title: Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.
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.
authors.affiliation: Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Fujii
authors.forename: Kurenai
authors.initials: K
authors.lastname: Kawasaki
authors.forename: Koji
authors.initials: K
authors.lastname: Sugiura
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Naito
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D002872:Chromosome Deletion; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D051379:Mice; D008813:Mice, Inbred ICR; D009154:Mutation; D016335:Zinc Fingers; D058727:RNA, Small Untranslated
chemical_list: D064130:CRISPR-Associated Proteins; D004706:Endodeoxyribonucleases; D058727:RNA, Small Untranslated