CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice

作者：Asami Oji, Taichi Noda, Yoshitaka Fujihara, Haruhiko Miyata, Yeon Joo Kim, Masanaga Muto, Kaori Nozawa, Takafumi Matsumura, Ayako Isotani, Masahito Ikawa · 发表于：Scientific Reports · 年份：2016 · DOI：10.1038/srep31666 · 被引用次数：104 · 研究领域：CRISPR and Genetic Engineering、Pluripotent Stem Cells Research、RNA regulation and disease

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.