Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes

CRISPR/Cas9-based gene editing is a recent advance that allows for the knockout or alteration of target genes within mammalian cells. Many variations of the technique exist, but here we describe two systems of plasmid-based CRISPR gene knockout which together allow for the selective knockout of virtually any gene target. Compared with other CRISPR-based systems, these plasmids have the advantages of delivering all the necessary components in one plasmid, choice of multiple selectable markers, and choice of route of administration into target cells. In addition, potential off-target effects from one system (dependent upon selection of target gene) can be overcome through use of the second system. Strategies for optimizing the knockout process and selection of finished cell lines are also presented.