US20140356956A1 - RNA-Guided Transcriptional Regulation - Google Patents

RNA-Guided Transcriptional Regulation Download PDF

Info

Publication number
US20140356956A1
US20140356956A1 US14/319,530 US201414319530A US2014356956A1 US 20140356956 A1 US20140356956 A1 US 20140356956A1 US 201414319530 A US201414319530 A US 201414319530A US 2014356956 A1 US2014356956 A1 US 2014356956A1
Authority
US
United States
Prior art keywords
nucleic acid
dna
cell
rnas
double stranded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US14/319,530
Other languages
English (en)
Inventor
George M. Church
Prashant G. MALI
Kevin M. Esvelt
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.)
Harvard University
Original Assignee
Harvard University
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=52008736&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20140356956(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Harvard University filed Critical Harvard University
Priority to US14/319,530 priority Critical patent/US20140356956A1/en
Assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE reassignment PRESIDENT AND FELLOWS OF HARVARD COLLEGE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHURCH, GEORGE M., ESVELT, KEVIN M., MALI, Prashant
Publication of US20140356956A1 publication Critical patent/US20140356956A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: HARVARD UNIVERSITY
Priority to US15/049,451 priority patent/US10640789B2/en
Priority to US16/441,209 priority patent/US10767194B2/en
Priority to US16/851,360 priority patent/US20200299732A1/en
Priority to US17/972,885 priority patent/US11981917B2/en
Priority to US18/425,219 priority patent/US20240175057A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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)
    • 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
    • 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)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • 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)

Definitions

  • CRISPR RNA crRNA fused to a normally trans-encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA.
  • trans-activating CRISPR RNA a normally trans-encoded tracrRNA fused to a normally trans-encoded tracrRNA
  • DNA binding proteins within the scope of the present disclosure include a protein that forms a complex with the guide RNA and with the guide RNA guiding the complex to a double stranded DNA sequence wherein the complex binds to the DNA sequence.
  • This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA.
  • a DNA binding protein-guide RNA complex may be used to localize a transcriptional regulator protein or domain at target DNA so as to regulate expression of target DNA.
  • a method of modulating expression of a target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucle
  • the foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein further encodes the transcriptional regulator protein or domain fused to the RNA guided nuclease-null DNA binding protein.
  • the foreign nucleic acid encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell or an animal cell.
  • the cell is a mammalian cell.
  • the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides.
  • the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition. According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
  • the one or more RNAs is a guide RNA.
  • the one or more RNAs is a tracrRNA-crRNA fusion.
  • the guide RNA includes a spacer sequence and a tracer mate sequence.
  • the guide RNA may also include a tracr sequence, a portion of which hybridizes to the tracr mate sequence.
  • the guide RNA may also include a linker nucleic acid sequence which links the tracer mate sequence and the tracr sequence to produce the tracrRNA-crRNA fusion.
  • the spacer sequence binds to target DNA, such as by hybridization.
  • the guide RNA includes a truncated spacer sequence. According to one aspect, the guide RNA includes a truncated spacer sequence having a 1 base truncation at the 5′ end of the spacer sequence. According to one aspect, the guide RNA includes a truncated spacer sequence having a 2 base truncation at the 5′ end of the spacer sequence. According to one aspect, the guide RNA includes a truncated spacer sequence having a 3 base truncation at the 5′ end of the spacer sequence. According to one aspect, the guide RNA includes a truncated spacer sequence having a 4 base truncation at the 5′ end of the spacer sequence. Accordingly, the spacer sequence may have a 1 to 4 base truncation at the 5′ end of the spacer sequence.
  • the spacer sequence may include between about 16 to about 20 nucleotides which hybridize to the target nucleic acid sequence. According to certain embodiments, the spacer sequence may include about 20 nucleotides which hybridize to the target nucleic acid sequence.
  • the linker nucleic acid sequence may include between about 4 and about 6 nucleic acids.
  • the tracr sequence may include between about 60 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 64 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 65 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 66 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 67 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 68 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 69 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 70 to about 500 nucleic acids.
  • the tracr sequence may include between about 80 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 90 to about 500 nucleic acids. According to certain aspects, the tracr sequence may include between about 100 to about 500 nucleic acids.
  • the tracr sequence may include between about 60 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 64 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 65 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 66 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 67 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 68 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 69 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 70 to about 200 nucleic acids.
  • the tracr sequence may include between about 80 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 90 to about 200 nucleic acids. According to certain aspects, the tracr sequence may include between about 100 to about 200 nucleic acids.
  • FIG. 5B An exemplary guide RNA is depicted in FIG. 5B .
  • the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
  • a method of modulating expression of a target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein of a Type II CRISPR System that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein of a Type II CRISPR System, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein of a Type II CRISPR System and the transcriptional regulator protein or domain are expressed, where
  • the foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein of a Type II CRISPR System further encodes the transcriptional regulator protein or domain fused to the RNA guided nuclease-null DNA binding protein of a Type II CRISPR System.
  • the foreign nucleic acid encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell or an animal cell.
  • the cell is a mammalian cell.
  • the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides.
  • the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition. According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
  • the one or more RNAs is a guide RNA. According to one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.
  • the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
  • a method of modulating expression of a target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid), wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.
  • RNAs ribon
  • the foreign nucleic acid encoding a nuclease-null Cas9 protein further encodes the transcriptional regulator protein or domain fused to the nuclease-null Cas9 protein.
  • the foreign nucleic acid encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell or an animal cell.
  • the cell is a mammalian cell.
  • the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides.
  • the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition. According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
  • the one or more RNAs is a guide RNA. According to one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.
  • the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
  • a cell includes a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes a target nucleic acid, a second foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein, and a third foreign nucleic acid encoding a transcriptional regulator protein or domain wherein the one or more RNAs, the RNA guided nuclease-null DNA binding protein and the transcriptional regulator protein or domain are members of a co-localization complex for the target nucleic acid.
  • the foreign nucleic acid encoding an RNA guided nuclease-null DNA binding protein further encodes the transcriptional regulator protein or domain fused to an RNA guided nuclease-null DNA binding protein.
  • the foreign nucleic acid encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell or an animal cell.
  • the cell is a mammalian cell.
  • the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides.
  • the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid to treat a disease or detrimental condition. According to one aspect, the target nucleic acid is associated with a disease or detrimental condition.
  • the one or more RNAs is a guide RNA. According to one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.
  • the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
  • the RNA guided nuclease-null DNA binding protein is an RNA guided nuclease-null DNA binding protein of a Type II CRISPR System. According to certain aspects, the RNA guided nuclease-null DNA binding protein is a nuclease-null Cas9 protein.
  • a method of altering a DNA target nucleic acid in a cell includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase and being guided by the two or more RNAs, wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase are expressed and wherein the at least one RNA guided DNA binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks.
  • a method of altering a DNA target nucleic acid in a cell includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase of a Type II CRISPR System and being guided by the two or more RNAs, wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System are expressed and wherein the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks.
  • a method of altering a DNA target nucleic acid in a cell includes introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one Cas9 protein nickase having one inactive nuclease domain and being guided by the two or more RNAs, wherein the two or more RNAs and the at least one Cas9 protein nickase are expressed and wherein the at least one Cas9 protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks.
  • the two or more adjacent nicks are on the same strand of the double stranded DNA.
  • the two or more adjacent nicks are on the same strand of the double stranded DNA and result in homologous recombination.
  • the two or more adjacent nicks are on different strands of the double stranded DNA.
  • the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks.
  • the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in nonhomologous end joining.
  • the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks resulting in nonhomologous end joining.
  • the method further includes introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence wherein the two or more nicks results in homologous recombination of the target nucleic acid with the donor nucleic acid sequence.
  • a method of altering a DNA target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase and being guided by the two or more RNAs, and wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase are expressed and wherein the at least one RNA guided DNA binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks, and wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in fragmentation of the target nucleic acid thereby preventing expression of the target nucleic acid
  • a method of altering a DNA target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase of a Type II CRISPR system and being guided by the two or more RNAs, and wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System are expressed and wherein the at least one RNA guided DNA binding protein nickase of a Type II CRISPR System co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks, and wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double strand
  • a method of altering a DNA target nucleic acid in a cell including introducing into the cell a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in the DNA target nucleic acid, introducing into the cell a second foreign nucleic acid encoding at least one Cas9 protein nickase having one inactive nuclease domain and being guided by the two or more RNAs, and wherein the two or more RNAs and the at least one Cas9 protein nickase are expressed and wherein the at least one Cas9 protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks, and wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in fragmentation of the target nucleic acid thereby preventing expression of the target
  • a cell including a first foreign nucleic acid encoding two or more RNAs with each RNA being complementary to an adjacent site in a DNA target nucleic acid, and a second foreign nucleic acid encoding at least one RNA guided DNA binding protein nickase, and wherein the two or more RNAs and the at least one RNA guided DNA binding protein nickase are members of a co-localization complex for the DNA target nucleic acid.
  • the RNA guided DNA binding protein nickase is an RNA guided DNA binding protein nickase of a Type II CRISPR System. According to one aspect, the RNA guided DNA binding protein nickase is a Cas9 protein nickase having one inactive nuclease domain.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell or an animal cell.
  • the cell is a mammalian cell.
  • the RNA includes between about 10 to about 500 nucleotides. According to one aspect, the RNA includes between about 20 to about 100 nucleotides.
  • the target nucleic acid is associated with a disease or detrimental condition.
  • the two or more RNAs are guide RNAs. According to one aspect, the two or more RNAs are tracrRNA-crRNA fusions.
  • the DNA target nucleic acid_ is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
  • FIG. 1A and FIG. 1B are schematics of RNA-guided transcriptional activation.
  • FIG. 1C is a design of a reporter construct (SEQ ID NOs:62 and 63).
  • FIG. 1D shows data demonstrating that Cas9N-VP64 fusions display RNA-guided transcriptional activation as assayed by both fluorescence-activated cell sorting (FACS) and immunofluorescence assays (IF).
  • FIG. 1E shows assay data by FACS and IF demonstrating gRNA sequence-specific transcriptional activation from reporter constructs in the presence of Cas9N, MS2-VP64 and gRNA bearing the appropriate MS2 aptamer binding sites.
  • FIG. 1F depicts data demonstrating transcriptional induction by individual gRNAs and multiple gRNAs.
  • FIG. 2A depicts a methodology for evaluating the landscape of targeting by Cas9-gRNA complexes and TALEs.
  • FIG. 2B depicts data demonstrating that a Cas9-gRNA complex is on average tolerant to 1-3 mutations in its target sequences.
  • FIG. 2C depicts data demonstrating that the Cas9-gRNA complex is largely insensitive to point mutations, except those localized to the PAM sequence.
  • FIG. 2D depicts heat plot data demonstrating that introduction of 2 base mismatches significantly impairs the Cas9-gRNA complex activity.
  • FIG. 2E depicts data demonstrating that an 18-mer TALE reveals is on average tolerant to 1-2 mutations in its target sequence.
  • FIG. 2F depicts data demonstrating the 18-mer TALE is, similar to the Cas9-gRNA complexes, largely insensitive to single base mismatched in its target.
  • FIG. 2G depicts heat plot data demonstrating that introduction of 2 base mismatches significantly impairs the 18-mer TALE activity.
  • FIG. 3A depicts a schematic of a guide RNA design.
  • FIG. 3B depicts data showing percentage rate of non-homologous end joining for off-set nicks leading to 5′ overhangs and off-set nicks leading to 3′ overhangs.
  • FIG. 3C depicts data showing percentage rate of targeting for off-set nicks leading to 5′ overhangs and off-set nicks leading to 3′ overhangs.
  • FIG. 4A is a schematic of a metal coordinating residue in RuvC PDB ID: 4EP4 (blue) position D7 (left), a schematic of HNH endonuclease domains from PDB IDs: 3M7K (orange) and 4H9D (cyan) including a coordinated Mg-ion (gray sphere) and DNA from 3M7K (purple) (middle) and a list of mutants analyzed (right).
  • FIG. 4B depicts data showing undetectable nuclease activity for Cas9 mutants m3 and m4, and also their respective fusions with VP64.
  • FIG. 4C is a higher-resolution examination of the data in FIG. 4B .
  • FIG. 5A is a schematic of a homologous recombination assay to determine Cas9-gRNA activity (SEQ ID NO:64).
  • FIG. 5B depicts guide RNAs with random sequence insertions and percentage rate of homologous recombination (SEQ ID NOs:65 and 66).
  • FIG. 6A is a schematic of guide RNAs for the OCT4 gene.
  • FIG. 6B depicts transcriptional activation for a promoter-luciferase reporter construct.
  • FIG. 6C depicts transcriptional activation via qPCR of endogenous genes.
  • FIG. 7A is a schematic of guide RNAs for the REX1 gene.
  • FIG. 7B depicts transcriptional activation for a promoter-luciferase reporter construct.
  • FIG. 7C depicts transcriptional activation via qPCR of endogenous genes.
  • FIG. 8A depicts in schematic a high level specificity analysis processing flow for calculation of normalized expression levels.
  • FIG. 8B depicts data of distributions of percentages of binding sites by numbers of mismatches generated within a biased construct library. Left: Theoretical distribution. Right: Distribution observed from an actual TALE construct library.
  • FIG. 8C depicts data of distributions of percentages of tag counts aggregated to binding sites by numbers of mismatches. Left: Distribution observed from the positive control sample. Right: Distribution observed from a sample in which a non-control TALE was induced.
  • FIG. 9A depicts data for analysis of the targeting landscape of a Cas9-gRNA complex showing tolerance to 1-3 mutations in its target sequence.
  • FIG. 9B depicts data for analysis of the targeting landscape of a Cas9-gRNA complex showing insensitivity to point mutations, except those localized to the PAM sequence.
  • FIG. 9C depicts heat plot data for analysis of the targeting landscape of a Cas9-gRNA complex showing that introduction of 2 base mismatches significantly impairs activity.
  • FIG. 9D depicts data from a nuclease mediated HR assay confirming that the predicted PAM for the S. pyogenes Cas9 is NGG and also NAG (SEQ ID NOs:67-69).
  • FIG. 10A depicts data from a nuclease mediated HR assay confirming that 18-mer TALEs tolerate multiple mutations in their target sequences (SEQ ID NOs:70-73).
  • FIG. 10B depicts data from analysis of the targeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-mer).
  • FIG. 10C depicts data for 10-mer TALEs show near single-base mismatch resolution.
  • FIG. 10D depicts heat plot data for 10-mer TALEs show near single-base mismatch resolution.
  • FIG. 11A depicts designed guide RNAs.
  • FIG. 11B depicts percentage rate of non-homologous end joining for various guide RNAs (SEQ ID NOs:74-87).
  • FIG. 10A depicts data from a nuclease mediated HR assay confirming that 18-mer TALEs tolerate multiple mutations in their target sequences.
  • FIG. 10B depicts data from analysis of the targeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-mer).
  • FIG. 10C depicts data for 10-mer TALEs show near single-base mismatch resolution.
  • FIG. 10D depicts heat plot data for 10-mer TALEs show near single-base mismatch resolution.
  • FIG. 11A depicts designed guide RNAs.
  • FIG. 11B depicts percentage rate of non-homologous end joining for various guide RNAs.
  • FIG. 12A depicts the Sox2 gene.
  • FIG. 12B depicts the Nanog gene.
  • FIGS. 13A-13F depict the targeting landscape of two additional Cas9-gRNA complexes.
  • FIG. 14A depicts the specificity profile of two gRNAs (wild-type (SEQ ID NO:88) and mutants (SEQ ID NOs:89-90). Sequence differences are highlighted in red.
  • FIGS. 15A-15D depict gRNA2 ( FIG. 15A-B ) and gRNA3 ( FIG. 15C-D ) bearing single or double-base mismatches (highlighted in red) in the spacer sequence versus the target. Sequences are set forth as SEQ ID NOs:91-131.
  • FIGS. 16A-16D depict a nuclease assay of two independent gRNA that were tested: gRNA1 ( FIG. 16A-B ) and gRNA3 ( FIG. 16C-D ) bearing truncations at the 5′ end of their spacer. Sequences are set forth as SEQ ID NOs:66, 185-186 and 133-140.
  • FIGS. 17A-17B depict a nuclease mediated HR assay that shows the PAM for the S. pyogenes Cas9 is NGG and also NAG. Sequences are set forth as SEQ ID NOs:67-69 and 141.
  • FIGS. 18A-18B depict a nuclease mediated HR assay that confirmed that 18-mer TALEs tolerate multiple mutations in their target sequences. Sequences are set forth as SEQ ID NOs:70-73.
  • FIGS. 19A-19C depict a comparison of TALE monomer specificity versus TALE protein specificity. Sequences are set forth as SEQ ID NOs:142-150.
  • FIGS. 20A-20B depict data related to off-set nicking. Sequences are set forth as SEQ ID NOs:151-158.
  • FIGS. 21A-21C depict off-set nicking and NHEJ profiles. Sequences are set forth as SEQ ID NOs:159-184.
  • Embodiments of the present disclosure are based on the use of DNA binding proteins to co-localize transcriptional regulator proteins or domains to DNA in a manner to regulate a target nucleic acid.
  • DNA binding proteins are readily known to those of skill in the art to bind to DNA for various purposes.
  • DNA binding proteins may be naturally occurring.
  • DNA binding proteins included within the scope of the present disclosure include those which may be guided by RNA, referred to herein as guide RNA.
  • guide RNA and the RNA guided DNA binding protein form a co-localization complex at the DNA.
  • the DNA binding protein may be a nuclease-null DNA binding protein.
  • the nuclease-null DNA binding protein may result from the alteration or modification of a DNA binding protein having nuclease activity.
  • DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems.
  • Cas9 proteins and Type II CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477 including all supplementary information hereby incorporated by reference in its entirety.
  • Exemplary DNA binding proteins having nuclease activity function to nick or cut double stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains with each domain responsible for cutting or nicking a particular strand of the double stranded DNA.
  • Exemplary polypeptide sequences having nuclease activity known to those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
  • exemplary DNA binding proteins are those that in nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
  • the DNA binding protein is altered or otherwise modified to inactivate the nuclease activity.
  • Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain.
  • Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the DNA binding protein.
  • a nuclease-null DNA binding protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the nuclease-null DNA binding protein retains the ability to bind to DNA even though the nuclease activity has been inactivated.
  • the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity.
  • the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may have one or more or all of the nuclease sequences exhibiting nuclease activity inactivated.
  • a DNA binding protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains.
  • a DNA binding protein nickase Such a modified or altered DNA binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding protein cuts or nicks only one strand of double stranded DNA.
  • the DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase.
  • An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System which lacks nuclease activity.
  • An exemplary DNA binding protein is a nuclease-null Cas9 protein.
  • An exemplary DNA binding protein is a Cas9 protein nickase.
  • Cas9 In S. pyogenes , Cas9 generates a blunt-ended double-stranded break 3 bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand.
  • PAM protospacer-adjacent motif
  • the Cas9 protein may be referred by one of skill in the art in the literature as Csn1.
  • the S. pyogenes Cas9 protein sequence that is the subject of experiments described herein is shown below. See Deltcheva et al., Nature 471, 602-607 (2011) hereby incorporated by reference in its entirety.
  • Cas9 is altered to reduce, substantially reduce or eliminate nuclease activity.
  • Cas9 nuclease activity is reduced, substantially reduced or eliminated by altering the RuvC nuclease domain or the HNH nuclease domain.
  • the RuvC nuclease domain is inactivated.
  • the HNH nuclease domain is inactivated.
  • the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • Cas9 proteins are provided where the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • nuclease-null Cas9 proteins are provided insofar as the RuvC nuclease domain and the HNH nuclease domain are inactivated.
  • a Cas9 nickase is provided where either the RuvC nuclease domain or the HNH nuclease domain is inactivated, thereby leaving the remaining nuclease domain active for nuclease activity. In this manner, only one strand of the double stranded DNA is cut or nicked.
  • nuclease-null Cas9 proteins are provided where one or more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins.
  • the amino acids include D10 and H840. See Jinke et al., Science 337, 816-821 (2012).
  • the amino acids include D839 and N863.
  • one or more or all of D10, H840, D839 and H863 are substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity.
  • one or more or all of D10, H840, D839 and H863 are substituted with alanine.
  • a Cas9 protein having one or more or all of D10, H840, D839 and H863 substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity, such as alanine is referred to as a nuclease-null Cas9 or Cas9N and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection.
  • nuclease activity for a Cas9N may be undetectable using known assays, i.e. below the level of detection of known assays.
  • the nuclease null Cas9 protein includes homologs and orthologs thereof which retain the ability of the protein to bind to the DNA and be guided by the RNA.
  • the nuclease null Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. pyogenes and having one or more or all of D10, H840, D839 and H863 substituted with alanine and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.
  • the nuclease null Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. pyogenes excepting the protein sequence of the RuvC nuclease domain and the HNH nuclease domain and also protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.
  • aspects of the present disclosure include the protein sequence responsible for DNA binding, for example, for co-localizing with guide RNA and binding to DNA and protein sequences homologous thereto, and need not include the protein sequences for the RuvC nuclease domain and the HNH nuclease domain (to the extent not needed for DNA binding), as these domains may be either inactivated or removed from the protein sequence of the naturally occurring Cas9 protein to produce a nuclease null Cas9 protein.
  • FIG. 4A depicts metal coordinating residues in known protein structures with homology to Cas9. Residues are labeled based on position in Cas9 sequence.
  • Left RuvC structure
  • PDB ID 4EP4 (blue) position D7, which corresponds to D10 in the Cas9 sequence, is highlighted in a Mg-ion coordinating position.
  • Middle Structures of HNH endonuclease domains from PDB IDs: 3M7K (orange) and 4H9D (cyan) including a coordinated Mg-ion (gray sphere) and DNA from 3M7K (purple).
  • Residues D92 and N113 in 3M7K and 4H9D positions D53 and N77, which have sequence homology to Cas9 amino acids D839 and N863, are shown as sticks.
  • FIG. 4B the Cas9 mutants: m3 and m4, and also their respective fusions with VP64 showed undetectable nuclease activity upon deep sequencing at targeted loci.
  • the plots show the mutation frequency versus genomic position, with the red lines demarcating the gRNA target.
  • FIG. 4C is a higher-resolution examination of the data in FIG. 4B and confirms that the mutation landscape shows comparable profile as unmodified loci.
  • an engineered Cas9-gRNA system which enables RNA-guided genome regulation in human cells by tethering transcriptional activation domains to either a nuclease-null Cas9 or to guide RNAs.
  • one or more transcriptional regulatory proteins or domains are joined or otherwise connected to a nuclease-deficient Cas9 or one or more guide RNA (gRNA).
  • the transcriptional regulatory domains correspond to targeted loci.
  • aspects of the present disclosure include methods and materials for localizing transcriptional regulatory domains to targeted loci by fusing, connecting or joining such domains to either Cas9N or to the gRNA.
  • a Cas9N-fusion protein capable of transcriptional activation is provided.
  • a VP64 activation domain (see Zhang et al., Nature Biotechnology 29, 149-153 (2011) hereby incorporated by reference in its entirety) is joined, fused, connected or otherwise tethered to the C terminus of Cas9N.
  • the transcriptional regulatory domain is provided to the site of target genomic DNA by the Cas9N protein.
  • a Cas9N fused to a transcriptional regulatory domain is provided within a cell along with one or more guide RNAs. The Cas9N with the transcriptional regulatory domain fused thereto bind at or near target genomic DNA.
  • the one or more guide RNAs bind at or near target genomic DNA.
  • the transcriptional regulatory domain regulates expression of the target gene.
  • a Cas9N-VP64 fusion activated transcription of reporter constructs when combined with gRNAs targeting sequences near the promoter, thereby displaying RNA-guided transcriptional activation.
  • a gRNA-fusion protein capable of transcriptional activation is provided.
  • a VP64 activation domain is joined, fused, connected or otherwise tethered to the gRNA.
  • the transcriptional regulatory domain is provided to the site of target genomic DNA by the gRNA.
  • a gRNA fused to a transcriptional regulatory domain is provided within a cell along with a Cas9N protein.
  • the Cas9N binds at or near target genomic DNA.
  • the one or more guide RNAs with the transcriptional regulatory protein or domain fused thereto bind at or near target genomic DNA.
  • the transcriptional regulatory domain regulates expression of the target gene.
  • a Cas9N protein and a gRNA fused with a transcriptional regulatory domain activated transcription of reporter constructs, thereby displaying RNA-guided transcriptional activation.
  • FIG. 5A is a schematic of a homologous recombination (HR) assay to determine Cas9-gRNA activity. As shown in FIG.
  • gRNAs bearing random sequence insertions at either the 5′ end of the crRNA portion or the 3′ end of the tracrRNA portion of a chimeric gRNA retain functionality, while insertions into the tracrRNA scaffold portion of the chimeric gRNA result in loss of function.
  • the points of insertion in the gRNA sequence are indicated by red nucleotides.
  • the increased activity upon random base insertions at the 5′ end may be due to increased half-life of the longer gRNA.
  • FIG. 1A is a schematic of RNA-guided transcriptional activation.
  • the VP64 activation domain was directly tethered to the C terminus of Cas9N.
  • FIG. 1B to generate gRNA tethers capable of transcriptional activation, two copies of the MS2 bacteriophage coat-protein binding RNA stem-loop were appended to the 3′ end of the gRNA. These chimeric gRNAs were expressed together with Cas9N and MS2-VP64 fusion protein.
  • FIG. 1C shows design of reporter constructs used to assay transcriptional activation.
  • the two reporters bear distinct gRNA target sites, and share a control TALE-TF target site.
  • Cas9N-VP64 fusions display RNA-guided transcriptional activation as assayed by both fluorescence-activated cell sorting (FACS) and immunofluorescence assays (IF).
  • FACS fluorescence-activated cell sorting
  • IF immunofluorescence assays
  • the Cas9N-VP64 fusion activates reporters in a gRNA sequence specific manner.
  • FIG. 1E gRNA sequence-specific transcriptional activation from reporter constructs only in the presence of all 3 components: Cas9N, MS2-VP64 and gRNA bearing the appropriate MS2 aptamer binding sites was observed by both FACS and IF.
  • an endogenous gene can be any desired gene, referred to herein as a target gene.
  • genes target for regulation included ZFP42 (REX1) and POU5F1 (OCT4), which are both tightly regulated genes involved in maintenance of pluripotency.
  • ZFP42 REX1
  • POU5F1 OCT4
  • FIG. 1F 10 gRNAs targeting a ⁇ 5 kb stretch of DNA upstream of the transcription start site (DNase hypersensitive sites are highlighted in green) were designed for the REX1 gene.
  • Transcriptional activation was assayed using either a promoter-luciferase reporter construct (see Takahashi et al., Cell 131 861-872 (2007) hereby incorporated by reference in its entirety) or directly via qPCR of the endogenous genes.
  • FIG. 6A-C is directed to RNA-guided OCT4 regulation using Cas9N-VP64.
  • 21 gRNAs targeting a ⁇ 5 kb stretch of DNA upstream of the transcription start site were designed for the OCT4 gene. The DNase hypersensitive sites are highlighted in green.
  • FIG. 6B shows transcriptional activation using a promoter-luciferase reporter construct.
  • FIG. 6C shows transcriptional activation directly via qPCR of the endogenous genes. While introduction of individual gRNAs modestly stimulated transcription, multiple gRNAs acted synergistically to stimulate robust multi-fold transcriptional activation.
  • FIG. 7A-C is directed to RNA-guided REX1 regulation using Cas9N, MS2-VP64 and gRNA+2X-MS2 aptamers.
  • 10 gRNAs targeting a ⁇ 5 kb stretch of DNA upstream of the transcription start site were designed for the REX1 gene. The DNase hypersensitive sites are highlighted in green.
  • FIG. 7B shows transcriptional activation using a promoter-luciferase reporter construct.
  • FIG. 7C shows transcriptional activation directly via qPCR of the endogenous genes. While introduction of individual gRNAs modestly stimulated transcription, multiple gRNAs acted synergistically to stimulate robust multi-fold transcriptional activation.
  • the absence of the 2X-MS2 aptamers on the gRNA does not result in transcriptional activation. See Maeder et al., Nature Methods 10, 243-245 (2013) and Perez-Pinera et al., Nature Methods 10, 239-242 (2013) each of which are hereby incorporated by reference in its entirety.
  • methods are directed to the use of multiple guide RNAs with a Cas9N protein and a transcriptional regulatory protein or domain to regulate expression of a target gene.
  • gRNA tethering approach in principle enables different effector domains to be recruited by distinct gRNAs so long as each gRNA uses a different RNA-protein interaction pair. See Karyer-Bibens et al., Biology of the Cell/Under the Auspices of the European Cell Biology Organization 100, 125-138 (2008) hereby incorporated by reference in its entirety.
  • different target genes may be regulated using specific guide RNA and a generic Cas9N protein, i.e. the same or a similar Cas9N protein for different target genes.
  • methods of multiplex gene regulation are provided using the same or similar Cas9N.
  • Methods of the present disclosure are also directed to editing target genes using the Cas9N proteins and guide RNAs described herein to provide multiplex genetic and epigenetic engineering of human cells.
  • Cas9-gRNA targeting being an issue (see Jiang et al., Nature Biotechnology 31, 233-239 (2013) hereby incorporated by reference in its entirety)
  • methods are provided for in-depth interrogation of Cas9 affinity for a very large space of target sequence variations. Accordingly, aspects of the present disclosure provide direct high-throughput readout of Cas9 targeting in human cells, while avoiding complications introduced by dsDNA cut toxicity and mutagenic repair incurred by specificity testing with native nuclease-active Cas9.
  • DNA binding proteins or systems in general for the transcriptional regulation of a target gene.
  • DNA binding systems need not have any nuclease activity, as with the naturally occurring Cas9 protein. Accordingly, such DNA binding systems need not have nuclease activity inactivated.
  • One exemplary DNA binding system is TALE.
  • TALE-FokI dimers are used, and for genome regulation TAEL-VP64 fusions have been shown to be highly effective. According to one aspect, TALE specificity was evaluated using the methodology shown in FIG. 2A .
  • a construct library in which each element of the library comprises a minimal promoter driving a dTomato fluorescent protein is designed. Downstream of the transcription start site m, a 24 bp (A/C/G) random transcript tag is inserted, while two TF binding sites are placed upstream of the promoter: one is a constant DNA sequence shared by all library elements, and the second is a variable feature that bears a ‘biased’ library of binding sites which are engineered to span a large collection of sequences that present many combinations of mutations away from the target sequence the programmable DNA targeting complex was designed to bind.
  • transcription of the dTomato reporter genes is stimulated with either a control-TF engineered to bind the shared DNA site, or the target-TF that was engineered to bind the target site.
  • the abundance of each expressed transcript tag is measured in each sample by conducting RNAseq on the stimulated cells, which is then mapped back to their corresponding binding sites using the association table established earlier.
  • the control-TF is expected to excite all library members equally since its binding site is shared across all library elements, while the target-TF is expected to skew the distribution of the expressed members to those that are preferentially targeted by it. This assumption is used in step 5 to compute a normalized expression level for each binding site by dividing the tag counts obtained for the target-TF by those obtained for the control-TF.
  • the targeting landscape of a Cas9-gRNA complex reveals that it is on average tolerant to 1-3 mutations in its target sequences.
  • the Cas9-gRNA complex is also largely insensitive to point mutations, except those localized to the PAM sequence. Notably this data reveals that the predicted PAM for the S. pyogenes Cas9 is not just NGG but also NAG. As shown in FIG. 2B , the targeting landscape of a Cas9-gRNA complex reveals that it is on average tolerant to 1-3 mutations in its target sequences.
  • the Cas9-gRNA complex is also largely insensitive to point mutations, except those localized to the PAM sequence. Notably this data reveals that the predicted PAM for the S. pyogenes Cas9 is not just NGG but also NAG. As shown in FIG.
  • TALE domains The mutational tolerance of another widely used genome editing tool, TALE domains, was determined using the transcriptional specificity assay described herein.
  • FIG. 2E the TALE off-targeting data for an 18-mer TALE reveals that it can tolerate on average 1-2 mutations in its target sequence, and fails to activate a large majority of 3 base mismatch variants in its targets.
  • FIG. 2F the 18-mer TALE is, similar to the Cas9-gRNA complexes, largely insensitive to single base mismatched in its target.
  • introduction of 2 base mismatches significantly impairs the 18-mer TALE activity. TALE activity is more sensitive to mismatches nearer the 5′ end of its target sequence (in the heat plot the target sequence positions are labeled from 1-18 starting from the 5′ end).
  • Results were confirmed using targeted experiments in a nuclease assay which is the subject of FIG. 10A-C directed to evaluating the landscape of targeting by TALEs of different sizes.
  • FIG. 10A using a nuclease mediated HR assay, it was confirmed that 18-mer TALEs tolerate multiple mutations in their target sequences.
  • FIG. 10B using the approach described in FIG. 2 , the targeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-mer) was analyzed. Shorter TALEs (14-mer and 10-mer) are progressively more specific in their targeting but also reduced in activity by nearly an order of magnitude. As shown in FIGS.
  • FIG. 8A-C is directed to high level specificity analysis processing flow for calculation of normalized expression levels illustrated with examples from experimental data.
  • construct libraries are generated with a biased distribution of binding site sequences and random sequence 24 bp tags that will be incorporated into reporter gene transcripts (top).
  • the transcribed tags are highly degenerate so that they should map many-to-one to Cas9 or TALE binding sequences.
  • the construct libraries are sequenced (3 rd level, left) to establish which tags co-occur with binding sites, resulting in an association table of binding sites vs. transcribed tags (4 th level, left).
  • construct libraries built for different binding sites may be sequenced at once using library barcodes (indicated here by the light blue and light yellow colors; levels 1-4, left).
  • a construct library is then transfected into a cell population and a set of different Cas9/gRNA or TALE transcription factors are induced in samples of the populations (2 nd level, right).
  • One sample is always induced with a fixed TALE activator targeted to a fixed binding site sequence within the construct (top level, green box); this sample serves as a positive control (green sample, also indicated by a + sign).
  • cDNAs generated from the reporter mRNA molecules in the induced samples are then sequenced and analyzed to obtain tag counts for each tag in a sample (3 rd and 4 th level, right).
  • FIG. 8B depicts example distributions of percentages of binding sites by numbers of mismatches generated within a biased construct library. Left: Theoretical distribution. Right: Distribution observed from an actual TALE construct library.
  • FIG. 8C depicts example distributions of percentages of tag counts aggregated to binding sites by numbers of mismatches. Left: Distribution observed from the positive control sample. Right: Distribution observed from a sample in which a non-control TALE was induced. As the positive control TALE binds to a fixed site in the construct, the distribution of aggregated tag counts closely reflects the distribution of binding sites in FIG.
  • binding specificity is increased according to methods described herein. Because synergy between multiple complexes is a factor in target gene activation by Cas9N-VP64, transcriptional regulation applications of Cas9N is naturally quite specific as individual off-target binding events should have minimal effect.
  • off-set nicks are used in methods of genome-editing. A large majority of nicks seldom result in NHEJ events, (see Certo et al., Nature Methods 8, 671-676 (2011) hereby incorporated by reference in its entirety) thus minimizing the effects of off-target nicking.
  • inducing off-set nicks to generate double stranded breaks (DSBs) is highly effective at inducing gene disruption.
  • 5′ overhangs generate more significant NHEJ events as opposed to 3′ overhangs.
  • 3′ overhangs favor HR over NHEJ events, although the total number of HR events is significantly lower than when a 5′ overhang is generated. Accordingly, methods are provided for using nicks for homologous recombination and off-set nicks for generating double stranded breaks to minimize the effects of off-target Cas9-gRNA activity.
  • FIG. 3A-C is directed to multiplex off-set nicking and methods for reducing the off-target binding with the guide RNAs.
  • the traffic light reporter was used to simultaneously assay for HR and NHEJ events upon introduction of targeted nicks or breaks. DNA cleavage events resolved through the HDR pathway restore the GFP sequence, whereas mutagenic NHEJ causes frameshifts rendering the GFP out of frame and the downstream mCherry sequence in frame.
  • FIG. 11A-B is directed to Cas9D10A nickase mediated NHEJ.
  • the traffic light reporter was used to assay NHEJ events upon introduction of targeted nicks or double-stranded breaks. Briefly, upon introduction of DNA cleavage events, if the break goes through mutagenic NHEJ, the GFP is translated out of frame and the downstream mCherry sequences are rendered in frame resulting in red fluorescence. 14 gRNAs covering a 200 bp stretch of DNA: 7 targeting the sense strand (U1-7) and 7 the antisense strand (D1-7) were designed. As shown in FIG.
  • methods are described herein of modulating expression of a target nucleic acid in a cell that include introducing one or more, two or more or a plurality of foreign nucleic acids into the cell.
  • the foreign nucleic acids introduced into the cell encode for a guide RNA or guide RNAs, a nuclease-null Cas9 protein or proteins and a transcriptional regulator protein or domain.
  • a guide RNA, a nuclease-null Cas9 protein and a transcriptional regulator protein or domain are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide RNA, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain bind to DNA and regulate expression of a target nucleic acid.
  • the foreign nucleic acids introduced into the cell encode for a guide RNA or guide RNAs and a Cas9 protein nickase.
  • a guide RNA and a Cas9 protein nickase are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide RNA and the Cas9 protein nickase bind to DNA and nick a target nucleic acid.
  • Cells according to the present disclosure include any cell into which foreign nucleic acids can be introduced and expressed as described herein. It is to be understood that the basic concepts of the present disclosure described herein are not limited by cell type.
  • Cells according to the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archael cells, eubacterial cells and the like.
  • Cells include eukaryotic cells such as yeast cells, plant cells, and animal cells.
  • Particular cells include mammalian cells.
  • cells include any in which it would be beneficial or desirable to regulate a target nucleic acid. Such cells may include those which are deficient in expression of a particular protein leading to a disease or detrimental condition. Such diseases or detrimental conditions are readily known to those of skill in the art.
  • the nucleic acid responsible for expressing the particular protein may be targeted by the methods described herein and a transcriptional activator resulting in upregulation of the target nucleic acid and corresponding expression of the particular protein. In this manner, the methods described herein provide therapeutic treatment.
  • Target nucleic acids include any nucleic acid sequence to which a co-localization complex as described herein can be useful to either regulate or nick.
  • Target nucleic acids include genes.
  • DNA such as double stranded DNA
  • a co-localization complex can bind to or otherwise co-localize with the DNA at or adjacent or near the target nucleic acid and in a manner in which the co-localization complex may have a desired effect on the target nucleic acid.
  • target nucleic acids can include endogenous (or naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids.
  • DNA includes genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.
  • Foreign nucleic acids i.e. those which are not part of a cell's natural nucleic acid composition
  • Methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
  • transfection transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
  • Transcriptional regulator proteins or domains which are transcriptional activators include VP16 and VP64 and others readily identifiable by those skilled in the art based on the present disclosure.
  • Diseases and detrimental conditions are those characterized by abnormal loss of expression of a particular protein. Such diseases or detrimental conditions can be treated by upregulation of the particular protein. Accordingly, methods of treating a disease or detrimental condition are provided where the co-localization complex as described herein associates or otherwise binds to DNA including a target nucleic acid, and the transcriptional activator of the co-localization complex upregulates expression of the target nucleic acid. For example upregulating PRDM16 and other genes promoting brown fat differentiation and increased metabolic uptake can be used to treat metabolic syndrome or obesity. Activating anti-inflammatory genes are useful in autoimmunity and cardiovascular disease. Activating tumor suppressor genes is useful in treating cancer. One of skill in the art will readily identify such diseases and detrimental conditions based on the present disclosure.
  • the Cas9 mutants were generated using the Quikchange kit (Agilent technologies).
  • the target gRNA expression constructs were either (1) directly ordered as individual gBlocks from IDT and cloned into the pCR-BluntII-TOPO vector (Invitrogen); or (2) custom synthesized by Genewiz; or (3) assembled using Gibson assembly of oligonucleotides into the gRNA cloning vector (plasmid #41824).
  • the vectors for the HR reporter assay involving a broken GFP were constructed by fusion PCR assembly of the GFP sequence bearing the stop codon and appropriate fragment assembled into the EGIP lentivector from Addgene (plasmid #26777).
  • TALENs used in this study were constructed using standard protocols. See Sanjana et al., Nature Protocols 7, 171-192 (2012) hereby incorporated by reference in its entirety. Cas9N and MS2 VP64 fusions were performed using standard PCR fusion protocol procedures. The promoter luciferase constructs for OCT4 and REX1 were obtained from Addgene (plasmid #17221 and plasmid #17222).
  • HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). Cells were maintained at 37° C. and 5% CO 2 in a humidified incubator.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • pen/streptomycin penicillin/streptomycin
  • NEAA non-essential amino acids
  • Transfections involving nuclease assays were as follows: 0.4 ⁇ 10 6 cells were transfected with 2 ⁇ g Cas9 plasmid, 2 ⁇ g gRNA and/or 2 ⁇ g DNA donor plasmid using Lipofectamine 2000 as per the manufacturer's protocols. Cells were harvested 3 days after transfection and either analyzed by FACS, or for direct assay of genomic cuts the genomic DNA of ⁇ 1 ⁇ 10 6 cells was extracted using DNAeasy kit (Qiagen). For these PCR was conducted to amplify the targeting region with genomic DNA derived from the cells and amplicons were deep sequenced by MiSeq Personal Sequencer (Illumina) with coverage >200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiencies.
  • transfections involving transcriptional activation assays 0.4 ⁇ 10 6 cells were transfected with (1) 2 ⁇ g Cas9N-VP64 plasmid, 2 ⁇ g gRNA and/or 0.25 ⁇ g of reporter construct; or (2) 2 ⁇ g Cas9N plasmid, 2 ⁇ g MS2-VP64, 2 ⁇ g gRNA-2XMS2aptamer and/or 0.25 ⁇ g of reporter construct.
  • Cells were harvested 24-48 hrs post transfection and assayed using FACS or immunofluorescence methods, or their total RNA was extracted and these were subsequently analyzed by RT-PCR.
  • standard taqman probes from Invitrogen for OCT4 and REX1 were used, with normalization for each sample performed against GAPDH.
  • 0.4 ⁇ 10 6 cells were transfected with (1) 2 ⁇ g Cas9N-VP64 plasmid, 2 ⁇ g gRNA and 0.25 ⁇ g of reporter library; or (2) 2 ⁇ g TALE-TF plasmid and 0.25 gm of reporter library; or (3) 2 ⁇ g control-TF plasmid and 0.25 ⁇ g of reporter library.
  • Cells were harvested 24 hrs post transfection (to avoid the stimulation of reporters being in saturation mode).
  • Total RNA extraction was performed using RNAeasy-plus kit (Qiagen), and standard RT-per performed using Superscript-III (Invitrogen). Libraries for next-generation sequencing were generated by targeted per amplification of the transcript-tags.
  • FIG. 8A The high-level logic flow for this process is depicted in FIG. 8A , and additional details are given here. For details on construct library composition, see FIGS. 8A (level 1) and 8 B.
  • construct library ( FIG. 8A , level 3, left) and reporter gene cDNA sequences ( FIG. 8A , level 3, right) were obtained as 150 bp overlapping paired end reads on an Illumina MiSeq, while for TALE experiments, corresponding sequences were obtained as 5 lbp non-overlapping paired end reads on an Illumina HiSeq.
  • novoalign V2.07.17 (world wide website novocraft.com/main/index/php) was used to align paired reads to a set of 250 bp reference sequences that corresponded to 234 bp of the constructs flanked by the pairs of 8 bp library barcodes (see FIG. 8A , 3 rd level, left).
  • the 23 bp degenerate Cas9 binding site regions and the 24 bp degenerate transcript tag regions were specified as Ns, while the construct library barcodes were explicitly provided.
  • Validity checking Novoalign output for comprised files in which left and right reads for each read pair were individually aligned to the reference sequences. Only read pairs that were both uniquely aligned to the reference sequence were subjected to additional validity conditions, and only read pairs that passed all of these conditions were retained.
  • SeqPrep (downloaded from world wide website github.com/jstjohn/SeqPrep) was first used to merge the overlapping read pairs to the 79 bp common segment, after which novoalign (version above) was used to align these 79 bp common segments as unpaired single reads to a set of reference sequences (see FIG. 8A , 3 rd level, right) in which (as for the construct library sequencing) the 24 bp degenerate transcript tag was specified as Ns while the sample barcodes were explicitly provided. Both TALE and Cas9 cDNA sequence regions corresponded to the same 63 bp regions of cDNA flanked by pairs of 8 bp sample barcode sequences.
  • Validity checking The same conditions were applied as for construct library sequencing (see above) except that: (a) Here, due prior SeqPrep merging of read pairs, validity processing did not have to filter for unique alignments of both reads in a read pair but only for unique alignments of the merged reads. (b) Only transcript tags appeared in the cDNA sequence reads, so that validity processing only applied these tag regions of the reference sequences and not also to a separate binding site region.
  • Custom perl was used to generate these tables from the validated construct library sequences ( FIG. 8A , 4 th level, left).
  • early analysis of binding site vs. tag associations revealed that a non-negligible fraction of tag sequences were in fact shared by multiple binding sequences, likely mainly caused by a combination of sequence errors in the binding sequences, or oligo synthesis errors in the oligos used to generate the construct libraries.
  • tags found associated with binding sites in validated read pairs might also be found in the construct library read pair reject file if it was not clear, due to barcode mismatches, which construct library they might be from.
  • the tag sequences themselves might contain sequence errors.
  • tags were categorized with three attributes: (i) safe vs. unsafe, where unsafe meant the tag could be found in the construct library rejected read pair file; shared vs. nonshared, where shared meant the tag was found associated with multiple binding site sequences, and 2+ vs. 1-only, where 2+ meant that the tag appeared at least twice among the validated construct library sequences and so presumed to be less likely to contain sequence errors.
  • Custom perl code was used to implement the steps indicated in FIG. 8A , levels 5-6.
  • tag counts obtained for each induced sample were aggregated for each binding site, using the binding site vs. transcript tag table previously computed for the construct library (see FIG. 8C ).
  • the aggregated tag counts for each binding site were then divided by the aggregated tag counts for the positive control sample to generate normalized expression levels. Additional considerations relevant to these calculations included:
  • the binding site built into the TALE construct libraries was 18 bp and tag associations were assigned based on these 18 bp sequences, but some experiments were conducted with TALEs programmed to bind central 14 bp or 10 bp regions within the 18 bp construct binding site regions. In computing expression levels for these TALEs, tags were aggregated to binding sites based on the corresponding regions of the 18 bp binding sites in the association table, so that binding site mismatches outside of this region were ignored.
  • the sgRNA (aptamer-modified single guide RNA) tethering approach described herein allows different effector domains to be recruited by distinct sgRNAs so long as each sgRNA uses a different RNA-protein interaction pair, enabling multiplex gene regulation using the same Cas9N— protein.
  • 10 gRNAs were designed targeting a ⁇ 1 kb stretch of DNA upstream of the transcription start site. The DNase hypersensitive sites are highlighted in green. Transcriptional activation via qPCR of the endogenous genes was assayed.
  • FIG. 13A-C the targeting landscape of two additional Cas9-gRNA complexes
  • FIG. 13D-F the targeting landscape of two additional Cas9-gRNA complexes
  • the two gRNAs have vastly different specificity profiles with gRNA2 tolerating up to 2-3 mismatches and gRNA3 only up to 1. These aspects are reflected in both the one base mismatch ( FIG. 13B , 13 E) and two base mismatch plots ( FIG. 13C , 13 F).
  • FIGS. 13A-C the targeting landscape of two additional Cas9-gRNA complexes
  • FIG. 14A-C specificity data was generated using two different sgRNA:Cas9 complexes. It was confirmed that the assay was specific for the sgRNA being evaluated, as a corresponding mutant sgRNA was unable to stimulate the reporter library.
  • FIG. 14A The specificity profile of two gRNAs (wild-type and mutant; sequence differences are highlighted in red) were evaluated using a reporter library designed against the wild-type gRNA target sequence.
  • FIG. 14B It was confirmed that this assay was specific for the gRNA being evaluated (data re-plotted from FIG. 13D ), as the corresponding mutant gRNA is unable to stimulate the reporter library.
  • Different sgRNAs can have different specificity profiles ( FIGS. 13A , 13 D), specifically, sgRNA2 tolerates up to 3 mismatches and sgRNA3 only up to 1. The greatest sensitivity to mismatches was localized to the 3′ end of the spacer, albeit mismatches at other positions were also observed to affect activity.
  • FIG. 15A-D it was confirmed by targeted experiments that single-base mismatches within 12 bp of the 3′ end of the spacer in the assayed sgRNAs resulted in detectable targeting. However, 2 bp mismatches in this region resulted in significant loss of activity.
  • 2 independent gRNAs were tested: gRNA2 ( FIG. 15A-B ) and gRNA3 ( FIG. 15C-D ) bearing single or double-base mismatches (highlighted in red) in the spacer sequence versus the target.
  • truncations in the 5′ portion of the spacer resulted in retention of sgRNA activity.
  • FIG. 18A-B Using a nuclease mediated HR assay ( FIG. 18A-B ) it was confirmed that 18-mer TALEs tolerate multiple mutations in their target sequences. As shown in FIG. 18A-B certain mutations in the middle of the target lead to higher TALE activity, as determined via targeted experiments in a nuclease assay.
  • FIG. 19A-C shows a comparison of TALE monomer specificity versus TALE protein specificity.
  • FIG. 19A Using a modification of approach described in FIG. 2 , the targeting landscape of 2 14-mer TALE-TFs bearing a contiguous set of 6 NI or 6 NH repeats was analyzed. In this approach, a reduced library of reporters bearing a degenerate 6-mer sequence in the middle was created and used to assay the TALE-TF specificity.
  • the expected target sequence is enriched (i.e. one bearing 6 As for NI repeats, and 6 Gs for NH repeats).
  • Each of these TALEs still tolerate 1-2 mismatches in the central 6-mer target sequence.
  • choice of monomers does contribute to base specificity, TALE specificity is also a function of the binding energy of the protein as a whole.
  • shorter engineered TALEs or TALEs bearing a composition of high and low affinity monomers result in higher specificity in genome engineering applications and FokI dimerization in nuclease applications allows for further reduction in off-target effects when using shorter TALEs.
  • FIG. 20A-B shows data related to off-set nicking.
  • off-set nicks were created to generate DSBs.
  • a large majority of nicks do not result in non-homologous end joining (NHEJ) mediated indels and thus when inducing off-set nicks, off-target single nick events will likely result in very low indel rates.
  • NHEJ non-homologous end joining
  • Inducing off-set nicks to generate DSBs is effective at inducing gene disruption at both integrated reporter loci and at the native AAVS1 genomic locus.
  • FIG. 20A The native AAVS1 locus with 8 gRNAs covering a 200 bp stretch of DNA was targeted: 4 targeting the sense strand (s1-4) and 4 the antisense strand (as1-4).
  • s1-4 sense strand
  • as1-4 antisense strand
  • FIG. 20B Using a Sanger sequencing based assay, it was observed that while single gRNAs did not induce detectable NHEJ events, inducing off-set nicks to generate DSBs is highly effective at inducing gene disruption.
  • FIG. 21A-C is directed to off-set nicking and NHEJ profiles. Representative Sanger sequencing results of three different off-set nicking combinations is shown with positions of the targeting gRNAs highlighted by boxes. Furthermore, consistent with the standard model for homologous recombination (HR) mediated repair, engineering of 5′ overhangs via off-set nicks generated more robust NHEJ events than 3′ overhangs ( FIG. 3B ). In addition to a stimulation of NHEJ, robust induction of HR was observed when the 5′ overhangs were created. Generation of 3′ overhangs did not result in improvement of HR rates ( FIG. 3C ).
  • HR homologous recombination
  • gRNA Targets for Endogenous Gene Regulation Targets in the REX1, OCT4, SOX2 and NANOG promoters used in Cas9-gRNA mediated activation experiments are listed (SEQ ID NOs: 11-61).
  • gRNA gRNA Target REX1 1 ctggcggatcactcgcggtt agg REX1 2 cctcggcctccaaaagtgct agg REX1 3 acgctgattcctgcagatca ggg REX1 4 ccaggaatacgtatccacca ggg REX1 5 gccacacccaagcgatcaaa tgg REX1 6 aaataatacattctaaggta agg REX1 7 gctactggggaggctgaggc agg REX1 8 tagcaatacagtcacattaa tgg REX1 9 ctcatgtgatccccccg
  • dTomato fluorescence based transcriptional activation reporter sequences are listed below (ISceI control-TF target, gRNA targets, minCMV promoter and FLAG tag+dTomato sequences are highlighted).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Lubricants (AREA)
US14/319,530 2013-06-04 2014-06-30 RNA-Guided Transcriptional Regulation Pending US20140356956A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US14/319,530 US20140356956A1 (en) 2013-06-04 2014-06-30 RNA-Guided Transcriptional Regulation
US15/049,451 US10640789B2 (en) 2013-06-04 2016-02-22 RNA-guided transcriptional regulation
US16/441,209 US10767194B2 (en) 2013-06-04 2019-06-14 RNA-guided transcriptional regulation
US16/851,360 US20200299732A1 (en) 2013-06-04 2020-04-17 RNA-Guided Transcriptional Regulation
US17/972,885 US11981917B2 (en) 2013-06-04 2022-10-25 RNA-guided transcriptional regulation
US18/425,219 US20240175057A1 (en) 2013-06-04 2024-01-29 RNA-Guided Transcriptional Regulation

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201361830787P 2013-06-04 2013-06-04
PCT/US2014/040868 WO2014197568A2 (en) 2013-06-04 2014-06-04 Rna-guideded transcriptional regulation
US14/319,530 US20140356956A1 (en) 2013-06-04 2014-06-30 RNA-Guided Transcriptional Regulation

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/040868 Continuation WO2014197568A2 (en) 2013-06-04 2014-06-04 Rna-guideded transcriptional regulation

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/049,451 Continuation US10640789B2 (en) 2013-06-04 2016-02-22 RNA-guided transcriptional regulation
US16/441,209 Continuation US10767194B2 (en) 2013-06-04 2019-06-14 RNA-guided transcriptional regulation

Publications (1)

Publication Number Publication Date
US20140356956A1 true US20140356956A1 (en) 2014-12-04

Family

ID=52008736

Family Applications (7)

Application Number Title Priority Date Filing Date
US14/319,530 Pending US20140356956A1 (en) 2013-06-04 2014-06-30 RNA-Guided Transcriptional Regulation
US14/319,289 Active US9267135B2 (en) 2013-06-04 2014-06-30 RNA-guided transcriptional regulation
US15/049,451 Active US10640789B2 (en) 2013-06-04 2016-02-22 RNA-guided transcriptional regulation
US16/441,209 Active US10767194B2 (en) 2013-06-04 2019-06-14 RNA-guided transcriptional regulation
US16/851,360 Pending US20200299732A1 (en) 2013-06-04 2020-04-17 RNA-Guided Transcriptional Regulation
US17/972,885 Active US11981917B2 (en) 2013-06-04 2022-10-25 RNA-guided transcriptional regulation
US18/425,219 Pending US20240175057A1 (en) 2013-06-04 2024-01-29 RNA-Guided Transcriptional Regulation

Family Applications After (6)

Application Number Title Priority Date Filing Date
US14/319,289 Active US9267135B2 (en) 2013-06-04 2014-06-30 RNA-guided transcriptional regulation
US15/049,451 Active US10640789B2 (en) 2013-06-04 2016-02-22 RNA-guided transcriptional regulation
US16/441,209 Active US10767194B2 (en) 2013-06-04 2019-06-14 RNA-guided transcriptional regulation
US16/851,360 Pending US20200299732A1 (en) 2013-06-04 2020-04-17 RNA-Guided Transcriptional Regulation
US17/972,885 Active US11981917B2 (en) 2013-06-04 2022-10-25 RNA-guided transcriptional regulation
US18/425,219 Pending US20240175057A1 (en) 2013-06-04 2024-01-29 RNA-Guided Transcriptional Regulation

Country Status (15)

Country Link
US (7) US20140356956A1 (cg-RX-API-DMAC10.html)
EP (4) EP3603679B1 (cg-RX-API-DMAC10.html)
JP (6) JP6621738B2 (cg-RX-API-DMAC10.html)
KR (3) KR102884236B1 (cg-RX-API-DMAC10.html)
CN (2) CN105451778B (cg-RX-API-DMAC10.html)
AU (4) AU2014274939B2 (cg-RX-API-DMAC10.html)
CA (2) CA3176690A1 (cg-RX-API-DMAC10.html)
ES (1) ES2930537T3 (cg-RX-API-DMAC10.html)
IL (3) IL302205B2 (cg-RX-API-DMAC10.html)
MX (3) MX385330B (cg-RX-API-DMAC10.html)
MY (3) MY208492A (cg-RX-API-DMAC10.html)
RU (2) RU2756865C2 (cg-RX-API-DMAC10.html)
SG (3) SG11201509962QA (cg-RX-API-DMAC10.html)
WO (1) WO2014197568A2 (cg-RX-API-DMAC10.html)
ZA (1) ZA201509025B (cg-RX-API-DMAC10.html)

Cited By (146)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150140664A1 (en) * 2013-11-19 2015-05-21 President And Fellows Of Harvard College Large Gene Excision and Insertion
US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
US9234213B2 (en) 2013-03-15 2016-01-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
WO2016114972A1 (en) 2015-01-12 2016-07-21 The Regents Of The University Of California Heterodimeric cas9 and methods of use thereof
WO2016196655A1 (en) 2015-06-03 2016-12-08 The Regents Of The University Of California Cas9 variants and methods of use thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
WO2017143071A1 (en) 2016-02-18 2017-08-24 The Regents Of The University Of California Methods and compositions for gene editing in stem cells
WO2017161043A1 (en) 2016-03-16 2017-09-21 The J. David Gladstone Institutes Methods and compositions for treating obesity and/or diabetes and for identifying candidate treatment agents
WO2017141173A3 (en) * 2016-02-15 2017-11-02 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes using cpf1 or csm1
US20170321214A1 (en) * 2014-12-12 2017-11-09 The Broad Institute Inc. Dead guides for crispr transcription factors
US9822370B2 (en) 2013-04-04 2017-11-21 President And Fellows Of Harvard College Method of making a deletion in a target sequence in isolated primary cells using Cas9 and two guide RNAs
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
WO2018005445A1 (en) 2016-06-27 2018-01-04 The Broad Institute, Inc. Compositions and methods for detecting and treating diabetes
WO2018064371A1 (en) 2016-09-30 2018-04-05 The Regents Of The University Of California Rna-guided nucleic acid modifying enzymes and methods of use thereof
US9970030B2 (en) 2014-08-27 2018-05-15 Caribou Biosciences, Inc. Methods for increasing CAS9-mediated engineering efficiency
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
WO2018112278A1 (en) 2016-12-14 2018-06-21 Ligandal, Inc. Methods and compositions for nucleic acid and protein payload delivery
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US20180320197A1 (en) * 2013-06-05 2018-11-08 Duke University Rna-guided gene editing and gene regulation
US10136649B2 (en) 2015-05-29 2018-11-27 North Carolina State University Methods for screening bacteria, archaea, algae, and yeast using CRISPR nucleic acids
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10208319B2 (en) 2013-07-09 2019-02-19 President And Fellows Of Harvard College Therapeutic uses of genome editing with CRISPR/Cas systems
US10316324B2 (en) 2017-08-09 2019-06-11 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
WO2019113132A1 (en) * 2017-12-05 2019-06-13 Caribou Biosciences, Inc. Modified lymphocytes
US10428319B2 (en) 2017-06-09 2019-10-01 Editas Medicine, Inc. Engineered Cas9 nucleases
WO2019195738A1 (en) 2018-04-06 2019-10-10 Children's Medical Center Corporation Compositions and methods for somatic cell reprogramming and modulating imprinting
US10450584B2 (en) 2014-08-28 2019-10-22 North Carolina State University Cas9 proteins and guiding features for DNA targeting and genome editing
US10494621B2 (en) 2015-06-18 2019-12-03 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
WO2020014528A1 (en) 2018-07-13 2020-01-16 The Regents Of The University Of California Retrotransposon-based delivery vehicle and methods of use thereof
WO2020041456A1 (en) 2018-08-22 2020-02-27 The Regents Of The University Of California Variant type v crispr/cas effector polypeptides and methods of use thereof
US10577630B2 (en) 2013-06-17 2020-03-03 The Broad Institute, Inc. Delivery and use of the CRISPR-Cas systems, vectors and compositions for hepatic targeting and therapy
US10584358B2 (en) 2013-10-30 2020-03-10 North Carolina State University Compositions and methods related to a type-II CRISPR-Cas system in Lactobacillus buchneri
US10669539B2 (en) 2016-10-06 2020-06-02 Pioneer Biolabs, Llc Methods and compositions for generating CRISPR guide RNA libraries
US10676726B2 (en) 2015-02-09 2020-06-09 Duke University Compositions and methods for epigenome editing
US10676735B2 (en) 2015-07-22 2020-06-09 Duke University High-throughput screening of regulatory element function with epigenome editing technologies
US10696986B2 (en) 2014-12-12 2020-06-30 The Board Institute, Inc. Protected guide RNAS (PGRNAS)
US10711256B2 (en) 2012-04-27 2020-07-14 Duke University Genetic correction of mutated genes
US10711267B2 (en) 2018-10-01 2020-07-14 North Carolina State University Recombinant type I CRISPR-Cas system
US10731181B2 (en) 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
WO2020163856A1 (en) 2019-02-10 2020-08-13 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Modified mitochondrion and methods of use thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10781444B2 (en) 2013-06-17 2020-09-22 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
US10787654B2 (en) 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
US10851357B2 (en) 2013-12-12 2020-12-01 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US10930367B2 (en) 2012-12-12 2021-02-23 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
US10946108B2 (en) 2013-06-17 2021-03-16 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for targeting disorders and diseases using viral components
US11008588B2 (en) 2013-06-17 2021-05-18 The Broad Institute, Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US11041173B2 (en) 2012-12-12 2021-06-22 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US11053482B2 (en) 2015-04-16 2021-07-06 Wageningen Universiteit Vector systems for nuclease-mediated genome editing
US11091798B2 (en) 2015-06-18 2021-08-17 The Broad Institute, Inc. CRISPR enzymes and systems
EP3877517A1 (en) 2018-11-09 2021-09-15 Inari Agriculture, Inc. Rna-guided nucleases and dna binding proteins
US11155795B2 (en) 2013-12-12 2021-10-26 The Broad Institute, Inc. CRISPR-Cas systems, crystal structure and uses thereof
US11155823B2 (en) 2015-06-15 2021-10-26 North Carolina State University Methods and compositions for efficient delivery of nucleic acids and RNA-based antimicrobials
WO2021224633A1 (en) 2020-05-06 2021-11-11 Orchard Therapeutics (Europe) Limited Treatment for neurodegenerative diseases
US11180792B2 (en) 2015-01-28 2021-11-23 The Regents Of The University Of California Methods and compositions for labeling a single-stranded target nucleic acid
DE212020000516U1 (de) 2019-03-07 2022-01-17 The Regents of the University of California CRISPR-CAS-Effektorpolypeptide
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
TWI758251B (zh) * 2015-06-18 2022-03-21 美商博得學院股份有限公司 新型ｃｒｉｓｐｒ酶以及系統
US11286478B2 (en) 2016-04-19 2022-03-29 The Broad Institute, Inc. Cpf1 complexes with reduced indel activity
US11286480B2 (en) 2015-09-28 2022-03-29 North Carolina State University Methods and compositions for sequence specific antimicrobials
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11407995B1 (en) 2018-10-26 2022-08-09 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US11407985B2 (en) 2013-12-12 2022-08-09 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for genome editing
US11427817B2 (en) 2015-08-25 2022-08-30 Duke University Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases
US11434477B1 (en) 2018-11-02 2022-09-06 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US11439712B2 (en) 2014-04-08 2022-09-13 North Carolina State University Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542466B2 (en) 2015-12-22 2023-01-03 North Carolina State University Methods and compositions for delivery of CRISPR based antimicrobials
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
US11578312B2 (en) 2015-06-18 2023-02-14 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
US11597949B2 (en) 2013-06-17 2023-03-07 The Broad Institute, Inc. Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11597919B2 (en) 2013-12-12 2023-03-07 The Broad Institute Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
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
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
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)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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
WO2024020346A2 (en) 2022-07-18 2024-01-25 Renagade Therapeutics Management Inc. Gene editing components, systems, and methods of use
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
US20240117383A1 (en) * 2020-05-04 2024-04-11 Editas Medicine, Inc. Selection by essential-gene knock-in
US11970710B2 (en) 2015-10-13 2024-04-30 Duke University Genome engineering with Type I CRISPR systems in eukaryotic cells
US12031155B2 (en) 2015-05-08 2024-07-09 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12098399B2 (en) 2022-06-24 2024-09-24 Tune Therapeutics, Inc. Compositions, systems, and methods for epigenetic regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression
US12110490B2 (en) 2015-12-18 2024-10-08 The Broad Institute, Inc. CRISPR enzymes and systems
US12110545B2 (en) 2017-01-06 2024-10-08 Editas Medicine, Inc. Methods of assessing nuclease cleavage
US12133884B2 (en) 2018-05-11 2024-11-05 Beam Therapeutics Inc. Methods of substituting pathogenic amino acids using programmable base editor systems
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12180263B2 (en) 2014-11-06 2024-12-31 President And Fellows Of Harvard College Cells lacking B2M surface expression and methods for allogeneic administration of such cells
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
US12203123B2 (en) 2018-10-01 2025-01-21 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for screening for variant cells
US12215345B2 (en) 2013-03-19 2025-02-04 Duke University Compositions and methods for the induction and tuning of gene expression
US12214054B2 (en) 2015-11-30 2025-02-04 Duke University Therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use
US12214056B2 (en) 2016-07-19 2025-02-04 Duke University Therapeutic applications of CPF1-based genome editing
WO2025049959A2 (en) 2023-09-01 2025-03-06 Renagade Therapeutics Management Inc. Gene editing systems, compositions, and methods for treatment of vexas syndrome
US12251429B2 (en) 2015-06-01 2025-03-18 Temple University—Of the Commonwealth System of Higher Education Methods and compositions for RNA-guided treatment of HIV infection
US12251450B2 (en) 2013-12-12 2025-03-18 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for HBV and viral diseases and disorders
WO2025064408A1 (en) 2023-09-18 2025-03-27 The Broad Institute, Inc. Compositions and methods for treating cardiovascular disease
US12264330B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for killing target cells
US12264313B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression
WO2025081042A1 (en) 2023-10-12 2025-04-17 Renagade Therapeutics Management Inc. Nickase-retron template-based precision editing system and methods of use
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12286727B2 (en) 2016-12-19 2025-04-29 Editas Medicine, Inc. Assessing nuclease cleavage
US12338436B2 (en) 2018-06-29 2025-06-24 Editas Medicine, Inc. Synthetic guide molecules, compositions and methods relating thereto
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
WO2025155753A2 (en) 2024-01-17 2025-07-24 Renagade Therapeutics Management Inc. Improved gene editing system, guides, and methods
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
WO2025174765A1 (en) 2024-02-12 2025-08-21 Renagade Therapeutics Management Inc. Lipid nanoparticles comprising coding rna molecules for use in gene editing and as vaccines and therapeutic agents
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12410416B2 (en) 2016-04-19 2025-09-09 The Broad Institute, Inc. CRISPR enzymes and systems with modified pam specificity
US12421506B2 (en) 2013-12-12 2025-09-23 The Broad Institute, Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation
US12428631B2 (en) 2016-04-13 2025-09-30 Duke University CRISPR/Cas9-based repressors for silencing gene targets in vivo and methods of use
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
US12435320B2 (en) 2014-12-24 2025-10-07 The Broad Institute, Inc. CRISPR having or associated with destabilization domains
US12454687B2 (en) 2012-12-12 2025-10-28 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
US12454694B2 (en) 2018-09-07 2025-10-28 Beam Therapeutics Inc. Compositions and methods for improving base editing
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
US12509492B2 (en) 2018-01-19 2025-12-30 Duke University Genome engineering with CRISPR-Cas systems in eukaryotes
US12522807B2 (en) 2018-07-09 2026-01-13 The Broad Institute, Inc. RNA programmable epigenetic RNA modifiers and uses thereof
US12529041B2 (en) 2018-09-07 2026-01-20 Beam Therapeutics Inc. Compositions and methods for delivering a nucleobase editing system
US12576151B2 (en) 2020-09-25 2026-03-17 Beam Therapeutics Inc. Fratricide resistant modified immune cells and methods of using the same
US12576110B2 (en) 2019-08-06 2026-03-17 The Children's Medical Center Corporation Methods and compositions for reconstituting microglia
US12594301B2 (en) 2019-09-27 2026-04-07 Beam Therapeutics Inc. Compositions and methods for treatment of liquid cancers
US12600971B2 (en) 2019-02-13 2026-04-14 Beam Therapeutics Inc. Modified immune cells having adenosine deaminase base editors for modifying a nucleobase in a target sequence
US12612618B2 (en) 2019-01-31 2026-04-28 Beam Therapeutics Inc. Nucleobase editors having reduced non-target deamination and assays for characterizing nucleobase editors
US12622931B2 (en) 2019-02-13 2026-05-12 Beam Therapeutics Inc. Compositions and methods for treating alpha-1 antitrypsin deficiency
US12630838B2 (en) 2020-11-13 2026-05-19 Egenesis, Inc. Cells, tissues, organs, and animals having one or more modified genes for enhanced xenograft survival and tolerance
US12629428B2 (en) 2019-04-14 2026-05-19 Duke University AAV vector-mediated deletion of large mutational hotspot for treatment of Duchenne muscular dystrophy

Families Citing this family (107)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2766498B1 (en) 2011-10-14 2019-06-19 President and Fellows of Harvard College Sequencing by structure assembly
ES2991004T3 (es) 2011-12-22 2024-12-02 Harvard College Métodos para la detección de analitos
US9637739B2 (en) 2012-03-20 2017-05-02 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
EP4397759A3 (en) * 2012-10-23 2024-10-09 Toolgen Incorporated 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
IL308158A (en) 2012-12-17 2023-12-01 Harvard College Rna-guided human genome engineering
US10138509B2 (en) 2013-03-12 2018-11-27 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
EP3603679B1 (en) 2013-06-04 2022-08-10 President and Fellows of Harvard College Rna-guided transcriptional regulation
ES2681622T3 (es) 2013-09-18 2018-09-14 Kymab Limited Métodos, células y organismos
EP3835419A1 (en) 2013-12-12 2021-06-16 The Regents of The University of California Methods and compositions for modifying a single stranded target nucleic acid
US11028388B2 (en) 2014-03-05 2021-06-08 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa
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
US9938521B2 (en) 2014-03-10 2018-04-10 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating leber's congenital amaurosis 10 (LCA10)
EP3126498A4 (en) * 2014-03-20 2017-08-23 Université Laval Crispr-based methods and products for increasing frataxin levels and uses thereof
EP3981876A1 (en) 2014-03-26 2022-04-13 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating sickle cell disease
EP3540061A1 (en) 2014-04-02 2019-09-18 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating primary open angle glaucoma
WO2016007839A1 (en) 2014-07-11 2016-01-14 President And Fellows Of Harvard College Methods for high-throughput labelling and detection of biological features in situ using microscopy
WO2016021973A1 (ko) * 2014-08-06 2016-02-11 주식회사 툴젠 캄필로박터 제주니 crispr/cas 시스템 유래 rgen을 이용한 유전체 교정
EP3180426B1 (en) * 2014-08-17 2019-12-25 The Broad Institute, Inc. Genome editing using cas9 nickases
WO2016049258A2 (en) * 2014-09-25 2016-03-31 The Broad Institute Inc. Functional screening with optimized functional crispr-cas systems
GB201418965D0 (cg-RX-API-DMAC10.html) 2014-10-24 2014-12-10 Ospedale San Raffaele And Fond Telethon
CA2969619A1 (en) 2014-12-03 2016-06-09 Agilent Technologies, Inc. Guide rna with chemical modifications
WO2016094874A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Escorted and functionalized guides for crispr-cas systems
WO2016149484A2 (en) * 2015-03-17 2016-09-22 Temple University Of The Commonwealth System Of Higher Education Compositions and methods for specific reactivation of hiv latent reservoir
CN107787367B (zh) 2015-04-06 2021-10-26 里兰斯坦福初级大学理事会 用于crispr/cas介导的基因调控的化学修饰的引导rna
EP4019975A1 (en) 2015-04-24 2022-06-29 Editas Medicine, Inc. Evaluation of cas9 molecule/guide rna molecule complexes
AU2016279062A1 (en) 2015-06-18 2019-03-28 Omar O. Abudayyeh Novel CRISPR enzymes and systems
JP2018519811A (ja) 2015-06-29 2018-07-26 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． 修飾ｃｒｉｓｐｒ ｒｎａ及び修飾単一ｃｒｉｓｐｒ ｒｎａならびにその使用
WO2017053729A1 (en) 2015-09-25 2017-03-30 The Board Of Trustees Of The Leland Stanford Junior University Nuclease-mediated genome editing of primary cells and enrichment thereof
EP3147363B1 (en) 2015-09-26 2019-10-16 B.R.A.I.N. Ag Activation of taste receptor genes in mammalian cells using crispr-cas-9
CN108474022A (zh) 2015-11-03 2018-08-31 哈佛学院董事及会员团体 用于包含三维核酸的基质容积成像的设备和方法
CA3005878A1 (en) 2015-11-19 2017-05-26 The Brigham And Women's Hospital, Inc. Lymphocyte antigen cd5-like (cd5l)-interleukin 12b (p40) heterodimers in immunity
KR101906491B1 (ko) * 2015-11-30 2018-12-05 기초과학연구원 F. novicida 유래 Cas9을 포함하는 유전체 교정용 조성물
US20190264186A1 (en) * 2016-01-22 2019-08-29 The Broad Institute Inc. Crystal structure of crispr cpf1
EP3433364A1 (en) 2016-03-25 2019-01-30 Editas Medicine, Inc. Systems and methods for treating alpha 1-antitrypsin (a1at) deficiency
AU2017257624B2 (en) 2016-04-25 2023-05-25 President And Fellows Of Harvard College Hybridization chain reaction methods for in situ molecular detection
DK3604527T3 (da) 2016-06-02 2021-05-31 Sigma Aldrich Co Llc Anvendelse af programmerbare dna-bindingsproteiner til at forbedre målrettet genommodifikation
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
US20190330603A1 (en) * 2016-06-17 2019-10-31 Genesis Technologies Limited Crispr-cas system, materials and methods
US11566263B2 (en) 2016-08-02 2023-01-31 Editas Medicine, Inc. Compositions and methods for treating CEP290 associated disease
GB2569252A (en) 2016-08-31 2019-06-12 Harvard College Methods of combining the detection of biomolecules into a single assay using fluorescent in situ sequencing
JP7256739B2 (ja) 2016-09-07 2023-04-12 サンガモ セラピューティクス， インコーポレイテッド 肝臓遺伝子のモジュレーション
EP4431607A3 (en) 2016-09-09 2024-12-11 The Board of Trustees of the Leland Stanford Junior University High-throughput precision genome editing
US12499971B2 (en) 2016-09-28 2025-12-16 The Broad Institute, Inc. Systematic screening and mapping of regulatory elements in non-coding genomic regions, methods, compositions, and applications thereof
WO2018083606A1 (en) 2016-11-01 2018-05-11 Novartis Ag Methods and compositions for enhancing gene editing
US11732258B2 (en) 2016-11-02 2023-08-22 President And Fellows Of Harvard College Engineered guide RNA sequences for in situ detection and sequencing
WO2018089386A1 (en) 2016-11-11 2018-05-17 The Broad Institute, Inc. Modulation of intestinal epithelial cell differentiation, maintenance and/or function through t cell action
CA3049980A1 (en) 2017-01-23 2018-07-26 Regeneron Pharmaceuticals, Inc. Hydroxysteroid 17-beta dehydrogenase 13 (hsd17b13) variants and uses thereof
TW201839136A (zh) 2017-02-06 2018-11-01 瑞士商諾華公司 治療血色素異常症之組合物及方法
EP3596217A1 (en) 2017-03-14 2020-01-22 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN110892069B (zh) 2017-03-30 2024-02-09 国立大学法人京都大学 基于基因组编辑的外显子跳跃诱导方法
WO2018191520A1 (en) 2017-04-12 2018-10-18 The Broad Institute, Inc. Respiratory and sweat gland ionocytes
CN116732106A (zh) 2017-04-20 2023-09-12 E开创生物技术股份有限公司 产生基因修改的动物的方法
WO2018195486A1 (en) 2017-04-21 2018-10-25 The Broad Institute, Inc. Targeted delivery to beta cells
US11591601B2 (en) 2017-05-05 2023-02-28 The Broad Institute, Inc. Methods for identification and modification of lncRNA associated with target genotypes and phenotypes
EP3622070A2 (en) 2017-05-10 2020-03-18 Editas Medicine, Inc. Crispr/rna-guided nuclease systems and methods
DK3635102T3 (da) 2017-06-05 2025-10-27 Regeneron Pharma B4galt1-varianter og anvendelser deraf
CN107266583B (zh) * 2017-06-27 2021-02-09 深圳大学 一种基于格氏嗜盐碱杆菌Argonaute靶向激活基因转录的方法
US20200140896A1 (en) 2017-06-30 2020-05-07 Novartis Ag Methods for the treatment of disease with gene editing systems
US11547614B2 (en) * 2017-10-31 2023-01-10 The Broad Institute, Inc. Methods and compositions for studying cell evolution
EP3704245A1 (en) 2017-11-01 2020-09-09 Novartis AG Synthetic rnas and methods of use
WO2019089910A1 (en) * 2017-11-01 2019-05-09 Ohio State Innovation Foundation Highly compact cas9-based transcriptional regulators for in vivo gene regulation
IL277234B2 (en) 2018-03-14 2026-03-01 Editas Medicine Inc Systems and methods for treating hemoglobin diseases
EP3592140A1 (en) 2018-03-19 2020-01-15 Regeneron Pharmaceuticals, Inc. Transcription modulation in animals using crispr/cas systems
AU2019255789B2 (en) 2018-04-19 2025-11-20 The Regents Of The University Of California Compositions and methods for gene editing
EP3575402A1 (en) * 2018-06-01 2019-12-04 Algentech SAS Gene targeting
WO2019246553A1 (en) * 2018-06-21 2019-12-26 Memorial Sloan Kettering Cancer Center Compositions & methods for monitoring dna repair
US12473533B2 (en) 2018-07-09 2025-11-18 The Regents Of The University Of California Gene targets for T-cell-based immunotherapy
EP3830256A2 (en) 2018-07-31 2021-06-09 The Broad Institute, Inc. Novel crispr enzymes and systems
GB201813011D0 (en) 2018-08-10 2018-09-26 Vib Vzw Means and methods for drought tolerance in crops
CN110951777A (zh) * 2018-09-26 2020-04-03 中国科学技术大学 一种基因转录调控体系及其应用
WO2020076976A1 (en) 2018-10-10 2020-04-16 Readcoor, Inc. Three-dimensional spatial molecular indexing
EP4317950B1 (en) 2019-03-18 2026-01-28 Regeneron Pharmaceuticals, Inc. Crispr/cas screening platform to identify genetic modifiers of tau seeding or aggregation
JP7389135B2 (ja) 2019-03-18 2023-11-29 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 タウ凝集に関連する遺伝的脆弱性を明らかにするためのｃｒｉｓｐｒ／ｃａｓドロップアウトスクリーニングプラットフォーム
CN114616002A (zh) 2019-09-13 2022-06-10 瑞泽恩制药公司 使用由脂质纳米颗粒递送的crispr/cas系统在动物中进行的转录调控
US20230025039A1 (en) 2019-09-20 2023-01-26 The Broad Institute, Inc. Novel type vi crispr enzymes and systems
WO2021108363A1 (en) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Crispr/cas-mediated upregulation of humanized ttr allele
IL271656A (en) 2019-12-22 2021-06-30 Yeda Res & Dev System and methods for identifying cells that have undergone genome editing
WO2021155063A1 (en) 2020-01-29 2021-08-05 Readcoor, Llc Compositions and methods for analyte detection
JP7525140B2 (ja) * 2020-01-31 2024-07-30 国立大学法人京都大学 タンパク質翻訳の制御システム
JP7837276B2 (ja) 2020-03-04 2026-03-30 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 免疫療法に対する腫瘍細胞の感作のための方法及び組成物
US20230096378A1 (en) 2020-03-12 2023-03-30 Institute For Basic Science Composition for inducing apoptosis of cells having genomic sequence variation and method for inducing apoptosis of cells by using composition
US20230102342A1 (en) 2020-03-23 2023-03-30 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use
US20220049303A1 (en) 2020-08-17 2022-02-17 Readcoor, Llc Methods and systems for spatial mapping of genetic variants
US20240002839A1 (en) 2020-12-02 2024-01-04 Decibel Therapeutics, Inc. Crispr sam biosensor cell lines and methods of use thereof
WO2023039586A1 (en) 2021-09-10 2023-03-16 Agilent Technologies, Inc. Guide rnas with chemical modification for prime editing
EP4426828A1 (en) 2021-11-01 2024-09-11 Tome Biosciences, Inc. Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo
AU2022408167A1 (en) 2021-12-08 2024-06-06 Regeneron Pharmaceuticals, Inc. Mutant myocilin disease model and uses thereof
US20250057980A1 (en) 2021-12-22 2025-02-20 Tome Biosciences, Inc. Co-Delivery of a Gene Editor Construct and a Donor Template
WO2023205744A1 (en) 2022-04-20 2023-10-26 Tome Biosciences, Inc. Programmable gene insertion compositions
AU2023269134A1 (en) 2022-05-09 2024-12-12 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
WO2023225670A2 (en) 2022-05-20 2023-11-23 Tome Biosciences, Inc. Ex vivo programmable gene insertion
WO2024020587A2 (en) 2022-07-22 2024-01-25 Tome Biosciences, Inc. Pleiopluripotent stem cell programmable gene insertion
WO2024023734A1 (en) 2022-07-26 2024-02-01 Bit Bio Limited MULTI-gRNA GENOME EDITING
CN120659627A (zh) 2022-07-29 2025-09-16 瑞泽恩制药公司 用于转铁蛋白受体（tfr）介导的脑和肌肉递送的组合物和方法
EP4598944A2 (en) 2022-10-03 2025-08-13 The Regents of the University of California Circumventing barriers to hybrid crops from genetically distant crosses
AU2023371614A1 (en) 2022-11-04 2025-05-15 Regeneron Pharmaceuticals, Inc. Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle
AU2023379457A1 (en) 2022-11-14 2025-05-15 Regeneron Pharmaceuticals, Inc. Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes
JP2025538387A (ja) 2022-11-16 2025-11-28 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 プロテアーゼ切断可能なリンカーを有する膜結合ｉｌ－１２を含むキメラタンパク質
WO2024138194A1 (en) 2022-12-22 2024-06-27 Tome Biosciences, Inc. Platforms, compositions, and methods for in vivo programmable gene insertion
WO2024193739A1 (en) * 2023-03-23 2024-09-26 Ostravska Univerzita Method for producing proteins in host cells
WO2024234006A1 (en) 2023-05-11 2024-11-14 Tome Biosciences, Inc. Systems, compositions, and methods for targeting liver sinusodial endothelial cells (lsecs)
WO2025050069A1 (en) 2023-09-01 2025-03-06 Tome Biosciences, Inc. Programmable gene insertion using engineered integration enzymes
EP4777526A1 (en) 2023-09-12 2026-07-22 Aadigen, LLC Methods and compositions for treating or preventing cancer
WO2025224107A1 (en) 2024-04-22 2025-10-30 Basecamp Research Ltd Method and compositions for detecting off-target editing
WO2025224182A2 (en) 2024-04-23 2025-10-30 Basecamp Research Ltd Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo
WO2026080515A2 (en) 2024-10-08 2026-04-16 Regeneron Pharmaceuticals, Inc. Crispr sam biosensor cells and cell lines and methods of use thereof

Citations (13)

* 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
WO2014093622A2 (en) * 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US20140179006A1 (en) * 2012-12-12 2014-06-26 Massachusetts Institute Of Technology Crispr-cas component systems, methods and compositions for sequence manipulation
WO2014113493A1 (en) * 2013-01-16 2014-07-24 Emory University Cas9-nucleic acid complexes and uses related thereto
US20140273226A1 (en) * 2013-03-15 2014-09-18 System Biosciences, Llc Crispr/cas systems for genomic modification and gene modulation
US20140295557A1 (en) * 2013-03-15 2014-10-02 The General Hospital Corporation Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing
US20140310830A1 (en) * 2012-12-12 2014-10-16 Feng Zhang CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
US20140315985A1 (en) * 2013-03-14 2014-10-23 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US8906616B2 (en) * 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8993233B2 (en) * 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US20160153006A1 (en) * 2013-06-17 2016-06-02 The Broad Institute Inc. Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation
US20160160210A1 (en) * 2012-12-17 2016-06-09 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
US20160355795A1 (en) * 2013-12-12 2016-12-08 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation

Family Cites Families (147)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4123610A (en) 1977-03-09 1978-10-31 The United States Government Nucleic acid crosslinking agent and affinity inactivation of nucleic acids therewith
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US4981985A (en) 1987-10-20 1991-01-01 Trustees Of The University Of Pennsylvania Synthesis of photolabile chelators for multivalent cations
US4844617A (en) 1988-01-20 1989-07-04 Tencor Instruments Confocal measuring microscope with automatic focusing
US5151189A (en) 1990-09-17 1992-09-29 Gelman Sciences, Inc. Cationic charge modified microporous membrane
JP3455217B2 (ja) 1992-02-13 2003-10-14 バイオ−メトリック システムズ インコーポレイテッド 架橋マトリックス中の化学種の固定
US5594235A (en) 1993-06-17 1997-01-14 Ultrapointe Corporation Automated surface acquisition for a confocal microscope
US5578832A (en) 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
SE9400522D0 (sv) 1994-02-16 1994-02-16 Ulf Landegren Method and reagent for detecting specific nucleotide sequences
US5750341A (en) 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5830708A (en) 1995-06-06 1998-11-03 Advanced Tissue Sciences, Inc. Methods for production of a naturally secreted extracellular matrix
US6284284B1 (en) 1995-06-06 2001-09-04 Advanced Tissue Sciences, Inc. Compositions and methods for production and use of an injectable naturally secreted extracellular matrix
WO1997046704A1 (en) 1996-06-06 1997-12-11 Lynx Therapeutics, Inc. Sequencing by ligation of encoded adaptors
US5948617A (en) 1997-06-13 1999-09-07 Biospeparations, Inc. Methods of in situ hybridization
US6083726A (en) 1998-02-03 2000-07-04 Lucent Technologies, Inc. Methods for polynucleotide synthesis and articles for polynucleotide hybridization
US7060431B2 (en) 1998-06-24 2006-06-13 Illumina, Inc. Method of making and decoding of array sensors with microspheres
ATE508200T1 (de) 1999-02-23 2011-05-15 Caliper Life Sciences Inc Sequenzierung durch inkorporation
US6534266B1 (en) 1999-04-22 2003-03-18 Albert Einstein College Of Medicine Of Yeshiva University Assay of transcription sites by multi-fluor fish
US20020015952A1 (en) 1999-07-30 2002-02-07 Anderson Norman G. Microarrays and their manufacture by slicing
EP1242131A4 (en) 1999-10-12 2006-05-03 Univ Ohio State REACTIVE POLYMER VALVE, DISPENSING UNITS AND METHODS THAT USE THIS VALVE
WO2001037266A1 (en) 1999-11-17 2001-05-25 The Research Foundation Of State University Of New York Three dimensional data storage device and method for reading
WO2001068673A1 (en) 2000-03-14 2001-09-20 Active Motif Oligonucleotide analogues, methods of synthesis and methods of use
US7033758B2 (en) 2000-06-02 2006-04-25 Bayer Corporation Highly sensitive gene detection and localization using in situ branched-DNA hybridization
JP2004537263A (ja) 2000-12-14 2004-12-16 ジェン−プローブ・インコーポレーテッド ポリヌクレオチドの会合速度を増強するための方法およびキット
EP2801624B1 (en) 2001-03-16 2019-03-06 Singular Bio, Inc Arrays and methods of use
US7473767B2 (en) 2001-07-03 2009-01-06 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
US20060248349A1 (en) 2001-09-24 2006-11-02 Rathjen Alicemarie G Method for preparing and using personal and genetic profiles
US20030148335A1 (en) 2001-10-10 2003-08-07 Li Shen Detecting targets by unique identifier nucleotide tags
GB2382137A (en) 2001-11-20 2003-05-21 Mats Gullberg Nucleic acid enrichment
US20090246879A1 (en) 2002-12-20 2009-10-01 Callida Genomics Materials and Methods Relating to Nano-Tags and Nano-Brands
DE602004026033D1 (de) 2003-01-29 2010-04-29 454 Corp Zweiendige sequenzierung
CN1791682B (zh) 2003-02-26 2013-05-22 凯利达基因组股份有限公司 通过杂交进行的随机阵列dna分析
ES2436597T3 (es) 2003-05-20 2014-01-03 Lucid, Inc. Microscopio confocal para la formación de imágenes de lugares seleccionados del cuerpo de un paciente
WO2005047543A2 (en) 2003-06-10 2005-05-26 Applera Corporation Ligation assay
US7193054B2 (en) 2003-08-26 2007-03-20 University Of Rochester Nanofabrication using actin filaments
WO2005049851A2 (en) 2003-11-14 2005-06-02 University Of Utah Research Foundation Methods, articles, and compositions for identifying oligonucleotides
US7381529B2 (en) 2003-12-31 2008-06-03 Intel Corporation Methods and compositions for detecting nucleic acids using scanning probe microscopy and nanocodes
US20050191687A1 (en) 2004-02-27 2005-09-01 Tianxin Wang Method for multiplexed analyte detection
CA2557841A1 (en) 2004-02-27 2005-09-09 President And Fellows Of Harvard College Polony fluorescent in situ sequencing beads
US7427479B2 (en) 2004-04-30 2008-09-23 Applera Corporation Methods and kits for identifying target nucleotides in mixed populations
US20060024711A1 (en) 2004-07-02 2006-02-02 Helicos Biosciences Corporation Methods for nucleic acid amplification and sequence determination
US7253947B2 (en) 2004-10-07 2007-08-07 New York University Portable automated confocal microscope
BRPI0515726A8 (pt) * 2004-11-09 2017-09-12 Santaris Pharma As Oligonucleotídeos de lna potentes para a inibição de expressão de hif-1a
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
ITRM20050068A1 (it) 2005-02-17 2006-08-18 Istituto Naz Per Le Malattie I Metodo per la rivelazione di acidi nucleici di agenti patogeni batterici o di parassiti nelle urine.
US20060234261A1 (en) 2005-03-08 2006-10-19 Pierce Niles A Colorimetric readout of hybridization chain reaction
US7727721B2 (en) 2005-03-08 2010-06-01 California Institute Of Technology Hybridization chain reaction amplification for in situ imaging
US20070020650A1 (en) 2005-04-01 2007-01-25 Avak Kahvejian Methods for detecting proteins
JP2009501511A (ja) 2005-04-08 2009-01-22 マイコソル，インコーポレイテッド スチルバジウム研究アッセイ
WO2007145612A1 (en) 2005-06-06 2007-12-21 454 Life Sciences Corporation Paired end sequencing
US7842793B2 (en) 2005-06-14 2010-11-30 The California Institute Of Technology Methods of making nucleic acid nanostructures
DK1907571T3 (en) 2005-06-15 2017-08-21 Complete Genomics Inc NUCLEIC ACID ANALYSIS USING INCIDENTAL MIXTURES OF NON-OVERLAPPING FRAGMENTS
EP3042963A1 (en) 2005-06-20 2016-07-13 Advanced Cell Diagnostics, Inc. Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations
US8486621B2 (en) 2005-08-11 2013-07-16 Cornell Research Foundation, Inc. Nucleic acid-based matrixes
WO2007076129A2 (en) 2005-12-23 2007-07-05 Nanostring Technologies, Inc. Nanoreporters and methods of manufacturing and use thereof
US7864996B2 (en) 2006-02-17 2011-01-04 Lucid, Inc. System for macroscopic and confocal imaging of tissue
US20090220968A1 (en) 2006-03-10 2009-09-03 President And Fellows Of Harvard College Methods and Apparatus for Near Field Irradiation
GB0605584D0 (en) 2006-03-20 2006-04-26 Olink Ab Method for analyte detection using proximity probes
WO2007111937A1 (en) 2006-03-23 2007-10-04 Applera Corporation Directed enrichment of genomic dna for high-throughput sequencing
US8975216B2 (en) 2006-03-30 2015-03-10 Pacific Biosciences Of California Articles having localized molecules disposed thereon and methods of producing same
SG170802A1 (en) 2006-03-31 2011-05-30 Solexa Inc Systems and devices for sequence by synthesis analysis
AU2007237909A1 (en) 2006-04-19 2007-10-25 Applied Biosystems, Llc. Reagents, methods, and libraries for gel-free bead-based sequencing
US7964347B2 (en) 2006-06-15 2011-06-21 Krassen Dimitrov Labels for electronic detection of individual molecules and methods for their detection
WO2007149696A1 (en) 2006-06-23 2007-12-27 Applera Corporation Systems and methods for cooling in biological analysis instruments
US7745129B1 (en) 2006-07-12 2010-06-29 Kenneth David Schatz Methods for sequencing of a necleic acid
WO2008033848A2 (en) 2006-09-11 2008-03-20 The Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Self-assembled combinatorial encoding nanoarrays for multiplexed biosensing
US20090208965A1 (en) 2006-10-25 2009-08-20 Ikonisys, Inc. Automated method for detecting cancers and high grade hyperplasias
US9201063B2 (en) 2006-11-16 2015-12-01 General Electric Company Sequential analysis of biological samples
GB2457402B (en) 2006-12-01 2011-10-19 Univ Columbia Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US20080269068A1 (en) 2007-02-06 2008-10-30 President And Fellows Of Harvard College Multiplex decoding of sequence tags in barcodes
MX2009009071A (es) 2007-03-02 2009-09-24 Danisco Cultivos con resistencia mejorada a fagos.
US8145677B2 (en) 2007-03-27 2012-03-27 Faleh Jassem Al-Shameri Automated generation of metadata for mining image and text data
WO2008144562A1 (en) 2007-05-16 2008-11-27 California Institute Of Technology A versatile nucleic acid hairpin motif for programming biomolecular self-assembly pathways
JP5745842B2 (ja) 2007-06-19 2015-07-08 ストラトス ゲノミクス インコーポレイテッド 拡張によるハイスループット核酸配列決定
EP2185725A4 (en) 2007-08-02 2011-01-12 Biodesic COMPOSITIONS AND METHODS FOR IDENTIFYING AND QUANTIFYING ANALYTES
US20090191553A1 (en) 2007-10-01 2009-07-30 Applied Biosystems Inc. Chase Ligation Sequencing
EA201000427A1 (ru) 2007-10-04 2010-10-29 Хэлсион Молекулар Секвенирование нуклеиново-кислотных полимеров с использованием электронной микроскопии
US20090139311A1 (en) 2007-10-05 2009-06-04 Applied Biosystems Inc. Biological Analysis Systems, Devices, and Methods
US20110020291A1 (en) 2007-11-17 2011-01-27 Debrabrata Banerjee Use of stem cells for wound healing
CN101586150B (zh) 2008-05-23 2016-09-28 陕西佰美基因股份有限公司 检测探针、通用寡核苷酸芯片及核酸检测方法及其用途
US8546553B2 (en) 2008-07-25 2013-10-01 University Of Georgia Research Foundation, Inc. Prokaryotic RNAi-like system and methods of use
CN102171234A (zh) 2008-08-05 2011-08-31 康奈尔大学 光交联核酸水凝胶
JP5836803B2 (ja) 2008-08-14 2015-12-24 ナノストリング テクノロジーズ， インコーポレイテッド 安定したナノレポーター
US9156010B2 (en) 2008-09-23 2015-10-13 Bio-Rad Laboratories, Inc. Droplet-based assay system
US20100076057A1 (en) * 2008-09-23 2010-03-25 Northwestern University TARGET DNA INTERFERENCE WITH crRNA
US20100087325A1 (en) 2008-10-07 2010-04-08 Illumina, Inc. Biological sample temperature control system and method
WO2010054108A2 (en) 2008-11-06 2010-05-14 University Of Georgia Research Foundation, Inc. Cas6 polypeptides and methods of use
US8309306B2 (en) 2008-11-12 2012-11-13 Nodality, Inc. Detection composition
EP3290531B1 (en) 2008-12-19 2019-07-24 President and Fellows of Harvard College Particle-assisted nucleic acid sequencing
JP4528345B2 (ja) 2009-01-28 2010-08-18 シャープ株式会社 動画再生装置、動画再生方法、動画再生方法をコンピュータで実現するためのプログラム及びそのプログラムを記録した記録媒体
FR2948475A1 (fr) 2009-07-24 2011-01-28 Bionext Procede de caracterisation d'objets tridimensionnels
US8431151B2 (en) 2009-08-06 2013-04-30 Syracuse University Antimicrobial nanostructured hydrogel web containing silver
US9714937B2 (en) 2009-10-13 2017-07-25 Nanostring Technologies, Inc. Protein detection via nanoreporters
WO2011056863A1 (en) 2009-11-03 2011-05-12 High Throughput Genomics, Inc. Quantitative nuclease protection sequencing (qnps)
US9089512B2 (en) 2009-12-18 2015-07-28 President And Fellows Of Harvard College Active scaffolds for on-demand drug and cell delivery
US20110257031A1 (en) 2010-02-12 2011-10-20 Life Technologies Corporation Nucleic acid, biomolecule and polymer identifier codes
US10267808B2 (en) 2010-03-08 2019-04-23 California Institute Of Technology Molecular indicia of cellular constituents and resolving the same by super-resolution technologies in single cells
US10087431B2 (en) 2010-03-10 2018-10-02 The Regents Of The University Of California Methods of generating nucleic acid fragments
GB201004292D0 (en) 2010-03-15 2010-04-28 Olink Ab Assay for localised detection of analytes
KR101866401B1 (ko) 2010-04-05 2018-06-11 프로그노시스 바이오사이언스, 인코포레이티드 공간적으로 엔코딩된 생물학적 검정
CA2798703A1 (en) 2010-05-10 2011-11-17 The Regents Of The University Of California Endoribonuclease compositions and methods of use thereof
WO2011143583A1 (en) 2010-05-13 2011-11-17 Illumina, Inc. Binding assays for markers
AU2011274642B2 (en) 2010-07-09 2015-05-21 Cergentis B.V. 3-d genomic region of interest sequencing strategies
WO2012056440A1 (en) 2010-10-28 2012-05-03 Nanodoc Ltd. COMPOSITIONS AND METHODS FOR ACTIVATING EXPRESSION BY A SPECIFIC ENDOGENOUS miRNA
WO2012058638A2 (en) 2010-10-29 2012-05-03 President And Fellows Of Harvard College Nucleic acid nanostructure barcode probes
WO2012067911A1 (en) 2010-11-16 2012-05-24 Nabsys, Inc. Methods for sequencing a biomolecule by detecting relative positions of hybridized probes
JP5881746B2 (ja) 2011-02-15 2016-03-09 ライカ バイオシステムズ ニューキャッスル リミテッド ｍＲＮＡの局在インサイチュー検出のための方法
JP5687514B2 (ja) 2011-02-17 2015-03-18 株式会社日立ハイテクノロジーズ 核酸配列解析装置
DK2500436T3 (en) 2011-03-17 2016-08-29 Pasteur Institut A method, probe and kit for in situ hybridization of DNA and use thereof
US20150086981A1 (en) 2011-05-04 2015-03-26 Genovoxx Gmbh Nucleoside-triphosphate conjugate and methods for the use thereof
CN103890191B (zh) 2011-05-27 2018-12-04 哈佛大学校长及研究员协会 单细胞全基因组扩增方法
US20140113376A1 (en) 2011-06-01 2014-04-24 Rotem Sorek Compositions and methods for downregulating prokaryotic genes
PL222511B1 (pl) 2011-06-08 2016-08-31 Międzynarodowy Inst Biologii Molekularnej I Komórkowej W Warszawie Endorybonukleazy dsRNA
EP2766498B1 (en) 2011-10-14 2019-06-19 President and Fellows of Harvard College Sequencing by structure assembly
GB201122458D0 (en) 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
EP2836226B1 (en) 2012-02-24 2017-06-28 Fred Hutchinson Cancer Research Center Compositions and methods for the treatment of hemoglobinopathies
EP3321378B1 (en) 2012-02-27 2021-11-10 Becton, Dickinson and Company Compositions for molecular counting
CA2865011C (en) 2012-02-29 2021-06-15 Sangamo Biosciences, Inc. Methods and compositions for treating huntington's disease
WO2013141680A1 (en) 2012-03-20 2013-09-26 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US9637739B2 (en) 2012-03-20 2017-05-02 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
SG196730A1 (en) * 2012-07-16 2014-02-13 Agency Science Tech & Res Methods for reading data from a storage medium using a reader and storage devices
US11225683B2 (en) 2012-07-20 2022-01-18 Lsi Medience Corporation Photocoupling method using probe containing photoresponsive nucleic acids
HK1207111A1 (en) 2012-08-03 2016-01-22 加利福尼亚大学董事会 Methods and compositions for controlling gene expression by rna processing
US9709791B2 (en) 2012-08-15 2017-07-18 Lucid, Inc. Systems and methods for imaging tissue
CN102908119A (zh) 2012-09-26 2013-02-06 温州医学院眼视光研究院 一种共焦扫描成像系统及其像差控制方法
EP4397759A3 (en) 2012-10-23 2024-10-09 Toolgen Incorporated 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
DK3363902T3 (da) * 2012-12-06 2020-01-02 Sigma Aldrich Co Llc Crispr-baseret genommodifikation og -regulering
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US9411930B2 (en) 2013-02-01 2016-08-09 The Regents Of The University Of California Methods for genome assembly and haplotype phasing
US10138509B2 (en) 2013-03-12 2018-11-27 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
US20140273230A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
US9330295B2 (en) 2013-03-15 2016-05-03 Brown University Spatial sequencing/gene expression camera
US10510435B2 (en) 2013-04-30 2019-12-17 California Institute Of Technology Error correction of multiplex imaging analysis by sequential hybridization
KR102368591B1 (ko) 2013-04-30 2022-02-25 캘리포니아 인스티튜트 오브 테크놀로지 순차적 하이브리드화 바코딩에 의한 분자의 멀티플렉스 표지화
DK3004349T3 (en) * 2013-05-29 2018-06-06 Cellectis Sa A method for producing precise DNA cleavage using CAS9 nickase activity
EP3603679B1 (en) * 2013-06-04 2022-08-10 President and Fellows of Harvard College Rna-guided transcriptional regulation
EP3058091B1 (en) 2013-10-18 2020-03-25 The Broad Institute, Inc. Spatial and cellular mapping of biomolecules in situ by high-throughput sequencing
GB201401885D0 (en) 2014-02-04 2014-03-19 Olink Ab Proximity assay with detection based on hybridisation chain reaction (HCR)
EP3108218A4 (en) 2014-02-21 2017-11-15 Massachusetts Institute Of Technology Expansion microscopy
AU2015349870B2 (en) 2014-11-21 2019-09-26 Bruker Spatial Biology, Inc. Enzyme- and amplification-free sequencing
WO2016138496A1 (en) 2015-02-27 2016-09-01 Cellular Research, Inc. Spatially addressable molecular barcoding
CN107406888A (zh) 2015-03-30 2017-11-28 赛卢拉研究公司 用于组合条形编码的方法和组合物
CN108474022A (zh) 2015-11-03 2018-08-31 哈佛学院董事及会员团体 用于包含三维核酸的基质容积成像的设备和方法
CN108700460B (zh) 2015-12-21 2020-11-03 威里利生命科学有限责任公司 成像系统和成像方法
US10844426B2 (en) 2016-03-17 2020-11-24 President And Fellows Of Harvard College Methods for detecting and identifying genomic nucleic acids
ES2908919T3 (es) 2016-07-05 2022-05-04 California Inst Of Techn Reacción en cadena de hibridación de iniciador fraccionario
CN110446719B (zh) 2017-03-31 2023-10-03 乌尔蒂维尤股份有限公司 Dna-抗原交换和扩增

Patent Citations (20)

* 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
US20170191078A1 (en) * 2012-12-12 2017-07-06 The Broad Institute Inc. CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
US20140179770A1 (en) * 2012-12-12 2014-06-26 Massachusetts Institute Of Technology Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US20140179006A1 (en) * 2012-12-12 2014-06-26 Massachusetts Institute Of Technology Crispr-cas component systems, methods and compositions for sequence manipulation
WO2014093622A2 (en) * 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US20150247150A1 (en) * 2012-12-12 2015-09-03 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US20140310830A1 (en) * 2012-12-12 2014-10-16 Feng Zhang CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
US20160340662A1 (en) * 2012-12-12 2016-11-24 The Broad Institute, Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8871445B2 (en) * 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8906616B2 (en) * 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8932814B2 (en) * 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8993233B2 (en) * 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8999641B2 (en) * 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US20160160210A1 (en) * 2012-12-17 2016-06-09 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
WO2014113493A1 (en) * 2013-01-16 2014-07-24 Emory University Cas9-nucleic acid complexes and uses related thereto
US20140315985A1 (en) * 2013-03-14 2014-10-23 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US20140273226A1 (en) * 2013-03-15 2014-09-18 System Biosciences, Llc Crispr/cas systems for genomic modification and gene modulation
US20140295557A1 (en) * 2013-03-15 2014-10-02 The General Hospital Corporation Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing
US20160153006A1 (en) * 2013-06-17 2016-06-02 The Broad Institute Inc. Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation
US20160355795A1 (en) * 2013-12-12 2016-12-08 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Sapranauskas et al (Nucleic Acids Research, 2011, 39:9275-9282) *

Cited By (318)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US11976307B2 (en) 2012-04-27 2024-05-07 Duke University Genetic correction of mutated genes
US20210040460A1 (en) 2012-04-27 2021-02-11 Duke University Genetic correction of mutated genes
US10711256B2 (en) 2012-04-27 2020-07-14 Duke University Genetic correction of mutated genes
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
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
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
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
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
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
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
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
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
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
US11970711B2 (en) 2012-05-25 2024-04-30 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
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
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
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
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
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
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
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
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
US12180504B2 (en) 2012-05-25 2024-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
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
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
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
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
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
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
US12180503B2 (en) 2012-05-25 2024-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
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
US12215343B2 (en) 2012-05-25 2025-02-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
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
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
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
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
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
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
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
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
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
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
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
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
US12123015B2 (en) 2012-05-25 2024-10-22 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
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
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
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
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
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
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
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
US10745716B2 (en) 2012-12-06 2020-08-18 Sigma-Aldrich Co. Llc CRISPR-based genome modification and regulation
US10731181B2 (en) 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
US12252707B2 (en) 2012-12-12 2025-03-18 The Broad Institute, Inc. Delivery, Engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US10930367B2 (en) 2012-12-12 2021-02-23 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
US11041173B2 (en) 2012-12-12 2021-06-22 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US12454687B2 (en) 2012-12-12 2025-10-28 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
US10202619B2 (en) 2013-03-15 2019-02-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
US9234213B2 (en) 2013-03-15 2016-01-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
US9738908B2 (en) 2013-03-15 2017-08-22 System Biosciences, Llc CRISPR/Cas systems for genomic modification and gene modulation
US12215345B2 (en) 2013-03-19 2025-02-04 Duke University Compositions and methods for the induction and tuning of gene expression
US9822370B2 (en) 2013-04-04 2017-11-21 President And Fellows Of Harvard College Method of making a deletion in a target sequence in isolated primary cells using Cas9 and two guide RNAs
US10745714B2 (en) * 2013-06-05 2020-08-18 Duke University RNA-guided gene editing and gene regulation
US20180320197A1 (en) * 2013-06-05 2018-11-08 Duke University Rna-guided gene editing and gene regulation
US10704060B2 (en) 2013-06-05 2020-07-07 Duke University RNA-guided gene editing and gene regulation
US11597949B2 (en) 2013-06-17 2023-03-07 The Broad Institute, Inc. Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
US10781444B2 (en) 2013-06-17 2020-09-22 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
US12441995B2 (en) 2013-06-17 2025-10-14 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
US12018275B2 (en) 2013-06-17 2024-06-25 The Broad Institute, Inc. Delivery and use of the CRISPR-CAS systems, vectors and compositions for hepatic targeting and therapy
US10577630B2 (en) 2013-06-17 2020-03-03 The Broad Institute, Inc. Delivery and use of the CRISPR-Cas systems, vectors and compositions for hepatic targeting and therapy
US12553065B2 (en) 2013-06-17 2026-02-17 The Broad Institute, Inc. Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
US10946108B2 (en) 2013-06-17 2021-03-16 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for targeting disorders and diseases using viral components
US11008588B2 (en) 2013-06-17 2021-05-18 The Broad Institute, Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US10208319B2 (en) 2013-07-09 2019-02-19 President And Fellows Of Harvard College Therapeutic uses of genome editing with CRISPR/Cas systems
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10227581B2 (en) 2013-08-22 2019-03-12 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 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
US12559737B2 (en) 2013-09-06 2026-02-24 President And Fellows Of Harvard College Cas9 variants and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
US12473573B2 (en) 2013-09-06 2025-11-18 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
US12584118B2 (en) 2013-09-06 2026-03-24 President And Fellows Of Harvard College Cas9 variants and uses thereof
US11499169B2 (en) 2013-10-30 2022-11-15 North Carolina State University Compositions and methods related to a type-II CRISPR-Cas system in Lactobacillus buchneri
US10584358B2 (en) 2013-10-30 2020-03-10 North Carolina State University Compositions and methods related to a type-II CRISPR-Cas system in Lactobacillus buchneri
US11390887B2 (en) 2013-11-07 2022-07-19 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9834791B2 (en) 2013-11-07 2017-12-05 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
US20150140664A1 (en) * 2013-11-19 2015-05-21 President And Fellows Of Harvard College Large Gene Excision and Insertion
US10787684B2 (en) * 2013-11-19 2020-09-29 President And Fellows Of Harvard College Large gene excision and insertion
US12215365B2 (en) 2013-12-12 2025-02-04 President And Fellows Of Harvard College Cas variants for gene editing
US11155795B2 (en) 2013-12-12 2021-10-26 The Broad Institute, Inc. CRISPR-Cas systems, crystal structure and uses thereof
US12410435B2 (en) 2013-12-12 2025-09-09 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US10851357B2 (en) 2013-12-12 2020-12-01 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US12258595B2 (en) 2013-12-12 2025-03-25 The Broad Institute, Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
US12421506B2 (en) 2013-12-12 2025-09-23 The Broad Institute, Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11597919B2 (en) 2013-12-12 2023-03-07 The Broad Institute Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11591581B2 (en) 2013-12-12 2023-02-28 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US11407985B2 (en) 2013-12-12 2022-08-09 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for genome editing
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US12251450B2 (en) 2013-12-12 2025-03-18 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for HBV and viral diseases and disorders
US10787654B2 (en) 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
US12698490B2 (en) 2014-01-24 2026-08-04 North Carolina State University Methods and compositions for sequences guiding Cas9 targeting
US11439712B2 (en) 2014-04-08 2022-09-13 North Carolina State University Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12398406B2 (en) 2014-07-30 2025-08-26 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US9970030B2 (en) 2014-08-27 2018-05-15 Caribou Biosciences, Inc. Methods for increasing CAS9-mediated engineering efficiency
US11753651B2 (en) 2014-08-28 2023-09-12 North Carolina State University Cas9 proteins and guiding features for DNA targeting and genome editing
US10450584B2 (en) 2014-08-28 2019-10-22 North Carolina State University Cas9 proteins and guiding features for DNA targeting and genome editing
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
US12180263B2 (en) 2014-11-06 2024-12-31 President And Fellows Of Harvard College Cells lacking B2M surface expression and methods for allogeneic administration of such cells
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US12571005B2 (en) 2014-12-12 2026-03-10 The Broad Institute, Inc. Protected guide RNAs (pgRNAs)
US20240093193A1 (en) * 2014-12-12 2024-03-21 The Broad Institute, Inc. Dead guides for crispr transcription factors
US11624078B2 (en) 2014-12-12 2023-04-11 The Broad Institute, Inc. Protected guide RNAS (pgRNAS)
US20170321214A1 (en) * 2014-12-12 2017-11-09 The Broad Institute Inc. Dead guides for crispr transcription factors
US10696986B2 (en) 2014-12-12 2020-06-30 The Board Institute, Inc. Protected guide RNAS (PGRNAS)
US12435320B2 (en) 2014-12-24 2025-10-07 The Broad Institute, Inc. CRISPR having or associated with destabilization domains
WO2016114972A1 (en) 2015-01-12 2016-07-21 The Regents Of The University Of California Heterodimeric cas9 and methods of use thereof
US11180792B2 (en) 2015-01-28 2021-11-23 The Regents Of The University Of California Methods and compositions for labeling a single-stranded target nucleic acid
US11155796B2 (en) 2015-02-09 2021-10-26 Duke University Compositions and methods for epigenome editing
US10676726B2 (en) 2015-02-09 2020-06-09 Duke University Compositions and methods for epigenome editing
US12215366B2 (en) 2015-02-09 2025-02-04 Duke University Compositions and methods for epigenome editing
US12534715B2 (en) 2015-04-16 2026-01-27 Wageningen Universiteit Nucleic acids for nuclease-mediated genome editing
US11760985B2 (en) 2015-04-16 2023-09-19 Wageningen Universiteit CRISPR-Cas complex
US11479761B2 (en) 2015-04-16 2022-10-25 Wageningen Universiteit Nuclease-mediated genome editing
US11053482B2 (en) 2015-04-16 2021-07-06 Wageningen Universiteit Vector systems for nuclease-mediated genome editing
US12497590B2 (en) 2015-05-08 2025-12-16 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12031154B2 (en) 2015-05-08 2024-07-09 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12662657B2 (en) 2015-05-08 2026-06-23 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12110500B2 (en) 2015-05-08 2024-10-08 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12421493B2 (en) 2015-05-08 2025-09-23 President And Fellows Of Harvard College Universal donor stem cells and related methods
US12031155B2 (en) 2015-05-08 2024-07-09 President And Fellows Of Harvard College Universal donor stem cells and related methods
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11261451B2 (en) 2015-05-29 2022-03-01 North Carolina State University Methods for screening bacteria, archaea, algae, and yeast using CRISPR nucleic acids
US10136649B2 (en) 2015-05-29 2018-11-27 North Carolina State University Methods for screening bacteria, archaea, algae, and yeast using CRISPR nucleic acids
US12251429B2 (en) 2015-06-01 2025-03-18 Temple University—Of the Commonwealth System of Higher Education Methods and compositions for RNA-guided treatment of HIV infection
WO2016196655A1 (en) 2015-06-03 2016-12-08 The Regents Of The University Of California Cas9 variants and methods of use thereof
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
US12606834B2 (en) 2015-06-15 2026-04-21 North Carolina State University Methods and compositions for efficient delivery of nucleic acids and rna-based antimicrobials
US11155823B2 (en) 2015-06-15 2021-10-26 North Carolina State University Methods and compositions for efficient delivery of nucleic acids and RNA-based antimicrobials
US11578312B2 (en) 2015-06-18 2023-02-14 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
US12091709B2 (en) 2015-06-18 2024-09-17 The Broad Institute, Inc. Crispr enzymes and systems
TWI758251B (zh) * 2015-06-18 2022-03-21 美商博得學院股份有限公司 新型ｃｒｉｓｐｒ酶以及系統
US12168789B2 (en) 2015-06-18 2024-12-17 The Broad Institute, Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
US12410468B2 (en) 2015-06-18 2025-09-09 The Broad Institute, Inc. CRISPR enzymes and systems
US12123032B2 (en) 2015-06-18 2024-10-22 The Broad Institute, Inc. CRISPR enzyme mutations reducing off-target effects
US12264359B2 (en) 2015-06-18 2025-04-01 The Broad Institute, Inc. CRISPR enzymes and systems
US11091798B2 (en) 2015-06-18 2021-08-17 The Broad Institute, Inc. CRISPR enzymes and systems
US11634755B2 (en) 2015-06-18 2023-04-25 The Broad Institute, Inc. Crispr enzymes and systems
US12371737B2 (en) 2015-06-18 2025-07-29 The Broad Institute, Inc. CRISPR enzymes and systems
US11781172B2 (en) 2015-06-18 2023-10-10 The Broad Institute, Inc. Crispr enzymes and systems
US11773432B2 (en) 2015-06-18 2023-10-03 The Broad Institute Inc. CRISPR enzymes and systems
US10494621B2 (en) 2015-06-18 2019-12-03 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
US10876100B2 (en) 2015-06-18 2020-12-29 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
US10676735B2 (en) 2015-07-22 2020-06-09 Duke University High-throughput screening of regulatory element function with epigenome editing technologies
US11427817B2 (en) 2015-08-25 2022-08-30 Duke University Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11286480B2 (en) 2015-09-28 2022-03-29 North Carolina State University Methods and compositions for sequence specific antimicrobials
US11970710B2 (en) 2015-10-13 2024-04-30 Duke University Genome engineering with Type I CRISPR systems in eukaryotic cells
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US12344869B2 (en) 2015-10-23 2025-07-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US12214054B2 (en) 2015-11-30 2025-02-04 Duke University Therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use
US12110490B2 (en) 2015-12-18 2024-10-08 The Broad Institute, Inc. CRISPR enzymes and systems
US11542466B2 (en) 2015-12-22 2023-01-03 North Carolina State University Methods and compositions for delivery of CRISPR based antimicrobials
US9896696B2 (en) 2016-02-15 2018-02-20 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
EP4063501A1 (en) * 2016-02-15 2022-09-28 Benson Hill, Inc. Compositions and methods for modifying genomes
WO2017141173A3 (en) * 2016-02-15 2017-11-02 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes using cpf1 or csm1
EP4306642A3 (en) * 2016-02-15 2024-07-03 Benson Hill Holdings, Inc. Compositions and methods for modifying genomes
US10113179B2 (en) 2016-02-15 2018-10-30 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
WO2017143071A1 (en) 2016-02-18 2017-08-24 The Regents Of The University Of California Methods and compositions for gene editing in stem cells
WO2017161043A1 (en) 2016-03-16 2017-09-21 The J. David Gladstone Institutes Methods and compositions for treating obesity and/or diabetes and for identifying candidate treatment agents
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US12049651B2 (en) 2016-04-13 2024-07-30 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US12428631B2 (en) 2016-04-13 2025-09-30 Duke University CRISPR/Cas9-based repressors for silencing gene targets in vivo and methods of use
US11286478B2 (en) 2016-04-19 2022-03-29 The Broad Institute, Inc. Cpf1 complexes with reduced indel activity
US12344838B2 (en) 2016-04-19 2025-07-01 The Broad Institute, Inc. CPF1 complexes with reduced indel activity
US12410416B2 (en) 2016-04-19 2025-09-09 The Broad Institute, Inc. CRISPR enzymes and systems with modified pam specificity
WO2018005445A1 (en) 2016-06-27 2018-01-04 The Broad Institute, Inc. Compositions and methods for detecting and treating diabetes
US12214056B2 (en) 2016-07-19 2025-02-04 Duke University Therapeutic applications of CPF1-based genome editing
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US12084663B2 (en) 2016-08-24 2024-09-10 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
WO2018064371A1 (en) 2016-09-30 2018-04-05 The Regents Of The University Of California Rna-guided nucleic acid modifying enzymes and methods of use thereof
US10669539B2 (en) 2016-10-06 2020-06-02 Pioneer Biolabs, Llc Methods and compositions for generating CRISPR guide RNA libraries
US12668815B2 (en) 2016-10-14 2026-06-30 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
WO2018112278A1 (en) 2016-12-14 2018-06-21 Ligandal, Inc. Methods and compositions for nucleic acid and protein payload delivery
US12123012B2 (en) 2016-12-14 2024-10-22 Ligandal, Inc. Methods and compositions for nucleic acid and protein payload delivery
US10975388B2 (en) 2016-12-14 2021-04-13 Ligandal, Inc. Methods and compositions for nucleic acid and protein payload delivery
US12286727B2 (en) 2016-12-19 2025-04-29 Editas Medicine, Inc. Assessing nuclease cleavage
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US12110545B2 (en) 2017-01-06 2024-10-08 Editas Medicine, Inc. Methods of assessing nuclease cleavage
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US12516308B2 (en) 2017-03-09 2026-01-06 President And Fellows Of Harvard College Suppression of pain by gene editing
US12435331B2 (en) 2017-03-10 2025-10-07 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
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
US12297466B2 (en) 2017-06-09 2025-05-13 Editas Medicine, Inc. Engineered Cas9 nucleases
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
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
US12359218B2 (en) 2017-07-28 2025-07-15 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
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)
US10837023B2 (en) 2017-08-09 2020-11-17 Benson Hill, Inc. Compositions and methods for modifying genomes
US12600975B2 (en) 2017-08-09 2026-04-14 Ricetec, Inc. Compositions and methods for modifying genomes
US12146141B2 (en) 2017-08-09 2024-11-19 Ricetec, Inc. Compositions and methods for modifying genomes
US11624070B2 (en) 2017-08-09 2023-04-11 Benson Hill, Inc. Compositions and methods for modifying genomes
US10316324B2 (en) 2017-08-09 2019-06-11 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
WO2019113132A1 (en) * 2017-12-05 2019-06-13 Caribou Biosciences, Inc. Modified lymphocytes
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12509492B2 (en) 2018-01-19 2025-12-30 Duke University Genome engineering with CRISPR-Cas systems in eukaryotes
WO2019195738A1 (en) 2018-04-06 2019-10-10 Children's Medical Center Corporation Compositions and methods for somatic cell reprogramming and modulating imprinting
US12133884B2 (en) 2018-05-11 2024-11-05 Beam Therapeutics Inc. Methods of substituting pathogenic amino acids using programmable base editor systems
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12338436B2 (en) 2018-06-29 2025-06-24 Editas Medicine, Inc. Synthetic guide molecules, compositions and methods relating thereto
US12522807B2 (en) 2018-07-09 2026-01-13 The Broad Institute, Inc. RNA programmable epigenetic RNA modifiers and uses thereof
WO2020014528A1 (en) 2018-07-13 2020-01-16 The Regents Of The University Of California Retrotransposon-based delivery vehicle and methods of use thereof
WO2020041456A1 (en) 2018-08-22 2020-02-27 The Regents Of The University Of California Variant type v crispr/cas effector polypeptides and methods of use thereof
US12454694B2 (en) 2018-09-07 2025-10-28 Beam Therapeutics Inc. Compositions and methods for improving base editing
US12529041B2 (en) 2018-09-07 2026-01-20 Beam Therapeutics Inc. Compositions and methods for delivering a nucleobase editing system
US12203123B2 (en) 2018-10-01 2025-01-21 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for screening for variant cells
US12264313B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression
US10711267B2 (en) 2018-10-01 2020-07-14 North Carolina State University Recombinant type I CRISPR-Cas system
US12264330B2 (en) 2018-10-01 2025-04-01 North Carolina State University Recombinant type I CRISPR-Cas system and uses thereof for killing target cells
US11680259B2 (en) 2018-10-01 2023-06-20 North Carolina State University Recombinant type I CRISPR-CAS system
US11407995B1 (en) 2018-10-26 2022-08-09 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12071641B2 (en) 2018-11-02 2024-08-27 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US11434477B1 (en) 2018-11-02 2022-09-06 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US12460189B2 (en) 2018-11-09 2025-11-04 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
EP3877517A1 (en) 2018-11-09 2021-09-15 Inari Agriculture, Inc. Rna-guided nucleases and dna binding proteins
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
US12612618B2 (en) 2019-01-31 2026-04-28 Beam Therapeutics Inc. Nucleobase editors having reduced non-target deamination and assays for characterizing nucleobase editors
WO2020163856A1 (en) 2019-02-10 2020-08-13 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Modified mitochondrion and methods of use thereof
US12622931B2 (en) 2019-02-13 2026-05-12 Beam Therapeutics Inc. Compositions and methods for treating alpha-1 antitrypsin deficiency
US12600971B2 (en) 2019-02-13 2026-04-14 Beam Therapeutics Inc. Modified immune cells having adenosine deaminase base editors for modifying a nucleobase in a target sequence
EP4219700A1 (en) 2019-03-07 2023-08-02 The Regents of the University of California Crispr-cas effector polypeptides and methods of use thereof
DE212020000516U1 (de) 2019-03-07 2022-01-17 The Regents of the University of California CRISPR-CAS-Effektorpolypeptide
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12624353B2 (en) 2019-03-19 2026-05-12 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12624354B2 (en) 2019-03-19 2026-05-12 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12570972B2 (en) 2019-03-19 2026-03-10 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12509680B2 (en) 2019-03-19 2025-12-30 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12281303B2 (en) 2019-03-19 2025-04-22 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12629428B2 (en) 2019-04-14 2026-05-19 Duke University AAV vector-mediated deletion of large mutational hotspot for treatment of Duchenne muscular dystrophy
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
US12576110B2 (en) 2019-08-06 2026-03-17 The Children's Medical Center Corporation Methods and compositions for reconstituting microglia
US12594301B2 (en) 2019-09-27 2026-04-07 Beam Therapeutics Inc. Compositions and methods for treatment of liquid cancers
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
US20240117383A1 (en) * 2020-05-04 2024-04-11 Editas Medicine, Inc. Selection by essential-gene knock-in
WO2021224633A1 (en) 2020-05-06 2021-11-11 Orchard Therapeutics (Europe) Limited Treatment for neurodegenerative diseases
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2020-05-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12576151B2 (en) 2020-09-25 2026-03-17 Beam Therapeutics Inc. Fratricide resistant modified immune cells and methods of using the same
US12637690B2 (en) 2020-11-13 2026-05-26 Egenesis, Inc. Cells, tissues, organs, and animals having one or more modified genes for enhanced xenograft survival and tolerance
US12630838B2 (en) 2020-11-13 2026-05-19 Egenesis, Inc. Cells, tissues, organs, and animals having one or more modified genes for enhanced xenograft survival and tolerance
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
US12098399B2 (en) 2022-06-24 2024-09-24 Tune Therapeutics, Inc. Compositions, systems, and methods for epigenetic regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression
WO2024020346A2 (en) 2022-07-18 2024-01-25 Renagade Therapeutics Management Inc. Gene editing components, systems, and methods of use
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2025049959A2 (en) 2023-09-01 2025-03-06 Renagade Therapeutics Management Inc. Gene editing systems, compositions, and methods for treatment of vexas syndrome
WO2025064408A1 (en) 2023-09-18 2025-03-27 The Broad Institute, Inc. Compositions and methods for treating cardiovascular disease
WO2025081042A1 (en) 2023-10-12 2025-04-17 Renagade Therapeutics Management Inc. Nickase-retron template-based precision editing system and methods of use
WO2025155753A2 (en) 2024-01-17 2025-07-24 Renagade Therapeutics Management Inc. Improved gene editing system, guides, and methods
WO2025174765A1 (en) 2024-02-12 2025-08-21 Renagade Therapeutics Management Inc. Lipid nanoparticles comprising coding rna molecules for use in gene editing and as vaccines and therapeutic agents

Also Published As

Publication number Publication date
BR112015030491A2 (pt) 2017-07-25
EP3003392A2 (en) 2016-04-13
NZ753951A (en) 2021-09-24
RU2015156198A (ru) 2017-07-14
CN113846096B (zh) 2025-05-09
EP4159243A1 (en) 2023-04-05
WO2014197568A2 (en) 2014-12-11
CN113846096A (zh) 2021-12-28
MX2026002033A (es) 2026-04-01
MY197877A (en) 2023-07-22
CA2914638C (en) 2026-03-24
IL284773B1 (en) 2023-05-01
MX385330B (es) 2025-03-18
EP3003392A4 (en) 2017-05-03
ES2930537T3 (es) 2022-12-16
RU2019114706A (ru) 2019-10-11
AU2020203977B2 (en) 2021-05-27
AU2021221488B2 (en) 2024-03-28
CA2914638A1 (en) 2014-12-11
JP6621738B2 (ja) 2019-12-18
JP2026021450A (ja) 2026-02-10
JP2022166077A (ja) 2022-11-01
CA3176690A1 (en) 2014-12-11
NZ715280A (en) 2021-06-25
JP7376650B2 (ja) 2023-11-08
SG10201913068PA (en) 2020-02-27
AU2024204479A1 (en) 2024-07-18
JP7036511B2 (ja) 2022-03-15
US20140356959A1 (en) 2014-12-04
IL302205B1 (en) 2025-04-01
AU2020203977A1 (en) 2020-07-02
JP2016521554A (ja) 2016-07-25
JP2021045153A (ja) 2021-03-25
KR102884236B1 (ko) 2025-11-12
AU2024204479B2 (en) 2026-08-06
KR102512979B1 (ko) 2023-03-22
ZA201509025B (en) 2025-09-25
RU2019114706A3 (cg-RX-API-DMAC10.html) 2020-09-25
IL302205B2 (en) 2025-08-01
BR112015030491A8 (pt) 2022-07-26
JP2019122384A (ja) 2019-07-25
EP3603679A1 (en) 2020-02-05
CN105451778A (zh) 2016-03-30
KR20210095235A (ko) 2021-07-30
IL284773A (en) 2021-08-31
KR102282990B1 (ko) 2021-07-28
RU2756865C2 (ru) 2021-10-06
JP2024012387A (ja) 2024-01-30
SG11201509962QA (en) 2016-01-28
US20240175057A1 (en) 2024-05-30
KR20230042154A (ko) 2023-03-27
IL302205A (en) 2023-06-01
CN105451778B (zh) 2021-08-03
WO2014197568A3 (en) 2015-03-12
EP4596565A2 (en) 2025-08-06
JP7768951B2 (ja) 2025-11-12
RU2690935C2 (ru) 2019-06-06
EP4596565A3 (en) 2025-11-05
JP7119055B2 (ja) 2022-08-16
MX2021009672A (es) 2021-09-08
NZ753950A (en) 2021-09-24
US20200024618A1 (en) 2020-01-23
US20200299732A1 (en) 2020-09-24
AU2014274939A1 (en) 2016-01-07
IL284773B2 (en) 2023-09-01
IL242959B (en) 2021-08-31
US9267135B2 (en) 2016-02-23
EP3603679B1 (en) 2022-08-10
US11981917B2 (en) 2024-05-14
US10640789B2 (en) 2020-05-05
KR20160014036A (ko) 2016-02-05
EP3003392B1 (en) 2019-10-23
MX2015016798A (es) 2016-10-26
US10767194B2 (en) 2020-09-08
RU2015156198A3 (cg-RX-API-DMAC10.html) 2018-05-08
MY177814A (en) 2020-09-23
AU2014274939B2 (en) 2020-03-19
SG10201710030QA (en) 2018-01-30
MY208492A (en) 2025-05-13
US20230131972A1 (en) 2023-04-27
AU2021221488A1 (en) 2021-09-16
US20160237456A1 (en) 2016-08-18

Similar Documents

Publication Publication Date Title
US11981917B2 (en) RNA-guided transcriptional regulation
AU2021273624C1 (en) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
CA2906553C (en) Rna-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
NZ753950B2 (en) Rna-guided transcriptional regulation
NZ715280B2 (en) Rna-guided transcriptional regulation
NZ753951B2 (en) Rna-guided transcriptional regulation

Legal Events

Date Code Title Description
AS Assignment

Owner name: PRESIDENT AND FELLOWS OF HARVARD COLLEGE, MASSACHU

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CHURCH, GEORGE M.;ESVELT, KEVIN M.;MALI, PRASHANT;SIGNING DATES FROM 20140924 TO 20141001;REEL/FRAME:034088/0230

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:HARVARD UNIVERSITY;REEL/FRAME:036897/0392

Effective date: 20151016

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED