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 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 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 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 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 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.