Wang Dan, Niu Cuili, Han Jingxin, Ma Dejun, Xi Zhen
Department of Chemical Biology, State Key Laboratory of Elemento-Organic Chemistry, National Engineering Research Center of Pesticide (Tianjin), College of Chemistry, Nankai University Tianjin 300071 China
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) Tianjin 300071 China.
RSC Adv. 2019 Mar 19;9(16):9067-9074. doi: 10.1039/c8ra10017a. eCollection 2019 Mar 15.
The RNA-guided CRISPR/Cas9 system could cleave double-stranded DNA at the on-target sites but also induce off-target mutations in unexpected genomic regions. The base-pairing interaction of sgRNA with off-target DNA was still not well understood and also lacked a direct cell-based assay. Herein we developed a fast target DNA mutagenesis-based fluorescence assay to directly detect the Cas9 activity at different off-target sites in living cells. The results showed that Cas9 nuclease had low tolerance to the nucleotide mismatches in the binding region adjacent to PAM sites, and a tradeoff between Cas9 activity and specificity was also observed compared with the high-fidelity Cas9 variant. The combination of computer-based predictions and this target DNA mutagenesis-based fluorescence assay could further provide accurate off-target prediction guidance to minimize off-target effects to enable safer genome engineering.
RNA引导的CRISPR/Cas9系统能够在目标位点切割双链DNA，但也会在意外的基因组区域诱导脱靶突变。目前，对于sgRNA与脱靶DNA的碱基配对相互作用仍未完全了解，并且也缺乏基于细胞的直接检测方法。在此，我们开发了一种基于快速目标DNA诱变的荧光检测方法，以直接检测活细胞中不同脱靶位点的Cas9活性。结果表明，Cas9核酸酶对PAM位点附近结合区域的核苷酸错配耐受性较低，并且与高保真Cas9变体相比，还观察到Cas9活性和特异性之间的权衡。基于计算机的预测与这种基于目标DNA诱变的荧光检测方法相结合，可以进一步提供准确的脱靶预测指导，以尽量减少脱靶效应，实现更安全的基因组工程。
