title: CRISPR/Cas9-Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation.
abstract: This unit describes a highly efficient and rapid procedure for brain-specific disruption of genes in the developing mouse brain using pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against target genes. The pX330 plasmids are delivered into the rodent brain using in utero electroporation. Focusing on the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor, we found that the introduction of pX330-Satb2 induced insertion/deletion (indel) mutations near the predicted cleavage site in the Satb2 gene, resulting in a dramatic reduction of Satb2 expression in post-mitotic neurons. Moreover, introduction of pX330-Satb2 induced abnormalities in axonal projection patterns, which was consistent with the phenotypes observed in Satb2 mutant mice. Thus, the procedure described here, combining the CRISPR/Cas9 system and in utero electroporation, is useful for knocking out genes of interest in the living rodent brain. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Department of Medical Neuroscience, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Shinmyo
authors.affiliation: Department of Medical Neuroscience, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japan.|Brain/Liver Interface Medicine Research Center, Kanazawa University, Ishikawa, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawasaki
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D018274:Electroporation / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods*; D054643:INDEL Mutation / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas9; cerebral cortex; gene knockout; in utero electroporation