Open AccessCRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice
Author(s) -
Asami Oji,
Taichi Noda,
Yoshitaka Fujihara,
Haruhiko Miyata,
Yeon Joo Kim,
Masanaga Muto,
Kaori Nozawa,
Takafumi Matsumura,
Ayako Isotani,
Masahito Ikawa
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep31666
Subject(s) - genome editing , biology , crispr , cas9 , gene , genetics , genome , subgenomic mrna , phenotype , embryonic stem cell , zygote , point mutation , mutation , embryogenesis
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.