WO2016080795A1 - 두 개의 벡터로부터 발현된 cas9 단백질을 이용한 유전자 발현 조절 방법 - Google Patents
두 개의 벡터로부터 발현된 cas9 단백질을 이용한 유전자 발현 조절 방법 Download PDFInfo
- Publication number
- WO2016080795A1 WO2016080795A1 PCT/KR2015/012503 KR2015012503W WO2016080795A1 WO 2016080795 A1 WO2016080795 A1 WO 2016080795A1 KR 2015012503 W KR2015012503 W KR 2015012503W WO 2016080795 A1 WO2016080795 A1 WO 2016080795A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- domain
- cas9 protein
- cas9
- vector
- cell
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
-
- 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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/635—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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
- C12N15/8645—Adeno-associated virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention provides a method of controlling genes, comprising introducing into a cell a recombinant vector expressing a first domain comprising a N-terminus of a Cas9 protein and a recombinant vector expressing a second domain comprising a C-terminus, respectively.
- the present invention relates to a composition comprising the recombinant vector, a kit for controlling gene expression, and a method for intracellular production of Cas9 protein.
- the present invention further relates to a composition
- a composition comprising a viral vector packaging the first domain and a transformed cell into which the viral vector packaging the second domain is introduced and a virus produced therefrom.
- restriction enzymes are one of the most important tools of current molecular biology research.
- various attempts have been made in response to the recognition of new DNA sequences and the need for a "rare cutter", that is, a restriction enzyme capable of recognizing and cutting 9 bp or more DNA, as a restriction enzyme useful for handling genome-sized DNA.
- meganucleases and zinc-finger nucleases are tools that can cause endogenous gene mutations, insertion of target genes, and chromosomal rearrangements in cells and organisms.
- artificial nucleases such as TAL-effector nucleases (TALENs) have been developed and can be usefully used in fields such as biotechnology and medicine as powerful and versatile tools in genetic engineering.
- RGEN RNA-guided Engineered Nuclease
- CRISPR / Cas system known as the immune system of microorganisms
- Such artificial nucleases recognize specific target sequences in cells and cause DNA double strand breaks (DSBs). Induced intracellular DSB can be repaired by two intrinsic DNA repair mechanisms of cells, distinguished by homologous recombination (HR) and nonhomologous end joining (NHEJ), wherein target specific mutations and Genetic modifications will occur.
- HR homologous recombination
- NHEJ nonhomologous end joining
- HR homologous recombination
- NHEJ nonhomologous end joining
- the Cas9 protein of the CRISPR / Cas system is a useful tool for designing genetic modifications in eukaryotic cells and organisms
- the size of the gene encoding it is large so that it can be inserted into the viral vector and delivered into the cell. Due to limitations, there is a problem in that virus production efficiency and delivery efficiency into cells are inferior. Thus, there is a need for research to express Cas9 proteins through viral vectors.
- the present inventors earnestly endeavored to overcome the packaging limitations of the viral vector and develop a system capable of expressing the Cas9 protein using the viral vector.
- the Cas9 protein is divided into two domains that can be packaged into the viral vector.
- the present invention was introduced by introducing the recombinant vector into a cell to confirm that each domain was fused to exhibit an Indel (insertion or deletion) effect on the target DNA on the genome. Completed.
- An object of the present invention includes introducing into a cell a recombinant vector expressing a first domain comprising the N-terminus of the Cas9 protein and a recombinant vector expressing a second domain comprising the C-terminus of the Cas9 protein, respectively.
- the present invention provides a composition comprising a recombinant vector expressing a first domain comprising the N-terminus of the Cas9 protein of the present invention and a recombinant vector expressing a second domain comprising the C-terminus of the Cas9 protein.
- Still another object of the present invention is to provide a kit for controlling gene expression comprising the composition.
- Still another object of the present invention is to provide a transgenic cell into which a viral vector packaging a first domain comprising the N-terminus of the Cas9 protein and a viral vector packaging a second domain comprising the C-terminus of the Cas9 protein are introduced. It is.
- Still another object of the present invention is to provide a composition comprising the culture solution or cell lysate of the cells.
- Another object of the present invention is to introduce into the cell a recombinant vector expressing a first domain comprising the N-terminus of the Cas9 protein and a recombinant vector expressing a second domain comprising the C-terminus of the Cas9 protein, respectively. It provides a method for intracellular production of Cas9 protein comprising.
- the present invention can improve the target specificity of the Cas9 protein, and also can be applied to the viral vector Cas9 protein, it can be usefully used for gene expression control using the Cas9 protein.
- FIG. 1 is a recombinant vector comprising a first domain and a second domain of the Cas9 protein, respectively, and then introduced into a cell, whereby each of these proteins is fused in the cell and the intact Cas9 protein (named Split-Cas9). It shows a schematic diagram forming.
- FIG. 2 shows the results confirmed by the T7E1 analysis that the Split-Cas9 protein is formed in the cell to work with sgRNA to induce Indel in all of the HPRT, DMD and CCR5 genes.
- Figure 3 shows the results of analyzing the mutation efficiency of the target gene of the Split-Cas9 protein through the next-generation sequencing.
- Figure 4 shows the results of analyzing the specificity of the target gene of the Split-Cas9 protein. Genetic mutation efficiency at on-target sites and at off-target sites in (a) Hela cells and (b) Hep1 cells is analyzed by next-generation sequencing. . Specificity was analyzed by the ratio of specificity ratios divided by the variance efficiencies of the target positions by the respective variability efficiencies at the four non-target positions.
- Figure 5 shows the results of confirming the vector production and function of split-Cas9.
- (a) and (b) Adenovirus vector construction that delivers split-Cas9 is modeled.
- a viral vector was packaged into a vector by inserting a U6 promoter, an sgRNA, an EFS promoter, and a first domain sequentially into an adenovirus vector.
- adeno-associated viruses were prepared that could include the second domain or package the second domain, U6 promoter, and sgRNA simultaneously.
- One embodiment of the present invention for achieving the above object is a recombinant vector expressing a first domain comprising the N-terminal of the Cas9 protein and a recombinant vector expressing a second domain comprising the C-terminal of the Cas9 protein, respectively Provided is a method of controlling gene expression, comprising the step of introducing into a cell.
- the term "gene expression control” refers to any action that increases or decreases the expression of a gene.
- the gene expression regulation may be performed by the Cas9 protein.
- any method for increasing or decreasing gene expression using Cas9 protein may be included without limitation in the scope of the present invention.
- the regulation of gene expression may mean genome correction, increased gene expression or reduced gene expression.
- genomic editing is a technique capable of introducing mutations targeted to genome sequences of animal and plant cells, including human cells, and knocks out or knocks down a specific gene. -in) or introducing a mutation into a non-coding DNA sequence that does not produce a protein.
- genome correction allows deletion, duplication, inversion, replacement or rearrangement of DNA on the genome.
- “deleted” refers to a mutation caused by missing part of a chromosome or part of a base on a DNA.
- “duplicate” means that two or more of the same genes are present in the genome.
- inversion refers to a portion of the genome that is placed upside down as compared to the original genome.
- replacement means that one nucleotide sequence is replaced with each other (i.e., replacement of informational sequences), and that only one polynucleotide is chemically or physically replaced with another polynucleotide. It does not mean.
- “rearrangement” means a structural change that causes a change in the position and sequence of genes on a chromosome, and includes translocation factors such as transposons or the like. In addition, it may include the conversion of genetic information by base rearrangement in the DNA molecule.
- Cas9 protein is a major protein component of the CRISPR / Cas9 system, complexes with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to form an activated endonuclease or nickase.
- Cas9 protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.
- the Cas9 protein may be encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto, as long as it can have target specific nuclease activity with guide RNA, all of the present invention May be included in the scope.
- Cas9 protein may also be linked to a protein transduction domain.
- the protein transfer domain may be, but is not limited to, poly-arginine or HIV derived TAT protein.
- the Cas9 protein may be appropriately linked to additional domains by those skilled in the art depending on the purpose.
- the Cas9 protein may include both wild type Cas9 as well as variants of Cas9 such as inactivated Cas9 (dCas9), or Cas9 nickases.
- the inactivated Cas9 may be an RNA-guided FokI Nuclease (RFN) linking a FokI nuclease domain to dCas9, or a transcription activator or repressor domain to dCas9,
- the Cas9 nickase may be, but is not limited to, D10A Cas9 or H840A Cas9.
- the Cas9 protein of the present invention is not limited in its origin either.
- the Cas9 protein is Streptococcus pyogenes , Francisella novicea novicida ), Streptococcus thermophilus , Legionella pneumophila ( Legionella) pneumophila ), Listeria innocua ( Listeria innocua ), or Streptococcus mutans .
- the Cas9 protein may be one that cannot be effectively expressed in a viral vector due to its size, but is not limited thereto.
- a vector capable of expressing a part of Cas9 was prepared to express Cas9 in a viral vector. That is, the Cas9 protein was divided into sizes that can be packaged in a viral vector and expressed in each vector.
- the first domain and the second domain of the Cas9 protein refer to a part of the Cas9 protein, and are expressed in a separate vector so that they are fused in a cell.
- the Cas9 protein produced in the above manner was named "split-Cas9" (FIG. 1).
- the split-Cas9 of the present invention is technically characterized in that the Cas9 protein, which is large in size and has not been packaged through a viral vector, is divided into packageable sizes and each of them is expressed through the vector so that its function is not lost in the cell. .
- first domain refers to a domain comprising the N-terminal region of the original Cas9 protein when expressing a portion of Cas9 cleaved for this purpose
- second domain refers to that of the original Cas9 protein.
- first domain and the second domain are fused to constitute the entire original Cas9 protein, the size of one domain is obtained by subtracting the size of the other domain from the size of the entire Cas9 protein.
- the first domain was introduced into the plasmid vector and the viral vector with 2.1 kbp and the second domain as 1.9 kbp.
- split-Cas9 was Indel at the target position. It was confirmed that can be induced.
- first domain and the second domain may be prepared by adding a nucleotide having a specific function by those skilled in the art according to the purpose, for example, a nuclear localization signal (NLS), a tag sequence, or a splice And may further comprise a splicing donor / splicing acceptor sequence and the like.
- NLS nuclear localization signal
- present invention is not limited thereto, but the first domain may be encoded by the nucleotide of SEQ ID NO: 3, and the second domain may be encoded by the nucleotide of SEQ ID NO: 5.
- vector refers to a gene construct that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert.
- operably linked refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function.
- a sequence encoding a DNA first domain or a DNA second domain of a nuclease of the present invention is operably linked to a promoter such that expression of the coding sequence is under the influence or control of this promoter.
- Two nucleic acid sequences (a sequence encoding a DNA first domain or a DNA second domain and a promoter region sequence at the 5 'end of the sequence) are operably linked when the coding sequence is transcribed by inducing promoter action, and Linkage properties between the two sequences do not induce frame-shift mutations and are operably linked when expression control sequences do not inhibit the ability to govern expression of each of these domains.
- Operable linkage with recombinant vectors can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can employ enzymes commonly known in the art.
- the vector of the present invention may include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and may be variously prepared according to the purpose. Can be.
- the promoter of the vector may be constitutive or inducible.
- the expression vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, may include a replication origin. Vectors can self replicate or integrate into host DNA.
- the vector may include a plasmid vector, a cosmid vector or a viral vector, and specifically, may be a viral vector.
- Viral vectors are retroviruses such as Human immunodeficiency virus HIV (Murine leukemia virus) Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV) and Mouse mammary (MMTV).
- tumor viruses such as, but not limited to, vectors derived from Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like.
- introducing into a cell may use any method known in the art, and may introduce foreign DNA into the cell by transfection or transduction.
- Transfections include sugars such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion. It can be carried out by various methods known in the art.
- each recombinant vector encoding the first domain and the second domain of the Cas9 protein was prepared (FIG. 1), and then introduced into the cell, thereby expressing each domain in the cell by integrating the intact cells. It was found to function as a Cas9 protein in form. Specifically, the Cas9 protein formed by fusion after expression of each half domain from recombinant vectors expressing each half domain of the first domain and the second domain acts together with the sgRNA to generate Indel (insertion or deletion) in all target genes. Induction was confirmed (FIGS. 2 and 3).
- split-Cas9 was expressed using an adeno-associated virus vector, and that Indel was effectively induced as a result of infecting the produced virus with cells (FIG. 5).
- Indel was effectively induced as a result of infecting the produced virus with cells
- sequence specific guide RNA when introduced into the cell, sequence specific guide RNA may be introduced. More specifically, the introduction of each vector, guide RNA may be performed simultaneously, sequentially or in reverse order.
- guide RNA may consist of two RNAs, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). Or sgRNA (single-chain RNA) made by fusion of crRNA and major portions of tracrRNA.
- the guide RNA may be dual RNA including crRNA and tracrRNA.
- RGEN known as third generation gene shears, may consist of Cas protein and dual RNA, or may consist of Cas protein and sgRNA.
- the guide RNA may comprise one or more additional nucleotides at the 5 'end of the crRNA of the sgRNA or dualRNA and may be delivered into the cell in the form of RNA or DNA encoding said RNA.
- compositions comprising a recombinant vector expressing a first domain comprising the N-terminus of the Cas9 protein and a recombinant vector expressing a second domain comprising the C-terminus of the Cas9 protein do.
- the composition may be introduced into cells to regulate expression of a desired gene.
- the composition may further comprise a sequence specific guide RNA.
- Cas9 protein and recombinant vector are as described above.
- a recombinant vector expressing the first domain and a recombinant vector expressing the second domain are introduced into a cell.
- This is for more efficient delivery and expression of Cas9 protein having a size difficult to package in the vector, it is possible to more easily express the Cas9 protein in the cell using the composition comprising the respective recombinant vector.
- the composition may include, without limitation, any material necessary for introducing the composition for the medium or the recombinant vector capable of maintaining the cell into the cell.
- kits for controlling gene expression comprising a recombinant vector expressing a first domain and a recombinant vector expressing a second domain.
- the kit may further comprise a sequence specific guide RNA.
- the kit is not only a composition for a medium for maintaining a substance or a cell that allows expression or promotion of a recombinant vector, but also a composition for facilitating the production of a recombinant vector or introduction into a cell, a recombinant vector. Instructions for the preparation or introduction into cells.
- Another embodiment of the present invention provides a transformed cell into which a viral vector packaging the first domain of the Cas9 protein and a viral vector packaging the second domain are introduced.
- transformed cell refers to a cell into which a polynucleotide of interest is introduced into a host cell. Transformation can be accomplished by the "introduction” method described above and can be carried out by selecting appropriate standard techniques depending on the host cell as is known in the art.
- the “host cell” refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced, and should be understood to refer not only to a particular subject cell but also to its progeny or potential progeny. Even if the progeny are not completely identical to the parent cell by mutation or environmental influence, they can still be included within the scope of the term as used herein.
- the transgenic cells are introduced with a viral vector encoding each half domain, from which a virus packaging the nucleotides encoding the respective domains of Cas9 can be obtained.
- the virus may be obtained from the culture solution of the transformed cells or the lysate of the cells.
- the cells may include prokaryotic cells such as E. coli , yeast, fungi, protozoa, higher plants, eukaryotic cells such as insects, mammalian cells such as CHO, HeLa, HEK293, and COS-1, and the like. It doesn't happen.
- somatic cells such as somatic cells, germ cells, induced pluripotent stem cells, adult stem cells.
- the somatic cell refers to all cells except germ cells obtained from the body of a child, adult as well as embryo, and may include genetically modified cells derived from them.
- the adult stem cells include cord blood stem cells, placenta stem cells, as well as all adult stem cells obtained from the body of human embryos, newborns and adults. Include extraembryonic stem cells such as witch stem cells, amniotic fluid stem cells, amniotic epithelial cells, and genetically modified cells derived therefrom. Can be.
- the cells may be cultured cells (in vitro), grafts and primary cultures (in vitro and ex vivo) and cells in vivo, without any limitation as the cells commonly used in the art.
- compositions comprising a culture solution or cell lysate of the transformed cells.
- Transformed cells, their cultures and lysates are as described above. Since the composition comprises a virus packaging a nucleotide encoding each domain, it can be used for gene expression control.
- the present invention overcomes the limitations of vector packaging for the Cas9 protein and produces a recombinant vector expressing each of the two cleaved sites of the Cas9 protein independently and delivers it into the cell to express the efficiency of delivery of the Cas9 protein into the cell. Is increased. Therefore, by using the principles of the present invention developed by the present inventors can be applied regardless of the type of cell, the type of Cas9 protein to increase the efficiency of delivery into the cell can efficiently regulate gene expression.
- Each half domain allowed independent expression to be induced by the CMV promoter.
- the stop codon was inserted into the cleavage 3'-end site of the first domain through PCR cloning to complete expression, and the start codon was inserted into the cleavage 5'-end site of the second domain. It was produced so that the expression can be started by connecting.
- HA tag and NLS are sequentially inserted below the start codon of the 5-end region of the first domain and between 3'end and stop codon of the second domain.
- the NLS site and the HA tag were sequentially inserted into the nucleus, and the protein expression level was measured using the HA antibody.
- Example 2 a recombinant vector expressing each half domain sgRNA Introduction into cells and target Gene Knockout (knock out) check
- Recombinant vectors expressing each half domain prepared in Example 1 and plasmids expressing sgRNA (single guide RNA) for each of CCR5, HPRT and DMD genes were transfected using lipofectamin. Delivery in Hela cells.
- the target sequence that can be used for allelic knockout of the CCR5 gene was a conserved sequence common to all humans, and the 5'- TGACATCAATTATTATACATCGG -3 'sequence (SEQ ID NO: 11) was targeted.
- 5'-GCCCCCCTTGAGCACACAGAGGG-3 'sequence (SEQ ID NO: 12) present in HPRT exon 3 was targeted as a target sequence that can be used for allele knockout of the HPRT gene.
- the 5'-TCCTACTCAGACTGTTACTCTGG-3 'sequence (SEQ ID NO: 13) present in DMD exon 51 was targeted.
- genomic DNA was extracted from Hela cells, target sequence sites in HPRT, DMD, and CCR5 genes were amplified by PCR.
- T7E1 T7 endonuclease I mutation detection assay
- FIG. 2 T7E1 assay was performed by a method known in the art.
- genomic DNA was isolated using the DNeasy Blood & Tissue Kit (G-DEX IIc Genomic extraction kit) according to the manufacturer's instructions.
- each half domain is fused after expression to form a Cas9 protein (named Split-Cas9) together with the sgRNA.
- Indel was induced in all of the HPRT, DMD and CCR5 genes.
- the target sequence region was amplified by PCR, and then analyzed by the next generation assay.
- 27.1% of HPRT gene, 23.75% of DMD gene and 20.27% of CCR5 gene showed Indel effect.
- each half domain can be fused after normal expression to form an intact Cas9 protein, thereby exhibiting an Indel effect on the target gene together with the sgRNA. I could see that.
- the first and second domains are independently introduced into cells for fusion of Cas9, which is less efficient for delivery into cells due to the size of the Cas9 expression cassette beyond the size that can be packaged in the viral vector, and then fused after each expression. The ability to function solves the problem of packaging Cas9 proteins into vectors.
- each of the first domain and the second domain was cloned into an Adeno-associated virus vector plasmid (FIGS. 5A and 5B).
- a splicing donor was linked to the C-terminal region of the first domain and a splicing acceptor was linked to the N-terminal region of the second domain.
- Viruses packaging each half domain were produced, harvested, delivered intracellularly, and analyzed for cleavage of the target sequence site.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims (25)
- Cas9 단백질의 N-말단을 포함하는 제1 도메인을 발현하는 재조합 벡터 및 Cas9 단백질의 C-말단을 포함하는 제2 도메인을 발현하는 재조합 벡터를 각각 세포 내에 도입하는 단계를 포함하는, 유전자 발현 조절 방법으로서,상기 Cas9 단백질은 상기 제1 도메인 및 제2 도메인으로 이루어지는 것인, 방법.
- 제1항에 있어서, 상기 유전자 발현 조절은 유전체 교정, 유전자 발현 증가 또는 유전자 발현 감소인 것인, 방법.
- 제2항에 있어서, 상기 유전체 교정은 유전자 낙인 (knock-in) 또는 낙아웃 (knock-out)인 것인, 방법.
- 제2항에 있어서, 상기 유전체 교정은 유전체 상에 표적화된 돌연변이를 도입하거나, 유전체 상의 DNA를 결실, 중복, 역위, 교체 또는 재배열시키는 것인, 방법.
- 제1항에 있어서, 상기 Cas9 단백질은 야생형 Cas9, 불활성화된 Cas9 (dCas9), 또는 Cas9 니케이즈 (nickase) 인 것인, 방법.
- 제5항에 있어서, 상기 불활성화된 Cas9은 dCas9에 FokI 뉴클레아제 도메인을 연결한 RFN (RNA-guided FokI Nuclease), 또는 dCas9에 전사활성인자 (transcription activator) 또는 억제자 도메인 (repressor domain)을 연결한 것인, 방법.
- 제5항에 있어서, 상기 Cas9 니케이즈는 D10A Cas9 또는 H840A Cas9인 것인, 방법.
- 제1항에 있어서, 상기 Cas9 단백질은 스트렙토코커스 피요제네스(Streptococcus pyogenes), 프란시셀라 노비시다 (Francisella novicida), 스트렙토코커스 써모필러스 (Streptococcus thermophilus), 레지오넬라 뉴모필라 (Legionella pneumophila), 리스테리아 이노큐아 (Listeria innocua), 및 스트렙토코커스 뮤탄스 (Streptococcus mutans)로 이루어진 군에서 선택되는 어느 하나의 유래인 것인, 방법.
- 제1항에 있어서, 상기 재조합 벡터는 플라스미드 벡터, 코즈미드 벡터, 또는 바이러스 벡터인 것인, 방법.
- 제9항에 있어서, 상기 바이러스 벡터는 레트로바이러스 (retrovirus) 벡터, 아데노바이러스 (adenovirus) 벡터, 아데노부속바이러스 (adeno-associated virus) 벡터, 및 헤르페스 심플렉스 바이러스 (herpes simplex virus) 벡터로 이루어진 군에서 선택되는 것인, 방법.
- 제1항에 있어서, 상기 도입된 각각의 벡터로부터 발현된 제1 도메인 및 제2 도메인이 융합되어, Cas9 단백질을 형성하는 단계를 추가로 포함하는 것인, 방법.
- 제1항에 있어서, 상기 제1 도메인 및 제2 도메인은 각각 400 bp (base pair) 내지 3.7 kbp (kilo base pair) 크기의 뉴클레오티드로 코딩되는 것인, 방법.
- 제1항에 있어서, 상기 제1 도메인 및 제2 도메인은 각각 NLS (nuclear localization signal), HA-태그, 스플라이싱 도너 (splicing donor) 서열, 스플라이싱 억셉터 (splicing acceptor) 서열 또는 이들의 조합을 추가로 포함하는 것인, 방법.
- 제1항에 있어서, 상기 제1 도메인은 서열번호 3의 뉴클레오티드로 코딩되는 것이고, 제2 도메인은 서열번호 5의 뉴클레오티드로 코딩되는 것인, 방법.
- 제1항에 있어서, 상기 세포 내에 도입하는 단계에서 서열 특이적인 가이드 RNA(guide RNA)를 추가로 도입하는 방법.
- 제15항에 있어서, 상기 각각의 벡터, 가이드 RNA의 도입은 동시, 순차 또는 역순으로 수행되는 것인 방법.
- Cas9 단백질의 N-말단을 포함하는 제1 도메인을 발현하는 재조합 벡터 및 Cas9 단백질의 C-말단을 포함하는 제2 도메인를 발현하는 재조합 벡터를 포함하는 조성물로서,상기 Cas9 단백질은 상기 제1 도메인 및 제2 도메인으로 이루어지는 것인, 조성물.
- 제17항에 있어서, 상기 조성물은 유전자 발현 조절용인 것인, 조성물.
- 제17항에 있어서, 상기 조성물은 서열 특이적인 가이드 RNA를 추가로 포함하는 것인, 조성물.
- 제17항의 조성물을 포함하는 유전자 발현 조절용 키트.
- 제20항에 있어서, 상기 키트는 서열 특이적인 가이드 RNA를 추가로 포함하는 것인, 키트.
- Cas9 단백질의 N-말단을 포함하는 제1 도메인을 패키징하는 바이러스 벡터 및 Cas9 단백질의 C-말단을 포함하는 제2 도메인을 패키징하는 바이러스 벡터가 도입된 형질전환 세포로서,상기 Cas9 단백질은 상기 제1 도메인 및 제2 도메인으로 이루어지는 것인, 세포.
- 제22항의 세포의 배양액 또는 세포 용해물을 포함하는, 조성물.
- 제23항에 있어서, 상기 조성물은 유전자 발현 조절용인 것인, 조성물.
- Cas9 단백질의 N-말단을 포함하는 제1 도메인을 발현하는 재조합 벡터 및 Cas9 단백질의 C-말단을 포함하는 제2 도메인을 발현하는 재조합 벡터를 각각 세포 내에 도입하는 단계를 포함하는, Cas9 단백질의 세포 내 제조 방법으로서,상기 Cas9 단백질은 상기 제1 도메인 및 제2 도메인으로 이루어지는 것인, 방법.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201580069646.8A CN107109422B (zh) | 2014-11-19 | 2015-11-19 | 使用由两个载体表达的拆分的Cas9的基因组编辑 |
US15/527,837 US10858662B2 (en) | 2014-11-19 | 2015-11-19 | Genome editing with split Cas9 expressed from two vectors |
JP2017527208A JP2017534294A (ja) | 2014-11-19 | 2015-11-19 | 二つのベクターから発現されたcas9タンパク質を利用した遺伝子発現調節方法 |
EP15861198.8A EP3222728B1 (en) | 2014-11-19 | 2015-11-19 | Method for regulating gene expression using cas9 protein expressed from two vectors |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2014-0161809 | 2014-11-19 | ||
KR20140161809 | 2014-11-19 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016080795A1 true WO2016080795A1 (ko) | 2016-05-26 |
Family
ID=56014239
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2015/012503 WO2016080795A1 (ko) | 2014-11-19 | 2015-11-19 | 두 개의 벡터로부터 발현된 cas9 단백질을 이용한 유전자 발현 조절 방법 |
Country Status (6)
Country | Link |
---|---|
US (1) | US10858662B2 (ko) |
EP (1) | EP3222728B1 (ko) |
JP (2) | JP2017534294A (ko) |
KR (2) | KR20160059994A (ko) |
CN (1) | CN107109422B (ko) |
WO (1) | WO2016080795A1 (ko) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
JP2021503279A (ja) * | 2017-11-16 | 2021-02-12 | アストラゼネカ・アクチエボラーグAstrazeneca Aktiebolag | Cas9ベースノックイン方針の効力を改善するための組成物及び方法 |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
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 |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
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 |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
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 |
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 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11168141B2 (en) | 2018-08-02 | 2021-11-09 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
EP4053284A4 (en) * | 2019-11-01 | 2024-03-06 | Suzhou Qi Biodesign biotechnology Company Limited | METHOD FOR TARGETED MODIFICATION OF PLANT GENOME SEQUENCE |
WO2022114815A1 (ko) * | 2020-11-25 | 2022-06-02 | 연세대학교 산학협력단 | 트랜스-스플라이싱 아데노-연관 바이러스 벡터를 포함하는 프라임에디팅용 조성물 |
WO2022169235A1 (ko) * | 2021-02-04 | 2022-08-11 | 연세대학교 산학협력단 | 교정 효율이 개선된 프라임에디팅용 조성물 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070161067A1 (en) * | 2006-01-10 | 2007-07-12 | Gambhir Sanjiv S | Split protein self complementing fragments, systems, and methods of use thereof |
WO2014089290A1 (en) * | 2012-12-06 | 2014-06-12 | Sigma-Aldrich Co. Llc | Crispr-based genome modification and regulation |
US20140273234A1 (en) * | 2012-12-12 | 2014-09-18 | The Board Institute, Inc. | Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation |
US20140295556A1 (en) * | 2013-03-15 | 2014-10-02 | The General Hospital Corporation | Using RNA-guided FokI Nucleases (RFNs) to Increase Specificity for RNA-Guided Genome Editing |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9637739B2 (en) * | 2012-03-20 | 2017-05-02 | Vilnius University | RNA-directed DNA cleavage by the Cas9-crRNA complex |
AU2014273085B2 (en) | 2013-05-29 | 2020-10-22 | Cellectis | New compact scaffold of Cas9 in the type II CRISPR system |
JP2016538782A (ja) | 2013-11-18 | 2016-12-08 | インタラクティブ・インテリジェンス・インコーポレイテッド | 通信のルーティングシステムおよび方法 |
KR101381368B1 (ko) | 2013-12-12 | 2014-04-04 | 유상근 | 휴대용 가스레인지의 폭발 방지장치 |
JP2017503485A (ja) * | 2013-12-12 | 2017-02-02 | ザ・ブロード・インスティテュート・インコーポレイテッド | 遺伝子産物の発現、構造情報、及び誘導性モジュラーcas酵素を変更するためのcrispr−cas系並びに方法 |
US20160304893A1 (en) * | 2013-12-13 | 2016-10-20 | Cellectis | Cas9 nuclease platform for microalgae genome engineering |
-
2015
- 2015-11-19 WO PCT/KR2015/012503 patent/WO2016080795A1/ko active Application Filing
- 2015-11-19 CN CN201580069646.8A patent/CN107109422B/zh active Active
- 2015-11-19 EP EP15861198.8A patent/EP3222728B1/en active Active
- 2015-11-19 JP JP2017527208A patent/JP2017534294A/ja active Pending
- 2015-11-19 KR KR1020150162799A patent/KR20160059994A/ko active Search and Examination
- 2015-11-19 US US15/527,837 patent/US10858662B2/en active Active
-
2018
- 2018-01-15 KR KR1020180005097A patent/KR102021585B1/ko active IP Right Grant
-
2020
- 2020-05-07 JP JP2020081896A patent/JP7335200B2/ja active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070161067A1 (en) * | 2006-01-10 | 2007-07-12 | Gambhir Sanjiv S | Split protein self complementing fragments, systems, and methods of use thereof |
WO2014089290A1 (en) * | 2012-12-06 | 2014-06-12 | Sigma-Aldrich Co. Llc | Crispr-based genome modification and regulation |
US20140273234A1 (en) * | 2012-12-12 | 2014-09-18 | The Board Institute, Inc. | Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation |
US20140295556A1 (en) * | 2013-03-15 | 2014-10-02 | The General Hospital Corporation | Using RNA-guided FokI Nucleases (RFNs) to Increase Specificity for RNA-Guided Genome Editing |
Non-Patent Citations (1)
Title |
---|
HYUN, YOUBONG ET AL.: "Site-Directed Mutagenesis in Arabidopsis Thaliana using Dividing Tissue-Targeted RGEN of the CRISPR/Cas System to Generate Heritable Null Alleles", PLANTA, vol. 241, no. 1, 1 October 2014 (2014-10-01), pages 271 - 284, XP035417502 * |
Cited By (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US12006520B2 (en) | 2011-07-22 | 2024-06-11 | 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 |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
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 |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
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 |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
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 |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | 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 |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11702651B2 (en) | 2016-08-03 | 2023-07-18 | 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 |
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 |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
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 |
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 |
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) |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | 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 |
JP7423520B2 (ja) | 2017-11-16 | 2024-01-29 | アストラゼネカ・アクチエボラーグ | Cas9ベースノックイン方針の効力を改善するための組成物及び方法 |
JP2021503279A (ja) * | 2017-11-16 | 2021-02-12 | アストラゼネカ・アクチエボラーグAstrazeneca Aktiebolag | Cas9ベースノックイン方針の効力を改善するための組成物及び方法 |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | 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 |
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 |
Also Published As
Publication number | Publication date |
---|---|
CN107109422B (zh) | 2021-08-13 |
KR102021585B1 (ko) | 2019-09-16 |
JP2017534294A (ja) | 2017-11-24 |
KR20180012834A (ko) | 2018-02-06 |
JP7335200B2 (ja) | 2023-08-29 |
KR20160059994A (ko) | 2016-05-27 |
CN107109422A (zh) | 2017-08-29 |
EP3222728B1 (en) | 2021-07-14 |
US20170349905A1 (en) | 2017-12-07 |
JP2020141683A (ja) | 2020-09-10 |
US10858662B2 (en) | 2020-12-08 |
EP3222728A1 (en) | 2017-09-27 |
EP3222728A4 (en) | 2018-09-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2016080795A1 (ko) | 두 개의 벡터로부터 발현된 cas9 단백질을 이용한 유전자 발현 조절 방법 | |
US20220017883A1 (en) | Variants of CRISPR from Prevotella and Francisella 1 (Cpf1) | |
JP6745391B2 (ja) | 遺伝子操作CRISPR−Cas9ヌクレアーゼ | |
KR102243243B1 (ko) | 신규한 cho 통합 부위 및 이의 용도 | |
ES2784754T3 (es) | Métodos y composiciones para modificar un locus objetivo | |
JP6700788B2 (ja) | Rna誘導性ヒトゲノム改変 | |
WO2017215648A1 (zh) | 基因敲除方法 | |
WO2016082135A1 (zh) | 一种利用定点切割系统对猪h11位点定点插入的方法 | |
WO2012093833A2 (en) | Genome engineering via designed tal effector nucleases | |
WO2019120193A1 (zh) | 拆分型单碱基基因编辑系统及其应用 | |
Valton et al. | Efficient strategies for TALEN-mediated genome editing in mammalian cell lines | |
JP7109009B2 (ja) | 遺伝子ノックアウト方法 | |
US20230193322A1 (en) | CAS9 Fusion Proteins and Related Methods | |
Dettmer et al. | Design and Derivation of Multi‐Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n‐Mediated Homology‐Directed Repair | |
JP2021164447A (ja) | ゲノム改変方法及びゲノム改変キット | |
JP2024501892A (ja) | 新規の核酸誘導型ヌクレアーゼ | |
WO2015182941A9 (ko) | 신규 카탈라아제 신호서열 및 이를 이용한 카탈라아제 발현방법 | |
TW202204611A (zh) | 基因組修飾方法及基因組修飾套組 | |
CN113355354A (zh) | 控制细胞行为的系统和方法 | |
Bao et al. | Genetic Engineering in Stem Cell Biomanufacturing | |
Roy | λ-integrase mediated seamless vector transgenesis platform | |
Sterckel | CRISPR-Cas genome engineering explained from A to T: Understanding CRISPR genome engineering via a rainbow human embryonic stem cell reporter line to identify pacemaker cells and a MEF2c construct production | |
WO2024038168A1 (en) | Novel rna-guided nucleases and nucleic acid targeting systems comprising such | |
WO2024042168A1 (en) | Novel rna-guided nucleases and nucleic acid targeting systems comprising such rna-guided nucleases | |
WO2024042165A2 (en) | Novel rna-guided nucleases and nucleic acid targeting systems comprising such rna-guided nucleases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15861198 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15527837 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2017527208 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2015861198 Country of ref document: EP |