title: Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.
abstract: 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.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yan
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Jiani
authors.initials: J
authors.lastname: Xing
authors.affiliation: Institute of applied biology, Shanxi University, 030006, Shanxi, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Wei
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Zhongtian
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Simin
authors.initials: S
authors.lastname: Shao
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D043244:Ribonuclease III / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; C517918:DROSHA protein, human; D043244:Ribonuclease III