title: CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation.
abstract: The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo.
authors: Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Yohei
authors: Y
authors: Shinmyo
authors: Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Satoshi
authors: S
authors: Tanaka
authors: Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Shinichi
authors: S
authors: Tsunoda
authors: Department of Bioinformatics and Genomics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Kazuyoshi
authors: K
authors: Hosomichi
authors: Department of Bioinformatics and Genomics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Atsushi
authors: A
authors: Tajima
authors: Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-8640, Japan.|Brain/Liver Interface Medicine Research Center, Kanazawa University, Ishikawa 920-8640, Japan.
authors: Hiroshi
authors: H
authors: Kawasaki
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002540:Cerebral Cortex / Q000196:embryology* / Q000378:metabolism; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005260:Female; D055786:Gene Knockout Techniques / Q000295:instrumentation / Q000379:methods*; D018014:Gene Transfer Techniques; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011247:Pregnancy; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014599:Uterus
chemical_list: D036961:Matrix Attachment Region Binding Proteins; C501473:SATB2 protein, mouse; D014157:Transcription Factors