protocol
CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos Yanjiao Shao1,3, Yuting Guan1,3, Liren Wang1,3, Zhongwei Qiu1, Meizhen Liu1, Yuting Chen1, Lijuan Wu1, Yongmei Li1, Xueyun Ma1, Mingyao Liu1,2 & Dali Li1 1Shanghai Key Laboratory of
Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA. 3These authors contributed equally to this work. Correspondence should be addressed to M.L. ([email protected]) or D.L. ([email protected]). 2Center for Cancer and Stem Cell Biology, Institute of
© 2014 Nature America, Inc. All rights reserved.
Published online 25 September 2014; doi:10.1038/nprot.2014.171
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
CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos Yanjiao Shao1,3, Yuting Guan1,3, Liren Wang1,3, Zhongwei Qiu1, Meizhen Liu1, Yuting Chen1, Lijuan Wu1, Yongmei Li1, Xueyun Ma1, Mingyao Liu1,2 & Dali Li1 1Shanghai Key Laboratory of
Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA. 3These authors contributed equally to this work. Correspondence should be addressed to M.L. ([email protected]) or D.L. ([email protected]). 2Center for Cancer and Stem Cell Biology, Institute of
© 2014 Nature America, Inc. All rights reserved.
Published online 25 September 2014; doi:10.1038/nprot.2014.171
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
