40 results about "Gene knockout technology" patented technology

The invention provides an IbFLZ9 gene and application thereof in regulation and control of growth of sweet potato vines, and belongs to the technical field of gene engineering and sweet potato breeding. The invention provides an IbFLZ9 gene for regulating and controlling growth of sweet potato vines. The nucleotide sequence of the IbFLZ9 gene is shown as SEQ ID NO. 1. Growth of sweet potato vines can be remarkably inhibited by overexpressing the IbFLZ9 gene, and growth of sweet potato vines can be remarkably promoted by inhibiting expression of the IbFLZ9 gene through a gene knockout technology. A new sweet potato variety which is moderate in vine length and convenient to mechanically harvest can be created by regulating and controlling the expression abundance of the IbFLZ9 gene, and a new gene resource and a new breeding technology are provided for improving the characters of the overground part of the sweet potato.

The invention relates to the technical field of biomedicine, in particular to a novel target spot for resisting chikungunya virus infection and application. According to the invention, human colon cancer cells (HCT116) are taken as target cells, and retinoic acid-induced protein expression of the target cells is reduced by adopting a gene knockout technology, so that host factors capable of effectively inhibiting CHIKV infection of the human colon cancer cells are found, and the purpose of blocking CHIKV infection from the source is achieved. It is found that retinoic acid induced protein 16 (RAI16) plays an important role in CHIKV infected HCT116 cells, and CHIKV infection can be obviously promoted by down-regulating expression of RAI16. The invention provides an application of RAI16 in preparation of a medicine for preventing or treating chikungunya virus infection, and provides a new target spot and a treatment scheme for clinically preventing and treating fever, arthralgia, arthrocele, muscular pain, headache, nausea, fatigue, rash and other diseases caused by CHIKV infection.

The invention relates to the technical field of gene engineering, in particular to SgRNA for targeted knockout of an MINOS1 gene and application of the SgRNA. According to the invention, a CRISPR / Cas9 gene knockout technology is adopted, a LentiCRISPR-V2 vector is constructed by using a CRISPR / Cas9 system, the LentiCRISPR-V2 vector is co-transferred into a porcine small intestine epithelial cell to obtain a positive cell clone, an MINOS1 gene in the positive cell clone is not expressed, the cell death rate under a hypoxia condition can be obviously reduced, and the positive cell clone has hypoxia tolerance. By regulating and controlling MINOS1 expression, the adaptive capacity of an organism to a low-oxygen environment can be enhanced, and a new thought is provided for preventing and relieving altitude stress and improving physiological tolerance in an extreme environment; besides, the hypoxia characteristic of the tumor microenvironment is a key factor causing chemoradiotherapy drug resistance, and the MINOS1 knockout related strategy can reverse the hypoxia tolerance phenotype of tumor cells, so that the sensitivity of the tumor cells to treatment is enhanced, and an innovative direction is provided for overcoming tumor drug resistance.

The application discloses a gene prdR for regulating myxobacteria predation and a protein coded by the gene, and belongs to the technical field of biotechnology. The mutant DK10 with significantly improved predation capacity is obtained by using the strategy of adaptive evolution; genetic variation sites of the mutant DK10 are analyzed by using the bacterial genome resequencing technology, and functions of the genetic variation sites are researched by using the gene knockout technology, and it is found that the gene deletion of MXAN_2902 (prdR, the nucleotide sequence is shown as SEQ ID NO. 1) significantly enhances the predation capacity of Myxococcus xanthus. The nucleic acid sequence and the amino acid sequence of the transcriptional activator PrdR for regulating the predation of Myxococcus xanthus are disclosed, and the functional strain of Myxococcus xanthus capable of efficiently predating Salmonella enteritidis can be developed by using the prdR gene, and a new strategy is provided for the prevention and control of drug-resistant HVPG pathogenic bacteria.

The invention relates to the technical field of genetic engineering breeding, in particular to application of ZmC2DP1 protein in regulation and control of drought resistance and salt tolerance of plants, the amino acid sequence of the ZmC2DP1 protein is as shown in SEQ ID NO.1, and the nucleotide sequence of a gene for coding the ZmC2DP1 protein is as shown in SEQ ID NO.2. After the ZmC2DP1 gene is knocked out, the drought resistance and the salt resistance of a mutant plant under the drought treatment or salt treatment condition are improved compared with those of a control group plant, and it is verified that the ZmC2DP1 protein has a negative regulation function on the drought resistance and the salt resistance of the plant. Compared with a traditional breeding mode, the method for regulating and controlling the drought resistance and the salt resistance of the plants has the advantages that the breeding time is short, the purposiveness is high, the drought resistance breeding period is remarkably shortened, and the crop stress resistance breeding efficiency is improved; a gene editing homozygous strain with improved drought resistance and salt tolerance is obtained by adopting a gene knockout technology, and gene resources are provided for cultivating and improving new varieties of stress-resistant plants; and a theoretical basis is provided for clarification of a molecular mechanism of the ZmC2DP1 protein in drought resistance and salt resistance of plants.

The invention belongs to the technical field of aquaculture biological growth, and particularly relates to a gene for regulating fish growth, application and a regulating method. The gene for regulating the growth of the grass carp is a myogenesis inhibition gene mdfi of a myogenesis regulatory factor (MRFs) family; the base sequence of the gene is shown in SEQ ID NO: 1. The invention discloses a method for promoting proliferation of fish muscle cells. The expression level or protein activity of mdfi genes in grass carp muscle cells is inhibited. The inhibiting effect of the mdfi gene in the muscle growth of the grass carp is systematically clarified for the first time, the individual growth of the grass carp can be effectively promoted by establishing an mdfi gene knockout technology, and a new technical means and a new gene target are provided for the genetic breeding of the grass carp.

The invention provides a construction method and application of an IgM gene knockout zebrafish antibody deletion model, and belongs to the technical field of zebrafish model construction.The method is based on a CRISPR gene knockout technology and specifically comprises the following operation steps that gRNA is synthesized for a CRISPR target located in a CH2 exon region of a zebrafish IgM gene, the gRNA and Cas9 mRNA / Cas9 protein are introduced into a wild type zebrafish embryo, and the zebrafish antibody deletion model is obtained. The P0-generation mutant and wild type zebra fish are hybridized to generate a heterozygote F1 generation, and then the heterozygote F1 generation is selfed to obtain a homozygote mutant, namely the IgM gene knockout zebra fish antibody deletion model. The invention provides the first IgM gene knockout zebrafish antibody deletion model, which is beneficial to exploring the biological function of the IgM gene and determining the influence of immunoglobulin antibody M synthesis disorder on the immune response of the zebrafish, and has application value for rapidly screening and evaluating vaccine adjuvant drugs and natural efficacy factors.

The application discloses a method for creating polyploid fish, which comprises the following steps of: knocking out a spo11 gene by using a CRISPR / Cas9 gene knockout technology, obtaining a spo11 homozygous knockout individual through genotyping and breeding, utilizing the characteristic that a spo11 homozygous knockout diploid female can produce part of unmeiotic oocytes to obtain a triploid, and further screening a spo11 homozygous knockout triploid 3N spo11 ‑ / ‑ / ‑ ; utilizing the characteristic that a 3N spo11 ‑ / ‑ / ‑ female can produce part of unmeiotic oocytes to further screen a heterozygous knockout tetraploid 4N spo11 + / ‑ / ‑ / ‑ ; utilizing the characteristic that a 4N spo11 + / ‑ / ‑ / ‑ can produce a large number of diploid gametes to further prepare a large number of triploid offspring. Compared with other polyploid breeding methods, the application has universality and genetic controllability, and provides important technical support for artificially inducing fish polyploidy.

The invention discloses an application of a tomato histone variant H2A. Z in improving low-temperature resistance of tomatoes, and is characterized in that the histone variant H2A. Z is coded by three genes of S1HTA8, S1HTA9 and S1HTA11, the nucleotide sequence of the S1HTA9 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the S1HTA11 is as shown in SEQ ID NO: 2. According to the application, the genes of the S1HTA9 and the S1HTA11 for coding the histone H2A. Z are knocked out through a gene knockout technology, and the tomato histone variant H2A. Z is obtained. The low-temperature resistance of the tomatoes is obviously improved. The invention discovers that the histone variant H2A. Z participates in regulating the low-temperature resistance of plants for the first time, provides a new gene resource for cultivating a new variety of low-temperature-resistant tomatoes, and has important potential application value.

The invention relates to the technical field of genetic engineering breeding, and discloses application of a protein ZmCHR17 or an expression substance for regulating and controlling a protein coding gene or a substance for regulating and controlling the activity or content of the protein in regulating and controlling the drought resistance of plants, the amino acid sequence of the protein is as shown in SEQ ID No.1, and the nucleotide sequence of the coding protein is as shown in SEQ ID No.2. After the ZmCHR17 gene is knocked out, the drought resistance of a mutant plant is improved compared with that of a wild plant under the drought treatment condition, and it is verified that the ZmCHR17 protein has the negative regulation function on the drought resistance of the plant. The invention also discloses a method for improving the drought resistance of plants, which comprises the step of knocking out the coding gene of the ZmCHR17 protein in a receptor plant to obtain a target plant with the drought resistance higher than that of the receptor plant. Compared with a traditional breeding mode, the method for regulating and controlling the drought resistance of the plants has the advantages that the breeding time is short, the purposiveness is high, the drought resistance breeding period is remarkably shortened, and the crop stress resistance breeding efficiency is improved; a gene editing homozygous strain with improved drought resistance and salt tolerance is obtained by adopting a gene knockout technology, and gene resources are provided for cultivating and improving new varieties of stress-resistant plants; and a theoretical basis is provided for clarification of a molecular mechanism of the ZmCHR17 protein in plant drought resistance.

The present invention discloses a method for constructing and applying a gene knockout non-human animal model. The model is constructed based on CRISPR / Cas9 gene knockout technology. The construction method comprises the following steps: Step 1: Designing sgRNA and Cas9 RNA, transcribing them into mRNA in vitro, and microinjecting the active sgRNA and Cas9 RNA into fertilized eggs of non-human animals to obtain a gene knockout non-human animal; Step 2: Identifying the gene knockout non-human animal model. The advantages of this method are as follows: Using CRISPR / Cas9 gene knockout technology, the present invention establishes the first gene knockout non-human animal model for Cry Me a Cat syndrome, providing a convenient and reliable animal model for studying and screening drugs for treating Cry Me a Cat syndrome.

The invention belongs to the technical field of oilseed rape genetic improvement and genetic engineering, and discloses application of a brassica napus BnaUGE2 gene in regulating and controlling the flowering period of oilseed rape. The BnaUGE2 gene has four homologous copies, and the four homologous copies are respectively as shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7. Experiments prove that the BnaUGE2 gene is over-expressed under the background of brassica napus B409, so that a plant can flower in advance, and the pod character is not influenced; on the contrary, the activity of the gene is inhibited through a gene knockout technology, so that flowering delay, pod length increase and grain number reduction of each pod can be caused. On the basis, through expression regulation of the BnaUGE2 gene, fine regulation of the flowering period of the rape can be realized, and new germplasm and gene resources are provided for breeding of a new variety of early-maturing rape.

The invention provides a method for increasing salt tolerance of zebrafish through a gene knockout technology and application, belongs to the technical field of gene engineering and molecular breeding, utilizes a gene editing technology and provides proper gRNA, a circpb gene can be directly knocked out, the zebrafish with salt tolerance is obtained, and the method is expected to be popularized to economic fish.

The invention discloses a method for increasing protein content and amino acid content of phaeodactylum tricornutum and application. According to the method, a DPH gene in wild type phaeodactylum tricornutum is knocked out through a gene knockout technology, so that the total protein content and the amino acid content of the phaeodactylum tricornutum are increased, a method for knocking out the DPH gene in the wild type phaeodactylum tricornutum comprises the steps that the DPH gene is knocked out in a targeted mode through a CRISPR / Cas9 editing carrier, the nucleotide sequence of gRNA of the DPH gene is knocked out in a targeted mode through the CRISPR / Cas9 editing carrier is shown as SEQ ID No.3, and the content of the total protein in the wild type phaeodactylum tricornutum is increased. The total protein content and the essential amino acid content of the DPH gene knockout phaeodactylum tricornutum strain are remarkably improved compared with those of a wild type strain. The transformed algal strain can be used as a high-quality protein source to be applied to the field of aquaculture, the nutritional value and the transformation efficiency of feed are effectively improved, and the growth performance and the health condition of bred animals are improved. Wide market prospects and important economic values are realized.

Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature mRNA sequence comprising the knockout guide RNA. An RNA-guided nuclease is used to excise the knockout guide RNA from the mRNA transcript, which then guides the RNA-guided nuclease to a second target locus where the RNA-guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the mRNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked.

The present invention uses gene knockout technology to construct a high-yield poly (γ-glutamic acid) (γ-PGA) engineered strain of Bacillus paralicheniformis that is independent of exogenous glutamate addition. By seamlessly knocking out the carbon metabolism repressor protein A gene on the genome of Bacillus paralicheniformis 285-3, which regulates carbon metabolism, ccpA , enabling the strain to enhance carbon metabolism pathways, increase the synthesis of the substrate glutamate, and ultimately enhance the synthesis of γ-PGA. The engineered Bacillus paralicheniformis strain constructed using this technology significantly increased γ-PGA production, paving the way for the subsequent use of this engineered strain to produce γ-PGA in a manner independent of exogenous glutamate addition. Compared to the starting strain 285-3, its γ-PGA production rate is higher, effectively saving costs such as labor, energy, and equipment usage and loss, thereby further reducing the production cost of γ-PGA.

The application discloses a kind of circSamd4 gene knockout mouse model construction method and application.CircSamd4 gene knockout mouse animal model of the construction method of the application includes the following steps: using sgRNA to knock out the gene of circSamd4 of mouse, obtains circSamd4 gene knockout mouse.The application uses Crispr-cas9 gene knockout technology, and knocks out mouse circRNA gene circSamd4, and the obtained mouse all shows obvious glaucoma phenotype such as thinning of retina thickness, retinal ganglion cell (GCL) loss, visual function damage.Can provide simple, reliable, economic animal model for the research of glaucoma pathogenesis, drug and clinical treatment strategy screening.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) gene editing vector targeting a wheat TaSnRK1 alpha gene and application of the CRISPR / Cas9 gene editing vector. PBUE414 is used as a skeleton vector of the vector, and the vector comprises a Cas9 expression cassette and an sgRNA (single guide ribonucleic acid) expression cassette; the Cas9 expression cassette is an expression cassette for regulating and controlling Cas9 expression by a Ubi promoter, and the sgRNA expression cassette sequentially comprises the following elements from upstream to downstream: a TaU3 promoter, sgRNA1 of TaSnRK1 alpha, a terminator OsU3t, a TaU3 promoter, sgRNA2 of TaSnRK1 alpha and a terminator TaU3t. The vector is suitable for reducing the high-molecular-weight glutelin content of wheat mature grains through a gene knockout technology, and a new technical means and germplasm resources are provided for wheat quality improvement.

The present invention discloses an sgRNA for targeted knockout of APP gene and its application, belonging to the field of animal genetic engineering. By using the CRISPR / Cas9 gene knockout technology, the sgRNA sequence is linked into the PX459 vector, and the PX459-APP targeting vector is transferred into porcine fibroblasts. After resistance screening and PCR sequencing identification, positive cell clones are obtained, and then cloned embryos are obtained through somatic cell nuclear transfer technology; the cloned embryos are transferred into the pig uterus for pregnancy to obtain cloned pigs. The present invention constructs APP gene knockout pigs for the first time, and makes the APP gene not express to obtain ASFV resistance, laying a foundation for the research on breeding against African swine fever.

The invention discloses a gene engineering bacterium for high yield of corac acid and a construction method and application thereof, and belongs to the technical field of gene engineering, the construction method comprises the following steps: sequentially knocking out a waaL gene cluster, a lon gene, an hns gene, a clsA gene, a clsB gene, a clsC gene, a mcbR gene and an opgDGH gene on an escherichia coli E.coli MG1655 genome, and then sequentially overexpressing an rcsA gene, a galU gene, a manA gene and a cpsG gene. According to the invention, through a multiple gene knockout technology and in combination with genome site-specific integration of an exogenous gene and an enhanced expression element, efficient and stable regulation and control of a corac acid synthesis pathway are realized, and the obtained strain has the capacity of high yield of corac acid and has high industrial application potential.

The invention relates to the technical field of D-ribose production, and provides a D-ribose production process which comprises the following steps: step 1, construction of engineering bacteria; step 2, fermentation and dynamic control of engineering bacteria; step 3, efficiently extracting and refining fermentation liquor; the first step comprises the steps of strengthening D-ribose synthesis, knocking out or weakening a byproduct synthesis route and introducing efflux transporter; the second step comprises the steps of inoculating the engineering bacteria into a seed culture medium, and culturing to the middle stage of the logarithmic phase; mixing the flora with the fermentation liquor for fermentation, and carrying out dynamic feeding control and dissolved oxygen segmented control; the third step comprises fermentation liquor pretreatment, membrane decoloration and desalination, continuous vacuum membrane crystallization and drying. A synthesis path of byproducts is blocked through a gene knockout technology, so that the content of acetic acid in fermentation liquor is controlled to be 1.0 g / L or below, the stability of a fermentation system is remarkably improved, a raw material system in the subsequent separation and refining process is directly optimized due to the great reduction of the byproducts, and the impurity separation difficulty is reduced.

The invention discloses a traceless gene knockout technology for indigenous bacillus velezensis difficult to genetically manipulate. The invention belongs to the technical field of biology, and particularly relates to a traceless gene knockout technology for indigenous bacillus velezensis difficult to genetically manipulate. The bacillus velezensis traceless gene knockout method comprises the following steps: 1) constructing a temperature-sensitive knockout plasmid, and carrying out in-vitro methylation on the temperature-sensitive knockout plasmid to obtain an in-vitro methylated temperature-sensitive knockout plasmid; (2) transforming bacillus velezensis competent cells by using the plasmid subjected to in-vitro methylation to obtain a positive transformant, and (3) carrying out homologous single exchange twice on the positive transformant obtained in the step (2), and screening to obtain a strain of which the target gene is knocked out.

The invention discloses a brown planthopper resistant gene BPH50 of rice and application of the brown planthopper resistant gene BPH50. The method comprises the following steps: carrying out GWAS analysis on brown planthopper resistance characters of a large number of rice varieties in the seedling stage by utilizing miniature core germplasm resources in a rice germplasm bank of the Ministry of Agriculture of America, screening out significant sites which are distributed on a No.6 chromosome and have the maximum insect-resistant effect, and comparing candidate genes in an NLR gene cluster where the sites are located, finally, the novel NLR gene ORF9 of Pi9, 920 and the NLR gene ORF9 of which no allele is detected in a Nipponbare reference genome are determined as candidate genes. A CRISPR-Cas9 gene knockout technology is utilized to find that an insect-resistant variety 551 can lose brown planthopper resistance by knocking out the ORF9 gene, so that the function of the ORF9 in brown planthopper resistance of rice is verified, and the ORF9 is named as BPH50. The invention provides a novel functional gene resource and a molecular tool for insect-resistant breeding of rice, and has important agricultural application value.

The invention discloses an attenuated vaccine for preventing toxoplasmosis and a preparation method, the attenuated vaccine for preventing toxoplasmosis is prepared by using a CRISPR-cas9 gene knockout technology, attenuated toxoplasmosis can be bred in a host in a limited manner, a natural infection process is simulated, cellular immunity and humoral immunity are effectively stimulated, and more comprehensive protection is provided; compared with an inactivated vaccine or a subunit vaccine, the attenuated vaccine can induce more lasting immunity; the attenuated strain contains a plurality of antigens, and can provide cross protection for parasites of similar species or different developmental stages; a CRISPR-cas9 gene knockout technology is used for preparation, complex antigen purification or adjuvants are not needed, and the method is suitable for being popularized in areas with limited resources; by immunologically preventing toxoplasmosis, the use of toxoplasmosis resisting drugs can be reduced, and the problem of drug resistance can be relieved.

The present invention discloses the use of OsDSK2a protein or its encoding gene in regulating rice blast resistance. The amino acid sequence of the OsDSK2a protein is shown in SEQ ID NO. 2. The present invention proves for the first time that the rice DSK2a gene (Os10g0542200) is a functional gene for rice blast susceptibility. The cloning and biological function verification of this gene are of great reference significance for the study of the molecular mechanism of rice blast resistance. The present invention provides a Cas9-mediated Os10g0542200 gene editing vector. After transformation of rice with this vector, the expression level of Os10g0542200 can be significantly reduced. With the reduction in expression level, the susceptibility of the transformed plants to rice blast is reduced and the disease resistance is significantly enhanced. In addition, the transgenic plants do not show obvious changes in growth status and agronomic traits. The Cas9-mediated Os10g0542200 gene knockout technology of the present invention can be applied to rice genetic engineering breeding and can be applied in production practice to improve rice blast resistance, thereby ensuring rice production safety under current climatic conditions where rice diseases frequently occur.

The invention provides a construction method and application of a KIF5C gene knockout animal model, and relates to the technical field of biological medicines. The sgRNA for knocking out the KIF5C gene is characterized in that the sequence of the sgRNA is selected from at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The invention further discloses a preparation method of the sgRNA for knocking out the KIF5C gene. The KIF5C gene knockout animal model disclosed by the invention can be used as an animal model for depression and disease research, the KIF5C gene knockout animal model is prepared by adopting a gene knockout technology, and animal behavioral detection shows that a mouse has depression-like symptoms (a sweet water preference experiment and a forced swimming experiment) and memory dysfunction (a Y maze experiment and a conditional fear experiment); the compound has depression symptoms, can be reversed through an antidepressant drug ketamine, and can be used for researching depression animal models and developing corresponding drugs.

The invention relates to the technical field of gene editing, in particular to a gRNA composition of a targeted CYP392E1 gene for preventing and treating tetranychus urticae and application of the gRNA composition. The CYP392E1 gene of which the nucleotide sequence is shown as SEQ ID NO.1 and any one of the following applications of gRNA of which the nucleotide sequence is shown as SEQ ID NO.2: 1) preventing and treating tetranychus urticae, and / or preparing a medicine for preventing and treating tetranychus urticae; and 2) reducing the resistance of the tetranychus urticae to the acaricide, and / or preparing a drug for reducing the resistance of the tetranychus urticae to the acaricide. The gRNA is designed by applying a gene knockout technology, especially aiming at a detoxification enzyme gene for regulating detoxification metabolism in a pest mite body, a compound consisting of the gRNA composition and cas9 protein is delivered into a tetranychus urticae body by utilizing a microinjection technology, and the gRNA composition loses the function of a target gene, reduces the detoxification metabolism capability of a medicament, and can be used for treating the pest mite body. The resistance reversion of the two-spotted spider mites is promoted, so that the aim of preventing and treating the two-spotted spider mites is

The application belongs to the technical field of genetic engineering and plant genetic breeding, and particularly relates to application of a new tetraploid rice fertility gene NTFR2. The application uses a gene knockout technology to determine the function of the fertility regulation gene NTFR2 in the new tetraploid rice, and finds that the NTFR2 affects the fertility of the new tetraploid rice. When the function of the NTFR2 gene is lost, the seed setting rate of the mutant is reduced, and the development of the female gametophyte sac exhibits abnormality. Meanwhile, the loss of the function of the NTFR2 gene leads to a significant decrease in the fertility of the new tetraploid rice, but has a smaller influence on the male gametes and other agronomic traits, which indicates that the NTFR2 is a new gene mainly affecting the fertility of the tetraploid rice, and has an important application prospect in the fields of creating a new female sterile line and further promoting the utilization of hybrid vigor of the tetraploid rice.

The present application relates to the field of bioengineering, and particularly relates to a mutant strain and application thereof.The present application provides a mutant strain, wherein a tsa2 gene of a chassis strain is knocked out; the chassis strain is NT64C; and the sequence of the tsa2 gene is shown as SEQ ID NO:7.The present application obtains a mutant strain capable of promoting protein misfolding by using a gene knockout technology, and a protein misfolding model can be efficiently obtained by using the strain.

The invention discloses a universal CAR-T cell based on Cas9-T6N and application, and relates to the technical field of gene engineering and tumor immunotherapy, the universal CAR-T cell comprises the CAR-T cell, a TRAC gene of the CAR-T cell is knocked out, the CAR-T cell expresses a chimeric antigen receptor containing a CD8 alpha signal peptide, c-Met scFv, a 4-1BB costimulatory domain and a CD3 zeta active domain, the TRAC gene knockout efficiency of the CAR-T cell is larger than or equal to 65%, and the CAR-T cell is a CAR-T cell. The killing efficiency of the CAR-T cell on c-Met positive tumor cells is greater than or equal to 80%; through an innovative Cas9-T6N protein mediated TRAC gene knockout technology and in combination with an optimized electroporation delivery system and c-Met CAR structural design, a universal CAR-T cell with efficient gene editing ability and remarkable anti-tumor activity is successfully developed; the technical bottlenecks of graft-versus-host disease risk, insufficient gene editing efficiency and the like in the traditional allogenic CAR-T therapy are effectively overcome, meanwhile, the functional integrity of cells is ensured through reasonable costimulatory signal design, and a brand new technical path is provided for ready-to-use immunotherapy of solid tumors.