Construction and Activity Evaluation of xCas9 and SpCas9-NG Nicking Mutants
ZHANG Ning1, QIN Huai-Yuan1, XIN Jing-Jing1, ZHAO Jin-Shan1, BAO Han-Xun2, LI He-Gang*
1 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China; 2 Jiaozhou Bureau of Agriculture and Rural Areas, Qingdao 266300, China
Abstract：The commonly used CRISPR/Cas9 system only recognizes targets with a NGG protospacer-adjacent motif (PAM) sequence, while the evolved xCas9 and SpCas9-NG systems can recognize targets with a NG PAM, which broadens the recognition range of CRISPR/Cas9. However, the off-target effects have always hindered the development and application of the CRISPR/Cas9 system. Previous studies showed that the double-nicking system could increase the specificity of DNA targeting. Four nicking mutants, named xCas9-D10A, SpCas9-NG-D10A, xCas9-H840A, and SpCas9-NG-H840A, and the corresponding single guide RNA (sgRNA) expression vectors were designed and constructed in this study to improve the specificity of gene editing of the CRISPR/Cas9 system. These vectors, with SSA-DKK1, SSA-DKK2 reporter plasmid, were co-transfected into sheep (Ovis aries) fibroblast cells through a specific plasmid combination, verifying cutting mutants activity with luciferase expression. The results showed that the SpCas9-NG-D10A and SpCas9-NG-H840A mutants had significantly greater reporter gene activity against most target vectors that recognize PAM as NG than the control group (P<0.05), and the xCas9-H840A variant had no activity at all.However, xCas9-D10A only showed cleavage activity in DKK1 gene. In contrast, the SpCas9-NG-D10A variant had stable activity and high efficiency in cutting DNA single strands, so this vector can be used for further research applications. The results of this study provides important reference data for the further research and application of NG PAM's Cas9 double-nicking system.
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