRNA adenosine deaminase (TadA) moiety contained in the adenine base editor ABE8e (Richter, 2020, Nature Biotechnology 38:883-891).
  • the TadA moiety of ABE8e comprises the following amino acid sequence:
  • an adenosine deaminase fusion partner comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% amino acid sequence identity with SEQ ID NO:166.
  • Type II Cas proteins of the disclosure in the form of a fusion protein comprising an adenosine deaminase can be used as an adenine base editor to change an “A” to a “G” in DNA.
  • Type II Cas proteins of the disclosure in the form of a fusion protein comprising a cytidine deaminase can be used as a cytosine base editor to change a “C” to a “T” in DNA.
  • a fusion protein of the disclosure comprises a means for deaminating adenosine, for example an adenosine deaminase, e.g., a TadA variant.
  • a fusion protein of the disclosure comprises a means for deaminating cytidine, for example a cytidine deaminase, e.g., cytidine deaminase 1 (CDA1) or an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase (Cheng et al., 2019, Nat Commun.
  • CDA1 cytidine deaminase 1
  • APOBEC apolipoprotein B mRNA-editing complex
  • a fusion protein of the disclosure comprises a means for synthesizing DNA from a single-stranded template, for example a reverse transcriptase.
  • Type II Cas proteins of the disclosure in the form of a fusion protein comprising a reverse transcriptase (RT) can be used as a prime editor to carry out precise base editing without double-stranded DNA breaks.
  • a fusion protein of the disclosure is a prime editor, e.g., a Type II Cas protein fused to a suitable RT (e.g., Moloney murine leukemia virus (M-MLV) RT or other RT enzyme).
  • a suitable RT e.g., Moloney murine leukemia virus (M-MLV) RT or other RT enzyme.
  • M-MLV Moloney murine leukemia virus
  • pegRNA prime editing guide RNA
  • a fusion protein of the disclosure comprises one or more nuclear localization signals positioned N-terminal and/or C-terminal to a Type II Cas protein sequence (e.g., an AEQH Type II Cas protein having a sequence of SEQ ID NO:1).
  • a fusion protein of the disclosure comprises an N-terminal and a C-terminal nuclear localization signal, for example each having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145).
  • the disclosure provides chimeric Type II Cas proteins comprising one or more domains of an AEQH Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an AAOF Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an ACEE Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an AQSL Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an ASWC Type II Cas protein and one or more domains of one or more different proteins (
  • the domain structures of wild-type AIK, BNK, HPLH, and ANAB Type II Cas proteins were inferred by multiple alignment with the amino acid sequences of Type II Cas proteins for which the crystal structure is known and for which it is thus possible to define the boundaries of each functional domain.
  • the domains identified in Type II Cas proteins are: the RuvC catalytic domain (discontinuous, represented by RuvC-l, RuvC-ll, and RuvC-lll domains), bridge helix (BH), recognition (REC) domain, HNH catalytic domain, wedge (WED) domain, and PAM-interacting domain (PID).
  • Tables 3A-3B below report the amino acid positions corresponding to the boundaries between different functional domains in wild-type AEQH (SEQ ID NO:2), AAOF (SEQ ID NO:8), ACEE (SEQ ID NO:14), AQSL (SEQ ID NO:20), ASWC (SEQ ID NO:26), AVFG (SEQ ID NO:32), AWIT (SEQ ID NO:38), AWMF (SEQ ID NO:44), BUMO (SEQ ID NQ:50), COIA (SEQ ID NO:56), DJQA (SEQ ID NO:62), and DWET (SEQ ID NO:68) Type II Cas proteins.
  • a chimeric Type II Cas protein can comprise one of more of the following domains (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more) from an AAOF Type II Cas protein, ACEE Type II Cas protein, AEQH Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, and/or DWET Type II Cas protein, and one or more domains from one or more other proteins, for example SaCas9, SpCas9 or a Type II Cas protein described in US 2020/0332273, US 2019/0169648, or 2015/0247150 (the contents of each of which are incorporated herein by reference in their entirety): RuvC-l, BH, REC, Ru
  • the PID domain can be swapped between different Type II Cas proteins to change the PAM specificity of the resulting chimeric protein (which is given by the donor PID domain). Swapping of other domains or portions of them is also within the scope of the disclosure ⁇ e.g., through protein shuffling).
  • a Type II Cas protein of the disclosure comprises one, two, three, four, five, six, seven, or eight of a RuvC-l domain, a BH domain, a REC domain, a RuvC-ll domain, a HNH domain, a RuvC-lll domain, a WED domain, and a PID domain arranged in the N-terminal to C-terminal direction.
  • all domains are from an AEQH Type II Cas protein (e.g., an AEQH Type II Cas protein whose amino acid sequence comprises SEQ ID NO:1 , 2, or 3).
  • all domains are from an AAOF Type II Cas protein (e.g., an AAOF Type II Cas protein whose amino acid sequence comprises SEQ ID NO:7, 8, or 9).
  • all domains are from an ACEE Type II Cas protein (e.g., an ACEE Type II Cas protein whose amino acid sequence comprises SEQ ID NO:13, 14, or 15).
  • all domains are from an AQSL Type II Cas protein (e.g., an AQSL Type II Cas protein whose amino acid sequence comprises SEQ ID NO:19, 20, or 21).
  • all domains are from an ASWC Type II Cas protein (e.g., an ASWC Type II Cas protein whose amino acid sequence comprises SEQ ID NO:25, 26, or 27). In some embodiments, all domains are from an AVFG Type II Cas protein (e.g., an AVFG Type II Cas protein whose amino acid sequence comprises SEQ ID NO:31 , 32, or 33). In some embodiments, all domains are from an AWIT Type II Cas protein (e.g., an AWIT Type II Cas protein whose amino acid sequence comprises SEQ ID NO:37, 38, or 39).
  • all domains are from an AWMF Type II Cas protein (e.g., an AWMF Type II Cas protein whose amino acid sequence comprises SEQ ID NO:43, 44, or 45). In some embodiments, all domains are from an BUMO Type II Cas protein (e.g., an BUMO Type II Cas protein whose amino acid sequence comprises SEQ ID NO:49, 50, or 51). In some embodiments, all domains are from an COIA Type II Cas protein (e.g., an COIA Type II Cas protein whose amino acid sequence comprises SEQ ID NO:55, 56, or 57).
  • all domains are from an DJQA Type II Cas protein (e.g., an DJQA Type II Cas protein whose amino acid sequence comprises SEQ ID NO:61 , 62, or 63). In some embodiments, all domains are from an DWET Type II Cas protein (e.g., an DWET Type II Cas protein whose amino acid sequence comprises SEQ ID NO:67, 68, or 69). In other embodiments, one or more domains (e.g., one domain), e.g., a PID domain, is from another Type II Cas protein.
  • one or more amino acid substitutions can be introduced in one or more domains to modify the properties of the resulting nuclease in terms of editing activity, targeting specificity or PAM recognition specificity.
  • one or more amino acid substitutions can be introduced to provide nickase activity.
  • Exemplary amino acid substitutions in SaCas9 providing nickase activity are the D10A substitution in the RuvC domain and the N580A substitution in the HNH domain. Combining both the D10A and N580A substitutions in SaCas9 provides a catalytically inactive nuclease.
  • Corresponding substitutions can be introduced into the Type II Cas nucleases of the disclosure to provide nickases and catalytically inactive Cas proteins.
  • an AEQH Type II Cas protein can include a D10A substitution (corresponding to D10A in SaCas9) or a N627A substitution (corresponding to N580A in SaCas9) to provide a nickase, or D10A and N627A substitutions to provide a catalytically inactive Cas protein, where the positions of the D13A and N589A substitutions are defined with respect to amino acid numbering of SEQ ID NO:2. Positions corresponding to D10 and N580 of SaCas9 for Type II Cas proteins of the disclosure as shown in Table 4. An exemplary amino acid substitution in CjCas9 providing nickase activity is the H559A substitution.
  • Type II Cas nucleases of the disclosure can be used, for example, in base editors comprising a cytosine or adenosine deaminase fusion partner.
  • Catalytically inactive Type II Cas proteins can also be used, for example, as fusion partners for transcriptional activators or repressors.
  • the disclosure provides gRNA molecules that can be used with Type II Cas proteins of the disclosure to edit genomic DNA, for example mammalian DNA, e.g., human DNA.
  • gRNAs of the disclosure typically comprise a spacer of 15 to 30 nucleotides in length. The spacer can be positioned 5’ of a crRNA scaffold to form a full crRNA. The crRNA can be used with a tracrRNA to effect cleavage of a target genomic sequence.
  • An exemplary crRNA scaffold sequence that can be used for AEQH Type II Cas gRNAs comprises GUUUUAGUACUCUGUUGGAUAUUGAUAAACUUACAC (SEQ ID NO:73) and an exemplary tracrRNA sequence that can be used for AEQH Type II Cas gRNAs comprises UGUGAGUUUAUCAAUAUCCAACAAUAGUUCUAAGAUAAGGCUAUUUAUGCCGUAGGGUAUGGCG GUAUCCCGUUAAUCCGCCUUUAAGCCAUUGCUUUGCAAUGGCUUA (SEQ ID NO:74).
  • An exemplary crRNA scaffold sequence that can be used for AAOF Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:75) and an exemplary tracrRNA sequence that can be used for AAOF Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUGCAGGUUACUAUGAUAAGGUAGUACACCGCAGAGCUCUAACGCC UCGCGUAAGCGGGGCGUUAUCUCU (SEQ ID NO:76).
  • An exemplary crRNA scaffold sequence that can be used for ACEE Type II Cas gRNAs comprises AUUGUAGUUCCCUAAUUUUUCUUGGUAUGUUAUAAU (SEQ ID NO:77) and an exemplary tracrRNA sequence that can be used for ACEE Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for AQSL Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:79) and an exemplary tracrRNA sequence that can be used for AQSL Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for ASWC Type II Cas gRNAs comprises GUUCUGGCCUAAGCUCAUUUCCUAACUGAUACAAUC (SEQ ID NO:81) and an exemplary tracrRNA sequence that can be used for ASWC Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for AVFG Type II Cas gRNAs comprises GUUAUAGUUCCUAGUAAAUUCUCGAUAUGCUAUAAU (SEQ ID NO:83) and an exemplary tracrRNA sequence that can be used for AVFG Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for AWIT Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:85) and an exemplary tracrRNA sequence that can be used for AWIT Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for AWMF Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:87) and an exemplary tracrRNA sequence that can be used for AWMF Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for BUMO Type II Cas gRNAs comprises GUUGUAGUUCCCUGAUGAUUCUUGGUAUGGUAUAAU (SEQ ID NO:89) and an exemplary tracrRNA sequence that can be used for BUMO Type II Cas gRNAs comprises
  • An exemplary crRNA scaffold sequence that can be used for COIA Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:91) and an exemplary tracrRNA sequence that can be used for COIA Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUU (SEQ ID NO:92).
  • An exemplary crRNA scaffold sequence that can be used for DJQA Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:93) and an exemplary tracrRNA sequence that can be used for DJQA Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUUAUGGUUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGCCGUUUGGCAGGGCGUCAUCUUU (SEQ ID NO:94).
  • An exemplary crRNA scaffold sequence that can be used for DWET Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:95) and an exemplary tracrRNA sequence that can be used for DWET Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUGCAGGUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCUAACGCC CCGCGUAAGCGGGGCGUUAUCUCU (SEQ ID NO:96).
  • gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise the spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold.
  • sgRNAs single guide RNAs
  • gRNAs can comprise separate crRNA and tracrRNA molecules.
  • the spacer sequence is partially or fully complementary to a target sequence found in a genomic DNA sequence, for example a human genomic DNA sequence.
  • a spacer sequence can be partially or fully complementary to a nucleotide sequence in a gene having a disease causing mutation.
  • a spacer that is partially complementary to a target sequence can have, for example, one, two, or three mismatches with the target sequence.
  • gRNAs of the disclosure can comprise a spacer that is 15 to 30 nucleotides in length (e.g., 15 to 25, 16 to 24, 17 to 23, 18 to 22, 19 to 21 , 18 to 30, 20 to 28, 22 to 26, or 23 to 25 nucleotides in length).
  • a spacer is 15 nucleotides in length.
  • a spacer is 16 nucleotides in length.
  • a spacer is 17 nucleotides in length.
  • a spacer is 18 nucleotides in length.
  • a spacer is 19 nucleotides in length.
  • a spacer is 20 nucleotides in length.
  • a spacer is 21 nucleotides in length. In other embodiments, a spacer is 22 nucleotides in length. In other embodiments, a spacer is 23 nucleotides in length. In other embodiments, a spacer is 24 nucleotides in length. In other embodiments, a spacer is 25 nucleotides in length. In other embodiments, a spacer is 26 nucleotides in length. In other embodiments, a spacer is 27 nucleotides in length. In other embodiments, a spacer is 28 nucleotides in length. In other embodiments, a spacer is 29 nucleotides in length. In other embodiments, a spacer is 30 nucleotides in length.
  • Type II Cas endonucleases require a specific sequence, called a protospacer adjacent motif (PAM) that is downstream (e.g., directly downstream) of the target sequence on the non-target strand.
  • PAM protospacer adjacent motif
  • spacer sequences for targeting a gene of interest can be identified by scanning the gene for PAM sequences recognized by the Type II Cas protein.
  • Exemplary PAM sequences for Type II Cas proteins are shown in Table 5A and Table 5B.
  • Example 3 describes exemplary sequences that can be used to target RHO genomic sequences.
  • Example 4 describes exemplary sequences that can be used to target TRAC, B2M, and PD1 genomic sequences.
  • a gRNA of the disclosure comprises a spacer sequence targeting RHO.
  • a gRNA of the disclosure comprises a spacer sequence targeting TRAC.
  • a gRNA of the disclosure comprises a spacer sequence targeting B2M.
  • a gRNA of the disclosure comprises a spacer sequence targeting PD1.
  • the RHO spacer sequences in Table 6A are useful for targeting a RHO gene in the vicinity of the rs7984 SNP, located in the 5’ untranslated region (UTR) of the RHO gene. Allele specific targeting can be achieved by using a gRNA targeting the SNP variant found in a cell or subject. For example, guides in Table 6 having “7984A” in their name can be used when the cell or subject has an “A” at the position of the rs7984 SNP, while guides having “7984G” in their name can be used when the cell or subject has a “G” at the position of the rs7984 SNP.
  • Such guides can be used, for example, with a guide RNA targeting RHO intron 1 (for example, having a spacer as shown in Table 6B) to knock-out expression of the mutated protein. Allele-specific targeting of RHO is described further in Example 3.
  • Exemplary combinations of guides include a first guide RNA having a spacer whose sequence is selected from SEQ ID NOS:326-334 and 336-346 and a second guide RNA having a spacer whose sequence is selected from SEQ ID NOs:402, 403, and 406.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence shown in Table 6A.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence shown in Table 6A.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence shown in Table 6A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 6A.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence shown in Table 6B.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence shown in Table 6B.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence shown in Table 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 or more consecutive nucleotides from a sequence shown in Table 6B.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence shown in Table 6C.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence shown in Table 6C.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence shown in Table 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 6C. 6.3.2. sgRNA Molecules
  • gRNAs of the disclosure can be single-guide RNA (sgRNA) molecules.
  • a sgRNA can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension can comprise one or more hairpins.
  • the sgRNA can comprise a variable length spacer sequence (e.g., 15 to 30 nucleotides) at the 5’ end of the sgRNA sequence and a 3’ sgRNA segment.
  • Type II Cas gRNAs typically comprise a repeat-antirepeat duplex and/or one or more stem-loops generated by the gRNA’s secondary structure.
  • the length of the repeat-antirepeat duplex and/or one or more stem-loops can be modified in order to modulate (e.g., increase) the editing efficacy of a Type II Cas nuclease, and/or to reduce the size of a guide RNA for easier vectorization in situations in which the cargo size of the vector is limiting (e.g., AAV vectors).
  • the repeat-antirepeat duplex (which in a sgRNA is fused through a synthetic linker to become an additional stem loop in the structure) can be trimmed at different lengths without generally having detrimental effects on nuclease function and in some cases even producing increased enzymatic activity. If bulges are present within this duplex they generally should be retained in the final guide RNA sequence.
  • base changes into the stems of the gRNA to increase their stability and folding.
  • Such base changes will preferably correspond to the introduction of G:C couples, which are known to generate the strongest Watson-Crick pairing.
  • these substitutions can consist in the introduction of a G or a C in a specific position of a stem together with a complementary substitution in another position of the gRNA sequence which is predicted to base pair with the former, for example according to available bioinformatic tools for RNA folding such as UNAfold or RNAfold.
  • Stem-loop trimming can also be exploited to stabilize desired secondary structures by removing portions of the guide RNA producing unwanted secondary structures through annealing with other regions of the RNA molecule.
  • Exemplary 3’ sgRNA scaffold sequences for Type 11 A Cas sgRNAs are shown in Table 7A.
  • Exemplary 3’ sgRNA scaffold sequences for Type 11 C Cas sgRNAs are shown in Table 7B.
  • the sgRNA (e.g., for use with AAOF Type II Cas protein, ACEE Type II Cas protein, AEQH Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, and/or DWET Type II Cas proteins) can comprise no uracil base at the 3’ end of the sgRNA sequence.
  • the sgRNA comprises one or more uracil bases at the 3’ end of the sgRNA sequence, for example to promote correct sgRNA folding.
  • the sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 2 uracil (UU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 3 uracil (UUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 4 uracil (UUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 6 uracil (UUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 7 uracil (UUUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence.
  • Different length stretches of uracil can be appended at the 3’end of a sgRNA as terminators.
  • the 3’ sgRNA sequences set forth in Table 7A and Table 7B can be modified by adding (or removing) one or more uracils at the end of the sequence.
  • a sgRNA scaffold for use with an AEQH Type II Cas protein comprises the sequence GUUUUAGUACUCUGUUGGAUAUUGAUAAACUUACAGAAAUGUGAGUUUAUCAAUAUCCAACAAUA GUUCUAAGAUAAGGCUAUUUAUGCCGUAGGGUAUGGCGGUAUCCCGUUAAUCCGCCUUUAAGCC AUUGCUUUGCAAUGGCUUAUUUUU (SEQ ID N0:121).
  • a sgRNA scaffold for use with an AEQH Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AAOF Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AAOF Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an ACEE Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an ACEE Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AQSL Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AQSL Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an ASWC Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an ASWC Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AVFG Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AVFG Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AWIT Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AWIT Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AWMF Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an AWMF Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an BUMO Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an BUMO Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an COIA Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an COIA Type II Cas protein comprises the sequence
  • a sgRNA scaffold for use with an DJQA Type II Cas protein comprises the sequence GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUUAUGG UUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGCCCUGCCGUUUGGCAGGGCGUCAUCU UUUUU (SEQ ID N0:141).
  • a sgRNA scaffold for use with an DJQA Type II Cas protein comprises the sequence GUCAUAGUUCCCUAAGAAAUUAUGGUUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGCCGUUUGGCAGGGCGUCAUCUUUUUU (SEQ ID NO:142).
  • a sgRNA scaffold for use with an DWET Type II Cas protein comprises the sequence GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUGCAG GUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCUAACGCCCCGCGUAAGCGGGGCGUUAUCUCU UUUUU (SEQ ID NO:143).
  • a sgRNA scaffold for use with an DWET Type II Cas protein comprises the sequence GUUGUAGUUCCCUGGUAGGAAACUAUGCAGGUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCU AACGCCCCGCGUAAGCGGGGCGUUAUCUCUUUUUU (SEQ ID NO:144).
  • Guide RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated, as described in the art.
  • the disclosed gRNA e.g., sgRNA) molecules can be unmodified or can contain any one or more of an array of chemical modifications.
  • RNAs While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
  • HPLC high-performance liquid chromatography
  • One approach that can be used for generating chemically modified RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Type II Cas endonuclease, are more readily generated enzymatically.
  • RNAs While fewer types of modifications are available for use in enzymatically produced RNAs, there are still modifications that can be used to, for instance, enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described herein and in the art.
  • modifications can comprise one or more nucleotides modified at the 2' position of the sugar, for instance a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide.
  • RNA modifications can comprise 2'-fluoro, 2'-amino or 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH, ⁇ N(CH3)-O-CH2 (known as a methylene(methylimino) or MMI backbone), CH2-O-N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P- O- CH,); amide backbones (see De Mesmaeker et al. 1995, Ace. Chem.
  • morpholino backbone structures see U.S. Patent No. 5,034,506
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 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'; see U.S.
  • Morpholino-based oligomeric compounds are described in Braasch and David Corey, 2002, Biochemistry, 41 (14):4503-4510; Genesis, Volume 30, Issue 3, (2001); Heasman, 2002, Dev. Biol., 243: 209-214; Nasevicius et al., 2000, Nat. Genet., 26:216-220; Lacerra et al., 2000, Proc. Natl. Acad. Sci., 97: 9591-9596; and U.S. Patent No. 5,034,506.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts; see U.S. Patent Nos.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 , OCH 3 O(CH 2 )n CH 3 , O(CH 2 )n NH 2 , or O(CH 2 )n CH 3 , where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or bi- alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group
  • a modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl)) (Martin et al., 1995, Helv. Chim. Acta, 78, 486).
  • Other modifications include 2'-methoxy (2'-O-CH 3 ), 2'-propoxy (2'- OCH 2 CH 2 CH 3 ) and 2'-fluoro (2 - F).
  • Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.
  • both a sugar and an internucleoside linkage (in the backbone) of the nucleotide units can be replaced with novel groups.
  • the base units can be maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • RNAs such as guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5- methylcytosine (also referred to as 5-methyl-2' deoxy cytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino) adenine or other heterosub stituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexy
  • Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouraci I, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouraci I, 8-halo, 8-amino, 8- thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluor
  • nucleobases can comprise those disclosed in U.S. Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science and Engineering', 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991 , 30, p. 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', 289-302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases can be useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by about 0.6-1 .2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, 276-278) and are aspects of base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • nucleobases are described in U.S. Patent No. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711 ; 5,552,540; 5,587,469; 5,596,091 ; 5,614,617; 5,681 ,941 ; 5,750,692; 5,763,588; 5,830,653;
  • a modified gRNA can include, for example, one or more non-natural sugars, internucleotide linkages and/or bases. It is not necessary for all positions in a given gRNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.
  • the guide RNAs and/or mRNA (or DNA) encoding an endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al. 1989, Proc. Natl. Acad. Sci. USA, 86: 6553-6556); cholic acid (Manoharan et al, 1994, Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S- tritylthiol
  • a thiocholesterol Olet al., 1992, Nucl.
  • Acids Res., 20: 533-538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al, 1990, FEBS Lett., 259: 327-330; Svinarchuk et al, 1993, Biochimie, 75: 49- 54); a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995, Tetrahedron Lett., 36: 3651-3654; and Shea et al, 1990, Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl-rac-g
  • Acids Res., 18: 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al, 1995, Nucleosides & Nucleotides, 14: 969-973); adamantane acetic acid (Manoharan et al, 1995, Tetrahedron Lett., 36: 3651-3654); a palmityl moiety (Mishra et al., 1995, Biochim. Biophys. Acta, 1264: 229- 237); or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al, 1996, J. Pharmacol. Exp.
  • Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites.
  • nucleotides such as cationic polysomes and liposomes
  • hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., 2014, Protein Pept Lett. 21 (10):1025-30.
  • ASGPRs asialoglycoprotein receptors
  • Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and/or complexes thereof to particular target cells of interest.
  • Targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups.
  • Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application Publication WO1993007883, and U.S. Patent No. 6,287,860.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -trityl thiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl- oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a
  • the disclosure provides systems comprising a Type II Cas protein of the disclosure (e.g., as described in Section 6.2) and a means for targeting the Type II Cas protein to a target genomic sequence.
  • the means for targeting the Type II Cas protein to a target genomic sequence can be a guide RNA (gRNA) (e.g., as described in Section 6.3).
  • gRNA guide RNA
  • the disclosure also provides systems comprising a Type II Cas protein of the disclosure (e.g., as described in Section 6.2) and a gRNA (e.g., as described in Section 6.3).
  • the systems can comprise a ribonucleoprotein particle (RNP) in which a Type II Cas protein is complexed with a gRNA, for example a sgRNA or separate crRNA and tracrRNA.
  • RNP ribonucleoprotein particle
  • Systems of the disclosure can in some embodiments further comprise genomic DNA complexed with the Type II Cas protein and the gRNA. Accordingly, the disclosure provides systems comprising a Type II Cas protein, a genomic DNA, and gRNA, all complexed with one another.
  • the systems of the disclosure can exist within a cell (whether the cell is in vivo, ex vivo, or in vitro) or outside a cell (e.g., in a particle our outside of a particle).
  • the disclosure provides nucleic acids (e.g., DNA or RNA) encoding Type II Cas proteins (e.g., AAOF Type II Cas proteins, ACEE Type II Cas proteins, AEQH Type II Cas proteins, AQSL Type II Cas proteins, ASWC Type II Cas proteins, AVFG Type II Cas proteins, AWIT Type II Cas proteins, AWMF Type II Cas proteins, BUMO Type II Cas proteins, COIA Type II Cas proteins, DJQA Type II Cas proteins, and DWET Type II Cas proteins), nucleic acids encoding gRNAs of the disclosure (e.g., a single gRNA or combination of gRNAs), nucleic acids encoding both Type II Cas proteins and gRNAs, and pluralities of nucleic acids, for example comprising a nucleic acid encoding a Type II Cas protein and a gRNA.
  • Type II Cas proteins e.g., AAOF Type II Cas proteins, ACEE Type II Ca
  • a nucleic acid encoding a Type II Cas protein and/or gRNA can be, for example, a plasmid or a viral genome (e.g., a lentivirus, retrovirus, adenovirus, or adeno-associated virus genome).
  • Plasmids can be, for example, plasmids for producing virus particles, e.g., lentivirus particles, or plasmids for propagating the Type II Cas and gRNA coding sequences in bacterial (e.g., E. coli) or eukaryotic (e.g., yeast) cells.
  • a nucleic acid encoding a Type II Cas protein can, in some embodiments, further encode a gRNA.
  • a gRNA can be encoded by a separate nucleic acid (e.g., DNA or mRNA).
  • Nucleic acids encoding a Type II Cas protein can be codon optimized, e.g., where at least one non-common codon or less-common codon has been replaced by a codon that is common in a host cell.
  • a codon optimized nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system.
  • a human codon-optimized polynucleotide encoding Type II Cas can be used for producing a Type II Cas polypeptide. Exemplary codon-optimized sequences are shown in Tables 1A-1G and Tables 2A-2C.
  • Nucleic acids of the disclosure can comprise one or more regulatory elements such as promoters, enhancers, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Such regulatory elements are described, for example, in Goeddel, 1990, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest or in particular cell types. Regulatory elements may also direct expression in a temporaldependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a nucleic acid of the disclosure comprises one or more pol III promoter (e.g., 1 , 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1 , 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1 , 2, 3, 4, 5, or more pol I promoters), or combinations thereof, e.g., to express a Type II Cas protein and a gRNA separately.
  • pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous Sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, 1985, Cell 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and EF1a promoters (for example, full length EF1a promoter and the EFS promoter, which is a short, intron-less form of the full EF1a promoter).
  • RSV Rous Sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Exemplary enhancer elements include WPRE; CMV enhancers; the R- U5' segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit p-globin. It will be appreciated by those skilled in the art that the design of an expression vector can depend on such factors as the choice of the host cell, the level of expression desired, etc.
  • vector refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked.
  • polynucleotide vector includes a "plasmid”, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • plasmid refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • viral vector Another type of polynucleotide vector; wherein additional nucleic acid segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors can be capable of directing the expression of nucleic acids to which they are operably linked. Such vectors can be referred to herein as “recombinant expression vectors", or more simply “expression vectors”, which serve equivalent functions.
  • operably linked means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence.
  • regulatory sequence is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.
  • Vectors can include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (e.g., AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, AAVrhIO), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors.
  • retrovirus e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcom
  • vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-l, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.
  • a vector can comprise one or more transcription and/or translation control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector.
  • the vector can be a selfinactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.
  • Non-limiting examples of suitable eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-l promoters (for example, the full EF1a promoter and the EFS promoter), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-l.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • LTRs long terminal repeats
  • human elongation factor-l promoters for example, the full EF1a promoter and the EFS promoter
  • CAG chicken beta-actin promoter
  • MSCV murine stem
  • An expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can also comprise appropriate sequences for amplifying expression.
  • the expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.
  • a promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.).
  • the promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter).
  • the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, for example a human RHO promoter or human rhodopsin kinase promoter (hGRK), a cell type specific promoter, etc.).
  • the disclosure further provides particles comprising a Type II Cas protein of the disclosure (e.g., an AAOF Type II Cas protein, an ACEE Type II Cas protein, an AEQH Type II Cas protein, an AQSL Type II Cas protein, an ASWC Type II Cas protein, an AVFG Type II Cas protein, an AWIT Type II Cas protein, an AWMF Type II Cas protein, a BUMO Type II Cas protein, a COIA Type II Cas protein, a DJQA Type II Cas protein, or a DWET Type II Cas protein), particles comprising a gRNA of the disclosure, particles comprising a system of the disclosure, and particles comprising a nucleic acid or plurality of nucleic acids of the disclosure.
  • a Type II Cas protein of the disclosure e.g., an AAOF Type II Cas protein, an ACEE Type II Cas protein, an AEQH Type II Cas protein, an AQSL Type II Cas protein, an ASWC Type II Cas protein
  • the particles can in some embodiments comprise or further comprise a gRNA, or a nucleic acid encoding the gRNA (e.g., DNA or mRNA).
  • the particles can comprise a RNP of the disclosure.
  • Exemplary particles include lipid nanoparticles, vesicles, viral-like particles (VLPs) and gold nanoparticles. See, e.g., WO 2020/012335, the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Type II Cas proteins to cells (e.g., complexed together as a RNP).
  • the disclosure provides particles (e.g., virus particles) comprising a nucleic acid encoding a Type II Cas protein of the disclosure.
  • the particles can further comprise a nucleic acid encoding a gRNA.
  • a nucleic acid encoding a Type II Cas protein can further encode a gRNA.
  • the disclosure further provides pluralities of particles (e.g., pluralities of virus particles).
  • Such pluralities can include a particle encoding a Type II Cas protein and a different particle encoding a gRNA.
  • a plurality of particles can comprise a virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 virus particle) encoding a Type II Cas protein and a second virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 virus particle) encoding a gRNA.
  • a plurality of particles can comprise a plurality of virus particles where each particle encodes a Type II Cas protein and a gRNA.
  • the disclosure further provides cells and populations of cells (e.g., ex vivo cells and populations of cells) that can comprise a Type II Cas protein (e.g., introduced to the cell as a RNP) or a nucleic acid encoding the Type II Cas protein (e.g., DNA or mRNA) (optionally also encoding a gRNA).
  • a Type II Cas protein e.g., introduced to the cell as a RNP
  • a nucleic acid encoding the Type II Cas protein e.g., DNA or mRNA
  • the disclosure further provides cells and populations of cells comprising a gRNA of the disclosure (optionally complexed with a Type II Cas protein) or a nucleic acid encoding the gRNA (e.g., DNA or mRNA) (optionally also encoding a Type II Cas protein).
  • the cells and populations of cells can be, for example, human cells such as a stem cell, e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS), or an embryonic stem cell.
  • a stem cell e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS), or an embryonic stem cell.
  • the cells and populations of cells are T cells.
  • Methods for introducing proteins and nucleic acids to cells are known in the art.
  • a RNP can be produced by mixing a Type II Cas protein and one or more guide RNAs in an appropriate buffer.
  • An RNP can be introduced to a cell, for example, via electroporation and other methods known in the art.
  • the cell populations of the disclosure can be cells in which gene editing by the systems of the disclosure has taken place, or cells in which the components of a system of the disclosure have been introduced or expressed but gene editing has not taken place, or a combination thereof.
  • a cell population can comprise, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have undergone gene editing by a system of the disclosure.
  • compositions and medicaments comprising a Type II Cas protein, gRNA, nucleic acid or plurality of nucleic acids, system, particle, or plurality of particles of the disclosure together with a pharmaceutically acceptable excipient.
  • Suitable excipients include, but are not limited to, salts, diluents, (e.g., Tris-HCI, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof.
  • Suitable pharmaceutically acceptable excipients can be selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • PEG polyethylene glycol
  • metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.
  • liposomes such as polyacetic acid, polyglycolic acid, hydrogels, etc.
  • Suitable dosage forms for administration include solutions, suspensions, and emulsions.
  • the components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3-butanediol.
  • formulations can include one or more tonicity agents to adjust the isotonic range of the formulation.
  • Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.
  • the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration.
  • Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
  • the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration.
  • the formulations can comprise a guide RNA and a Type II Cas protein in a pharmaceutically effective amount sufficient to edit a gene in a cell.
  • the pharmaceutical compositions can be formulated for medical and/or veterinary use.
  • the disclosure further provides methods of using the Type II Cas proteins, gRNAs, nucleic acids (including pluralities of nucleic acids), systems, and particles (including pluralities of particles) of the disclosure for altering cells.
  • a method of altering a cell comprises contacting a eukaryotic cell (e.g., a human cell) with a nucleic acid, particle, system or pharmaceutical composition described herein.
  • a eukaryotic cell e.g., a human cell
  • Contacting a cell with a disclosed nucleic acid, particle, system or pharmaceutical composition can be achieved by any method known in the art and can be performed in vivo, ex vivo, or in vitro.
  • the methods can include obtaining one or more cells from a subject prior to contacting the cell(s) with a herein disclosed nucleic acid, particle, system or pharmaceutical composition.
  • the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.
  • Type II Cas and gRNA as well as nucleic acids encoding Type II Cas and gRNAs can be delivered to a cell by any means known in the art, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.
  • a polynucleotide encoding Type II Cas and a gRNA can be delivered to a cell (ex vivo or in vivo) by a lipid nanoparticle (LNP).
  • LNPs can have, for example, a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs can be made from cationic, anionic, neutral lipids, and combinations thereof.
  • Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability.
  • LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Lipids and combinations of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC- cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE- polyethylene glycol (PEG).
  • DOTMA DOSPA
  • DOTAP DOTAP
  • DMRIE DC- cholesterol
  • DOTAP-cholesterol DOTAP-cholesterol
  • GAP-DMORIE-DPyPE GAP-DMORIE-DPyPE
  • PEG polyethylene glycol
  • Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 , and 7C1 .
  • Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM.
  • Examples of PEG- modified lipids are: PEG-DMG, PEG- CerCI4, and PEG-CerC20.
  • Lipids can be combined in any number of molar ratios to produce a LNP.
  • the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
  • Type II Cas and/or gRNAs can be delivered to a cell via an adeno-associated viral vector (e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype), or by another viral vector.
  • an adeno-associated viral vector e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype
  • another viral vector e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype
  • viral vectors include, but are not limited to lentivirus, adenovirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.
  • a Type II Cas mRNA is formulated in a lipid nanoparticle, while a sgRNA is delivered to a cell in an AAV or other viral vector.
  • one or more AAV vectors are used to deliver both a sgRNA and a Type II Cas.
  • a Type II Cas and a sgRNA are delivered using separate vectors.
  • a Type II Cas and a sgRNA are delivered using a single vector.
  • BNK Type II Cas and AIK Type II Cas with their relatively small size, can be delivered with a gRNA (e.g., sgRNA) using a single AAV vector.
  • compositions and methods for delivering Type II Cas and gRNAs to a cell and/or subject are further described in PCT Patent Application Publications WO 2019/102381 , WO 2020/012335, and WO 2020/053224, each of which is incorporated by reference herein in its entirety.
  • DNA cleavage can result in a single-strand break (SSB) or double-strand break (DSB) at particular locations within the DNA molecule.
  • SSB single-strand break
  • DSB double-strand break
  • Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-dependent repair (HDR) and non-homologous endjoining (NHEJ).
  • HDR homology-dependent repair
  • NHEJ non-homologous endjoining
  • These repair processes can edit the targeted polynucleotide by introducing a mutation, thereby resulting in a polynucleotide having a sequence which differs from the polynucleotide’s sequence prior to cleavage by a Type II Cas.
  • NHEJ and HDR DNA repair processes consist of a family of alternative pathways.
  • Non- homologous end-joining refers to the natural, cellular process in which a double-stranded DNA- break is repaired by the direct joining of two non-homologous DNA segments. See, e.g. Cahill et al., 2006, Front. Biosci. 11 :1958-1976.
  • DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair.
  • NHEJ repair mechanisms can introduce mutations into the coding sequence which can disrupt gene function.
  • NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with a modification of the polynucleotide sequence such as a loss of or addition of nucleotides in the polynucleotide sequence.
  • the modification of the polynucleotide sequence can disrupt (or perhaps enhance) gene expression.
  • Homology-dependent repair utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point.
  • the homologous sequence can be in the endogenous genome, such as a sister chromatid.
  • the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double- stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.
  • a third repair mechanism includes microhomology-mediated end joining (MMEJ), also referred to as “Alternative NHEJ (ANHEJ)”, in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site.
  • MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.
  • Modifications of a cleaved polynucleotide by HDR, NHEJ, and/or ANHEJ can result in, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation.
  • the aforementioned process outcomes are examples of editing a polynucleotide.
  • Advantages of ex vivo cell therapy approaches include the ability to conduct a comprehensive analysis of the therapeutic prior to administration.
  • Nuclease-based therapeutics can have some level of off-target effects.
  • Performing gene correction ex vivo allows a method user to characterize the corrected cell population prior to implantation, including identifying any undesirable off-target effects. Where undesirable effects are observed, a method user may opt not to implant the cells or cell progeny, may further edit the cells, or may select new cells for editing and analysis.
  • Other advantages include ease of genetic correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells that will be required for a cell-based therapy. Furthermore, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic correction, without risking a decrease in viability.
  • Additional promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid.
  • the amount of time that delivered protein and RNA remain in the cell can also be adjusted using treatments or domains added to change the half-life.
  • In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing.
  • In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment.
  • An advantage of in vivo gene therapy can be the ease of therapeutic production and administration.
  • the same therapeutic approach and therapy has the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele.
  • ex vivo cell therapy typically requires using a subject’s own cells, which are isolated, manipulated and returned to the same patient.
  • Progenitor cells are capable of both proliferation and giving rise to more progenitor cells, which in turn have the ability to generate a large number of cells that can in turn give rise to differentiated or differentiable daughter cells.
  • the daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • stem cell refers then to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating.
  • progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.
  • Cellular differentiation is a complex process typically occurring through many cell divisions.
  • a differentiated cell can derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types that each can give rise to can vary considerably.
  • Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors.
  • stem cells can also be "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required.
  • Human cells described herein can be induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • An advantage of using iPSCs in the methods of the disclosure is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then differentiated into a progenitor cell to be administered to the subject (e.g., an autologous cell). Because progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.
  • Mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, 2006, Cell 126(4): 663-76.
  • iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape.
  • mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3(6):595-605), and tetrapioid complementation.
  • iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., 2014, Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57; Barrett et al, 2014, Stem Cells Trans Med 3: 1-6 sctm.2014-0121 ; Focosi et al, 2014, Blood Cancer Journal 4: e211 .
  • the production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.
  • iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell.
  • reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., 2010, Cell Stem Cell, 7(5):6I8- 30.
  • Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3/4 or Pouf5l), Soxl, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2, Klf4, Klf5, NR5A2, c- Myc, 1- Myc, n-Myc, Rem2, Tert, and LIN28.
  • Reprogramming using the methods and compositions described herein can further comprise introducing one or more of Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell.
  • the methods and compositions described herein can further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming.
  • the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein.
  • the reprogramming is not affected by a method that alters the genome.
  • reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.
  • Efficiency of reprogramming (the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al., 2008, Cell-Stem Cell 2:525-528; Huangfu et al., 2008, Nature Biotechnology 26(7):795-797; and Marson et al., 2008, Cell-Stem Cell 3: 132-135.
  • an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patientspecific or disease-specific iPSCs.
  • agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HD AC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
  • reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g ., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(l,3-Dioxo-IH,3H- benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or
  • reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g, catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs.
  • inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
  • BIOMOL International Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
  • Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdfi, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl.
  • a cell that expresses Oct4 or Nanog is identified as pluripotent.
  • Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. Detection can involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be best identified via RT-PCR, or protein detection methods such as immunocytochemistry, while cell surface markers are readily identified, e.g., by immunocytochemistry.
  • Pluripotency of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers.
  • teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
  • the cells can be introduced into nude mice and histology and/or immunohistochemistry can be performed on a tumor arising from the cells.
  • the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
  • Patient-specific iPS cells or cell line can be created.
  • the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell.
  • the set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1 , SOX2, SOX3, SOX15, SOX18, NANOG, KLF1 , KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.
  • a biopsy or aspirate of a subject’s bone marrow can be performed.
  • a biopsy or aspirate is a sample of tissue or fluid taken from the body.
  • biopsies or aspirates There are many different kinds of biopsies or aspirates. Nearly all of them involve using a sharp tool to remove a small amount of tissue. If the biopsy will be on the skin or other sensitive area, numbing medicine can be applied first.
  • a biopsy or aspirate can be performed according to any of the known methods in the art. For example, in a bone marrow aspirate, a large needle is used to enter the pelvis bone to collect bone marrow.
  • a mesenchymal stem cell can be isolated from a subject.
  • Mesenchymal stem cells can be isolated according to any method known in the art, such as from a subject’s bone marrow or peripheral blood.
  • marrow aspirate can be collected into a syringe with heparin.
  • Cells can be washed and centrifuged on a PercollTM density gradient.
  • Cells, such as blood cells, liver cells, interstitial cells, macrophages, mast cells, and thymocytes can be separated using density gradient centrifugation media, PercollTM.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the Type II Cas proteins and gRNAs of the disclosure can be used to alter various genomic targets.
  • the methods of altering a cell are methods for altering a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence.
  • the methods of altering a cell are methods of altering a TRAC, B2M, PD1, or LAG3 genomic sequence.
  • Reference sequences of RHO, TRAC, B2M, PD1, and LAG3 are available in public databases, for example those maintained by NCBI.
  • RHO has the NCBI gene ID: 6010
  • TRAC has the NCBI gene ID:28755
  • B2M has the NCBI gene ID: 567
  • PD1 has the NCBI gene ID:5133
  • LAG3 has the NCBI gene ID: 3902.
  • the methods of altering a cell are methods for altering a hemoglobin subunit beta (HBB) gene.
  • HBB mutations are associated with p-thalassemia and SCD. Dever et al., 2016 Nature 539(7629):384-389.
  • the methods of altering a cell are methods for altering a CCR5 gene.
  • CCR5 has demonstrated involvement in several different disease states including, but not limited to, human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • 2018/119359 describes CCR5 editing by CRISPR-Cas to make loss of function CCR5 in order to provide protection against HIV infection, decrease one or more symptoms of HIV infection, halt or delay progression of HIV to AIDS, and/or decrease one or more symptoms of AIDS.
  • the methods of altering a cell are methods for altering a PD1 , B2M gene, TRAC gene, or a combination thereof.
  • CAR-T cells having PD1 , B2M and TRAC genes disrupted by CRISPR-Type II Cas have demonstrated enhanced activity in preclinical glioma models. Choi et al., 2019, Journal for ImmunoTherapy of Cancer 7:309.
  • the methods of altering a cell are methods for altering an USH2A gene. Mutations in the USH2A gene can cause Usher syndrome type 2A, which is characterized by progressive hearing and vision loss.
  • the methods of altering a cell are methods for altering a RHO gene. Mutations in the RHO gene can cause retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • Allele specific editing of human RHO alleles having pathogenic mutations can be achieved using guide RNA (gRNA) molecules targeting the rs7984 SNP (for example having spacers as shown in Table 6A) located in the 5’ untranslated region (UTR) of the RHO gene.
  • gRNA guide RNA
  • SNPs are very common in the human population, and a significant proportion of subjects are heterozygous for the rs7984 SNP.
  • allele specific editing of the RHO allele having the pathogenic mutation can be achieved through the use of a gRNA targeting the SNP variant found in the subject’s RHO allele having the pathogenic mutation.
  • This allele-specific editing strategy which does not directly target a specific pathogenic RHO gene mutation, advantageously allows editing of RHO genes having a variety of different pathogenic mutations.
  • a rs7984 SNP targeting gRNA of the disclosure can be used in combination with a second gRNA targeting a second site in the RHO gene, for example a site in intron 1 (e.g., a gRNA having a spacer as shown in Table 6B), to promote two cuts in the RHO gene having the pathogenic mutation. Cleaving the RHO gene having the pathogenic mutation at two sites can promote a deletion in the RHO gene having the pathogenic mutation, which can result in reduced mutant RHO protein expression.
  • a site intron 1 e.g., a gRNA having a spacer as shown in Table 6B
  • Editing a subject’s RHO allele can comprise editing a RHO allele in one or more cells from the subject (e.g., photoreceptor cells or retinal progenitor cells) or one or more cells derived from a cell of the subject (e.g., an induced pluripotent stem cell (iPSC)).
  • iPSC induced pluripotent stem cell
  • one or more cells from the subject or one or more cells derived from a cell of the subject can be contacted with a nucleic acid, system, or particle of the disclosure ex vivo, and cells having an edited RHO gene or progeny thereof can subsequently be implanted into the subject.
  • Edited iPSCs can be differentiated, for instance into photoreceptor cells or retinal progenitor cells.
  • resultant differentiated cells can be implanted into the subject.
  • implantation of edited cells can proceed without an intervening differentiation step.
  • An in vivo method of RHO allele editing can comprise editing a RHO allele having a pathogenic mutation in a cell of a subject, such as photoreceptor cells or retinal progenitor cells.
  • the in vivo methods comprise administering one or more pharmaceutical compositions of the disclosure to or near the eye of a subject, e.g., by sub-retinal injection or intravitreal injection.
  • a single pharmaceutical composition comprising one or more AAV particles encoding one or more gRNAs (e.g., a gRNA targeting the rs7984 SNP and a gRNA targeting RHO intron 1) and a Type II Cas protein of the disclosure can be used; or alternatively, multiple pharmaceutical compositions can be used, for example a first pharmaceutical composition comprising an AAV particle encoding the gRNA(s) and a second, separate pharmaceutical composition comprising a second AAV particle encoding the Type II Cas protein.
  • they are preferably administered sufficiently close in time so that the gRNA(s) and Type II Cas protein provided by the pharmaceutical compositions are present together in vivo.
  • Targeting of (one or more of) human TRAC, human B2M, human PD1, and human LAG3 genes can be used, for example, in the engineering of chimeric antigen receptor (CAR) T cells.
  • CAR chimeric antigen receptor
  • CRISPR/Cas technology has been used to deliver CAR-encoding DNA sequences to loci such as TRAC and PD1 (see, e.g., Eyquem et al., 2017, Nature 543(7643):113-117; Hu et al., 2023, eClinicalMedicine 60:102010), while TRAC, B2M, PD1, and LAG3 knockout CAR T-cells have been reported (see, e.g., Dimitri et al., 2022, Molecular Cancer 21 :78; Liu et al., 2016, Cell Research 27:154-157; Ren et al., 2017, Clin Cancer Res.
  • Type II Cas proteins and TRAC, B2M, PD1, and LAG3 guides of the disclosure can be used for targeted knock-in of an exogenous DNA sequence to a desired genomic site in a human cell and/or knock-out of TRAC, B2M, PD1, or LAG3 in a human cell, for example a human T cell.
  • T cells are edited ex vivo to produce CAR-T cells and subsequently administered to a subject in need of CAR-T cell therapy.
  • the methods of altering a cell are methods for altering a DNMT1 gene.
  • DNMT1 -related disorder which is a degenerative disorder of the central and peripheral nervous systems.
  • DNMT1 -related disorder is characterized by sensory impairment, loss of sweating, dementia, and hearing loss.
  • Example 1 Identification and Characterization of Type II Cas Proteins [0249] This Example describes studies performed to identify and characterize AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins.
  • MAGs metagenome-assembled genomes
  • cas1, cas2 and cas9 genes were identified from the protein annotation, performed with Prokka version 1.12 (Seemann, 2014, Bioinformatics 30(14):2068-2069).
  • CRISPR arrays were identified using MinCED version 0.4.2 (with default parameters) (Bland, et al., 2007, BMC bioinformatics 8:209).
  • RNIE Rho-independent transcription terminators
  • This Example describes studies performed to further characterize AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins.
  • Type II Cas proteins were expressed in mammalian cells from a plasmid vector characterized by an EF1 alpha-driven cassette. Each Type II Cas protein coding sequence was human codon-optimized and modified by the addition of an SV5 tag at the N-terminus and two bipartite nuclear localization signals (1 at the N-term and 1 at the C-term). The sgRNA were expressed from a U6-driven cassette located on an independent plasmid construct. The human codon-optimized coding sequence of the Type II Cas proteins, as well as the sgRNA scaffolds, were obtained by synthesis from Twist Bioscience.
  • Spacer sequences were cloned into the sgRNA plasmid as annealed DNA oligonucleotides (Eurofins Genomics) using a double Bsal site present in the plasmid.
  • the list of spacer sequences and relative cloning oligonucleotides used in the present example is reported in Table 8. In all cases in which a spacer did not contain a matching native 5’-G, this nucleotide was appended upstream the targeting sequence in order to allow efficient transcription from a U6 promoter.
  • U2OS-EGFP cells harboring a single integrated copy of an EGFP reporter gene, were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 2 mM L-Glutamine (Life Technologies) and penicillin/streptomycin (Life Technologies). All cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. All cells tested mycoplasma negative (PlasmoTest, Invivogen).
  • the sgRNAs to perform the assay were obtained by in vitro transcription of the guide using the High Yield T7 RNA Synthesis Kit (Jena Bioscience) starting from a PCR template generated by amplification from each sgRNA expression construct, as commonly done in the field.
  • the primers used to generate the IVT templates are reported in Table 9.
  • In vitro transcribed gRNAs were subsequently purified using the MEGACIear Transcription Clean-up kit (Thermo Fisher Scientific).
  • the in vitro transcription and translation reaction for Cas expression was performed according to the manufacturer’s protocol (1-Step Human High-Yield Mini IVT Kit, Thermo Fisher Scientific).
  • the nuclease- guide RNA RNP complex was assembled by combining 20 pL of the supernatant containing the soluble Type II Cas protein with 1 pL of RiboLock RNase Inhibitor (Thermo Fisher Scientific) and 2pg of guide RNA (previously transcribed in vitro).
  • the RNP complex was used to digest 1 ug of a PAM plasmid DNA library (containing a defined target sequence flanked at the 3’-end by a randomized 8 nucleotide PAM sequence) for 1 hour at 37°C.
  • a double stranded DNA adapter (Table 10) was ligated to the DNA ends generated by the targeted Cas cleavage and the final ligation product was purified using a GeneJet PCR Purification Kit (Thermo Fisher Scientific).
  • the library was analysed with a 71-bp single read sequencing, using a flow cell v2 micro, on an Illumina MiSeq® sequencer.
  • PAM sequences were extracted from Illumina MiSeq reads and used to generate PAM sequence logos, using Logomaker version 0.8.
  • PAM heatmaps were used to display PAM enrichment, computed dividing the frequency of PAM sequences in the cleaved library by the frequency of the same sequences in a control uncleaved library.
  • U2OS-EGFP cells were nucleofected with 500 ng of nuclease-expressing plasmid and 250 ng of sgRNA-expressing plasmid containing a guide designed to target EGFP using the 4D-NucleofectorTM SE Kit (Lonza), DN-100 program, according to the manufacturer’s protocol. After electroporation, cells were plated in a 24-well plate. EGFP knock-out was analysed 4 days after nucleofection using a BD FACSymphony A1 (BD) flow cytometer.
  • BD BD FACSymphony A1
  • the assay uses in vitro translated Type II Cas proteins coupled with an in vitro synthesized sgRNA to generate a functional ribonucleoprotein complex to cleave a plasmid library characterized by a defined target sequence followed by a randomized 8 nt stretch corresponding to the putative PAMs. Cleaved PAMs can then be recovered after library preparation by next generation sequencing.
  • Table 12 reported here below contains the PAM preferences as determined based on the assay outcome. The PAM logos and the PAM heatmaps reporting the nucleotide preferences for specific positions along the PAMs are reported in FIG. 5A-FIG. 8F.
  • the remaining AWMF Type II Cas protein did not show editing levels above the background of the assay against the currently evaluated targets in the EGFP coding sequence.
  • Example 3 Allele Specific RHO Editing with AAOF, ACEE, AEQH, AVFG, BUMO, DJQA, and DWET Type II Cas
  • This Example describes the design and evaluation of a mutation independent allele-specific strategy to selectively inactivate mutated RHO alleles.
  • the RHO gene which encodes for the photopigment rhodopsin, is one of the most frequently mutated genes in autosomal dominant retinitis pigmentosa and more than one hundred mutations have been described in the art.
  • the great heterogeneity of mutations in affected patients and the overall low prevalence of most of these mutations makes a mutation independent approach to target the disease particularly desirable.
  • effective knock-out of diseased alleles can be effectively obtained using gene editing tools, such as Type II Cas enzymes. Key for the success of the approach is the ability to preferentially downregulate RHO mutated alleles while sparing the wild-type counterpart, in order to preserve photoreceptor function.
  • the strategy described in this Example exploits a commonly occurring non-pathogenic SNP in the RHO gene, rs7984, located in the 5 -UTR of the gene and common in the general population, to selectively target only the one RHO allele containing dominant negative mutations, independently of the exact nature of the mutation. Only patients which are heterozygous for the rs7984 SNP are potentially eligible for this targeting strategy, which is based on the exact knowledge of the phase between the SNP alleles and the mutation affecting each patient. Allele-selectivity is achieved selectively targeting the rs7984 allele which is in phase with the patient’s mutation.
  • a second cut in RHO intron 1 can be introduced to remove the entire exon 1 and knock-out expression of the mutated protein.
  • This second cut which has to occur synchronously with the cut on the rs7984 locus to produce the desired deletion, can be bi-allelic, targeting a site present on both RHO alleles.
  • EF1 alpha-driven expression plasmids were used to express AAOF, ACEE, AEQH, AVFG, BUMO, DJQA, AND DWET Type II Cas protein in mammalian cells. Briefly, the human codon-optimized coding sequences of the different Type II Cas were cloned into the aforementioned expression plasmid.
  • the sgRNA scaffold of each Type II Cas (trimmed scaffold reported in Table 7A or Table 7B, with added 3’ uracils) was cloned into an expression plasmid containing a human U6 promoter to drive guide RNA expression in mammalian cells.
  • Each Type II Cas coding sequence modified by the addition of an SV5 tag at the N-terminus and two bipartite nuclear localization signals (1 at the N-terminus and 1 at the C- terminus) and human codon-optimized, as well as the sgRNA expression cassettes (U6 promoter + sgRNA scaffolds), were obtained as synthetic constructs from Twist Bioscience. Spacer sequences were cloned into the sgRNA expression plasmids as annealed DNA oligonucleotides using a double Bsal site present in the plasmid. The list of spacer sequences and relative cloning oligonucleotides used in the present example is reported in Table 13.
  • HEK293T cells obtained from ATCC
  • HEK293-rs7984G cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 2 mM L-Glutamine (Life Technologies) and penicillin/streptomycin (Life Technologies).
  • HEK293-rs7984G cells which are homozygous for the rs7984G SNP allele, were obtained by base editing and single clones were subsequently isolated, expanded and characterized to select the one to be used for further studies. All cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. All cells tested mycoplasma negative (PlasmoTestTM, Invivogen).
  • HEK293T or HEK293-rs7984G cells were seeded in a 24-well plate 24 hours before transfection. Cells were then transfected with 500 ng of nuclease-expressing plasmid together with 250 ng of sgRNA expression vector targeting the locus of interest using the TranslT®-LT1 reagent (Mirus Bio) according to the manufacturer’s protocol. Cell pellets were collected three days from transfection for analysis.
  • a set of sgRNAs associated with PAMs strongly recognized by AAOF, ACEE, AEQH, AVFG, BUMO, DJQA or DWET Type II Cas spanning the rs7984 SNP were designed (FIG. 10).
  • the editing activity of the selected guides in combination with the respective nucleases was evaluated by transient transfection of HEK293T cells. These cells are homozygous for the rs7984A allele of the SNP and sgRNAs targeting the rs7984A allele were used. As shown in FIG.
  • HEK293T cells were transiently transfected with two alternative versions of each selected sgRNA targeting either the rs7984A or rs7984G alleles together with the corresponding Type II Cas. Since HEK293T cells are homozygous for rs7984A, the on-target editing activity was measured by using rs7984A-targeting guides (FIG. 13A, left bars), while the allele specificity (G vs A direction) was evaluated by using rs7984G-targeting sgRNAs (FIG. 13A, right bars).
  • an engineered HEK293T cell clone modified by base editing to be homozygous for the rs7984G SNP allele (HEK293T-rs7984G) was transfected with the same combination of constructs to evaluate the on-target cleavage activity towards the rs7984G allele (FIG. 13B, left bars) and to verify the allelic specificity in the A vs G direction (FIG. 13B, right bars). AVFG Type II Cas was not included in these evaluations.
  • HEK293T cells were transfected with either ACEE or DJQA Type II Cas together with a panel of sgRNAs targeting the first half of RHO intron 1 in order to generate a deletion below 1000bp in size, when used in combination with the selected sgRNAs targeting the rs7984 SNP for each nuclease (ACEE: g1/g3; DJQA: g1).
  • AZA g1/g3
  • DJQA g1
  • Variable levels of indel formation were observed with the different sgRNAs, with some of the guides failing to generate appreciable editing at the target site in this study (FIGS. 15A-15B).
  • highly efficient guide RNAs were identified.
  • ddPCR very sensitive droplet digital PCR
  • HEK293T cells (homozygous rs7984A) were transiently transfected with either ACEE Type II Cas in combination with the g1 or g3 SNP-targeting guides (rs7984A version) together with intron guides g481 , g543, g546, g794 or g948 or DJQA Type II Cas in combination with the g1 SNP-targeting guide (rs7984A version) together with intron guides g825 or g884. Deletion and inversion formation was then quantified by ddPCR, showing efficient modification of the target locus for most of the evaluated candidates (FIG. 17).
  • ACEE Type II Cas showed a generally higher efficacy in generating the desired edits at the RHO target locus, with SNP g1/g3 in combination with intron g543, g546 or g794 being among the top performers (FIG. 17).
  • sgRNAs targeting the RHO gene were identified that generate high levels of desired deletions/inversions of the first exon of the gene and also have selectivity for the desired RHO copy.
  • Table 19 shows protospacer and oligo sequences used for cloning sgRNA spacers.
  • Table 20 shows oligos used for TIDE analysis.
  • a Type II Cas protein comprising an amino acid sequence having at least 50% sequence identity to:
  • the reference protein sequence is SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • the Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • the Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • the Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • the Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 31 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the BH domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • the Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 61 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the REC domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • the Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the WED domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the PID domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the PID domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the PID domain of the reference protein sequence.
  • Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the PID domain of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the full length of the reference protein sequence.
  • the Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the full length of the reference protein sequence.
  • the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the full length of the reference protein sequence.
  • the Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the full length of the reference protein sequence.
  • Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the full length of the reference protein sequence.
  • the Type II Cas protein of embodiment 1 wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the full length of the reference protein sequence.
  • the Type II Cas protein of embodiment 137 which comprises one or more nuclear localization signals.
  • the Type II Cas protein of embodiment 138 which comprises two or more nuclear localization signals.
  • Type II Cas protein of embodiment 138 or embodiment 139 which comprises an N- terminal nuclear localization signal.
  • the Type II Cas protein of any one of embodiments 138 to 141 which comprises an N- terminal nuclear localization signal and a C-terminal nuclear localization signal.
  • the Type II Cas protein of any one of embodiments 138 to 142, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145), PKKKRKV (SEQ ID NO:146), PKKKRRV (SEQ ID NO:147), KRPAATKKAGQAKKKK (SEQ ID NO:148), YGRKKRRQRRR (SEQ ID NO:149), RKKRRQRRR (SEQ ID NQ:150), PAAKRVKLD (SEQ ID NO:151), RQRRNELKRSP (SEQ ID NO:152), VSRKRPRP (SEQ ID NO:153), PPKKARED (SEQ ID NO:154), PQPKKKPL (SEQ ID NO:155), SALIKKKKKMAP (SEQ ID NO:156), PKQKKRK (SEQ ID NO:157), RKLKKKIKKL (SEQ ID NO:158), REKKKFLKRR (SEQ ID NO
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRKV (SEQ ID NO:146).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRRV (SEQ ID NO:147).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO:148).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO:149).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKKRRQRRR (SEQ ID NQ:150).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PAAKRVKLD (SEQ ID NO:151).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RQRRNELKRSP (SEQ ID NO:152).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence VSRKRPRP (SEQ ID NO:153).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PPKKARED (SEQ ID NO:154).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PQPKKKPL (SEQ ID NO:155).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence SALIKKKKKMAP (SEQ ID NO:156).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKQKKRK (SEQ ID NO:157).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKLKKKIKKL (SEQ ID NO:158).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence REKKKFLKRR (SEQ ID NO:159).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160).
  • Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKCLQAGMNLEARKTKK (SEQ ID NO:161).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:162).
  • the Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:163).
  • Type II Cas protein of any one of embodiments 136 to 164 which comprises a means for deaminating adenosine, optionally wherein the means for deaminating adenosine is an adenosine deaminase.
  • the Type II Cas protein of any one of embodiments 136 to 164 which comprises a fusion partner which is an adenosine deaminase, optionally wherein the amino acid sequence of the adenosine deaminase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:166, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e.
  • the Type II Cas protein of any one of embodiments 136 to 164 which comprises a means for deaminating cytidine, optionally wherein the means for deaminating cytidine is a cytidine deaminase.
  • the Type II Cas protein of any one of embodiments 136 to 164 which comprises a means for synthesizing DNA from a single-stranded template, optionally wherein the means for synthesizing DNA from a single-stranded template is a reverse transcriptase.
  • Type II Cas protein of any one of embodiments 136 to 164 which comprises a fusion partner which is a reverse transcriptase.
  • the Type II Cas protein of embodiment 171 wherein the tag is a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:164) or IPNPLLGLD (SEQ ID NO:165).
  • Type II Cas protein of embodiment 173 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1 .
  • Type II Cas protein of embodiment 173 or embodiment 174 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:3.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:7.
  • the Type II Cas protein of embodiment 178 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:7.
  • 180 The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:8.
  • Type II Cas protein of embodiment 178 or embodiment 179 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:9.
  • the Type II Cas protein of embodiment 183 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:13.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:14.
  • Type II Cas protein of any one of embodiments 183 to 185 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:14.
  • the Type II Cas protein of embodiment 183 or embodiment 184 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:15.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:19.
  • the Type II Cas protein of embodiment 188 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:19.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NQ:20.
  • Type II Cas protein of any one of embodiments 188 to 190, whose amino acid sequence comprises the amino acid sequence of SEQ ID NQ:20.
  • the Type II Cas protein of embodiment 188 or embodiment 189 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:21 .
  • Type II Cas protein of embodiment 193 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:25.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:26.
  • Type II Cas protein of any one of embodiments 193 to 195 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:26.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:31 .
  • the Type II Cas protein of embodiment 198 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:31 .
  • Type II Cas protein of any one of embodiments 199 to 200, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:32.
  • the Type II Cas protein of embodiment 198 or embodiment 199 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:33.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:37.
  • the Type II Cas protein of embodiment 203 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:37.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:38.
  • Type II Cas protein of any one of embodiments 203 to 205 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:38.
  • the Type II Cas protein of embodiment 203 or embodiment 204 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:39.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:43.
  • Type II Cas protein of embodiment 208 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:43.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:44.
  • Type II Cas protein of any one of embodiments 208 to 210 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:44.
  • the Type II Cas protein of embodiment 208 or embodiment 209 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:45.
  • the Type II Cas protein of embodiment 213 or embodiment 214 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:51 .
  • the Type II Cas protein of embodiment 218 or embodiment 219 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:57.
  • Type II Cas protein of any one of embodiments 223 to 225 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:62.
  • Type II Cas protein of embodiment 223 or embodiment 224 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:63.
  • Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:68.
  • the Type II Cas protein of embodiment 228 or embodiment 229 whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:69.
  • a Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of embodiments 1 to 232 except for one or more amino acid substitutions relative to the reference sequence that provide nickase activity, optionally wherein the one or more amino acid substitutions comprise a substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4).
  • a substiution e.g., alanine substitution
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions that provide nickase activity are in a RuvC or HNH domain.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N627A substitution, wherein the position of the N627A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H604A substitution, wherein the position of the H604A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.
  • Type II Cas of embodiment 233 wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N613A substitution, wherein the position of the N613A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N633A substitution, wherein the position of the N633A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H610A substitution, wherein the position of the H610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D12A substitution, wherein the position of the D12A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N607A substitution, wherein the position of the N607A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H584A substitution, wherein the position of the H584A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D8A substitution, wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.
  • the Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.
  • Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.
  • a Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of embodiments 1 to 232 except for one or more amino acid substitutions relative to the reference sequence that render the Type II Cas protein catalytically inactive, optionally wherein the one or more amino acid substitutions comprise a substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4), or a combination thereof.
  • a substiution e.g., alanine substitution
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N627A substitutions, wherein the positions of the D10A and N627A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:2.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H604A substitutions, wherein the positions of the D10A and H604A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:2.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N610A substitutions, wherein the positions of the D9A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:8.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:8.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and N613A substitutions, wherein the positions of the D8A and N613A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:14. 277.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and H590A substitutions, wherein the positions of the D8A and H590A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:14.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N610A substitutions, wherein the positions of the D9A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:20.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NQ:20.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D15A and N633A substitutions, wherein the positions of the D15A and N633A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:26.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D15A and H610A substitutions, wherein the positions of the D15A and H610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:26
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D12A and N607A substitutions, wherein the positions of the D12A and N607A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:32.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D12A and H584A substitutions, wherein the positions of the D12A and H584A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:32
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:38.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:38.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:44. 287.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:44.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and N610A substitutions, wherein the positions of the D8A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:50.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and H587A substitutions, wherein the positions of the D8A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NQ:50.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:56.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:56.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:62.
  • Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:62.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N615A substitutions, wherein the positions of the D9A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:68.
  • the Type II Cas protein of embodiment 271 wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:68.
  • gRNA Cas guide RNA
  • gRNA Cas guide RNA
  • gRNA Cas guide RNA
  • gRNA AQSL Type II Cas guide RNA
  • gRNA ASWC Type II Cas guide RNA
  • gRNA AVFG Type II Cas guide RNA
  • gRNA Cas guide RNA
  • gRNA Cas guide RNA
  • gRNA Cas guide RNA
  • a COIA Type II Cas guide RNA (gRNA) molecule A COIA Type II Cas guide RNA (gRNA) molecule.
  • a DJQA Type II Cas guide RNA (gRNA) molecule A DJQA Type II Cas guide RNA (gRNA) molecule.
  • a DWET Type II Cas guide RNA (gRNA) molecule A DWET Type II Cas guide RNA (gRNA) molecule.
  • the gRNA of any one of embodiments 296 to 307 which is a gRNA for editing a human RHO gene.
  • the gRNA of any one of embodiments 296 to 307 which is a gRNA for editing a human B2/W gene.
  • the gRNA of any one of embodiments 296 to 307 which is a gRNA for editing a human PD1 gene.
  • a guide RNA (gRNA) molecule for editing a human RHO gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:314-399.
  • a guide RNA (gRNA) molecule for editing a human RHO gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID N0s:400-419.
  • a guide RNA (gRNA) molecule for editing a human B2M gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NQs:420-441 .
  • a guide RNA (gRNA) molecule for editing a human TRAC gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:442-461 .
  • a guide RNA (gRNA) molecule for editing a human PD1 gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:462-482.
  • the gRNA of embodiment 318, wherein the spacer is 15 to 25 nucleotides in length.
  • the gRNA of embodiment 318, wherein the spacer is 16 to 24 nucleotides in length.
  • the gRNA of embodiment 318, wherein the spacer is 20 nucleotides in length.
  • gRNA of any one of embodiments 313 to 331 wherein the reference sequence is a reference sequence having at least 23 nucleotides and the spacer comprises 23 consecutive nucleotides of the reference sequence.
  • gRNA of any one of embodiments 296 to 517 which is a single guide RNA (sgRNA).
  • a gRNA comprising a spacer and a sgRNA scaffold which is optionally a gRNA according to any one of embodiments 296 to 518, wherein:
  • the nucleotide sequence of the sgRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is any one of SEQ ID NQS:97-120.
  • a gRNA comprising a means for binding a target mammalian genomic sequence and a sgRNA scaffold, optionally wherein the means for binding a target mammalian genomic sequence is a spacer, wherein:
  • the nucleotide sequence of the sgRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is any one of SEQ ID NQS:97-120.
  • gRNA of embodiment 523 wherein the trimmed stem loop sequence comprises a GAAA tetraloop in place of a longer stem loop sequence in the reference scaffold sequence.
  • 528. The gRNA of embodiment 527, wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 60% identical to the reference scaffold sequence.
  • the gRNA of embodiment 527, wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 65% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 70% identical to the reference scaffold sequence.
  • the gRNA of embodiment 527, wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 75% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 80% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 85% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 90% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 95% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 96% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 97% identical to the reference scaffold sequence.
  • gRNA of embodiment 527, wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 98% identical to the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 99% identical to the reference scaffold sequence.
  • sgRNA scaffold comprises a nucleotide sequence that has no more than 5 nucleotide mismatches with the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that has no more than 3 nucleotide mismatches with the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that has no more than 2 nucleotide mismatches with the reference scaffold sequence.
  • gRNA of embodiment 527 wherein the sgRNA scaffold comprises a nucleotide sequence that has no more than 1 nucleotide mismatches with the reference scaffold sequence.
  • the gRNA of embodiment 519or embodiment 520, wherein the sgRNA scaffold comprises a nucleotide sequence that is 100% identical to the reference scaffold sequence.
  • gRNA of embodiment 549, wherein the reference scaffold sequence is SEQ ID NO:99.
  • the gRNA of embodiment 549, wherein the reference scaffold sequence is SEQ ID NQ:100.
  • gRNA of any one of embodiments 519to 545, wherein the reference scaffold sequence is SEQ ID NQ:101 or SEQ ID NQ:102.
  • the gRNA of embodiment 552, wherein the reference scaffold sequence is SEQ ID NQ:101.
  • the gRNA of embodiment 552, wherein the reference scaffold sequence is SEQ ID NQ:102.
  • gRNA of any one of embodiments 519to 545, wherein the reference scaffold sequence is SEQ ID NQ:103 or SEQ ID NQ:104.
  • the gRNA of embodiment 555, wherein the reference scaffold sequence is SEQ ID NQ:103.
  • the gRNA of embodiment 555, wherein the reference scaffold sequence is SEQ ID NQ:104.
  • the gRNA of embodiment 558, wherein the reference scaffold sequence is SEQ ID NQ:105.
  • the gRNA of embodiment 558, wherein the reference scaffold sequence is SEQ ID NQ:106.
  • gRNA of any one of embodiments 519to 545, wherein the reference scaffold sequence is SEQ ID NQ:107 or SEQ ID NQ:108.
  • the gRNA of embodiment 561 wherein the reference scaffold sequence is SEQ ID NQ:107.
  • the gRNA of embodiment 561 wherein the reference scaffold sequence is SEQ ID NQ:108.

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Family Cites Families (123)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
JPS5927900A (ja) 1982-08-09 1984-02-14 Wakunaga Seiyaku Kk 固定化オリゴヌクレオチド
FR2540122B1 (fr) 1983-01-27 1985-11-29 Centre Nat Rech Scient Nouveaux composes comportant une sequence d'oligonucleotide liee a un agent d'intercalation, leur procede de synthese et leur application
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5430136A (en) 1984-10-16 1995-07-04 Chiron Corporation Oligonucleotides having selectably cleavable and/or abasic sites
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
FR2575751B1 (fr) 1985-01-08 1987-04-03 Pasteur Institut Nouveaux nucleosides de derives de l'adenosine, leur preparation et leurs applications biologiques
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
JPS638396A (ja) 1986-06-30 1988-01-14 Wakunaga Pharmaceut Co Ltd ポリ標識化オリゴヌクレオチド誘導体
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
CA1340032C (en) 1987-06-24 1998-09-08 Jim Haralambidis Lucleoside derivatives
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US4924624A (en) 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
DE3738460A1 (de) 1987-11-12 1989-05-24 Max Planck Gesellschaft Modifizierte oligonukleotide
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
WO1989009221A1 (en) 1988-03-25 1989-10-05 University Of Virginia Alumni Patents Foundation Oligonucleotide n-alkylphosphoramidates
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5457183A (en) 1989-03-06 1995-10-10 Board Of Regents, The University Of Texas System Hydroxylated texaphyrins
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5177198A (en) 1989-11-30 1993-01-05 University Of N.C. At Chapel Hill Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5587470A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. 3-deazapurines
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
AU7579991A (en) 1990-02-20 1991-09-18 Gilead Sciences, Inc. Pseudonucleosides and pseudonucleotides and their polymers
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
DE69032425T2 (de) 1990-05-11 1998-11-26 Microprobe Corp., Bothell, Wash. Teststreifen zum Eintauchen für Nukleinsäure-Hybridisierungsassays und Verfahren zur kovalenten Immobilisierung von Oligonucleotiden
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5614617A (en) 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
PT98562B (pt) 1990-08-03 1999-01-29 Sanofi Sa Processo para a preparacao de composicoes que compreendem sequencias de nucleo-sidos com cerca de 6 a cerca de 200 bases resistentes a nucleases
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
AU662298B2 (en) 1990-09-20 1995-08-31 Gilead Sciences, Inc. Modified internucleoside linkages
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
EP0556301B1 (de) 1990-11-08 2001-01-10 Hybridon, Inc. Verbindung von mehrfachreportergruppen auf synthetischen oligonukleotiden
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
CA2122030C (en) 1991-10-24 1997-03-04 Muthiah Manoharan Derivatized oligonucleotides having improved uptake and other properties
TW393513B (en) 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
ATE160572T1 (de) 1993-03-31 1997-12-15 Sanofi Sa Oligonucleotide mit amidverkettungen die phosphoesterverkettungen einsetzen
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US6287860B1 (en) 2000-01-20 2001-09-11 Isis Pharmaceuticals, Inc. Antisense inhibition of MEKK2 expression
US20030158403A1 (en) 2001-07-03 2003-08-21 Isis Pharmaceuticals, Inc. Nuclease resistant chimeric oligonucleotides
MY189533A (en) 2012-05-25 2022-02-16 Univ California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
CN119752887A (zh) 2012-12-12 2025-04-04 布罗德研究所有限公司 用于序列操纵的系统、方法和优化的指导组合物的工程化
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
MA50877A (fr) 2017-11-21 2020-09-30 Bayer Healthcare Llc Matériaux et méthodes pour le traitement de la rétinite pigmentaire autosomique dominante
CA3105925A1 (en) 2018-07-10 2020-01-16 Alia Therapeutics S.R.L. Vesicles for traceless delivery of guide rna molecules and/or guide rna molecule/rna-guided nuclease complex(es) and a production method thereof
WO2020053224A1 (en) 2018-09-11 2020-03-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for increasing fetal hemoglobin content in eukaryotic cells and uses thereof for the treatment of hemoglobinopathies
AU2019398351A1 (en) * 2018-12-14 2021-06-03 Pioneer Hi-Bred International, Inc. Novel CRISPR-Cas systems for genome editing
US10913941B2 (en) 2019-02-14 2021-02-09 Metagenomi Ip Technologies, Llc Enzymes with RuvC domains
CN114934031B (zh) * 2022-05-25 2023-08-01 广州瑞风生物科技有限公司 新型Cas效应蛋白、基因编辑系统及用途

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