Extended Data Fig. 2: Structure-informed saturation mutagenesis and bacterial positive selections for SpCas9 PAM variant enzymes.

(a) Structural representation of the PAM-interacting (PI) ___domain of SpCas9 showing amino acid residues interacting with a canonical NGG PAM (from PDB ID: 4UN3)¹⁰. (b) Schematic of the bacterial positive selection assay. A plasmid encoding the SpCas9(6AA) library (with randomized NNS codons at SpCas9 positions D1135, S1136, G1218, E1219, R1335, and T1337), a sgRNA expression cassette, and chloramphenicol resistance gene is transfected into an E. coli strain harboring a selection plasmid encoding an inducible toxic gene and the Cas9 target site (with protospacer adjacent to a non-canonical 4 nt PAM of interest). Selections were performed similar to previously described^14,44,73, where the ccdB gene (encoding a DNA gyrase toxin) on the selection plasmid is induced by plating on arabinose-containing media. Bacterial colonies survive the selection when they harbor a plasmid that expresses an SpCas9 enzyme variant capable of cleaving the selection plasmid (by recognizing a non-canonical PAM). The schematic of the flask with yellow liquid was adapted from Clker (https://www.clker.com). (c) Summary of the SpCas9 enzymes that survived the bacterial positive selections using selection plasmids encoding each of the 16 NGNN PAMs. The heatmaps depict the percent of SpCas9 enzymes from each of the 16x selections that contain each possible amino acid substitution at each of the six SpCas9(6AA) library positions. Each heatmap is labeled based on the PAM utilized in that set of bacterial selections; the number of enzymes selected from each set of selections is indicated. The bottom panel represents a summary of the composition of amino acid residues at each of the six positions of the SpCas9(6AA) library.