1622 results about "Gene knockout" patented technology

Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing greater than 50 weight percent of eicosapentaenoic acid [“EPA”], an ω-3 polyunsaturated fatty acid, in the total oil fraction are described. These strains over-express heterologous Δ9 elongases, Δ8 desaturases, Δ5 desaturases, Δ17 desaturases, Δ12 desaturases and C16 / 18 elongases, and optionally over-express diacylglycerol cholinephosphotransferases. Preferred gene knockouts are also described. Production host cells, methods for producing EPA within said host cells, and products comprising EPA from the optimized Yarrowia lipolytica strains are claimed.

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 in vivo siRNA expression system according to this invention is a system that intracellularly expresses small interfering (si) RNAs and comprises antisense and sense code DNAs coding for antisense and sense RNAs targeting any region of a target gene mRNA and one or more promoters that function to express the antisense and sense RNAs from the antisense and sense code DNAS, respectively.

The invention belongs to the field of gene engineering and particularly relates to a method for specifically knocking out a human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting the CCR5 gene as well as an intermediate carrier and application thereof. The sgRNA for specifically targeting the human CCR5 gene, prepared by the method disclosed by the invention, can be used for accurately targeting the human CCR5 gene and realizing gene knockout. The preparation method is simple in steps and good in sgRNA targeting property; the knockout efficiency of a CRISPR-Cas 9 system is high.

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 genetic engineering, particularly relates to a method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting a CTLA4 gene, and provides a method for human CTLA4 gene specific knockout through CRISPR-Cas9 and sgRNA(single guide RNA)for specially targeting a CTLA4 gene. The sgRNA(single guide RNA)for specially targeting a human CTLA4 gene, prepared through adopting the method provided by the invention, can be used for accurately targeting the human CTLA4 gene and realizes gene knockout; the preparing method has simple steps, the targeting performance of the sgRNA is good, and the knockout efficiency of a CRISPR-Cas9 system is high.

A method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout comprises steps as follows: design of a CRISPR / Cas9 gene knockout target site: a gRNA expression carrier is established and gRNA is synthesized in vitro; micro-injection of a zebra fish embryo; detection of effectiveness of the target site with a T7E1 method through Sanger sequencing; tail cutting identification according to the identification steps after two months of injection; TA cloning of a target sequence; Sanger sequencing of plasmids; obtaining of heritable F1 generation of a zebra fish mutant; obtaining of F2 generation homozygote of the zebra fish mutant, F3 generation pure line inheritance of the gene-deleted zebra fish with the above method, and obtaining of a new zebra fish strain.

The invention discloses a CAS9-carrying recombinant adenovirus and application thereof. The invention protects a recombinant adenovirus carrying coding gene of CAS9 protein; and the CAS9 protein is disclosed as Sequence 7 in the sequence table. The coding gene of the CAS9 protein is disclosed as 789th-5045th nucleotides from the 5' terminal of Sequence 6 in the sequence table. The invention also protects application of the recombinant adenovirus in preparing a kit for knocking out target gene. The CRISPR (clustered regularly interspaced short palindromic repeat) method has the characteristics of high efficiency, high speed and low cost. The CAS9 recombinant adenovirus disclosed by the invention can be used in a protein function cell platform (1) for quick gene modification and high flux screening, a liver protein function platform (2) for implementing quick and specific gene knock-out at the liver, and a mouse gene modification platform (3) for preparing a gene knock-out / knock-in mouse.

The invention provides statla gene deletion type zebra fish. After an experiment design region is knocked out, the sequence is represented as SEQ ID No.1; an experiment comprises the following steps: designing a CRISPR / Cas9 gene knockout target point: constructing a gRNA expression vector and synthesizing gRNA in vitro; carrying out micro-injection of zebra fish embryos; detecting the effectiveness of the target point by a T7E1 method and Sanger sequencing; two months after injection, carrying out tail shearing and identification and carrying out identification steps above; carrying out TA cloning of a target sequence; carrying out Sanger sequencing of plasmids; obtaining an F1 generation of descendible zebra fish mutants; obtaining F2 generation homozygotes of the zebra fish mutants; and carrying out F3 generation homozygous heredity of the gene deletion type zebra fish according to the method above to obtain a new zebra fish line.

The invention relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

The subject invention pertains to materials and methods for preparing multi-potential stem cells having a pre-selected expression of MHC antigens. Stem cells of the subject invention can be used to generate histocompatible tissues / organs for transplantation. The process of the subject invention comprises the use of targeting vectors capable of gene knockout, insertion of site-specific recombination cassettes, and the replacement of histocompatibility alleles in the stem cell. Novel knockout vectors are used to delete designated regions of one chromosome. Recombination cassette vectors are then used to delete the same region on the second chromosome and deposit a site-specific recombination cassette which can be utilized by replacement vectors for inserting the new MHC genes on the chromosome of the engineered cell. The subject invention also pertains to cells, tissues, and transgenic mammal prepared using the methods and materials of the invention.

The invention discloses a preparation method of a staphylococcus aureus CRISPR / Cas9 system and an application of the system in constructing a genetically modified mouse model. The staphylococcus aureus CRISPR / Cas9 system is composed of two components, namely Cas9 mRNA and gRNA, wherein a preparation method of the Cas9 mRNA is achieved by adding a T7 promoter to the upstream region of original Cas9 coding DNA, and a preparation method of the gRNA is achieved by adding the T7 promoter to the upstream region of an original gRNA coding sequence. The staphylococcus aureus Cas9 mRNA and gRNA, which are injected to mouse fertilized embryos through micro-injection, can achieve gene editing and modification of various types, such as single-gene knockout, multi-gene knockout and / or gene knock-in and the like, on the mouse fertilized embryos; therefore, the CRISPR / Cas9 system has a good application prospect in the aspects of fertilized embryo gene editing and modification of such animals as mouse and the like as well as construction of animal models.

The invention provides a breeding method for knocking out fish MC4R gene by using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. The method comprises the following steps: determination of MC4R gene targeted site, preparation of gRNA, in-vitro microinjection and knock-out verification, screening of MC4R-gene-knock-out fish, culture of hybrid fish, and the like. The breeding method is suitable for all commercial fishes, and can implement quick growth and breeding of fishes by knocking out the fish MC4R gene. Compared with the traditional breeding process, the method provided by the invention has the characteristics of high accuracy, low cost and short pure line acquisition time. Compared with the transgenic breeding, the gene knock-out can cause gene malfunction of the fish, and can not bring in any external gene, thereby avoiding the problem of transgenic safety.

The invention relates to a CRISPR-Cas9 system sgRNA action target screening method. The method comprises the steps of (1) obtaining segments with 5'-Nx-NGG-3' sequences (x is an integer ranging from 19 to 22 and N represents A / T / C / G) in genomes by utilizing whole genome sequences and gene annotation information of published species to serve as candidate targets of CRISPR-Cas9 system sgRNA; (2) fragmenting the genomes into fragments of 22-25bp and screening sequences ending with NGG and non-repeated on the genomes; and (3) comparing the sequences of the candidate targets in the step (1) with the screened sequences in the step (2), and performing screening and sorting on corresponding optimal sequences according to mismatch information and a selection formula to obtain an optimal genome sgRNA action target set. The invention furthermore provides an apparatus used for realizing the screening method. The method is suitable for all species with known genomes and gene annotation information, and a universal sgRNA sequence set of whole genome level of the species is quickly and efficiently obtained to build a gene knockout mutant library or a gene knockout animal model.

The invention provides a cultivation method of Cyp gene knocked-out rats, and a preparation method of liver microsome of the rats. The Cyp gene knock-out herein includes Cyp single gene knock-out and Cyp multiple gene knock-out. In the method, firstly a Cyp gene knocked-out rat is constructed by means of a CRISPR / Cas system, which includes selection of a knocked-out target site, in-vitro synthesis and transcription of sg RNA and Cas9m RNA, preparation of a pseudopregnant female rat, in-vitro micro-injection and transplanting of a single-cell embryo, and cultivation of the rat, and finally, a homozygote Cyp gene knocked-out rat can be cultured. Furthermore, the liver of the Cyp gene knocked-out rat is extracted and is subjected to homogenization and differential centrifugation to prepare the liver microsome of the rat in Cyp gene deletion. The invention also provides an application of the Cyp gene knocked-out rats and the liver microsome thereof in study on drug metabolism.

The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR / Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR / Cas9 gene knockout vector construction; fourthly, CRISPR / Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR / Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR / Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.

The invention relates to the field of genetic engineering and genetic modification, in particular to a method for obtaining an MC4R gene knockout pig by using a CRISPR / Cas9 system for editing MC4R genes through a somatic cell nuclear transfer technology. sgRNA is designed by aiming at two sites (a 6133-6152bp sequence and a 7127-7146 bp sequence in a CDS region of the MC4R gene) in the CDS region of the pig MC4R gene for the first time; in addition, the CRISPR / Cas9 system is used for simultaneously cutting the two sites; the large segment deletion of the MC4R gene is realized; in addition, the gene knockout individual pig subjected to large segment deletion is obtained; a feasible method is provided for studying the pig MC4R gene.

The present invention relates to the technical field of biology, particularly to development and applications of a heat shock induced Cas9 enzyme transgene danio rerio. The present invention firstly provides a Cas9 enzyme expression vector, which utilizes a heat shock induced promoter HSP70 to drive the expression of a downstream Cas9 gene. According to the present invention, the Cas9 enzyme expression vector and Tol2 mRNA are co-injected into wild type danio rerio single cell fertilized egg, and selection is performed to obtain the heat shock induced Cas9 enzyme transgene danio rerio, such that the gene editing research of the CRISPR-Cas9 system in the danio rerio is successfully achieved, and the know-out of the MC4R gene in the transgene danio rerio is firstly achieved; and the Cas9 enzyme expression vector is further suitable for heat shock induced gene knockout, gene knock-in, gene expression modification and other applications of other fishes.

The invention discloses a method for preparing T cells with knockout of double genes TCR and HLA. The method comprises the following steps: co-transfecting T cells by plasmids of CRISPR / Cas9-HLA and CRISPR / CAs9-TCR, and carrying out knockout of double genes TCR and HLA to obtain the T cells with knockout of double genes TCR and HLA. The method can be used for allotransplantation without causing immunological rejection.

The invention relates to an animal model for obese rats and an establishing method, and belongs to the field of animal genetic engineering and genetic modification. The animal model for obese rats and the establishing method are characterized in that rats with LEP and LEPR genes knocked out are obtained through the CRISPR / Cas9 technology. The rats with LEP and LEPR genes knocked out have a typical obesity characteristic, the reliable animal model can be provided for studying the obesity disease of people, and the rats with LEP and LEPR genes knocked out are expected to become a standard experiment animal of obese rats.

The invention discloses a method for pig CMAH gene specific knockout through CRISPR-Cas9 and sgRNA for specially targeting a CMAH gene. A target sequence of the sgRNA for specially targeting the CMAH gene on the CMAH gene meets a 5'-N(20)NGG-3' sequence arrangement rule, wherein the N(20) represents 20 continuous bases, and each N represents A, T, C or G; the target sequence on the CMAH gene locates at a 5-exon coding area or a junction position of adjacent introns of an N end of the CMAH gene; and the target sequence on the CMAH gene is unique. The sgRNA is used in a method for pig CMAH gene specific knockout through the CRISPR-Cas9, the pig CMAH gene can be rapidly, accurately, efficiently and specifically knocked out, and the problems of the long period and high cost of construction of a CMAH gene knockout pig are effectively solved.

The present invention discloses a pig SLA-1 gene knockout method using CRISPR-Cas9 specificity and sgRNA used for specific targeting SLA-1 gene. The target sequence of the sgRNA used for specific targeting SLA-1 gene in the SLA-1 gene complies with a 5'-N (20) NGG-3' sequence arrangement rule, wherein N (20) represents 20 consecutive basic groups, wherein each N represents a A or T or C or G; the target sequence in the SLA-1 gene is located at the four exon coding regions of the N-terminal of the SLA-1 gene or the junction of adjacent introns; and the target sequence in the SLA-1 gene is unique. The sgRNA used in the pig SLA-1 gene knockout method using CRISPR-Cas9 specificity, may fast, accurately, efficiently, and specifically knockout pig SLA-1 gene, effectively solve long cycle and high cost in construction of SLA-1 gene knockout pig.

The invention discloses a method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for the method. The sgRNA combination consists of sgRNAMSTN-1 and sgRNAFGF5-1, wherein sgRNAMSTN-1 is sgRNA which can realize specific targeted modification of sheep MSTN gene and is RNA shown in the second to 21st nucleotides in a sequence 6 or RNA with the second to 21st nucleotides of the sequence 6; sgRNAFGF5-1 is sgRNA which can realize specific targeted modification of sheep FGF5 gene and is RNA shown in the second to the 21st nucleotides in a sequence 8 or RNA with the second to 21st nucleotides of the sequence 8. According to the method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for the method, the CRISPR / Cas9 genome editing technology and the micro-injection technology are combined, so that the sheep targeting efficiency is higher and more accurate, sheep double-gene knockout is realized for the first time in the generation, improvement on sheep meat production and wool production is greatly promoted, and a larger space and a more effective technical tool are provided for breeding of new sheep varieties.

The invention relates to the genetic manipulation of non-human animals. More particularly, the invention relates to genetic manipulation of non-human animals to be used for xenotransplantation. The invention provides viable gene knockout swine including swine in which the α(1,3)-galactosyltransferase gene has been disrupted, methods for making such swine, and methods of using the tissues and organs of such swine for xenotransplantation.

The invention discloses a method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout and belongs to the field of gene knockout. According to the method, construction of a gRNA expression vector and gRNA in-vitro synthesis are performed through design of a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated 9) gene knockout target site, micro-injection is performed on an embryo of the zebra fish, the effectiveness of the target site is detected, tail cutting identification is performed, TA cloning is performed on a target sequence, plasmids are subjected to Sanger sequencing, an F1 generation of heritable zebra fish mutants is obtained, the same mutant female fish and male fish are picked from mutants of the F1 generation, hybridization is performed, an F2 generation of the zebra fish mutants is obtained, F2 generation homozygote is picked from the F2 generation of the zebra fishmutants, F3 generation pure-line inheritance is performed, and an rmnd5b gene deletion type zebra fish strain is obtained. The method is lower in off-target rate and has good medical research value inresearch of the correlation between rmnd5b gene deletion and development of other organs.

The invention discloses a method for targeted knockout of a gene ALK6 by using CRISPR-Cas9. The method comprises the steps of carrying out homologous comparison on a plurality of species so as to select two pairs of gene ALK6 target sequences, synthesizing DNA double strands, of which two pairs of target sequences are different and expressed proteins are the same and which have the same BsmBI cohesive ends, by using PAM design principles of gRNA, connecting the double strands with plasmid pPDNA330 so as to obtain recombinant vectors carrying ALK6 homologous genes of a plurality of species, detecting gene knockout efficiency of the recombinant vectors by using a fluorescin expression method, carrying out transfection on a vector with higher knockout efficiency with recipient cells of a plurality of species, and carrying out gene knockout and verification, thereby completing simple, efficient and accurate gene knockout of genes ALK6 of a plurality of species.

The invention discloses a process of knocking out Wnt3a gene and a verification method thereof. The knockout and verification of Wnt3a gene are finished through the following steps: establishment of a Cas9 lentiviral vector for Wnt3a gene, culture and passage of HepG2 cell, lentivirus infection and screening of target cell, verification of gene knockout efficiency through a mispairing enzyme method, cell protein analysis and cell proliferation detection by a CCK-8 method. The invention has the following advantages: the Wnt3a gene is knocked out by establishing a Cas9 double-vector lentivirus system for the first time; Crispr / Cas9 is a technology for accurately editing specific site of the genome of any species, and the cell-level single gene or multiple genes can be knocked out by the technology; compared with other gene editing technologies, the method has the advantages that the targeting accuracy is higher; only if the RNA target sequence is completely matched with the genome sequence, can the Cas9 cut the DNA and realize simultaneous knockout of multiple sites of the target gene; and moreover, the experimental period of vector establishment is short, the time and the cost are remarkably saved, and species limit is avoided.

The invention discloses a method using CRISPR-Cas9 to specifically knock off pig PDX1 gene and sgRNA of PDX1 gene for specific targeting. The target sequence of sgRNA of PDX1 gene for specific targeting on PDX1 gene is accord with the sequence alignment rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous bases, and each N represent A, T, C or G; the target sequence of PDX1 gene is arranged in the first exon coding region on the N terminal of PDX1 gene or a junction between the coding region and neighbored intron, and the target sequence of PDX1 gene is unique. The provided method can rapidly, precisely, efficiently and specifically knock off PDX1 gene of pigs, and solves the problems of long period and high cost of pig PDX1 gene knocking.

The invention relates to a cell line gene knock-out method for obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the field of genetic engineering and genetic modification. A pX458 vector is modified, so that the pX458 vector is provided with DsRed2 and ECFP (Enhanced Cyan Fluorescence Protein); then, a plurality of specific sgRNA sites are designed by aiming at a target gene and are connected into the modified vector; after the cell line transfection, cell groups are sorted by a flow cytometry; single cells subjected to genome editing can be very fast obtained; then, a single-cell DNA (Deoxyribonucleic Acid) sequence is subjected to PCR (Polymerase Chain Reaction) amplification; and single cells with large fragment deletion can be picked out from the single cells through gel electrophoresis. The CRISPR / Cas9 system, the flow cytometry single-cell sorting and fluorescent protein screening on the expression vector are combined, so that the positive monoclonal cells with the large fragment deletion can be obtained in a short time; and the work efficiency of the cell line gene knock-out is greatly improved.