Journal Article

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

Tanaka, Satoshi, Tsunoda, Shinichi, Shinmyo, Yohei, Kawasaki, Hiroshi, Hosomichi, Kazuyoshi, Tajima, Atsushi

2016

Overview

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 .

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 42/41 / 631/1647 / 631/378 / Animals / Axon guidance / Cerebral cortex / Cerebral Cortex - embryology / Cerebral Cortex - metabolism / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / Electroporation / Electroporation - instrumentation / Electroporation - methods / Female / Gene Knockout Techniques - instrumentation / Gene Knockout Techniques - methods / Gene Transfer Techniques / Humanities and Social Sciences / Matrix Attachment Region Binding Proteins - genetics / Matrix Attachment Region Binding Proteins - metabolism / Mice / multidisciplinary / Plasmids / Pregnancy / Rodents / Science / Transcription Factors - genetics / Transcription Factors - metabolism / Uterus