A Positive, Growth-Based PAM Screen Reveals Non-Canonical Motifs Recognized By the S. Pyogenes Cas9

CRISPR technologies have overwhelmingly relied on the ubiquitous and extensively studied Cas9 from Streptococcus pyogenes (SpCas9). One well-defined feature of SpCas9 is its recognition of the canonical NGG and less-preferred NAG and NGA protospacer-adjacent motifs (PAMs) as the complete set used for target selection and off-target predictions. Here, we report that SpCas9 recognizes additional motifs at levels similar to that of the less-preferred PAMs. These motifs were identified using a positive, growth-based screen in E. coli that surprisingly enriched N(A/C/T)GG sequences over the consensus NGG. The enriched N(A/C/T)GG motif effectively represents an extra N between the target and consensus NGG. DNA binding and cleavage assays in bacteria validated the screen’s enriched sequences while indel formation and subsequent GFP disruption validated the N(C/T)GG motif in mammalian cells. Within these experiments, we found that the first two PAM nucleotides contributed to the overall targeting activity. We also discovered that high-fidelity SpCas9’s recognized the N(C/T)GG motif, while the editing location for SpCas9-derived base editors could be shifted by selecting (C/T)GG or N(C/T)GG PAMs. Finally, we found that these motifs were present but overlooked in high-throughput spacer acquisition and off-target assays. SpCas9 therefore exhibits greater targeting flexibility than previously recognized, with implications for precise editing, off-target predictions, and CRISPR-based immunity.