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

• Published in: Scientific reports Vol. 6; no. 1; p. 38970
• Main Authors: Yan, Qiang, Xu, Kun, Xing, Jiani, Zhang, Tingting, Wang, Xin, Wei, Zehui, Ren, Chonghua, Liu, Zhongtian, Shao, Simin, Zhang, Zhiying
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 12.12.2016
• Subjects: CRISPR; CRISPR-Cas Systems; DNA ligase (ATP); Editing; Gene Editing - methods; Genome editing; Genomes; HEK293 Cells; Homology; Humans; LIG4 protein; Mammalian cells; Puromycin; Ribonuclease III - genetics; RNA, Small Interfering - genetics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep38970

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 homology-directed 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.