Figure 1: CRISPR-Cas9 genome editing is inhibited by increasing target site PAM density.

(a) Schematic representation of engineered target sites tested for repair efficiency by CRISPR-Cas9. Blue highlight denotes the target site PAM, red highlights additional PAMs in the target site gRNA homology region, while underlined purple text denotes PAMs on the opposite (and non-homologous to the gRNA) strand. (b) TLR flow cytometry analysis. Shown are representative fluorescence scatter plots, with RFP+ cells (NHEJ repair events) versus GFP+ cells (HDR repair events) for the indicated cell lines. One plot is for cells transfected with Cas9 and a gRNA that targets the engineered site compared to a plot for the same cells transfected with Cas9 and gRNA that targets a upstream control site (VF2468). (c) Cumulative inhibitory fold-difference in Cas9-driven genome editing efficiency observed at the engineered target site for cell lines expressing TLR loci with increasing number of PAMs relative to the VF2468 control upstream site. N=4±s.e.m. (d) Cas9 protein levels cannot account for inhibitory fold-difference effects seen in the TLR assay. Expression of Cas9 for the various transfected ‘all-in-one’ vectors. Shown is a representative western blot measuring FLAG-epitope expression of the Cas9 protein in the various TLR lines (eEF2 serves as a loading control). (e) Differences in gRNA levels cannot account for the inhibitory effects observed in the TLR assay. Northern blot of RNA extracted from the indicated cell lines, each transfected with donor plasmid alone or with Cas9 and the labelled gRNA, either control VF2468 or 0x or 6x gRNAs. The probed transcripts are indicated on the right. Quantification of the fold-difference between the gRNA levels relative to control U6 RNA is shown underneath.