Coevolutionary Couplings Unravel PAM-Proximal Constraints of CRISPR-SpCas9

The clustered regularly interspaced short palindromic repeats (CRISPR) system, an immune system analog found in prokaryotes, allows a single-guide RNA to direct a CRISPR-associated protein (Cas) with combined helicase and nuclease activity to DNA. The presence of a specific protospacer adjacent motif (PAM) next to the DNA target site plays a crucial role in determining both efficacy and specificity of gene editing. Herein, we introduce a coevolutionary framework to computationally unveil nonobvious molecular interactions in CRISPR systems and experimentally probe their functional role. Specifically, we use direct coupling analysis, a statistical inference framework used to infer direct coevolutionary couplings, in the context of protein/nucleic acid interactions. Applied to Streptococcus pyogenes Cas9, a Hamiltonian metric obtained from coevolutionary relationships reveals, to our knowledge, novel PAM-proximal nucleotide preferences at the seventh position of S. pyogenes Cas9 PAM (5'-NGRNNNT-3'), which was experimentally confirmed by in vitro and functional assays in human cells. We show that coevolved and conserved interactions point to specific clues toward rationally engineering new generations of Cas9 systems and may eventually help decipher the diversity of this family of proteins.