Energetics of Expanded PAM Readability by Engineered Cas9-NG

Abstract

The energetic basis for the enhanced PAM (protospacer adjacent motif) readability in engineered Cas9-NG (a variant of Cas9 from Streptococcus pyogenes (SpCas9)) with seven mutations: (R1335V, E1219F, D1135V, L1111R, T1337R, G1218R, and A1322R) remains a fundamental unsolved problem. Utilizing the X-ray structure of the precatalytic complex (SpCas9:sgRNA:dsDNA) as a template, we calculated the changes in PAM (TGG, TGA, TGT, or TGC) binding affinity (ΔΔG) associated with each of the seven mutations in SpCas9 through rigorous alchemical simulations (sampling ∼ 53 μs). The underlying thermodynamics (ΔΔG) accounts for the experimentally observed differences in DNA cleavage activity between SpCas9 and Cas9-NG across various DNA substrates. The interaction energies between SpCas9 and DNA are significantly influenced by the type and location of the amino acid mutations. Notably, the R1335V mutation disfavors DNA binding by disrupting critical interactions with the PAM. However, the destabilizing effect of the R1335V mutation is mitigated by four advantageous mutations (E1219F, D1135V, L1111R, and T1337R), which primarily introduce nonbase-specific interactions and enhance PAM readability. The hydrophobic substitutions (E1219F and D1135V) are particularly impactful, as they exclude solvent from the PAM binding pocket, strengthening electrostatic interactions in the low dielectric medium and increasing the stability of the noncognate PAM complexes by ∼2–5 kcal/mol. Additionally, L1111R and T1337R facilitate DNA binding by forming direct electrostatic contacts. In contrast, the charge mutations G1218R and A1322R do not effectively promote interactions with the negatively charged DNA, clearly demonstrating that the location of mutations is crucial in shaping these interaction energetics. We demonstrated that stabilization of the Cas9-NG: noncognate PAM complexes enables broader PAM recognition. This is primarily achieved through two mechanisms: (1) the establishment of new nonbase-specific interactions between the protein and nucleotides and (2) the enhancement of electrostatic interactions within a relatively dry and hydrophobic pocket. The findings revealed that mutation-induced desolvation can improve the recognition of noncognate PAMs, paving the way for the rational and innovative design of SpCas9 mutants.