Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• CRISPR-Cas9 genome editing requires precise control, driving demand for anti-CRISPR molecules.
• Small-molecule inhibitors offer a feasible method for regulating CRISPR-Cas9 activity.
• The precise binding site and inhibitory mechanism of small molecules on Cas9 remain unclear.

Purpose of the Study:

• To identify the ligand binding site on CRISPR-Cas9.
• To elucidate the molecular mechanism by which small molecules inhibit Cas9 function.
• To provide a basis for developing improved and novel small-molecule CRISPR-Cas9 inhibitors.

Main Methods:

• Integrative computational protocol including binding site mapping, molecular docking, molecular dynamics simulations, and free energy calculations.
• Utilized the small-molecule inhibitor BRD0539 as a probe.
• Analyzed dynamics trajectories to identify ligand binding sites and structural changes.

Main Results:

• Discovered a novel Cas9 ligand binding site within the carboxyl-terminal domain (CTD), which recognizes the protospacer adjacent motif (PAM).
• Demonstrated that BRD0539 binding induces CTD structural rearrangements, leading to an incompetent conformation for PAM DNA engagement.
• Observed that the inhibitory mechanism aligns with experimental data.

Conclusions:

• The study reveals a hidden CTD binding site crucial for Cas9 function and inhibition by small molecules.
• The findings provide a structural and mechanistic understanding of small-molecule inhibition of CRISPR-Cas9.
• This work supports the rational design of more potent inhibitors and safer CRISPR-Cas9 technologies.