Understanding Improved Thermostability of Mutated BrCas12b Using Molecular Dynamics Simulations

In the wake of the COVID-19 pandemic, the landscape of CRISPR-based diagnostics has undergone a major evolution, promising transformative advancements in the field of infectious disease detection. The innovative fusion of pre-amplification techniques, notably reverse transcription-loop-mediated isothermal amplification (RT-LAMP), with CRISPR-Cas reactions has helped create a seamless one-pot diagnostic assay. While this integrated approach holds the potential for rapid pathogen detection, it introduces challenges associated with the elevated operational temperatures necessitated by RT-LAMP. To surmount this challenge, CRISPR-Cas proteins have been engineered to confer enhanced thermostability. A recent example of this is BrCas12b, which, following strategic mutagenesis, has emerged as a robust contender capable of withstanding high temperatures with high efficacies in single-pot assays for infectious diseases is evident. However, the underlying mechanisms that bolster this stability remain unclear. Such insights are imperative, not only for understanding the inherent attributes of BrCas12b but also for guiding the precise tailoring of other resilient Cas proteins tailored explicitly for detection purposes.

To address this, we employ all-atom molecular dynamics (MD) simulations aimed at elucidating the unfolding dynamics of the wild-type CrCas12b protein at elevated temperatures. Comparative analysis is conducted with a mutated variant as proposed by Nguyen et al. (Cell Rep. Med., 2023). Our assessment of unfolding dynamics employs a range of structural metrics, including root-mean-square deviation (RMSD), hydrogen bond count, and solvent accessible surface area (SASA). Notably, BrCas12b is divided into individual functional domains, to help capture the sequential order of their destabilization. Furthermore, we explore the thermal stability contributions of the N-terminus PEGylated BrCas12b and provide insights into its reinforcing effects. Looking ahead, we anticipate the correlation of our findings with experimental data to substantiate the predictive utility of MD simulations in devising effective strategies for enhancing stability. This study underscores the role of molecular dynamics simulations in unraveling the complexities of protein stability and offers a pathway for the rational design of thermally robust Cas proteins tailored for advanced diagnostics.