title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.
abstract: 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.
  
  
  
  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.
  
  
  
  Our results provide a technical platform for high-throughput knock-in.
authors.affiliation: 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.|Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan. aidat@mit.edu.|Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, 43 Vassar St., Cambridge, MA, 02139, USA. aidat@mit.edu.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Aida
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakade
authors.affiliation: 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.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Animal Risk Management, Chiba Institute of Science, 3 Shiomi-cho, Choshi, Chiba, 288-0025, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Izu
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.|Present address: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Ayu
authors.initials: A
authors.lastname: Oishi
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Mochida
authors.affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan.|Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan.
authors.forename: Harumi
authors.initials: H
authors.lastname: Ishikubo
authors.affiliation: Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Usami
authors.affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan.|Department of Neurobiology, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Aizawa
authors.affiliation: 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.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: 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.|The Center for Brain Integration Research (CBIR), TMDU, Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.
authors.forename: Kohichi
authors.identifier: 0000-0001-6929-4228
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002875:Chromosomes; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005090:Exodeoxyribonucleases / Q000378:metabolism; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D056426:Genetic Loci; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D000069896:Transcription Activator-Like Effector Nucleases; D015053:Zygote
chemical_list: D005090:Exodeoxyribonucleases; D000069896:Transcription Activator-Like Effector Nucleases; C028450:exodeoxyribonuclease I
keywords: CRISPR/Cas; Cloning-free; Exo1; Flox; Gene cassette; High throughput; Knock-in; MMEJ; Mouse; Reporter