title: Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9.
abstract: Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hara
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Moe
authors.initials: M
authors.lastname: Tamano
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Saito
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Inui
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Takada
mesh_terms: D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific