Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

BMC Genomics

Methodology Article

Yayoi Izu¹  Keiji Mochida²  Takashi Yamamoto²  Shota Nakade²  Tetsushi Sakuma²  Ayu Oishi³  Hidenori Aizawa⁴  Harumi Ishikubo⁵  Tomomi Aida⁶  Kohichi Tanaka⁷  Takako Usami⁸ 

[1] Department of Animal Risk Management, Chiba Institute of Science, 3 Shiomi-cho, 288-0025, Choshi, Chiba, Japan;Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, 739-8526, Higashi-Hiroshima, Hiroshima, Japan;Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, 739-8526, Higashi-Hiroshima, Hiroshima, Japan;Present address: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, 464-8601, Nagoya, Japan;Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, 113-8510, Tokyo, Japan;Department of Neurobiology, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, 734-8553, Hiroshima, Japan;Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, 113-8510, Tokyo, Japan;Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, 101-0062, Tokyo, Japan;Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, 113-8510, Tokyo, Japan;Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, 101-0062, Tokyo, Japan;Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, 43 Vassar St., 02139, Cambridge, MA, USA;Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, 113-8510, Tokyo, Japan;The Center for Brain Integration Research (CBIR), TMDU, 113-8510, Tokyo, Japan;Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, 101-0062, Tokyo, Japan;

关键词: MMEJ;    CRISPR/Cas;    Gene cassette;    Reporter;    Flox;    Knock-in;    Mouse;    Exo1;    High throughput;    Cloning-free;   

DOI  :  10.1186/s12864-016-3331-9

 received in 2016-09-03, accepted in 2016-11-22,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.

【 授权许可】

CC BY   
© The Author(s). 2016