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CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells. • Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons. • Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout. • PCR screening allows relatively fast and efficient determination of isoform-specific deletion.