Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

作者：Yan, Qiang; Xu, Kun; Xing, Jiani; Zhang, Tingting; Wang, Xin; Wei, Zehui; Ren, Chonghua; Liu, Zhongtian; Shao, Simin; Zhang, Zhiying^*

来源：Scientific Reports, 2016, 6(1): 38970.

DOI：10.1038/srep38970

• RNA
• REPAIR
• CELLS
• RECOMBINATION
• ENDONUCLEASE
• CRISPR-CAS9
• GENERATION
• ENRICHMENT
• EFFICIENCY
• REPORTERS

The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homologydirected repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells.

• 出版日期2016-12-12
• 单位山西大学