Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• CRISPR-Cas nucleases are crucial for molecular diagnostics.
• Thermal stability is essential for high-temperature assays like isothermal amplification.
• Understanding protein stabilization mechanisms informs tool design.

Purpose of the Study:

• To elucidate the atomistic mechanism of thermal stabilization in an engineered BrCas12b variant.
• To investigate the impact of specific mutations on BrCas12b protein dynamics and stability.
• To provide insights for designing improved CRISPR-based diagnostic tools.

Main Methods:

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

Main Results:

• Mutant BrCas12b exhibited greater flexibility in the PAM-interacting domain at high temperatures.
• Marginal structural and flexibility changes were observed in other mutated domains.
• MD simulations provided insights into the stabilizing effects of specific mutations.

Conclusions:

• Specific mutations enhance the thermal stability of BrCas12b by altering protein dynamics.
• Understanding these stabilizing effects can guide the rational design of CRISPR-Cas nucleases.
• This research contributes to the development of more robust CRISPR-based diagnostic platforms.