2084 results about "Cas9" patented technology

The present invention relates to the fields of genetic engineering and plant genetic breeding, and specifically to the application of wheat TaPND gene. In the present invention, by taking gene TaPND as a target, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas, a plant binary expression vector capable of knocking out the TaPND gene in wheat is constructed; by genetically transforming wheat, offspring with TaPND gene knockout are obtained, and wheat mutants with early flowering time are obtained. Experiments have confirmed that transgenic wheat with TaPND gene knockout achieves the advancement of wheat flowering time. The technical scheme of the present invention provides a practical method for realizing rapid wheat breeding using genetic engineering technology, which has important breeding application value and broad market application prospects.

The invention discloses a porcine muscle stem cell line capable of being cultured in vitro for a long time and application of the porcine muscle stem cell line, and belongs to the technical field of stem cells and cell culture meat. The porcine muscle stem cell line is prepared by the following steps: performing CDKN2A gene knockout on porcine muscle stem cells by using a CRISPR / Cas9 gene editing technology, performing monoclonal screening and genotype identification to obtain a cell strain with successful CDKN2A knockout, and then performing carrier-free and serum-free suspension culture domestication on the cell strain. And a carrier-free and serum-free porcine muscle stem cell line capable of being subjected to in-vitro long-term suspension culture is obtained. Verification shows that the CDKN2A knockout porcine muscle stem cell line subjected to carrier-free and serum-free suspension culture domestication not only can be subjected to carrier-free and serum-free suspension culture in a shake flask for a long time, but also can maintain a good differentiation characteristic. The porcine muscle stem cell line which can be cultured on a large scale, is derived from edible animals and can be cultured in vitro for a long time is created, and excellent cell resources are provided for commercial production of cultured meat.

The invention discloses a site for stably expressing protein in a CHO cell gene NW023276806.1 and application of the site, and belongs to the technical field of biological genes. The site belongs to a fixed position in a CHO cell genome, different protein genes are introduced based on a micro-homologous end connection mechanism through a CRISPR / Cas9 tool, and stable expression is carried out. By adopting a site-specific integration method, a target gene is integrated to a stable expression area in a site-specific manner, repeated high-expression monoclonal screening is effectively avoided, and an MMEJ mechanism is introduced to integrate a donor fragment, so that the research and development time for constructing a stable expression cell strain in biological pharmacy can be effectively shortened, and the cost is reduced.

The invention discloses a tea tree CsU3 gene promoter and application thereof, and belongs to the technical field of plant biotechnology and gene engineering. Tea tree RNA polymerase III type promoters-tea tree endogenous U3 gene promoters CsU3a, CsU3b, CsU3c and CsU3d are successfully cloned for the first time, a tea tree U3 gene promoter activity detection carrier is successfully constructed, the transcriptional activities of the four CsU3 promoters are compared through instantaneous tobacco leaf conversion, GUS dyeing and GUS enzyme activity determination, and the CsU3 gene promoter activity detection carrier can be used for detecting the activity of the tea tree U3 gene promoter. CsU3a and CsU6b promoters with strong transcriptional activity are selected to be used for constructing a tea tree gene editing carrier, the feasibility of the promoter applied to a tea tree CRISPR / Cas9 gene editing system is verified in a tea tree protoplast, and CRISPR / Cas9 mediated tea tree genome targeted editing is realized.

The invention relates to the field of biotechnology and plant breeding, and particularly provides a method for fixing rice heterosis by using a DMP gene. Aiming at the problems of low maturing rate and complex operation of the existing apomixis system, the haploid induction functions of the rice OsDMP8 and OsDMP13 genes are verified for the first time, the OsPAIR1, OsREC8, OsOSD1 and OsDMP genes are synchronously knocked out through a CRISPR / Cas9 technology, a'MiMe-OsDMP 'four-gene editing system is constructed, and a new thought is provided for construction of the rice apomixis system. A new solution is provided for fixing heterosis of rice apomixis, the efficiency and quality of agricultural production can be greatly improved, and the method has high economic value and wide application prospects.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The invention relates to the field of gene therapy, in particular to an RBM25 gene epigenetics activation technology based on a CRISPR / dCas9-SunTag system and application of the RBM25 gene epigenetics activation technology in ischemic heart failure treatment, the RBM25 gene epigenetics activation technology comprises the following steps: (a) protein containing inactivated Cas9 (dCas9) fused with a plurality of GCN4 peptide repetitive sequences, the number of the GCN4 peptide repetitive sequences is 8-12, and the dCas9 contains D10A and H840A double mutations; (b) a protein containing fusion of an anti-GCN4 single-chain antibody fragment (scFv) and a TET1 catalytic structural domain; (c) at least one gRNA of a target RBM25 gene promoter region, wherein a target sequence of the gRNA is selected from a sequence as shown in SEQ ID NO: 1-3; and (d) a lipid nanoparticle (LNP) system for delivery of said (a), (b), and (c).

The invention discloses a cotton GhSNL1 protein as well as a coding gene and application thereof. The cotton GhSNL1 protein coding gene (GhA01G1757; according to the present invention, the CRISPR-Cas9 gene editing material of the GhSNL1 gene is subjected to phenotypic analysis to discover that the GhSNL1 gene knockout material has the phenotype of fiber start slowing, fiber density shortening and mature plant boll regeneration, such that the GhSNL1 gene participates in the cotton fiber development process, and the cotton fiber development efficiency is significantly improved; the method provides a theoretical basis for cotton variety improvement, and can be used for cultivating new cotton varieties with excellent fiber quality and high yield.

The invention provides a grass carp sourced U6 promoter. The sequence of the grass carp sourced U6 promoter is shown as one of SEQ ID NO: 1-3. Three grass carp-derived U6 promoters are cloned, the transcriptional activity and cross-species adaptability of the three promoters are analyzed, results show that the three promoters can show transcriptional activity in fish cells and human cells, the transcriptional activity of ciU6-1 is remarkably higher than that of the other two promoters, heterologous DNA can be transcribed in various cells, and the expression of the ciU6-1 in the fish cells and the human cells is improved. And the method can be used for gene editing means based on Cas9.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

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.

Provided herein are systems, compositions, and methods for generating immunogenic peptides or epitopes from tumor associated antigens (e.g., in vivo or ex vivo). Polynucleotides (e.g., genes) encoding the tumor associated antigens may be edited at selected target sites by nucleobase editors comprising a catalytically-inactive Cas9 and a cytosine deaminase, leading to the expression of heteroclitic or cryptic peptides that are more immunogenic than the native peptide derived from the tumor associated antigens. The heteroclitic or cryptic peptide elicit strong tumor-specific immune response (e.g., T-cell response or B-cell response), which inhibits tumor growth and metastasis.

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 proteins or protein 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 proteins or domains, are provided.

The present invention relates to a method for constructing an eight-gene-edited xenogeneic organ transplantation donor pig, belonging to the field of animal biotechnology. In the wild-type porcine fetal fibroblast cell line, the GGTA1, β4GalNT2, and CMAH genes are knocked out by using the CRISPR / Cas9 gene editing technology, and the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transfected. Combining with somatic cell cloning technology, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene-edited cloned pigs are constructed. Further, through genotype, mRNA, protein expression identification and functional analysis, eight-gene-edited xenogeneic organ transplantation donor pigs are obtained. The present invention solves the technical problems of high production difficulty, low efficiency, and low survival rate of donor pigs for multi-gene-edited xenogeneic organ transplantation, and maximally solves the common problems of immune rejection reaction and complement dysregulation faced during xenogeneic organ transplantation, laying a foundation for more targeted development of donor pigs suitable for different tissue and organ xenotransplantation, and having important value for promoting the clinical transformation of xenogeneic organ transplantation.

The invention discloses an application of a soybean nucleolus GTP binding protein gene GmNSN1 in soybean resistance to soybean mosaic virus diseases. The invention relates to an application of a soybean nucleolus GTP binding protein gene GmNSN1 as shown in SEQ ID NO.1 and SEQ ID NO.2 in genetic engineering modification of soybean mosaic virus resistance. The soybean nucleolus GTP binding protein gene GmNSN1 can negatively regulate and control the resistance of soybeans to diseased soybean mosaic virus diseases, and the antiviral response of the soybeans can be activated due to function deficiency of the soybean nucleolus GTP binding protein gene GmNSN1. The gene is knocked out through a CRISPR / Cas9 gene editing technology, so that the resistance of soybeans to the soybean mosaic virus disease can be remarkably improved.

The invention discloses a toxoplasma gondii attenuated vaccine strain RH delta rop67 as well as a construction method and application thereof, and belongs to the technical field of parasitic disease prevention and control and biological product preparation. The attenuated vaccine strain is constructed by performing targeted knockout on the ROP67 gene in a toxoplasma gondii strain RH delta ku80 through a CRISPR / Cas9 mediated gene editing technology. Compared with a wild type strain, the attenuated vaccine strain shows remarkable attenuation characteristic and good immunogenicity. A test result shows that the attenuated vaccine strain can induce a host to generate specific immune response mainly based on cellular immunity, maintains a protection effect on toxoplasma gondii infection in a relatively long immune period, and has a protection effect on tachyzoite infection and a chronic infection stage of toxoplasma gondii strains with different virulence; the survival ability of a host to tachyzoite infection can be improved, and the formation level of cysts in tissues is reduced. The invention provides a technical scheme with long-term immune potential for research and development of toxoplasma gondii attenuated vaccines.

The invention discloses a rice salt stress resistant gene OsHAK11 coding protein and application thereof, and belongs to the field of plant genetic engineering. According to the method, a rice OsHAK11 gene (the nucleotide sequence is shown as SEQ ID NO.1) is knocked out through a CRISPR / Cas9 gene editing technology, and a mutant with significantly enhanced salt tolerance is obtained. The gene editing vector pEGCas9Pubi-B-OsHAK11 contains sgRNA of a region as shown in a target SEQ ID NO.4, a rice receptor material is transformed through agrobacterium tumefaciens mediation, and a plant with the OsHAK11 gene subjected to frame shift mutation is obtained through screening. A salt stress experiment shows that the survival rate of the mutant oshak11 is obviously higher than that of a wild type. The invention provides a new gene resource and an efficient technical means for salt-tolerant breeding of rice.

The invention discloses a toxoplasma gondii attenuated strain as well as a construction method and application thereof. The toxoplasma gondii attenuated strain delta MIC1 delta MIC3 disclosed by the invention is obtained by knocking out MIC1 and MIC3 genes from a TIR1 strain by utilizing a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology. The toxoplasma gondii attenuated strain delta MIC1 delta MIC3 can effectively enhance macrophage functions including phagocytic ability, proinflammatory factor secretion and the like, and provides theoretical basis and technical support for clarification of an immunoregulation network of toxoplasma gondii-host interaction and development of vaccine design based on attenuated strains.

The invention discloses an application of an OsPES gene and an encoding protein thereof in regulating and controlling the tiller number of rice, and belongs to the technical field of biology. According to the application disclosed by the invention, two OsPES BRCT structural domain homozygous knockout strains Ospesc-cas9-1 and Ospesc-cas9-5, two OsPES overexpression strains OE-OsPES-3 and OE-OsPES-7 and two RNAi (Ribonucleic Acid Interference) interference strains RNAi-OsPES-1 and RNAi-OsPES-6 are respectively obtained by taking japonica rice'cloud guide 'as a background material and utilizing related technologies. Phenotypic observation and statistical analysis find that compared with a wild type, the tiller number of an overexpressed OsPES strain is increased, and the tiller number of an OsPES interference strain and the tiller number of a BRCT structural domain knockout strain are reduced. It is found for the first time that OsPES can positively regulate the tiller number of rice and has application significance in regulation of the tiller number of rice and new strain breeding.

The invention belongs to the technical field of plant gene engineering, and particularly relates to an OsNUC1 gene and application of a gene mutant constructed by the OsNUC1 gene to regulation and control of rice plant height, leaf length and panicle type. The regulation is to regulate the plant height and leaf growth of the rice and influence the shape of the ear part. Compared with the prior art, the OsNUC1 gene mutant has the following advantages: (1) the OsNUC1 is directionally edited by utilizing a CRISPR / Cas9 gene editing technology, an OsNUC1 gene mutant rice material is obtained, the biological phenotype of the OsNUC1 gene mutant rice material is investigated, the phenotype investigation on T1-generation seedlings of the OsNUC1 shows that the plant heights and the leaf lengths of one-week-old and two-week-old seedlings of the mutant are obviously higher than those of wild seedlings, the leaf widths have no obvious difference, and the OsNUC1 gene mutant rice material has a good application prospect; the result shows that the OsNUC1 can regulate the plant height and the leaf growth of the rice and influence the shape of the ear part. (2) it is proved that the nucleolin gene OsNUC1 is negatively regulated by a transcription factor OsARF7, and the phenotypes such as the plant height, the leaf length, the grain length and the grain width of the OsNUC1 / OsARF7 mutant are more obvious than those of the OsNUC1 mutant. The invention further proves the application of the OsNUC1 gene in regulation and control of rice plant height and leaf growth.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The invention discloses an application of a soybean ARGONAUTE family gene GmAGO5 in soybean resistance to soybean mosaic virus diseases. The invention also discloses application of the soybean ARGONAUTE family gene GmAGO5 as shown in SEQ ID NO.1 and SEQ ID NO.2 in genetic engineering modification of soybean mosaic virus resistance. The soybean ARGONAUTE family gene GmAGO5 can negatively regulate and control the resistance of soybeans to diseased soybean mosaic virus diseases, and the antiviral response of the soybeans can be activated due to function deficiency of the soybean ARGONAUTE family gene GmAGO5. The gene is knocked out through a CRISPR / Cas9 gene editing technology, so that the resistance of soybeans to the soybean mosaic virus disease can be remarkably improved.

The invention belongs to the technical field of gene editing, and discloses a gene editing system and application. The invention provides a gene editing system. The gene editing system comprises Cas9 nickase, sgRNA (small guide ribonucleic acid) and annular petRNA. According to the present invention, the target chain cutting mediated reverse pilot editing (Split Reverse Prime Editing, srPE) realizes the precise editing of the non-target chain cutting site upstream sequence, such that the gene editing coverage range is expanded from the specific region only limited to the non-target chain cutting site downstream to the whole genome range, the stable and efficient editing ability is maintained in different cell types, and the target chain cutting mediated reverse pilot editing can be provided for the target chain cutting site. And meanwhile, the off-target risk is reduced. The srPE gene editing technology breaks through the fundamental limitation of the prior art, and provides a more comprehensive and more efficient technical tool for precise medicine and gene therapy.

The invention relates to the field of rice gene engineering, and discloses an application of a GSE3.1 protein and a coding gene thereof in regulating and controlling the grain length, the grain width and the grain weight of rice seeds. According to the invention, a GSE3.1 gene is edited based on a CRISPR / Cas9 technology, a japonica rice variety Zhonghua 11 (ZH11) is introduced by using an agrobacterium-mediated method, and a knockout mutant is obtained through screening. The homozygous knockout mutation of the GSE3.1 gene in the invention leads to reduction of the grain width and grain length of rice seeds and reduction of thousand grain weight. According to the invention, the constructed plant overexpression vector ProActin: GSE3.1 is expressed in wild type ZH11, so that compared with the wild type plant, the seed grain length and grain width of the transgenic plant are obviously increased, and the thousand grain weight is obviously increased. Therefore, the GSE3.1 gene and the encoding protein thereof can regulate and control the size and the weight of the rice seeds, and are of great significance to cultivation of high-yield rice varieties.

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

The invention relates to the field of rice gene engineering, and discloses an application of OsMYB36 protein and a coding gene thereof in regulation and control of rice salt tolerance. The gene of the OsMYB36 protein is edited on the basis of a CRISPR / Cas9 technology, an agrobacterium-mediated method is utilized to introduce a japonica rice variety Zhonghua 11, and a knockout mutant is obtained through screening; tests prove that the survival rate of the rice mutant with the OsMYB36 gene knocked out is remarkably higher than that of a wild-type Zhonghua 11 plant after the rice mutant is subjected to stress treatment for 11 days by 150 mM NaCl and 200 mM NaCl salt and then rehydrated for 7 days after the rice mutant is subjected to two-leaf and one-core stage. And a simple and effective technical means is provided for rapidly creating a new salt-tolerant rice strain. Therefore, the OsMYB36 protein and the coding gene thereof disclosed by the invention can regulate and control the salt tolerance of the rice, and are of great significance to the cultivation of salt-tolerant transgenic rice.

The present disclosure provides Crispr / cas9-based repressors for silencing gene targets in vivo and methods of use.

The invention belongs to the technical field of biotechnology and agricultural biological breeding, and relates to a sorghum double haploid breeding method which comprises the following steps: (1) performing gene mutation on SbMTL and SbDMP to obtain a sorghum double-gene mutant strain according to any one of claims 1 to 3; (2) screening and identifying sorghum haploids by using GFP (Green Fluorescent Protein) marker and Ruby marker genes; and (3) naturally doubling the obtained haploid plant to obtain the double haploid of sorghum. SbMTL and SbDMP genes of sorghum are knocked out by utilizing a CRISPR / Cas9 technology so as to realize high-frequency haploid induction, meanwhile, an effective haploid selection marker is introduced, the induction frequency of a haploid induction system is evaluated, and after the obtained haploid plant is naturally doubled, a double-haploid system of sorghum is successfully obtained. The induction efficiency of the sorghum haploid induction line reaches up to 18% or above, the economic value is high, and the process of sorghum breeding is remarkably promoted.

The invention discloses an application of a BnaMYB7 gene or an encoded protein thereof in regulating and controlling the oil content of rape, and belongs to the fields of genetic engineering and crop genetic breeding. The nucleotide sequence of the BnaMYB7 gene is shown as SEQ ID NO: 1 or SEQ ID NO: 2, an agrobacterium-mediated genetic transformation method is used, an overexpression vector of the BnaMYB7 gene and a CRISPR / Cas9 gene editing vector are respectively transformed into a genome of the rape, the rape germplasm with overexpression and function deletion of the BnaMYB7 gene is obtained, and the rape germplasm with overexpression and function deletion of the BnaMYB7 gene is determined by measuring the oil content of seeds of overexpression and mutant materials. It is found that the BnaMYB7 gene positively regulates the oil content of the rape, so that rape varieties with different oil contents can be obtained by using the gene to perform molecular breeding on the rape. The feed additive is of great significance in relieving grain and oil tension and guaranteeing feed safety.

The invention discloses a method for detecting a diallele editing cell based on a CRISPR / Cas12a technology. The invention provides crRNA, and the nucleotide sequence of a binding target of the crRNA is SEQ ID NO.6. The crRNA is obtained by in-vitro transcription of a transcription template; the transcription template is a product obtained by annealing a single-stranded DNA (Deoxyribose Nucleic Acid) molecule as shown in SEQ ID NO.10 and a single-stranded DNA molecule as shown in SEQ ID NO.13. The invention provides crRNA of the CD71 gene, a CRISPR / Cas12a system is constructed by using the crRNA, CRISPR / Cas12a detection is performed, screening of CRISPR / Cas9 induced CD71 gene diallele editing cells is realized, and the method is simple, convenient, quick and convenient for economically and effectively screening a large number of mutant cells.