体外定点突变体或CRISPR/Cas9介导的敲入突变体快速筛选方法的建立

ESTABLISHMENT OF A RAPID SCREENING METHOD FOR IN VITRO SITE-DIRECTED MUTANTS OR CRISPR/Cas9-MEDIA-TED KNOCK-IN MUTANTS

目的 探讨建立一种体外定点突变体或CRISPR/cas9介导的敲入突变体的快速筛选方法,以提高突变体筛选效率,降低实验成本。方法 针对构建的体外定点突变体或CRISPR/Cas9介导的敲入突变体,采用3′端第1、2和3个核苷酸分别与突变序列相匹配的筛选引物进行聚合酶链式反应(PCR),通过琼脂糖凝胶电泳检测是否有特异性扩增条带。结果 利用特异性突变筛选引物进行PCR在正确的突变体中有效地扩增出了DNA条带,而在未成功突变的样本中则无法扩增,琼脂糖凝胶电泳显示有DNA扩增条带,证明正确突变体筛选成功。结论本研究建立的突变体快速筛选方法能够简便快速地筛选出正确突变体,明显简化了突变体的鉴定过程,可有效降低实验成本,提高工作效率。

Objective To establish a rapid screening method for in vitro site-directed mutants or CRISPR/Cas9-mediated knock-in mutants,and to improve mutant screening efficiency and reduce experimental costs.Methods Polymerase chain reactions(PCRs)were carried out with primers designed with the last one,two,and three nucleotides in the 3′end matching the mutated DNA sequences for the constructed in vitro site-directed mutants or CRISPR/Cas9-mediated knock-in mutants,and agarose gel electrophoresis was used to detect specific amplification bands.Results PCR using specific mutation screening primers effectively amplified DNA bands in correct mutants but failed in unsuccessfully mutated samples.The presence of DNA amplification bands revealed by agarose gel electrophoresis confirmed the successful screening for desired mutants.Conclusion The rapid screening method for mutants introduced in this study can easily and quickly identify correct mutants,which significantly simplifies the identification process of mutations,and thereby reduces experimental costs and improves work efficiency.