2396 results about "Cas9" patented technology

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific recombination, using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

The present invention relates to an isothermal method for detecting in a sample a target nucleic acid strand.

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR/Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant Caspase-9 protein to correct a point mutation associated with a disease or disorder, e.g., with neuroblastoma. The methods provided are useful for correcting a Caspase-9 point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

The invention provides a method for constructing a eukaryon gene knockout library by using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system. The method comprises the following steps: expressing genes encoded with Cas9 and OCT1 (Organic Cation Transporter 1) proteins into a eukaryon cell line; screening to obtain a cell line which stably expresses Cas9; and carrying out library construction and functional screening. The method has the greatest advantages that the method can be applied to most of eukaryon cell lines and is not limited by the specific cell line. Furthermore, the functional screening positive rate is high and the background value is low. According to a large-scale screening method, the cost is greatly reduced; the problems that the time is long and the labor cost is high due to the fact that a single gene knockout cell is prepared are solved.

The present disclosure relates to methods of and systems for modifying the transcriptional regulation of stem or progenitor cells to promote their differentiation or reprogramming of somatic cells. Further, the labeling and editing of human genomic loci in live cells with three orthogonal CRISPR/Cas9 components allow multicolor detection of genomic loci with high spatial resolution, which provides an avenue for barcoding elements of the human genome in the living state.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant PI3KCA protein to correct a point mutation associated with a disease or disorder, e.g., with a neoplastic disorder. The methods provided are useful for correcting a PI3KCA point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

The invention belongs to the field of genetic engineering, and particularly relates to a method for specifically knocking out hepatitis b virus cccDNA by using CRISPR-Cas9 and sgRNA for specifically targeting the hepatitis b virus cccDNA. The invention provides a method for specifically knocking out hepatitis b virus cccDNA by using CRISPR-Cas9 and sgRNA for specifically targeting the hepatitis b virus cccDNA. The sgRNA of specific targeted hepatitis b virus cccDNA prepared according to the invention can precisely target hepatitis b virus cccDNA and realize gene knockout. A preparation method is simple in steps and good in sgRNA targeting, and the knockout efficiency of a CRISPR-Cas9 system is high.

The invention belongs to the field of pharmaceutical and biological engineering, and relates to a CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and a lentivirus of the CRISPR/Cas9 recombinant lentiviral vector. The recombinant lentiviral vector is prepared by carrying out enzyme digestion on a lentiviral vectorlentiCRISPR by BsmBI and connecting into a BsmBI cohesive end-containing CXCR4 specific target sequence to recombine; the obtained CRISPR/Cas9 recombinant lentiviral vector is capable of mutating gene sequences at four different loci of ahuman immunodeficiency virusco-receptor CXCR4 and themutatuin rate is high and up to 25-75%. The cells transformed by the recombinant lentiviral vector cannot be infected by the human immunodeficiency virus. Compared with theRNAi-Knockdown, ZFN and TALEN technologies, the method has higher efficiency of suppressing the human immunodeficiency virus replication; the system is rapid to construct, simple and low in cost, is capable of preventing the invasion of the human immunodeficiency virus and is suitable for human immunodeficiency virus gene therapy.

The invention relates to a breeding method for prolongation of rice fertility stage, and the breeding method comprises the following steps: selecting a target fragment in a rice fertility stage determining gene Ehd3 exon region and constructing a plant CRISPR / Cas9 targeting recombinant vector, introducing into rice cells for regeneration to sprout; using an expression frame in the vector to realize shearing of Ehd3 exon region DNA in the rice cells so as to cause self healing of the rice cells; sequencing regeneration strain genome target fragments to obtain an afunction-mutational strain carrying two equipotential Ehd3 genes, and confirming the prolongation of the regeneration plant fertility stage by phenotypic identification. Experiments show that the method can quickly obtain fertility stage prolonged rice materials.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant von Willebrand Factor protein to correct a point mutation associated with a disease or disorder, e.g., with von Willebrand disease. The methods provided are useful for correcting a vWF point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

The invention discloses a method for knocking out a PD-1 gene by utilizing a CRISPR/Cas9 technology to construct an MSLN-targeted novel CAR-T cell. According to the method, a CAR-T cell is synchronously infected with a virus solution carrying sgRNA and Cas9-HF nuclease of a human PD-1 gene by utilizing the CRISPR/Cas9 technology to knock out the human PD-1 gene, so that the MSLN-targeted novel CAR-T cell is obtained. The novel CAR-T cell can be used for preparing a preparation used for treating solid tumors. A preparation method is simple in step, and the obtained CAR-T cell has high killing rate for solid tumor cells.

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

The invention discloses a CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof. The targeted knockout vector is prepared through the following steps: after a pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid is subjected to enzyme digestion and filling-in by using EcoRI and SacII, connecting the pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid with a pAdTrack-CMV plasmid subjected to enzyme digestion and filling-in by using BstXI; after the obtained product is linearized by using BbsI, connecting the obtained product to a specific target sequence of a desired gene; and after the obtained object is linearized by using PmeI and dephosphorylated by using CIAP alkaline phosphatase, recombining the obtained product with a pAdEasy-1 plasmid. The targeted knockout vector can mutate gene sequences in target sequence areas, and the mutation rate is high, up to 30.6-45.8%, therefore, the targeted knockout vector can be used for gene site-directed mutation, and lays a foundation for gene therapy.

The invention provides a method for establishing a humanized rat drug evaluation animal model. According to the method, a multidrug resistance gene 1 (Abcb1)-knocked-out genetically engineered rat is obtained through a microinjection method by virtue of a CRISPR/Cas9 gene knockout technology and 153kb bacterial artificial chromosome (BAC) fragments containing a humanized Abcb1 promoter and cDNA is simultaneously inoculated into the rat genome through the microinjection method by virtue of a large fragment transgenic technology to obtain a transgenic rat capable of stably expressing human Abcb1 and the genetically engineered rat and the transgenic rat are hybridized to establish the humanized rat drug evaluation animal model. RT-PCR analysis shows that Abcb1 expression profiles of humanized Abcb1 rat are significantly different from those of the rat endogenous Abcb1. The method has the beneficial effects that the humanized rat capable of expressing human Abcb1 is obtained and the rat is used for expressing human Abcb1 genes and has closer expression profiles to those of human so that the model can be well used for the efficacy evaluation of newly developed drugs.

The invention relates to a novel molecular cloning method, and in particular to a method for obtaining recombinant vectors by modifying initial vectors through combined utilization of a CRISPR/Cas9 system and a saccharomyces cerevisiae cell endogenous gene homologous recombination system; only through one-time transformation and selection operations, the method can simultaneously complete insertion of ore or more target DNA fragments into the initial vectors, deletion of one or more DNA fragments from the initial vectors and/or substitution of one or more DNA fragments on the vectors for one or more target DNA fragments. The invention further relates to a reagent kit for conveniently and effectively applying the method provided by the invention.

The invention discloses a method for obtaining a temperature-sensitive sterile line by performing site-specific mutagenesis on P/TMS12-1 through a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. The method comprises the following steps: cloning and controlling a Pei'ai 64S temperature-sensitive sterile major gene P/TMS12-1 fragment; designing a target sequence according to the P/TMS12-1 sequence; constructing a pU3-gRNA carrier of the target-containing sequence fragment; constructing a pCRISPR/Cas9 carrier; obtaining a positive transgenic seedling by utilizing the pCRISPR/Cas9 carrier containing the target sequence fragment; screening a mutant plant from the positive transgenic seeding; performing subculture planting on the mutant plant to obtain the temperature-sensitive sterile line without transgenic components. According to the method disclosed by the invention, the CRISPR/Cas9 system is utilized to completely inactivate P/TMS12-1 non-coding RNA, and the temperature-sensitive sterile line without transgenic components is artificially cultivated. The method disclosed by the invention has the advantages of being strong in purposiveness, small in genome damages and capable of avoiding transgenic interference.