35results about How to "High mutation efficiency" patented technology

The invention discloses a method for site-specific mutagenesis of medicago sativa genes by employing a CRISPR/Cas9 system. First of all, a binary expression vector MsCRISPR/Cas9 is constructed; the vector is designed and constructed for a target gene; then, agrobacterium tumefaciens-mediated transformation is used to achieve site-specific mutagenesis of a specific gene, and a mutant transformed plant with the target gene knocked out is obtained by screening; the method uses the CRISPR/Cas9 technology for cross-pollination autotetraploid plant medicago sativa for the first time, and obtains a mutant plant; a MtU6 promoter is used for the first time to initiate sgRNA transcription in the medicago sativa, thereby improving the transcription efficiency; the mutation rate can reach 52%; the method realizes direct mutation on the coding sequence of the gene, and opens up a new field for gene editing in the medicago sativa, laying a technical foundation for simultaneous silencing of a plurality of genes, deletion of large chromosome fragments, precise insertion of foreign genes into targets, gene regulation and the like.

The invention relates to a site-directed mutagenesis method based on re-engineering. Firstly, a downstream segment, another resistant gene and a DNA (deoxyribose nucleic acid) modification segment are obtained by the aid of overlap-extension polymerase chain reaction, the downstream segment contains a left-end 50bp homologous arm, a mutagenesis site and a target gene mutagenesis site, the left-end 50bp homologous arm is consistent with an upstream 50bp sequence of a target site to be mutated, the target site to be mutated is cloned on a plasmid, the another resistant gene contains a resistant gene different from that of the plasmid, and the DNA modification segment with a right-end 50bp homologous arm is positioned on the downstream of a resistant gene of a carrier. Secondly, the integrated segment is electrically transformed in Escherichia coli expressing recombinase and containing the plasmid with a cloned target gene, and the target site with site-directed mutagenesis and recombinant clone with the resistant gene on the original plasmid replaced by the resistant gene carried on the modification segment are obtained by means of recombinase mediated homologous recombination.

The invention discloses a construction method for gene site-specific mutagenesis in an embryonic cell of a mouse. A gene site-specific mutagenesis mouse is constructed by introducing a specific nucleotide sequence into the embryonic cell of the mouse. The construction method comprises the following steps: confirming a target sequence in a target genome sequence of the mouse; constructing a TALEN nucleotide sequence capable of identifying and cutting a target gene, according to the sequence of the target sequence; introducing the TALEN nucleotide sequence into the embryonic cell of the mouse; and applying the obtained embryonic cell of the mouse to culture in vitro or directly implanting to a female mouse, expressing the TALEN and cutting the target genome, thereby selecting the gene site-specific mutagenesis mouse. The construction method provided by the invention has the advantages that a homologous recombined target carrier need not be constructed, the ES target cell selection is unnecessary, the mutant proportion is high, the operation is convenient, the test cost and period are greatly reduced, and the like.

The invention provides a CHO cell for protein display and expression, an expression cassette is integrated in a genome of the CHO cell, and the expression cassette is integrated behind a termination codon of a YWHAE gene of the genome of the CHO cell; the expression cassette comprises a promoter, a first recombinase recognition sequence, a target gene, a second recombinase recognition sequence anda terminator. In the cell, the fluorescent protein gene and the antibody gene can be highly transcribed and continuously and stably highly expressed under the condition of no antibiotic maintenance,and the antibody transcription level is remarkably improved compared with that of a CHO-puro cell strain established before. The experiments prove that the AID mutation efficiency of the site is significantly higher than that of a random insertion site of CHO-puro.

The invention discloses a method for producing coenzyme Q10 by combining plasma action with oxygen limitation, which comprises the following steps: uniformly covering the surface of a slide with a bacterial suspension, putting the slide in an ARTP mutagenesis instrument, and carrying out mutagenesis treatment and elution to obtain the mutagenized bacterial suspension; diluting the mutated bacterial suspension under an aseptic condition, coating the diluted bacterial suspension on a flat plate containing a flat plate culture medium added with anhydrous sodium sulfite, and culturing to obtain aflat plate with a single colony; picking single colonies from the flat plate with the single colonies, putting the single colonies into a 24-pore plate, adding a seed culture medium, covering the 24-pore plate with a cover, and culturing to obtain a seed solution; and fermenting to produce the coenzyme Q10. The rhodobacter sphaeroides production strain with improved specific growth rate, oxygen affinity and product synthesis rate can be obtained and screened by combining plasma action with an oxygen limitation model, and the fermentation level of the coenzyme Q10 is effectively improved.

The invention belongs to the technical field of animal genome editing and particularly relates to sgRNA for preparing a skeletal dysplasia swine model and application thereof, and aims to solve the technical problem that existing rat skeletal dysplasia animal models have greater difference from human in skeleton structure and function. The technical means provides the sgRNA for preparing the skeletal dysplasia swine model. The sgRNA recognizes a target site located on an editing gene of a swine fibroblast growth factor receptor 3 and having a nucleotide sequence of 5'-GAAAGCCCAGCCCGTAGCTGAGG-3'. On the basis of obtaining the sgRNA, the matched donor DNA is designed, an Fgfr3 c.1132G>A precise mutant swine model can be prepared efficiently, an FGFR p.G378R mutant swine system with cartilage dysplasia is cultured, and a large animal model closer to human is provided for skeletal development study and clinical tretament of skeletal diseases.

The invention discloses a method for screening high-yield cellulase filamentous fungi through self-adaptive mutagenesis and an obtained mutant strain, and particularly relates to mutation of Trichoderma harzianum. The method comprises the following specific steps: preparing a wild type African trichoderma harzianum spore suspension; carrying out three rounds of mutagenesis on the wild type African trichoderma harzianum; screening mutant strains; and determining the enzyme activity stability of the excellent mutant strain. The strain is mutagenized through three rounds of self-adaptive mutagenesis, so that the mutagenesis purposiveness is strong, and the practicability is high. According to the method disclosed by the invention, a mutant strain MEA-12 of which the cellulase activity is remarkably improved (FPA is improved by 3.17 times, CMCase is improved by 4.77 times, pNPCase is improved by 3.86 times and pNPGase is improved by 2.97 times) and the genetic character is stable is finally obtained through mutagenesis screening, and the mutant strain MEA-12 has important value in industrial cellulase preparation production.

The invention discloses a rice gene directional editing method based on pollen tube channel introduction, and belongs to the technical field of biology. The method is characterized in that by utilizing a pollen tube channel, a CRISPR/Cas9 vector is introduced into a rice embryonic sac, and directional editing of a tms5 target gene is realized. The gene directional editing method base on the pollentube channel does not depend on genetic transformation of calluses, target gene mutation can be obtained, and the operation process is simple and reliable.

The invention discloses a special gene therapy drug device comprising a body and a radiation cavity arranged in the body. A first turntable is rotatingly matched and connected in the radiation cavity.The bottom of the first turntable is in power connection with a first motor, and an outer surface of the first motor is embedded in an inner wall of the bottom of the radiation cavity and is fixedlyconnected to the radiation cavity. The top end surface of the first turntable is provided with a groove with an upward opening, left and right ends of the groove are provided with symmetrical first cavities, and the first cavities are provided with connected second cavities away from the inner wall of the groove. First rotating shaft extending left and right are rotatingly matched and connected between the groove and the first cavities, and one end of each of the first rotating shafts close to the groove extends into the groove and a first tooth rotating wheel is arranged at an end. The special gene therapy drug device has the advantages of a simple structure, convenient operation, convenient storage and high safety and reliability, and the production efficiency of gene drug is improved.

The invention discloses a method for preparing a single mutant on the basis of a SaKKHn-pBE system. The preparation method for the single mutant comprises the following steps: introducing a SaKKHn-pBEbase editing system into an organism or a biological cell, and editing a genome target sequence through the SaKKHn-pBE base editing system so as to obtain the single mutant, wherein the single mutantis a biological mutant of which a single site C in a target sequence is mutated into T; and the SaKKHn-pBE base editing system comprises a SaKKHn protein, tRNA-sgRNA and a PmCDA1 protein. Results ofa test on 23 rice genome target sequences through the method disclosed by the invention show that almost all targets can obtain single mutants of a unit point C with highest mutation efficiency, and the proportions of the single mutants mostly exceed 50%.

The invention discloses a sgRNA and use thereof in repairing abnormal splicing of introns. The sgRNA is used for repairing abnormal splicing of introns caused by a HBB (a beta-globin gene) IVS2-654 C>T mutation and comprises a sgRNA-U and a sgRNA-D. A targeting site of the sgRNA-U is located in a range from an upstream 21 bp of the IVS2-654 C>T site to a starting site of a second intron of the HBBgene where the IVS2-654 C>T site is located. A targeting site of the sgRNA-D is located in a range from a downstream 71bp of the IVS2-654 C>T site to a termination site of the second intron of the HBB gene where the IVS2-654 C>T site is located. An existing gene editing technology can be used for repairing a blood transfusion-dependent beta-thalassemia IVS2-654 C>T, has high editing efficiency, and can efficiently modify a persistent and balanced hematopoietic system for autologous hematopoietic stem cells.

The invention provides a large primer PCR site-specific mutagenesis method, which comprises the following operation steps: (1) designing primers according to a target gene sequence to obtain two flanking primers which are an upper flanking primer and a lower flanking primer; (2) designing a mutation primer according to the sequence position of the mutation base, wherein the mutation site is positioned in the middle of the mutation primer sequence; (3) carrying out a first-step PCR reaction on the mutation primer and the flanking primer far away from the mutation site to obtain a large primer;and (4) adding two flanking primers into the obtained large primer to carry out second-step PCR amplification so as to obtain a mutant target gene sequence. According to the method, when the mutationprimer is designed, the mutation site is designed in the middle of a mutation primer sequence, so that the mutation primer is favorably combined with a template in the subsequent PCR reaction, and themutation rate is improved.

The invention discloses an intron abnormal splicing repair method. The method is a technology for targeted knockout of an abnormal mutation site IVS2-654 C> T in beta-thalassemia (thalassemia) by using a CRISPR-Cas9 gene editing technology, and comprises the following steps: designing and synthesizing a guide RNA sequence (sgRNA) capable of identifying and guiding a Cas9 protein to a target sequence of a target gene; And electrically transferring and introducing an sgRNA and Cas9 protein mixture into beta-thalassemia IVS2-654 C>T hematopoietic stem cells to efficiently destroy an abnormal splicing mutation site, so that normal shearing and expression of a beta-globin gene are recovered. The blood transfusion dependent beta-thalassemia IVS2-654 C>T can be edited by utilizing the existing gene editing technology, the editing efficiency is high, and the edited hematopoietic stem cells of the patient can reconstruct a blood system of the patient and treat thalassemia diseases after autotransplantation.

The invention relates to the technical field of genetic engineering and molecular genetic breeding, in particular to a method for improving the CRISPR/Cas9 mediated biallelic mutation efficiency and an application thereof. A CRISPR/Cas9 gene editing method provided by the invention comprises the following steps of introducing gene editing nucleic acid into a prokaryotic embryo, wherein the gene editing nucleic acid comprises Cas9 mRNA and sgRNA, and in the gene editing nucleic acid, the molar concentration ratio of the Cas9 mRNA to the sgRNA is 1:10 to 1:20. According to the gene editing method provided by the invention, the efficiency of biallelic mutation is remarkably improved, so that the proportion of gene editing chimeric individuals is effectively reduced, and the proportion of biallelic mutation individuals is improved; and the gene editing method has important application value for construction and genetic breeding of animals with target traits.