FIG 4.

RGN-directed mutagenesis of either the E6 or E7 locus induces cell cycle arrest in G₁. (A) S. pyogenes Cas9-mediated disruption of either the HPV-18 E6 or E7 gene results in the expected inhibition of HeLa cell growth. The number of GFP-positive cells transfected with vectors encoding S. pyogenes Cas9, a control or HPV-specific sgRNA, and the gfp gene is shown. (B) Cell cycle analysis of HeLa cells expressing E6- or E7-specific RGNs using BrdU incorporation and PI staining followed by fluorescence-activated cell sorter analysis. Results from four separate biological replicates of the transfected eGFP-positive HeLa cell population are shown. (C) Representative flow cytometry plots are shown for the sgRNAs indicated. The percentage of GFP-positive HeLa cells in each phase of the cell cycle was quantitated and is indicated. NS, nonspecific; APC, allophycocyanin.