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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • Chromosome Deletion
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Genetic Engineering / methods*
  • Genome
  • Mice
  • Mice, Inbred ICR
  • Mutation
  • RNA, Guide / chemistry
  • RNA, Guide / metabolism*
  • Zinc Fingers


  • CRISPR-Associated Proteins
  • RNA, Guide
  • Endodeoxyribonucleases