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Sci Rep. 2016 Aug 17;6:31666. doi: 10.1038/srep31666.

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

Oji A1,2,3, Noda T1,3, Fujihara Y1, Miyata H1, Kim YJ1, Muto M1,2,3, Nozawa K1,3,4, Matsumura T1,2, Isotani A5, Ikawa M1,2,4,5.

Author information

1
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871 Japan.
2
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871 Japan.
3
Research Fellow of Japan Society for the Promotion of Science (JSPS), Tokyo 1020083, Japan.
4
Graduate School of Medicine, Osaka University, Suita, Osaka 5650871 Japan.
5
Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871 Japan.

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.

PMID:
27530713
PMCID:
PMC4987700
DOI:
10.1038/srep31666
[Indexed for MEDLINE]
Free PMC Article

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