Cdk5 sequencing, CRISPR/Cas9-mediated knockout of the Cdk5 gene in the ferret cerebral cortex.. Mustela putorius furo

We recently found that genes of interest could be knocked out with high efficiency in the developing cerebral cortex by combining the CRISPR/Cas9 system and in utero electroporation (IUE) in mice. This result raised the possibility that gene knockout can also be achieved in the cerebral cortex of ferrets. We therefore tried knockout of the Cdk5 gene in the developing ferret cerebral cortex using our method. We cloned target sequences of Cdk5 in the pX330 plasmid, in which humanized Cas9 and synthetic chimeric guide RNA are simultaneously expressed under the chicken b-actin hybrid and human U6 promoters, respectively. To investigate the mutations caused by pX330-Cdk5, we introduced pCAG-EGFP and pX330-Cdk5 into the developing ferret cortex using IUE at E31. We then extracted genomic DNA from EGFP-positive areas of the cerebral cortex at postnatal day 0. The target site of the Cdk5 gene was amplified with PCR and sequenced using a next-generation sequencer. We found several kinds of deletion mutations near the predicted cleavage site in the Cdk5 gene. This result suggests that introduction of pX330-Cdk5 induces insertion and deletion (indel) mutations in the Cdk5 gene, leading to disruption of endogenous Cdk5 function.