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EBioMedicine. 2017 Jun;20:19-26. doi: 10.1016/j.ebiom.2017.05.015. Epub 2017 May 11.

CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms.

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; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
2
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, 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.
4
The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Huhhot 010070, China; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA.
5
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: huangpy@shanghaitech.edu.cn.
6
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

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah-/- liver failure mice, offering an efficient approach for precisely targeted gene therapies.

KEYWORDS:

CRISPR/Cas9; Fah(−/−) mice; Gene therapy; In vivo targeted integration; MMEJ

PMID:
28527830
PMCID:
PMC5478232
DOI:
10.1016/j.ebiom.2017.05.015
[Indexed for MEDLINE]
Free PMC Article

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