921 results about "Cas9" patented technology

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 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 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 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 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 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 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 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.

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 discloses recombinant escherichia coli capable of producing salidroside and application, exogenous genes are introduced by utilizing a CRISPR / Cas9 technology, the recombinant escherichia coli capable of improving the fermentation yield of the salidroside is obtained by modifying and screening key genes, and finally, the salidroside is obtained by optimizing a fermentation tank process. The recombinant Escherichia coli can reach the highest yield of salidroside produced by fermentation of the scale at present on a 5L fermentation tank. The salidroside is fermented in a 5L fermentation tank for 104 hours, so that the maximum accumulation amount of the salidroside is 33.68 g / L, and industrial enlarged production is facilitated.

The invention provides a method for overspeed preparation of gene edited goldfish. The method comprises the following steps: S1, obtaining gobiocypris rarus without primordial germ cells as receptor fish; s2, designing a target spot for the goldfish amh gene, introducing a Cas9 / gRNA complex into a goldfish fertilized egg, performing sequencing verification to obtain a mutant individual, and feeding the mutant individual as a donor fish; s3, taking gonads of donor fish, cutting into pieces, performing enzymolysis digestion, filtering, and performing density gradient centrifugation to enrich donor germline stem cells; s4, transplanting the germline stem cells of the donor fish into the receptor fish body, and feeding to obtain the gene edited goldfish sperms and ova. S5, hybridizing and feeding goldfish amh mutant sperms and ova produced by gobiocypris rarus to obtain gene edited goldfish individuals. According to the invention, gobiocypris gobiocypris is used as a receptor, the cross-subfamily gene edited goldfish is used as a donor, and an efficient, stable and easy-to-popularize method for preparing the gene edited goldfish is provided by utilizing a belly borrowing reproduction technology.

The invention discloses sgRNAs of a specific targeting grass carp RIOK3 gene and a construction method and application of an RIOK3 gene knockout cell line, and belongs to the technical field of gene engineering. The invention provides sgRNAs of a specific targeting grass carp RIOK3 gene and a sequence of a primer group, and also provides a method for constructing a large-fragment knockout cell line of the RIOK3 gene in combination with the sgRNAs, and application of the gene knockout cell line in research on an anti-virus infection mechanism of grass carp. The invention discloses the construction of the grass carp gene in-vitro research model by knocking out the antiviral negative regulatory factor RIOK3 of the fish cells by using the CRISPR-Cas9 technology for the first time, provides an in-vitro research material and a new way for the analysis of an antiviral immune regulation molecular mechanism of the grass carp, and particularly has important guiding significance for the research of resisting grass carp reovirus infection.

The present disclosure provides evolved and engineered reverse transcriptase variants and Cas9 variants with improved properties (e.g., improved editing efficiency when used in a guided editor context). The disclosure also provides fusion proteins comprising the reverse transcriptase variants and Cas9 variants described herein, including, for example, a guided editor. The disclosure also provides polynucleotides encoding the reverse transcriptase variants, Cas9 variants, and guide editors provided herein, as well as vectors comprising such polynucleotides. The disclosure also provides pharmaceutical compositions and cells comprising the reverse transcriptase variants, Cas9 variants, and a guided editor described herein. The present disclosure also provides methods and uses relating to the reverse transcriptase variants, Cas9 variants, and guide editors described herein.

The invention belongs to the technical field of gene editing, and particularly relates to a construction method and application of a needle mushroom CRISPR / Cas9 gene editing vector. The CRISPR / Cas9 gene editing vector for flammulina velutipes is characterized in that the vector takes pFgnpt as a skeleton vector, and further comprises a Cas9 expression cassette and an sgRNA expression cassette; the nucleotide sequence of the Cas9 expression cassette is as shown in SEQ ID NO. 10. The nucleotide sequence of the sgRNA expression cassette is as shown in SEQ ID NO. 11. According to the present invention, with the agrobacterium-mediated transformation technology, the stable expression of Cas9 and sgRNA in the flammulina velutipes body is achieved, the efficient and stable CRISPR / Cas9 gene editing system is established, and the technical support is provided for the flammulina velutipes variety improvement, the functional gene research and the industrial upgrading.

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

The invention provides a valine production strain as well as a construction method and application thereof, the strain is constructed by utilizing a CRIPSR / Cas9 system gene editing technology based on allele exchange, an artificial operon is designed and constructed, the artificial operon is constructed at a ygaY site, a strong promoter Ptrc is used for controlling ilvIH (G14N S17F)-pykF to carry out synchronous multi-copy expression, and the valine production strain can be used for producing valine. On the basis of the strain, an artificial operon is constructed at an ycgH site, a strong promoter Ptrc is used for controlling ilvC-ilvD to carry out synchronous multi-copy expression, meanwhile, feedback inhibition of valine on ilvIH is relieved, and an ilvIH natural promoter is replaced by the strong promoter Ptrc, so that more pyruvic acid flows to valine; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for efficiently synthesizing valine from the beginning by taking glucose as a substrate.

The invention belongs to the technical field of genetic engineering application and plant breeding, and particularly relates to application of a rice OsHGO gene to improvement of high-temperature tolerance of rice. The OsHGO gene in japonica rice Zhonghua 11 is knocked out through a CRISPR / Cas9 system, and a homozygous mutant plant with the OsHGO gene knocked out is obtained. The phenotypes of the wild type and mutant plants after high-temperature treatment are observed, and it is found that the wild type has more serious leaf wilt than the mutant plants. It is shown that after the OsHGO is knocked out, the influence of the high temperature on plant growth is relieved, and the OsHGO gene regulates and controls the tolerance of the rice to the high temperature. Through mutation of the gene, high-temperature-resistant rice can be cultivated.

The invention belongs to the technical field of genetic engineering, and relates to the field of wheat genetic engineering, in particular to a gene TaABCG42 for regulating and controlling cadmium accumulation of wheat as well as an encoding protein and application of the gene TaABCG42. According to the invention, a TaABCG42 gene is knocked out by using CRISPR / Cas9, a TaABCG42 gene overexpression plant is constructed at the same time, and a homozygous mutant is obtained by using a Hi-Tom sequencing technology and PCR (Polymerase Chain Reaction) successive generation screening. A wild type TaABCG42 homozygous overexpression and knockout plant treated with a 40 [mu] mol / L Cd solution for 5 days is subjected to analysis such as metal content determination, subcellular component metal determination, Cd chemical form determination and Cd ion flow determination, and the Cd absorption and transport approach of TaABCG42 is analyzed, so that the physiological and molecular mechanism of the TaABCG42 metal transporter for absorbing and transporting Cd is explored, and the application prospect is wide. The understanding on the wheat ABC metal transporter is enriched, and a theoretical basis is provided for cultivating high-quality wheat with low Cd accumulation.

The invention relates to the field of plant genetic engineering, and discloses an application of OsAAT2 protein and a coding gene thereof in regulating and controlling the salt tolerance of rice. The gene of the OsAAT2 protein is edited on the basis of a CRISPR / Cas9 technology, a rice ZH11 variety is introduced by utilizing an agrobacterium-mediated method, and a knockout mutant is obtained through screening; tests prove that the rice mutant with the OsAAT2 gene knocked out is rehydrated for 4 days after being subjected to stress treatment of 150 mM NaCl salt, and the survival rate is remarkably higher than that of a wild type ZH11 plant. And a simple and effective technical means is provided for rapidly creating a new salt-tolerant rice strain. Therefore, the OsAAT2 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 invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

Metastatic uveal melanomas are highly resistant to all existing treatments. To identify actionable vulnerabilities, the inventors conducted a CRISPR-Cas9 knockout screen using a library composed of chromatin remodelers. They revealed that the histone H3 methyltransferase SETDB1 plays a critical role in metastatic uveal melanoma cell proliferation and survival. Functionally, SETDB1 knockdown triggers decreased expression of genes related to replication and cell cycle and promotes growth arrest associated with increased markers for DNA damage and senescence entry. Using pre-clinical model, they further demonstrated that anti-SETDB1 therapy tumor growth in vivo. The inventors identify SETDB1 as a new relevant therapeutic target for the treatment of metastatic uveal melanomas. The present invention relates to a method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of SETDB1 inhibitor.

The invention discloses application of an OsGIT2 gene in rice blast resistance of rice. The applicant constructs a CRISPR carrier of the gene and transforms a japonica rice variety Kitaake of rice to obtain a transgenic line material, and the resistance of the transgenic line material to magnaporthe oryzae is obviously higher than that of a wild type material. The mutant rice is obtained by an applicant through a CRISPR-Cas9 technology, the mutant shows resistance to rice blast, and the technical scheme of the invention is of great significance to creation of broad-spectrum disease-resistant high-yield excellent new germplasm.

The invention discloses application of fluorinated ionizable lipid as a gene drug delivery carrier, and belongs to the technical field of biological medicine. The gene drug delivery carrier provided by the invention is prepared from fluorinated ionizable lipids, cationic lipids, auxiliary lipids, structural lipids and PEG lipids, wherein the molar percentage of the fluorinated ionizable lipids in the gene drug delivery carrier is 1%-10%. According to the invention, the molar percentage of the fluorinated ionizable lipid in the total lipid is optimized to 1-10%, so that the cytotoxicity is obviously reduced, and more excellent nucleic acid transfection efficiency is realized in vivo and in vitro; various nucleic acid drugs including DNA, mRNA, siRNA and CRISPR-Cas9 ribonucleoprotein can be efficiently delivered, action targets of the nucleic acid drugs are expanded to cell nucleuses and cytoplasm, and the nucleic acid drugs are more accordant with the action mechanism of modern gene therapy and editing.

The invention belongs to the technical field of plant genetic engineering and breeding, and particularly relates to application of a lettuce LsGAI1 gene in lettuce growth trait screening breeding. 1, and the protein coded by the LsGAI1 gene is shown as SEQ ID NO. 2. The LsGAI1 gene is knocked out from the asparagus lettuce through a CRISPR-Cas9 technology, so that internode elongation and plant height increase can be caused; and overexpression of the LsGAI1 enables internode shortening and plant height reduction. The cloning and functional verification of the LsGAI1 gene provide important gene resources and theoretical basis for plant type improvement and high-yield breeding of asparagus lettuce.

This invention belongs to the field of molecular genetics and discloses a method and application for controlling fish fertility using in vivo gene editing technology. The applicant, for the first time, cloned a carp-specific U6 promoter and used this promoter to construct the U6 gene. MOVIE gRNA transgenic vectors and water The Cas9 vector allows for the establishment of an in vivo editing system. This strategy enables the heritability of sterility, a reproductive control trait, through hybridization of fertile parents to produce sterile offspring. The operation is highly efficient, simple, and environmentally friendly. Furthermore, since high-copy-rate short, scattered repetitive sequences exist in all fish species, this strategy has broad applicability across species.

This invention discloses the application of the DNAL1 gene as a target in screening drugs for the prevention and treatment of Zika virus or dengue virus type 2 infection, specifically the application of screening drugs for the prevention and / or treatment of Zika virus or dengue virus type 2 infection with the aim of inhibiting or knocking out the DNAL1 gene. The invention has found that the DNAL1 gene is a key host factor promoting the replication of Zika virus or dengue virus type 2, and that inhibiting the DNAL1 gene through siRNA or knocking out the DNAL1 gene through CRISPR / Cas9 can inhibit the replication of Zika virus or dengue virus type 2. The DNAL1 gene is a potential host factor for flaviviruses, and this invention provides a potential target for the prevention and / or treatment of Zika virus or dengue virus type 2 infection.

The invention discloses construction of myocardial cells differentiated from propionemia stem cells based on a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology. The model constructed by the invention has the metabolic phenotype of propionemia and can also be differentiated into myocardial cells. A reliable and effective experimental model is provided for research of propionemia disease related targets and screening of drugs for preventing / treating propionemia, and the method has a wide application prospect.

The invention belongs to the technical field of gene engineering, and discloses application of an F-box protein gene OsFBX235 in improvement of bacterial blight resistance. The invention specifically discloses an application of knocking out OsFBX235 gene in improving the bacterial blight resistance of rice or cultivating a bacterial blight resistant rice variety. The F-box protein coding gene OsFBX235 is separated and cloned from rice, and the gene is proved to participate in the defensive reaction of the rice to the bacterial blight for the first time through functional analysis, and is an important negative regulation factor for regulating the resistance of the rice bacterial blight. The gene OsFBX235 is knocked out through target gene screening in combination with a CRISPR / Cas9 technology, and the importance of the gene OsFBX235 on resistance regulation and control of the rice bacterial blight is proved. Meanwhile, knockout of the gene can be used for creating rice germplasm resources with high bacterial leaf blight resistance and can be applied to breeding of new varieties with the bacterial leaf blight resistance of the rice.

The invention discloses application of beta-nicotinamide mononucleotide in regulation and control of gene editing efficiency, belongs to the field of gene editing treatment, and finds that the beta-nicotinamide mononucleotide (NMN) can efficiently inhibit the activity of CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13 systems in a broad-spectrum manner for the first time. The application comprises emergency blocking of off-target effect in gene editing clinical treatment, biological safety prevention and control of a virus vector gene editing system, and CRISPR activity regulation and control of in-vitro non-diagnostic purpose. Experiments show that NMN can inhibit CRISPR-mediated gene damage and cell death in a cell model, the inhibition efficiency in an in-vitro enzyme digestion system reaches 68.7%, and cell growth or transfection efficiency is not affected. The invention provides an innovative solution for safe application of CRISPR (clustered regularly interspaced short palindromic repeats) technology, and the NMN is approved to be taken orally as a health care product, so that the NMN has extremely strong clinical application potential. Compared with the existing CRISPR (clustered regularly interspaced short palindromic repeats)-resistant protein or synthetic small-molecule inhibitor, the NMN has the advantages of endogenous property, high biocompatibility, good oral safety and the like.