title: Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.
abstract: Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.
authors: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Masafumi
authors: M
authors: Inui
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Mami
authors: M
authors: Miyado
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Maki
authors: M
authors: Igarashi
authors: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Moe
authors: M
authors: Tamano
authors: Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.
authors: Atsushi
authors: A
authors: Kubo
authors: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Satoshi
authors: S
authors: Yamashita
authors: 1] Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan [2] Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan [3] CREST, Japan Science and Technology Agency (JST), Saitama 332-0011, Japan [4] Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
authors: Hiroshi
authors: H
authors: Asahara
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Maki
authors: M
authors: Fukami
authors: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors: Shuji
authors: S
authors: Takada
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation / Q000235:genetics*; D014162:Transfection / Q000379:methods*