Cas9-sgRNA共质粒系统提高在AAVS1位点的打靶效率

摘要

Objective: CRISPR/Cas9 has been widely used for gene targeting. To further improve the targeting effi⁃ciency, we compared the effects of different vector designs on the Cas9-sgRNA mediated gene targeting efficiency. Methods: We designed sgRNAs targeting the AAVS1 locus and compared the Cas9-sgRNA all-in-one vector vs. the two-plasmid system. After transfection of 293T cells with plasmids, the cleavage or targeting efficiency was de⁃termined by T7E1 assay. Results: We found that T7E1 endonuclease can effectively cleave the PCR products with⁃out denaturing and annealing. We also found that the all-in-one Cas9-sgRNA vector significantly increases gene targeting efficiency when compared with the two-plasmid system. Conclusion: The T7E1 assay can be simplified by omitting the denaturing and annealing steps. Co-expression of Cas9 and sgRNA in one vector is the better de⁃sign for achieving high gene targeting efficiency.%目的：为进一步提高CRISPR/Cas9系统的打靶效率，比较共质粒或双质粒表达Cas9内切酶和小向导RNA （sgRNA）的不同载体设计对其打靶效率的影响。方法：将针对AAVS1位点的2个sgRNA序列分别插入表达Cas9的质粒pSpCas9上，再将其构建为Cas9-sgRNA二合一的共质粒以及独立表达sgRNA和Cas9的双质粒系统；质粒转染293T细胞后，用T7E1酶切方法检测打靶效率。结果：不经过变性退火的PCR片段也能直接进行T7E1酶切；Cas9-sgRNA共质粒系统的打靶效率显著高于双质粒系统。结论：T7E1酶切方法可去除变性退火步骤，简化了传统的检测方法。采用Cas9-sgRNA共表达的载体设计可显著提高打靶效率。