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

by Selleckchem | Aug 31 2023

The need for precision control of CRISPR-Cas9 genome editing has created a demand for anti-CRISPR molecules. Recently, the first class of small-molecule Cas9 inhibitors has been identified, verifying the feasibility of regulating CRISPR-Cas9 activity using direct-acting small molecules. However, it remains enigmatic as to the location of the ligand binding site(s) on CRISPR-Cas9 and how the ligand binding leads to Cas9 functional inhibition. Here, we established an integrative computational protocol, including massive binding site mapping, molecular docking, molecular dynamics simulations, and free energy calculations. Ultimately, a Cas9 ligand binding site was discovered from the dynamics trajectories that is hidden within its carboxyl-terminal domain (CTD), a domain recognizing the protospacer adjacent motif (PAM). Using the top inhibitor BRD0539 as a probe, we demonstrated that the ligand binding induces significant CTD structural rearrangements toward an incompetent conformation for PAM DNA engagement. The revealed molecular mechanism of BRD0539 inhibiting Cas9 is in well agreement with the experimental data. This study provides a structural and mechanistic basis for the potency improvement of existing ligands and the rational discovery of novel small-molecule brakes for developing safer CRISPR-Cas9 technologies.

Comments:

The statement you provided describes a research study focused on identifying and understanding the binding site and mechanism of action of small-molecule inhibitors of CRISPR-Cas9, a genome editing tool. CRISPR-Cas9 has revolutionized the field of genetic engineering by enabling precise modification of DNA sequences. However, the need for improved control over CRISPR-Cas9 activity has led to the search for molecules that can inhibit its function.

In this study, the researchers employed a computational approach that involved several techniques such as binding site mapping, molecular docking, molecular dynamics simulations, and free energy calculations. By applying this integrative computational protocol, they were able to identify a binding site for small molecules on the carboxyl-terminal domain (CTD) of Cas9, which is responsible for recognizing a specific DNA sequence called the protospacer adjacent motif (PAM).

Using a specific inhibitor called BRD0539 as a probe, the researchers demonstrated that the binding of this molecule induced significant structural rearrangements in the CTD, rendering it unable to engage with the PAM DNA sequence effectively. This finding provided a molecular understanding of how BRD0539 inhibits Cas9 and was consistent with experimental data.

Overall, this study not only identified a specific binding site for small molecules on Cas9 but also elucidated the mechanism by which ligand binding leads to the inhibition of Cas9 function. This knowledge could be valuable for improving the potency of existing inhibitors and facilitating the rational discovery of new small molecules that can regulate CRISPR-Cas9 activity, thereby enhancing the safety and precision of CRISPR-based genome editing technologies.