title: Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9.
abstract: The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice.
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Terao
authors.forename: Moe
authors.initials: M
authors.lastname: Tamano
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hara
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kato
authors.forename: Masato
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Shuji
authors.initials: S
authors.lastname: Takada
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D064130:CRISPR-Associated Proteins / Q000008:administration & dosage; D064113:CRISPR-Cas Systems; D003850:Deoxyribonuclease I; D000072669:Gene Editing / Q000379:methods*; D008817:Mice, Mutant Strains / Q000235:genetics*; D008845:Microinjections; D012313:RNA / Q000008:administration & dosage / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D015053:Zygote
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D003850:Deoxyribonuclease I