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BMC Genomics. 2016 Nov 28;17(1):979.

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

Author information

1
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan. aidat@mit.edu.
2
Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan. aidat@mit.edu.
3
Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, 43 Vassar St., Cambridge, MA, 02139, USA. aidat@mit.edu.
4
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
5
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
6
Department of Animal Risk Management, Chiba Institute of Science, 3 Shiomi-cho, Choshi, Chiba, 288-0025, Japan.
7
Present address: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
8
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan.
9
Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan.
10
Department of Neurobiology, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.
11
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
12
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.
13
The Center for Brain Integration Research (CBIR), TMDU, Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.

Abstract

BACKGROUND:

Although 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.

RESULTS:

Here, 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.

CONCLUSIONS:

Our results provide a technical platform for high-throughput knock-in.

KEYWORDS:

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

PMID:
27894274
PMCID:
PMC5126809
DOI:
10.1186/s12864-016-3331-9
[Indexed for MEDLINE]
Free PMC Article

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