34results about How to "Achieve knockout" patented technology

The invention discloses a primer used for gene knockout, which comprises five groups of primer, and can be respectively shown in a SEQ NO:1, a SEQ NO:2, a SEQ NO:3, a SEQ NO:4, a SEQ NO:5, a SEQ NO:6, a SEQ NO:7, a SEQ NO:8, SEQ NO:9 and a SEQ NO:10. The invention also discloses an application of the primer in gene knockout aspect. The invention also discloses a conditional gene knockout method based on a CRISPR / Cas9 technology. The method can performing condition specificity, space-time specificity, and medicine-induced type gene modification; harm due to other cells can be reduced, function of constitutive expression gene in a specific tissue can be researched; tissue and space specificity gene knockout or induction type gene knockout can be realized by only using Cas9 tool mice; test period is short, and time and cost are saved.

The invention belongs to the technical field of gene engineering, in particular to a pCr-NHEJ (non-homologous end joining) carrier as well as a construction method of the pCr-NHEJ carrier and an application of the pCr-NHEJ carrier in site-specific knockout of bacterial genes. The sequence of the pCr-NHEJ carrier is shown as the SEQ ID NO.1. The application of the pCr-NHEJ carrier in site-specific knockout of the bacterial genes is provided. The technical principle is as follows: NHEJ and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) are jointly applied, so that after DNA (deoxyribonucleic acid) is broken when a CRISPR-Cas9 system cuts bacterial genomic DNA, the NHEJ system can be connected with a broken DNA end automatically, bacteria are survived, double-strand DNA having homology with a target sequence is not required to be introduced artificially to repair the broken DNA end, and the operation steps of the CRISPR-Cas9 technique are simplified.

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 invention provides an SgRNA (Small Guide Ribonucleic Acid) targeting sequence of specific target pig IRS1 (Insulin Receptor Substrate 1) gene and application thereof, and relates to the sgRNA targeting sequence and the application. The sgRNA targeting sequence is CGTAGTACTCGAGGCGCGCG. According to the sgRNA targeting sequence provided by the invention, the IRS1 gene can be knocked out or edited through a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated Protein) 9 system, so that expression of IRS1 can be removed, and the foundation is laid for preparing IRS1 transgenic pigs.

The invention relates to a sgRNA targeting sequence of specific target pig MC4R gene and applications of the sgRNA targeting sequence, relating to a gRNA targeting sequence and applications. The sgRNA targeting sequence is CGTCTCGCGCTTGGACTCAG. The sgRNA targeting sequence can knock out or edit MC4R gene through a CRISPR / Cas9 system, and further eliminate the expression of MC4R, thus providing basis for preparing MC4R transgenic pigs. The sgRNA targeting sequence is applied to the field of genetic engineering.

The invention relates to a sgRNA targeting sequence and application, in particular to a specifically-targeted swine IGFBP3 gene sgRNA targeting sequence and application. The sgRNA targeting sequence is CGTCTCGCGCTTGGACTCAG. By the sgRNA targeting sequence, an IGFBP3 gene can be knocked out or edited through a CTISPR / Cas9 system so as to eliminate IGFBP3 expression to lay the foundation for preparation of IGFBP3 transgenic swine. The specifically-targeted swine IGFBP3 gene sgRNA targeting sequence is applied to the field of gene engineering.

The invention discloses sgRNA for specific targeting of a human CXCR4 gene, a sgRNA-containing staphylococcus aureus CRISPR / SaCas9 system and a method for specific human CXCR4 gene knockout by the method. A target sequence of sgRNA is shown as any one of SEQ ID NO:1-4. sgRNA for specific targeting of the human CXCR4 gene and AAV-CRISPR / SaCas9 plasmids are connected to form a carrier and packaged into AAV infection cells, simplicity, convenience, high efficiency and specificity in CXCR4 gene knockout are realized, and accordingly the problem of limitation in adoption of SpCas9 targeted CXCR4 for treatment of acquired immune deficiency syndrome is effectively solved.

The invention provides an establishing method of a recoverable immortalized hepatic cell line. The establishing method comprises the following steps: separating a hepatic progenitor cell from liver in a mouse which is 12.5-14.5 days old in an embryonic period; guiding a retrovirus containing genes SV40T and HSV-TK into the hepatic progenitor cell; screening a monoclonal cell strain having a hepatic progenitor cell marker and having a function of differing to a mature hepatic cell; then guiding a retrovirus containing CD gene into the cell strain so as to obtain recoverable immortalized hepatic cell carrying double suicide genes. The cell strain can multiply in vitro to obtain the phenotype and functions of a normal hepatic cell, the safety of the immortalized cell becomes adjustable through controllable modes such as locus recombination and drug screening; when the cell is used in a human body, the biological safety of the immortalized cell is ensured to the greatest extent and the dangerousness of the immortalized cell is reduced; therefore, a reliable, safe and ideal hepatic cell material is provided for bioartificial liver technology.

The invention discloses a preparation method of liver cells with low expression or no expression of PERV. The preparation method includes the following steps: (1), respectively preparing a recombinant vector containing siRNA applied to RNA interference technology, a recombinant vector containing gRNA applied to CRIPRS / Cas9 technology and a vector containing Cas9 applied to CRIPRS / Cas9 technology; (2), jointly transfecting the three vectors to porcine somatic cells, culturing the transfected porcine somatic cells, and screening and identifying to obtain monoclonal genetically-modified porcine somatic cells; (3), utilizing somatic cell nuclear transfer technology to obtain genetically-modified pig containing nuclear genes of the genetically-modified porcine somatic cells; (4), extracting liver cells of the genetically-modified pig. According to the preparation method, knockout before transcription and inhibition after transcription of PERV genes are realized, and the technical scheme is effective and feasible.

The invention relates to an artificial gene editing system for the gene editing of rice. The artificial gene editing system comprises a regulation element I and a regulation element II, wherein the regulation element I comprises a nucleotide sequence which can encode an amino acid sequence I; the amino acid sequence I comprises one of amino acid sequence I-1, amino acid sequence I-2 and amino acidsequence I-3; the regulation element II comprises a nucleotide sequence II-1 and a nucleotide sequence II-2 in series connection in turn from the end 5' to the end 3'; the nucleotide sequence II-1 comprises a target nucleotide sequence; the target nucleotide sequence is derived from the genome of a target organism and contains the to-be-mutated target site in the genome of the target organism; the nucleotide sequence II-2 comprises sgRNA nucleotide sequence derived from streptococcus pyogenes; the nucleotide sequence II-1 and the nucleotide sequence II-2 are subjected to transcriptional fusion.

The invention discloses a primer for gene knockout, including five sets of primers, such as SEQ NO:1 and SEQ NO:2, SEQ NO:3 and SEQ NO:4, SEQ NO:5 and SEQ NO:6 , SEQ NO:7 and SEQ NO:8, SEQ NO:9 and SEQ NO:10. The invention also discloses the application of the primer in gene knockout. The invention also discloses a conditional gene knockout method based on CRISPR / Cas9 technology. The invention can carry out condition specificity, space-time specificity, and drug-induced gene modification; reduce the harm to other cells, and study the function of constitutively expressed genes in specific tissues; only the Cas9 tool mouse can realize tissue and space specificity Sexual gene knockout or inducible gene knockout; the test cycle is short, saving time and cost.

The invention provides NDUFA13 and application of a gene of NDUFA13 as a hepatitis or liver fibrosis drug or a diagnosis target. Research results show that the NDUFA13 and the gene thereof are closelyrelated to hepatitis or hepatic fibrosis; and a brand-new target and a novel diagnosis, prevention and treatment means and thought are provided for later research and development of hepatitis or hepatic fibrosis drugs and diagnostic reagents. Meanwhile, an NDUFA13 <flox / ->Alb-Cre mouse strain is constructed for the first time in the invention; the liver-specific NDUFA13 hybrid knockout mouse canstably generate non-infectious spontaneous liver inflammation that is developed into a liver fibrosis phenotype in the later period, so that a spontaneous hepatitis-liver fibrosis mouse model is formed and can be applied to liver inflammation-liver fibrosis mechanism research, new drug research and development evaluation and the like.

The invention relates to a method for knocking in a terminator to achieve efficient knockout of a transcription factor (including protein editing genes, non-encoding genes, and the like) by using a gene editing technology. Specifically, DNA (deoxyribonucleic acid) break is caused behind a transcription start site (TSS) of a gene to be knocked out by using a gene editing technology, meanwhile, a DNA donor with a transcription termination sequence (terminator) is introduced into cells, the doner does not comprise a target gene homologous sequence, the DNA donor is also linearized by using the gene editing technology, then after the terminator is knocked into a TSS of a target gene through a non-homologous repairing way, endogenous transcription of the target gene is terminated ahead of time,and thus knockout of the target gene is achieved. The method is wide in application scope, and all transcription facts can be knocked out by the method. The doner DNA has universality, has high efficiency, and in addition, has resistance genes, and the method is high in efficiency and has certain practical value.

The invention provides a mitochondrion-targeted gene editing complex, a preparation method and application thereof and a mitochondrion genome editing method. The gene editing complex comprises a mitochondria-targeted cationic liposome and an mtCRISPR / Cas9 system whihc are a mitochondrial TERT gene double-stranded homologous template, a crRNA of a specific targeting TERT, an RNA homologous recombination repair template and a Cas9 protein. A gene editing technology based on CRISPR / Cas9 is combined with a mitochondrial targeting means, the mitochondrion-targeted gene editing complex is provided,a normal mitochondrial TERT gene functional fragment is introduced to replace a mismatched gene, and the oxidative stress level is improved by up-regulating a mitochondrial TERT gene.

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.

Fungus has wide applications in biotechnology. CRISPR / Cas9 system is a powerful genome editing method and is successfully applied to a plurality of genomes, but the system is not widely used in fungus, and thus, a convenient, effective and widely applicable method which uses CRISPR / Cas9 system to perform gene editing in fungus is provided.

The invention provides a sgRNA combination of a targeted AHRR gene and application of the sgRNA combination, the sgRNA combination of the targeted AHRR gene comprises sgRNA1 and / or sgRNA2, the sgRNA1 comprises a nucleic acid sequence as shown in SEQ ID No.1, and the sgRNA2 comprises a nucleic acid sequence as shown in SEQ ID No.2. The invention also provides an AHRR gene editing system, a recombinant cell and a construction method thereof, and a construction method of an obese animal model, through gene editing and screening, the obtained homozygous mouse without the AHRR gene has typical obesity signs, larger body type, heavier body weight, higher fat content in vivo, and can be used for screening blood sugar and lipid reducing related drugs, and has extremely wide application value.

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

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 provides an establishing method of a recoverable immortalized hepatic cell line. The establishing method comprises the following steps: separating a hepatic progenitor cell from liver in a mouse which is 12.5-14.5 days old in an embryonic period; guiding a retrovirus containing genes SV40T and HSV-TK into the hepatic progenitor cell; screening a monoclonal cell strain having a hepatic progenitor cell marker and having a function of differing to a mature hepatic cell; then guiding a retrovirus containing CD gene into the cell strain so as to obtain recoverable immortalized hepatic cell carrying double suicide genes. The cell strain can multiply in vitro to obtain the phenotype and functions of a normal hepatic cell, the safety of the immortalized cell becomes adjustable through controllable modes such as locus recombination and drug screening; when the cell is used in a human body, the biological safety of the immortalized cell is ensured to the greatest extent and the dangerousness of the immortalized cell is reduced; therefore, a reliable, safe and ideal hepatic cell material is provided for bioartificial liver technology.

The invention discloses a myxococcus flavus large-fragment knockout plasmid and a knockout method thereof. According to the method, a promoter fragment PpilA of a pilA gene and an ORF fragment of a sacB gene are fused to obtain a PpilA-SacB module, a tetracycline resistance gene is amplified to obtain a PpilA-SacB module, and the PpilA-SacB module and the Ptet-TCR module are constructed to BamHI and XbaI sites of a pBJ113 vector to obtain the plasmid pBJ116. According to the invention, the plasmid pBJ116 contains a kanamycin positive screening marker, a galactose negative screening marker, a tetracycline positive screening module and a sucrose negative screening module, and large fragments on a myxococcus flavus genome are knocked out through a positive and negative screening system twice; tests find that the siderophore generation level of the Myxochelin A coding gene delta MXAN_3618 mutant obtained by the method disclosed by the invention is remarkably reduced, and the capability of preying on pseudomonas aeruginosa is remarkably reduced; and the time required by the method is short.

The invention discloses a method for increasing the ovalbumin expression of Bacillus subtilis, and belongs to the field of genetic engineering. The invention improves the ovalbumin expression of Bacillus subtilis by increasing the specific growth rate of Bacillus subtilis. In the present invention, the wild-type 168 is used as the starting strain, and a plurality of recombinant strains with increased specific growth rate can be obtained by using the method of knockout of growth-related genes, adaptive evolution (Adaptive Laboratory Evolution, ALE) and the combination of the two. The specific growth rate of Bacillus to increase the expression of Bacillus subtilis ovalbumin provides a new idea and method for increasing the expression of Bacillus subtilis ovalbumin.

The invention belongs to the field of gene engineering, and particularly relates to construction of an RIPLET knockout cell line and application of the RIPLET knockout cell line as a picornaviridae virus vaccine production cell line. Firstly, it is found that inhibition of RIPLET gene expression in host cells can promote replication of picornaviridae viruses, especially foot and mouth disease viruses and Seneca viruses. Secondly, the invention provides sgRNA specifically targeting RIPLET. The sgRNA can specifically target the RIPLET gene, the knockout of the RIPLET gene is realized by combining a CRISPR-Cas9 technology, and an obtained monoclonal cell line can significantly promote the replication of picornaviridae viruses, especially foot-and-mouth disease viruses and Seneca viruses. The production quantity and the antigen expression quantity of picornaviridae virus vaccines are improved, and the cell line can be used as a picornaviridae virus vaccine production cell line and has a wide application prospect.

The invention discloses three newly isolated fast-growing Vibrio strains and applications thereof, and relates to the field of bioengineering. The three Vibrio strains are named Vibrio sp.FA1, Vibrio sp.FA2 and Vibrio sp.FA3 respectively, and were published on August 2019. It was deposited on the 5th in the China Center for Type Culture Collection, address: Wuhan University, China, and the deposit numbers are CCTCC NO: M 2019603, CCTCC NO: M 2019604, CCTCC NO: M 2019605. Compared with the known fastest growing Vibrio natriegens ATCC 14048, these three Vibrio strains have obvious growth advantages. Through genome-wide comparative analysis, it is found that they also have more DNA replication-related genes, amino acid synthesis-related genes and resistance Inversion-related genes; these three Vibrio strains can grow rapidly using a variety of carbon sources under the condition of chemically synthesized medium, which is more conducive to industrial application. In addition, the transformation plasmids and gene knockout by electrotransformation proved the genetic operability of these strains, which provided an important basis and basis for the application of these three strains of Vibrio in the field of industrial technology.

The present invention provides a combination of sgRNA targeting AHRR gene and application thereof, the combination of sgRNA targeting AHRR gene includes sgRNA1 and / or sgRNA2, and the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No. 1, and the sgRNA2 includes the nucleic acid sequence shown in SEQ ID No.2. The present invention also provides an AHRR gene editing system, a recombinant cell and a method for constructing the same, and a method for constructing an obese animal model. Obesity signs, larger body size, heavier body weight, and higher body fat content can be used in the screening of hypoglycemic and lipid-lowering related drugs, and have extremely wide application value.

The invention relates to the technical field of biology, in particular to a method for increasing the yield of yeast astaxanthin by inactivating ubiquitin ligase and a recombinant strain. The recombinant strain is obtained by genetic modification and knockout of at least one E3 ubiquitin ligase of an original strain phaffia rhodozyma, and the yield of astaxanthin of phaffia rhodozyma is increased by the obtained recombinant strain. The phaffia rhodozyma E3 ubiquitin ligase coding gene is knocked out, and the negative regulation effect of E3 ubiquitin ligase astaxanthin synthesis is eliminated, so that the yield of astaxanthin is increased. Compared with a control strain, the astaxanthin cell content of the E3 ubiquitin ligase gene knockout strain can be obviously improved by 8.3 times. The method can be applied to construction of astaxanthin high-yield strains and increase of astaxanthin yield.

The present application relates to a set of artificial gene editing system for gene editing of rice, comprising: a regulatory element I comprising a nucleotide sequence capable of encoding an amino acid sequence I, the amino acid sequence I comprising one of an amino acid sequence I-1, an amino acid sequence I-2 and an amino acid sequence I-3; and a regulatory element II which comprises an II-1 nucleotide sequence and an II-2 nucleotide sequence which are sequentially connected in series from the 5' end to the 3' end. The II-1 nucleotide sequence comprises a target nucleotide sequence; the target nucleotide sequence is derived from a genome of a target organism, and the target nucleotide sequence contains a target site to be mutated in the genome of the target organism; the (II-2)th nucleotide sequence comprises an sgRNA nucleic acid sequence derived from streptococcus pyogenes; and the (II-1)th nucleotide sequence and the (II-2)th nucleotide sequence are transcribed and fused.

The invention belongs to the field of biotechnology and provides a systemic Plin1 gene knockout animal model construction and identification method thereof. The steps of the construction method are: designing sgRNA sequences according to the sequence before and after exon 2 of the Plin1 gene, constructing a Plin1 gene knockout vector, and performing in vitro transcription to obtain sgRNA; mixing sgRNA and Cas9 protein and then microinjecting it into mouse fertilized eggs to obtain whole body Sexual Plin1 knockout mice. The beneficial effect of this project is that a pair of sgRNA sequences with the highest scores were screened out, which ensured the success rate of the project; a large fragment of exon 2 of the Plin1 gene was knocked out, and the knockout efficiency was improved; the knockout region was designed 3 The two primers can successfully identify various genotypes of mice, avoiding the situation that two primers may not be able to distinguish genotypes when large fragments are knocked out. It provides a convenient, reliable and economical animal model and a good foundation for studying the biological function of PLIN1 and the pathogenesis of obesity-related metabolic diseases.