Exploring the Thermostability of CRISPR-Cas12b using Molecular Dynamics Simulations

Area of Science:

• Biotechnology
• Molecular Biology
• Structural Biology

Background:

• CRISPR-based diagnostics offer rapid detection of infectious diseases.
• Integrating reverse transcription-loop-mediated isothermal amplification (RT-LAMP) with CRISPR-Cas systems creates advanced one-pot assays.
• Thermophilic Cas12 proteins, like BrCas12b, enhance assay sensitivity and thermal stability.

Purpose of the Study:

• To elucidate the stabilization mechanism of a mutant BrCas12b protein using all-atom molecular dynamics (MD) simulations.
• To understand the dynamic alterations induced by mutations in BrCas12b.
• To provide insights for the rational design of Cas12 protein-based diagnostic and therapeutic platforms.

Main Methods:

• All-atom molecular dynamics (MD) simulations were performed on wild-type and mutant BrCas12b.
• Simulations were conducted at both ambient and elevated temperatures.
• Comparative essential dynamics analysis was used to compare protein dynamics.

Main Results:

• High-temperature simulations showed increased flexibility in the PAM-interacting domain of mutant BrCas12b.
• Mutations conferred enhanced thermal stability to BrCas12b.
• MD simulations revealed specific dynamic motions in BrCas12b crucial for its function.

Conclusions:

• Mutations in BrCas12b enhance its thermal stability through specific dynamic alterations.
• Understanding these dynamics is key for developing next-generation CRISPR diagnostic tools.
• The findings support the rational design of improved Cas12 protein variants for various applications.