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

Author(s)
Aida, Tomomi; Nakade, Shota; Sakuma, Tetsushi; Izu, Yayoi; Oishi, Ayu; Mochida, Keiji; Ishikubo, Harumi; Usami, Takako; Aizawa, Hidenori; Yamamoto, Takashi; Tanaka, Kohichi; ... Show more Show less
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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.
Date issued
2016-11
URI
http://hdl.handle.net/1721.1/112735
Department
McGovern Institute for Brain Research at MIT
Journal
BMC Genomics
Publisher
Biomed Central Ltd
Citation
Aida, Tomomi et al. "Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ." BMC Genomics 17 (November 2016): 979 © 2016 The Author(s)
Version: Final published version
ISSN
1471-2164

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