title: Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions.
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.forename: Tomoko
authors.initials: T
authors.lastname: Kato
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Souichirou
authors.initials: S
authors.lastname: Kubota
authors.forename: Moe
authors.initials: M
authors.lastname: Tamano
authors.forename: Miho
authors.initials: M
authors.lastname: Terao
authors.forename: Shuji
authors.initials: S
authors.lastname: Takada
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D003062:Codon; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D016678:Genome; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008845:Microinjections; D009154:Mutation; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics
chemical_list: D003062:Codon; D018398:Homeodomain Proteins; C501599:Irx3 protein, mouse; C501601:Irx5 protein, mouse; D014157:Transcription Factors