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Dev Cell. 2018 May 21;45(4):526-536.e5. doi: 10.1016/j.devcel.2018.04.021.

Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells.

Author information

1
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: xyao@ion.ac.cn.
2
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China.
3
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
4
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
5
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China.
6
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
7
Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China; National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, China; The Key Laboratory for Reproductive Endocrinology of Ministry of Education, Jinan, Shandong 250021, China.
8
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China. Electronic address: liweiping@renji.com.
9
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China; Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China; National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, China; The Key Laboratory for Reproductive Endocrinology of Ministry of Education, Jinan, Shandong 250021, China. Electronic address: chenzijiang@hotmail.com.
10
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: huiyang@ion.ac.cn.

Abstract

The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.

KEYWORDS:

CRISPR/Cas9; Tild-CRISPR; gene editing; genetically modified mice; human embryo; knockin

PMID:
29787711
DOI:
10.1016/j.devcel.2018.04.021
[Indexed for MEDLINE]
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