36results about How to "High homologous recombination efficiency" patented technology

The invention discloses a CRISPR-Cas9 system used for assembling DNA and a DNA assembly method. The CRISPR-Cas9 system includes the following parts: a plasmid used for expressing Cas9 gene, a first guide RNA, and / or a plasmid used for expressing the first guide RNA, wherein the first guide RNA has a CRISPR site. The CRISPR site is combined with a first reporter gene carried by a semisynthetic chromosome used for assembling according to base complementation pairing rule. The CRISPR-Cas9 system has advantages of high replacement success rate of homologous recombination, less species of guide RNA which needs to design and use, less harmful effect subjected by the genomic sequence, and low off-target rate.

The invention discloses a method and application for improving application efficiency of a gene targeting technique in aspergillus terreus, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, by taking Aspergillus terreus as an initial bacterium, knocking off a ku80 gene or an lig4 gene so as to increase the exogenous DNA homologous recombination probability of a strain; secondly, establishing a pyrG gene deletion uracil auxotroph stain, establishing a inheritance conversion system based on a pyrG gene as a screening tag; and finally, cutting off the screening tag by using a Cre / LoxP specific binding site recombinant system, thereby obtaining a uracil auxotroph stain which can be applied to genetic modification again. By adopting the method disclosed by the invention, an efficient aspergillus terreus gene targeting platform can be established, the method has the advantages that high homologous recombination efficiency can be achieved, the bidirectional screening of the conversion system can be achieved, a screening tag cutting method is simple and feasible, the screening tag can be recycled, and the like, and basic support can be provided for efficient genetic modification of aspergillus terreus by using the gene targeting technique.

The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of: (a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell; (b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and / or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and (c) in said cell with high homologous recombination efficiency, causing deletion and / or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.

The invention discloses an anti-acid-stress component and application thereof, belonging to the field of bioengineering technology. According to the invention, two recombinant Lactococcus lactis strains with substantially improved resistance to acid stress, i.e., L. lactis NZ9000 (pNZ8148 / RecT-A76) and L. lactis NZ9000 (pNZ8148 / RecT-MG1655), are obtained by overexpression of recT genes, originated from Lactococcus lactis L. lactis A76 and Escherichia coli E. coli MG1655, in Lactococcus lactis NZ9000, respectively. After the two recombinant Lactococcus lactis strains suffer from stress in an environment with a pH value of 4.0 for 3h, the survival rates of the two strains are 10.5 times and 1.4 times the survival rates of control strains, respectively; and after the two strains suffer from stress caused by 20% (v / v) ethanol for 6 h, the survival rates of the two strains are 3.7 times and 2.1 times the survival rates of control strains, respectively. The invention also provides a method for improving resistance to acid stress. The method has good industrial application value.

The invention discloses a porcine myostatin gene editing site and application thereof, and belongs to the fields of biotechnology and biomedical medicine engineering. A gene editing target site is separated from a porcine myostatin gene editing area, the sequence of the gene editing target site is 5'-GGCTGTGTAATGCATGTATGTGG-3', and the gene editing target site is located at a first exon of an MSTN (myostatin) coding area. The site can be recognized by Cas9 endonuclease specifically, so that double strands are medicated to break, homologous recombination between the double strands and shooting carriers is achieved, and mutant genes or selectable maker genes are integrated to a pre-positioned site of a recipient cell genome. A statistical result shows that shooting efficiency of the site is 80.5%. The porcine myostatin gene editing site has the advantages that an effective drone is provided to accurate porcine myostatin gene editing, a new strategy is provided to development of new species of live pigs with high lean meat percentage, and reliable measures and materials are provided to study on molecular mechanism and signal channel of myostatin.

The invention relates to an efficient and precise gene point mutation restoration gene editing method. Different fixed point cutting capability on the genome DNA (deoxyribonucleic acid) during the combination of nuclease with sgRNA with different lengths is used, the targeting transcriptional control factor is used in an auxiliary way; the homologous recombination reaction efficiency is obviously improved; the gene mutation accurate restoration can be accurately and efficiently performed, or exogenous genes can be guided into a targeted gene. The invention also discloses a gene editing composition, an RNA (ribonucleic acid) aptamer sequence and ssgRNA coupling molecule, and a purpose used for gene editing.

The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of:(a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell;(b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and / or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and(c) in said cell with high homologous recombination efficiency, causing deletion and / or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.

The invention discloses a pig muscle myostatin gene editing site 864-883 in a pig genome and application thereof, and belongs to the field of biological engineering. One gene editing target site is separated out from a pig muscle myostatin gene region; the sequence of the target site is 5'-GGATTTTGAAGCTTTTGGATGGG-3'; the site is positioned at a No.3 exon of an MSTN (myostatin) editing region. The site can be specifically identified by Cas9 endonuclease, so that mediated double chains fracture and then generate homologous recombination with targeting vectors; mutant genes or selectable maker gene and the like are integrated to the preset sites of a recipient cell genome. Statistical results show that the site targeting efficiency is 86.7 percent. The pig muscle myostatin gene editing site provided by the invention has the advantages that the effective target is provided for the precise embedding of the gene; new strategies are provided for developing live pig new varieties with high meat factor; reliable means and materials can be provided for developing myostatin molecular mechanisms and signal channels.

The invention discloses a method for improving homologous recombination efficiency and recombination virus screening of pseudorabies virus. According to the method, a pseudorabies virus AH strain transfer vector containing an EGFP gene complete expression box is subjected to enzyme digestion linearization, and then subjected to homologous recombination with the pseudorabies virus AH strain; and the obtained recombination virus screening method comprises the steps of separating fluorescent single host cells with pathological changes, and then performing further screening by improved plaque experiment. After recombinant plasmid enzyme is subjected to digestion linearization, the enzyme is subjected to cell transfection; next, virus homologous recombination is performed, so that the homologous recombination efficiency is improved; by virtue of the method in combination with single cell absorption and plaque purification, the screening and purifying time of the recombination viruses is greatly shortened; and therefore, the method is of great significance to improvement of the virus homologous recombination efficiency, has high application prospect in single cell separation, particularly recombination virus screening, and can be widely applied to the recombination virus screening.

The invention provides recombinant yarrowia lipolytica with high homologous recombination efficiency and a construction method thereof, and relates to the field of biological engineering. The recombinant yarrowia lipolytica with the high homologous recombination efficiency is obtained by inserting an Rad52 gene expression cassette derived from a saccharomyces cerevisiae Rad52 epistatic gene cluster into a yarrowia lipolytica genome. The recombinant yarrowia lipolytica can have high homologous recombination efficiency, and the construction method is efficient, and simple to operate.

The invention relates to a method for improving the homology directed efficiency in gene editing and plasmids used in the method. The method for improving the homology directed efficiency in gene editing comprises the following steps: firstly, conducting target gene editing and design and molecular cloning; and secondly, adding a compound RS-1 into a cell culture medium, conducting culture at 37 DEG C for 24 hours, and then introducing a Cas9 gene segment, an AUNIP gene segment and a target gene gRNA segment into a CRISPR-Cas9 gene editing system by adopting an electroporation transfection method or a micro-injection technology. By means of the method for improving the homology directed efficiency in gene editing, precise gene editing can be conducted on eukaryotic cells, especially mammalian cells, is suitable for any target genes, and includes introduction of point mutation, precise knockout or insertion of small fragments, and the like, and compared with the conventional CRISPR-Cas9system, the method for improving the homology directed efficiency in gene editing has the advantages that the homology directed repair efficiency can be improved by at least 50%, and the homology directed repair efficiency of certain gene loci can even be improved by 2-5 times.

The invention discloses a construction method of gene engineered bacteria for arteannuic acid production, and the construction method comprises the following steps: A) construction of a gene expression module including an artemisinic acid production related gene, an upstream promoter of the artemisinic acid production related gene, and a downstream terminator of the artemisinic acid production related gene; and B) co-transformation of the gene expression module into saccharomyces cerevisiae. The method has the advantages of being simple and fast and efficient, avoids multiple cloning, and does not rely on enzyme digestion sites, many fragments can be together transformed, the homologous recombination efficiency is high, and the method can significantly shorten the construction time of the gene engineered bacteria.

The invention discloses an efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof. The method comprises the following steps: (1) biotin labeling is performed on donor DNA to obtain biotin-labeled donor DNA; (2) fusion protein of Cas9 protein and monovalent streptavidin, sgRNA and the biotin-labeled donor DNA are evenly mixed and undergo standing to obtain a CRISPR RNP-donor DNA complex; and (3) cell nucleus transformation is performed on the CRISPR RNP-donor DNA complex to realize gene insertion or replacement. According to the method, by utilizing the characteristic that the fusion protein can be combined with the biotin-labeled donor DNA, the CRISPRRNP and the donor DNA appear at a target locus together, and precise insertion of the donor DNA at the target locus or accurate replacement of a gene at the target locus is realized; and the method issuitable for the aspect of crop and livestock breeding.

The invention discloses a construction method of porcine delta coronavirus infectious clone plasmids, and belongs to the technical field of animal infectious disease prevention and treatment and biology. According to the invention, a reverse genetic system of a recombinant virus of a PDCoV CHN-HN-2014 strain is rescued based on a BAC system and a homologous recombination one-step cloning method, wherein five gene segments of the strain are cloned to an intermediate vector pBAC-M-PDCoV in one step, and a recombinant plasmid pBAC-CHN-HN-2014 containing full-length cDNA of the PDCoV CHN-HN-2014 strain is obtained, and the recombinant virus rCHN-HN-2014 is rescued after cells are transfected; therefore, a porcine delta coronavirus reverse genetic manipulation platform is established, and a foundation is laid for in-depth study on infection and pathogenic mechanisms of PDCoV. Meanwhile, aiming at the practical problem that no available PDCoV vaccine exists in China at present, the PDCoV infectious cloning platform disclosed by the invention can also be used for developing a safer and more efficient novel PDCoV genetic engineering vaccine. The platform can also be used as a live virus vector and is used for developing a multi-combined vaccine, so that the PDCoV infectious cloning platform disclosed by the invention has a great value for the research related to the virus.

The invention discloses a method for improving the homologous recombination efficiency of Gluconobacter oxydans, belonging to the field of genetic engineering. The present invention expresses exogenous recombinase in Gluconobacter oxydans, improves the homologous recombination efficiency of the strain, and realizes gene knockout and screening by constructing homologous fragments with screening markers and homologous fragments without screening markers Mark removal. The obtained gluconobacterium oxidans expressing the Red recombinase will play an important role in the construction of vitamin C one-step production bacteria.

The invention provides a method for improving homologous recombination efficiency based on a CRISPR gene editing system, which comprises the following steps: introducing a Cas9 plasmid fused with streptavidin and a corresponding homologous donor with biotin modification into a host cell, and further introducing an NHEJ fluorescent reporter plasmid into the host cell. According to the invention, a Cas9 plasmid fused with streptavidin, a corresponding biotin-modified homologous donor and an NHEJ fluorescent reporter plasmid are introduced into a CRISPR / Cas9 system for the first time to improve the homologous recombination efficiency, and the NHEJ-based fluorescent reporter plasmid can indicate all elements subjected to homologous recombination; preliminary flow analysis results show that the homologous recombination efficiency in enriched cells can be improved to 50-80%.

The invention discloses an engineering bacterium for efficiently producing patchouli alcohol, and a construction method and application of the engineering bacterium; the engineering bacterium is obtained by inserting optimized patchouli alcohol synthase coding genes PS1 and tHMGR into a genome of yarrowia lipolytica serving as an original strain; the optimized patchouli alcohol synthase coding gene PS1 is as shown in SEQ ID NO. 1; and the tHMGR coding gene is as shown in SEQ ID NO. 2. The engineering bacterium disclosed by the invention can efficiently synthesize the patchouli alcohol; the construction method is efficient and easy to operate; in addition, the method for collecting the product provided by the invention adopts a method of extracting while fermenting; the produced patchouli alcohol is extracted into an organic phase in time; the pressure of intracellular dissolution of the patchouli alcohol is relieved; the yield of the patchouli alcohol is effectively increased; and a foundation is laid for synthesizing the patchouli alcohol by artificial cells.

The invention discloses a method and application for improving application efficiency of a gene targeting technique in aspergillus terreus, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, by taking Aspergillus terreus as an initial bacterium, knocking off a ku80 gene or an lig4 gene so as to increase the exogenous DNA homologous recombination probability of a strain; secondly, establishing a pyrG gene deletion uracil auxotroph stain, establishing a inheritance conversion system based on a pyrG gene as a screening tag; and finally, cutting off the screening tag by using a Cre / LoxP specific binding site recombinant system, thereby obtaining a uracil auxotroph stain which can be applied to genetic modification again. By adopting the method disclosed by the invention, an efficient aspergillus terreus gene targeting platform can be established, the method has the advantages that high homologous recombination efficiency can be achieved, the bidirectional screening of the conversion system can be achieved, a screening tag cutting method is simple and feasible, the screening tag can be recycled, and the like, and basic support can be provided for efficient genetic modification of aspergillus terreus by using the gene targeting technique.

The invention is applicable to the field of biotechnology, and provides a preparation method of an Escherichia coli capable of automatically degrading nucleic acids during cracking. The method comprises steps of: connecting a pelB signal peptide sequence with an SNase sequence to obtain a first combined sequence; connecting the first sequence with a Km gene sequence to obtain a second combined sequence; substituting a ptsG gene of Escherichia coli with the second combined sequence to obtain Escherichia coli with deletion of ptsG gene; and knocking out the Km gene from the second sequence and in the Escherichia coli with deletion of ptsG gene. The preparation method of Escherichia coli provided by the invention has the advantages of high efficiency of homologous recombination; the SNase expressed by the prepared Escherichia coli can secret into a periplasmic space, and a product expressed by the SNase gene does not influence the expression of other protein; and the method can reduce the accumulation of acetic acid in a fermentation process and promote thallus growth.

The invention discloses a genetically engineered bacterium of coleophoma sp. with efficient homologous recombination as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of gene engineering. The invention aims to solve the problem of how to improve the homologous recombination efficiency of Coleophoma sp.. According to the Coleophoma sp. genetically engineered bacterium for improving the homologous recombination efficiency through ku80 gene deletion, the Coleophoma sp. genetically engineered bacterium serves as a starting bacterium, an exogenous gene in a targeting element is integrated to a genome in a homologous recombination mode in a fixed point mode, the homologous recombination efficiency reaches 90% or above, and a good theoretical basis is provided for an application of a gene targeting technology in filamentous fungi.

A method for improving homologous recombination efficiency in gene editing and a plasmid used in the method, comprising the following steps: first, carrying out target gene editing design and molecular cloning; secondly, adding compound RS-1 to a cell culture medium, and Cultivate at 37°C for 24 hours, then introduce Cas9 gene fragments, AUNIP gene fragments, and target gene gRNA fragments into the CRISPR-Cas9 gene editing system by electroporation transfection or microinjection technology; the method of the present invention improves homology in gene editing The method of recombination efficiency can carry out precise gene editing on eukaryotic cells, especially mammalian cells, and is suitable for any target gene, such as introducing point mutations, precise knockouts or inserting small fragments, etc. Compared with the conventional CRISPR‑Cas9 system, it can improve The homologous recombination repair efficiency is at least 50%, and it can even achieve a 2-5 times improvement for certain gene loci.

The invention discloses a method for remarkably improving fish genome editing efficiency. The method for remarkably improving fish genome editing efficiency comprises the following steps: designing a genome editing tool for specifically identifying and cutting a specified site sequence of the fish genome, designing a homologous donor corresponding to the specified site sequence and containing a knock-in exogenous gene fragment, introducing the genome editing tool, the homologous donor and mRNA for specifically and stably expressing fluorescent protein in primordial germ cells into fish animal embryos by using a codominant microinjection method, and selecting the embryos by detecting fluorescent protein expressed by the fluorescent protein mRNA and obtaining stable inheritable characters. The efficiency of the method for knocking the exogenous gene fragment into the specified site of the fish genome so as to obtain a first filial generation of fish with the knock-in exogenous gene fragment is remarkably higher than that of the prior art.

The invention provides a method for preparing a DNA vaccine with herpes simplex virus type I (HSV-1) UL5 gene deletion. The method includes the following steps: S1, carrying out homologous recombination knockout of a herpes simplex virus type I UL5 gene, to obtain a recombinant strain containing no UL5 gene and no exogenous gene, wherein a homologous-recombination-knockout recombination knockout system is a host strain containing a BAC-HSV-1 vector and a pREDI recombinant plasmid; and S2, cutting a BAC vector and obtaining the DNA vaccine with HSV-1 UL5 gene deletion. The invention belongs to the technical field of biological medicine; the attenuated live vaccine can be prepared by the method provided by the invention, is safe and effective and less prone to virulence reversion, and can effectively prevent and treat HSV-1 infection.

The invention discloses an efficient CRISPR RNP and donor DNA colocalization-mediated gene insertion or replacement method and its application. The method comprises the following steps: (1) biotin-labeling donor DNA to obtain biotin-labeled donor DNA; (2) fusion protein of Cas9 protein and monovalent streptavidin, sgRNA and biotin-labeled The donor DNA is mixed evenly and left to stand to obtain the CRISPR RNP-donor DNA complex; (3) the CRISPR RNP-donor DNA complex is subjected to nuclear transformation to realize gene insertion or replacement. In the present invention, the fusion protein can be combined with the characteristics of biotin-labeled donor DNA, so that CRISPR RNP and donor DNA co-appear at the target site, so as to realize the precise insertion of the donor DNA at the target site or the gene expression of the target site. Precise replacement, suitable for crop and livestock breeding.

The invention relates to the technical field of gene engineering, in particular to a method for constructing a thermophilic mycelia strain with high homologous recombination efficiency and application of the thermophilic mycelia strain. According to the mutant strain delta Ku80 obtained by the invention, the homologous recombination efficiency of a trichoderma thermophile CRISPR-Cas9 gene editing system can be improved to 86% from 57% of a wild type, the workload of genetic transformation of the trichoderma thermophile is reduced, and the homologous recombination efficiency is improved.

The invention provides an auxiliary protein for improving DNA repair efficiency and a gene editing vector and application thereof, and belongs to the technical field of gene editing and gene therapy. The nucleotide sequence of the auxiliary protein for improving DNA repair efficiency provided by the invention is as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The auxiliary protein fragment provided by the invention is shorter, and compared with an RAD51-Cas9n system, the homologous recombination efficiency is higher, and the auxiliary protein fragment is more suitable for transgenic virus vector packaging; compared with a Prime edging system, the method has the advantages that the multi-base and large-fragment DNA variation can be repaired, and the repairing function is stronger.

The invention provides a method for preparing a DNA vaccine with herpes simplex virus type I (HSV-1) UL5 gene deletion. The method includes the following steps: S1, carrying out homologous recombination knockout of a herpes simplex virus type I UL5 gene, to obtain a recombinant strain containing no UL5 gene and no exogenous gene, wherein a homologous-recombination-knockout recombination knockout system is a host strain containing a BAC-HSV-1 vector and a pREDI recombinant plasmid; and S2, cutting a BAC vector and obtaining the DNA vaccine with HSV-1 UL5 gene deletion. The invention belongs to the technical field of biological medicine; the attenuated live vaccine can be prepared by the method provided by the invention, is safe and effective and less prone to virulence reversion, and can effectively prevent and treat HSV-1 infection.

The invention relates to a conditional gene knockout method constructed by using a CRISPR / Cas9 system. LoxP-antibiotic-LoxP and FRT-antibiotic-FRT-LoxP sequence insertion sites are selected from leftand right sides of a to-be-knocked-out area respectively; left and right homologous arms are selected from upstream and downstream positions of to-be-inserted sites, amplification, purification, enzyme digestion, linking and conversion are performed, and left and right targeting plasmids are obtained; Cas9 plasmid, the left or right targeting plasmid of the knockout area and corresponding gRNA expression plasmids are sequentially or simultaneously introduced into cells, screening is performed with antibiotics after transfection, and target cells with left and right targeting sequences insertedare screened out simultaneously; 4-OHT and DOX are used for inducing Cre-LoxP and FLP-FRT medicated homologous recombination, so that loxp sites are introduced in left and right sides of the to-be-knocked-out area; 4-OHT is used for inducing Cre-LoxP medicated homologous recombination, and cloning of target fragment knockout is obtained.