1541 results about "Cas9" patented technology

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.

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 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 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 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 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 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 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 provides a method for constructing a rice epigenetic recombinant inbred line, and belongs to the technical field of molecular biology and genetic breeding. According to the method for constructing the rice epigenetic recombination inbred line, a fertile rice DDM1 gene function weakening mutant is obtained through a CRISPR / Cas9 gene editing technology and hybridized with wild type rice with the same background to obtain F1, then the F1 and the wild type rice are subjected to backcrossing to obtain BC1F1, single-plant offspring selfing with the DDM1 genotype being the wild type is selected, selfing is conducted for multiple generations through a single-grain transmission method, and the rice epigenetic recombination inbred line is obtained. According to the present invention, the strain population with stable inheritance is constructed, such that the rice epigenetic recombinant inbred line is obtained, and has characteristics of rich phenotype, genetic and epigenetic variation so as to provide important population resources for batch identification and the like of important agronomic shape functional sites of rice.

The invention provides a uracil production strain and a construction method and application thereof.According to the strain, on an E.coli UR14 genome by means of a CRIPSR / Cas9 gene editing technology, firstly, psuG genes, preTA genes, rutA genes and upp genes are knocked out, so that decomposition of uracil is blocked; then, a uridine phosphorylase gene udp and a pyrimidine-5 '-nucleotide nucleotidase gene ppnN are subjected to overexpression, and synthesis and accumulation of uracil are synergistically enhanced; and finally, overexpression of the ribose phosphate mutase gene pgm further enhances the conversion of a by-product ribose phosphate 1-precursor 5-ribose phosphate 1-pyrophosphate and improves the carbon utilization rate, and the obtained strain has good genetic stability and high fermentation yield, can stably produce uracil, and has wide application prospects.

The invention provides an application of WNT7B in construction of a myopia animal model. Homologous genes wnt7ba and wnt7bb of the WNT7B in the zebra fish are knocked out or knocked down by applying a CRISPR / Cas9 gene editing technology, an animal model of which the eye axis length is remarkably increased, the eyeball movement frequency is remarkably reduced and the light response is reduced is obtained, and the result shows that the WNT7B is related to the high myopia. The model provides a favorable tool for pathogenesis and drug screening of high myopia, and has a good application prospect.

The invention discloses an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter gene is shown as SEQ ID NO.1. The oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, is obtained by cloning in an oil palm genome for the first time, and the promoter gene has high transcriptional activity and can drive downstream fluorescent protein mNeonGreen expression. The candidate oil palm endogenous U6 promoter gene can be provided for subsequently establishing a high-efficiency oil palm gene editing technology system based on a CRISPR / cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) system.

The invention discloses application of a gene CsPOD7 in cucumber haploid breeding, and belongs to the technical field of molecular breeding. According to the invention, a parthenogenesis haploid induction gene CsPOD7 is cloned from cucumber, the CsPOD7 is knocked out through a CRISPR-Cas9 gene editing technology, and a cspod7 mutant is used as a male parent to be hybridized with other cucumber materials as a female parent, so that parthenogenesis haploid can be generated. The invention is the first application of the gene CsPOD7 in developing a parthenogenesis haploid induction line in dicotyledon, realizes the application of the haploid induction line based on the gene CsPOD7 in cucumber genetic breeding, accelerates the cucumber breeding process, and has huge potential application value and market prospect.

The present invention relates to the fields of biotechnology and plant breeding, specifically providing a method for fixing heterosis in rice using the DMP gene. To address the low fruit set rate and complex operation issues of existing apomictic reproduction systems, the present invention verifies for the first time the haploid-inducing function of the rice genes OsDMP8 and OsDMP13. Furthermore, the CRISPR / Cas9 technology is used to simultaneously knock out the OsPAIR1, OsREC8, OsOSD1, and OsDMP genes, constructing a "MiMe-OsDMP" four-gene editing system. This system provides new ideas for constructing apomictic reproduction systems in rice and offers a new solution for fixing heterosis in rice apomictic reproduction. This system can significantly improve the efficiency and quality of agricultural production, and has significant economic value and broad application prospects.

The invention provides a system and a method for screening Cas protein mutants capable of identifying different PAM sequences, and belongs to the technical field of bioengineering. The system comprises a Cas protein mutant screening vector and an sgRNA expression vector, the Cas protein mutant screening vector comprises the following expression elements: a Cas protein coding gene, a plasmid replicon and a first resistance screening tag expression cassette; the sgRNA expression vector comprises the following expression elements: a plasmid replicon, a second resistance screening tag expression cassette, a third resistance screening tag expression cassette and an sgRNA expression cassette. According to the system and the method, aiming at the problem that the efficiency of identifying PAM sequences except NGG by wild type spCas9 protein is low, an active Cas9 mutant capable of identifying PAM sites except NGG is screened out, and the application field and the editing efficiency of a CRISPR gene editing system can be greatly expanded.

The invention relates to the technical field of fish genetic breeding, in particular to a method for improving grass carp reovirus resistance of grass carp. Four specific knockout targets of the herc4 gene are disclosed, the grass carp herc4 gene is specifically knocked out by utilizing a CRISPR / Cas9 gene editing technology, and a herc4 gene knockout individual, namely the high-resistance grass carp reovirus fish, is obtained. According to the invention, the function of the herc4 in the grass carp is verified for the first time, a new grass carp germplasm with improved grass carp reovirus resistance is rapidly obtained by knocking out the single gene of the herc4, and the herc4 has important industrial value.

The invention belongs to the technical field of gene engineering, and discloses a CRISPR / Cas9 delivery system and application thereof. The CRISPR / Cas9 delivery system is obtained by loading the CRISPR / Cas9 system with the fluorinated polyethyleneimine, and the fluorinated polyethyleneimine can deliver the CRISPR / Cas9 carrier into cells, has excellent transmembrane delivery efficiency, and can efficiently mediate the CRISPR / Cas9 system to enter target cells. The CRISPR / Cas9 delivery system is applied to large-scale breeding of aquatic animals, the use safety is high, the biocompatibility is excellent, and the delivery system has excellent gene editing efficiency and has wide application prospects in the field of gene editing breeding.

The invention discloses a male sterility gene ZmUCE4 and application thereof in creating a male sterility line of corn, and belongs to the field of plant biotechnology breeding. According to the invention, site-directed mutagenesis of the ZmUCE4 gene in wild type corn is carried out through a CRISPR / Cas9 gene editing technology, pollen development abnormality and abortion can be caused, complete male sterility is caused, and it is proved that the ZmUCE4 gene has a regulation and control function on corn male reproductive development. Through offspring screening, a sterile line without transgenic components can be obtained, a stable maize male sterile line is created, and the method has important significance on maize male fertility control and hybrid seed production. The invention also designs a functional molecular marker aiming at the obtained uce4 male sterility mutant gene, and has important application value in maize male sterility line cultivation, sterile hybrid seed production and molecular marker-assisted selection.

The invention relates to the fields of biology, medicine and clinic. In particular to application of PAICS as a target spot in developing or screening reagents for preventing and / or treating orthopoxvirus infection, and application of inhibiting a synthetic pathway of purine nucleotide and / or inhibiting a metabolic pathway of folic acid in preparing reagents for treating and / or preventing orthopoxvirus infection diseases. In order to restrain the infection of orthopoxvirus, the inventor team uses a CRISPR / Cas9 technology to carry out generic genome screening on host cells infected by orthopoxvirus, determines a host-dependent gene PAICS gene in the orthopoxvirus infection process, and verifies and clarifies the virus-dependent mechanism of the gene; in order to deal with the global continuous orthopoxvirus infection problem, the invention plays an important role in the development and screening of future related prevention or treatment drugs, and also expands the new clinical application of existing drugs including methotrexate, mercaptopurine, mizoribine and the like.

The invention relates to the field of plant breeding, and particularly provides a method for fixing rice heterosis by using an OsZFPP gene. The method comprises the following steps: firstly, constructing an OsZFPP1 gene or OsZFPP2 gene expression cassette driven by an OsECA1 promoter; then, an expression box of rice with three target points of OsPAIR1, OsREC8 and OsOSD1 being knocked out of CRISPR / Cas9 is constructed; integrating the expression cassette into the same vector to transform hybrid rice; screening three-gene homozygous mutation and OsZFPP positive plants, and identifying diploid cloned offspring through flow cytometry and genome sequencing. According to the method, the rice OsZFPP gene is combined with MiMe to provide a novel method for fixing the heterosis of the rice through apomixis, the heterosis of the rice can be successfully fixed through the method, an apomixis system with the high maturing rate can be obtained, and a novel solution is provided for fixing the heterosis of the rice through the apomixis.

The invention relates to the field of gene engineering and molecular breeding, and discloses application of a ZmTCP5 gene in regulation and control of heat resistance of corn. According to the invention, three types of knockout mutants of the gene are obtained by using a CRISPR / Cas9 technology, and three overexpression materials are created. At normal temperature (25-28 DEG C), the material has no obvious phenotypic difference with a wild type; however, under high temperature stress (45 DEG C), the ZmTCP5 knockout mutant has enhanced heat resistance, reduced reactive oxygen species (ROS) accumulation, and significantly increased ROS scavenging enzyme activity, chlorophyll content and photochemical efficiency; on the contrary, the ZmTCP5 overexpression strain is more sensitive to heat stress, which indicates that the ZmTCP5 negatively regulates the heat resistance of the corn. The research provides a theoretical basis for analyzing a heat-resistant molecular mechanism of the corn, and also provides an important gene resource for creating a new germplasm of the heat-resistant corn.

Disclosed in the present invention is a use of genes NAT1 and bHLH110 in improving high-temperature resistance of rice, for use in using gene editing technology to obtain a rice material having enhanced high-temperature resistance. The base sequence of a rice gene NAT1 is as shown in SEQ ID NO. 1, and the base sequence of a gene bHLH110 is as shown in SEQ ID NO. 4. A base of a rice gene NAT1 of rice is replaced, deleted or inserted through gene editing technology to release inhibition on the rice gene bHLH110, and the expression of wax synthesis genes CER1 and CER1L is promoted by the rice gene bHLH110 to enhance the high-temperature resistance of a plant, thereby preparing a rice material having enhanced high-temperature resistance. According to the present invention, a NAT1 mutant rice material Nipponbare obtained by using CRISPR-Cas9 gene editing technology has remarkably enhanced high-temperature resistance, including an improved survival rate in a seedling stage and improved agronomic traits in a reproductive stage.

The invention belongs to the technical field of synthetic biology, and particularly relates to recombinant escherichia coli for efficiently producing O-acetyl-L-homoserine as well as a construction method and application of the recombinant escherichia coli. According to the invention, CRISPR-Cas9 gene editing is combined with metabolic modification, so as to precisely integrate OAH biosynthesis key nodes: introducing and overexpressing a mutant metX gene at a genome pseudogene site to enhance the expression of homoserine acetyltransferase, knocking out byproducts such as poxB and mgsA to synthesize genes, and carrying out high-yield synthesis of OAH. Corynebacterium glutamicum thrA (anti-feedback inhibition), aspB and bacillus subtilis acsA-acuA genes (construction of an acetic acid switch) are introduced, gene expression of gltA, tpiA and the like is optimized to increase supply of precursors such as acetyl CoA and the like, and finally a stable strain without plasmids, antibiotics and inducers is obtained. The production cost is effectively reduced, and the method has the advantage of high genetic stability and shows a good application prospect in OAH industrial production.

The invention relates to the technical field of gene editing, in particular to a Cas9 protein mutant and application thereof. According to the present invention, the mutation is introduced at the position far away from the catalytic site to induce the Cas9 protein conformation change so as to obtain the new Cas9 protein mutant, only the Cas9-sgRNA target chain is cut, and the Cas9-sgRNA non-target chain is not cut, such that the new Cas9 protein mutant can be used for constructing the novel base editor, and the high-efficiency base editing can be performed on the mammalian cell genome. The Cas9 protein mutant disclosed by the invention is fused with a mutant TX with DNA binding deletion of 3 '-> 5' exonuclease TREX2, so that the gene editing efficiency of a 3'terminal generated by paired single-nick enzyme cutting can be greatly improved, and the safety of the Cas9 protein mutant is maintained.