CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation

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.