title: CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos.
abstract: Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
authors.affiliation: 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2].
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Shao
authors.affiliation: 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2].
authors.forename: Yuting
authors.initials: Y
authors.lastname: Guan
authors.affiliation: 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2].
authors.forename: Liren
authors.initials: L
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Qiu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Meizhen
authors.initials: M
authors.lastname: Liu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Wu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Li
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Xueyun
authors.initials: X
authors.lastname: Ma
authors.affiliation: 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2] Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA.
authors.forename: Mingyao
authors.initials: M
authors.lastname: Liu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Dali
authors.initials: D
authors.lastname: Li
mesh_terms: D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004622:Embryo, Mammalian / Q000166:cytology*; D055879:Gene Knock-In Techniques / Q000379:methods; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000295:instrumentation / Q000379:methods*; D016678:Genome; D059765:Homologous Recombination; D008845:Microinjections; D009154:Mutation; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic