一种新型的构建CRISPR/Cas9 KnockOut载体的方法

A Novel Method for KnockOut Vector Constraction of CRISPR/Cas9 System

利用CRISPR/Cas9系统这一基于细菌核酸酶Cas9的新型基因编辑工具,可以在原核细胞和真核细胞中实现基因敲除的功能.首先使用CRISPR设计工具设计靶点,退火来制备sgRNA双链,用Bsm BⅠ酶切割gRNA质粒,构建Lenti CRISPRv2的重组质粒.通过U6启动子上的LKO1.5引物对每个菌落序列进行了测序验证,结果表明利用此新方法可以成功构建CRISPR/Cas9系统的Knock Out载体.

A new tool based on a bacterial using CRISPR-associated protein-9 nuclease(Cas9)has generated to facilitate efficient genome engineering in procaryotic cell and eukaryotic cells.Firstly gRNA targeting sequence were designed using CRISPR design tool and the sgRNA oligos were prepared by annealing.The sgRNA oligos were constructed into LentiCRISPRv2 by Bsm BⅠcut site and transformed into a competent E.coli strain.The colony was sequenced from the U6 promoter using LKO1.5 primer.the results show that the KnockOut vector of CRISPR/Cas9 system is successfully constructed by the new method.