42results about How to "Efficient knockout" patented technology

The invention discloses a CRISPR-Cas9 system for knocking out the gene GRIN2D and its application. The CRISPR-Cas9 system comprises Cas9 and gRNA specifically targeting to the gene GRIN2D. The invention also discloses application of the CRISPR-Cas9 system to preparation of drugs used for treating cancer and application of the gene GRIN2D to diagnostic kits and medicines used for precise treatmentof stomach cancer and other tumors. In virtue of a clinical sample tissue chip of stomach cancer, the gene GRIN2D is proved to be increased in expression in stomach cancer tissue and be related to prognosis according to immunohistochemical results. The CRISPR-Cas9 system provided by the invention can highly efficiently knock out the highly-expressed gene GRIN2D in stomach cancer and inhibit the proliferation and migration / invasion of gastric cancer cells, and is simple to operate and high in knockout efficiency. The CRISPR-Cas9 system is expected to be applied to diagnosis and treatment of stomach cancer and other tumors with over-expressed GRIN2D. The CRISPR-Cas9 system is applicable to a plurality of tumors with abnormal expression of GRIN2D.

The invention discloses an unmarked gene knock-out method of extremely acidophilic thiobacillus ferrooxidans based on the principle of homologous recombination. The method comprises the following steps of: constructing initial plasmids, suicide plasmids containing homologous fragments at upstream and downstream parts of target genes to be knocked out, and induction plasmids containing yeast endonuclease I-SceI genes; jointing and transferring the suicide plasmids and the induction plasmids to acidophilic thiobacillus ferrooxidans; and screening and identifying single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time. The method disclosed by the invention realizes the unmarked gene knock-out of acidophilic thiobacillus ferrooxidans for the first time, can realize the purpose of quickly, stably and efficiently knocking out the genes of the thiobacillus ferrooxidans, and can be used for researching the functional and metabolic mechanisms of the genes of the thiobacillus ferrooxidans, improving the genetic characters and constructing efficient bioleaching engineering bacteria; and moreover, the obtained mutant strain does not carry any resistance gene, thus the obtained mutant strain not only can be used as an original strain to knock out and improve genes in subsequent different sites, but also can be safely used for large-scale industrial production.

The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.

The invention provides sgRNA or PTG (Polycistronic tRNA-gRNA), through combination with a CRISPR-Cas9 technique, a T cell antigen receptor (TCR) can be knocked out, or one or more of genes relevant tomajor histocompatibility complex (MHCI) and immunosuppression molecules can be knocked out. The invention also provides a general type CAR-T cell which can express chimeric antigen receptor (CAR) butcannot express TCR, and a preparation method of the general type CAR-T cell. The general type CAR-T cell prepared by the preparation method is suitable for heterogeneity antigen, has generality and has higher killability at the same time.

The invention discloses a porcine myostatin gene editing site and application thereof, and belongs to the fields of biotechnology and biomedical medicine engineering. A gene editing target site is separated from a porcine myostatin gene editing area, the sequence of the gene editing target site is 5'-GGCTGTGTAATGCATGTATGTGG-3', and the gene editing target site is located at a first exon of an MSTN (myostatin) coding area. The site can be recognized by Cas9 endonuclease specifically, so that double strands are medicated to break, homologous recombination between the double strands and shooting carriers is achieved, and mutant genes or selectable maker genes are integrated to a pre-positioned site of a recipient cell genome. A statistical result shows that shooting efficiency of the site is 80.5%. The porcine myostatin gene editing site has the advantages that an effective drone is provided to accurate porcine myostatin gene editing, a new strategy is provided to development of new species of live pigs with high lean meat percentage, and reliable measures and materials are provided to study on molecular mechanism and signal channel of myostatin.

The invention provides a CAR-T cell and a preparation method. The CAR-T cell restrains a T cell antigen receptor (TCR), a major histocompatibility complex (MHCI) and an immunosuppression molecule related gene. The CAR-T cell expresses a chimeric antigen receptor (CAR), does not express the TCR, MHCI and an immunosuppression molecule PD1, is suitable for a heterogenous receptor, has generality, andmeanwhile has higher killability.

The invention discloses a pig muscle myostatin gene editing site 864-883 in a pig genome and application thereof, and belongs to the field of biological engineering. One gene editing target site is separated out from a pig muscle myostatin gene region; the sequence of the target site is 5'-GGATTTTGAAGCTTTTGGATGGG-3'; the site is positioned at a No.3 exon of an MSTN (myostatin) editing region. The site can be specifically identified by Cas9 endonuclease, so that mediated double chains fracture and then generate homologous recombination with targeting vectors; mutant genes or selectable maker gene and the like are integrated to the preset sites of a recipient cell genome. Statistical results show that the site targeting efficiency is 86.7 percent. The pig muscle myostatin gene editing site provided by the invention has the advantages that the effective target is provided for the precise embedding of the gene; new strategies are provided for developing live pig new varieties with high meat factor; reliable means and materials can be provided for developing myostatin molecular mechanisms and signal channels.

The invention belongs to the technical field of molecular biology and particularly relates to a preparation method of a PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation. The preparation method comprises steps of construction of a PD-1 knockout vector, separation and activation of T-lymphocytes, electrotransfection of the T-lymphocytes, identification by T7E1 restriction enzyme digestion, flow cytometer screening and sequencing analysis. By means production of the gene deficiency type immune cell preparation, on one hand, the cell production routine is simplified, and on the other hand, the preparation obtaining speed is increased.

The invention discloses sgRNA used for targeted knockout of a human NKG2A / KLRC1 gene, and belongs to the technical field of genetic engineering. The sgRNA has an arbitrary nucleotide sequence as shownin SEQ ID NO.1-56. The invention further discloses a sgRNA compound, expression vector, CRISPR / Cas9 system and kit used for targeted knockout of the human NKG2A / KLRC1 gene, and application of the sgRNA compound in preparing an immune cell medicine for treating neoplastic diseases. By means of the prepared specific targeted human KNG2A / KLRC1 gene, the human NKG2A / KLRC1 gene can be precisely targeted, effective knockout can be achieved, and meanwhile, an obtained human NKG2A / KLRC1 gene knockout immune cell is suitable for multiple tumor cell models; and a preparation method has simple steps, the targeting specificity of the sgRNA is good, and the knockout efficiency of the CRISPR / Cas9 system is high.

The invention discloses a method for screening an unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans. The method comprises introducing a knockout plasmid for construction of a target gene and an induction plasmid carrying an I-SceI gene into acidithiobacillus thiooxidans by conjugational transfer, carrying out single recon screening by a resistance maker, and carrying out double recon preliminary screening by blue-white selection. The method discloses the method for unmarked gene knockout of acidithiobacillus thiooxidans first, utilizes beta-galactosidase blue-white selection for double recon screening, can fast, stably and efficiently knock out a target gene, solves the problem that in the existing unmarked gene knockout process, selective pressure lacks so that screening is difficult, shortens double recon screening time, improves screening efficiency, reduces a screening cost and provides a novel method for stable genetic modification of acidithiobacillus thiooxidans.

The invention provides a method for terminating lncRNA diallele transcription. The method comprises the following steps of 1) constructing CMV-antibiotic screening gene-PolyA DNA fragments containing two different antibiotic screening genes; 2) designing gRNA according to different target genes, and constructing Cas9 / gRNA expression vectors; 3) inserting the CMV-antibiotic screening gene-PolyA DNA fragments obtained in the step 1) into the target genes, and constructing homologous recombinant vectors containing the target genes; 4) introducing cells: co-transfecting the pair of homologous recombinant vectors containing the target genes obtained in the step 3) and the Cas9 / gRNA expression vectors containing the target genes obtained in the step 2) to the cells; and 5) performing screening for at least 2 weeks by using related antibiotics used in the step 1), and obtaining polyclonal cell strains of which lncRNA dialleles are knocked out. According to the method, the purpose of quickly obtaining the cell strains of which the lncRNA dialleles are knocked out at the same time while realizing efficient fixed-point insertion can be achieved.

The invention relates to the technical field of biology and in particular discloses a method for knocking out a saccharomyces cerevisiae chromosome. According to the method, a Vika / vox technology is utilized for removing a complete yeast chromosome, and chromosome centromeres are specifically recombined into a ring by virtue of loci, so that knockout of the whole chromosome is realized. Compared with a method for realizing elimination of the whole chromosome in a reduction mitosis manner, the method disclosed by the invention has the advantages that cutting of the saccharomyces cerevisiae chromosome is more simply, efficiently and rapidly realized, crossing over of sister chromatids is avoided, and a saccharomyces cerevisiae homozygous diploid bacterial strain with the chromosome knocked out is obtained.

The invention provides a method for enhancing the genome editing efficiency of Streptomyces and its application. By adding elements that control Cas9 activity, the triple control of Cas9 protein activity at the transcription, translation, and protein levels can be achieved, the cytotoxicity of Cas9 protein to Streptomyces can be inhibited, and the transformation efficiency of plasmids in Streptomyces can be improved. Achieve efficient gene editing. The present invention establishes a gene editing system that is both induced by small chemical molecules and regulated by inhibitory proteins, which can inhibit the toxicity of Cas9 in vivo, and realize the efficient introduction of genetic elements without affecting the transformation efficiency of edited plasmids and the growth and metabolism of host cells. and subsequent gene editing. Small-molecule chemical inducers are easy to add, and can realize efficient and precise genome editing based on double-strand breaks and directional repair. The present invention can be applied to Streptomyces genetic engineering and genetic modification including model or industrial Streptomyces.

The invention discloses a spc-PcopA-yoeBVp gene cassette and a preparation method thereof, and belongs to the technical field of gene engineering, and the spc-PcopA-yoeBVp gene cassette comprises a positive selection marker spc and a negative selection marker PcopA-yoeBVp. The negative selection marker expresses YoeBVp toxin under the induction of copper sulfate and can inhibit the growth of an intermediate strain. By utilizing the gene box, the streptococcus suis traceless gene knockout can be efficiently realized. The gene knockout mutant strain does not contain any resistance marker, so that the polar effect caused by the resistance marker is avoided. In addition, because the gene knockout mutant strain does not contain a resistance marker, the gene knockout mutant strain can be used for subsequent vaccine research.

The invention provides a method for enhancing streptomyces genome editing efficiency and application of the method. By adding an element for controlling Cas9 activity, triple control of Cas9 protein activity on transcription, translation and protein levels is realized, cytotoxicity of Cas9 protein to streptomycete is inhibited, transformation efficiency of plasmids in streptomycete is improved, and efficient gene editing can be realized under induction conditions. According to the invention, a gene editing system which is induced by chemical small molecules and regulated and controlled by inhibitory proteins is established, in-vivo toxicity of Cas9 is inhibited, and efficient introduction of genetic elements and efficient editing of subsequent genes are realized on the premise of not influencing transformation efficiency of editing plasmids and growth and metabolism of host cells. The small molecule chemical inducer is convenient to add, and efficient and accurate genome editing based on double-strand breakage and directional repair can be achieved. The method of the inventioon can be applied to gene engineering and genetic modification transformation of streptomycetes including mode or industrial streptomycetes.

The invention relates to a preparation and an application for an engineered immune cell with a CD52 gene knocked out. Specifically, an sgRNA molecule is selected and can target the CD52 gene with highspecificity, so the efficiency of gene editing is improved; and preparation of universal CAR cells is facilitated.

The invention provides sgRNA for knocking out a rice OsPLS4 gene. An sgRNA sequence based on CRISPR / Cas9 is designed for the rice OsPLS4 gene, a DNA fragment containing the coded sgRNA sequence is connected to a carrier carrying the CRISPR / Cas9, rice calluses are transformed with an agrobacterium-mediated method, and the rice OsPLS4 gene is knocked out through screening and identification, whereinthe nucleotide sequence of the sgRNA action site is as shown in SEQ ID NO. 1. The rice endogenous gene OsPLS4 is edited through a CRISPR-CAS9 technology, and an OsPLS4 knockout mutant is obtained. The sgRNA prepared by the invention can efficiently, quickly and accurately target the rice OsPLS4 gene, and has certain significance in fundamental research (rice premature senility molecular mechanism) and production practice (rice premature senility variety improvement and stress-resistant breeding).

The present invention provides an adeno-associated virus recombinant vector for knocking out the CXCL12 gene, the recombinant vector at least includes the following sequence elements operably linked: 5' terminal inverted repeat sequence, CMV promoter, sacas9 sequence, polyA signal sequence, U6 promoter sequence, gRNA sequence, 3'-end inverted repeat sequence. The inventors found that the CXCL12 gene knockout vector can effectively inhibit the growth of transplanted tumors and tumor angiogenesis in the U87 cell glioma model, and can effectively inhibit the tumor growth and tumor angiogenesis in the U87 cell subcutaneous glioma model in nude mice. It shows that the technical scheme of the present invention can be applied to the treatment of glioma diseases.

The invention provides a method for terminating biallelic gene transcription. The method comprises the steps of 1, building a SA-T2A-antibiotic screening gene-Poly DNA fragment respectively containing two different antibiotic screening genes; 2, designing gRNA according to different targeting genes, and building a Cas9 / gRNA expression vector; 3, inserting the SA-T2A-antibiotic screening gene-Poly DNA fragment obtained in step 1 into the targeting genes, and building homologous recombinant vectors containing the targeting genes; 4, transferring into cells: transfecting the cells with both a pair of homologous recombinant vectors containing the targeting genes obtained in step 3 and the Cas9 / gRNA expression vector containing the targeting genes obtained in step 2; 5, performing screening on the relevant antibiotic of the antibiotic genes adopted in step 1 for at least two weeks to obtain biallelic gene knock-out polyclonal cell lines. The method for terminating biallelic gene transcription can achieve the purpose that when performing inserting into an efficient fixed point, biallelic gene simultaneously knock-out cell lines can be quickly obtained.

Provided are an sgRNA, a lentivirus vector constructed therefrom, and an application thereof. The nucleotide sequence of the sgRNA is as shown in SEQ ID NOs. 1-3. The present invention uses the currently most efficient gene-editing tool, CRISPR / Cas9, and the designed HIF-1α gene sgRNA locus has better gene knockout activity than other loci reported in previous studies. By combining with TAE / TACE surgery for the first time, the present invention can be used in HCC tumor treatment.

The invention discloses a method for screening an unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans. The method comprises introducing a knockout plasmid for construction of a target gene and an induction plasmid carrying an I-SceI gene into acidithiobacillus thiooxidans by conjugational transfer, carrying out single recon screening by a resistance maker, and carrying out double recon preliminary screening by blue-white selection. The method discloses the method for unmarked gene knockout of acidithiobacillus thiooxidans first, utilizes beta-galactosidase blue-white selection for double recon screening, can fast, stably and efficiently knock out a target gene, solves the problem that in the existing unmarked gene knockout process, selective pressure lacks so that screening is difficult, shortens double recon screening time, improves screening efficiency, reduces a screening cost and provides a novel method for stable genetic modification of acidithiobacillus thiooxidans.

The invention provides a method for efficiently and specifically removing plasmids in bacteria by utilizing an integrated suicide vector. The method comprises the following steps: (1) constructing theintegrated suicide vector targeting to-be-knocked-out plasmids, (2) screening and converting the integrated suicide vector, (3) carrying out first-round screening on the bacteria, and (4) carrying out second-round screening on the bacteria. According to the method for efficiently and specifically removing the plasmids in the bacteria by utilizing the integrated suicide vector, the stability of genomes of the bacteria is not influenced, the method is verified in various strains, the plasmids with the sizes of 24kbp to 340kbp can be efficiently knocked out, the method is simple and effective, and the removal rate is as high as 100 percent.

The invention provides a CAR-T cell and a preparation method. The CAR-T cell restrains a T cell antigen receptor (TCR), a major histocompatibility complex (MHCI) and an immunosuppression molecule related gene. The CAR-T cell expresses a chimeric antigen receptor (CAR), does not express the TCR, MHCI and an immunosuppression molecule PD1, is suitable for a heterogenous receptor, has generality, andmeanwhile has higher killability.

The invention belongs to the field of biotechnology, and in particular relates to an sgRNA targeting the APOBEC3G gene and a method for knocking out the APOBEC3G gene in cynomolgus monkeys. The present invention establishes a target sequence for mRNA of cynomolgus monkey APOBEC3G gene and synthesizes corresponding sgRNA, then constructs a knockout vector, co-transfects cynomolgus monkey embryonic fibroblasts, and obtains cynomolgus monkey embryonic fibroblasts with knockout of APOBEC3G gene, Or mixed injection of sgRNA and Cas9 mRNA into cynomolgus monkey embryos to obtain APOBEC3G knockout cynomolgus monkey embryos. The invention can effectively realize gene knockout of APOBEC3G gene on cynomolgus monkey cells and cynomolgus monkey embryos through detection and identification of knockout effect, and lays a solid foundation for establishing AIDS animal models.

The invention discloses a CRISPR-Cas9 system for knocking out the GRIN2D gene and its application. The CRISPR-Cas9 system includes Cas9 and a gRNA specifically targeting the GRIN2D gene. The invention also discloses its application in the preparation of drugs for treating cancer and the application of GRIN2D gene in the preparation of diagnostic kits and drugs for precision medicine of gastric cancer and other tumors; it is confirmed by immunohistochemistry through tissue chips of clinical samples of gastric cancer, The expression of GRIN2D is increased in gastric cancer and is related to prognosis; the CRISPR-Cas9 system of the present invention can efficiently knock out the GRIN2D gene highly expressed in gastric cancer, and inhibit the proliferation, invasion and migration of gastric cancer cells. The system is simple to operate and has high knockout efficiency. It is expected to be applied in the diagnosis and treatment of gastric cancer and other tumors that overexpress GRIN2D. The system of the present invention is applicable to various tumors with abnormal expression of GRIN2D.

The invention discloses construction of an efficient gene editing system based on CRISPR-Cpf1, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, representing a Cas protein Cpf1 from F.novicida, and integrating FnCpf1 to a lacA site of a bacillus subtilis genome; the crRNA targeting different genes is placed at the downstream of a strong constitutive promoter Pveg from bacillus subtilis and is used for high-intensity expression of the corresponding crRNA. By selecting crRNA of different target genes, the genome editing method is verified. A bacterial colony PCR (Polymerase Chain Reaction) result shows that the sacA gene, the ligDV gene and the pps gene cluster can be efficiently knocked out. Meanwhile, in order to verify the knock-in efficiency of the system, the exogenous gene sfGFP is efficiently knocked out to the sacA site. In order to further verify the polygene knockout efficiency of the system, sacA and aprE are selected as targets, and a bacterial colony PCR result shows high knockout efficiency.

The present invention provides an adeno-associated virus recombinant vector for knocking out the CXCL12 gene. The recombinant vector comprises at least the following operably linked sequence elements:5 '-terminal inverse repeat sequence, CMV promoter, sacas9 sequence, polyA signal sequence, U6 promoter sequence, gRNA sequence, 3'-terminal inverse repeat sequence. CXCL12 gene knockout vector can effectively inhibit tumor growth and tumor angiogenesis of U87 cell glioma model, and can effectively inhibit tumor growth and tumor angiogenesis of U87 cell subcutaneous glioma model in nude mice. Thetechnical proposal of the invention can be applied to the treatment of glioma diseases.