US20150284727A1 - Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof - Google Patents

Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof Download PDF

Info

Publication number
US20150284727A1
US20150284727A1 US14/438,098 US201314438098A US2015284727A1 US 20150284727 A1 US20150284727 A1 US 20150284727A1 US 201314438098 A US201314438098 A US 201314438098A US 2015284727 A1 US2015284727 A1 US 2015284727A1
Authority
US
United States
Prior art keywords
guide rna
composition
cas protein
dna
cas9
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.)
Abandoned
Application number
US14/438,098
Inventor
Jin-soo Kim
Seung Woo Cho
Sojung Kim
Jong Min Kim
Seokjoong Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toolgen Inc
Original Assignee
Toolgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=50544909&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20150284727(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Toolgen Inc filed Critical Toolgen Inc
Priority to US14/438,098 priority Critical patent/US20150284727A1/en
Assigned to TOOLGEN INCORPORATED reassignment TOOLGEN INCORPORATED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHO, SEUNG WOO, KIM, JONG MIN, KIM, SOJUNG, KIM, JIN-SOO, KIM, Seokjoong
Publication of US20150284727A1 publication Critical patent/US20150284727A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
  • CRISPRs are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.
  • CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages.
  • the CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.
  • Cas9 an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells.
  • crRNA is transcribed from the CRISPR element in the host genome, which was previously captured from such foreign invaders.
  • Jinek et al. (1) demonstrated that a single-chain chimeric RNA produced by fusing an essential portion of crRNA and tracrRNA could replace the two RNAs in the Cas9/RNA complex to form a functional endonuclease.
  • CRISPR/Cas systems offer an advantage to zinc finger and transcription activator-like effector DNA-binding proteins, as the site specificity in nucleotide binding CRISPR-Cas proteins is governed by a RNA molecule instead of the DNA-binding protein, which can be more challenging to design and synthesize.
  • RFLP Restriction fragment length polymorphism
  • Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive T7 endonuclease I (T7E1) or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, Dideoxy sequencing, and deep sequencing.
  • T7E1 and Surveyor assays are widely used but are cumbersome.
  • theses enzymes tend to underestimate mutation frequencies because mutant sequences can form homoduplexes with each other and cannot distinguish homozygous bi-allelic mutant clones from wildtype cells.
  • RFLP is free of these limitations and therefore is a method of choice. Indeed, RFLP was one of the first methods to detect engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction sites. It is possible that no restriction sites are available at the target site of interest.
  • the present inventors have made many efforts to develop a genome editing method based on CRISPR/Cas system and finally established a programmable RNA-guided endonuclease that cleave DNA in a targeted manner in eukaryotic cells and organisms.
  • RGENs RNA-guided endonucleases
  • It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • RNA-guided endonuclease RGEN
  • the RGEN comprises a guide RNA specific for target DNA and Cas protein.
  • RGEN RNA-guided endonuclease
  • It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • RGEN RNA-guided endonuclease
  • RGEN RNA-guided endonuclease
  • the present composition for cleaving a target DNA or inducing a targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, the kit comprising the composition, and the method for inducing targeted mutagenesis provide a new convenient genome editing tools.
  • custom RGENs can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP, therefore, the composition and method of the present invention may be used in detection and cleaving naturally-occurring variations and mutations.
  • FIG. 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro.
  • FIG. 2 shows Cas9-induced mutagenesis at an episomal target site.
  • (b) Flow cytometry of cells transfected with Cas9. The percentage of cells that express the RFP-GFP fusion protein is indicated.
  • FIG. 3 shows RGEN-driven mutations at endogenous chromosomal sites.
  • FIG. 4 shows that RGEN-driven off-target mutations are undetectable.
  • On-target and potential off-target sequences The human genome was searched in silico for potential off-target sites. Four sites were identified, each of which carries 3-base mismatches with the CCR5 on-target site. Mismatched bases are underlined.
  • the T7E1 assay was used to investigate whether these sites were mutated in cells transfected with the Cas9/RNA complex. No mutations were detected at these sites. N/A (not applicable), an intergenic site.
  • Cas9 did not induce off-target-associated chromosomal deletions. The CCR5-specific RGEN and ZFN were expressed in human cells. PCR was used to detect the induction of the 15-kb chromosomal deletions in these cells.
  • FIG. 5 shows RGEN-induced Foxn1 gene targeting in mice.
  • (a) A schematic diagram depicting a sgRNA specific to exon 2 of the mouse Foxn1 gene. PAM in exon 2 is shown in red and the sequence in the sgRNA that is complementary to exon 2 is underlined. Triangles indicate cleavage sites.
  • (b) Representative T7E1 assays demonstrating gene-targeting efficiencies of Cas9 mRNA plus Foxn1-specific sgRNA that were delivered via intra-cytoplasmic injection into one-cell stage mouse embryos. Numbers indicate independent founder mice generated from the highest dose. Arrows indicate bands cleaved by T7E1.
  • (c) DNA sequences of mutant alleles observed in three Foxn1 mutant founders identified in b.
  • FIG. 6 shows Foxn1 gene targeting in mouse embryos by intra-cytoplasmic injection of Cas9 mRNA and Foxn1-sgRNA.
  • FIG. 7 shows Foxn1 gene targeting in mouse embryos using the recombinant Cas9 protein: Foxn1-sgRNA complex.
  • (a) and (b) are representative T7E1 assays results and their summaries. Embryos were cultivated in vitro after they underwent pronuclear (a) or intra-cytoplasmic injection (b). Numbers in red indicate T7E1-positive mutant founder mice.
  • FIG. 8 shows Germ-line transmission of the mutant alleles found in Foxn1 mutant founder #12.
  • FIG. 9 shows Genotypes of embryos generated by crossing Prkdc mutant founders. Prkdc mutant founders ⁇ 25 and ⁇ 15 were crossed and E13.5 embryos were isolated.
  • FIG. 10 shows Cas9 protein/sgRNA complex induced targeted mutation.
  • FIG. 11 shows recombinant Cas9 protein-induced mutations in Arabidopsis protoplasts.
  • FIG. 12 shows recombinant Cas9 protein-induced mutant sequences in the Arabidopsis BRI1 gene.
  • FIG. 13 shows T7E1 assay showing endogenous CCR5 gene disruption in 293 cells by treatment of Cas9-mal-9R4L and sgRNA/C9R4LC complex.
  • FIG. 14 ( a, b ) shows mutation frequencies at on-target and off-target sites of RGENs reported in Fu et al. (2013).
  • T7E1 assays analyzing genomic DNA from K562 cells (R) transfected serially with 20 ⁇ g of Cas9-encoding plasmid and with 60 ⁇ g and 120 ⁇ g of in vitro transcribed GX19 crRNA and tracrRNA, respectively (1 ⁇ 10 6 cells), or (D) co-transfected with 1 ⁇ g of Cas9-encoding plasmid and 1 ⁇ g of GX 19 sgRNA expression plasmid (2 ⁇ 10 5 cells).
  • FIG. 15 ( a, b ) shows comparison of guide RNA structure. Mutation frequencies of the RGENs reported in Fu et al. (2013) were measured at on-target and off-target sites using the T7E1 assay. K562 cells were co-transfected with the Cas9-encoding plasmid and the plasmid encoding GX19 sgRNA or GGX20 sgRNA. Off-target sites (OT1-3 etc.) are labeled as in Fu et al. (2013).
  • FIG. 16 shows that in vitro DNA cleavage by Cas9 nickases.
  • (a) Schematic overview of the Cas9 nuclease and the paired Cas9 nickase. The PAM sequences and cleavage sites are shown in box.
  • (c) Schematic overview of DNA cleavage reactions. FAM dyes (shown in box) were linked to both 5′ ends of the DNA substrate.
  • DSBs and SSBs analyzed using fluorescent capillary eletrophoresis. Fluorescentlylabeled DNA substrates were incubated with Cas9 nucleases or nickases before electrophoresis.
  • FIG. 17 shows comparison of Cas9 nuclease and nickase behavior.
  • FIG. 18 shows paired Cas9 nickases tested at other endogenous human loci.
  • (a,c) The sgRNA target sites at human CCR5 and BRCA2 loci. PAM sequences are indicated in red.
  • (b,d) Genome editing activities at each target site were detected by the T7E1 assay. The repair of two nicks that would produce 5′ overhangs led to the formation of indels much more frequently than did those producing 3′ overhangs.
  • FIG. 19 shows that paired Cas9 nickases mediate homologous recombination.
  • FIG. 20 shows DNA splicing induced by paired Cas9 nickases.
  • FIG. 21 shows that paired Cas9 nickases do not induce translocations.
  • FIG. 22 shows a conceptual diagram of the T7E1 and RFLP assays.
  • FIG. 23 shows in vitro cleavage assay of a linearized plasmid containing the C4BPB target site bearing indels.
  • DNA sequences of individual plasmid substrates (upper panel).
  • the PAM sequence is underlined. Inserted bases are shown in box.
  • Arrows (bottom panel) indicate expected positions of DNA bands cleaved by the wild-type-specific RGEN after electrophoresis.
  • FIG. 24 shows genotyping of mutations induced by engineered nucleases in cells via RGEN-mediated RFLP.
  • FIG. 25 shows genotyping of RGEN-induced mutations via the RGEN-RFLP technique.
  • FIG. 26 shows genotyping of mutations induced by engineered nucleases in organisms via RGEN-mediated RFLP.
  • FIG. 27 shows RGEN-mediated genotyping of ZFN-induced mutations.
  • the ZFN target site is shown in box.
  • Black arrows indicate DNA bands cleaved by T7E1.
  • FIG. 28 shows polymorphic sites in a region of the human HLA-B gene.
  • the sequence, which surrounds the RGEN target site, is that of a PCR amplicon from HeLa cells. Polymorphic positions are shown in box.
  • the RGEN target site and the PAM sequence are shown in dashed and bolded box, respectively. Primer sequences are underlined.
  • FIG. 29 shows genotyping of oncogenic mutations via RGEN-RFLP analysis.
  • a recurrent mutation c.133-135 deletion of TCT
  • HCT116 cells was detected by RGENs.
  • HeLa cells were used as a negative control.
  • Genotyping of the KRAS substitution mutation c.34 G>A
  • Mismatched nucleotides are shown in box.
  • HeLa cells were used as a negative control.
  • Arrows indicate DNA bands cleaved by RGENs. DNA sequences confirmed by Sanger sequencing are shown.
  • FIG. 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells via RGEN-RFLP analysis.
  • FIG. 31 shows genotyping of a KRAS point mutation (c.34 G>A).
  • Plasmids harboring either the wild-type or mutant KRAS sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
  • FIG. 32 shows genotyping of a PIK3CA point mutation (c.3140 A>G).
  • Plasmids harboring either the wild-type or mutant PIK3CA sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
  • FIG. 33 shows genotyping of recurrent point mutations in cancer cell lines.
  • Genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are shown in box. Black arrows indicate DNA bands cleaved by RGENs.
  • the present invention provides a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the present invention provides a use of the composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the composition is also referred to as a RNA-guided endonuclease (RGEN) composition.
  • RGEN RNA-guided endonuclease
  • ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequencies obtained with individual nucleases are widely different from each other. Furthermore, some ZFNs and TALENs fail to show any genome editing activities. DNA methylation may limit the binding of these engineered nucleases to target sites. In addition, it is technically challenging and time-consuming to make customized nucleases.
  • the present inventors have developed a new RNA-guided endonuclease composition based on Cas protein to overcome the disadvantages of ZFNs and TALENs.
  • an endonuclease activity of Cas proteins has been known. However, it has not been known whether the endonuclease activity of Cas protein would function in an eukaryotic cell because of the complexity of the eukaryotic genome. Further, until now, a composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA specific for the target DNA to cleave a target DNA in eukaryotic cells or organisms has not been developed.
  • the present RGEN composition based on Cas protein can be more readily customized because only the synthetic guide RNA component is replaced to make a new genome-editing nuclease. No sub-cloning steps are involved to make customized RNA guided endonucleases.
  • the relatively small size of the Cas gene for example, 4.2 kbp for Cas9 as compared to a pair of TALEN genes ( ⁇ 6 kbp) provides an advantage for this RNA-guided endonuclease composition in some applications such as virus-mediated gene delivery. Further, this RNA-guided endonuclease does not have off-target effects and thus does not induce unwanted mutations, deletion, inversions, and duplications.
  • RNA-guided endonuclease composition a scalable, versatile, and convenient tools for genome engineering in eukaryotic cells and organisms.
  • RGEN can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP.
  • the specificity of RGENs is determined by the RNA component that hybridizes with a target DNA sequence of up to 20 base pairs (bp) in length and by the Cas9 protein that recognize the protospacer-adjacent motif (PAM).
  • PAM protospacer-adjacent motif
  • RGENs are readily reprogrammed by replacing the RNA component. Therefore, RGENs provide a platform to use simple and robust RFLP analysis for various sequence variations.
  • the target DNA may be an endogenous DNA, or artificial DNA, preferably, endogenous DNA.
  • Cas protein refers to an essential protein component in the CRISPR/Cas system, forms an active endonuclease or nickase when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • the information on the gene and protein of Cas are available from GenBank of National Center for Biotechnology Information (NCBI), without limitation.
  • CRISPR-associated (cas) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. There are three types of CRISPR-Cas system. Among them, Type II CRISPR/Cas system involving Cas9 protein and crRNA and tracrRNA is representative and is well known. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes ( E. coli , Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube).
  • the Cas protein may be linked to a protein transduction domain.
  • the protein transduction domain may be poly-arginine or a TAT protein derived from HIV, but it is not limited thereto.
  • the present composition may comprise Cas component in the form of a protein or in the form of a nucleic acid encoding Cas protein.
  • Cas protein may be any Cas protein provided that it has an endonuclease or nickase activity when complexed with a guide RNA.
  • Cas protein is Cas9 protein or variants thereof.
  • the variant of the Cas9 protein may be a mutant form of Cas9 in which the cataytic asapartate residue is changed to any other amino acid.
  • the other amino acid may be an alanine, but it is not limited thereto.
  • Cas protein may be the one isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogens or a recombinant protein, but it is not limited thereto.
  • the Cas protein derived from Streptococcus pyogens may recognizes NGG trinucleotide.
  • the Cas protein may comprise an amino acid sequence of SEQ ID NO: 109, but it is not limited thereto.
  • recombinant when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • a recombinant Cas protein may be generated by reconstituting Cas protein-encoding sequence using the human codon table.
  • Cas protein-encoding nucleic acid may be a form of vector, such as plasmid comprising Cas-encoding sequence under a promoter such as CMV or CAG.
  • Cas protein is Cas9
  • Cas9 encoding sequence may be derived from Streptococcus sp., and preferably derived from Streptococcus pyogenes .
  • Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID. NO: 1.
  • Cas9 encoding nucleic acid may comprise the nucleotide sequence having homology of at least 50% to the sequence of SEQ ID NO: 1, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% to the SEQ ID NO:1, but it is not limited thereto.
  • Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NOs. 108, 110, 106, or 107.
  • guide RNA refers to a RNA which is specific for the target DNA and can form a complex with Cas protein and bring Cas protein to the target DNA.
  • the guide RNA may consist of two RNA, i.e., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) or be a single-chain RNA (sgRNA) produced by fusion of an essential portion of crRNA and tracrRNA.
  • crRNA CRISPR RNA
  • tracrRNA transactivating crRNA
  • sgRNA single-chain RNA
  • the guide RNA may be a dualRNA comprising a crRNA and a tracrRNA.
  • the guide RNA comprises the essential portion of crRNA and tracrRNA and a portion complementary to a target, any guide RNA may be used in the present invention.
  • the crRNA may hybridize with a target DNA.
  • the RGEN may consist of Cas protein, and dualRNA (invariable tracrRNA and target-specific crRNA), or Cas protein and sgRNA (fusion of an essential portion of invariable tracrRNA and target-specific crRNA), and may be readily reprogrammed by replacing crRNA.
  • the guide RNA further comprises one or more additional nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
  • the guide RNA further comprises 2-additional guanine nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
  • the guide RNA may be transferred into a cell or an organism in the form of RNA or DNA that encodes the guide RNA.
  • the guide RNA may be in the form of an isolated RNA, RNA incorporated into a viral vector, or is encoded in a vector.
  • the vector may be a viral vector, plasmid vector, or agrobacterium vector, but it is not limited thereto.
  • a DNA that encodes the guide RNA may be a vector comprising a sequence coding for the guide RNA.
  • the guide RNA may be transferred into a cell or organism by transfecting the cell or organism with the isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
  • the guide RNA may be transferred into a cell or organism using virus-mediated gene delivery.
  • the guide RNA When the guide RNA is transfected in the form of an isolated RNA into a cell or organism, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art.
  • the guide RNA is preferably transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
  • isolated RNA may be interchangeable to “naked RNA”. This is cost- and time-saving because it does not require a step of cloning.
  • the use of plasmid DNA or virus-mediated gene delivery for transfection of the guide RNA is not excluded.
  • the present RGEN composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA can specifically cleave a target DNA due to a specificity of the guide RNA for a target and an endonuclease or nickase activity of Cas protein.
  • cleavage refers to the breakage of the covalent backbone of a nucleotide molecule.
  • a guide RNA may be prepared to be specific for any target which is to be cleaved. Therefore, the present RGEN composition can cleave any target DNA by manipulating or genotyping the target-specific portion of the guide RNA.
  • the guide RNA and the Cas protein may function as a pair.
  • the term “paired Cas nickase” may refer to the guide RNA and the Cas protein functioning as a pair.
  • the pair comprises two guide RNAs.
  • the guide RNA and Cas protein may function as a pair, and induce two nicks on different DNA strand.
  • the two nicks may be separated by at least 100 bps, but are not limited thereto.
  • paired Cas nickase allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells.
  • paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations.
  • paired nickases did not promote unwanted translocations associated with off-target DNA cleavages.
  • paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
  • the composition may be used in the genotyping of a genome in the eukaryotic cells or organisms in vitro.
  • the guide RNA may comprise the nucleotide sequence of Seq ID. No. 1, wherein the portion of nucleotide position 3 ⁇ 22 is a target-specific portion and thus, the sequence of this portion may be changed depending on a target.
  • a eukaryotic cell or organism may be yeast, fungus, protozoa, plant, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation.
  • Cas9 protein/single-chain guide RNA could generate site-specific DNA double-strand breaks in vitro and in mammalian cells, whose spontaneous repair induced targeted genome mutations at high frequencies.
  • composition comprising Cas protein and a guide RNA may be used to develop therapeutics or value-added crops, livestock, poultry, fish, pets, etc.
  • the present invention provides a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the present invention provides a use of the composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • the present invention provides a kit for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • the kit may comprise a guide RNA and Cas protein-encoding nucleic acid or Cas protein as separate components or as one composition.
  • the present kit may comprise some additional components necessary for transferring the guide RNA and Cas component to a cell or an organism.
  • the kit may comprise an injection buffer such as DEPC-treated injection buffer, and materials necessary for analysis of mutation of a target DNA, but are not limited thereto.
  • the present invention provides a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • a Cas protein-encoding nucleic acid or Cas protein and a guide RNA or DNA that encodes the guide RNA may be transferred into a cell by various methods known in the art, such as microinjection, electroporation, DEAEdextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection in protoplast, and so on, but are not limited thereto.
  • a Cas protein encoding nucleic acid or Cas protein and a guide RNA may be transferred into an organism by various method known in the art to administer a gene or a protein such as injection.
  • a Cas protein-encoding nucleic acid or Cas protein may be transferred into a cell in the form of complex with a guide RNA, or separately. Cas protein fused to a protein transduction domain such as Tat can also be delivered efficiently into cells.
  • the eukarotic cell or organisms is co-transfected or serial-transfected with a Cas9 protein and a guide RNA.
  • serial-transfection may be performed by transfection with Cas protein-encoding nucleic acid first, followed by second transfection with naked guide RNA.
  • the second transfection is after 3, 6, 12, 18, 24 hours, but it is not limited thereto.
  • the present invention provides a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the eukaryotic cells or organisms may be prepared by transferring the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
  • the eukaryotic cell may be yeast, fungus, protozoa, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation.
  • the organism may be yeast, fungus, protozoa, plant, higher plant, insect, amphibian, or mammal.
  • the present invention provides a method for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a step of treating a cell or organism comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the step of treating a cell or organism with the composition may be performed by transferring the present composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
  • such transfer may be performed by microinjection, transfection, electroporation, and so on.
  • the present invention provides an embryo comprising a genome edited by the present RGEN composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • the embryo may be an embryo of a mouse.
  • the embryo may be produced by injecting PMSG (Pregnant Mare Serum Gonadotropin) and hCG (human Coldinic Gonadotropin) into a female mouse of 4 to 7 weeks and the super-ovulated female mouse may be mated to males, and the fertilized embryos may be collected from oviduts.
  • PMSG Pregnant Mare Serum Gonadotropin
  • hCG human Cold-ovulated female mouse may be mated to males, and the fertilized embryos may be collected from oviduts.
  • the present RGEN composition introduced into an embryo can cleave a target DNA complementary to the guide RNA by the action of Cas protein and cause a mutation in the target DNA.
  • the embryo into which the present RGEN composition has been introduced has an edited genome.
  • the present RGEN composition could cause a mutation in a mouse embryo and the mutation could be transmitted to offsprings.
  • a method for introducing the RGEN composition into the embryo may be any method known in the art, such as microinjection, stem cell insertion, retrovirus insertion, and so on.
  • a microinjection technique can be used.
  • the present invention provides a genome-modified animal obtained by transferring the embryo comprising a genome edited by the present RGEN composition into the oviducts of an animal.
  • the term “genome-modified animal” refers to an animal of which genome has been modified in the stage of embryo by the present RGEN composition and the type of the animal is not limited.
  • the genome-modified animal has mutations caused by a targeted mutagenesis based on the present RGEN composition.
  • the mutations may be any one of deletion, insertion, translocation, inversion.
  • the site of mutation depends on the sequence of guide RNA of the RGEN composition.
  • the genome-modified animal having a mutation of a gene may be used to determine the function of the gene.
  • the present invention provides a method of preparing a genome-modified animal comprising a step of introducing the present RGEN composition comprising a guide RNA specific for the target DNA or DNA that encodes the guide RNA and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • the step of introducing the present RGEN composition may be accomplished by any method known in the art such as microinjection, stem cell insertion, retroviral insertion, and so on.
  • the present invention provides a plant regenerated form the genome-modified protoplasts prepared by the method for eukaryotic cells comprising the RGEN composition.
  • the present invention provides a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence Cas protein.
  • the present invention provides a composition for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • genotyping refers to the “Restriction fragment length polymorphism (RFLP) assay”.
  • RFLP may be used in 1) the detection of indel in cells or organisms induced by the engineered nucleases, 2) the genotyping naturally-occurring mutations or variations in cells or organisms, or 3) the genotyping the DNA of infected pathogenic microorganisms including virus or bacteria, etc.
  • the mutations or variation may be induced by engineered nucleases in cells.
  • the engineered nuclease may be a Zinc Finger Nuclease (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), or RGENs, but it is not limited thereto.
  • ZFNs Zinc Finger Nuclease
  • TALENs Transcription Activator-Like Effector Nucleases
  • RGENs RGENs
  • biological sample includes samples for analysis, such as tissues, cells, whole blood, semm, plasma, saliva, sputum, cerbrospinal fluid or urine, but is not limited thereto
  • the mutations or variation may be a naturally-occurring mutations or variations.
  • the mutations or variations are induced by the pathogenic microorganisms. Namely, the mutations or variation occur due to the infection of pathogenic microorganisms, when the pathogenic microorganisms are detected, the biological sample is identified as infected.
  • the pathogenic microorganisms may be virus or bacteria, but are not limited thereto.
  • Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescent PCR, DNA melting analysis, and Sanger and deep sequencing.
  • T7E1 and Surveyor assays are widely used but often underestimate mutation frequencies because the assays detect heteroduplexes (formed by the hybridization of mutant and wild-type sequences or two different mutant sequences); they fail to detect homoduplexes formed by the hybridization of two identical mutant sequences. Thus, these assays cannot distinguish homozygous bialleic mutant clones from wild-type cells nor heterozygous biallelic mutants from heterozygous monoalleic mutants ( FIG. 22 ).
  • sequence polymorphisms near the nuclease target site can produce confounding results because the enzymes can cleave heteroduplexes formed by hybridization of these different wild-type alleles.
  • RFLP analysis is free of these limitations and therefore is a method of choice. Indeed, RFLP analysis was one of the first methods used to detect engineered nuclease-mediated mutations. Unfortunately, however, it is limited by the availability of appropriate restriction sites.
  • the present invention provides a kit for genotyping mutations or variations in an isolated biological sample, comprising the composition for genotyping mutations or variations in an isolated biological sample.
  • the present invention provides a kit for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • the present invention provides a method of genotyping mutations or variations in an isolated biological sample, using the composition for genotyping mutations or variations in an isolated biological sample.
  • the present invention provides a method of genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • a Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or a chimeric guide RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas9 itself ( FIG. 1A ).
  • the Cas9-coding sequence (4,104 bp), derived from Streptococcus pyogenes strain M1 GAS (NC — 002737.1), was reconstituted using the human codon usage table and synthesized using oligonucleotides.
  • 1-kb DNA segments were assembled using overlapping ⁇ 35-mer oligonucleotides and Phusion polymerase (New England Biolabs) and cloned into T-vector (SolGent).
  • SolGent SolGent
  • a full-length Cas9 sequence was assembled using four 1-kbp DNA segments by overlap PCR.
  • the Cas9-encoding DNA segment was subcloned into p3s, which was derived from pcDNA3.1 (Invitrogen).
  • a peptide tag (NH2-GGSGPPKKKRKVYPYDVPDYA-COOH, SEQ ID NO: 2) containing the HA epitope and a nuclear localization signal (NLS) was added to the C-terminus of Cas9. Expression and nuclear localization of the Cas9 protein in HEK 293T cells were confirmed by western blotting using anti-HA antibody (Santa Cruz).
  • the Cas9 cassette was subcloned into pET28-b(+) and transformed into BL21(DE3).
  • the expression of Cas9 was induced using 0.5 mM IPTG for 4 h at 25° C.
  • the Cas9 protein containing the His6-tag at the C terminus was purified using Ni-NTA agarose resin (Qiagen) and dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol (1).
  • Cas9 cleaved the plasmid DNA efficiently at the expected position only in the presence of the synthetic RNA and did not cleave a control plasmid that lacked the target sequence ( FIG. 1B ).
  • a RFP-GFP reporter was used to investigate whether the Cas9/guide RNA complex can cleave the target sequence incorporated between the RFP and GFP sequences in mammalian cells.
  • the GFP sequence is fused to the RFP sequence out-of-frame (2).
  • the active GFP is expressed only when the target sequence is cleaved by site-specific nucleases, which causes frameshifting small insertions or deletions (indels) around the target sequence via error-prone non-homologous end-joining (NHEJ) repair of the double-strand break (DSB) ( FIG. 2 ).
  • the RFP-GFP reporter plasmids used in this study were constructed as described previously (2). Oligonucleotides corresponding to target sites (Table 1) were synthesized (Macrogen) and annealed. The annealed oligonucleotides were ligated into a reporter vector digested with EcoRI and BamHI.
  • HEK 293T cells were co-transfected with Cas9-encoding plasmid (0.8 ⁇ g) and the RFP-GFP reporter plasmid (0.2 ⁇ g) in a 24-well plate using Lipofectamine 2000 (Invitrogen).
  • chimeric RNA (1 ⁇ g) prepared by in vitro transcription was transfected using Lipofectamine 2000.
  • transfected cells were subjected to flow cytometry and cells expressing both RFP and GFP were counted.
  • GFP-expressing cells were obtained only when the cells were transfected first with the Cas9 plasmid and then with the guide RNA 12 h later ( FIG. 2 ), demonstrating that RGENs could recognize and cleave the target DNA sequence in cultured human cells.
  • GFP-expressing cells were obtained by serial-transfection of the Cas9 plasmid and the guide RNA rather than co-transfection.
  • T7E1 T7 endonuclease I
  • K562 cells were transfected with 20 ⁇ g of Cas9-encoding plasmid using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol.
  • K562 ATCC, CCL-243 cells were grown in RPMI1640 with 10% FBS and the penicillin/streptomycin mix (100 U/ml and 100 ⁇ g/ml, respectively).
  • in vitro transcribed chimeric RNA was nucleofected into 1 ⁇ 10 6 K562 cells.
  • the in vitro transcribed chimeric RNA had been prepared as described in the Example 1-2.
  • Mutation frequencies (Indels (%) in FIG. 3A ) estimated from the relative DNA band intensities were RNA-dosage dependent, ranging from 1.3% to 5.1%.
  • DNA sequencing analysis of the PCR amplicons corroborated the induction of RGEN-mediated mutations at the endogenous sites. Indels and microhomologies, characteristic of error-prone NHEJ, were observed at the target site.
  • the most striking off-target sites associated with these CCR5-specific engineered nucleases reside in the CCR2 locus, a close homolog of CCR5, located 15-kbp upstream of CCR5.
  • the present inventors intentionally chose the target site of our CCR5-specific RGEN to recognize a region within the CCR5 sequence that has no apparent homology with the CCR2 sequence.
  • the present inventors investigated whether the CCR5-specific RGEN had off-target effects. To this end, we searched for potential off-target sites in the human genome by identifying sites that are most homologous to the intended 23-bp target sequence. As expected, no such sites were found in the CCR2 gene. Instead, four sites, each of which carries 3-base mismatches with the on-target site, were found ( FIG. 4A ). The T7E1 assays showed that mutations were not detected at these sites (assay sensitivity, ⁇ 0.5%), demonstrating extraordinar specificities of RGENs ( FIG. 4B ).
  • PCR was used to detect the induction of chromosomal deletions in cells separately transfected with plasmids encoding the ZFN and RGEN specific to CCR5. Whereas the ZFN induced deletions, the RGEN did not ( FIG. 4C ).
  • RGENs was reprogrammed by replacing the CCR5-specific guide RNA with a newly-synthesized RNA designed to target the human C4BPB gene, which encodes the beta chain of C4b-binding protein, a transcription factor.
  • RGENs can be delivered into cells in many different forms.
  • RGENs consist of Cas9 protein, crRNA, and tracrRNA.
  • the two RNAs can be fused to form a single-chain guide RNA (sgRNA).
  • sgRNA single-chain guide RNA
  • a plasmid that encodes Cas9 under a promoter such as CMV or CAG can be transfected into cells.
  • crRNA, tracrRNA, or sgRNA can also be expressed in cells using plasmids that encode these RNAs.
  • Use of plasmids however, often results in integration of the whole or part of the plasmids in the host genome.
  • the bacterial sequences incorporated in plasmid DNA can cause unwanted immune response in vivo.
  • Plasmid DNA can persist in cells for several days post-transfection, aggravating off-target effects of RGENs.
  • Recombinant Cas9 protein complexed with in vitro transcribed guide RNA to induce targeted disruption of endogenous genes in human cells.
  • Recombinant Cas9 protein fused with the hexa-histidine tag was expressed in and purified from E. coli using standard Ni ion affinity chromatography and gel filtration. Purifed recombinant Cas9 protein was concentrated in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol).
  • Cas9 protein/sgRNA complex was introduced directly into K562 cells by nucleofection: 1 ⁇ 10 6 K562 cells were transfected with 22.5-225 (1.4-14 ⁇ M) of Cas9 protein mixed with 100 ug (29 ⁇ M) of in vitro transcribed sgRNA (or crRNA 40 ug and tracrRNA 80 ug) in 1000 solution using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. After nucleofection, cells were placed in growth media in 6-well plates and incubated for 48 hr.
  • Cas9 protein/sgRNA complex induced targeted mutation at the CCR5 locus at frequencies that ranged from 4.8 to 38% in a sgRNA or Cas9 protein dose-dependent manner, on par with the frequency obtained with Cas9 plasmid transfection (45%).
  • Cas9 protein/crRNA/tracrRNA complex was able to induce mutations at a frequency of 9.4%.
  • Cas9 protein alone failed to induce mutations.
  • the forkhead box N1 (Foxn1) gene which is important for thymus development and keratinocyte differentiation (Nehls et al., 1996), and the protein kinase, DNA activated, catalytic polypeptide (Prkdc) gene, which encodes an enzyme critical for DNA DSB repair and recombination (Taccioli et al., 1998) were used.
  • Cas9 mRNA and sgRNAs were synthesized in vitro from linear DNA templates using the mMESSAGE mMACHINE T7 Ultra kit (Ambion) and MEGAshortscript T7 kit (Ambion), respectively, according to the manufacturers' instructions, and were diluted with appropriate amounts of diethyl pyrocarbonate (DEPC, Sigma)-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4). Templates for sgRNA synthesis were generated using oligonucleotides listed in Table 3. Recombinant Cas9 protein was obtained from ToolGen, Inc.
  • Cas9 mRNA and sgRNAs in M2 medium were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei using a Piezo-driven micromanipulator (Prime Tech).
  • the recombinant Cas9 protein: Foxn1-sgRNA complex was diluted with DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4) and injected into male pronuclei using a TransferMan NK2 micromanipulator and a FemtoJet microinjector (Eppendorf).
  • the manipulated embryos were transferred into the oviducts of pseudo-pregnant foster mothers to produce live animals, or were cultivated in vitro for further analyses.
  • T7E1 assays were performed as previously described using genomic DNA samples from tail biopsies and lysates of whole embryos (Cho et al., 2013).
  • the genomic region encompassing the RGEN target site was PCR-amplified, melted, and re-annealed to form heteroduplex DNA, which was treated with T7 endonuclease 1 (New England Biolabs), and then analyzed by agarose gel electrophoresis. Potential off-target sites were identified by searching with bowtie 0.12.9 and were also similarly monitored by T7E1 assays.
  • the primer pairs used in these assays were listed in Tables 4 and 5.
  • mutant fractions (the number of mutant embryos/the number of total embryos) were dose-dependent, ranging from 33% (1 ng/ ⁇ l sgRNA) to 91% (100 ng/ ⁇ l) ( FIG. 6 b ).
  • Sequence analysis confirmed mutations in the Foxn1 gene; most mutations were small deletions ( FIG. 6 c ), reminiscent of those induced by ZFNs and TALENs (Kim et al., 2013).
  • mutant fractions were proportional to the doses of Foxn1-sgRNA, and reached up to 93% (100 ng/ ⁇ l Foxn1-sgRNA) (Tables 6 and 7, FIG. 5 b ).
  • Prkdc-targeted mice To generate Prkdc-targeted mice, we applied a 5-fold higher concentration of Cas9 mRNA (50 ng/ ⁇ l) with increasing doses of Prkdc-sgRNA (50, 100, and 250 ng/ ⁇ l). Again, the birth rates were very high, ranging from 51% to 60%, enough to produce a sufficient number of newborns for the analysis (Table 6). The mutant fraction was 57% (21 mutant founders among 37 newborns) at the maximum dose of Prkdc-sgRNA. These birth rates obtained with RGENs were approximately 2- to 10-fold higher than those with TALENs reported in our previous study (Sung et al., 2013). These results demonstrate that RGENs are potent gene-targeting reagents with minimal toxicity.
  • Genotyping Summary Detected alleles 58* 1 not determined ⁇ 11 19 100 bi-allelic ⁇ 60/+1 20 100 bi-allelic ⁇ 67/ ⁇ 19 13 100 bi-allelic ⁇ 18/+455 32 10 bi-allelic (heterozygote) ⁇ 13/ ⁇ 15 + 1 115 10 bi-allelic (heterozygote) ⁇ 18/ ⁇ 5 111 10 bi-allelic (heterozygote) ⁇ 11/+1 110 10 bi-allelic (homozygote) ⁇ 8/ ⁇ 8 120 10 bi-allelic (homozygote) +2/+2 81 100 heterozygote +1/WT 69 100 homozygote ⁇ 11/ ⁇ 11 55 1 mosaic ⁇ 18/ ⁇ 1/ +1/+3 56 1 mosaic ⁇ 127/ ⁇ 41 / ⁇ 2/ +1 127 1 mosaic ⁇ 18 / +1/WT 53 1 mosaic ⁇ 11
  • the Cas9 coding sequence (4104 bps), derived from Streptococcus pyogenes strain M1 GAS (NC — 002737.1), was cloned to pET28-b(+) plasmid.
  • a nuclear targeting sequence (NLS) was included at the protein N terminus to ensure the localization of the protein to the nucleus.
  • pET28-b(+) plasmid containing Cas9 ORF was transformed into BL21(DE3).
  • Cas9 was then induced using 0.2 mM IPTG for 16 hrs at 18° C. and purified using Ni-NTA agarose beads (Qiagen) following the manufacturer's instructions. Purified Cas9 protein was concentrated using Ultracel—100K (Millipore).
  • the genomic sequence of the Arabidopsis gene encoding the BRI1 was screened for the presence of a NGG motif, the so called protospacer adjacent motif (PAM), in an exon which is required for Cas9 targeting
  • PAM protospacer adjacent motif
  • sgRNAs were produced in vitor using template DNA. Each template DNA was generated by extension with two partially overlapped oligonucleotides (Macrogen, Table X1) and Phusion polymerase (Thermo Scientific) using the following conditions—98° C. 30 sec ⁇ 98° C. 10 sec, 54° C. 20 sec, 72° C. 2 min ⁇ 20, 72° C. 5 min.
  • Oligonucleotides for the production of the template DNA for in vitro transcription SEQ Oligonuc- ID leotides Sequence (5′-3′) NO BRI1 target 1 GAAATTAATACGACTCACTATAGGTTTGAA 73 (Forward) AGATGGAAGCGCGGGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCG BRI1 target 2 GAAATTAATACGACTCACTATAGGTGAAAC 74 (Forward) TAAACTGGTCCACAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCG Universal AAAAAAGCACCGACTCGGTGCCACTTTTTC 75 (Reverse) AAGTTGATAACGGACTAGCCTTATTTTAAC TTGC
  • the extended DNA was purified and used as a template for the in vitro production of the guide RNA's using the MEGAshortscript T7 kit (Life Technologies). Guide RNA were then purified by Phenol/Chloroform extraction and ethanol precipitation.
  • 10 ul of purified Cas9 protein (12 ⁇ g/ ⁇ l) and 4 ul each of two sgRNAs (11 ⁇ g/ ⁇ l) were mixed in 20 ⁇ l NEB3 buffer (New England Biolabs) and incubated for 10 min at 37° C.
  • the leaves of 4-week-old Arabidopsis seedlings grown aseptically in petri dishes were digested in enzyme solution (1% cellulose R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salt) for 8 ⁇ 16 hrs at 25° C. with 40 rpm shaking in the dark. Enzyme/protoplast solutions were filtered and centrifuged at 100 ⁇ g for 3 ⁇ 5 min. Protoplasts were re-suspended in CPW solution after counting cells under the microscope ( ⁇ 100) using a hemacytometer.
  • enzyme solution 1% cellulose R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salt
  • protoplasts were re-suspended at 1 ⁇ 10 6 /ml in MMG solution (4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2).
  • MMG solution 4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2.
  • Cas9/sgRNA complex 200 ⁇ L (200,000 protoplasts) of the protoplast suspension were gently mixed with 3.3 or 10 uL of Cas9/sgRNA complex [Cas9 protein (6 ⁇ g/ ⁇ L) and two sgRNAs (2.2 ⁇ g/ ⁇ L each)] and 200 ul of 40% polyethylene glycol transfection buffer (40% PEG4000, 200 mM mannitol and 100 mM CaCl2) in 2 ml tubes.
  • Cas9 with a cysteine at the C-terminal was prepared by PCR amplification using the previously described Cas9 plasmid ⁇ Cho, 2013 #166 ⁇ as the template and cloned into pET28-(a) vector (Novagen, Merk Millipore, Germany) containing His-tag at the N-terminus.
  • 293T Human embryonic kidney cell line
  • HeLa human ovarian cancer cell line
  • DMEM Gibco-BRL Rockville
  • E. coli BL21 cells were transformed with thepET28-(a) vector encoding Cas9 and plated onto Luria-Bertani (LB) agar medium containing 50 ⁇ g/mL kanamycin (Amresco, Solon, Ohio). Next day, a single colony was picked and cultured in LB broth containing 50 ⁇ g/mL kanamycin at 37° C. overnight. Following day, this starter culture at 0.1 OD600 was inoculated into Luria broth containing 50 ⁇ g/mL kanamycin and incubated for 2 hrs at 37° C. until OD600 reached to 0.6-0.8. To induce Cas9 protein expression, the cells were cultured at 30° C. overnight after addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) (Promega, Madison, Wis.) to the final concentration of 0.5 mM.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the cells were collected by centrifugation at 4000 rpm for 15-20 mins, resuspendedin a lysis buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, 1 ⁇ protease inhibitor cocktail, 1 mg/ml lysozyme), and lysed by sonication (40% duty, 10 sec pulse, 30 sec rest, for 10 mins on ice).
  • the soluble fraction was separated as the supernatant after centrifugation at 15,000 rpm for 20 mins at 4° C.
  • Cas9 protein was purified at 4° C. using a column containing Ni-NTA agarose resin (QIAGEN) and AKTA prime instrument (AKTA prime, GE Healthcare, UK).
  • soluble protein fractions were loaded onto Ni-NTA agarose resin column (GE Healthcare, UK) at the flow rate of 1 mL/min.
  • the column was washed with a washing buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, lx protease inhibitor cocktail) and the bound protein was eluted at the flow rate of 0.5 ml/min with an elution buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 250 mM imidazole, 1 ⁇ protease inhibitor cocktail).
  • the pooled eluted fraction was concentrated and dialyzed against storage buffer (50 mM Tris-HCl, pH8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% Glycerol). Protein concentration was quantitated by Bradford assay (Biorad, Hercules, Calif.) and purity was analyzed by SDS-PAGE using bovine serum albumin as the control.
  • sgRNA (1 ⁇ g) was gently added to various amounts of C9R4LC peptide (ranging from 1 to 40 weight ratio) in 100 ⁇ l of DPBS (pH 7.4). This mixture was incubated at room temperature for 30 mins and diluted to 10 folds using RNAse-free deionized water. The hydrodynamic diameter and z-potential of the formed nanoparticles were measured using dynamic light scattering (Zetasizer-nano analyzer ZS; Malvern instruments, Worcestershire, UK).
  • Cas9-9R4L and sgRNA-C9R4LC were treated to the cells as follows: 1 ⁇ g of sgRNA and 15 ⁇ g of C9R4LC peptide were added to 250 mL of OPTIMEM medium and incubated at room temperature for 30 mins. At 24 hrs after seeding, cells were washed with OPTIMEM medium and treated with sgRNA-C9R4LC complex for 4 hrs at 37° C. Cells were washed again with OPTIMEM medium and treated with Cas9-9R4L for 2 hrs at 37° C. After treatment, culture media was replaced with serum-containing complete medium and incubated at 37° C. for 24 hrs before the next treatment. Same procedure was followed for multiple treatments of Cas9 and sgRNA for three consecutive days.
  • Cas9-9R4L and sgRNA-9R4L can Edit Endogenous Genes in Cultured Mammalian Cells without the Use of Additional Delivery Tools
  • our guide RNA had two additional guanine nucleotides at the 5′ end, which are required for efficient transcription by T7 polymerase in vitro. No such additional nucleotides were included in the sgRNA used by others.
  • the RNA sequence of our guide RNA can be shown as 5′-GGX 20 , whereas 5′-GX 19 , in which X 20 or GX 19 corresponds to the 20-bp target sequence, represents the sequence used by others.
  • the first guanine nucleotide is required for transcription by RNA polymerase in cells. To test whether off-target RGEN effects can be attributed to these differences, we chose four RGENs that induced off-target mutations in human cells at high frequencies (13).
  • SSBs single-strand breaks
  • HDR homology-directed repair
  • nickase-induced targeted mutagenesis via HDR is much less efficient than is nuclease-induced mutagenesis.
  • paired Cas9 nickases would produce composite DSBs, which trigger DNA repair via NHEJ or HDR, leading to efficient mutagenesis ( FIG. 16A ).
  • paired nickases would double the specificity of Cas9-based genome editing.
  • Cas9 nucleases and nickases designed to target sites in the AAVS1 locus ( FIG. 16B ) in vitro via fluorescent capillary electrophoresis.
  • Cas9 nickases composed of guide RNA and a mutant form of Cas9 in which a catalytic aspartate residue is changed to an alanine (D10A Cas9) cleaved only one strand, producing site-specific nicks (FIG. 16 C,D).
  • some nickases AS1, AS2, AS3, and S6 in FIG.
  • the Cas9 nuclease complexed with the S2 sgRNA was equally efficient at this site and the on-target site.
  • D10A Cas9 complexed with the S2 and AS2 sgRNAs discriminated this site from the on-target site by a factor of 270 fold.
  • This paired nickase also discriminated the AS2 off-target sites (Off-1 and Off-9 in FIG. 17B ) from the on-target site by factors of 160 fold and 990 fold, respectively.
  • FIG. 21A We then investigated whether Cas9 nucleases and nickases can induce unwanted chromosomal translocations that result from NHEJ repair of on-target and off-target DNA cleavages.
  • FIG. 21B We were able to detect translocations induced by Cas9 nucleases using PCR (FIG. 21 B,C). No such PCR products were amplified using genomic DNA isolated from cells transfected with the plasmids encoding the AS2+S3 Cas9 nickase pair. This result is in line with the fact that both AS2 and S3 nickases, unlike their corresponding nucleases, did not produce indels at off-target sites ( FIG. 17B ).
  • paired Cas9 nickases allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells.
  • paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations.
  • paired nickases did not promote unwanted translocations associated with off-target DNA cleavages.
  • paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
  • paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
  • One caveat to this approach is that two highly active sgRNAs are needed to make an efficient nickase pair, limiting targetable sites.
  • RGENs can be used in Restriction fragment length polymorphism (RFLP) analysis, replacing conventional restriction enzymes.
  • Engineered nucleases including RGENs induce indels at target sites, when the DSBs caused by the nucleases are repaired by the error-prone non-homologous end-joining (NHEJ) system.
  • NHEJ error-prone non-homologous end-joining
  • crRNA and tracrRNA were prepared by in vitro transcription using MEGAshortcript T7 kit (Ambion) according to the manufacturer's instruction. Transcribed RNAs were resolved on a 8% denaturing urea-PAGE gel. The gel slice containing RNA was cut out and transferred to elution buffer. RNA was recovered in nuclease-free water followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry.
  • Templates for crRNA were prepared by annealing an oligonucleotide whose sequence is shown as 5′-GAAATTAATACGACTCACTATAGGX 20 GTTTTAGAGCTATGCTGTTTTG-3′ (SEQ ID NO: 76), in which X 20 is the target sequence, and its complementary oligonucleotide.
  • the template for tracrRNA was synthesized by extension of forward and reverse oligonucleotides (5′-GAAATTAATACGACTCACTATAGGAACCATTCAAAACAGCATAGCAAG TTAAAATAAGGCTAGTCCG-3′ (SEQ ID NO: 77) and 5′-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAG CCTTATTTTAACTTGCTATG-3′(SEQ ID NO: 78)) using Phusion polymerase (New England Biolabs).
  • the Cas9 DNA construct used in our previous Example which encodes Cas9 fused to the His6-tag at the C terminus, was inserted in the pET-28a expression vector.
  • the recombinant Cas9 protein was expressed in E. coli strain BL21(DE3) cultured in LB medium at 25° C. for 4 hour after induction with 1 mM IPTG. Cells were harvested and resuspended in buffer containing 20 mM Tris PH 8.0, 500 mM NaCl, 5 mM immidazole, and 1 mM PMSF. Cells were frozen in liquid nitrogen, thawed at 4° C., and sonicated.
  • the Cas9 protein in the lysate was bound to Ni-NTA agarose resin (Qiagen), washed with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 20 mM immidazole, and eluted with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 250 mM immidazole.
  • Purified Cas9 protein was dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol and analyzed by SDS-PAGE.
  • the T7E1 assay was performed as following. In brief, PCR products amplified using genomic DNA were denatured at 95° C., reannealed at 16° C., and incubated with 5 units of T7 Endonuclease I (New England BioLabs) for 20 min at 37° C. The reaction products were resolved using 2 to 2.5% agarose gel electrophoresis.
  • PCR products (100-150 ng) were incubated for 60 min at 37° C. with optimized concentrations (Table 10) of Cas9 protein, tracrRNA, crRNA in 10 ⁇ l NEB buffer 3 (1 ⁇ ). After the cleavage reaction, RNase A (4 ⁇ g) was added, and the reaction mixture was incubated for 30 min at 37° C. to remove RNA. Reactions were stopped with 6 ⁇ stop solution buffer containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1-2.5% agarose gel electrophoresis and visualized with EtBr staining.
  • Restriction enzyme-treated linearized plasmid 100 ng was incubated for 60 min at 37° C. with Cas9 protein (0.1 ⁇ g), tracrRNA (60 ng), and crRNA (25 ng) in 10 ⁇ l NEB 3 buffer (1 ⁇ ). Reactions were stopped with 6 ⁇ stop solution containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1% agarose gel electrophoresis and visualized with EtBr staining.
  • New RGENs with desired DNA specificities can be readily created by replacing crRNA; no de novo purification of custom proteins is required once recombinant Cas9 protein is available.
  • Engineered nucleases, including RGENs induce small insertions or deletions (indels) at target sites when the DSBs caused by the nucleases are repaired by error-prone non-homologous end-joining (NHEJ).
  • NHEJ error-prone non-homologous end-joining
  • RGENs can differentially cleave plasmids that contain wild-type or modified C4BPB target sequences that harbor 1- to 3-base indels at the cleavage site. None of the six plasmids with these indels were cleaved by a C4BPB-specific RGEN5 composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein ( FIG. 23 ). In contrast, the plasmid with the intact target sequence was cleaved efficiently by this RGEN.
  • Target sequence of RGENs used in this study Gene Target sequence SEQ ID NO human AATGACCACTACATCCTCAA 104 C4BPB GGG mouse Pibf1 AGATGATGTCTCATCATCAG 105 AGG
  • C4BPB mutant clones used in this study have various mutations ranging from 94 bp deletion to 67 bp insertion ( FIG. 24A ). Importantly, all mutations occurred in mutant clones resulted in the loss of RGEN target site. Among 6 C4BPB clones analyzed, 4 clones have both wildtype and mutant alleles (+/ ⁇ ) and 2 clones have only mutant alleles ( ⁇ / ⁇ ).
  • PCR products spanning the RGEN target site amplified from wildtype K562 genomic DNA were digested completely by the RGEN composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein expressed in and purified from E. coli (FIG. 24 B/Lane 1).
  • RGEN target-specific crRNA
  • tracrRNA tracrRNA
  • Cas9 protein expressed in and purified from E. coli FIG. 24 B/Lane 1
  • PCR amplicons of +/ ⁇ clones that contained both wild-type and mutant alleles were partially digested, and those of ⁇ / ⁇ cloned that did not contain the wildtype allele were not digested at all, yielding no cleavage products corresponding to the wildtype sequence ( FIG. 24B ).
  • RGEN-RFLP analysis is a quantitative method. Genomic DNA samples isolated from the C4BPB null clone and the wild-type cells were mixed at various ratios and used for PCR amplifications. The PCR products were subjected to RGEN genotyping and the T7E1 assay in parallel ( FIG. 25 b ). As expected, DNA cleavage by the RGEN was proportional to the wild type to mutant ratio. In contrast, results of the T7E1 assay correlated poorly with mutation frequencies inferred from the ratios and were inaccurate, especially at high mutant %, a situation in which complementary mutant sequences can hybridize with each other to form homoduplexes.
  • RGEN genotyping in short
  • RGEN genotyping was applied to the analysis of mutant mouse founders that had been established by injection of TALENs into mouse one-cell embryos.
  • FIG. 26A We designed and used an RGEN that recognized the TALEN target site in the Pibf1 gene (Table 10).
  • Genomic DNA was isolated from a wildtype mouse and mutant mice and subjected to RGEN genotyping after PCR amplification.
  • RGEN genotyping successfully detected various mutations, which ranged from one to 27-bp deletions ( FIG. 26B ).
  • RGEN genotyping enabled differential detection of +/ ⁇ and ⁇ / ⁇ founder.
  • RGENs to detect mutations induced in human cells by a CCR5-specific ZFN, representing yet another class of engineered nucleases ( FIG. 27 ). These results show that RGENs can detect mutations induced by nucleases other than RGENs themselves. In fact, we expect that RGENs can be designed to detect mutations induced by most, if not all, engineered nucleases.
  • the only limitation in the design of an RGEN genotyping assay is the requirement for the GG or AG (CC or CT on the complementary strand) dinucleotide in the PAM sequence recognized by the Cas9 protein, which occurs once per 4 bp on average.
  • Indels induced anywhere within the seed region of several bases in crRNA and the PAM nucleotides are expected to disrupt RGEN-catalyzed DNA cleavage. Indeed, we identified at least one RGEN site in most (98%) of the ZFN and TALEN sites.
  • RGEN-RFLP analysis has applications beyond genotyping of engineered nuclease-induced mutations.
  • HCT116 human colorectal cancer cell line
  • PCR products amplified from HCT116 genomic DNA were cleaved partially by both wild-type-specific and mutant-specific RGENs, in line with the heterozygous genotype in HCT116 cells ( FIG. 29 a ).
  • PCR products amplified from DNA from HeLa cells harboring only wild-type alleles were digested completely by the wild-type-specific RGEN and were not cleaved at all by the mutation-specific RGEN.
  • HEK293 cells harbor the 32-bp deletion (del32) in the CCR5 gene, which encodes an essential co-receptor of HIV infection: Homozygous del32 CCR5 carriers are immune to HIV infection.
  • the wild-type-specific RGEN cleaved the PCR products obtained from K562, SKBR3, or HeLa cells (used as wild-type controls) completely but those from HEK293 cells partially ( FIG. 30 a ), confirming the presence of the uncleavable del32 allele in HEK293 cells.
  • the del32-specific RGEN cleaved the PCR products from wild-type cells as efficiently as those from HEK293 cells.
  • this RGEN had an off-target site with a single-base mismatch immediately downstream of the on-target site ( FIG. 30 ).
  • RGENs that contained the perfectly-matched guide RNA specific to the wild-type sequence or mutant sequence cleaved both sequences ( FIGS. 31 a and 32 a ).
  • RGENs that contained a single-base mismatched guide RNA distinguished the two sequences, enabling genotyping of three recurrent oncogenic point mutations in the KRAS, PIK3CA, and IDH1 genes in human cancer cell lines ( FIG. 29 b and FIGS. 33 a, b ).
  • RGENs as providing a platform to use simple and robust RFLP analysis for various sequence variations.
  • RGENs can be used to detect various genetic variations (single nucleotide variations, small insertion/deletions, structural variations) such as diseaserelated recurring mutations, genotypes related to drug-response by a patient and also mutations induced by engineered nucleases in cells.
  • RGEN genotyping to detect mutations induced by engineered nucleases in cells and animals. In principle, one could also use RGENs that will specifically detect and cleave naturally-occurring variations and mutations.

Abstract

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

Description

    TECHNICAL FIELD
  • The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
    • BACKGROUND ART
  • CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.
  • Cas9, an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells. crRNA is transcribed from the CRISPR element in the host genome, which was previously captured from such foreign invaders. Recently, Jinek et al. (1) demonstrated that a single-chain chimeric RNA produced by fusing an essential portion of crRNA and tracrRNA could replace the two RNAs in the Cas9/RNA complex to form a functional endonuclease.
  • CRISPR/Cas systems offer an advantage to zinc finger and transcription activator-like effector DNA-binding proteins, as the site specificity in nucleotide binding CRISPR-Cas proteins is governed by a RNA molecule instead of the DNA-binding protein, which can be more challenging to design and synthesize.
  • However, until now, a genome editing method using the RNA-guided endonuclease (RGEN) based on CRISPR/Cas system has not been developed.
  • Meanwhile, Restriction fragment length polymorphism (RFLP) is one of the oldest, most convenient, and least expensive methods of genotyping that is still used widely in molecular biology and genetics but is often limited by the lack of appropriate sites recognized by restriction endonucleases.
  • Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive T7 endonuclease I (T7E1) or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, Dideoxy sequencing, and deep sequencing. The T7E1 and Surveyor assays are widely used but are cumbersome. Furthermore, theses enzymes tend to underestimate mutation frequencies because mutant sequences can form homoduplexes with each other and cannot distinguish homozygous bi-allelic mutant clones from wildtype cells. RFLP is free of these limitations and therefore is a method of choice. Indeed, RFLP was one of the first methods to detect engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction sites. It is possible that no restriction sites are available at the target site of interest.
    • DISCLOSURE OF INVENTION Technical Problem
  • Until now, a genome editing and genotyping method using the RNA-guided endonuclease (RGEN) based on CRISPR/Cas system has not been developed.
  • Under these circumstances, the present inventors have made many efforts to develop a genome editing method based on CRISPR/Cas system and finally established a programmable RNA-guided endonuclease that cleave DNA in a targeted manner in eukaryotic cells and organisms.
  • In addition, the present inventors have made many efforts to develop a novel method of using RNA-guided endonucleases (RGENs) in RFLP analysis. They have used RGENs to genotype recurrent mutations found in cancer and those induced in cells and organisms by engineered nucleases including RGENs themselves, thereby completing the present invention.
    • Solution to Problem
  • It is an object of the present invention to provide a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is another object of the present invention to provide a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
  • It is still another object of the present invention to provide a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide an embryo, a genome-modified animal, or genome-modified plant comprising a genome edited by a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • It is still another object of the present invention to provide a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence Cas protein.
  • It is still another object of the present invention to provide a
  • method of using a RNA-guided endonuclease (RGEN) to genotype mutations induced by engineered nucleases in cells or naturally-occurring mutations or variations, wherein the RGEN comprises a guide RNA specific for target DNA and Cas protein.
  • It is still another object of the present invention to provide a kit for genotyping mutations induced by engineered nucleases in cells or naturally-occurring mutations or variations, comprising a RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for target DNA and Cas protein.
  • It is an object of the present invention to provide a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is another object of the present invention to provide a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
  • It is still another object of the present invention to provide a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide an embryo, a genome-modified animal, or genome-modified plant comprising a genome edited by a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • It is still another object of the present invention to provide a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence Cas protein.
  • It is still another object of the present invention to provide a composition for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • It is still another object of the present invention to provide a kit for genotyping mutations or variations in an isolated biological sample, comprising the compostion, specifically comprising a RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for target DNA and Cas protein.
  • It is still another object of the present invention to provide a method of genotyping mutations or variations in an isolated biological sample, using the composition, specifically comprising a RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for target DNA and Cas protein.
    • Advantageous Effects of Invention
  • The present composition for cleaving a target DNA or inducing a targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, the kit comprising the composition, and the method for inducing targeted mutagenesis provide a new convenient genome editing tools. In addition, because custom RGENs can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP, therefore, the composition and method of the present invention may be used in detection and cleaving naturally-occurring variations and mutations.
    • BRIEF DESCRIPTION OF DRAWINGS
  • FIG. 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (a) Schematic representation of target DNA and chimeric RNA sequences. Red triangles indicate cleavage sites. The PAM sequence recognized by Cas9 is shown in bold. The sequences in the guide RNA derived from crRNA and tracrRNA are shown in box and underlined, respectively. (b) In vitro cleavage of plasmid DNA by Cas9. An intact circular plasmid or ApaLI-digested plasmid was incubated with Cas9 and guide RNA.
  • FIG. 2 shows Cas9-induced mutagenesis at an episomal target site. (a) Schematic overview of cell-based assays using a RFP-GFP reporter. GFP is not expressed from this reporter because the GFP sequence is fused to the RFP sequence out-of-frame. The RFP-GFP fusion protein is expressed only when the target site between the two sequences is cleaved by a site-specific nuclease. (b) Flow cytometry of cells transfected with Cas9. The percentage of cells that express the RFP-GFP fusion protein is indicated.
  • FIG. 3 shows RGEN-driven mutations at endogenous chromosomal sites. (a) CCR5 locus. (b) C4BPB locus. (Top) The T7E1 assay was used to detect RGEN-driven mutations. Arrows indicate the expected position of DNA bands cleaved by T7E1. Mutation frequencies (Indels (%)) were calculated by measuring the band intensities. (Bottom) DNA sequences of the CCR5 and C4BPB wild-type (WT) and mutant clones. The region of the target sequence complementary to the guide RNA is shown in boc. The PAM sequence is shown in bold. Triangles indicate the cleavage site. Bases corresponding to microhomologies are underlined. The column on the right indicates the number of inserted or deleted bases.
  • FIG. 4 shows that RGEN-driven off-target mutations are undetectable. (a) On-target and potential off-target sequences. The human genome was searched in silico for potential off-target sites. Four sites were identified, each of which carries 3-base mismatches with the CCR5 on-target site. Mismatched bases are underlined. (b) The T7E1 assay was used to investigate whether these sites were mutated in cells transfected with the Cas9/RNA complex. No mutations were detected at these sites. N/A (not applicable), an intergenic site. (c) Cas9 did not induce off-target-associated chromosomal deletions. The CCR5-specific RGEN and ZFN were expressed in human cells. PCR was used to detect the induction of the 15-kb chromosomal deletions in these cells.
  • FIG. 5 shows RGEN-induced Foxn1 gene targeting in mice. (a) A schematic diagram depicting a sgRNA specific to exon 2 of the mouse Foxn1 gene. PAM in exon 2 is shown in red and the sequence in the sgRNA that is complementary to exon 2 is underlined. Triangles indicate cleavage sites. (b) Representative T7E1 assays demonstrating gene-targeting efficiencies of Cas9 mRNA plus Foxn1-specific sgRNA that were delivered via intra-cytoplasmic injection into one-cell stage mouse embryos. Numbers indicate independent founder mice generated from the highest dose. Arrows indicate bands cleaved by T7E1. (c) DNA sequences of mutant alleles observed in three Foxn1 mutant founders identified in b. The number of occurrences is shown in parentheses. (d) PCR genotyping of F1 progenies derived from crossing Foxn1 founder #108 and wild-type FVB/NTac. Note the segregation of the mutant alleles found in Foxn1 founder #108 in the progenies.
  • FIG. 6 shows Foxn1 gene targeting in mouse embryos by intra-cytoplasmic injection of Cas9 mRNA and Foxn1-sgRNA. (a) A representative result of a T7E1 assay monitoring the mutation rate after injecting the highest dose. Arrows indicate bands cleaved by T7E1. (b) A summary of T7E1 assay results. Mutant fractions among in vitro cultivated embryos obtained after intra-cytoplasmic injection of the indicated RGEN doses are indicated. (c) DNA sequences of Foxn1 mutant alleles identified from a subset of T7E1-positive mutant embryos. The target sequence of the wild-type allele is denoted in box.
  • FIG. 7 shows Foxn1 gene targeting in mouse embryos using the recombinant Cas9 protein: Foxn1-sgRNA complex. (a) and (b) are representative T7E1 assays results and their summaries. Embryos were cultivated in vitro after they underwent pronuclear (a) or intra-cytoplasmic injection (b). Numbers in red indicate T7E1-positive mutant founder mice. (c) DNA sequences of Foxn1 mutant alleles identified from the in vitro cultivated embryos that were obtained by the pronucleus injection of recombinant Cas9 protein: Foxn1-sgRNA complex at the highest dose. The target sequence of the wild-type allele is denoted in box.
  • FIG. 8 shows Germ-line transmission of the mutant alleles found in Foxn1 mutant founder #12. (a) fPCR analysis. (b) PCR genotyping of wild-type FVB/NTac, the founder mouse, and their F1 progenies.
  • FIG. 9 shows Genotypes of embryos generated by crossing Prkdc mutant founders. Prkdc mutant founders ♂25 and ♀15 were crossed and E13.5 embryos were isolated. (a) fPCR analysis of wild-type, founder ♂25, and founder ♀15. Note that, due to the technical limitations of fPCR analysis, these results showed small differences from the precise sequences of the mutant alleles; e.g., from the sequence analysis, Δ269/Δ61/WT and Δ5+1/+7/+12/WT were identified in founders ♂25 and ♀15, respectively. (b) Genotypes of the generated embryos.
  • FIG. 10 shows Cas9 protein/sgRNA complex induced targeted mutation.
  • FIG. 11 shows recombinant Cas9 protein-induced mutations in Arabidopsis protoplasts.
  • FIG. 12 shows recombinant Cas9 protein-induced mutant sequences in the Arabidopsis BRI1 gene.
  • FIG. 13 shows T7E1 assay showing endogenous CCR5 gene disruption in 293 cells by treatment of Cas9-mal-9R4L and sgRNA/C9R4LC complex.
  • FIG. 14 (a, b) shows mutation frequencies at on-target and off-target sites of RGENs reported in Fu et al. (2013). T7E1 assays analyzing genomic DNA from K562 cells (R) transfected serially with 20 μg of Cas9-encoding plasmid and with 60 μg and 120 μg of in vitro transcribed GX19 crRNA and tracrRNA, respectively (1×106 cells), or (D) co-transfected with 1 μg of Cas9-encoding plasmid and 1 μg of GX19 sgRNA expression plasmid (2×105 cells).
  • FIG. 15 (a, b) shows comparison of guide RNA structure. Mutation frequencies of the RGENs reported in Fu et al. (2013) were measured at on-target and off-target sites using the T7E1 assay. K562 cells were co-transfected with the Cas9-encoding plasmid and the plasmid encoding GX19 sgRNA or GGX20 sgRNA. Off-target sites (OT1-3 etc.) are labeled as in Fu et al. (2013).
  • FIG. 16 shows that in vitro DNA cleavage by Cas9 nickases. (a) Schematic overview of the Cas9 nuclease and the paired Cas9 nickase. The PAM sequences and cleavage sites are shown in box. (b) Target sites in the human AAVS1 locus. The position of each target site is shown in triangle. (c) Schematic overview of DNA cleavage reactions. FAM dyes (shown in box) were linked to both 5′ ends of the DNA substrate. (d) DSBs and SSBs analyzed using fluorescent capillary eletrophoresis. Fluorescentlylabeled DNA substrates were incubated with Cas9 nucleases or nickases before electrophoresis.
  • FIG. 17 shows comparison of Cas9 nuclease and nickase behavior. (a) On-target mutation frequencies associated with Cas9 nucleases (WT), nickases (D10A), and paired nickases. Paired nickases that would produce 5′ overhangs or 3′ overhangs are indicated. (b) Analysis of off-target effects of Cas9 nucleases and paired nickases. A total of seven potential off-target sites for three sgRNAs were analyzed.
  • FIG. 18 shows paired Cas9 nickases tested at other endogenous human loci. (a,c) The sgRNA target sites at human CCR5 and BRCA2 loci. PAM sequences are indicated in red. (b,d) Genome editing activities at each target site were detected by the T7E1 assay. The repair of two nicks that would produce 5′ overhangs led to the formation of indels much more frequently than did those producing 3′ overhangs.
  • FIG. 19 shows that paired Cas9 nickases mediate homologous recombination. (a) Strategy to detect homologous recombination. Donor DNA included an XbaI restriction enzyme site between two homology arms, whereas the endogenous target site lacked this site. A PCR assay was used to detect sequences that had undergone homologous recombination. To prevent amplification of contaminating donor DNA, primers specific to genomic DNA were used. (b) Efficiency of homologous recombination. Only amplicons of a region in which homologous recombination had occurred could be digested with XbaI; the intensities of the cleavage bands were used to measure the efficiency of this method.
  • FIG. 20 shows DNA splicing induced by paired Cas9 nickases. (a) The target sites of paired nickases in the human AAVS1 locus. The distances between the AS2 site and each of the other sites are shown. Arrows indicate PCR primers. (b) Genomic deletions detected using PCR. Asterisks indicate deletion-specific PCR products. (c) DNA sequences of deletion-specific PCR products obtained using AS2 and L1 sgRNAs. Target site PAM sequences are shown in box and sgRNA-matching sequences are shown in capital letters. Intact sgRNA-matching sequences are underlined. (d) A schematic model of paired Cas9 nickase-mediated chromosomal deletions. Newly-synthesized DNA strands are shown in box.
  • FIG. 21 shows that paired Cas9 nickases do not induce translocations. (a) Schematic overview of chromosomal translocations between the on-target and off-target sites. (b) PCR amplification to detect chromosomal translocations. (c) Translocations induced by Cas9 nucleases but not by the nickase pair.
  • FIG. 22 shows a conceptual diagram of the T7E1 and RFLP assays. (a) Comparison of assay cleavage reactions in four possible scenarios after engineered nuclease treatment in a diploid cell: (A) wild type, (B) a monoallelic mutation, (C) different biallelic mutations (hetero), and (D) identical biallelic mutations (homo). Black lines represent PCR products derived from each allele; dashed and dotted boxes indicate insertion/deletion mutations generated by NHEJ. (b) Expected results of T7E1 and RGEN digestion resolved by electrophoresis.
  • FIG. 23 shows in vitro cleavage assay of a linearized plasmid containing the C4BPB target site bearing indels. DNA sequences of individual plasmid substrates (upper panel). The PAM sequence is underlined. Inserted bases are shown in box. Arrows (bottom panel) indicate expected positions of DNA bands cleaved by the wild-type-specific RGEN after electrophoresis.
  • FIG. 24 shows genotyping of mutations induced by engineered nucleases in cells via RGEN-mediated RFLP. (a) Genotype of C4BPB mutant K562 cell clones. (b) Comparison of the mismatch-sensitive T7E1 assay with RGEN-mediated RFLP analysis. Black arrows indicate the cleavage product by treatment of T7E1 enzyme or RGENs.
  • FIG. 25 shows genotyping of RGEN-induced mutations via the RGEN-RFLP technique. (a) Analysis of C4BPB-disrupted clones using RGEN-RFLP and T7E1 assays. Arrows indicate expected positions of DNA bands cleaved by RGEN or T7E1. (b) Quantitative comparison of RGEN-RFLP analysis with T7E1 assays. Genomic DNA samples from wild-type and C4BPB-disrupted K562 cells were mixed in various ratios and subjected to PCR amplification. (c) Genotyping of RGEN-induced mutations in the HLA-B gene in HeLa cells with RFLP and T7E1 analyses.
  • FIG. 26 shows genotyping of mutations induced by engineered nucleases in organisms via RGEN-mediated RFLP. (a) Genotype of Pibf1 mutant founder mice. (b) Comparison of the mismatch-sensitive T7E1 assay with RGEN-mediated RFLP analysis. Black arrows indicate the cleavage product by treatment of T7E1 enzyme or RGENs.
  • FIG. 27 shows RGEN-mediated genotyping of ZFN-induced mutations. The ZFN target site is shown in box. Black arrows indicate DNA bands cleaved by T7E1.
  • FIG. 28 shows polymorphic sites in a region of the human HLA-B gene. The sequence, which surrounds the RGEN target site, is that of a PCR amplicon from HeLa cells. Polymorphic positions are shown in box. The RGEN target site and the PAM sequence are shown in dashed and bolded box, respectively. Primer sequences are underlined.
  • FIG. 29 shows genotyping of oncogenic mutations via RGEN-RFLP analysis. (a) A recurrent mutation (c.133-135 deletion of TCT) in the human CTNNB1 gene in HCT116 cells was detected by RGENs. HeLa cells were used as a negative control. (b) Genotyping of the KRAS substitution mutation (c.34 G>A) in the A549 cancer cell line with RGENs that contain mismatched guide RNA. Mismatched nucleotides are shown in box. HeLa cells were used as a negative control. Arrows indicate DNA bands cleaved by RGENs. DNA sequences confirmed by Sanger sequencing are shown.
  • FIG. 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells via RGEN-RFLP analysis. (a) RGEN-RFLP assays of cell lines. K562, SKBR3, and HeLa cells were used as wild-type controls. Arrows indicate DNA bands cleaved by RGENs. (b) DNA sequence of wild-type and delta32 CCR5 alleles. Both on-target and off-target sites of RGENs used in RFLP analysis are underlined. A single-nucleotide mismatch between the two sites is shown in box. The PAM sequence is underlined. (c) In vitro cleavage of plasmids harboring WT or del32 CCR5 alleles using the wild-type-specific RGEN. (d) Confirming the presence of an off-target site of the CCR5-delta32-specific RGEN at the CCR5 locus. In vitro cleavage assays of plasmids harboring either on-target or off-target sequences using various amounts of the del32-specific RGEN.
  • FIG. 31 shows genotyping of a KRAS point mutation (c.34 G>A). (a) RGEN-RFLP analysis of the KRAS mutation (c.34 G>A) in cancer cell lines. PCR products from HeLa cells (used as a wild-type control) or A549 cells, which are homozygous for the point mutation, were digested with RGENs with perfectly matched crRNA specific to the wild-type sequence or the mutant sequence. KRAS genotypes in these cells were confirmed by Sanger sequencing. (b) Plasmids harboring either the wild-type or mutant KRAS sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
  • FIG. 32 shows genotyping of a PIK3CA point mutation (c.3140 A>G). (a) RGEN-RFLP analysis of the PIK3CA mutation (c.3140 A>G) in cancer cell lines. PCR products from HeLa cells (used as a wild-type control) or HCT116 cells that are heterozygous for the point mutation were digested with RGENs with perfectly matched crRNA specific to the wild-type sequence or the mutant sequence. PIK3CA genotypes in these cells were confirmed by Sanger sequencing. (b) Plasmids harboring either the wild-type or mutant PIK3CA sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
  • FIG. 33 shows genotyping of recurrent point mutations in cancer cell lines. (a) RGEN-RFLP assays of recurrent oncogenic point mutations in IDH (c.394c>T), (b) PIK3CA (c.3140A>G), (c) NRAS (c.181C>A), (d) and BRAF genes (c.1799T>A). Genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are shown in box. Black arrows indicate DNA bands cleaved by RGENs.
    •  BEST MODE FOR CARRYING OUT THE INVENTION
  • In accordance with one aspect of the invention, the present invention provides a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein. In addition, the present invention provides a use of the composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • In the present invention, the composition is also referred to as a RNA-guided endonuclease (RGEN) composition.
  • ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequencies obtained with individual nucleases are widely different from each other. Furthermore, some ZFNs and TALENs fail to show any genome editing activities. DNA methylation may limit the binding of these engineered nucleases to target sites. In addition, it is technically challenging and time-consuming to make customized nucleases.
  • The present inventors have developed a new RNA-guided endonuclease composition based on Cas protein to overcome the disadvantages of ZFNs and TALENs.
  • Prior to the present invention, an endonuclease activity of Cas proteins has been known. However, it has not been known whether the endonuclease activity of Cas protein would function in an eukaryotic cell because of the complexity of the eukaryotic genome. Further, until now, a composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA specific for the target DNA to cleave a target DNA in eukaryotic cells or organisms has not been developed.
  • Compared to ZFNs and TALENs, the present RGEN composition based on Cas protein can be more readily customized because only the synthetic guide RNA component is replaced to make a new genome-editing nuclease. No sub-cloning steps are involved to make customized RNA guided endonucleases. Furthermore, the relatively small size of the Cas gene (for example, 4.2 kbp for Cas9) as compared to a pair of TALEN genes (˜6 kbp) provides an advantage for this RNA-guided endonuclease composition in some applications such as virus-mediated gene delivery. Further, this RNA-guided endonuclease does not have off-target effects and thus does not induce unwanted mutations, deletion, inversions, and duplications. These features make the present RNA-guided endonuclease composition a scalable, versatile, and convenient tools for genome engineering in eukaryotic cells and organisms. In addition, RGEN can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP. The specificity of RGENs is determined by the RNA component that hybridizes with a target DNA sequence of up to 20 base pairs (bp) in length and by the Cas9 protein that recognize the protospacer-adjacent motif (PAM). RGENs are readily reprogrammed by replacing the RNA component. Therefore, RGENs provide a platform to use simple and robust RFLP analysis for various sequence variations.
  • The target DNA may be an endogenous DNA, or artificial DNA, preferably, endogenous DNA.
  • As used herein, the term “Cas protein” refers to an essential protein component in the CRISPR/Cas system, forms an active endonuclease or nickase when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
  • The information on the gene and protein of Cas are available from GenBank of National Center for Biotechnology Information (NCBI), without limitation.
  • The CRISPR-associated (cas) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. There are three types of CRISPR-Cas system. Among them, Type II CRISPR/Cas system involving Cas9 protein and crRNA and tracrRNA is representative and is well known. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (E. coli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube).
  • The Cas protein may be linked to a protein transduction domain. The protein transduction domain may be poly-arginine or a TAT protein derived from HIV, but it is not limited thereto.
  • The present composition may comprise Cas component in the form of a protein or in the form of a nucleic acid encoding Cas protein.
  • In the present invention, Cas protein may be any Cas protein provided that it has an endonuclease or nickase activity when complexed with a guide RNA.
  • Preferably, Cas protein is Cas9 protein or variants thereof.
  • The variant of the Cas9 protein may be a mutant form of Cas9 in which the cataytic asapartate residue is changed to any other amino acid. Preferably, the other amino acid may be an alanine, but it is not limited thereto.
  • Further, Cas protein may be the one isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogens or a recombinant protein, but it is not limited thereto.
  • The Cas protein derived from Streptococcus pyogens may recognizes NGG trinucleotide. The Cas protein may comprise an amino acid sequence of SEQ ID NO: 109, but it is not limited thereto.
  • The term “recombinant” when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant Cas protein may be generated by reconstituting Cas protein-encoding sequence using the human codon table.
  • As for the present invention, Cas protein-encoding nucleic acid may be a form of vector, such as plasmid comprising Cas-encoding sequence under a promoter such as CMV or CAG. When Cas protein is Cas9, Cas9 encoding sequence may be derived from Streptococcus sp., and preferably derived from Streptococcus pyogenes. For example, Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID. NO: 1. Moreover, Cas9 encoding nucleic acid may comprise the nucleotide sequence having homology of at least 50% to the sequence of SEQ ID NO: 1, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% to the SEQ ID NO:1, but it is not limited thereto. Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NOs. 108, 110, 106, or 107.
  • As used herein, the term “guide RNA” refers to a RNA which is specific for the target DNA and can form a complex with Cas protein and bring Cas protein to the target DNA.
  • In the present invention, the guide RNA may consist of two RNA, i.e., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) or be a single-chain RNA (sgRNA) produced by fusion of an essential portion of crRNA and tracrRNA.
  • The guide RNA may be a dualRNA comprising a crRNA and a tracrRNA.
  • If the guide RNA comprises the essential portion of crRNA and tracrRNA and a portion complementary to a target, any guide RNA may be used in the present invention.
  • The crRNA may hybridize with a target DNA.
  • The RGEN may consist of Cas protein, and dualRNA (invariable tracrRNA and target-specific crRNA), or Cas protein and sgRNA (fusion of an essential portion of invariable tracrRNA and target-specific crRNA), and may be readily reprogrammed by replacing crRNA.
  • The guide RNA further comprises one or more additional nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
  • Preferably, the guide RNA further comprises 2-additional guanine nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
  • The guide RNA may be transferred into a cell or an organism in the form of RNA or DNA that encodes the guide RNA. The guide RNA may be in the form of an isolated RNA, RNA incorporated into a viral vector, or is encoded in a vector. Preferably, the vector may be a viral vector, plasmid vector, or agrobacterium vector, but it is not limited thereto.
  • A DNA that encodes the guide RNA may be a vector comprising a sequence coding for the guide RNA. For example, the guide RNA may be transferred into a cell or organism by transfecting the cell or organism with the isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
  • Alternatively, the guide RNA may be transferred into a cell or organism using virus-mediated gene delivery.
  • When the guide RNA is transfected in the form of an isolated RNA into a cell or organism, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA. As used herein, the term “isolated RNA” may be interchangeable to “naked RNA”. This is cost- and time-saving because it does not require a step of cloning. However, the use of plasmid DNA or virus-mediated gene delivery for transfection of the guide RNA is not excluded.
  • The present RGEN composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA can specifically cleave a target DNA due to a specificity of the guide RNA for a target and an endonuclease or nickase activity of Cas protein.
  • As used herein, the term “cleavage” refers to the breakage of the covalent backbone of a nucleotide molecule.
  • In the present invention, a guide RNA may be prepared to be specific for any target which is to be cleaved. Therefore, the present RGEN composition can cleave any target DNA by manipulating or genotyping the target-specific portion of the guide RNA.
  • The guide RNA and the Cas protein may function as a pair. As used herein, the term “paired Cas nickase” may refer to the guide RNA and the Cas protein functioning as a pair. The pair comprises two guide RNAs. The guide RNA and Cas protein may function as a pair, and induce two nicks on different DNA strand. The two nicks may be separated by at least 100 bps, but are not limited thereto.
  • In the Example, the present inventors confirmed that paired Cas nickase allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells. Importantly, paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations. Furthermore, unlike nucleases, paired nickases did not promote unwanted translocations associated with off-target DNA cleavages. In principle, paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
  • In the present invention, the composition may be used in the genotyping of a genome in the eukaryotic cells or organisms in vitro.
  • In one specific embodiment, the guide RNA may comprise the nucleotide sequence of Seq ID. No. 1, wherein the portion of nucleotide position 3˜22 is a target-specific portion and thus, the sequence of this portion may be changed depending on a target.
  • As used herein, a eukaryotic cell or organism may be yeast, fungus, protozoa, plant, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation.
  • In one specific embodiment, it was found that Cas9 protein/single-chain guide RNA could generate site-specific DNA double-strand breaks in vitro and in mammalian cells, whose spontaneous repair induced targeted genome mutations at high frequencies.
  • Moreover, it was found that gene-knockout mice could be induced by the injection of Cas9 protein/guide RNA complexes or Cas9 mRNA/guide RNA into one-cell stage embryo and germ-line transmittable mutations could be generated by Cas9/guide RNA system.
  • Using Cas protein rather than a nucleic acid encoding Cas protein to induce a targeted mutagenesis is advantageous because exogeneous DNA is not introduced into an organism. Thus, the composition comprising Cas protein and a guide RNA may be used to develop therapeutics or value-added crops, livestock, poultry, fish, pets, etc.
  • In accordance with another aspect of the invention, the present invention provides a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein. In addition, the present invention provides a use of the composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • In accordance with another aspect of the invention, the present invention provides a kit for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • The kit may comprise a guide RNA and Cas protein-encoding nucleic acid or Cas protein as separate components or as one composition.
  • The present kit may comprise some additional components necessary for transferring the guide RNA and Cas component to a cell or an organism. For example, the kit may comprise an injection buffer such as DEPC-treated injection buffer, and materials necessary for analysis of mutation of a target DNA, but are not limited thereto.
  • In accordance with another aspect, the present invention provides a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • In the present invention, a Cas protein-encoding nucleic acid or Cas protein and a guide RNA or DNA that encodes the guide RNA may be transferred into a cell by various methods known in the art, such as microinjection, electroporation, DEAEdextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection in protoplast, and so on, but are not limited thereto. Also, a Cas protein encoding nucleic acid or Cas protein and a guide RNA may be transferred into an organism by various method known in the art to administer a gene or a protein such as injection. A Cas protein-encoding nucleic acid or Cas protein may be transferred into a cell in the form of complex with a guide RNA, or separately. Cas protein fused to a protein transduction domain such as Tat can also be delivered efficiently into cells.
  • Preferably, the eukarotic cell or organisms is co-transfected or serial-transfected with a Cas9 protein and a guide RNA.
  • The serial-transfection may be performed by transfection with Cas protein-encoding nucleic acid first, followed by second transfection with naked guide RNA. Preferably, the second transfection is after 3, 6, 12, 18, 24 hours, but it is not limited thereto.
  • In accordance with another aspect, the present invention provides a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • The eukaryotic cells or organisms may be prepared by transferring the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
  • The eukaryotic cell may be yeast, fungus, protozoa, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation. Further the organism may be yeast, fungus, protozoa, plant, higher plant, insect, amphibian, or mammal.
  • In accordance with another aspect of the invention, the present invention provides a method for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a step of treating a cell or organism comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • The step of treating a cell or organism with the composition may be performed by transferring the present composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
  • As described in the above, such transfer may be performed by microinjection, transfection, electroporation, and so on.
  • In accordance with another aspect of the invention, the present invention provides an embryo comprising a genome edited by the present RGEN composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
  • Any embryo can be used in the present invention, and for the present invention, the embryo may be an embryo of a mouse. The embryo may be produced by injecting PMSG (Pregnant Mare Serum Gonadotropin) and hCG (human Choirinic Gonadotropin) into a female mouse of 4 to 7 weeks and the super-ovulated female mouse may be mated to males, and the fertilized embryos may be collected from oviduts.
  • The present RGEN composition introduced into an embryo can cleave a target DNA complementary to the guide RNA by the action of Cas protein and cause a mutation in the target DNA. Thus, the embryo into which the present RGEN composition has been introduced has an edited genome.
  • In one specific embodiment, it was found that the present RGEN composition could cause a mutation in a mouse embryo and the mutation could be transmitted to offsprings.
  • A method for introducing the RGEN composition into the embryo may be any method known in the art, such as microinjection, stem cell insertion, retrovirus insertion, and so on. Preferably, a microinjection technique can be used.
  • In accordance with another aspect, the present invention provides a genome-modified animal obtained by transferring the embryo comprising a genome edited by the present RGEN composition into the oviducts of an animal.
  • In the present invention, the term “genome-modified animal” refers to an animal of which genome has been modified in the stage of embryo by the present RGEN composition and the type of the animal is not limited.
  • The genome-modified animal has mutations caused by a targeted mutagenesis based on the present RGEN composition. The mutations may be any one of deletion, insertion, translocation, inversion. The site of mutation depends on the sequence of guide RNA of the RGEN composition.
  • The genome-modified animal having a mutation of a gene may be used to determine the function of the gene.
  • In accordance with another aspect of the invention, the present invention provides a method of preparing a genome-modified animal comprising a step of introducing the present RGEN composition comprising a guide RNA specific for the target DNA or DNA that encodes the guide RNA and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
  • The step of introducing the present RGEN composition may be accomplished by any method known in the art such as microinjection, stem cell insertion, retroviral insertion, and so on.
  • In accordance with another aspect of the invention, the present invention provides a plant regenerated form the genome-modified protoplasts prepared by the method for eukaryotic cells comprising the RGEN composition.
  • In accordance with another aspect of the invention, the present invention provides a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence Cas protein. In addrion, the present invention provides a composition for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • As used herein the term “genotyping” refers to the “Restriction fragment length polymorphism (RFLP) assay”.
  • RFLP may be used in 1) the detection of indel in cells or organisms induced by the engineered nucleases, 2) the genotyping naturally-occurring mutations or variations in cells or organisms, or 3) the genotyping the DNA of infected pathogenic microorganisms including virus or bacteria, etc.
  • The mutations or variation may be induced by engineered nucleases in cells.
  • The engineered nuclease may be a Zinc Finger Nuclease (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), or RGENs, but it is not limited thereto.
  • As used herein the term “biological sample” includes samples for analysis, such as tissues, cells, whole blood, semm, plasma, saliva, sputum, cerbrospinal fluid or urine, but is not limited thereto
  • The mutations or variation may be a naturally-occurring mutations or variations.
  • The mutations or variations are induced by the pathogenic microorganisms. Namely, the mutations or variation occur due to the infection of pathogenic microorganisms, when the pathogenic microorganisms are detected, the biological sample is identified as infected.
  • The pathogenic microorganisms may be virus or bacteria, but are not limited thereto.
  • Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescent PCR, DNA melting analysis, and Sanger and deep sequencing. The T7E1 and Surveyor assays are widely used but often underestimate mutation frequencies because the assays detect heteroduplexes (formed by the hybridization of mutant and wild-type sequences or two different mutant sequences); they fail to detect homoduplexes formed by the hybridization of two identical mutant sequences. Thus, these assays cannot distinguish homozygous bialleic mutant clones from wild-type cells nor heterozygous biallelic mutants from heterozygous monoalleic mutants (FIG. 22). In addition, sequence polymorphisms near the nuclease target site can produce confounding results because the enzymes can cleave heteroduplexes formed by hybridization of these different wild-type alleles. RFLP analysis is free of these limitations and therefore is a method of choice. Indeed, RFLP analysis was one of the first methods used to detect engineered nuclease-mediated mutations. Unfortunately, however, it is limited by the availability of appropriate restriction sites.
  • In accordance with another aspect of the invention, the present invention provides a kit for genotyping mutations or variations in an isolated biological sample, comprising the composition for genotyping mutations or variations in an isolated biological sample. In addition, the present invention provides a kit for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
  • In accordance with another aspect of the invention, the present invention provides a method of genotyping mutations or variations in an isolated biological sample, using the composition for genotyping mutations or variations in an isolated biological sample. In addtion, the present invention provides a method of genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
  • A guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
    • Mode for the Invention
  • Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
    • Example 1 Genome Editing Assay
  • 1-1. DNA Cleavage Activity of Cas9 Protein
  • Firstly, the DNA cleavage activity of Cas9 derived from Streptococcus pyogenes in the presence or absence of a chimeric guide RNA in vitro was tested.
  • To this end, recombinant Cas9 protein that was expressed in and purified from E. coli was used to cleave a predigested or circular plasmid DNA that contained the 23-base pair (bp) human CCR5 target sequence. A Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or a chimeric guide RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas9 itself (FIG. 1A).
  • Specifically, the Cas9-coding sequence (4,104 bp), derived from Streptococcus pyogenes strain M1 GAS (NC—002737.1), was reconstituted using the human codon usage table and synthesized using oligonucleotides. First, 1-kb DNA segments were assembled using overlapping ˜35-mer oligonucleotides and Phusion polymerase (New England Biolabs) and cloned into T-vector (SolGent). A full-length Cas9 sequence was assembled using four 1-kbp DNA segments by overlap PCR. The Cas9-encoding DNA segment was subcloned into p3s, which was derived from pcDNA3.1 (Invitrogen). In this vector, a peptide tag (NH2-GGSGPPKKKRKVYPYDVPDYA-COOH, SEQ ID NO: 2) containing the HA epitope and a nuclear localization signal (NLS) was added to the C-terminus of Cas9. Expression and nuclear localization of the Cas9 protein in HEK 293T cells were confirmed by western blotting using anti-HA antibody (Santa Cruz).
  • Then, the Cas9 cassette was subcloned into pET28-b(+) and transformed into BL21(DE3). The expression of Cas9 was induced using 0.5 mM IPTG for 4 h at 25° C. The Cas9 protein containing the His6-tag at the C terminus was purified using Ni-NTA agarose resin (Qiagen) and dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol (1). Purified Cas9 (50 nM) was incubated with supercoiled or pre-digested plasmid DNA (300 ng) and chimeric RNA (50 nM) in a reaction volume of 20 μl in NEB buffer 3 for 1 h at 37° C. Digested DNA was analyzed by electrophoresis using 0.8% agarose gels.
  • Cas9 cleaved the plasmid DNA efficiently at the expected position only in the presence of the synthetic RNA and did not cleave a control plasmid that lacked the target sequence (FIG. 1B).
  • 1-2. DNA Cleavage by Cas9/Guide RNA Complex in Human Cells
  • A RFP-GFP reporter was used to investigate whether the Cas9/guide RNA complex can cleave the target sequence incorporated between the RFP and GFP sequences in mammalian cells.
  • In this reporter, the GFP sequence is fused to the RFP sequence out-of-frame (2). The active GFP is expressed only when the target sequence is cleaved by site-specific nucleases, which causes frameshifting small insertions or deletions (indels) around the target sequence via error-prone non-homologous end-joining (NHEJ) repair of the double-strand break (DSB) (FIG. 2).
  • The RFP-GFP reporter plasmids used in this study were constructed as described previously (2). Oligonucleotides corresponding to target sites (Table 1) were synthesized (Macrogen) and annealed. The annealed oligonucleotides were ligated into a reporter vector digested with EcoRI and BamHI.
  • HEK 293T cells were co-transfected with Cas9-encoding plasmid (0.8 μg) and the RFP-GFP reporter plasmid (0.2 μg) in a 24-well plate using Lipofectamine 2000 (Invitrogen).
  • Meanwhile, the in vitro transcribed chimeric RNA had been prepared as follows. RNA was in vitro transcribed through run-off reactions using the MEGAshortscript T7 kit (Ambion) according to the manufacturer's manual. Templates for RNA in vitro transcription were generated by annealing two complementary single strand DNAs or by PCR amplification (Table 1). Transcribed RNA was resolved on a 8% denaturing urea-PAGE gel. The gel slice containing RNA was cut out and transferred to probe elution buffer. RNA was recovered in nuclease-free water followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNAs were quantified by spectrometry.
  • At 12 h post transfection, chimeric RNA (1 μg) prepared by in vitro transcription was transfected using Lipofectamine 2000.
  • At 3 d post-transfection, transfected cells were subjected to flow cytometry and cells expressing both RFP and GFP were counted.
  • It was found that GFP-expressing cells were obtained only when the cells were transfected first with the Cas9 plasmid and then with the guide RNA 12 h later (FIG. 2), demonstrating that RGENs could recognize and cleave the target DNA sequence in cultured human cells. Thus GFP-expressing cells were obtained by serial-transfection of the Cas9 plasmid and the guide RNA rather than co-transfection.
  • TABLE 1
    [Table 1]
    SEQ ID
    Gene sequence (5′ to 3′) NO.
    Oligonucleotides used for the construction of the
    reporter plasmid
    CCR5 F AATTCATGACATCAATTATTATACATCGGAG 3
    GAG
    R GATCCTCCTCCGATGTATAATAATTGATGTC
    4
    ATG
    Primers used in the T7E1 assay
    CCR5 F1 CTCCATGGTGCTATAGAGCA 5
    F2 GAGCCAAGCTCTCCATCTAGT 6
    R GCCCTGTCAAGAGTTGACAC 7
    C4BPB F1 TATTTGGCTGGTTGAAAGGG 8
    R1 AAAGTCATGAAATAAACACACCCA 9
    F2 CTGCATTGATATGGTAGTACCATG 10
    R2 GCTGTTCATTGCAATGGAATG 11
    Primers used for the amplification of off-target
    sites
    ADCY5 F1 GCTCCCACCTTAGTGCTCTG 12
    R1 GGTGGCAGGAACCTGTATGT 13
    F2 GTCATTGGCCAGAGATGTGGA 14
    R2 GTCCCATGACAGGCGTGTAT 15
    KCNJ6 F GCCTGGCCAAGTTTCAGTTA 16
    R1 TGGAGCCATTGGTTTGCATC
    17
    R2 CCAGAACTAAGCCGTTTCTGAC 18
    CNTNAP2 F1 ATCACCGACAACCAGTTTCC 19
    F2 TGCAGTGCAGACTCTTTCCA 20
    R AAGGACACAGGGCAACTGAA 21
    N/A Chr. F1 TGTGGAACGAGTGGTGACAG 22
    5 R1 GCTGGATTAGGAGGCAGGATTC 23
    F2 GTGCTGAGAACGCTTCATAGAG 24
    R2 GGACCAAACCACATTCTTCTCAC 25
    Primers used for the detection of chromosomal
    deletions
    Deletion F CCACATCTCGTTCTCGGTTT 26
    R TCACAAGCCCACAGATATTT 27
  • 1-3. Targeted Disruption of Endogeneous Genes in Mammalian Cells by RGEN
  • To test whether RGENs could be used for targeted disruption of endogenous genes in mammalian cells, genomic DNA isolated from transfected cells using T7 endonuclease I (T7E1), a mismatch-sensitive endonuclease that specifically recognizes and cleaves heteroduplexes formed by the hybridization of wild-type and mutant DNA sequences was analyzed (3).
  • To introduce DSBs in mammalian cells using RGENs, 2×106 K562 cells were transfected with 20 μg of Cas9-encoding plasmid using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. For this experiment, K562 (ATCC, CCL-243) cells were grown in RPMI1640 with 10% FBS and the penicillin/streptomycin mix (100 U/ml and 100 μg/ml, respectively).
  • After 24 h, 10-40 μg of in vitro transcribed chimeric RNA was nucleofected into 1×106 K562 cells. The in vitro transcribed chimeric RNA had been prepared as described in the Example 1-2.
  • Cells were collected two days after RNA transfection and genomic DNA was isolated. The region including the target site was PCR-amplified using the primers described in Table 1. The amplicons were subjected to the T7E1 assay as described previously (3). For sequencing analysis, PCR products corresponding to genomic modifications were purified and cloned into the T-Blunt vector using the T-Blunt PCR Cloning Kit (SolGent). Cloned products were sequenced using the M13 primer.
  • It was found that mutations were induced only when the cells were transfected serially with Cas9-encoding plasmid and then with guide RNA (FIG. 3). Mutation frequencies (Indels (%) in FIG. 3A) estimated from the relative DNA band intensities were RNA-dosage dependent, ranging from 1.3% to 5.1%. DNA sequencing analysis of the PCR amplicons corroborated the induction of RGEN-mediated mutations at the endogenous sites. Indels and microhomologies, characteristic of error-prone NHEJ, were observed at the target site. The mutation frequency measured by direct sequencing was 7.3% (=7 mutant clones/96 clones), on par with those obtained with zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs).
  • Serial-transfection of Cas9 plasmid and guide RNA was required to induce mutations in cells. But when plasmids that encode guide RNA, serial transfection was unnecessary and cells were co-transfected with Cas9 plasmid and guide RNA-encoding plasmid.
  • In the meantime, both ZFNs and TALENs have been successfully developed to disrupt the human CCR5 gene (3-6), which encodes a G-protein-coupled chemokine receptor, an essential co-receptor of HIV infection. A CCR5-specific ZFN is now under clinical investigation in the US for the treatment of AIDS (7). These ZFNs and TALENs, however, have off-target effects, inducing both local mutations at sites whose sequences are homologous to the on-target sequence (6, 8-10) and genome rearrangements that arise from the repair of two concurrent DSBs induced at on-target and off-target sites (11-12). The most striking off-target sites associated with these CCR5-specific engineered nucleases reside in the CCR2 locus, a close homolog of CCR5, located 15-kbp upstream of CCR5. To avoid off-target mutations in the CCR2 gene and unwanted deletions, inversions, and duplications of the 15-kbp chromosomal segment between the CCR5 on-target and CCR2 off-target sites, the present inventors intentionally chose the target site of our CCR5-specific RGEN to recognize a region within the CCR5 sequence that has no apparent homology with the CCR2 sequence.
  • The present inventors investigated whether the CCR5-specific RGEN had off-target effects. To this end, we searched for potential off-target sites in the human genome by identifying sites that are most homologous to the intended 23-bp target sequence. As expected, no such sites were found in the CCR2 gene. Instead, four sites, each of which carries 3-base mismatches with the on-target site, were found (FIG. 4A). The T7E1 assays showed that mutations were not detected at these sites (assay sensitivity, ˜0.5%), demonstrating exquisite specificities of RGENs (FIG. 4B). Furthermore, PCR was used to detect the induction of chromosomal deletions in cells separately transfected with plasmids encoding the ZFN and RGEN specific to CCR5. Whereas the ZFN induced deletions, the RGEN did not (FIG. 4C).
  • Next, RGENs was reprogrammed by replacing the CCR5-specific guide RNA with a newly-synthesized RNA designed to target the human C4BPB gene, which encodes the beta chain of C4b-binding protein, a transcription factor. This RGEN induced mutations at the chromosomal target site in K562 cells at high frequencies (FIG. 3B). Mutation frequencies measured by the T7E1 assay and by direct sequencing were 14% and 8.3% (=4 mutant clones/48 clones), respectively. Out of four mutant sequences, two clones contained a single-base or two-base insertion precisely at the cleavage site, a pattern that was also observed at the CCR5 target site. These results indicate that RGENs cleave chromosomal target DNA at expected positions in cells.
    • Example 2 Proteinaceous RGEN-Mediated Genome Editing
  • RGENs can be delivered into cells in many different forms. RGENs consist of Cas9 protein, crRNA, and tracrRNA. The two RNAs can be fused to form a single-chain guide RNA (sgRNA). A plasmid that encodes Cas9 under a promoter such as CMV or CAG can be transfected into cells. crRNA, tracrRNA, or sgRNA can also be expressed in cells using plasmids that encode these RNAs. Use of plasmids, however, often results in integration of the whole or part of the plasmids in the host genome. The bacterial sequences incorporated in plasmid DNA can cause unwanted immune response in vivo. Cells transfected with plasmid for cell therapy or animals and plants derived from DNA-transfected cells must go through a costly and lengthy regulation procedure before market approval in most developed countries. Furthermore, plasmid DNA can persist in cells for several days post-transfection, aggravating off-target effects of RGENs.
  • Here, we used recombinant Cas9 protein complexed with in vitro transcribed guide RNA to induce targeted disruption of endogenous genes in human cells. Recombinant Cas9 protein fused with the hexa-histidine tag was expressed in and purified from E. coli using standard Ni ion affinity chromatography and gel filtration. Purifed recombinant Cas9 protein was concentrated in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol). Cas9 protein/sgRNA complex was introduced directly into K562 cells by nucleofection: 1×106 K562 cells were transfected with 22.5-225 (1.4-14 μM) of Cas9 protein mixed with 100 ug (29 μM) of in vitro transcribed sgRNA (or crRNA 40 ug and tracrRNA 80 ug) in 1000 solution using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. After nucleofection, cells were placed in growth media in 6-well plates and incubated for 48 hr. When 2×105 K562 cells were transfected with ⅕ scale-downed protocol, 4.5-45 μg of Cas9 protein mixed with 6-60 ug of in vitro transcribed sgRNA (or crRNA 8 μg and tracrRNA 16 μg) were used and nucleofected in 20 μl solution. Nucleofected cell were then placed in growth media in 48-well plates. After 48 hr, cells were collected and genomic DNA was isolated. The genomic DNA region spanning the target site was PCR-amplified and subjected to the T7E1 assay.
  • As shown in FIG. 10, Cas9 protein/sgRNA complex induced targeted mutation at the CCR5 locus at frequencies that ranged from 4.8 to 38% in a sgRNA or Cas9 protein dose-dependent manner, on par with the frequency obtained with Cas9 plasmid transfection (45%). Cas9 protein/crRNA/tracrRNA complex was able to induce mutations at a frequency of 9.4%. Cas9 protein alone failed to induce mutations. When 2×105 cells were transfected with 1/5 scale-downed doses of Cas9 protein and sgRNA, mutation frequencies at the CCR5 locus ranged from 2.7 to 57% in a dose-dependent manner, greater than that obtained with co-transfection of Cas9 plasmid and sgRNA plasmid (32%).
  • We also tested Cas9 protein/sgRNA complex that targets the ABCC11 gene and found that this complex induced indels at a frequency of 35%, demonstrating general utility of this method.
  • TABLE 2
    [Table 2]
    Sequences of guide RNA
    SEQ
    Tar- RNA ID
    get type RNA sequence (5′ to 3′) Length NO
    CCR5 sgRNA GGUGACAUCAAUUAUUAUACAUGUUU 104 bp 28
    UAGAGCUAGAAAUAGCAAGUUAAAAU
    AAGGCUAGUCCGUUAUCAACUUGAAAA
    AGUGGCACCGAGUCGGUGCUUUUUUU
    crRNA GGUGACAUCAAUUAUUAUACAUGUUU
     44 bp 29
    UAGAGCUAUGCUGUUUUG
    tracrRNA GGAACCAUUCAAAACAGCAUAGCAAGU  86 bp 30
    UAAAAUAAGGCUAGUCCGUUAUCAACU
    UGAAAAAGUGGCACCGAGUCGGUGCUU
    UUUUU
    • Example 3 RNA-Guided Genome Editing in Mice
  • To examine the gene-targeting potential of RGENs in pronuclear (PN)-stage mouse embryos, the forkhead box N1 (Foxn1) gene, which is important for thymus development and keratinocyte differentiation (Nehls et al., 1996), and the protein kinase, DNA activated, catalytic polypeptide (Prkdc) gene, which encodes an enzyme critical for DNA DSB repair and recombination (Taccioli et al., 1998) were used.
  • To evaluate the genome-editing activity of the Foxn1-RGEN, we injected Cas9 mRNA (10-ng/μl solution) with various doses of the sgRNA (FIG. 5 a) into the cytoplasm of PN-stage mouse embryos, and conducted T7 endonuclease I (T7E1) assays (Kim et al. 2009) using genomic DNAs obtained from in vitro cultivated embryos (FIG. 6 a).
  • Alternatively, we directly injected the RGEN in the form of recombinant Cas9 protein (0.3 to 30 ng/μl) complexed with the two-fold molar excess of Foxn1-specific sgRNA (0.14 to 14 ng/μl) into the cytoplasm or pronucleus of one-cell mouse embryos, and analyzed mutations in the Foxn1 gene using in vitro cultivated embryos (FIG. 7).
  • Specifically, Cas9 mRNA and sgRNAs were synthesized in vitro from linear DNA templates using the mMESSAGE mMACHINE T7 Ultra kit (Ambion) and MEGAshortscript T7 kit (Ambion), respectively, according to the manufacturers' instructions, and were diluted with appropriate amounts of diethyl pyrocarbonate (DEPC, Sigma)-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4). Templates for sgRNA synthesis were generated using oligonucleotides listed in Table 3. Recombinant Cas9 protein was obtained from ToolGen, Inc.
  • TABLE 3
    [Table 3]
    SEQ
    Direc- ID
    RNA Name tion Sequence (5′ to 3′) NO
    Foxn1 #1 F GAAATTAATACGACTCACTATAGG CA 31
    sgRNA GTCTGACGTCACACTTCCGTTTTAGAGC
    TAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCG
    Foxn1 #2 F GAAATTAATACGACTCACTATAGG ACT 32
    sgRNA TCCAGGCTCCACCCGACGTTTTAGAGC
    TAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCG
    Foxn1 #3 F GAAATTAATACGACTCACTATAGG CCA 33
    sgRNA GGCTCCACCCGACTGGAGTTTTAGAGC
    TAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCG
    Foxn1 #4 F GAAATTAATACGACTCACTATAGG ACT 34
    sgRNA GGAGGGCGAACCCCAAGGTTTTAGAGC
    TAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCG
    Foxn1 #5 F GAAATTAATACGACTCACTATAGG ACC 35
    sgRNA CCAAGGGGACCTCATGCGTTTTAGAGC
    TAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCG
    Prkdc #1 F GAAATTAATACGACTCACTATAGG TTA 36
    sgRNA GTTTTTTCCAGAGACTT GTTTTAGAGCT
    AGAAATAGCAAGTTAAAATAAGGCTA
    GTCCG
    Prkdc #2 F GAAATTAATACGACTCACTATAGG TTG 37
    sgRNA GTTTGCTTGTGTTTATC GTTTTAGAGCT
    AGAAATAGCAAGTTAAAATAAGGCTA
    GTCCG
    Prkdc #3 F GAAATTAATACGACTCACTATAGG CAC 38
    sgRNA AAGCAAACCAAAGTCTCGTTTTAGAGCT
    AGAAATAGCAAGTTAAAATAAGGCTA
    GTCCG
    Prkdc #4 F GAAATTAATACGACTCACTATAGG CCT 39
    sgRNA CAATGCTAAGCGACTTCGTTTTAGAGCT
    AGAAATAGCAAGTTAAAATAAGGCTA
    GTCCG
  • All animal experiments were performed in accordance with the Korean Food and Drug Administration (KFDA) guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of the Laboratory Animal Research Center at Yonsei University (Permit Number: 2013-0099). All mice were maintained in the specific pathogen-free facility of the Yonsei Laboratory Animal Research Center. FVB/NTac (Taconic) and ICR mouse strains were used as embryo donors and foster mothers, respectively. Female FVB/NTac mice (7-8 weeks old) were super-ovulated by intra-peritoneal injections of 5 IU pregnant mare serum gonadotropin (PMSG, Sigma) and 5 IU human chorionic gonadotropin (hCG, Sigma) at 48-hour intervals. The super-ovulated female mice were mated to FVB/NTac stud males, and fertilized embryos were collected from oviducts.
  • Cas9 mRNA and sgRNAs in M2 medium (Sigma) were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei using a Piezo-driven micromanipulator (Prime Tech).
  • In the case of injection of recombinant Cas9 protein, the recombinant Cas9 protein: Foxn1-sgRNA complex was diluted with DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4) and injected into male pronuclei using a TransferMan NK2 micromanipulator and a FemtoJet microinjector (Eppendorf).
  • The manipulated embryos were transferred into the oviducts of pseudo-pregnant foster mothers to produce live animals, or were cultivated in vitro for further analyses.
  • To screen F0 mice and in vitro cultivated mouse embryos with RGEN-induced mutations, T7E1 assays were performed as previously described using genomic DNA samples from tail biopsies and lysates of whole embryos (Cho et al., 2013).
  • Briefly, the genomic region encompassing the RGEN target site was PCR-amplified, melted, and re-annealed to form heteroduplex DNA, which was treated with T7 endonuclease 1 (New England Biolabs), and then analyzed by agarose gel electrophoresis. Potential off-target sites were identified by searching with bowtie 0.12.9 and were also similarly monitored by T7E1 assays. The primer pairs used in these assays were listed in Tables 4 and 5.
  • TABLE 4
    [Table 4]
    Primers used in the T7E1 assay
    SEQ
    Direc- ID
    Gene tion Sequence(5′ to 3′) NO
    Foxn 1 F1 GTCTGTCTATCATCTCTTCCCTTCTCTCC 40
    F2 TCCCTAATCCGATGGCTAGCTCCAG 41
    R1 ACGAGCAGCTGAAGTTAGCATGC 42
    R2 CTACTCAATGCTCTTAGAGCTACCAGGCTTG 43
    C
    Prkdc F GACTGTTGTGGGGAGGGCCG 44
    F2 GGGAGGGCCGAAAGTCTTATTTTG
    45
    R1 CCTGAAGACTGAAGTTGGCAGAAGTGAG 46
    R2 CTTTAGGGCTTCTTCTCTACAATCACG 47
  • TABLE 5
    [Table 5]
    Primers used for amplification of off-target sites
    SEQ
    Direc- ID
    Gene Notation tion Sequence(5′ to 3′) NO
    Foxn1 off 1 F CTCGGTGTGTAGCCCTGAC 48
    R AGACTGGCCTGGAACTCACA 49
    G
    off 2 F CACTAAAGCCTGTCAGGAAG 50
    CCG
    R CTGTGGAGAGCACACAGCAG 51
    C
    off 3 F GCTGCGACCTGAGACCATG 52
    R CTTCAATGGCTTCCTGCTTAG 53
    GCTAC
    off 4 F GGTTCAGATGAGGCCATCCTT 54
    TC
    R CCTGATCTGCAGGCTTAACCC 55
    TTG
    Prkdc off 1 F CTCACCTGCACATCACATGTG 56
    G
    R GGCATCCACCCTATGGGGTC
    57
    off 2 F GCCTTGACCTAGAGCTTAAAG 58
    AGCC
    R GGTCTTGTTAGCAGGAAGGAC 59
    ACTG
    off 3 F AAAACTCTGCTTGATGGGATA 60
    TGTGGG
    R CTCTCACTGGTTATCTGTGCT
    61
    CCTTC
    off 4 F GGATCAATAGGTGGTGGGGG 62
    ATG
    R GTGAATGACACAATGTGACA 63
    GCTTCAG
    off 5 F CACAAGACAGACCTCTCAAC 64
    ATTCAGTC
    R GTGCATGCATATAATCCATTC 65
    TGATTGCTCTC
    off 6 F1 GGGAGGCAGAGGCAGGT 66
    F2 GGATCTCTGTGAGTTTGAGGC 67
    CA
    R1 GCTCCAGAACTCACTCTTAGG 68
    CTC
  • Mutant founders identified by the T7E1 assay were further analyzed by fPCR. Appropriate regions of genomic DNA were sequenced as described previously (Sung et al., 2013). For routine PCR genotyping of F1 progenies, the following primer pairs were used for both wild-type and mutant alleles:
  • (SEQ ID NO: 69)
    5′-CTACTCCCTCCGCAGTCTGA-3′
    and
    (SEQ ID NO: 70)
    5′-CCAGGCCTAGGTTCCAGGTA-3′ for the Foxn1
    gene,
    (SEQ ID NO: 71)
    5′-CCCCAGCATTGCAGATTTCC-3′
    and
    (SEQ ID NO: 72)
    5′-AGGGCTTCTTCTCTACAATCACG-3′ for Prkdc gene.
  • In the case of injection of Cas9 mRNA, mutant fractions (the number of mutant embryos/the number of total embryos) were dose-dependent, ranging from 33% (1 ng/μl sgRNA) to 91% (100 ng/μl) (FIG. 6 b). Sequence analysis confirmed mutations in the Foxn1 gene; most mutations were small deletions (FIG. 6 c), reminiscent of those induced by ZFNs and TALENs (Kim et al., 2013).
  • In the case of injection of Cas9 protein, these injection doses and methods minimally affected the survival and development of mouse embryos in vitro: over 70% of RGEN-injected embryos hatched out normally in both experiments. Again, mutant fractions obtained with Cas9 protein injection were dose-dependent, and reached up to 88% at the highest dose via pronucleus injection and to 71% via intra-cytoplasmic injection (FIGS. 7 a and 7 b). Similar to the mutation patterns induced by Cas9 mRNA plus sgRNA (FIG. 6 c), those induced by the Cas9 protein-sgRNA complex were mostly small deletions (FIG. 7 c). These results clearly demonstrate that RGENs have high gene-targeting activity in mouse embryos.
  • Encouraged by the high mutant frequencies and low cytotoxicity induced by RGENs, we produced live animals by transferring the mouse embryos into the oviducts of pseudo-pregnant foster mothers.
  • Notably, the birth rates were very high, ranging from 58% to 73%, and were not affected by the increasing doses of Foxn1-sgRNA (Table 6).
  • TABLE 6
    RGEN-mediated gene-targeting in FVB/NTac mice
    Cas9
    mRNA + Transferred Total Live
    Target sgRNA Injected embryos newborns newborns* Founders†
    Gene (ng/μL) embryos (%) (%) (%) (%)
    Foxn1 10 + 1 76 62 (82) 45 (73) 31 (50) 12 (39)
    10 + 10 104 90 (87) 52 (58) 58 (64) 33 (57)
    10 + 100 100 90 (90) 62 (69) 58 (64) 54 (93)
    Total 280 242 (86) 159 (66) 147 (61) 99 (67)
    Prkdc 50 + 50 73 58 (79) 35 (60) 33 (57) 11 (33)
    50 + 100 79 59 (75) 22 (37) 21 (36) 7 (33)
    50 + 250 94 73 (78) 37 (51) 37 (51) 21 (57)
    Total 246 190 (77) 94 (49) 91 (48) 39 (43)
  • Out of 147 newborns, we obtained 99 mutant founder mice. Consistent with the results observed in cultivated embryos (FIG. 6 b), mutant fractions were proportional to the doses of Foxn1-sgRNA, and reached up to 93% (100 ng/μl Foxn1-sgRNA) (Tables 6 and 7, FIG. 5 b).
  • TABLE 7
    [Table 7]
    DNA sequences of Foxn1 mutant alleles identified from a subset of T7E1-positive
    mutant founders
    ACTTCCAGGCTCCACCCGACTGGAGGGCGAACC Founder
    CCAAGGGGACCTCATGCAGG del + ins # mice
    ACTTCCAGGC-------------------AACCCCAAGGGGAC Δ19 1 20
    CTCATGCAGG
    ACTTCCAGGC------------------GAACCCCAAGGGGA Δ18 1 115
    CCTCATGCAGG
    ACTTCCAGGCTCC---------------------------------------- Δ60 1 19
    ACTTCCAGGCTCC---------------------------------------- Δ44 1 108
    ACTTCCAGGCTCC---------------------CAAGGGGACC Δ21 1 64
    TCATGCAGG
    ACTTCCAGGCTCC------------TTAGGAGGCGAACCC Δ12 + 6 1 126
    CAAGGGGACCTCA
    ACTTCCAGGCTCCACC----------------------------TCAT Δ28 1 5
    GCAGG
    ACTTCCAGGCTCCACCC---------------------CCAAGG Δ21 + 4 1 61
    GACCTCATG
    ACTTCCAGGCTCCACCC------------------AAGGGGAC Δ18 2 95, 29
    CTCATGCAGG
    ACTTCCAGGCTCCACCC-----------------CAAGGGGA Δ17 7 12, 14, 27,
    CCTCATGCAGG 66, 108,
    114, 126
    ACTTCCAGGCTCCACCC---------------ACCCAAGGG Δ15 + 1 1 32
    GACCTCATGCAG
    ACTTCCAGGCTCCACCC---------------CACCCAAGG Δ15 + 2 1 124
    GGACCTCATGCA
    ACTTCCAGGCTCCACCC-------------ACCCCAAGGG Δ13 1 32
    GACCTCATGCAGG
    ACTTCCAGGCTCCACCC--------GGCGAACCCCAAG Δ8 1 110
    GGGACCTCATGCAGG
    ACTTCCAGGCTCCACCCT-------------------GGGGAC Δ20 + 1 1 29
    CTCATGCAGG
    ACTTCCAGGCTCCACCCG-----------AACCCCAAGG Δ11 1 111
    GGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGA----------------------ACCT Δ22 1 79
    CATGCAGG
    ACTTCCAGGCTCCACCCGA------------------GGGGAC Δ18 2 13, 127
    CTCATGCAGG
    ACTTCCAGGCTCCACCCCA-----------------AGGGGA Δ17 1 24
    CCTCATGCAGG
    ACTTCCAGGCTCCACCCGA-----------ACCCCAAGG Δ11 5 14, 53, 58,
    GGACCTCATGCAGG 69, 124
    ACTTCCAGGCTCCACCCGA----------GACCCCAAGG Δ10 1 14
    GGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGA-----GGGCGAACCCCA Δ5 3 53, 79, 115
    AGGGGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGAC-----------------------CTC Δ23 1 108
    ATGCAGG
    ACTTCCAGGCTCCACCCGAC-----------CCCCAAGG Δ11 1 3
    GGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGAC-----------GAAGGGCC Δ11 + 6 1 66
    CCAAGGGGACCTCA
    ACTTCCAGGCTCCACCCGAC--------GAACCCCAAG Δ8 2 3,66
    GGGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGAC-----GGCGAACCCCA Δ5 1 27
    AGGGGACCTCATGCAGG
    ACTTCCAGGCTCCACCCGAC--GTGCTTGAGGGCG Δ2 + 6 2 5
    AACCCCAAGGGGACCTCA
    ACTTCCAGGCTCCACCCGACT------CACTATCTTC Δ6 + 25 2 21,114
    TGGGCTCCTCCATGTC
    ACTTCCAGGCTCCACCCGACT----TGGCGAACCCC Δ4 + 1 1 53
    AAGGGGACCTCATGCAG
    ACTTCCAGGCTCCACCCGACT--TGCAGGGCGAAC Δ2 + 3 1 126
    CCCAAGGGGACCTCATGC
    ACTTCCAGGCTCCACCCGACTTGGAGGGCGAAC +1 15 3, 5, 12, 19,
    CCCAAGGGGACCTCATGCAG 29, 55, 56,
    61, 66, 68,
    81, 108,
    111, 124,
    127
    ACTTCCAGGCTCCACCCGACTTTGGAGGGCGAAC +2 2 79, 120
    CCCAAGGGGACCTCATGCA
    ACTTCCAGGCTCCACCCGACTGTTGGAGGGCGA +3 1 55
    ACCCCAAGGGGACCTCATGC
    ACTTCCAGGCTCCACCCGACTGGAG(+455)GGCG +455 1 13
    AACCCCAAGGGGACCTCC
  • To generate Prkdc-targeted mice, we applied a 5-fold higher concentration of Cas9 mRNA (50 ng/μl) with increasing doses of Prkdc-sgRNA (50, 100, and 250 ng/μl). Again, the birth rates were very high, ranging from 51% to 60%, enough to produce a sufficient number of newborns for the analysis (Table 6). The mutant fraction was 57% (21 mutant founders among 37 newborns) at the maximum dose of Prkdc-sgRNA. These birth rates obtained with RGENs were approximately 2- to 10-fold higher than those with TALENs reported in our previous study (Sung et al., 2013). These results demonstrate that RGENs are potent gene-targeting reagents with minimal toxicity.
  • To test the germ-line transmission of the mutant alleles, we crossed the Foxn1 mutant founder #108, a mosaic with four different alleles (FIG. 5 c, and Table 8) with wild-type mice, and monitored the genotypes of F1 offspring.
  • TABLE 8
    Genotypes of Foxn1 mutant mice
    Founder sgRNA
    NO. (ng/ml) Genotyping Summary Detected alleles
     58* 1 not determined Δ11
    19 100 bi-allelic Δ60/+1
    20 100 bi-allelic Δ67/Δ19
    13 100 bi-allelic Δ18/+455
    32 10 bi-allelic (heterozygote) Δ13/Δ15 + 1
    115  10 bi-allelic (heterozygote) Δ18/Δ5
    111  10 bi-allelic (heterozygote) Δ11/+1
    110  10 bi-allelic (homozygote) Δ8/Δ8
    120  10 bi-allelic (homozygote) +2/+2
    81 100 heterozygote +1/WT
    69 100 homozygote Δ11/Δ11
    55 1 mosaic Δ18/Δ1/+1/+3
    56 1 mosaic Δ127/Δ41 /Δ2/+1
    127  1 mosaic Δ18/+1/WT
    53 1 mosaic Δ11/Δ5/Δ4 + 1/WT
    27 10 mosaic Δ17/Δ5/WT
    29 10 mosaic Δ18/Δ20 + 1/+1
    95 10 mosaic Δ18/Δ14/Δ8/Δ4
    108  10 mosaic +1/Δ17/Δ23/Δ44
    114  10 mosaic Δ17/Δ8/Δ6 + 25
    124  10 mosaic Δ11/Δ15 + 2/+1
    126  10 mosaic Δ17/Δ2 + 3/Δ12 + 6
    12 100 mosaic Δ30/Δ28/Δ17/+1
     5 100 mosaic Δ28/Δ11/Δ2 + 6/+1
    14 100 mosaic Δ17/Δ11/Δ10
    21 100 mosaic Δ127/Δ41/Δ2/Δ6 + 25
    24 100 mosaic Δ17/+1/WT
    64 100 mosaic Δ31/Δ21/+1/WT
    68 100 mosaic Δ17/Δ11/+1/WT
    79 100 mosaic Δ22/Δ5/+2/WT
    61 100 mosaic Δ21 + 4/Δ6/+1/+9
     66** 100 mosaic Δ17/Δ8/Δ11 + 6/+1/WT
     3 100 mosaic Δ11/Δ8/+1
    Underlined alleles were sequenced.
    Alleles in red, detected by sequencing, but not by fPCR.
    *only one clone sequenced.
    **Not determined by fPCR.
  • As expected, all the progenies were heterozygous mutants possessing the wild-type allele and one of the mutant alleles (FIG. 5 d). We also confirmed the germ-line transmission in independent founder mice of Foxn1 (FIG. 8) and Prkdc (FIG. 9). To the best of our knowledge, these results provide the first evidence that RGEN-induced mutant alleles are stably transmitted to F1 progenies in animals.
    • Example 4 RNA-Guided Genome Editing in Plants
  • 4-1. Production of Cas9 Protein
  • The Cas9 coding sequence (4104 bps), derived from Streptococcus pyogenes strain M1 GAS (NC—002737.1), was cloned to pET28-b(+) plasmid. A nuclear targeting sequence (NLS) was included at the protein N terminus to ensure the localization of the protein to the nucleus. pET28-b(+) plasmid containing Cas9 ORF was transformed into BL21(DE3). Cas9 was then induced using 0.2 mM IPTG for 16 hrs at 18° C. and purified using Ni-NTA agarose beads (Qiagen) following the manufacturer's instructions. Purified Cas9 protein was concentrated using Ultracel—100K (Millipore).
  • 4-2. Production of Guide RNA
  • The genomic sequence of the Arabidopsis gene encoding the BRI1 was screened for the presence of a NGG motif, the so called protospacer adjacent motif (PAM), in an exon which is required for Cas9 targeting To disrupt the BRI1 gene in Arabidopsis, we identified two RGEN target sites in an exon that contain the NGG motif. sgRNAs were produced in vitor using template DNA. Each template DNA was generated by extension with two partially overlapped oligonucleotides (Macrogen, Table X1) and Phusion polymerase (Thermo Scientific) using the following conditions—98° C. 30 sec {98° C. 10 sec, 54° C. 20 sec, 72° C. 2 min}×20, 72° C. 5 min.
  • TABLE 9
    [Table 9]
    Oligonucleotides for the production of the
    template DNA for in vitro transcription
    SEQ
    Oligonuc- ID
    leotides Sequence (5′-3′) NO
    BRI1 target 1 GAAATTAATACGACTCACTATAGGTTTGAA 73
    (Forward) AGATGGAAGCGCGGGTTTTAGAGCTAGAA
    ATAGCAAGTTAAAATAAGGCTAGTCCG
    BRI1 target
     2 GAAATTAATACGACTCACTATAGGTGAAAC 74
    (Forward) TAAACTGGTCCACAGTTTTAGAGCTAGAAA
    TAGCAAGTTAAAATAAGGCTAGTCCG
    Universal AAAAAAGCACCGACTCGGTGCCACTTTTTC 75
    (Reverse) AAGTTGATAACGGACTAGCCTTATTTTAAC
    TTGC
  • The extended DNA was purified and used as a template for the in vitro production of the guide RNA's using the MEGAshortscript T7 kit (Life Technologies). Guide RNA were then purified by Phenol/Chloroform extraction and ethanol precipitation. To prepare Cas9/sgRNA complexes, 10 ul of purified Cas9 protein (12 μg/μl) and 4 ul each of two sgRNAs (11 μg/μl) were mixed in 20 μl NEB3 buffer (New England Biolabs) and incubated for 10 min at 37° C.
  • 4-3. Transfection of Cas9/sgRNA Complex to Protoplast
  • The leaves of 4-week-old Arabidopsis seedlings grown aseptically in petri dishes were digested in enzyme solution (1% cellulose R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salt) for 8˜16 hrs at 25° C. with 40 rpm shaking in the dark. Enzyme/protoplast solutions were filtered and centrifuged at 100×g for 3˜5 min. Protoplasts were re-suspended in CPW solution after counting cells under the microscope (×100) using a hemacytometer. Finally, protoplasts were re-suspended at 1×106/ml in MMG solution (4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2). To transfect the protoplasts with Cas9/sgRNA complex, 200 μL (200,000 protoplasts) of the protoplast suspension were gently mixed with 3.3 or 10 uL of Cas9/sgRNA complex [Cas9 protein (6 μg/μL) and two sgRNAs (2.2 μg/μL each)] and 200 ul of 40% polyethylene glycol transfection buffer (40% PEG4000, 200 mM mannitol and 100 mM CaCl2) in 2 ml tubes. After 5-20 min incubation at room temperature, transfection was stopped by adding wash buffer with W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaC12 and 5 mM KCl). Protoplasts were then collected by centrifugation for 5 min at 100×g, washed with 1 ml of W5 solution, centrifuged for another 5 min at 100×g. The density of protoplasts was adjusted to 1×105/ml and they were cultured in modified KM 8p liquid medium with 400 mM glucose.
  • 4-4. Detection of Mutations in Arabidopsis Protoplasts and Plants
  • After 24 hr or 72 hr post-transfection, protoplasts were collected and genomic DNA was isolated. The genomic DNA region spanning the two target sites was PCR-amplified and subjected to the T7E1 assay. As shown in FIG. 11, indels were induced by RGENs at high frequencies that ranged from 50% to 70%. Surprisingly, mutations were induced at 24 hr post-transfection. Apparently, Cas9 protein functions immediately after transfection. PCR products were purified and cloned into T-Blunt PCR Cloning Kit (Solgent). Plasmids were purified and subjected to Sanger sequencing with M13F primer. One mutant sequence had a 7-bp deletion at one site (FIG. 12). The other three mutant sequences had deletions of ˜220-bp DNA segments between the two RGEN site.
    • Example 5 Cas9 Protein Transduction Using a Cell-Penetrating Peptide or Protein Transduction Domain
  • 5-1. Construction of his-Cas9-Encoding Plasmid
  • Cas9 with a cysteine at the C-terminal was prepared by PCR amplification using the previously described Cas9 plasmid {Cho, 2013 #166} as the template and cloned into pET28-(a) vector (Novagen, Merk Millipore, Germany) containing His-tag at the N-terminus.
  • 5-2. Cell Culture
  • 293T (Human embryonic kidney cell line), and HeLa (human ovarian cancer cell line) were grown in DMEM (GIBCO-BRL Rockville) supplemented with 10% FBS and 1% penicillin and streptomycin.
  • 5-3. Expression and Purification of Cas9 Protein
  • To express the Cas9 protein, E. coli BL21 cells were transformed with thepET28-(a) vector encoding Cas9 and plated onto Luria-Bertani (LB) agar medium containing 50 μg/mL kanamycin (Amresco, Solon, Ohio). Next day, a single colony was picked and cultured in LB broth containing 50 μg/mL kanamycin at 37° C. overnight. Following day, this starter culture at 0.1 OD600 was inoculated into Luria broth containing 50 μg/mL kanamycin and incubated for 2 hrs at 37° C. until OD600 reached to 0.6-0.8. To induce Cas9 protein expression, the cells were cultured at 30° C. overnight after addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (Promega, Madison, Wis.) to the final concentration of 0.5 mM.
  • The cells were collected by centrifugation at 4000 rpm for 15-20 mins, resuspendedin a lysis buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, 1× protease inhibitor cocktail, 1 mg/ml lysozyme), and lysed by sonication (40% duty, 10 sec pulse, 30 sec rest, for 10 mins on ice). The soluble fraction was separated as the supernatant after centrifugation at 15,000 rpm for 20 mins at 4° C. Cas9 protein was purified at 4° C. using a column containing Ni-NTA agarose resin (QIAGEN) and AKTA prime instrument (AKTA prime, GE Healthcare, UK). During this chromatography step, soluble protein fractions were loaded onto Ni-NTA agarose resin column (GE Healthcare, UK) at the flow rate of 1 mL/min. The column was washed with a washing buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, lx protease inhibitor cocktail) and the bound protein was eluted at the flow rate of 0.5 ml/min with an elution buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 250 mM imidazole, 1× protease inhibitor cocktail). The pooled eluted fraction was concentrated and dialyzed against storage buffer (50 mM Tris-HCl, pH8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% Glycerol). Protein concentration was quantitated by Bradford assay (Biorad, Hercules, Calif.) and purity was analyzed by SDS-PAGE using bovine serum albumin as the control.
  • 5-4. Conjugation of Cas9 to 9R4L
  • 1 mg Cas9 protein diluted in PBS at the concentration of 1 mg/mL and 50 μg of maleimide-9R4L peptide in 25 μL DW (Peptron, Korea) were gently mixed using a rotor at room temperature for 2 hrs and at 4° C. overnight. To remove unconjugated maleimide-9R4L, the samples were dialyzed using 50kDa molecular weight cutoff membrane against of DPBS (pH 7.4) at 4° C. for 24 hrs. Cas9-9R4L protein was collected from the dialysis membrane and the protein amount was determined using Bradford assay.
  • 5-5. Preparation of sgRNA-9R4L
  • sgRNA (1 μg) was gently added to various amounts of C9R4LC peptide (ranging from 1 to 40 weight ratio) in 100 μl of DPBS (pH 7.4). This mixture was incubated at room temperature for 30 mins and diluted to 10 folds using RNAse-free deionized water. The hydrodynamic diameter and z-potential of the formed nanoparticles were measured using dynamic light scattering (Zetasizer-nano analyzer ZS; Malvern instruments, Worcestershire, UK).
  • 5-6. Cas9 Protein and sgRNA Treatments
  • Cas9-9R4L and sgRNA-C9R4LC were treated to the cells as follows: 1 μg of sgRNA and 15 μg of C9R4LC peptide were added to 250 mL of OPTIMEM medium and incubated at room temperature for 30 mins. At 24 hrs after seeding, cells were washed with OPTIMEM medium and treated with sgRNA-C9R4LC complex for 4 hrs at 37° C. Cells were washed again with OPTIMEM medium and treated with Cas9-9R4L for 2 hrs at 37° C. After treatment, culture media was replaced with serum-containing complete medium and incubated at 37° C. for 24 hrs before the next treatment. Same procedure was followed for multiple treatments of Cas9 and sgRNA for three consecutive days.
  • 5-7. Cas9-9R4L and sgRNA-9R4L can Edit Endogenous Genes in Cultured Mammalian Cells without the Use of Additional Delivery Tools
  • To determine whether Cas9-9R4L and sgRNA-9R4L can edit endogenous genes in cultured mammalian cells without the use of additional delivery tools, we treated 293 cells with Cas9-9R4L and sgRNA-9R4L targeting the CCR5 gene and analyzed the genomic DNA. T7E1 assay showed that 9% of CCR5 gene was disrupted in cells treated with both Cas9-9R4L and sgRNA-9R4L and that the CCR5 gene disruption was not observed in control cells including those untreated, treated with either Cas9-9R or sgRNA-9R4L, or treated with both unmodified Cas-9 and sgRNA (FIG. 13), suggesting that the treatment with Cas9-9R4L protein and sgRNA conjugated with 9R4L, but not unmodified Cas9 and sgRNA, can lead to efficient genome editing in mammalian cells.
    • Example 6 Control of Off-Target Mutation According to Guide RNA Structure
  • Recently, three groups reported that RGENs had off-target effects in human cells. To our surprise, RGENs induced mutations efficiently at off-target sites that differ by 3 to 5 nucleotides from on-target sites. We noticed, however, that there were several differences between our RGENs and those used by others. First, we used dualRNA, which is crRNA plus tracrRNA, rather than single-guide RNA (sgRNA) that is composed of essential portions of crRNA and tracrRNA. Second, we transfected K562 cells (but not HeLa cells) with synthetic crRNA rather than plasmids encoding crRNA. HeLa cells were transfected with crRNA-encoding plasmids. Other groups used sgRNA-encoding plasmids. Third, our guide RNA had two additional guanine nucleotides at the 5′ end, which are required for efficient transcription by T7 polymerase in vitro. No such additional nucleotides were included in the sgRNA used by others. Thus, the RNA sequence of our guide RNA can be shown as 5′-GGX20, whereas 5′-GX19, in which X20 or GX19 corresponds to the 20-bp target sequence, represents the sequence used by others. The first guanine nucleotide is required for transcription by RNA polymerase in cells. To test whether off-target RGEN effects can be attributed to these differences, we chose four RGENs that induced off-target mutations in human cells at high frequencies (13). First, we compared our method of using in vitro transcribed dualRNA with the method of transfecting sgRNA-encoding plasmids in K562 cells and measured mutation frequencies at the on-target and off-target sites via the T7E1 assay. Three RGENs showed comparable mutation frequencies at on-target and off-target sites regardless of the composition of guide RNA. Interestingly, one RGEN (VEFGA site 1) did not induce indels at one validated off-target site, which differs by three nucleotides from the on-target site (termed OT1-11, FIG. 14), when synthetic dualRNA was used. But the synthetic dualRNA did not discriminate the other validated off-target site (OT1-3), which differs by two nucleotides from the on-target site.
  • Next, we tested whether the addition of two guanine nucleotides at the 5′ end of sgRNA could make RGENs more specific by comparing 5′-GGX20 (or 5′-GGGX19) sgRNA with 5′-GX19 sgRNA. Four GX19 sgRNAs complexed with Cas9 induced indels equally efficiently at on-target and off-target sites, tolerating up to four nucleotide mismatches. In sharp contrast, GGX20 sgRNAs discriminated off-target sites effectively. In fact, the T7E1 assay barely detected RGEN-induced indels at six out of the seven validated off-target sites when we used the four GGX20 sgRNAs (FIG. 15). We noticed, however, that two GGX20 sgRNAs (VEGFA sites 1 and 3) were less active at on-target sites than were the corresponding GX19 sgRNAs. These results show that the extra nucleotides at the 5′ end can affect mutation frequencies at on-target and off-target sites, perhaps by altering guide RNA stability, concentration, or secondary structure.
  • These results suggest that three factors—the use of synthetic guide RNA rather than guide RNA-encoding plasmids, dualRNA rather than sgRNA, and GGX20 sgRNA rather than GX19 sgRNA-have cumulative effects on the discrimination of off-target sites.
    • Example 7 Paired Cas9 Nickases
  • In principle, single-strand breaks (SSBs) cannot be repaired by error-prone NHEJ but still trigger high fidelity homology-directed repair (HDR) or base excision repair. But nickase-induced targeted mutagenesis via HDR is much less efficient than is nuclease-induced mutagenesis. We reasoned that paired Cas9 nickases would produce composite DSBs, which trigger DNA repair via NHEJ or HDR, leading to efficient mutagenesis (FIG. 16A). Furthermore, paired nickases would double the specificity of Cas9-based genome editing.
  • We first tested several Cas9 nucleases and nickases designed to target sites in the AAVS1 locus (FIG. 16B) in vitro via fluorescent capillary electrophoresis. Unlike Cas9 nucleases that cleaved both strands of DNA substrates, Cas9 nickases composed of guide RNA and a mutant form of Cas9 in which a catalytic aspartate residue is changed to an alanine (D10A Cas9) cleaved only one strand, producing site-specific nicks (FIG. 16C,D). Interestingly, however, some nickases (AS1, AS2, AS3, and S6 in FIG. 17A) induced indels at target sites in human cells, suggesting that nicks can be converted to DSBs, albeit inefficiently, in vivo. Paired Cas9 nickases producing two adjacent nicks on opposite DNA strands yielded indels at frequencies that ranged from 14% to 91%, comparable to the effects of paired nucleases (FIG. 17A). The repair of two nicks that would produce 5′ overhangs led to the formation of indels much more frequently than those producing 3′ overhangs at three genomic loci (FIG. 17A and FIG. 18). In addition, paired nickases enabled targeted genome editing via homology-directed repair more efficiently than did single nickases (FIG. 19).
  • We next measured mutation frequencies of paired nickases and nucleases at off-target sites using deep sequencing. Cas9 nucleases complexed with three sgRNAs induced off-target mutations at six sites that differ by one or two nucleotides from their corresponding on-target sites with frequencies that ranged from 0.5% to 10% (FIG. 17B). In contrast, paired Cas9 nickases did not produce indels above the detection limit of 0.1% at any of the six off-target sites. The S2 Off-1 site that differs by a single nucleotide at the first position in the PAM (i.e., N in NGG) from its on-target site can be considered as another on-target site. As expected, the Cas9 nuclease complexed with the S2 sgRNA was equally efficient at this site and the on-target site. In sharp contrast, D10A Cas9 complexed with the S2 and AS2 sgRNAs discriminated this site from the on-target site by a factor of 270 fold. This paired nickase also discriminated the AS2 off-target sites (Off-1 and Off-9 in FIG. 17B) from the on-target site by factors of 160 fold and 990 fold, respectively.
    • Example 8 Chromosomal DNA Splicing Induced by Paired Cas9 Nickases
  • Two concurrent DSBs produced by engineered nucleases such as ZFNs and TALENs can promote large deletions of the intervening chromosomal segments has reproted. We tested whether two SSBs induced by paired Cas9 nickases can also produce deletions in human cells. We used PCR to detect deletion events and found that seven paired nickases induced deletions of up to 1.1-kbp chromosomal segments as efficiently as paired Cas9 nucleases did (FIG. 20A,B). DNA sequences of the PCR products confirmed the deletion events (FIG. 20C). Interestingly, the sgRNA-matching sequence remained intact in two out of seven deletion-specific PCR amplicons (underlined in FIG. 20C). In contrast, Cas9 nuclease pairs did not produce sequences that contained intact target sites. This finding suggests that two distant nicks were not converted to two separate DSBs to promote deletions of the intervening chromosomal segment. In addition, it is unlikely that two nicks separated by more than a 100 bp can produce a composite DSB with large overhangs under physiological conditions because the melting temperature is very high.
  • We propose that two distant nicks are repaired by strand displacement in a head-to-head direction, resulting in the formation of a DSB in the middle, whose repair via NHEJ causes small deletions (FIG. 20D). Because the two target sites remain intact during this process, nickases can induce SSBs again, triggering the cycle repeatedly until the target sites are deleted. This mechanism explains why two offset nicks producing 5′ overhangs but not those producing 3′ overhangs induced indels efficiently at three loci.
  • We then investigated whether Cas9 nucleases and nickases can induce unwanted chromosomal translocations that result from NHEJ repair of on-target and off-target DNA cleavages (FIG. 21A). We were able to detect translocations induced by Cas9 nucleases using PCR (FIG. 21B,C). No such PCR products were amplified using genomic DNA isolated from cells transfected with the plasmids encoding the AS2+S3 Cas9 nickase pair. This result is in line with the fact that both AS2 and S3 nickases, unlike their corresponding nucleases, did not produce indels at off-target sites (FIG. 17B).
  • These results suggest that paired Cas9 nickases allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells. Importantly, paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations. Furthermore, unlike nucleases, paired nickases did not promote unwanted translocations associated with off-target DNA cleavages. In principle, paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy. One caveat to this approach is that two highly active sgRNAs are needed to make an efficient nickase pair, limiting targetable sites. As shown in this and other studies, not all sgRNAs are equally active. When single clones rather than populations of cells are used for further studies or applications, the choice of guide RNAs that represent unique sequences in the genome and the use of optimized guide RNAs would suffice to avoid off-target mutations associated with Cas9 nucleases. We propose that both Cas9 nucleases and paired nickases are powerful options that will facilitate precision genome editing in cells and organisms.
    • Example 9 Genotyping with CRISPR/Cas-Derived RNA-Guided Endonucleases
  • Next, We reasoned that RGENs can be used in Restriction fragment length polymorphism (RFLP) analysis, replacing conventional restriction enzymes. Engineered nucleases including RGENs induce indels at target sites, when the DSBs caused by the nucleases are repaired by the error-prone non-homologous end-joining (NHEJ) system. RGENs that are designed to recognize the target sequences cannot cleave mutant sequences with indels but will cleave wildtype target sequences efficiently.
  • 9-1. RGEN Components
  • crRNA and tracrRNA were prepared by in vitro transcription using MEGAshortcript T7 kit (Ambion) according to the manufacturer's instruction. Transcribed RNAs were resolved on a 8% denaturing urea-PAGE gel. The gel slice containing RNA was cut out and transferred to elution buffer. RNA was recovered in nuclease-free water followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry. Templates for crRNA were prepared by annealing an oligonucleotide whose sequence is shown as 5′-GAAATTAATACGACTCACTATAGGX20GTTTTAGAGCTATGCTGTTTTG-3′ (SEQ ID NO: 76), in which X20 is the target sequence, and its complementary oligonucleotide. The template for tracrRNA was synthesized by extension of forward and reverse oligonucleotides (5′-GAAATTAATACGACTCACTATAGGAACCATTCAAAACAGCATAGCAAG TTAAAATAAGGCTAGTCCG-3′ (SEQ ID NO: 77) and 5′-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAG CCTTATTTTAACTTGCTATG-3′(SEQ ID NO: 78)) using Phusion polymerase (New England Biolabs).
  • 9-2. Recombinant Cas9 Protein Purification
  • The Cas9 DNA construct used in our previous Example, which encodes Cas9 fused to the His6-tag at the C terminus, was inserted in the pET-28a expression vector. The recombinant Cas9 protein was expressed in E. coli strain BL21(DE3) cultured in LB medium at 25° C. for 4 hour after induction with 1 mM IPTG. Cells were harvested and resuspended in buffer containing 20 mM Tris PH 8.0, 500 mM NaCl, 5 mM immidazole, and 1 mM PMSF. Cells were frozen in liquid nitrogen, thawed at 4° C., and sonicated. After centrifugation, the Cas9 protein in the lysate was bound to Ni-NTA agarose resin (Qiagen), washed with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 20 mM immidazole, and eluted with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 250 mM immidazole. Purified Cas9 protein was dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol and analyzed by SDS-PAGE.
  • 9-3. T7 Endonuclease I Assay
  • The T7E1 assay was performed as following. In brief, PCR products amplified using genomic DNA were denatured at 95° C., reannealed at 16° C., and incubated with 5 units of T7 Endonuclease I (New England BioLabs) for 20 min at 37° C. The reaction products were resolved using 2 to 2.5% agarose gel electrophoresis.
  • 9-4. RGEN-RFLP Assay
  • PCR products (100-150 ng) were incubated for 60 min at 37° C. with optimized concentrations (Table 10) of Cas9 protein, tracrRNA, crRNA in 10 μl NEB buffer 3 (1×). After the cleavage reaction, RNase A (4 μg) was added, and the reaction mixture was incubated for 30 min at 37° C. to remove RNA. Reactions were stopped with 6× stop solution buffer containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1-2.5% agarose gel electrophoresis and visualized with EtBr staining.
  • TABLE 10
    Concentration of RGEN components in RFLP assays
    tracrRNA
    Target Name Cas9 (ng/μl) crRNA (ng/μl) (ng/μl)
    C4BPB 100 25 60
    PIBF-NGG-RGEN 100 25 60
    HLA-B 1.2 0.3 0.7
    CCR5-ZFN 100 25 60
    CTNNB1 Wild type specific 30 10 20
    CTNNB1 mutant specific 30 10 20
    CCR5 WT-specific 100 25 60
    CCR5 Δ32-specific 10 2.5 6
    KRAS WT specific (wt) 30 10 20
    KRAS mutant specific (m8) 30 10 20
    KRAS WT specific (m6) 30 10 20
    KRAS mutant specific (m6, 8) 30 10 20
    PIK3CA WT specific (wt) 100 25 60
    PIK3CA mutant specific (m4) 30 10 20
    PIK3CA WT specific (m7) 100 25 60
    PIK3CA mutant 30 10 20
    specific (m4, 7)
    BRAF WT-specific 30 10 20
    BRAF mutant-specific 100 25 60
    NRAS WT-specific 100 25 60
    NRAS mutant-specific 30 10 20
    IDH WT-specific 30 10 20
    IDH mutant-specific 30 10 20
    PIBF-NAG-RGEN 30 10 60
  • TABLE 11
    [Table 11]
    Primers
    SEQ
    Gene Direc- ID
    (site) tion Sequence(5′ to 3′) NO
    CCR5 F1 CTCCATGGTGCTATAGAGCA 79
    (RGEN) F2 GAGCCAAGCTCTCCATCTAGT 80
    R GCCCTGTCAAGAGTTGACAC 81
    CCR5 F GCACAGGGTGGAACAAGATGGA 82
    (ZFN) R GCCAGGTACCTATCGATTGTCAGG 83
    CCR5 F GAGCCAAGCTCTCCATCTAGT 84
    (del32) R ACTCTGACTG GGTCACCAGC 85
    C4BPB F1 TATTTGGCTGGTTGAAAGGG 86
    R1 AAAGTCATGAAATAAACACACCCA 87
    F2 CTGCATTGATATGGTAGTACCATG 88
    R2 GCTGTTCATTGCAATGGAATG 89
    CTNNB1 F ATGGAGTTGGACATGGCCATGG 90
    R ACTCACTATCCACAGTTCAGCATTTAC 91
    C
    KRAS F TGGAGATAGCTGTCAGCAACTTT 92
    R CAACAA AGCAAAG- 93
    GTAAAGTTGGTAATAG
    PIK3CA F GGTTTCAGGAGATGTGTTACAAGGC 94
    R GATTGTGCAATTCCTATGCAATCGGTC 95
    NRAS F CACTGGGTACTTAATCTGTAGCCTC 96
    R GGTTCCAAGTCATTCCCAGTAGC 97
    IDH1 F CATCACTGCAGTTGTAGGTTATAACTATC 98
    C
    R TTGAAAACCACAGATCTGGTTGAACC
    99
    BRAF F GGAGTGCCAAGAGAATATCTGG 100
    R CTGAAACTGGTTTCAAAATATTCGTTTTA 101
    AGG
    PIBF F GCTCTGTATGCCCTGTAGTAGG 102
    R TTTGCATCTGACCTTACCTTTG
    103
  • 9-5. Plasmid Cleavage Assay
  • Restriction enzyme-treated linearized plasmid (100 ng) was incubated for 60 min at 37° C. with Cas9 protein (0.1 μg), tracrRNA (60 ng), and crRNA (25 ng) in 10 μl NEB 3 buffer (1×). Reactions were stopped with 6× stop solution containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1% agarose gel electrophoresis and visualized with EtBr staining.
  • 9-6. Strategy of RFLP
  • New RGENs with desired DNA specificities can be readily created by replacing crRNA; no de novo purification of custom proteins is required once recombinant Cas9 protein is available. Engineered nucleases, including RGENs, induce small insertions or deletions (indels) at target sites when the DSBs caused by the nucleases are repaired by error-prone non-homologous end-joining (NHEJ). RGENs that are designed to recognize the target sequences cleave wild-type sequences efficiently but cannot cleave mutant sequences with indels (FIG. 22).
  • We first tested whether RGENs can differentially cleave plasmids that contain wild-type or modified C4BPB target sequences that harbor 1- to 3-base indels at the cleavage site. None of the six plasmids with these indels were cleaved by a C4BPB-specific RGEN5 composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein (FIG. 23). In contrast, the plasmid with the intact target sequence was cleaved efficiently by this RGEN.
  • 9-7. Detection of Mutations Induced by the Same RGENs Using RGEN-Mediated RFLP
  • Next, to test the feasibility of RGEN-mediated RFLP for detection of mutations induced by the same RGENs, we utilized gene-modified K562 human cancer cell clones established using an RGEN targeting C4BPB gene (Table 12).
  • TABLE 12
    [Table 12]
    Target sequence of RGENs used in this study
    Gene Target sequence SEQ ID NO
    human AATGACCACTACATCCTCAA 104
    C4BPB GGG
    mouse Pibf1 AGATGATGTCTCATCATCAG 105
    AGG
  • C4BPB mutant clones used in this study have various mutations ranging from 94 bp deletion to 67 bp insertion (FIG. 24A). Importantly, all mutations occurred in mutant clones resulted in the loss of RGEN target site. Among 6 C4BPB clones analyzed, 4 clones have both wildtype and mutant alleles (+/−) and 2 clones have only mutant alleles (−/−).
  • The PCR products spanning the RGEN target site amplified from wildtype K562 genomic DNA were digested completely by the RGEN composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein expressed in and purified from E. coli (FIG. 24B/Lane 1). When the C4BPB mutant clones were subjected to RFLP analysis using the RGEN, PCR amplicons of +/− clones that contained both wild-type and mutant alleles were partially digested, and those of −/− cloned that did not contain the wildtype allele were not digested at all, yielding no cleavage products corresponding to the wildtype sequence (FIG. 24B). Even a single-base insertion at the target site blocked the digestion (#12 and #28 clones) of amplified mutant alleles by the C4BPB RGEN, showing the high specificity of RGEN-mediated RFLP. We subjected the PCR amplicons to the mismatch-sensitive T7E1 assay in parallel (FIG. 24B). Notably, the T7E1 assay was not able to distinguish −/− clones from +/− clones. To make it matters worse, the T7E1 assay cannot distinguish homozygous mutant clones that contain the same mutant sequence from wildtype clones, because annealing of the same mutant sequence will form a homoduplex. Thus, RGEN-mediated RFLP has a critical advantage over the conventional mismatch-sensitive nuclease assay in the analysis of mutant clones induced by engineered nucleases including ZFNs, TALENs and RGENs.
  • 9-8. Quantitative Assay for RGEN-RFLP Analysis
  • We also investigated whether RGEN-RFLP analysis is a quantitative method. Genomic DNA samples isolated from the C4BPB null clone and the wild-type cells were mixed at various ratios and used for PCR amplifications. The PCR products were subjected to RGEN genotyping and the T7E1 assay in parallel (FIG. 25 b). As expected, DNA cleavage by the RGEN was proportional to the wild type to mutant ratio. In contrast, results of the T7E1 assay correlated poorly with mutation frequencies inferred from the ratios and were inaccurate, especially at high mutant %, a situation in which complementary mutant sequences can hybridize with each other to form homoduplexes.
  • 9-9. Analysis of Mutant Mouse Founders Using a RGEN-Mediated RFLP Genotyping
  • We also applied RGEN-mediated RFLP genotyping (RGEN genotyping in short) to the analysis of mutant mouse founders that had been established by injection of TALENs into mouse one-cell embryos (FIG. 26A). We designed and used an RGEN that recognized the TALEN target site in the Pibf1 gene (Table 10). Genomic DNA was isolated from a wildtype mouse and mutant mice and subjected to RGEN genotyping after PCR amplification. RGEN genotyping successfully detected various mutations, which ranged from one to 27-bp deletions (FIG. 26B). Unlike the T7E1 assay, RGEN genotyping enabled differential detection of +/− and −/− founder.
  • 9-10. Detection of Mutations Induced in Human Cells by a CCR5-Specific ZFN Using RGENs
  • In addition, we used RGENs to detect mutations induced in human cells by a CCR5-specific ZFN, representing yet another class of engineered nucleases (FIG. 27). These results show that RGENs can detect mutations induced by nucleases other than RGENs themselves. In fact, we expect that RGENs can be designed to detect mutations induced by most, if not all, engineered nucleases. The only limitation in the design of an RGEN genotyping assay is the requirement for the GG or AG (CC or CT on the complementary strand) dinucleotide in the PAM sequence recognized by the Cas9 protein, which occurs once per 4 bp on average. Indels induced anywhere within the seed region of several bases in crRNA and the PAM nucleotides are expected to disrupt RGEN-catalyzed DNA cleavage. Indeed, we identified at least one RGEN site in most (98%) of the ZFN and TALEN sites.
  • 9-11. Detection of Polymorphisms or Variations Using RGEN
  • Next, we designed and tested a new RGEN that targets a highly polymorphic locus, HLA-B, that encodes Human Leukocyte Antigen B (a.k.a. MHC class I protein) (FIG. 28). HeLa cells were transfected with RGEN plasmids, and the genomic DNA was subjected to T7E1 and RGEN-RFLP analyses in parallel. T7E1 produced false positive bands that resulted from sequence polymorphisms near the target site (FIG. 25 c). As expected, however, the same RGEN used for gene disruption cleaved PCR products from wild-type cells completely but those from RGEN-transfected cells partially, indicating the presence of RGEN-induced indels at the target site. This result shows that RGEN-RFLP analysis has a clear advantage over the T7E1 assay, especially when it is not known whether target genes have polymorphisms or variations in cells of interest.
  • 9-12. Detection of Recurrent Mutations Found in Cancer and Naturally-Occurring Polymorphisms Through RGEN-RFLP Analysis
  • RGEN-RFLP analysis has applications beyond genotyping of engineered nuclease-induced mutations. We sought to use RGEN genotyping to detect recurrent mutations found in cancer and naturally-occurring polymorphisms. We chose the human colorectal cancer cell line, HCT116, which carries a gain-of-function 3-bp deletion in the oncogenic CTNNB1 gene encoding beta-catenin. PCR products amplified from HCT116 genomic DNA were cleaved partially by both wild-type-specific and mutant-specific RGENs, in line with the heterozygous genotype in HCT116 cells (FIG. 29 a). In sharp contrast, PCR products amplified from DNA from HeLa cells harboring only wild-type alleles were digested completely by the wild-type-specific RGEN and were not cleaved at all by the mutation-specific RGEN.
  • We also noted that HEK293 cells harbor the 32-bp deletion (del32) in the CCR5 gene, which encodes an essential co-receptor of HIV infection: Homozygous del32 CCR5 carriers are immune to HIV infection. We designed one RGEN specific to the del32 allele and the other to the wild-type allele. As expected, the wild-type-specific RGEN cleaved the PCR products obtained from K562, SKBR3, or HeLa cells (used as wild-type controls) completely but those from HEK293 cells partially (FIG. 30 a), confirming the presence of the uncleavable del32 allele in HEK293 cells. Unexpectedly, however, the del32-specific RGEN cleaved the PCR products from wild-type cells as efficiently as those from HEK293 cells. Interestingly, this RGEN had an off-target site with a single-base mismatch immediately downstream of the on-target site (FIG. 30). These results suggest that RGENs can be used to detect naturally-occurring indels but cannot distinguish sequences with single nucleotide polymorphisms or point mutations due to their off-target effects.
  • To genotype oncogenic single-nucleotide variations using RGENs, we attenuated RGEN activity by employing a single-base mismatched guide RNA instead of a perfectly-matched RNA. RGENs that contained the perfectly-matched guide RNA specific to the wild-type sequence or mutant sequence cleaved both sequences (FIGS. 31 a and 32 a). In contrast, RGENs that contained a single-base mismatched guide RNA distinguished the two sequences, enabling genotyping of three recurrent oncogenic point mutations in the KRAS, PIK3CA, and IDH1 genes in human cancer cell lines (FIG. 29 b and FIGS. 33 a, b). In addition, we were able to detect point mutations in the BRAF and NRAS genes using RGENs that recognize the NAG PAM sequence (FIGS. 33 c, d). We believe that we can use RGEN-RFLP to genotype almost any, if not all, mutations or polymorphisms in the human and other genomes.
  • The above data proposes RGENs as providing a platform to use simple and robust RFLP analysis for various sequence variations. With high flexibility in reprogramming target sequence, RGENs can be used to detect various genetic variations (single nucleotide variations, small insertion/deletions, structural variations) such as diseaserelated recurring mutations, genotypes related to drug-response by a patient and also mutations induced by engineered nucleases in cells. Here, we used RGEN genotyping to detect mutations induced by engineered nucleases in cells and animals. In principle, one could also use RGENs that will specifically detect and cleave naturally-occurring variations and mutations.
  • Based on the above description, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the technical idea or essential features of the invention as defined in the following claims. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all of the modifications or modified form derived from the meaning and scope of the following claims or its equivalent concepts.
    • REFERENCES
    • 1. M. Jinek et al., Science 337, 816 (Aug. 17, 2012).
    • 2. H. Kim, E. Um, S. R. Cho, C. Jung, J. S. Kim, Nat Methods 8, 941 (November, 2011).
    • 3. H. J. Kim, H. J. Lee, H. Kim, S. W. Cho, J. S. Kim, Genome Res 19, 1279 (July, 2009).
    • 4. E. E. Perez et al., Nat Biotechnol 26, 808 (July, 2008).
    • 5. J. C. Miller et al., Nat Biotechnol 29, 143 (February, 2011).
    • 6. C. Mussolino et al., Nucleic Acids Res 39, 9283 (November, 2011).
    • 7. J. Cohen, Science 332, 784 (May 13, 2011).
    • 8. V. Pattanayak, C. L. Ramirez, J. K. Joung, D. R. Liu, Nat Methods 8, 765 (September, 2011).
    • 9. R. Gabriel et al., Nat Biotechnol 29, 816 (September, 2011).
    • 10. E. Kim et al., Genome Res, (Apr. 20, 2012).
    • 11. H. J. Lee, J. Kweon, E. Kim, S. Kim, J. S. Kim, Genome Res 22, 539 (March, 2012).
    • 12. H. J. Lee, E. Kim, J. S. Kim, Genome Res 20, 81 (January, 2010).
    • 13. Fu Y, Foden J A, Khayter C, Maeder M L, Reyon D, Joung J K, Sander J D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotech advance online publication (2013)

Claims (21)

1. A composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
2. The composition of claim 1, wherein the target DNA is an endogenous target DNA.
3. The composition of claim 1, wherein the guide RNA is a dualRNA comprising a crRNA and a tracrRNA.
4. The composition of claim 1, wherein the guide RNA is a single-chain guide RNA (sgRNA).
5. The composition of claim 4, wherein the single-chain guide RNA comprises portion of a crRNA and a tracrRNA.
6. The composition of claim 1, wherein the guide RNA further comprises one or more additional nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
7. The composition of claim 1, wherein the guide RNA further comprises 2 additional guanine nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
8. The composition of claim 1, which induces targeted mutagenesis in eukaryotic cells or organisms.
9. The composition of claim 1, for use in the genotyping of a genome in the eukaryotic cells or organisms in vitro.
10. The composition of claim 1, wherein the guide RNA and the Cas protein function as a pair, and wherein the pair comprises two guide RNAs which induce two nicks on different strands.
11. The composition of claim 1, wherein the guide RNA is in the form of an isolated RNA, or is encoded in a vector, wherein the vector is a viral vector, plasmid vector, or agrobacterium vector.
12. The composition of claim 1, comprising a guide RNA specific for the target DNA and Cas9 protein.
13. The composition of claim 1, for cleaving the target DNA in eukaryotic cells or organisms ex vivo or in vivo.
14. The composition of claim 1, wherein the Cas protein-encoding nucleic acid or the Cas protein is derived from the genus Streptococcus.
15. The composition of claim 14, wherein the genus Streptococcus is Streptococcus pyogenes.
16. The composition of claim 15, wherein the Cas protein recognizes the NGG trinucleotide.
17. The composition of claim 1, wherein the Cas protein is Cas9 protein or variant thereof.
18. The composition of claim 1, wherein the Cas protein is linked to a protein transduction domain.
19. The composition of claim 17, wherein the variant of Cas9 protein is a mutant form of Cas9 in which the catalytic aspartate residue is changed to any other amino acid.
20. The composition of claim 19, wherein the amino acid is an alanine.
21-57. (canceled)
US14/438,098 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof Abandoned US20150284727A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/438,098 US20150284727A1 (en) 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201261717324P 2012-10-23 2012-10-23
US201361803599P 2013-03-20 2013-03-20
US201361837481P 2013-06-20 2013-06-20
US14/438,098 US20150284727A1 (en) 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
PCT/KR2013/009488 WO2014065596A1 (en) 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

Publications (1)

Publication Number Publication Date
US20150284727A1 true US20150284727A1 (en) 2015-10-08

Family

ID=50544909

Family Applications (9)

Application Number Title Priority Date Filing Date
US14/438,098 Abandoned US20150284727A1 (en) 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US14/685,568 Active US10851380B2 (en) 2012-10-23 2015-04-13 Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US14/685,510 Pending US20150344912A1 (en) 2012-10-23 2015-04-13 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,338 Pending US20210047647A1 (en) 2012-10-23 2020-08-27 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,355 Pending US20210047648A1 (en) 2012-10-23 2020-08-27 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/314,050 Pending US20230374525A1 (en) 2012-10-23 2023-05-08 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/313,946 Pending US20230374524A1 (en) 2012-10-23 2023-05-08 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/467,967 Pending US20240026367A1 (en) 2012-10-23 2023-09-15 Compositions Comprising Cas9/guide RNA Complexes for Inducing Targeted Disruption of Endogenous Genes in Eukaryotic Cells
US18/467,952 Pending US20240052356A1 (en) 2012-10-23 2023-09-15 Cas9/RNA Complexes for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells

Family Applications After (8)

Application Number Title Priority Date Filing Date
US14/685,568 Active US10851380B2 (en) 2012-10-23 2015-04-13 Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US14/685,510 Pending US20150344912A1 (en) 2012-10-23 2015-04-13 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,338 Pending US20210047647A1 (en) 2012-10-23 2020-08-27 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,355 Pending US20210047648A1 (en) 2012-10-23 2020-08-27 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/314,050 Pending US20230374525A1 (en) 2012-10-23 2023-05-08 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/313,946 Pending US20230374524A1 (en) 2012-10-23 2023-05-08 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/467,967 Pending US20240026367A1 (en) 2012-10-23 2023-09-15 Compositions Comprising Cas9/guide RNA Complexes for Inducing Targeted Disruption of Endogenous Genes in Eukaryotic Cells
US18/467,952 Pending US20240052356A1 (en) 2012-10-23 2023-09-15 Cas9/RNA Complexes for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells

Country Status (16)

Country Link
US (9) US20150284727A1 (en)
EP (6) EP2912175B1 (en)
JP (7) JP6517143B2 (en)
KR (5) KR101656236B1 (en)
CN (8) CN110592089B (en)
AU (5) AU2013335451B2 (en)
BR (1) BR112015008708A2 (en)
CA (2) CA2888190A1 (en)
DE (2) DE202013012597U1 (en)
DK (1) DK2912175T3 (en)
ES (3) ES2886448T3 (en)
HK (4) HK1212732A1 (en)
PL (1) PL2912175T3 (en)
PT (1) PT2912175T (en)
SG (3) SG10201809564YA (en)
WO (1) WO2014065596A1 (en)

Cited By (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150050699A1 (en) * 2012-03-20 2015-02-19 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US20150376587A1 (en) * 2014-06-25 2015-12-31 Caribou Biosciences, Inc. RNA Modification to Engineer Cas9 Activity
US20160002670A1 (en) * 2012-12-17 2016-01-07 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
US20160168592A1 (en) * 2013-07-09 2016-06-16 President And Fellows Of Harvard College Multiplex RNA-Guided Genome Engineering
US20160186208A1 (en) * 2013-04-16 2016-06-30 Whitehead Institute For Biomedical Research Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
US9677090B2 (en) 2015-10-23 2017-06-13 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US9771600B2 (en) 2015-12-04 2017-09-26 Caribou Biosciences, Inc. Engineered nucleic acid-targeting nucleic acids
US9856497B2 (en) 2016-01-11 2018-01-02 The Board Of Trustee Of The Leland Stanford Junior University Chimeric proteins and methods of regulating gene expression
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10000772B2 (en) 2012-05-25 2018-06-19 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10166255B2 (en) 2015-07-31 2019-01-01 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US10190137B2 (en) 2013-11-07 2019-01-29 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
WO2019051419A1 (en) * 2017-09-08 2019-03-14 University Of North Texas Health Science Center Engineered cas9 variants
EP3461894A1 (en) 2015-08-07 2019-04-03 Caribou Biosciences, Inc. Engineered crispr-cas9 compositions and methods of use
US10286084B2 (en) 2014-02-18 2019-05-14 Duke University Compositions for the inactivation of virus replication and methods of making and using the same
US10336807B2 (en) 2016-01-11 2019-07-02 The Board Of Trustees Of The Leland Stanford Junior University Chimeric proteins and methods of immunotherapy
US10428319B2 (en) 2017-06-09 2019-10-01 Editas Medicine, Inc. Engineered Cas9 nucleases
US10450576B2 (en) 2015-03-27 2019-10-22 E I Du Pont De Nemours And Company Soybean U6 small nuclear RNA gene promoters and their use in constitutive expression of small RNA genes in plants
US10519457B2 (en) 2013-08-22 2019-12-31 E I Du Pont De Nemours And Company Soybean U6 polymerase III promoter and methods of use
US10731181B2 (en) 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
US10912797B2 (en) 2016-10-18 2021-02-09 Intima Bioscience, Inc. Tumor infiltrating lymphocytes and methods of therapy
US20210054371A1 (en) * 2019-08-19 2021-02-25 Minghong Zhong Conjugates of Guide RNA-Cas Protein Complex
US10934536B2 (en) 2018-12-14 2021-03-02 Pioneer Hi-Bred International, Inc. CRISPR-CAS systems for genome editing
US20210147879A1 (en) * 2013-11-19 2021-05-20 President And Fellows Of Harvard College Large Gene Excision and Insertion
US11078481B1 (en) 2016-08-03 2021-08-03 KSQ Therapeutics, Inc. Methods for screening for cancer targets
US11078483B1 (en) 2016-09-02 2021-08-03 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
US11149281B2 (en) 2015-10-06 2021-10-19 Institute For Basic Science Method for producing genome-modified plants from plant protoplasts at high efficiency
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11312953B2 (en) 2013-03-14 2022-04-26 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11414657B2 (en) 2015-06-29 2022-08-16 Ionis Pharmaceuticals, Inc. Modified CRISPR RNA and modified single CRISPR RNA and uses thereof
US11466271B2 (en) 2017-02-06 2022-10-11 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US11549126B2 (en) 2015-06-03 2023-01-10 Board Of Regents Of The University Of Nebraska Treatment methods using DNA editing with single-stranded DNA
US11572574B2 (en) 2017-09-28 2023-02-07 Toolgen Incorporated Artificial genome manipulation for gene expression regulation
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation

Families Citing this family (318)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6261500B2 (en) 2011-07-22 2018-01-17 プレジデント アンド フェローズ オブ ハーバード カレッジ Evaluation and improvement of nuclease cleavage specificity
US11021737B2 (en) 2011-12-22 2021-06-01 President And Fellows Of Harvard College Compositions and methods for analyte detection
GB201122458D0 (en) 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
RU2645475C2 (en) 2012-04-25 2018-02-21 Регенерон Фармасьютикалз, Инк. Nuclease-mediated targeting with large targeting vectors
EP3597741A1 (en) 2012-04-27 2020-01-22 Duke University Genetic correction of mutated genes
US20150225734A1 (en) 2012-06-19 2015-08-13 Regents Of The University Of Minnesota Gene targeting in plants using dna viruses
EP3808844A1 (en) * 2012-07-25 2021-04-21 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
DE202013012597U1 (en) 2012-10-23 2017-11-21 Toolgen, Inc. A composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and a Cas protein-encoding nucleic acid or Cas protein, and their use
EP3031921A1 (en) 2012-12-12 2016-06-15 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
ES2553782T3 (en) 2012-12-12 2015-12-11 The Broad Institute, Inc. Systems engineering, methods and guide compositions optimized for sequence manipulation
DK3064585T3 (en) 2012-12-12 2020-04-27 Broad Inst Inc DESIGN AND OPTIMIZATION OF IMPROVED SYSTEMS, PROCEDURES AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
SG10201707569YA (en) 2012-12-12 2017-10-30 Broad Inst Inc Delivery, Engineering and Optimization of Systems, Methods and Compositions for Sequence Manipulation and Therapeutic Applications
EP2931892B1 (en) 2012-12-12 2018-09-12 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
US20140310830A1 (en) 2012-12-12 2014-10-16 Feng Zhang CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
EP2840140B2 (en) 2012-12-12 2023-02-22 The Broad Institute, Inc. Crispr-Cas based method for mutation of prokaryotic cells
DK2931898T3 (en) 2012-12-12 2016-06-20 Massachusetts Inst Technology CONSTRUCTION AND OPTIMIZATION OF SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS
US10138509B2 (en) 2013-03-12 2018-11-27 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
US20140273235A1 (en) * 2013-03-15 2014-09-18 Regents Of The University Of Minnesota ENGINEERING PLANT GENOMES USING CRISPR/Cas SYSTEMS
EP4286517A3 (en) * 2013-04-04 2024-03-13 President and Fellows of Harvard College Therapeutic uses of genome editing with crispr/cas systems
SI2986729T1 (en) 2013-04-16 2019-02-28 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
WO2014186686A2 (en) * 2013-05-17 2014-11-20 Two Blades Foundation Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases
WO2014191518A1 (en) * 2013-05-29 2014-12-04 Cellectis A method for producing precise dna cleavage using cas9 nickase activity
US9267135B2 (en) 2013-06-04 2016-02-23 President And Fellows Of Harvard College RNA-guided transcriptional regulation
SG11201509962QA (en) * 2013-06-04 2016-01-28 Harvard College Rna-guideded transcriptional regulation
AU2014279694B2 (en) 2013-06-14 2020-07-23 Cellectis Methods for non-transgenic genome editing in plants
MX2015017312A (en) 2013-06-17 2017-04-10 Broad Inst Inc Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy.
DK3011029T3 (en) 2013-06-17 2020-03-16 Broad Inst Inc ADMINISTRATION, MODIFICATION AND OPTIMIZATION OF TANDEM GUIDE SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION
WO2014204727A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof
WO2014204725A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation
EP3597755A1 (en) 2013-06-17 2020-01-22 The Broad Institute, Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
JP2016528890A (en) 2013-07-09 2016-09-23 プレジデント アンド フェローズ オブ ハーバード カレッジ Therapeutic use of genome editing using the CRISPR / Cas system
US10563225B2 (en) 2013-07-26 2020-02-18 President And Fellows Of Harvard College Genome engineering
US20150044192A1 (en) 2013-08-09 2015-02-12 President And Fellows Of Harvard College Methods for identifying a target site of a cas9 nuclease
KR102304167B1 (en) 2013-08-16 2021-09-24 메사추세츠 인스티튜트 오브 테크놀로지 Selective delivery of material to cells
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
EP3418379B1 (en) 2013-09-18 2020-12-09 Kymab Limited Methods, cells & organisms
WO2015065964A1 (en) 2013-10-28 2015-05-07 The Broad Institute Inc. Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof
WO2015066119A1 (en) 2013-10-30 2015-05-07 North Carolina State University Compositions and methods related to a type-ii crispr-cas system in lactobacillus buchneri
RU2685914C1 (en) 2013-12-11 2019-04-23 Регенерон Фармасьютикалс, Инк. Methods and compositions for genome targeted modification
PL3080279T3 (en) 2013-12-11 2019-03-29 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a genome
WO2015089364A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crystal structure of a crispr-cas system, and uses thereof
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
EP3080271B1 (en) 2013-12-12 2020-02-12 The Broad Institute, Inc. Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems
CA2932436A1 (en) 2013-12-12 2015-06-18 The Broad Institute, Inc. Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
WO2015089427A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crispr-cas systems and methods for altering expression of gene products, structural information and inducible modular cas enzymes
MX2016007328A (en) 2013-12-12 2017-07-19 Broad Inst Inc Delivery, use and therapeutic applications of the crispr-cas systems and compositions for genome editing.
US10787654B2 (en) 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
CN111705365A (en) 2014-02-11 2020-09-25 科罗拉多州立大学董事会（法人团体） CRISPR-supported multiplexed genome engineering
EP3971283A1 (en) 2014-02-27 2022-03-23 Monsanto Technology LLC Compositions and methods for site directed genomic modification
WO2015134812A1 (en) 2014-03-05 2015-09-11 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating usher syndrome and retinitis pigmentosa
DK3116997T3 (en) 2014-03-10 2019-08-19 Editas Medicine Inc CRISPR / CAS-RELATED PROCEDURES AND COMPOSITIONS TO TREAT LEVER'S CONGENITAL AMAUROSIS 10 (LCA10)
US11141493B2 (en) 2014-03-10 2021-10-12 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
US11339437B2 (en) 2014-03-10 2022-05-24 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
WO2015148863A2 (en) 2014-03-26 2015-10-01 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating sickle cell disease
CA2944978C (en) 2014-04-08 2024-02-13 North Carolina State University Methods and compositions for rna-directed repression of transcription using crispr-associated genes
EP3152221A4 (en) * 2014-05-20 2018-01-24 Regents of the University of Minnesota Method for editing a genetic sequence
KR101886381B1 (en) * 2014-05-28 2018-08-10 기초과학연구원 A method for isolating target DNA using inactivated site-specific nuclease
WO2015188056A1 (en) * 2014-06-05 2015-12-10 Sangamo Biosciences, Inc. Methods and compositions for nuclease design
HUE049776T2 (en) 2014-06-06 2020-10-28 Regeneron Pharma Methods and compositions for modifying a targeted locus
WO2015191693A2 (en) * 2014-06-10 2015-12-17 Massachusetts Institute Of Technology Method for gene editing
EP3154694A1 (en) 2014-06-13 2017-04-19 Children's Medical Center Corporation Products and methods to isolate mitochondria
SI3354732T1 (en) 2014-06-23 2020-07-31 Regeneron Pharmaceuticals, Inc. Nuclease-mediated dna assembly
WO2015200555A2 (en) * 2014-06-25 2015-12-30 Caribou Biosciences, Inc. Rna modification to engineer cas9 activity
MX2016017317A (en) 2014-06-26 2017-08-24 Regeneron Pharma Methods and compositions for targeted genetic modifications and methods of use.
AU2015288157A1 (en) 2014-07-11 2017-01-19 E. I. Du Pont De Nemours And Company Compositions and methods for producing plants resistant to glyphosate herbicide
CA2954791A1 (en) * 2014-07-14 2016-01-21 The Regents Of The University Of California Crispr/cas transcriptional modulation
AU2015294354B2 (en) * 2014-07-21 2021-10-28 Illumina, Inc. Polynucleotide enrichment using CRISPR-Cas systems
CA2956224A1 (en) 2014-07-30 2016-02-11 President And Fellows Of Harvard College Cas9 proteins including ligand-dependent inteins
CA2957441A1 (en) 2014-08-06 2016-02-11 Toolgen Incorporated Genome editing using campylobacter jejuni crispr/cas system-derived rgen
CN106536721B (en) * 2014-08-06 2020-12-04 车医科学大学校产学协力团 Nuclease-mediated generation of immune-compatible cells by editing HLA-encoding genes
US11071289B2 (en) 2014-08-14 2021-07-27 Biocytogen Boston Corp DNA knock-in system
CA2959070C (en) * 2014-08-27 2020-11-10 Caribou Biosciences, Inc. Methods for increasing cas9-mediated engineering efficiency
WO2016033298A1 (en) 2014-08-28 2016-03-03 North Carolina State University Novel cas9 proteins and guiding features for dna targeting and genome editing
EP3188763B1 (en) 2014-09-02 2020-05-13 The Regents of The University of California Methods and compositions for rna-directed target dna modification
RU2017112324A (en) 2014-09-12 2018-10-15 Пайонир Хай-Бред Интернэшнл, Инк. CREATION OF WEBSITES OF SITE-SPECIFIC INTEGRATION FOR COMPLEX SIGNS LOCUSES IN CORN AND SOY, AND ALSO WAYS OF APPLICATION
CN104212836A (en) * 2014-09-18 2014-12-17 东华大学 Method for knocking out mir-505 from mammal cell line
WO2016061416A1 (en) 2014-10-15 2016-04-21 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
RU2706965C2 (en) 2014-10-15 2019-11-21 Регенерон Фармасьютикалз, Инк. Methods and compositions for producing or maintaining pluripotent cells
SG10201903912XA (en) 2014-10-31 2019-05-30 Massachusetts Inst Technology Delivery of biomolecules to immune cells
DK3215611T3 (en) * 2014-11-06 2019-11-25 Du Pont Peptide-Mediated Administration of RNA-Guided ENDONUCLEASE IN CELLS
US11352666B2 (en) 2014-11-14 2022-06-07 Institute For Basic Science Method for detecting off-target sites of programmable nucleases in a genome
WO2016076672A1 (en) * 2014-11-14 2016-05-19 기초과학연구원 Method for detecting off-target site of genetic scissors in genome
CN113444747A (en) 2014-11-21 2021-09-28 瑞泽恩制药公司 Methods and compositions for targeted genetic modification using paired guide RNAs
EP3227447A4 (en) 2014-12-03 2018-07-11 Agilent Technologies, Inc. Guide rna with chemical modifications
EP3230451B1 (en) 2014-12-12 2021-04-07 The Broad Institute, Inc. Protected guide rnas (pgrnas)
WO2016100272A1 (en) * 2014-12-16 2016-06-23 Danisco Us Inc Fungal genome modification systems and methods of use
CN107208096B (en) * 2014-12-18 2021-02-12 综合基因技术公司 CRISPR-based compositions and methods of use
EP3653048B9 (en) 2014-12-19 2023-10-04 Regeneron Pharmaceuticals, Inc. Methods and compositions for targeted genetic modification through single-step multiple targeting
US10196613B2 (en) 2014-12-19 2019-02-05 Regeneron Pharmaceuticals, Inc. Stem cells for modeling type 2 diabetes
US11125739B2 (en) 2015-01-12 2021-09-21 Massachusetts Institute Of Technology Gene editing through microfluidic delivery
US20180163232A1 (en) * 2015-03-16 2018-06-14 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences A method for making site-directed modification to plant genomes by using non-inheritable materials
EP3271461A1 (en) * 2015-03-20 2018-01-24 Danmarks Tekniske Universitet Crispr/cas9 based engineering of actinomycetal genomes
EP3851530A1 (en) * 2015-03-26 2021-07-21 Editas Medicine, Inc. Crispr/cas-mediated gene conversion
KR20240038141A (en) 2015-04-06 2024-03-22 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 Chemically modified guide rnas for crispr/cas-mediated gene regulation
KR102535217B1 (en) 2015-04-24 2023-05-19 에디타스 메디신, 인코포레이티드 Assessment of CAS9 Molecule/Guide RNA Molecule Complexes
KR101785847B1 (en) 2015-05-12 2017-10-17 연세대학교 산학협력단 Targeted genome editing based on CRISPR/Cas9 system using short linearized double-stranded DNA
EP3095870A1 (en) 2015-05-19 2016-11-23 Kws Saat Se Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable therefrom
JP2018516563A (en) 2015-05-29 2018-06-28 ノース カロライナ ステート ユニバーシティＮｏｒｔｈ Ｃａｒｏｌｉｎａ Ｓｔａｔｅ Ｕｎｉｖｅｒｓｉｔｙ Method for screening bacteria, archaea, algae, and yeast using CRISPR nucleic acid
CN115537421A (en) 2015-06-15 2022-12-30 北卡罗来纳州立大学 Methods and compositions for efficient delivery of nucleic acids and RNA-based antimicrobials
SG10201912329YA (en) 2015-06-18 2020-02-27 Broad Inst Inc Crispr Enzyme Mutations Reducing Off-Target Effects
WO2016205759A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of cas9 orthologs and variants for sequence manipulation
JP6925984B2 (en) 2015-07-09 2021-08-25 マサチューセッツ インスティテュート オブ テクノロジー Delivery of substance to anucleated cells
DK3329001T3 (en) * 2015-07-28 2021-12-20 Danisco Us Inc RE-EDITING SYSTEMS AND APPLICATION PROCEDURES
JP2018529759A (en) 2015-08-14 2018-10-11 ザ ユニバーシティー オブ シドニー Therapeutic connexin 45 inhibitor
US11427817B2 (en) * 2015-08-25 2022-08-30 Duke University Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases
US11613759B2 (en) 2015-09-04 2023-03-28 Sqz Biotechnologies Company Intracellular delivery of biomolecules to cells comprising a cell wall
EP3352795B1 (en) * 2015-09-21 2020-08-12 The Regents of The University of California Compositions and methods for target nucleic acid modification
WO2017058751A1 (en) 2015-09-28 2017-04-06 North Carolina State University Methods and compositions for sequence specific antimicrobials
MX2018004809A (en) * 2015-10-20 2018-06-08 Pionner Hi Bred Int Inc Methods and compositions for marker-free genome modification.
IL310721A (en) 2015-10-23 2024-04-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
MX2018005611A (en) 2015-11-03 2018-11-09 Harvard College Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix.
KR101885901B1 (en) 2015-11-13 2018-08-07 기초과학연구원 RGEN RNP delivery method using 5'-phosphate removed RNA
WO2017087979A1 (en) * 2015-11-20 2017-05-26 Washington University Preparative electrophoretic method for targeted purification of genomic dna fragments
EP3397261A4 (en) 2015-11-30 2019-07-03 Flagship Pioneering Innovations V, Inc. Methods and compositions relating to chondrisomes
CN106811479B (en) * 2015-11-30 2019-10-25 中国农业科学院作物科学研究所 The system and its application of Herbicide Resistant Rice are obtained using CRISPR/Cas9 system pointed decoration als gene
CN106845151B (en) * 2015-12-07 2019-03-26 中国农业大学 The screening technique and device of CRISPR-Cas9 system sgRNA action target spot
WO2017099494A1 (en) * 2015-12-08 2017-06-15 기초과학연구원 Genome editing composition comprising cpf1, and use thereof
WO2017112620A1 (en) 2015-12-22 2017-06-29 North Carolina State University Methods and compositions for delivery of crispr based antimicrobials
EP3402327A1 (en) * 2016-01-15 2018-11-21 The Jackson Laboratory Genetically modified non-human mammals by multi-cycle electroporation of cas9 protein
US11512311B2 (en) 2016-03-25 2022-11-29 Editas Medicine, Inc. Systems and methods for treating alpha 1-antitrypsin (A1AT) deficiency
CA3018978A1 (en) * 2016-03-30 2017-10-05 Intellia Therapeutics, Inc. Lipid nanoparticle formulations for crispr/cas components
CN109415761B (en) 2016-04-25 2022-09-20 哈佛学院董事及会员团体 Hybrid chain reaction method for in situ molecular detection
CN107326046A (en) * 2016-04-28 2017-11-07 上海邦耀生物科技有限公司 A kind of method for improving foreign gene homologous recombination efficiency
JP7324583B2 (en) 2016-05-20 2023-08-10 リジェネロン・ファーマシューティカルズ・インコーポレイテッド Methods for breaking immune tolerance using multiple guide RNAs
MX2018014599A (en) * 2016-05-27 2019-02-28 Aadigen Llc Peptides and nanoparticles for intracellular delivery of genome-editing molecules.
PL3604527T3 (en) 2016-06-02 2021-09-06 Sigma-Aldrich Co., Llc Using programmable dna binding proteins to enhance targeted genome modification
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
US20190177710A1 (en) * 2016-06-15 2019-06-13 Toolgen Incorporated Method For Screening Target Specific Nuclease Using Multi-Target System Of On-Target And Off-Target And Use Thereof
CN109642231A (en) * 2016-06-17 2019-04-16 博德研究所 VI type CRISPR ortholog and system
EP3472321A2 (en) * 2016-06-17 2019-04-24 Genesis Technologies Limited Crispr-cas system, materials and methods
CA3027869A1 (en) * 2016-06-20 2017-12-28 Keygene N.V. Method for targeted dna alteration in plant cells
AU2017280353B2 (en) 2016-06-24 2021-11-11 Inscripta, Inc. Methods for generating barcoded combinatorial libraries
US20200069818A1 (en) * 2016-07-05 2020-03-05 The Johns Hopkins University Crispr/cas9-based compositions and methods for treating cancer
EP3484994A4 (en) * 2016-07-13 2020-01-22 DSM IP Assets B.V. A crispr-cas system for an algal host cell
WO2018015995A1 (en) * 2016-07-19 2018-01-25 株式会社バイオダイナミクス研究所 Method for preparing long-chain single-stranded dna
KR101961332B1 (en) 2016-07-28 2019-03-22 기초과학연구원 Pharmaceutical Composition for Treating or Preventing Eye Disease Comprising Cas9 Protein and Guide RNA
BR112019001783A2 (en) 2016-07-29 2019-05-07 Regeneron Pharmaceuticals, Inc. nonhuman mammal, and methods for producing the nonhuman mammal and screening a compound.
AU2017305404B2 (en) 2016-08-02 2023-11-30 Editas Medicine, Inc. Compositions and methods for treating CEP290 associated disease
KR20230095129A (en) 2016-08-03 2023-06-28 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Adenosine nucleobase editors and uses thereof
CN109804066A (en) 2016-08-09 2019-05-24 哈佛大学的校长及成员们 Programmable CAS9- recombination enzyme fusion proteins and application thereof
KR101710026B1 (en) 2016-08-10 2017-02-27 주식회사 무진메디 Composition comprising delivery carrier of nano-liposome having Cas9 protein and guide RNA
KR101856345B1 (en) * 2016-08-24 2018-06-20 경상대학교산학협력단 Method for generation of APOBEC3H and APOBEC3CH double-knocked out cat using CRISPR/Cas9 system
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US20190225974A1 (en) 2016-09-23 2019-07-25 BASF Agricultural Solutions Seed US LLC Targeted genome optimization in plants
EP3523427A4 (en) 2016-10-07 2020-09-02 Integrated DNA Technologies, Inc. S. pyogenes cas9 mutant genes and polypeptides encoded by same
US11242542B2 (en) 2016-10-07 2022-02-08 Integrated Dna Technologies, Inc. S. pyogenes Cas9 mutant genes and polypeptides encoded by same
KR101997116B1 (en) 2016-10-14 2019-07-05 연세대학교 산학협력단 Guide RNA complementary to KRAS gene and use thereof
KR20240007715A (en) 2016-10-14 2024-01-16 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Aav delivery of nucleobase editors
WO2018144097A1 (en) 2016-11-04 2018-08-09 Akeagen Llc Genetically modified non-human animals and methods for producing heavy chain-only antibodies
EP4256951A3 (en) 2016-11-04 2023-12-06 Flagship Pioneering Innovations V. Inc. Novel plant cells, plants, and seeds
EP3896162A1 (en) * 2016-11-14 2021-10-20 Toolgen Incorporated Artificially engineered sc function control system
AU2017364084A1 (en) 2016-11-22 2019-05-16 Integrated Dna Technologies, Inc. CRISPR/Cpf1 systems and methods
WO2018111947A1 (en) 2016-12-12 2018-06-21 Integrated Dna Technologies, Inc. Genome editing enhancement
KR101748575B1 (en) * 2016-12-16 2017-06-20 주식회사 엠젠플러스 INSulin gene knockout diabetes mellitus or diabetic complications animal model and a method for producing the same
CN110325643A (en) * 2016-12-22 2019-10-11 株式会社图尔金 The plant and its production method rich in oleic acid with the FAD2 through gene modification
WO2018119359A1 (en) 2016-12-23 2018-06-28 President And Fellows Of Harvard College Editing of ccr5 receptor gene to protect against hiv infection
CN110300802A (en) * 2016-12-23 2019-10-01 基础科学研究院 Composition and base edit methods for animal embryo base editor
JP6994730B2 (en) * 2016-12-26 2022-02-04 独立行政法人酒類総合研究所 Genome editing method for filamentous fungus genome editing by direct introduction of protein
US11859219B1 (en) 2016-12-30 2024-01-02 Flagship Pioneering Innovations V, Inc. Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
KR102515727B1 (en) * 2017-01-11 2023-03-30 주식회사 툴젠 Composition and method for inserting specific nucleic acid sequence into target nucleic acid using overlapping guide nucleic acid
SG11201905508VA (en) 2017-01-23 2019-08-27 Regeneron Pharma Hsd17b13 variants and uses thereof
SG11201906795SA (en) 2017-01-28 2019-08-27 Inari Agriculture Inc Novel plant cells, plants, and seeds
EP3594339A4 (en) * 2017-03-06 2020-12-30 Institute For Basic Science Composition containing c2cl endonuclease for dielectric calibration and method for dielectric calibration using same
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
WO2018165629A1 (en) 2017-03-10 2018-09-13 President And Fellows Of Harvard College Cytosine to guanine base editor
WO2018170184A1 (en) 2017-03-14 2018-09-20 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
IL269458B2 (en) 2017-03-23 2024-02-01 Harvard College Nucleobase editors comprising nucleic acid programmable dna binding proteins
AU2018250330A1 (en) 2017-04-07 2019-09-19 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification
CA3059643A1 (en) 2017-04-13 2018-10-18 Cellectis New sequence specific reagents targeting ccr5 in primary hematopoietic cells
US11913015B2 (en) 2017-04-17 2024-02-27 University Of Maryland, College Park Embryonic cell cultures and methods of using the same
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
CN108977442B (en) * 2017-06-05 2023-01-06 广州市锐博生物科技有限公司 System for DNA editing and application thereof
US10738284B2 (en) 2017-06-05 2020-08-11 Regeneron Pharmaceuticals, Inc. B4GALT1 cDNA variants and compositions comprising the same
EP3638797A1 (en) 2017-06-13 2020-04-22 Flagship Pioneering Innovations V, Inc. Compositions comprising curons and uses thereof
EP3638317A4 (en) 2017-06-15 2021-03-17 The Regents of The University of California Targeted non-viral dna insertions
US10011849B1 (en) 2017-06-23 2018-07-03 Inscripta, Inc. Nucleic acid-guided nucleases
US9982279B1 (en) 2017-06-23 2018-05-29 Inscripta, Inc. Nucleic acid-guided nucleases
CN107488710B (en) * 2017-07-14 2020-09-22 上海吐露港生物科技有限公司 Application of Cas protein, and detection method and kit of target nucleic acid molecule
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
KR20200032693A (en) 2017-07-31 2020-03-26 리제너론 파마슈티칼스 인코포레이티드 Cas-transformed mouse embryonic stem cells and mice and uses thereof
BR112019027673A2 (en) 2017-07-31 2020-09-15 Regeneron Pharmaceuticals, Inc. non-human animal, and, methods to test the recombination induced by crispr / cas and to optimize the ability of crispr / cas
JP2020533957A (en) 2017-07-31 2020-11-26 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． CRISPR Reporter Non-Human Animals and Their Use
JP7207665B2 (en) 2017-08-04 2023-01-18 北京大学 TALE RVDs that specifically recognize DNA bases modified by methylation and uses thereof
CN111278983A (en) 2017-08-08 2020-06-12 北京大学 Gene knockout method
EP3676376A2 (en) 2017-08-30 2020-07-08 President and Fellows of Harvard College High efficiency base editors comprising gam
TW201920661A (en) 2017-09-18 2019-06-01 大陸商博雅輯因（北京）生物科技有限公司 Gene editing T cell and use thereof
CN111527208A (en) * 2017-09-28 2020-08-11 株式会社图尔金 Artificial genomic manipulation for gene expression regulation
US20190098879A1 (en) 2017-09-29 2019-04-04 Regeneron Pharmaceuticals, Inc. Non-Human Animals Comprising A Humanized TTR Locus And Methods Of Use
KR102207352B1 (en) * 2017-09-29 2021-01-26 서울대학교산학협력단 klotho knock-out porcine Models for klotho deficiency disease and the Use thereof
CN111405912A (en) 2017-09-29 2020-07-10 因特利亚治疗公司 Polynucleotides, compositions and methods for genome editing
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN109722415B (en) 2017-10-27 2021-01-26 博雅辑因（北京）生物科技有限公司 Hematopoietic stem cell culture composition, culture medium and hematopoietic stem cell culture method
KR20230034416A (en) 2017-10-27 2023-03-09 더 리전트 오브 더 유니버시티 오브 캘리포니아 Targeted replacement of endogenous t cell receptors
CN108192956B (en) * 2017-11-17 2021-06-01 东南大学 Cas9 nuclease-based DNA detection and analysis method and application thereof
CN111836894B (en) 2017-11-21 2023-11-10 基恩科雷有限责任公司 Compositions for genome editing using CRISPR/Cpf1 systems and uses thereof
CN111566121A (en) 2018-01-12 2020-08-21 巴斯夫欧洲公司 Gene for determining number of spikelets per ear QTL on wheat 7a chromosome
US20210032621A1 (en) * 2018-01-23 2021-02-04 Institute For Basic Science Extended single guide rna and use thereof
US11926835B1 (en) 2018-01-29 2024-03-12 Inari Agriculture Technology, Inc. Methods for efficient tomato genome editing
KR102086689B1 (en) * 2018-02-06 2020-03-09 주식회사 진씨커 Kit for isolation of mutant cell free DNA and method for isolation of mutant cell free DNA using the same
WO2019156475A1 (en) * 2018-02-06 2019-08-15 주식회사 진씨커 Mutant cell-free dna isolation kit and mutant cell-free dna isolation method using same
CN111885915B (en) 2018-03-19 2023-04-28 瑞泽恩制药公司 Transcription modulation of animals using CRISPR/CAS systems
KR20210003819A (en) 2018-04-19 2021-01-12 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 Compositions and methods for gene editing
WO2019206233A1 (en) * 2018-04-25 2019-10-31 中国农业大学 Rna-edited crispr/cas effector protein and system
WO2019222545A1 (en) 2018-05-16 2019-11-21 Synthego Corporation Methods and systems for guide rna design and use
US11866719B1 (en) 2018-06-04 2024-01-09 Inari Agriculture Technology, Inc. Heterologous integration of regulatory elements to alter gene expression in wheat cells and wheat plants
KR102035654B1 (en) * 2018-07-23 2019-10-24 대한민국 Novel mutant insect cell line producing antimicrobial peptide or a preparation method thereof
US11479762B1 (en) 2018-08-31 2022-10-25 Inari Agriculture Technology, Inc. Compositions, systems, and methods for genome editing
CN112955550A (en) * 2018-09-12 2021-06-11 基础科学研究院 Composition for inducing death of cell having mutant gene and method for inducing death of cell having mutant gene by using the same
US10711267B2 (en) 2018-10-01 2020-07-14 North Carolina State University Recombinant type I CRISPR-Cas system
KR102081962B1 (en) * 2018-10-11 2020-02-26 충남대학교 산학협력단 Composition for cleaving amyloid precursor protein gene
JP2022505402A (en) 2018-10-18 2022-01-14 インテリア セラピューティクス，インコーポレーテッド Compositions and Methods for Expression of Introduced Genes from the Albumin Locus
JP2022505381A (en) 2018-10-18 2022-01-14 インテリア セラピューティクス，インコーポレーテッド Compositions and Methods for Treating Alpha-1 Antitrypsin Deficiency
EP3867380A2 (en) 2018-10-18 2021-08-25 Intellia Therapeutics, Inc. Compositions and methods for expressing factor ix
CA3116580A1 (en) 2018-10-18 2020-04-23 Intellia Therapeutics, Inc. Nucleic acid constructs and methods of use
CA3118251A1 (en) * 2018-10-29 2020-05-07 China Agricultural University Novel crispr/cas12f enzyme and system
JP2022505440A (en) * 2018-11-01 2022-01-14 キージーン ナムローゼ フェンノートシャップ Dual guide RNA for CRISPR / Cas genome editing in plant cells
EP3650557B1 (en) * 2018-11-07 2021-10-20 Justus-Liebig-Universität Gießen Method for determination of restriction efficiency of endonucleases mediating double-strand breaks in dna target sequences
US20210324420A1 (en) * 2018-11-16 2021-10-21 Osaka University Method for producing genome-edited cell
KR20200071198A (en) 2018-12-10 2020-06-19 네오이뮨텍, 인코퍼레이티드 Development of new adoptive T cell immunotherapy by modification of Nrf2 expression
US11166996B2 (en) 2018-12-12 2021-11-09 Flagship Pioneering Innovations V, Inc. Anellovirus compositions and methods of use
US20220025405A1 (en) * 2018-12-12 2022-01-27 Kyushu University, National University Corporation Production method for genome-edited cells
KR20210105914A (en) 2018-12-20 2021-08-27 리제너론 파마슈티칼스 인코포레이티드 Nuclease-mediated repeat expansion
WO2020146290A1 (en) * 2019-01-07 2020-07-16 Crisp-Hr Therapeutics, Inc. A non-toxic cas9 enzyme and application thereof
CN109868275A (en) * 2019-03-12 2019-06-11 中国农业大学 The method of sheep FGF5 gene knockout and site-directed integration MTNR1A gene that CRISPR/Cas9 is mediated
IL286359B1 (en) 2019-03-18 2024-02-01 Regeneron Pharma Crispr/cas dropout screening platform to reveal genetic vulnerabilities associated with tau aggregation
CN113631700A (en) 2019-03-18 2021-11-09 瑞泽恩制药公司 CRISPR/Cas screening platform for identifying genetic modification factors for tau vaccination or aggregation
WO2020191248A1 (en) 2019-03-19 2020-09-24 The Broad Institute, Inc. Method and compositions for editing nucleotide sequences
CA3133361A1 (en) 2019-04-03 2020-10-08 Regeneron Pharmaceuticals, Inc. Methods and compositions for insertion of antibody coding sequences into a safe harbor locus
WO2020206139A1 (en) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized coagulation factor 12 locus
CA3133359C (en) 2019-04-04 2023-04-11 Regeneron Pharmaceuticals, Inc. Methods for scarless introduction of targeted modifications into targeting vectors
EA202192931A1 (en) 2019-04-30 2022-02-22 Эдиджен Инк. METHOD FOR PREDICTION OF THE EFFICIENCY OF HEMOGLOBINOPATHY TREATMENT
US11692197B2 (en) 2019-05-06 2023-07-04 Inari Agriculture Technology, Inc. Delivery of biological molecules to plant cells
CN111304180B (en) * 2019-06-04 2023-05-26 山东舜丰生物科技有限公司 Novel DNA nucleic acid cutting enzyme and application thereof
CN113874510A (en) 2019-06-04 2021-12-31 瑞泽恩制药公司 Non-human animals including humanized TTR loci with beta glide mutations and methods of use
TW202112229A (en) 2019-06-07 2021-04-01 美商雷傑納榮製藥公司 Non-human animals comprising a humanized albumin locus
JP2022536606A (en) 2019-06-14 2022-08-18 リジェネロン・ファーマシューティカルズ・インコーポレイテッド Model of tauopathy
US11041172B2 (en) 2019-06-25 2021-06-22 Inari Agriculture, Inc. Homology dependent repair genome editing
CN114269941A (en) 2019-09-04 2022-04-01 博雅辑因（北京）生物科技有限公司 Method for evaluating gene editing therapy based on off-target assessment
KR20220062079A (en) 2019-09-13 2022-05-13 리제너론 파마슈티칼스 인코포레이티드 Transcriptional Regulation in Animals Using the CRISPR/Cas System Delivered by Lipid Nanoparticles
EP4041894A1 (en) 2019-09-23 2022-08-17 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4a) GENE EXPRESSION
WO2021061707A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein b (apob) gene expression
WO2021067788A1 (en) 2019-10-03 2021-04-08 Artisan Development Labs, Inc. Crispr systems with engineered dual guide nucleic acids
WO2021092130A1 (en) * 2019-11-05 2021-05-14 Pairwise Plants Services, Inc. Compositions and methods for rna-encoded dna-replacement of alleles
US20230001019A1 (en) 2019-11-08 2023-01-05 Regeneron Pharmaceuticals, Inc. Crispr and aav strategies for x-linked juvenile retinoschisis therapy
JP7448120B2 (en) * 2019-11-14 2024-03-12 国立研究開発法人農業・食品産業技術総合研究機構 Method for introducing genome editing enzymes into plant cells using plasma
WO2021108363A1 (en) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Crispr/cas-mediated upregulation of humanized ttr allele
WO2021108442A2 (en) * 2019-11-27 2021-06-03 The Regents Of The University Of California Modulators of cas9 polypeptide activity and methods of use thereof
EP4086340A1 (en) 2019-12-30 2022-11-09 Edigene Biotechnology Inc. Universal car-t targeting t-cell lymphoma cell and preparation method therefor and use thereof
JP2023516538A (en) 2019-12-30 2023-04-20 博雅▲緝▼因（北京）生物科技有限公司 Methods and applications for purifying UCART cells
AU2021208254A1 (en) * 2020-01-14 2022-07-28 Toolgen Incorporated Cells having high adaptability under hypoxic conditions, and use thereof
EP4091635A4 (en) 2020-01-14 2024-02-21 Toolgen Inc Pharmaceutical composition for preventing or treating alzheimer's and use thereof
WO2021163515A1 (en) * 2020-02-12 2021-08-19 Temple University - Of The Commonwealth System Of Higher Education Crispr-cas9 mediated disruption of alcam gene inhibits adhesion and trans-endothelial migration of myeloid cells
KR20210109384A (en) 2020-02-27 2021-09-06 주식회사 엘지화학 Method for increasing gene editing efficiency in plant using protoplasts
KR20230004456A (en) 2020-03-04 2023-01-06 리제너론 파아마슈티컬스, 인크. Methods and compositions for sensitization of tumor cells to immunotherapy
EP4118207A1 (en) 2020-03-11 2023-01-18 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
EP4125348A1 (en) 2020-03-23 2023-02-08 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use
US20210319851A1 (en) 2020-04-03 2021-10-14 Creyon Bio, Inc. Oligonucleotide-based machine learning
JP2023525304A (en) 2020-05-08 2023-06-15 ザ ブロード インスティテュート，インコーポレーテッド Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN111690720B (en) * 2020-06-16 2021-06-15 山东舜丰生物科技有限公司 Method for detecting target nucleic acid using modified single-stranded nucleic acid
WO2022120022A1 (en) 2020-12-02 2022-06-09 Regeneron Pharmaceuticals, Inc. Crispr sam biosensor cell lines and methods of use thereof
KR20220081949A (en) * 2020-12-09 2022-06-16 재단법인 아산사회복지재단 Guide RNA of which on-target activity is maintained and off-target activity decrease and use thereof
JP2024501287A (en) 2020-12-23 2024-01-11 フラッグシップ パイオニアリング イノベーションズ ブイ， インコーポレイテッド In vitro assembly of RNA-confined Anellovirus capsids
EP4271799A1 (en) 2020-12-30 2023-11-08 Intellia Therapeutics, Inc. Engineered t cells
KR102285906B1 (en) * 2021-02-18 2021-08-04 주식회사 엔에이피 Composition for inducing large mouth bass sterility for management of disturbing fish species in aquatic ecosystem
CA3218511A1 (en) 2021-05-10 2022-11-17 Sqz Biotechnologies Company Methods for delivering genome editing molecules to the nucleus or cytosol of a cell and uses thereof
KR102616916B1 (en) * 2021-05-11 2023-12-21 주식회사 애이마 Guide RNA for detecting oncogenic mutations and uses thereof
WO2022251644A1 (en) 2021-05-28 2022-12-01 Lyell Immunopharma, Inc. Nr4a3-deficient immune cells and uses thereof
WO2022256448A2 (en) 2021-06-01 2022-12-08 Artisan Development Labs, Inc. Compositions and methods for targeting, editing, or modifying genes
AU2022286947A1 (en) 2021-06-02 2023-12-14 Lyell Immunopharma, Inc. NR4A-deficient cells expressing c-Jun and uses thereof
KR102618072B1 (en) 2021-06-15 2023-12-28 대한민국 Guide RNA for editing vernalization gene and use thereof
CA3223311A1 (en) 2021-06-18 2022-12-22 Andrea BARGHETTI Compositions and methods for targeting, editing or modifying human genes
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
CN117716026A (en) 2021-07-09 2024-03-15 株式会社图尔金 Mesenchymal stem cells with oxidative stress resistance, preparation method and application thereof
CN117730144A (en) 2021-07-29 2024-03-19 株式会社图尔金 Mesenchymal stem cells with blood compatibility, preparation method and application thereof
CN113584134B (en) * 2021-09-06 2024-01-30 青岛金斯达生物技术有限公司 Isothermal nucleic acid detection system based on CRISPR-Cas9, and method and application thereof
CA3230927A1 (en) 2021-09-10 2023-03-16 Agilent Technologies, Inc. Guide rnas with chemical modification for prime editing
WO2023064924A1 (en) 2021-10-14 2023-04-20 Codiak Biosciences, Inc. Modified producer cells for extracellular vesicle production
WO2023077053A2 (en) 2021-10-28 2023-05-04 Regeneron Pharmaceuticals, Inc. Crispr/cas-related methods and compositions for knocking out c5
WO2023077148A1 (en) 2021-11-01 2023-05-04 Tome Biosciences, Inc. Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo
WO2023108047A1 (en) 2021-12-08 2023-06-15 Regeneron Pharmaceuticals, Inc. Mutant myocilin disease model and uses thereof
WO2023122764A1 (en) 2021-12-22 2023-06-29 Tome Biosciences, Inc. Co-delivery of a gene editor construct and a donor template
WO2023122800A1 (en) 2021-12-23 2023-06-29 University Of Massachusetts Therapeutic treatment for fragile x-associated disorder
WO2023129974A1 (en) 2021-12-29 2023-07-06 Bristol-Myers Squibb Company Generation of landing pad cell lines
WO2023150181A1 (en) 2022-02-01 2023-08-10 President And Fellows Of Harvard College Methods and compositions for treating cancer
WO2023150623A2 (en) 2022-02-02 2023-08-10 Regeneron Pharmaceuticals, Inc. Anti-tfr:gaa and anti-cd63:gaa insertion for treatment of pompe disease
WO2023167882A1 (en) 2022-03-01 2023-09-07 Artisan Development Labs, Inc. Composition and methods for transgene insertion
CN114410609B (en) * 2022-03-29 2022-07-12 舜丰生物科技(海南)有限公司 Cas protein with improved activity and application thereof
WO2023205744A1 (en) 2022-04-20 2023-10-26 Tome Biosciences, Inc. Programmable gene insertion compositions
WO2023205844A1 (en) * 2022-04-26 2023-11-02 Peter Maccallum Cancer Institute Nucleic acids and uses thereof
WO2023212677A2 (en) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification of tissue-specific extragenic safe harbors for gene therapy approaches
WO2023215831A1 (en) 2022-05-04 2023-11-09 Tome Biosciences, Inc. Guide rna compositions for programmable gene insertion
WO2023220603A1 (en) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
WO2023225665A1 (en) 2022-05-19 2023-11-23 Lyell Immunopharma, Inc. Polynucleotides targeting nr4a3 and uses thereof
WO2023225410A2 (en) 2022-05-20 2023-11-23 Artisan Development Labs, Inc. Systems and methods for assessing risk of genome editing events
WO2023225670A2 (en) 2022-05-20 2023-11-23 Tome Biosciences, Inc. Ex vivo programmable gene insertion
WO2023230549A2 (en) 2022-05-25 2023-11-30 Flagship Pioneering Innovations Vii, Llc Compositions and methods for modulation of tumor suppressors and oncogenes
WO2023230566A2 (en) 2022-05-25 2023-11-30 Flagship Pioneering Innovations Vii, Llc Compositions and methods for modulating cytokines
WO2023230573A2 (en) 2022-05-25 2023-11-30 Flagship Pioneering Innovations Vii, Llc Compositions and methods for modulation of immune responses
WO2023230570A2 (en) 2022-05-25 2023-11-30 Flagship Pioneering Innovations Vii, Llc Compositions and methods for modulating genetic drivers
WO2023230578A2 (en) 2022-05-25 2023-11-30 Flagship Pioneering Innovations Vii, Llc Compositions and methods for modulating circulating factors
WO2023235726A2 (en) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Crispr interference therapeutics for c9orf72 repeat expansion disease
WO2023235725A2 (en) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Crispr-based therapeutics for c9orf72 repeat expansion disease
WO2024006955A1 (en) 2022-06-29 2024-01-04 Intellia Therapeutics, Inc. Engineered t cells
EP4299739A1 (en) 2022-06-30 2024-01-03 Inari Agriculture Technology, Inc. Compositions, systems, and methods for genome editing
WO2024005864A1 (en) 2022-06-30 2024-01-04 Inari Agriculture Technology, Inc. Compositions, systems, and methods for genome editing
WO2024005863A1 (en) 2022-06-30 2024-01-04 Inari Agriculture Technology, Inc. Compositions, systems, and methods for genome editing
EP4299733A1 (en) 2022-06-30 2024-01-03 Inari Agriculture Technology, Inc. Compositions, systems, and methods for genome editing
WO2024020587A2 (en) 2022-07-22 2024-01-25 Tome Biosciences, Inc. Pleiopluripotent stem cell programmable gene insertion
WO2024026474A1 (en) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
WO2024031053A1 (en) 2022-08-05 2024-02-08 Regeneron Pharmaceuticals, Inc. Aggregation-resistant variants of tdp-43
CN115386597A (en) * 2022-09-19 2022-11-25 国家纳米科学中心 Transfection auxiliary reagent for improving transfection efficiency and/or transfection precision of gene vector and application thereof
WO2024064958A1 (en) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Methods for culturing nr4a-deficient cells
WO2024064952A1 (en) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Methods for culturing nr4a-deficient cells overexpressing c-jun
WO2024073606A1 (en) 2022-09-28 2024-04-04 Regeneron Pharmaceuticals, Inc. Antibody resistant modified receptors to enhance cell-based therapies

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140068797A1 (en) * 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Family Cites Families (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0262516A2 (en) * 1986-09-29 1988-04-06 Miles Inc. Genetic transformation of lactic acid bacteria
US5350689A (en) * 1987-05-20 1994-09-27 Ciba-Geigy Corporation Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells
NZ228948A (en) 1988-05-13 1991-06-25 Phillips Petroleum Co Hbv particles containing s and pre-s 2 proteins and recombinant processes for their production in yeast cells
US5767367A (en) * 1990-06-23 1998-06-16 Hoechst Aktiengesellschaft Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US6040295A (en) * 1995-01-13 2000-03-21 Genemedicine, Inc. Formulated nucleic acid compositions and methods of administering the same for gene therapy
JPH1080274A (en) 1996-09-06 1998-03-31 Hirofumi Hamada Tetracycline transactivator bearing nuclear transport signal bonded thereto
FR2753204B1 (en) 1996-09-11 1998-12-04 Transgene Sa PROCESS FOR THE PREPARATION OF PLASMID DNA
JP2002538842A (en) 1999-03-16 2002-11-19 デイナ−ファーバー キャンサー インスティチュート，インコーポレイテッド Lentiviral vector system for high volume screening
AU2002254007B2 (en) 2001-02-22 2006-09-21 Purdue Research Foundation Compositions based on vanilloid-catechin synergies for prevention and treatment of cancer
DE10290663D2 (en) 2001-02-24 2004-04-15 Mologen Forschungs Entwicklung Local pain relief
AU2011203213A1 (en) 2002-08-28 2011-08-04 Chromocell Corporation Selection and Isolation of Living Cells Using mRNA-Binding Probes
JP2005006578A (en) * 2003-06-20 2005-01-13 Nippon Genetech Co Ltd Method for producing functional rna molecule by utilizing in-vitro transcription reaction
DK1667678T3 (en) 2003-10-03 2009-08-24 Veijlen N V animal feed composition
AU2004294567A1 (en) 2003-11-26 2005-06-16 University Of Massachusetts Sequence-specific inhibition of small RNA function
US20050220796A1 (en) 2004-03-31 2005-10-06 Dynan William S Compositions and methods for modulating DNA repair
US20070048741A1 (en) 2005-08-24 2007-03-01 Getts Robert C Methods and kits for sense RNA synthesis
JP2009506025A (en) 2005-08-26 2009-02-12 国立大学法人京都大学 Antiviral agent and viral replication inhibitor
SI2126130T1 (en) 2007-03-02 2015-10-30 Dupont Nutrition Biosciences Aps Cultures with improved phage resistance
JP5702144B2 (en) 2007-09-27 2015-04-15 ダウ アグロサイエンシィズ エルエルシー Modified zinc finger protein targeting the 5-enolpyruvylshikimate-3-phosphate synthase gene
US20100067918A1 (en) 2008-04-18 2010-03-18 New Jersey Institute Of Technology Ultra-miniaturized thz communication device and system
WO2010001189A1 (en) 2008-07-03 2010-01-07 Cellectis The crystal structure of i-dmoi in complex with its dna target, improved chimeric meganucleases and uses thereof
US20100076057A1 (en) * 2008-09-23 2010-03-25 Northwestern University TARGET DNA INTERFERENCE WITH crRNA
EP2184292A1 (en) 2008-11-10 2010-05-12 Ulrich Kunzendorf Anti-Apoptotic Fusion Proteins
US8388824B2 (en) 2008-11-26 2013-03-05 Enthone Inc. Method and composition for electrodeposition of copper in microelectronics with dipyridyl-based levelers
KR20100080068A (en) * 2008-12-31 2010-07-08 주식회사 툴젠 A novel zinc finger nuclease and uses thereof
WO2011007193A1 (en) 2009-07-17 2011-01-20 Cellectis Viral vectors encoding a dna repair matrix and containing a virion-associated site specific meganuclease for gene targeting
WO2011056186A1 (en) * 2009-10-26 2011-05-12 Albert Einstein College Of Medicine Of Yeshiva University Microrna affinity assay and uses thereof
US10087431B2 (en) * 2010-03-10 2018-10-02 The Regents Of The University Of California Methods of generating nucleic acid fragments
US9315825B2 (en) 2010-03-29 2016-04-19 The Trustees Of The University Of Pennsylvania Pharmacologically induced transgene ablation system
WO2011130346A1 (en) 2010-04-13 2011-10-20 Sigma-Aldrich Co. Methods for generating endogenously tagged protein
WO2011146121A1 (en) 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Novel dna-binding proteins and uses thereof
US9512444B2 (en) 2010-07-23 2016-12-06 Sigma-Aldrich Co. Llc Genome editing using targeting endonucleases and single-stranded nucleic acids
CN102358902B (en) * 2011-04-02 2013-01-02 西南大学 Silkworm fibroin heavy-chain gene mutation sequence and mutation method and application
CA3111953C (en) 2011-04-05 2023-10-24 Cellectis Method for the generation of compact tale-nucleases and uses thereof
GB201122458D0 (en) 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
US9637739B2 (en) 2012-03-20 2017-05-02 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
WO2013141680A1 (en) 2012-03-20 2013-09-26 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
WO2013176776A1 (en) 2012-05-23 2013-11-28 Carrier Corporation Wall panel for climate controlled cargo container
CA2876076A1 (en) * 2012-06-12 2013-12-19 Soren WARMING Methods and compositions for generating conditional knock-out alleles
US20150225734A1 (en) 2012-06-19 2015-08-13 Regents Of The University Of Minnesota Gene targeting in plants using dna viruses
EP3808844A1 (en) * 2012-07-25 2021-04-21 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
EP2880171B1 (en) 2012-08-03 2018-10-03 The Regents of The University of California Methods and compositions for controlling gene expression by rna processing
DK2890780T3 (en) * 2012-08-29 2020-09-21 Sangamo Therapeutics Inc METHODS AND COMPOSITIONS FOR TREATING A GENETIC CONDITION
BR112015004995B1 (en) 2012-09-07 2023-05-02 Sangamo Biosciences, Inc. METHOD FOR MODIFYING THE GENOME OF A CELL, USE OF A CELL, SEED OR PLANT OBTAINED BY THE SAID METHOD AND SITE-SPECIFIC ZINC FINGER NUCLEASE
UA118090C2 (en) * 2012-09-07 2018-11-26 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
UA119135C2 (en) 2012-09-07 2019-05-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Engineered transgene integration platform (etip) for gene targeting and trait stacking
DE202013012597U1 (en) 2012-10-23 2017-11-21 Toolgen, Inc. A composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and a Cas protein-encoding nucleic acid or Cas protein, and their use
KR102145760B1 (en) 2012-12-06 2020-08-19 시그마-알드리치 컴퍼니., 엘엘씨 Crispr-based genome modification and regulation
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20140310830A1 (en) 2012-12-12 2014-10-16 Feng Zhang CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
EP2840140B2 (en) 2012-12-12 2023-02-22 The Broad Institute, Inc. Crispr-Cas based method for mutation of prokaryotic cells
EP2931899A1 (en) 2012-12-12 2015-10-21 The Broad Institute, Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
US8889559B2 (en) 2012-12-12 2014-11-18 Micron Technology, Inc. Methods of forming a pattern on a substrate
EP2931892B1 (en) 2012-12-12 2018-09-12 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
DK3064585T3 (en) 2012-12-12 2020-04-27 Broad Inst Inc DESIGN AND OPTIMIZATION OF IMPROVED SYSTEMS, PROCEDURES AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION
SG10201707569YA (en) 2012-12-12 2017-10-30 Broad Inst Inc Delivery, Engineering and Optimization of Systems, Methods and Compositions for Sequence Manipulation and Therapeutic Applications
ES2553782T3 (en) 2012-12-12 2015-12-11 The Broad Institute, Inc. Systems engineering, methods and guide compositions optimized for sequence manipulation
EP3031921A1 (en) 2012-12-12 2016-06-15 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
DK2931898T3 (en) 2012-12-12 2016-06-20 Massachusetts Inst Technology CONSTRUCTION AND OPTIMIZATION OF SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS
CA3081054A1 (en) 2012-12-17 2014-06-26 President And Fellows Of Harvard College Rna-guided human genome engineering
ES2953523T3 (en) * 2012-12-27 2023-11-14 Keygene Nv Method for inducing a directed translocation in a plant
CN111454951A (en) * 2013-03-14 2020-07-28 卡里布生物科学公司 Compositions and methods for nucleic acid-targeted nucleic acids
US20140273235A1 (en) 2013-03-15 2014-09-18 Regents Of The University Of Minnesota ENGINEERING PLANT GENOMES USING CRISPR/Cas SYSTEMS
US20140273230A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
EP2999788B1 (en) * 2013-05-22 2020-07-08 Northwestern University Rna-directed dna cleavage and gene editing by cas9 enzyme from neisseria meningitidis
US20150067922A1 (en) * 2013-05-30 2015-03-05 The Penn State Research Foundation Gene targeting and genetic modification of plants via rna-guided genome editing
SG11201509962QA (en) * 2013-06-04 2016-01-28 Harvard College Rna-guideded transcriptional regulation
DK3011029T3 (en) 2013-06-17 2020-03-16 Broad Inst Inc ADMINISTRATION, MODIFICATION AND OPTIMIZATION OF TANDEM GUIDE SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION
MX2015017312A (en) 2013-06-17 2017-04-10 Broad Inst Inc Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy.
WO2014204723A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Oncogenic models based on delivery and use of the crispr-cas systems, vectors and compositions
WO2014204725A1 (en) * 2013-06-17 2014-12-24 The Broad Institute Inc. Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation
EP3597755A1 (en) 2013-06-17 2020-01-22 The Broad Institute, Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
KR20160019553A (en) 2013-06-17 2016-02-19 더 브로드 인스티튜트, 인코퍼레이티드 Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells
WO2015026887A1 (en) 2013-08-22 2015-02-26 E. I. Du Pont De Nemours And Company A soybean u6 polymerase iii promoter and methods of use
EP3188763B1 (en) * 2014-09-02 2020-05-13 The Regents of The University of California Methods and compositions for rna-directed target dna modification

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140068797A1 (en) * 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Chiu et al in Engineered GFP as a viral reporter in plants (Curr. Biol. Vol 6, No 3, pages 325-330). *
Jinek et al A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity (Science Vol 337, published online June 28, 2012). *
Perez-Rodriquez (Dissertation Abstract first available to public on June 17, 2010). *
Perez-Rodriquez (Dissertation, first available to public on June 17, 2010). *

Cited By (145)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150050699A1 (en) * 2012-03-20 2015-02-19 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US11555187B2 (en) 2012-03-20 2023-01-17 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
US10844378B2 (en) 2012-03-20 2020-11-24 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
US10774344B1 (en) 2012-05-25 2020-09-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10570419B2 (en) 2012-05-25 2020-02-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11814645B2 (en) 2012-05-25 2023-11-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982230B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11674159B2 (en) 2012-05-25 2023-06-13 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11634730B2 (en) 2012-05-25 2023-04-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982231B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988782B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11549127B2 (en) 2012-05-25 2023-01-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10415061B2 (en) 2012-05-25 2019-09-17 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10000772B2 (en) 2012-05-25 2018-06-19 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10752920B2 (en) 2012-05-25 2020-08-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988780B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10113167B2 (en) 2012-05-25 2018-10-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11479794B2 (en) 2012-05-25 2022-10-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11473108B2 (en) 2012-05-25 2022-10-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10676759B2 (en) 2012-05-25 2020-06-09 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11401532B2 (en) 2012-05-25 2022-08-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10669560B2 (en) 2012-05-25 2020-06-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10227611B2 (en) 2012-05-25 2019-03-12 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10640791B2 (en) 2012-05-25 2020-05-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11332761B2 (en) 2012-05-25 2022-05-17 The Regenis of Wie University of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11001863B2 (en) 2012-05-25 2021-05-11 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10266850B2 (en) 2012-05-25 2019-04-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10626419B2 (en) 2012-05-25 2020-04-21 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11293034B2 (en) 2012-05-25 2022-04-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11274318B2 (en) 2012-05-25 2022-03-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10301651B2 (en) 2012-05-25 2019-05-28 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10308961B2 (en) 2012-05-25 2019-06-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11242543B2 (en) 2012-05-25 2022-02-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10337029B2 (en) 2012-05-25 2019-07-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10351878B2 (en) 2012-05-25 2019-07-16 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358659B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358658B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10385360B2 (en) 2012-05-25 2019-08-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10400253B2 (en) 2012-05-25 2019-09-03 The Regents Of The University Of California Methods and compositions or RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10407697B2 (en) 2012-05-25 2019-09-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10612045B2 (en) 2012-05-25 2020-04-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10793878B1 (en) 2012-05-25 2020-10-06 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10421980B2 (en) 2012-05-25 2019-09-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11186849B2 (en) 2012-05-25 2021-11-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10428352B2 (en) 2012-05-25 2019-10-01 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10597680B2 (en) 2012-05-25 2020-03-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10443076B2 (en) 2012-05-25 2019-10-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11028412B2 (en) 2012-05-25 2021-06-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008589B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10487341B2 (en) 2012-05-25 2019-11-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008590B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10513712B2 (en) 2012-05-25 2019-12-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10577631B2 (en) 2012-05-25 2020-03-03 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10519467B2 (en) 2012-05-25 2019-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10526619B2 (en) 2012-05-25 2020-01-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10533190B2 (en) 2012-05-25 2020-01-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10550407B2 (en) 2012-05-25 2020-02-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10563227B2 (en) 2012-05-25 2020-02-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10900054B2 (en) 2012-05-25 2021-01-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10731181B2 (en) 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
US10745716B2 (en) 2012-12-06 2020-08-18 Sigma-Aldrich Co. Llc CRISPR-based genome modification and regulation
US11535863B2 (en) 2012-12-17 2022-12-27 President And Fellows Of Harvard College RNA-guided human genome engineering
US10717990B2 (en) 2012-12-17 2020-07-21 President And Fellows Of Harvard College RNA-guided human genome engineering
US20160002670A1 (en) * 2012-12-17 2016-01-07 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
US11359211B2 (en) 2012-12-17 2022-06-14 President And Fellows Of Harvard College RNA-guided human genome engineering
US11365429B2 (en) 2012-12-17 2022-06-21 President And Fellows Of Harvard College RNA-guided human genome engineering
US9970024B2 (en) 2012-12-17 2018-05-15 President And Fellows Of Harvard College RNA-guided human genome engineering
US11512325B2 (en) 2012-12-17 2022-11-29 President And Fellows Of Harvard College RNA-guided human genome engineering
US10273501B2 (en) * 2012-12-17 2019-04-30 President And Fellows Of Harvard College RNA-guided human genome engineering
US11236359B2 (en) 2012-12-17 2022-02-01 President And Fellows Of Harvard College RNA-guided human genome engineering
US10435708B2 (en) 2012-12-17 2019-10-08 President And Fellows Of Harvard College RNA-guided human genome engineering
US11312953B2 (en) 2013-03-14 2022-04-26 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US20160186208A1 (en) * 2013-04-16 2016-06-30 Whitehead Institute For Biomedical Research Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
US20160168592A1 (en) * 2013-07-09 2016-06-16 President And Fellows Of Harvard College Multiplex RNA-Guided Genome Engineering
US11459585B2 (en) * 2013-07-09 2022-10-04 President And Fellows Of Harvard College Multiplex RNA-guided genome engineering
US10519457B2 (en) 2013-08-22 2019-12-31 E I Du Pont De Nemours And Company Soybean U6 polymerase III promoter and methods of use
US11773400B2 (en) 2013-08-22 2023-10-03 E.I. Du Pont De Nemours And Company Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof
US11390887B2 (en) 2013-11-07 2022-07-19 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US10640788B2 (en) 2013-11-07 2020-05-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAs
US10190137B2 (en) 2013-11-07 2019-01-29 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US20210147879A1 (en) * 2013-11-19 2021-05-20 President And Fellows Of Harvard College Large Gene Excision and Insertion
US10286084B2 (en) 2014-02-18 2019-05-14 Duke University Compositions for the inactivation of virus replication and methods of making and using the same
US20150376587A1 (en) * 2014-06-25 2015-12-31 Caribou Biosciences, Inc. RNA Modification to Engineer Cas9 Activity
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10993419B2 (en) 2014-12-10 2021-05-04 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US11234418B2 (en) 2014-12-10 2022-02-01 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10278372B2 (en) 2014-12-10 2019-05-07 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10450576B2 (en) 2015-03-27 2019-10-22 E I Du Pont De Nemours And Company Soybean U6 small nuclear RNA gene promoters and their use in constitutive expression of small RNA genes in plants
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11549126B2 (en) 2015-06-03 2023-01-10 Board Of Regents Of The University Of Nebraska Treatment methods using DNA editing with single-stranded DNA
US11555208B2 (en) 2015-06-03 2023-01-17 Board Of Regents Of The University Of Nebraska DNA editing using relatively long single-stranded DNA and CRISPR/Cas9 to increase success rate in methods for preparing transgenic embryos and animals
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
US11414657B2 (en) 2015-06-29 2022-08-16 Ionis Pharmaceuticals, Inc. Modified CRISPR RNA and modified single CRISPR RNA and uses thereof
US10166255B2 (en) 2015-07-31 2019-01-01 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11147837B2 (en) 2015-07-31 2021-10-19 Regents Of The University Of Minnesota Modified cells and methods of therapy
US11583556B2 (en) 2015-07-31 2023-02-21 Regents Of The University Of Minnesota Modified cells and methods of therapy
US11642374B2 (en) 2015-07-31 2023-05-09 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
US10406177B2 (en) 2015-07-31 2019-09-10 Regents Of The University Of Minnesota Modified cells and methods of therapy
US11642375B2 (en) 2015-07-31 2023-05-09 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
US11266692B2 (en) 2015-07-31 2022-03-08 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11903966B2 (en) 2015-07-31 2024-02-20 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11925664B2 (en) 2015-07-31 2024-03-12 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
EP3461894A1 (en) 2015-08-07 2019-04-03 Caribou Biosciences, Inc. Engineered crispr-cas9 compositions and methods of use
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11149281B2 (en) 2015-10-06 2021-10-19 Institute For Basic Science Method for producing genome-modified plants from plant protoplasts at high efficiency
US9957490B1 (en) 2015-10-23 2018-05-01 Caribou Biosciences, Inc. Cells comprising engineered nucleic-acid targeting nucleic acids
US9816081B1 (en) 2015-10-23 2017-11-14 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10196619B1 (en) 2015-10-23 2019-02-05 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10711258B2 (en) 2015-10-23 2020-07-14 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US9677090B2 (en) 2015-10-23 2017-06-13 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10501728B2 (en) 2015-10-23 2019-12-10 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US9745562B2 (en) 2015-10-23 2017-08-29 Caribou Biosciences, Inc. Methods of using engineered nucleic-acid targeting nucleic acids
US10023853B1 (en) 2015-10-23 2018-07-17 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10125354B1 (en) 2015-10-23 2018-11-13 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10138472B2 (en) 2015-10-23 2018-11-27 Caribou Biosciences, Inc. Engineered nucleic-acid targeting nucleic acids
US10100333B2 (en) 2015-12-04 2018-10-16 Caribou Biosciences, Inc. Engineered nucleic acid-targeting nucleic acids
US9771600B2 (en) 2015-12-04 2017-09-26 Caribou Biosciences, Inc. Engineered nucleic acid-targeting nucleic acids
US11505808B2 (en) 2015-12-04 2022-11-22 Caribou Biosciences, Inc. Engineered nucleic acid-targeting nucleic acids
US9970029B1 (en) 2015-12-04 2018-05-15 Caribou Biosciences, Inc. Engineered nucleic acid-targeting nucleic acids
US11111287B2 (en) 2016-01-11 2021-09-07 The Board Of Trustees Of The Leland Stanford Junior University Chimeric proteins and methods of immunotherapy
US10336807B2 (en) 2016-01-11 2019-07-02 The Board Of Trustees Of The Leland Stanford Junior University Chimeric proteins and methods of immunotherapy
US11773411B2 (en) 2016-01-11 2023-10-03 The Board Of Trustees Of The Leland Stanford Junior University Chimeric proteins and methods of regulating gene expression
US9856497B2 (en) 2016-01-11 2018-01-02 The Board Of Trustee Of The Leland Stanford Junior University Chimeric proteins and methods of regulating gene expression
US10457961B2 (en) 2016-01-11 2019-10-29 The Board Of Trustees Of The Leland Stanford Junior University Chimeric proteins and methods of regulating gene expression
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11078481B1 (en) 2016-08-03 2021-08-03 KSQ Therapeutics, Inc. Methods for screening for cancer targets
US11912987B2 (en) 2016-08-03 2024-02-27 KSQ Therapeutics, Inc. Methods for screening for cancer targets
US11078483B1 (en) 2016-09-02 2021-08-03 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
US11946163B2 (en) 2016-09-02 2024-04-02 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
US11154574B2 (en) 2016-10-18 2021-10-26 Regents Of The University Of Minnesota Tumor infiltrating lymphocytes and methods of therapy
US10912797B2 (en) 2016-10-18 2021-02-09 Intima Bioscience, Inc. Tumor infiltrating lymphocytes and methods of therapy
US11466271B2 (en) 2017-02-06 2022-10-11 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US10428319B2 (en) 2017-06-09 2019-10-01 Editas Medicine, Inc. Engineered Cas9 nucleases
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11713452B2 (en) 2017-09-08 2023-08-01 University Of North Texas Health Science Center Engineered CAS9 variants
WO2019051419A1 (en) * 2017-09-08 2019-03-14 University Of North Texas Health Science Center Engineered cas9 variants
US11572574B2 (en) 2017-09-28 2023-02-07 Toolgen Incorporated Artificial genome manipulation for gene expression regulation
US11807878B2 (en) 2018-12-14 2023-11-07 Pioneer Hi-Bred International, Inc. CRISPR-Cas systems for genome editing
US10934536B2 (en) 2018-12-14 2021-03-02 Pioneer Hi-Bred International, Inc. CRISPR-CAS systems for genome editing
US20210054371A1 (en) * 2019-08-19 2021-02-25 Minghong Zhong Conjugates of Guide RNA-Cas Protein Complex

Also Published As

Publication number Publication date
CN110066775A (en) 2019-07-30
SG10201809566SA (en) 2018-11-29
CN105441440B (en) 2020-12-15
HK1212732A1 (en) 2016-06-17
CN110643600A (en) 2020-01-03
EP3733847B1 (en) 2022-06-01
US20240052356A1 (en) 2024-02-15
KR101656237B1 (en) 2016-09-12
KR20150101478A (en) 2015-09-03
EP3372679A1 (en) 2018-09-12
AU2013335451A1 (en) 2015-05-07
EP3346003B1 (en) 2021-06-09
SG10201809564YA (en) 2018-11-29
AU2017254854A1 (en) 2017-11-16
ES2926021T3 (en) 2022-10-21
US20240026367A1 (en) 2024-01-25
CA2888190A1 (en) 2014-05-01
AU2017254854B2 (en) 2019-12-05
HK1223125A1 (en) 2017-07-21
JP2023166401A (en) 2023-11-21
US20150322457A1 (en) 2015-11-12
CN110592089B (en) 2022-11-01
US10851380B2 (en) 2020-12-01
HK1257301A1 (en) 2019-10-18
CN110669758A (en) 2020-01-10
KR20150101446A (en) 2015-09-03
DE202013012597U1 (en) 2017-11-21
EP2912175B1 (en) 2018-08-22
DE202013012610U1 (en) 2017-11-24
CN105441440A (en) 2016-03-30
DK2912175T3 (en) 2018-10-22
KR101706085B1 (en) 2017-02-14
EP4180526A2 (en) 2023-05-17
EP3733847A1 (en) 2020-11-04
BR112015008708A2 (en) 2017-09-26
KR20150101476A (en) 2015-09-03
US20210047648A1 (en) 2021-02-18
US20210047647A1 (en) 2021-02-18
EP2912175A4 (en) 2015-12-09
CN116064532A (en) 2023-05-05
JP2018019698A (en) 2018-02-08
KR20150101477A (en) 2015-09-03
CN104968784A (en) 2015-10-07
JP2023166400A (en) 2023-11-21
CN110592089A (en) 2019-12-20
CN110066775B (en) 2024-03-19
CN116064533A (en) 2023-05-05
EP2912175A1 (en) 2015-09-02
CN110669746A (en) 2020-01-10
US20230374525A1 (en) 2023-11-23
CA3233048A1 (en) 2014-05-01
PT2912175T (en) 2018-11-05
JP6517143B2 (en) 2019-05-22
EP3346003A1 (en) 2018-07-11
JP2022095663A (en) 2022-06-28
KR102052286B1 (en) 2019-12-06
ES2886448T3 (en) 2021-12-20
WO2014065596A1 (en) 2014-05-01
EP4180526A3 (en) 2023-06-14
US20230374524A1 (en) 2023-11-23
CN104968784B (en) 2019-11-05
JP2016027807A (en) 2016-02-25
HK1257302A1 (en) 2019-10-18
EP4342987A2 (en) 2024-03-27
SG11201503059XA (en) 2015-06-29
JP2020078303A (en) 2020-05-28
KR101656236B1 (en) 2016-09-12
US20150344912A1 (en) 2015-12-03
KR20180029092A (en) 2018-03-19
AU2013335451B2 (en) 2016-09-15
PL2912175T3 (en) 2019-03-29
ES2690386T3 (en) 2018-11-20
KR102182847B1 (en) 2020-11-27
AU2022204902A1 (en) 2022-08-18
JP2016500003A (en) 2016-01-07
AU2020201485A1 (en) 2020-03-19
AU2015218519A1 (en) 2015-09-17

Similar Documents

Publication Publication Date Title
US20240052356A1 (en) Cas9/RNA Complexes for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells
KR20190137932A (en) Composition for cleaving a target DNA comprising a guideRNA specific for the target DNA and Cas protein-encoding nucleicacid or Cas protein, and use thereof

Legal Events

Date Code Title Description
AS Assignment

Owner name: TOOLGEN INCORPORATED, KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIM, JIN-SOO;CHO, SEUNG WOO;KIM, SOJUNG;AND OTHERS;SIGNING DATES FROM 20150515 TO 20150520;REEL/FRAME:035733/0913

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION