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Nucleic Acids Res. 2013 Nov;41(20):e187. doi: 10.1093/nar/gkt772. Epub 2013 Aug 30.

Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.

Author information

1
Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.

Abstract

The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (~10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.

PMID:
23997119
PMCID:
PMC3814358
DOI:
10.1093/nar/gkt772
[Indexed for MEDLINE]
Free PMC Article

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