In-silico study of molecular adaptations in halophilic Cas9.

This study explores the structural adaptations of the CRISPR-Cas9 system in halophilic bacteria, focusing on Cas9 protein of halophilic bacterium Salicibibacter cibi. Protein sequences were analysed using different tools such as ExPASy ProtParam for different physicochemical properties, PONDR web server for disordered regions, and InterPro server and WebLogo for domains. Protein structures were generated using the AlphaFold database, and the quality of the modelled structure was checked through PROCHECK. The protein surface's amino acids and electrostatic potential were visualized using PyMOL, APBS server and UCSF chimera. Comparative analysis revealed that halophilic Cas9 proteins possess a higher abundance of acidic residues, resulting in enhanced stability and hydration in saline conditions; halophilic Cas9 proteins also shows higher intrinsically disordered regions. Electrostatic potential maps confirmed that S. cibi Cas9 proteins maintain a highly negative surface charge, crucial for adaptation to salt-rich environments. These findings provide insights into the molecular mechanisms driving the structural and functional adaptations of Cas9 in salty environment, highlighting its potential applications in genome editing-based biotechnological approaches in extreme conditions.