132results about How to "High knockout efficiency" patented technology

The invention belongs to the technical field of gene engineering, in particular to a pCr-NHEJ (non-homologous end joining) carrier as well as a construction method of the pCr-NHEJ carrier and an application of the pCr-NHEJ carrier in site-specific knockout of bacterial genes. The sequence of the pCr-NHEJ carrier is shown as the SEQ ID NO.1. The application of the pCr-NHEJ carrier in site-specific knockout of the bacterial genes is provided. The technical principle is as follows: NHEJ and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) are jointly applied, so that after DNA (deoxyribonucleic acid) is broken when a CRISPR-Cas9 system cuts bacterial genomic DNA, the NHEJ system can be connected with a broken DNA end automatically, bacteria are survived, double-strand DNA having homology with a target sequence is not required to be introduced artificially to repair the broken DNA end, and the operation steps of the CRISPR-Cas9 technique are simplified.

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR genes. The system comprises sgRNA for specifically targeting KRAS genes and sgRNA for specifically targeting EGFR genes, wherein a corresponding DNA sequence of the sgRNA for specifically targeting KRAS genes is shown as SEQ ID NO.1 or / and SEQ ID NO.2; and a corresponding DNA sequence of the sgRNA for specifically targeting EGFR genes is shown as SEQ ID NO.11 or / and SEQ ID NO.12. The invention further discloses application of the system in preparation of medicines for treating cancers. The CRISPR-Cas9 system disclosed by the invention is capable of simultaneously and efficiently knocking out two cancer driving factors KRAS and EGFR which are highly-expressed in lung cancer. The system is simple in operation and high in knockout efficiency and is expected to be applied to treatment of the lung cancer. The system disclosed by the invention is applicable to multiple cancers with abnormal expressions of the EGFR and KRAS.

The invention discloses a MiR-126 full-length gene knockout kit based on a CRISPR-Cas9 technology and an application thereof. CRISPR-Cas9 target sequences upstream and downstream a MiR-126 gene are preferably selected, and sgRNA single strands are designed and synthesized for the target sequences and built into a carrier. Through 293T cell strain transfection, sgRNA and trRNA constitute a specific recognition structure. Thus, Cas9 enzyme is guided to specifically shear the corresponding sequences at the two ends of the MiR-126 gene. Drug sieving is carried out constantly to get a MiR-126 full-length gene knockout cell strain. An optimal upstream and downstream sgRNA combination is obtained through drug sieving cell strain sequencing verification. The knockout efficiency of the combination is as high as 90% above. The kit built based on the combination can be used to carry out specific MiR-126 full-length gene knockout on a variety of cell lines such as 293T, a lung cancer cell line A 549 and a vascular endothelial cell HUVEC line.

The invention provides sgRNA and a gene vector for inhibiting bladder cancer by targeting human lncRNA-UCA1 and an application of the sgRNA; specifically, sgRNA sequences of an lncRNA-UCA1 gene and a PD-1 gene suitable for CRISPR-Cas9 targeted splicing are designed; by transforming plasmids of specific splicing lncRNA-UCA1 gene and CRISPR-Cas9 nuclease gene into human bladder cancer cells, the expression of the lncRNA-UCA1 gene drops so as to inhibit the growth of tumor cells; and the plasmid, together with a human PD-1 gene targeted knockout vector, is transformed into a humanized mouse model of bladder cancer transplanted tumor, so as to obviously inhibit the growth of tumors. According to the invention, the vector is simple in preparing steps, the sgRNA is good in targeting property and the CRISPR-Cas9 is high in knockout efficiency.

The invention discloses gRNA for knockout of a wild type T cell TCR [beta] strand and a method. The sequence of the gRNA is as shown in SEQ ID NO: 1, by utilizing a CRISPR / Cas9 technology, the gRNA and CRISPR / Cas9 jointly infect a T cell, the wild type T cell TCR [beta] strand is knocked out, and the T cell lacking the wild type TCR [beta] strand is constructed and used for CAR-T or TCR-T cellular immunotherapy. According to the gRNA for knockout of the wild type T cell TCR [beta] strand and the method, the knockout efficiency is high, the preparation method is relatively simple and easy, and T cells lacking wild type TCR [beta] strands can be provided for clinic rapidly and efficiently.

The invention provides a method for performing GINS2 gene editing on mesenchymal stem cells by adopting a CRISPR-cas 9 system, and particularly relates to a method for constructing a GINS2 gene-knocked mesenchymal stem cell line. A new enhancin CREnhancer 1.0 is used, so that the editing efficiency of the CRISPR-cas 9 gene in cells can be remarkably improved. The GINS2 knockout plasmid for the mesenchymal stem cells provided by the invention has high genetic stability.

The invention discloses a goat TLR4 gene knock-out vector and a construction method thereof. The construction method comprises the following steps: firstly, designing an sgRNA fragment of the TLR4 gene by adopting a CRISPR / cas9 system, synthesizing an sgRNA nucleotide sequence, constructing and simultaneously expressing the sgRNA and plasmid PYSY-sgRNA of Cas9 D10A, connecting and transforming to an Escherichia coli DH5 alpha competent cell, and verifying the transformant; and judging and proving by enzyme digestion and sequencing that the TLR4 gene knock-out vector is constructed correctly. The invention adopts the CRISPR / cas9 for constructing the vector, and provides a theoretical basis for subsequently acquiring a goat TLR4 gene deletion type alveolar epithelial cell system, and studying the immune response molecular mechanism of mycoplasma pneumonia infection.

The invention discloses a gRNA subjected to wild type T cell TCR alpha chain knockout and a method. The sequence of the gRNA is shown as SEQ ID NO:1. By using a CRISPR / Cas 9 technology, the gRNA and the CRISPR / Cas9 perform co-infection on T cells; the wild type T cell TCR alpha chain knockout is performed; the T cells lack of wild type T cell TCR alpha chains are built; the gRNA can be used for CAR-T or TCR-T cellular immunity treatment. The gRNA has high knockout rate; the preparation method is relatively simple and easy; the T cells lack of wild type T cell TCR alpha chains can be fast and efficiently provided for clinics.

The invention provides a method using a CRISPR-Cas system to perform GING2 gene editing on an epidermal stem cell and particularly relates to a method for building an epidermal stem cell line with theGING2 gene being knocked out. Two specific gRNAs are built and acquired, and the GINS2 gene editing efficiency of CRISPR / Cas9 in the epidermal stem cell can be increased evidently. An epidermal stemcell GINS2 knockout plasmid is good in hereditary stability and high in targeting efficiency.

The invention provides CASP3 gene editing performed on a mesenchymal stem cell through a zsystem, and particularly relates to the establishment of a mesenchymal stem cell line for constructing CASP3 gene knockout. Novel enhancing CREnhancer1.0 is adopted, and CRISPR-cas9 gene editing efficiency in a cell can be obviously improved. Bone mesenchymal stem cell CASP3 gene knocked out plasmid has goodhereditary stability.

The invention discloses a CRISPR-Cas9 system for knocking out the gene GRIN2D and its application. The CRISPR-Cas9 system comprises Cas9 and gRNA specifically targeting to the gene GRIN2D. The invention also discloses application of the CRISPR-Cas9 system to preparation of drugs used for treating cancer and application of the gene GRIN2D to diagnostic kits and medicines used for precise treatmentof stomach cancer and other tumors. In virtue of a clinical sample tissue chip of stomach cancer, the gene GRIN2D is proved to be increased in expression in stomach cancer tissue and be related to prognosis according to immunohistochemical results. The CRISPR-Cas9 system provided by the invention can highly efficiently knock out the highly-expressed gene GRIN2D in stomach cancer and inhibit the proliferation and migration / invasion of gastric cancer cells, and is simple to operate and high in knockout efficiency. The CRISPR-Cas9 system is expected to be applied to diagnosis and treatment of stomach cancer and other tumors with over-expressed GRIN2D. The CRISPR-Cas9 system is applicable to a plurality of tumors with abnormal expression of GRIN2D.

The invention provides a method for Card3 gene editing for epidermal stem cells through a CRISPR-Cas system and particularly relates to a method for building an epidermal stem cell line of which the Card3 gene is knocked out. The method constructs two specific gRNAs and significantly improves the gene editing efficiency of CRISPR / Cas9 in epidermal stem cells to Card3. The epidermal stem cell Card3knockout plasmid provided by the invention has good genetic stability and high targeting efficiency.

The invention relates to a CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method. The method comprises the following steps: establishing a CRISPER-Cas9-system-based gRNA (guide ribonucleic acid) expression vector according to a sheep MSTN gene sequence, establishing an exogenous-gene-containing donor plasmid capable of being integrated into a host genome according to the acting site of the gRNA, and transforming the optimized CRISPER-Cas9 vector, the gRNA expression vector and the linearized donor plasmid into sheep fibroblasts, thereby obtaining the sheep MSTN gene knock-out and exogenous gene site-specific integrated cells. The CRISPER-Cas9-mediated targeted vector provides a simple quick safe way for sheep MSTN gene knock-out and exogenous gene site-specific integration, thereby greatly enhancing the screening efficiency of the transgenic cell line. The method can screen the site-specific integrated exogenous gene cell line without adding any selection marker, thereby greatly enhancing the safety of the transgenic animals and having important value for sheep genetic breeding.

The invention discloses a specific target site for site-directed knockout of gene Myostatin (MSTN) by zinc finger nuclease (ZFN). The invention provides a DNA molecule, which can be as shown in the following (1) or (2) or (3): (1) is the DNA molecule shown in sequence 1 of a sequence table; (2) is the DNA molecule which hybridizes with a DNA sequence restricted by (1) under a strict condition and encodes an associated protein with the same function. Experiments of the invention prove that, the invention positions a specific target sequence site DNA fragment in the MSTN gene and constructs ZFN plasmids to the specific site, and nucleofaction of the plasmids to porcine embryonic fibroblasts can realize site-directed knockout or mutation of the specific target sequence in the MSTN gene, and can thoroughly destroy the expression of the MSTN gene.

The invention discloses a double gRNA, a double gRNA library, and a double gRNA vector library, a preparation method and applications thereof. The double gRNA is formed by sequentially linking a first promoter, gRNA-a, a crRNA scaffold sequence, a second promoter, and gRNA-b. The double gRNA library comprises a plurality of double gRNAs for a target gene. The double gRNA vector library is formed by linking the double gRNA library and a vector. According to the present invention, in the double gRNA, the independent expressions of the gRNA-a and the gRNA-b are controlled respectively by different promoters, and the gRNA-a and the gRNA-b are respectively complemented and paired with two target sites on the target gene, such that the double gRNA can recruit the Cas9 nuclease to cleave the target gent at the two sgRNA action sites so as to effectively improve the knockout efficiency of the target gene.

The present invention provides a gene knockout vector, a method for preparing a CD13 knockout porcine fibroblast or gene-edited pig, and the CD13 knockout porcine fibroblast prepared by the method. The gene knockout vector includes a gene editing vector backbone and a DNA segment linked to the vector backbone, and the vector backbone is selected from CRISPR / Cas9, CRISPR / Cas9n, CRISPR / Cpf1 or CRISPR / C2c2. The nucleotide sequence of the DNA segment is as shown in any one of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO: 3, preferably as shown in the SEQ ID NO:1. CD13 gene can be rapidly and accuratelyknocked out by the gene knockout vector and the method to obtain the CD13 gene homozygous knockout porcine fibroblast and gene-edited pig without an exogenous marker gene, a research platform is provided for porcine epidemic diarrhea, and the CD13 gene homozygous knockout porcine fibroblast and gene-edited pig have very high breeding value for disease resistance.

The invention discloses a novel method for efficiently knocking out low-transfection-efficiency cells on the basis of lentivirus expression CRISPR / Cas9. The method is characterized in that the method relates to two lentiviruses which include a lentivirus for expressing sgRNA and Cas9 and a lentivirus for expressing Cre protein; the multigene sgRNA and Cas9 are simultaneously cloned to a position between two same-direction loxp of a lentivirus vector, simultaneous multigene knockout in the low-transfection-efficiency cells is achieved, the Cas9 and sgRNA integrated in a genome are effectively removed by using the lentivirus expressing Cre, and an off-target effect caused by Cas9 and sgRNA overexpressing is prevented. The method has the advantages that the method combines the features that the lentiviruses can effective infect cleavage and non-cleavage cells and an loxp-Cre system can perform efficient removing reaction, and the system is low in toxicity, high in gene knockout efficiency, high in accuracy, short in cycle and significant to the realizing of multigene knockout in a low-transfection-efficiency cell line.

The invention relates to a design method of an sgRNA and a lentivirus carrier formed by the sgRNA and plasmids. In the method, the design length of the sgRNA is 17bp or 18bp, and the tail end of the sgRNA is provided with a G or A; the target sequence is (N16)GNGG, (N16)ANGG, (N16)CNCC or (N16)TNCC; when a first basic group is A, C or T, g is additionally arranged in front of the first basic group. Whether a similar sequence exists in a gene group is searched in a TagScan database; when the sgRNA with the length of 17bp is additionally provided with a completely-matched sequence, the sgRNA is abandoned, and other appropriate sequences are tested; for the sgRNA which is 18bp long, when only one non-matched nucleotide is provided, the sgRNA is also abandoned, and other sequences with the designed sgRNA are continuously tested. The method for designing the sgRNA is optimized, the high knock-out efficiency and low target missing or no target missing can be realized.

The invention discloses an MCIR (MelanoCortin 1 Receptor) gene carrier and a construction method thereof. The MCIR gene carrier comprises MC1R-neo and MC1R-GFP (Green Fluorescent Protein), wherein the nucleotide sequence of the MC1R-neo is SEQ ID NO:1, and the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2. A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system is adopted to construct an MC1R gene knock-out vector, and only a sgRNA (Ribonucleic Acid) of which the length is about 20bp needs to be designed by aiming at a gene knock-out point to be connected with a universal Cas9 gene. Therefore, compared with traditional gene knock-out technologies including ZFNs (Zinc finger nuclease), TALENs (Transcription Activator-Like Effector Nucleases) and the like, the construction method disclosed by the invention adopting the CRISPR / Cas9 to construct the gene knock-out vector is simpler and more convenient and is more convenient to be further popularized and applied to subsequent experiments.

The invention discloses a Pichia pastoris gene knockout and resistance gene recovery carrier, and a construction method and application thereof, and belongs to the technical field of microbial geneticengineering. A template carrier of the Pichia pastoris gene knockout comprises a mazf gene expression cassette regulated by a methanol type promoter AOX1 of the Pichia pastoris, specific recombinaserecognition sites of lox71 and lox66 at two ends and a bleomycin resistance gene expression cassette; the Pichia pastoris gene resistance gene recovery carrier comprises the mazf gene expression cassette regulated by the methanol type promoter AOX1 of the Pichia pastoris, a cre gene expression cassette constitutively expressed by using a GAP promoter and a HygB resistance gene expression cassette.The Pichia pastoris gene knockout and the resistance gene recovery carrier and the construction method thereof greatly improve the knockout efficiency of a Pichia pastoris gene. The invention provides a method for recovering antibiotic markers after a gene knockout is conducted, the method for recovering the antibiotic markers after the gene knockout is conducted provides great convenience for efficient knockout of multiple genes, and has high knockout efficiency, and a used homology arm is short.

The invention discloses a method for knocking out a cytidine deaminase (cdd) gene in escherichia coli by utilizing a CRISPR-Cas9 technology and application. The method comprises the following steps: 1, making escherichia coli BL21 (DE3) competence containing a Cas9 plasmid; 2, designing and synthesizing a mutant sgRNA; 3, constructing Donor DNA; 4, transforming the sgRNA plasmid and the Donor DNAinto the escherichia coli competence carrying the Cas9 plasmid by employing an electro transformation method, and knocking out the cdd gene; and 5, introducing a target plasmid pET28a-UCK into a knock-out strain to produce cytidylic acid. The method has the advantages that operation is simple, the knockout success rate of the cdd gene is high, and the conversion rate of the knock-out strain on a substrate is improved, and is suitable for industrial production of the cytidine acid and the like. Compared with producing the cytidylic acid without strain knockout, producing the cytidylic acid withstrain knockout improves the conversion rate of the substrate cytidine and adenosine triphosphate (ATP) by 15%, and the conversion rate reaches 99%.

The invention discloses a paired sgRNA for gene editing and an application thereof. A plurality of sgRNAs are designed according to the specific editing requirements of a target gene or a target genomic site, and two sgRNAs with specific spacing and corresponding PAM specific position combinations are selected as paired sgRNAs. The paired Cas9-sgRNA of the invention can improve gene editing efficiency and precision, and can be used for quantitative analysis of NHEJ in cells and animals, which includes total NHEJ efficiency (ie, total editing efficiency), frequency and proportion of precise NHEJ and mutant NHEJ, addition and deletion direction, length and frequency of a mutant NHEJ interface, and the availability of micro-homologous sequences of the mutant NHEJ interface. The technology ismore flexible, simple, and fast, the paired sgRNA can be easily transplanted into various cells and tissues, and the accuracy is not affected.

The invention discloses a construction method for a high-gene-knockout-efficiency Aspergillus chevalieri var. intermedius mutant engineering bacterial strain. The construction method includes cloning the 3' end DNA segment and the 5'end DNA segment of the ku70 gene, constructing a knockout vector for the ku70 gene, performing agrobacterium tumefaciens-mediated transformation, and indentifying a ku70 gene knockout mutant strain. Compared with the parent Aspergillus chevalieri var. intermedius mutant, the mutant engineering bacterial strain obtained by the invention has a slightly slow grow rate, no significant change in other phenotypes and genetic stability, and can be used as a receptor strain to perform knockout verification of gene functions. When the mutant engineering bacterial strain obtained by the invention is used as the receptor strain, the gene knockout efficiency is high, the gene knockout mutant strain can be obtained easily and heavy screening work is reduced.

The invention discloses a method for increasing S-adenosyl-L-methionine yield by saccharomyces cerevisiae genetic engineering. The method includes: replacing a GLC3 (glycogen branching enzyme gene) allelic gene on a chromosome of a saccharomyces cerevisiae strain with a G418 resistance gene according to a gene replacement method, and using a spore isolation method for obtaining a haploid with the gene GLC3 replaced only, so that a homozygote with gene GLC3 mutated is obtained. According to fermentation in 10L and 500L fermentation tanks, yields of ademetionine produced with mutant strains reach 7.93g / L and 8.35g / L and are increased by 15.1% and 24.7% respectively as compared with that of ademetionine produced with original strains.

The invention relates to a construction method of an immunodeficiency rat animal model and an application. The method comprises the following steps of separately constructing sgRNA expression vectorsof the 2nd exon of a rat Rag 1, the 3rd exon of an Rag2 gene, and the 1st exon of the I12rg, through in vitro transcription expression, obtaining rats of which the Rag1 gene and the Rag2 gene are knocked out, obtaining rats of which the I12rg gene is knocked out, and performing hybridizing, so as to obtain the rats of which the Rag1, the Rag2 and the I12rg are all knocked out. The rats SD-RG constructed by the method have immunodeficiency phenotype, transplant of various tumors is convenient, quick growth is promoted, and the method can be widely applied to fields of a humanized animal model,stem cells and tumor research and the like.

The invention belongs to the field of gene engineering, and particularly relates to SgRNA for targeted knockout of an RPSA gene and a construction method of an RPSA gene knockout cell line. Accordingto the sgRNA of a specific targeted RPSA gene, the sgRNA can specifically target an RPSA gene, the complete knockout of the RPSA gene in a host cell is realized by applying a CRISPR-Cas9 technology, and the knockout efficiency is high; the invention also provides a method for transfecting the sgRNA into a host cell by virtue of a CRISPR-Cas9 technology to construct an RPSA gene knockout cell line.Knocking out the RPSA gene in the host cell not only can promote replication of the FMDV virus, but also can be used for improving the production quantity and antigen expression quantity of the FMDVvaccine; in addition, it is accidentally found that by knocking out the RPSA gene in the host cell, replication of the Seneca valley virus in the cell can be remarkably inhibited; a research tool anda material are provided for further researching a molecular mechanism of the RPSA gene for regulating and controlling pathogenic microorganism replication in cells.

The invention relates to the field of biotechnology, and in particular, relates to a method for knocking out a pig GOT1 gene by a CRISPR / Cas9 system. The invention provides an sgRNA specifically targeting to the pig GOT1 gene, a CRISPR / Cas9 gene knock-out vector containing the sgRNA, a method for knocking out the pig GOT1 gene by the vector, and a detection method for the pig GOT1 gene. The CRISPR / Cas9 knockout vector provided by the invention can achieve high efficiency of the pig GOT1 gene knockout, significantly improve the construction efficiency of GOT1 gene knockout cells and GOT1 gene knockout pigs, and provide an effective method and basis for the function research and application of the pig GOT1 gene.

The invention relates to a preparation method of a rrbp1 gene knock-out xenopus tropicalis model, and an application. The preparation method comprises the steps of selecting a rrbp1 gene knock-out target point, designing sgRNA, synthetizing a double-stranded DNA fragment, constructing a sgRNA expression vector, performing in vitro transcription to obtain the sgRNA, mixing the sgRNA with Cas9 proteins, performing microinjection into fertilized eggs, and performing screening so as to obtain the rrbp1 gene knock-out xenopus tropicalis model. According to the preparation method disclosed by the invention, a reliable genetic modification animal model is provided for clarifying the functions of a rrbp1 gene in xenopus tropicalis, and a powerful research tool is provided for further studying tumor and cardiovascular diseases related to the rrbp1 gene based on the model; and besides, the method disclosed by the invention provides technical support for innovation of xenopus tropicalis mode animal germplasm resources.

The invention relates to a method to knock out goat EDAR (ectodysplasin-areceptor) gene via CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system mediation; the method comprises: constructing two gRNA expression vectors based on CRISPER-Cas9 according to goat's EDAR gene sequences, transferring optimized CRISPER-Cas9 vectors and the constructed gRNA expression vectors jointly into fibroblasts of a goat fetus to obtain goat cells with EDAR gene knocked out. The CRISPER-Cas9-mediated targeting vectors constructed herein provide a simple, quick and safe means to knock out goat EDAR gene. The method involves no selection marker genes during cell line screening, the safety of transgenic animals is improved greatly, and the method is of great value to the genetic breeding of goat and genetic function studies.

In order to solve the problems that pig Fah and Rag2 double genes are low in knockout efficiency and difficult to screen, the invention provides a target sequence of a group of Fah and Rag2 double genes and application of the target sequence in CRISPR-Cas9 specific knockout of the pig Fah and Rag2 double genes. The invention also provides a method for knocking out pig Fah and Rag2 double genes byusing CRISPR-Cas9. Through verification, the porcine Fah and Rag2 double genes can be effectively knocked out, the double knockout efficiency is as high as 50%, and the screening is very simple.