2652 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 wheat salt-tolerant gene and application thereof. Specifically, the invention discloses a protein TaSGR (SEQ ID NO: 3, 6 and / or 9) and an application of a coding gene of the protein TaSGR in regulation and control of plant salt tolerance and / or plant height. According to the invention, a homozygous gene editing plant of which the TaSGR gene is knocked out is successfully created by utilizing a CRISPR / Cas9 technology. The result shows that after the TaSGR gene is knocked out, the plant height of the wheat is obviously reduced, and the salt tolerance is obviously enhanced. The invention discloses the application of the protein TaSGR and the coding gene thereof for the first time. The salt tolerance of the plant is increased and / or the plant height of the plant is reduced by reducing the content and / or activity of the protein TaSGR (for example, the TaSGR gene is knocked out or silenced). According to the invention, a good gene resource is provided for salt-tolerant breeding of wheat, a new field is developed for application of the TaSGR gene, and the TaSGR gene has important significance for cultivation of novel salt-tolerant wheat.

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 discloses an endogenous tRNA (transfer ribonucleic acid) promoter. The nucleotide sequence of the endogenous tRNA promoter is shown as SEQ ID NO: 1; an expression cassette containing the endogenous tRNA promoter and a recombinant vector; the invention also relates to application of the endogenous tRNA promoter, the expression cassette or the recombinant vector in thraustochytriaceae microorganism gene editing, and a thraustochytriaceae microorganism gene editing method. The CRISPR / Cas9 gene editing system based on the endogenous tRNA promoter is successfully established, convenient, rapid and efficient fixed-point editing in thraustochytriaceae microorganisms is achieved, and the CRISPR / Cas9 gene editing system has high application value; a transformation system for screening the recombinant thraustochytriaceae microorganisms by using sodium acetate as an auxotroph is established, the transformation screening efficiency of the recombinant thraustochytriaceae microorganisms obtained based on an agrobacterium transformation method is remarkably improved, and rapid gene editing is assisted.

The invention belongs to the technical field of plant genetic engineering, and discloses an application of a corn gene ZmGG1 in control of corn yield, a protein encoded by the ZmGG1 is shown in SEQ ID NO.2, and the gene is located in the ninth chromosome of corn and used for controlling the row number and the ear weight of corn ears. The gene is knocked out by using a CRISPR / Cas9 technology, gene expression is inhibited, and the ear row number and the ear weight of corn can be increased.

The invention discloses a rice OsTCP11 gene and application of the rice OsTCP11 gene in rice disease resistance, and the nucleotide sequence of the OsTCP11 gene is as shown in SEQ ID NO.1; the method is realized by knocking out a rice OsTCP11 gene; the knockout is carried out by utilizing a CRISPR / Cas9 gene editing technology; the gene OsTCP11 is knocked out through a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) technology, and a knockout mutant ostcp11 is obtained; it is found through rice blast resistance identification that compared with wild type rice, the mutant ostcp11 is more resistant to diseases and small in disease spot area, the number of pyricularia grisea carried by diseased leaves is small, and it is indicated that OsTCP11 negatively regulates the resistance of rice to rice blast. The bacterial leaf blight resistance identification finds that compared with wild rice, the mutant ostcp11 is more resistant to diseases and shorter in disease spots, which indicates that the OsTCP11 negatively regulates the resistance of the rice to the bacterial leaf blight. The method is beneficial to cultivation of rice blast and bacterial leaf disease resistant rice varieties, and provides a basis for later screening of high-resistance and broad-spectrum-resistance rice varieties.

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 a breeding method of large-seed and high-oil type brassica napus L. The disclosed method comprises the following steps: knocking out a BnaWIP2 gene in brassica napus L., so that the BnaWIP2 gene is mutated to obtain the large-seed and high-oil type brassica napus L.. According to the method, six copies of the BnaWIP2 gene are knocked out at the same time, the large-seed and high-oil brassica napus CRISPR / Cas9 gene editing material is obtained, and the gene editing material provides precious gene resources and germplasm resources for breeding of high-oil and high-yield brassica napus varieties.

The invention discloses regulation and control of a GmERECTAs gene (GmERs for short) on a soybean plant height and application of the GmERECTAs gene (GmERs for short). The invention belongs to the technical field of biology. An sgRNA sequence of a GmERs gene is recombined to a knockout vector pGES401 through a Golden Gate cloring technology. According to the method, four homologous genes of GmERs are knocked out by using a CRISPR / Cas9 technology, the four genes comprise GmERa, GmERb, GmERc and GmERd, and offspring screening is performed through herbicide resistance endowed by a Bar marker gene; after a positive result is obtained through screening, the GmERs gene knockout condition is determined through sequencing, and the plant height of a gene knockout soybean plant is remarkably reduced compared with that of a contrast. The invention has important significance for developing and breeding new soybean germplasm resources.

The invention relates to a rice OsABCB7 gene as well as an encoding protein and application thereof. The inventor utilizes a CRISPR / Cas9 gene editing system to obtain two homozygous knockout mutants osabcb7-1 and osabcb7-9 of an OsABCB7 gene, wherein the two homozygous knockout mutants osabcb7-1 and osabcb7-9 take japonica rice Nipponbare as a background and have different editing conditions. Many-year and multi-generation agronomic trait investigation shows that under the normal field planting condition, the grain length, grain width, thousand seed weight, single plant yield and the like in the mature period are all remarkably increased compared with those of a wild type contrast, and the maturing rate has no obvious difference; however, under the continuous high-temperature condition, the maturing rate and the yield of a single plant of the mutant are obviously higher than those of a wild type control. Besides, the mutant rice after salt treatment shows enhanced salt tolerance, so that the OsABCB7 gene can be applied to rice crossbreeding and molecular design breeding to improve the rice yield and enhance the salt tolerance and high temperature resistance of the rice.

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).

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.

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 belongs to the technical field of gene editing, and particularly relates to a poa pratensis gene editing method. The invention provides a bluegrass CRISPR / Cas9 gene editing system, which comprises a pCAMBIA1300-Cas9 vector, a bluegrass gene PpTCP4 target sequence and a primer pair for amplifying the bluegrass gene PpTCP4 target sequence, the gene PpTCP4 of the bluegrass is directionally edited by utilizing a CRISPR / Cas9 gene editing technology, gene editing of a base mutation type is realized, a gene editing plant is successfully obtained, and the gene editing effect is good. The gene editing efficiency is 0.67%, and new research basis and technical reference are provided for developing the directional improvement of the poa pratensis to create high-quality germplasm resources.

The invention provides a recombinant engineering bacterium for producing lumbrukinase and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, when the lumbrukinase F238 is expressed, one or more genes in TDA3 and KRE9 genes are knocked out in a targeted manner by utilizing a CRISPR-Cas9 gene editing technology, and / or an SGA1 gene is overexpressed and integrated into a strain, so that the expression quantity of the lumbrukinase F238 is increased. According to the recombinant engineering bacterium, the yield of the lumbrukinase can be obviously increased and can reach 235% at most, meanwhile, the activity of the lumbrukinase is obviously enhanced and can reach 699.6 U / mL at most, and the recombinant engineering bacterium has a wide application prospect.

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.

The invention discloses a method for constructing a Ddx58-R109C point mutation mouse model by using a CRISPR / Cas9 gene editing technology and an application of the Ddx58-R109C point mutation mouse model. The method comprises the following steps: (1) target construction: searching a CDS region of a Ddx58 gene, determining an exon part, and determining a gene mutation site; (2) obtaining a fertilized egg, and injecting Cas9mRNA, gRNA and donor oligo into the fertilized egg of the C57BL / 6J mouse together in vitro through a microinjection technology to obtain an F0-generation hybrid mouse; (3) mating the F0-generation hybrid mouse with the wild type mouse to obtain an F1-generation mouse; (4) the F1-generation heterozygous mice which are identified to be positive are subjected to mating, and homozygous Ddx58-R109C mutant type and wild type mice can be obtained by breeding obtained offspring; (5) phenotypic analysis shows that Ddx58-R109C point mutation mice can have pathological changes of systemic lupus erythematosus and lupus nephritis; the invention can provide an effective research approach and method for researchers to deeply explore the action mechanism of Ddx58-R109C mutation in diseases, and has important significance for explaining the pathogenesis of systemic lupus erythematosus and researching and developing targeted therapeutic drugs.

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 discloses a nano antibody against BCMA and its preparation method and application, and relates to the field of biomedical technology. The present invention uses CRISPR / Cas9 technology to knock out B2M and TRAC genes in T cells to prepare universal CAR-T cells; the nano antibody targeting BCMA and the natural ligand SIRPα extracellular N-terminal V region of CD47, the transmembrane region of CD28, the intracellular signal region of 4-1BB and the intracellular signal region of CD3ζ are connected in series to form a dual-targeted chimeric antigen receptor for the treatment of multiple myeloma, so as to solve the high recurrence problem faced by CAR-T cell therapy in the treatment of multiple myeloma. The universal CAR-T cells prepared by the present invention have strong killing activity against multiple myeloma cells, effectively improve the treatment effect, and provide a key material for the subsequent development of adoptive immunotherapy with high efficiency and lasting anti-tumor activity.

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. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and / or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs. In some embodiments, methods for targeted nucleic acid editing 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 an MDH2 knockout system or inhibitor in reducing toxicity of T-2 toxin, and belongs to the technical field of biological agents. An MDH2 knockout IPEC-J2 cell line is constructed through a CRISPR / Cas9 gene editing system, and it is found that the cell activity of the cell line is remarkably improved under the condition of 20 ng / mLT-2 toxin concentration, the cell death rate is remarkably reduced, and the cell line has obvious resistance to T-2 toxin. The MDH2 inhibitor LW6 can also enhance the resistance of the cells to T-2 toxin when being used for treating the cells. Therefore, the toxicity of the T-2 toxin in the cell can be reduced by knocking out the MDH2 gene in the IPEC-J2 cell and inhibiting the activity of the MDH2, the tolerance of the cell to the T-2 toxin is improved, and a theoretical basis is provided for prevention and control of harm of the T-2 toxin to human and animals.

The invention belongs to the technical field of plant breeding and biology, and particularly relates to how to reduce the cadmium content of rice grains through a gene OsCT1 and application of the gene OsCT1 to creation of new germplasm of low-cadmium-accumulation rice. The invention discloses a nucleotide sequence of a gene OsCT1 coding region, a coded protein sequence, a CRISPR-Cas9 knockout target site nucleotide sequence, a nucleotide sequence of a knockout mutant and a protein sequence of the knockout mutant, as shown in an ST.26 standard sequence nucleotide or amino acid sequence table. After the gene OsCT1 is knocked out by utilizing a CRISPR-Cas9 technology, the cadmium content of grains of the OsCT1-knocked-out transgenic plant is obviously lower than that of a wild type variety. In addition, in the seedling stage, the plant height and the root length of the OsCT1-knocked-out transgenic plant are remarkably larger than those of a wild type variety; after the OsCT1 gene is subjected to overexpression, the grain cadmium content of an overexpressed transgenic plant is slightly higher than that of a wild type variety. In addition, in the seedling stage, the plant height and the root length of the overexpressed OsCT1 transgenic plant are obviously smaller than those of a wild type variety. According to the invention, the OsCT1 gene can be utilized to create a novel rice germplasm with low cadmium content in grains.