Thermodynamics of PAM Recognition by Cas9 of <i>Streptococcus pyogenes</i>

The CRISPR/Cas9 system from <i>Streptococcus pyogenes</i> (<i>Sp</i>Cas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of <i>Sp</i>Cas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. <i>Sp</i>Cas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. <i>Sp</i>Cas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of <i>Sp</i>Cas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of <i>Sp</i>Cas9 to differentiate between noncanonical and canonical PAMs (ΔΔ<i>G</i>) is directly related to the number of direct interactions between <i>Sp</i>Cas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔ<i>G</i>. The calculated ΔΔ<i>G</i> adequately explains the observed differences in DNA cleavage activity of <i>Sp</i>Cas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in <i>Sp</i>Cas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.