Fig. 3: Identification of D1135V/S1136R substitutions that augment NGG discrimination within the KG background.

a List of non-synonymous PID substitutions identified in a dual-selection ω-dCas9 library experiment with random mutagenesis in the KG variant’s PID. The reporter plasmid’s PAM configuration is displayed above with the counterselection PAM in brackets. Cyan and violet lettering was used to emphasize the S1136R and D1135V substitutions, respectively, which were multiply represented. b GFP activation assays comparing Wt to the mKG-d4 isolate and its single-reversion (Δ) derivatives as indicated, with normalization as in Fig. 1b. The PAMs present in each single-target reporter plasmid is indicated below. Error bars, mean ± s.d. (n = 3, biological replicates). c Plasmid cleavage assays comparing catalytically intact Wt, KG, and VRKG plasmids co-transformed with the single-target plasmids tested in Fig. 1c. 10-fold serial dilutions were plated in parallel on rich media with antibiotics to select for both plasmids and a minimally inducing concentration of IPTG (2.5 µM) for Cas9 expression. Source data are available in the Source Data file.