通过CRISPR/Cas系统构建miR-17-92基因簇双切载体

摘要

通过CRISPR/Cas系统来构建miR-17-92基因簇的双切载体,研究结果表明:在miR-17-92基因簇的序列上找到了2个PAM(TGG和GGG)位点,从而得到了2个原间隔序列,设计合成基因簇双链crRNA;合成该片段并将其插入到pX260载体,使得在同一个载体上虽然只有一个g-RNA,但能够同时切割两个不同的靶位点;将该载体转染NIH3T3细胞验证切割效率,PCR结果和测序结果显示,所构建的CRISPR系统可以对基因片段进行有效切割.%We constructed double-cut carrier of miR-17-92 gene cluster using CRISPR/Cas9 system.We identified two PAM and two protospacesare,and designed pre-crRNAs.The synthesized pre-crRNAs(that was the designed annealing oligonucleotides based on the sequence specific)were inserted between the pX260 carrier.Therefore,there is only one g-RNA in the same carrier,cutting two different target sites at the same time.NIH3T3 cells were transfected by plasmid of pX260 carrier including pre-crRNAs for the cutting effect.The miR-17-92 gene was cut by CRISPR/Cas system.