title: Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.
abstract: The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.
authors.affiliation: 1] Division of Cellular and Molecular Toxicology, Biological Safety Research Centre, National Institute of Health Sciences (NIHS), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan [2] Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Ono
authors.affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishii
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fujihara
authors.affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Moe
authors.initials: M
authors.lastname: Kitazawa
authors.affiliation: Facility for Recombinant Mice, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Usami
authors.affiliation: School of Health Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kaneko-Ishino
authors.affiliation: Division of Cellular and Molecular Toxicology, Biological Safety Research Centre, National Institute of Health Sciences (NIHS), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Kanno
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
authors.affiliation: 1] Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan [2] Global Centre of Excellence Programme for International Research Centre for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Fumitoshi
authors.initials: F
authors.lastname: Ishino
mesh_terms: D000818:Animals; D004249:DNA Damage; D004260:DNA Repair; D051379:Mice; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D018626:Retroelements; D048348:Reverse Transcription; D015053:Zygote / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D018626:Retroelements