title: A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.
abstract: The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, 210061, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Microbiology and Parasitology, Anhui Medical University, Hefei, 230032, China.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Du
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, 210061, China.
authors.forename: Xueyan
authors.initials: X
authors.lastname: He
authors.affiliation: School of Life Science and Technology, Shanghai Tech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China. huangxx@shanghaitech.edu.cn.
authors.forename: Xingxu
authors.initials: X
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, 210061, China. yunshi@nju.edu.cn.
authors.forename: Yun S
authors.initials: YS
authors.lastname: Shi
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D055786:Gene Knockout Techniques / Q000379:methods; D023061:Gene Order; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D018773:Genes, erbB-1; D005822:Genetic Vectors / Q000235:genetics; D019426:Integrases; D008264:Macrophages / Q000378:metabolism; D051258:MafB Transcription Factor / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D051257:Proto-Oncogene Proteins c-maf / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems; D012680:Sensitivity and Specificity
chemical_list: C495608:Maf protein, mouse; D051258:MafB Transcription Factor; C490406:Mafb protein, mouse; D051257:Proto-Oncogene Proteins c-maf; D000094704:RNA, Guide, CRISPR-Cas Systems; C045073:Cre recombinase; D019426:Integrases