Fig. 4: PAM repertoire profiling for the KG and VRKG variants.
From: Engineered dual selection for directed evolution of SpCas9 PAM specificity
a Plasmid cleavage assays with a randomized PAM library. Wt, KG, or VRKG Cas9 plasmids programmed with a targeting spacer in their sgRNA scaffold (Spacer 1 or Spacer 2) were co-delivered with the PAM library and plated with 10 µM IPTG and antibiotics to select for both plasmids. After deep sequencing, PAM frequencies were calculated for each pool of double transformants and normalized to their frequencies in the pre-depletion PAM library. For each spacer (X or Y axis), normalized PAM frequencies were plotted with binning for all possible sequences at PAM positions two through four. Gray dashed lines denote a normalized frequency value of 0.2 (fivefold depletion). Red dashed lines denote a normalized frequency value of 0.977; none of the normalized frequency values from Wt dCas9 control experiments with either spacer were lower than 0.97 when rounded to the nearest hundredth (Supplementary Data 1). b Same data from experiments in panel a, but re-plotted with binning for all possible sequences at PAM positions one through three. Source data are available in the Source Data file.
