title: CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.
abstract: Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871 Japan.|Research Fellow of Japan Society for the Promotion of Science (JSPS), Tokyo 1020083, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Oji
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Research Fellow of Japan Society for the Promotion of Science (JSPS), Tokyo 1020083, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Noda
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fujihara
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Miyata
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Yeon Joo
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871 Japan.|Research Fellow of Japan Society for the Promotion of Science (JSPS), Tokyo 1020083, Japan.
authors.forename: Masanaga
authors.initials: M
authors.lastname: Muto
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Research Fellow of Japan Society for the Promotion of Science (JSPS), Tokyo 1020083, Japan.|Graduate School of Medicine, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Nozawa
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Matsumura
authors.affiliation: Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Isotani
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.|Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871 Japan.|Graduate School of Medicine, Osaka University, Suita, Osaka 5650871 Japan.|Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871 Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D064113:CRISPR-Cas Systems; D002678:Chimera / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005260:Female; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques; D005804:Genes, Lethal; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics; D006849:Hydrocephalus / Q000235:genetics / Q000473:pathology; D054643:INDEL Mutation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D018832:Molecular Chaperones; D017354:Point Mutation; D011247:Pregnancy; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013081:Sperm Motility / Q000235:genetics; D013094:Spermatozoa / Q000002:abnormalities
chemical_list: D051017:Apoptosis Regulatory Proteins; C531170:DNAJB13 protein, mouse; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones; D000094704:RNA, Guide, CRISPR-Cas Systems