Exploring the Thermostability of CRISPR Cas12b using Molecular Dynamics Simulations

TL;DR

This study uses molecular dynamics simulations to understand how specific mutations enhance the thermal stability of the CRISPR enzyme BrCas12b, aiding the development of more robust diagnostic tools.

Contribution

It provides detailed insights into the stabilization mechanisms of mutant BrCas12b through all-atom MD simulations and dynamic analysis.

Findings

Mutations increase flexibility in the PAM-interacting domain.

Structural changes are minimal in other mutated domains.

Mutations confer enhanced thermal stability to BrCas12b.

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat)- based diagnostics are at the forefront of rapid detection platforms of infectious diseases. The integration of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) with CRISPR-Cas protein systems has led to the creation of advanced one-pot assays. The sensitivity of these assays has been bolstered by the utilization of a thermophilic Cas12 protein, BrCas12b, and its engineered variant, which exhibits enhanced thermal stability and allows for broader operation temperatures of the assay. Here, we perform all-atom molecular dynamics (MD) simulations on wild-type and mutant BrCas12b to reveal the mechanism of stabilization conferred by the mutation. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings not only offer a comprehensive insight into the dynamic alterations induced by mutations but reveal important motions in BrCas12b, important for the rational design of diagnostic and therapeutic platforms of Cas12 proteins.