Energetics of Expanded PAM Readability by Engineered Cas9-NG

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Computational Chemistry

Background:

• The CRISPR-Cas9 system is a powerful gene-editing tool, but its targeting specificity is limited by the protospacer adjacent motif (PAM) sequence.
• Engineered Cas9 variants, such as Cas9-NG, have been developed to expand PAM recognition, but the energetic basis for this enhanced readability is not fully understood.

Purpose of the Study:

• To elucidate the energetic contributions of seven specific mutations in Cas9-NG that enhance protospacer adjacent motif (PAM) recognition.
• To understand the thermodynamic basis for the altered DNA cleavage activity and broader PAM recognition observed in Cas9-NG compared to wild-type SpCas9.

Main Methods:

• Utilized the X-ray structure of the SpCas9:sgRNA:dsDNA precatalytic complex as a structural template.
• Performed rigorous alchemical simulations (sampling ~53 μs) to calculate the changes in PAM binding affinity (ΔΔG) for each of the seven mutations.
• Analyzed the interaction energies between SpCas9 and DNA to correlate mutation type and location with binding affinity.

Main Results:

• The R1335V mutation was found to destabilize DNA binding by disrupting critical PAM interactions.
• Four mutations (E1219F, D1135V, L1111R, and T1337R) mitigated the destabilizing effect of R1335V by introducing non-base-specific interactions and enhancing PAM readability.
• Hydrophobic substitutions (E1219F, D1135V) were particularly effective, excluding solvent to strengthen electrostatic interactions and increase non-cognate PAM complex stability by 2-5 kcal/mol.

Conclusions:

• The stabilization of Cas9-NG: non-cognate PAM complexes, driven by mutation-induced desolvation and enhanced electrostatic interactions in hydrophobic pockets, enables broader PAM recognition.
• The location of amino acid mutations is crucial for shaping interaction energetics and improving the recognition of non-canonical PAM sequences.
• These findings provide a foundation for the rational design of SpCas9 mutants with improved and tailored PAM recognition capabilities for gene editing applications.