title: Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.
abstract: The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.
authors.affiliation: Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: mhashimo@fbs.osaka-u.ac.jp.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Laboratory for Embryology, Institute for Advanced Medical Sciences, Tokushima University, 3-18-15 kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yamashita
authors.affiliation: Laboratory for Embryology, Institute for Advanced Medical Sciences, Tokushima University, 3-18-15 kuramoto-cho, Tokushima 770-8503, Japan. Electronic address: takemoto.tatsuya@tokushima-u.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Takemoto
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing; D055879:Gene Knock-In Techniques / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009030:Mosaicism / Q000196:embryology*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D015053:Zygote / Q000166:cytology*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D004720:Endonucleases
keywords: CRISPR/Cas9 system; Cas9 protein; Electroporation; Genome editing