12results about How to "High knockout efficiency" patented technology

The invention relates to the technical field of grass carp (Ctenopharyngodon idella), in particular to a method for knocking out a grass carp SMYD3 gene and application of the grass carp SMYD3 gene. The nucleotide sequence of the gRNA is as shown in SEQ ID NO: 1. The method comprises the following steps: synthesizing gRNA of a target gene through in-vitro transcription, then mixing the gRNA with Cas9 protein, introducing the mixture into an animal pole in a grass carp fertilized egg single-cell period by adopting a microinjection mode, and hatching to obtain the grass carp F0 generation with the target gene mutation. According to the application, the knockout of the grass carp gene is efficiently realized, and an effective technical means is provided for developing grass carp breeding and gene function research.

The invention belongs to the technical field of gene engineering, and particularly relates to application of a TRIM3 gene or protein serving as a target spot in screening of drugs for inhibiting foot and mouth disease virus replication. The method specifically comprises the following steps: firstly, constructing a TRIM3 knockout cell line by utilizing a CRISPR / Cas9 system, wherein the cell line obviously inhibits the replication of FMDV; secondly, according to the invention, a TRIM3 stable expression cell line is constructed by using a lentivirus packaging system, and the cell line promotes the replication of FMDV. Therefore, the inhibition or silencing of the TRIM3 gene / protein can inhibit the replication of the foot-and-mouth disease virus, can be used as a target spot for screening drugs for inhibiting the replication of the foot-and-mouth disease virus, provides some theoretical bases for preventing or inhibiting FMDV infection in the future, and has a wide application prospect.

The invention discloses a hansenula polymorpha efficient gene editing system based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 and application of the hansenula polymorpha efficient gene editing system. The hansenula polymorpha gene editing system comprises: (1) a Cpf1 integration plasmid, wherein the Cpf1 integration plasmid is expressed by an endogenous constitutive promoter, and the Cpf1 is integrated into a hansenula polymorpha genome; (2) a crRNA expression plasmid, wherein the crRNA expression plasmid comprises a crRNA expression cassette; wherein the crRNA expression cassette is composed of an RNA pol III type promoter and a terminator.

The invention belongs to the technical field of gene engineering, and particularly relates to application of LULL1 gene / protein as a target spot in screening of drugs for inhibiting foot and mouth disease virus replication. The method specifically comprises the following steps: firstly, constructing an LULL1 knockout cell line by utilizing a CRISPR / Cas9 system, wherein the cell line remarkably inhibits the replication of FMDV; secondly, a lentivirus packaging system is utilized to construct an LULL1 stable expression cell line, and the cell line promotes the replication of FMDV. Therefore, the inhibition or silencing of the LULL1 gene / protein can inhibit the replication of the foot-and-mouth disease virus, can be used as a target spot for screening drugs for inhibiting the replication of the foot-and-mouth disease virus, provides some theoretical bases for preventing or inhibiting FMDV infection in the future, and has a wide application prospect.

The invention discloses a method for knocking out a pig E4F1 gene by using a CRISPR / Cas9 system, and belongs to the technical field of gene editing. The method comprises the following steps: constructing a CRISPR / Cas9 gene knockout vector containing sgRNA by utilizing an sgRNA composition (a pair of sgRNA) of a specific targeting pig E4F1 gene, and then carrying out pig E4F1 gene knockout by utilizing the vector. According to the method disclosed by the invention, high-efficiency knockout of the pig E4F1 gene can be realized, so that the construction efficiency of E4F1 gene knockout cells and E4F1 gene knockout pigs is remarkably improved, and an effective method and basis are provided for functional research and application of the pig E4F1 gene.

The utility model relates to the technical field of monocrystalline silicon manufacturing, in particular to rapid cleaning equipment for a single crystal furnace pier crucible material, which comprises a bottom plate, four I-shaped steels are fixedly connected onto the bottom plate, the tops of the I-shaped steels are fixedly connected with a fixed frame, a movable plate is arranged in a groove between two adjacent I-shaped steels in a sliding manner, and the movable plate is fixedly connected with the fixed frame. Two driving machines are mounted on the movable plate, a clamping part is arranged between the output ends of the two driving machines, the clamping part is used for clamping a crucible edge with a hole in the bottom, a pushing part is arranged on the fixed frame, and when the clamping part clamps the crucible edge, the driving machines can drive the crucible edge to rotate. According to the utility model, through the matching of the related structures, the crucible bottom material in the crucible edge can be slowly pushed out, so that the phenomena that the crucible edge needs to be cooled during manual knockout, the high-temperature crucible edge scalds a knocker and the crucible bottom material is likely to splash to hurt a worker in the knockout process are avoided, and the knockout efficiency is improved.

The present invention provides a sgRNA for targeted knockout of bovine mammary epithelial cell TARDBP gene and a cell line constructed thereof. The sgRNA includes sgRNA1 and sgRNA2, and the nucleotide sequences of the sgRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The sgRNA of the present invention can accurately guide the Cas9 protein to target and cut the bovine mammary epithelial cell TARDBP gene, and the expression vector cell conversion rate obtained by constructing the sgRNA is high, which greatly improves the success rate of knocking out the bovine mammary epithelial cell TARDBP gene, and obtains the TARDBP gene knockout mammary epithelial cell line. In order to facilitate the study of the function and mechanism of the TARDBP gene in milk fat secretion and milk production of dairy cows, a cell model is provided, which lays the foundation for dairy cow biological breeding.

The present invention provides a method for constructing an abortion mouse model, the method comprising constructing an abortion mouse model by specifically knocking out the Klk8 gene of mouse liver cells. The mouse model constructed by the present invention has phenotypes such as embryo absorption, abortion, and reduced embryo number, which are consistent with the symptoms and pathological process of human recurrent abortion, and has good controllability and stability. It can be used to study the occurrence and development mechanism and prevention and treatment measures of recurrent abortion, as well as for the development and research of new drugs.

The application discloses a double-sgRNA site knockout cassette suitable for fusarium oxysporum, a CRISPR / Cas9 system and a construction method and application thereof, and is based on a filamentous fungus gene knockout plasmid pFC332 to construct a new vector pFRCas9-G418 which is more suitable for gene knockout of the fusarium oxysporum. The application provides a method for knocking out genes by using the double-sgRNA site CRISPR / Cas9 system suitable for the fusarium oxysporum, the method can realize rapid knockout of genes of the fusarium oxysporum, the knockout period is shortened to 20 days, and the knockout efficiency is as high as 77.8%. The application successfully verifies the influence of the FCC1 gene knockout on the morphology of the fusarium oxysporum, the center of the strain presents a yellow solid, and the growth morphology of the strain is small, and the FCC1 gene can be used as a phenotype reporter gene of the fusarium oxysporum in the future, so as to verify gene editing.

The present application relates to an application of an inducible promoter in Clostridium beijerinckii gene editing, and belongs to the technical field of biotechnology and genetic engineering. The rhamnose inducible promoter from Clostridium beijerinckii is applied to the CRISPR / Cas9 gene editing system of Clostridium beijerinckii, and the gene knockout efficiency is significantly improved, and an efficient and controllable promoter tool is provided for genetic modification of Clostridium beijerinckii. The present application provides a solution for improving the gene editing technology of solventogenic Clostridium, and significantly improves the efficiency and feasibility of genetic operation in the industrial strain.

The invention provides construction and application of a ZXDB gene knockout mouse. The invention provides a method for knocking out a ZXDB gene in a mouse cell, the method is carried out by adopting a CRISPR / Cas9 system, the CRISPR / Cas9 system comprises sgRNA targeting the ZXDB gene, and the sgRNA comprises sgRNA1 and sgRNA2; two sgRNA loci are designed aiming at the ZXDB gene with high GC content, the ZXDB gene of a mouse cell is knocked out, the ZXDB gene knockout mouse model is constructed, the method has the advantages of being high in knockout efficiency, simple in method and easy and convenient to operate, and a good foundation is laid for further utilizing the ZXDB gene knockout mouse model to carry out in-vivo function verification on a biological mechanism in which the ZXDB gene participates.

The invention relates to the technical field of medicine and health, in particular to a method for improving the adaptability of T cells to a tumor microenvironment, and the key point of the technical scheme is that the method comprises the following steps: S1, preparing tumor neoantigen mRNA; s2, collecting and separating PBMCs (peripheral blood mononuclear cells) to obtain initial T cells; s3, inducing by using the initial T cells to obtain DC cells; s4, modifying the DC cells by using the tumor neoantigen mRNA; s5, carrying out electric transfer knockout on the PD-1 of the initial T cell through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9); and S6, adding the modified DC cells into the T cells of which the PD-1 is knocked out, and carrying out co-incubation amplification, so as to obtain the treated T cells. The tumor neoantigen mRNA is used for modifying the DC cell, the CRISPR-Cas9 is combined for modifying the T cell, then the DC cell and the T cell are incubated and amplified together, and the method has the advantages that the safety is improved, and the adaptability of the T cell to the tumor microenvironment is improved.