Method for achieving HMGCR gene knockout based on CRISPR/Cas9 technology
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A gene knockout and technology technology, applied in the field of genetic engineering, can solve problems such as HMGCR gene knockout that have not yet been seen
Active Publication Date: 2018-08-17
HUNAN AGRICULTURAL UNIV
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[0005] However, so far, there has been no report on the spec
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Embodiment 1
[0029] Embodiment 1: vector construction
[0030] (1) HMGCR target optimization design
[0031]For the HMGCR gene (gene name HMGCR, gene ID number: 100144446, see https: / / www.ncbi.nlm.nih.gov / gene / ?term=DQ432054.1 for gene details), retrieve and download HMGCR from the Genebank website Partial genome sequence (SEQ ID NO: 1): TTCTGAAgCTACAATGTTGTCAAGACTTCTTCCGAATGCATGGCCTCTTTGTGGCCTCCCATCCCTGGGAAGTCATAGTGGGGACAGTGACACTGACCATCTGTATGATGTCCATGAACATGTTTACTGGTAACGATAAGATCTGTGGTTG.
[0032] Using the online software Feng Zhang lab's Target Finder( http: / / crispr.mit.edu / ) to design gRNA, input the 150bp of the above HMGCR genome sequence, set and retrieve several gRNA sequences, and analyze the position of the gRNA on the gene sequence and the off-target (off-target) information of the gRNA, see figure 1 , respectively select the optimal upstream target sequence, as shown in SEQ ID NO: 3; the downstream 1 target sequence, as shown in SEQ ID NO: 2, specifically as follows:
[0033...
Embodiment 2
[0083] Example 2: Test knockout efficiency and construct HMGCR knockout PK15 cell line
[0084] (1) Plasmid amplification
[0085] 1) Take out 100ul competent cells from the ultra-low temperature refrigerator at -80°C, place them on ice, and gently suspend the cells evenly after thawing completely;
[0086]2) Add 1 μl of plasmid (PX459-HMGCR-gRNA1, PX459-HMGCR-gRNA2) and mix gently, and place on ice for 30 minutes;
[0087] 3) Heat shock in a water bath at 42°C for 60 seconds, and place on ice for 2 minutes;
[0088] 4) Add 500 μl of SOC medium (containing MgCL2), culture at 37°C, 225rpm for 1 hour to recover;
[0089] 5) Mix the bacterial solution with a pipette tip, take 100 μl (up to 200 μl), and spread the bacteria on the ampicillin plate;
[0090] 6) The plate was placed upright at 37° C. for 10 minutes to absorb excess liquid, and then cultured upside down overnight (about 12 hours).
[0091] 7) Pick a single colony from the ampicillin culture plate, infuse it into a...
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Abstract
The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR/Cas9 technology. The method is characterized in that two CRISPR/Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for knocking out the HMGCR gene based on CRISPR / Cas9 technology. Background technique [0002] CRISPR / Cas system (clustered, regularly interspaced, short palindromic repeats-associated protein systems) is an adaptive immune mechanism evolved by bacteria and archaea to resist virus and plasmid invasion. Due to the many advantages of this system, it has been widely used in gene editing technology, and its powerful and efficient genome editing function has been successfully applied to genetic modification of various organisms, including bacteria, plants, Caenorhabditis elegans, zebrafish, mice , rats, pigs, and even higher non-human primates. Most studies have shown that CRISPR / Cas system can better The target gene is identified, and the structure of the CRISPR / Cas9 system is simple, so its construction only needs to design and synthesize a pair of primers. ...
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