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INTERNATIONAL CONGRESS OF

CancerGenetics

• Thermostabilization and Structural Optimization of Cas9 Variants Using Protein Stability Predictions and AlphaFold Modeling

• Mohammad Mehdi Sadehsani,^1,* Hosna Moghadasi,² Setayesh Alikhani,³ Zahra Eslami,⁴ Bahare Shadafza,⁵
  1. Department of Cellular and Molecular Biology, Islamic Azad University of Sari, Iran
  2. Alimohammadi Research Institute, Sari, Mazandaran, Iran
  3. Department of Cellular and Molecular Biology, Islamic Azad University of Sari, Iran
  4. Department of Cellular and Molecular Biology, Islamic Azad University of Sari, Iran
  5. Department of Cellular and Molecular Biology, Islamic Azad University Tehran Medical Branch, Tehran, Iran
  
  
  
• Introduction: CRISPR-Cas9 genome editing is an extremely effective tool; however, its use in thermophilic organisms and industrial processes is still quite far due to the instability of the protein at high temperatures. Here we have chosen the Streptococcus pyogenes Cas9 structure (PDB ID: Q99ZW2), and have done in silico thermostability prediction using ThermoMN. Based on these predictions, 25 rationally designed single-point mutations were generated to confer thermal stability to the protein while maintaining the functional domains. The thermodynamic study showed a large reduction in Gibbs free energy (ΔG) for the mutated protein, which means that protein stability was improved. These findings validate the utilization of computationally guided mutagenesis for the Cas9 thermostability that is a prerequisite for the implementation of thermophilic and industrial genome-editing platforms.
• Methods: The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), used the structure Q99ZW2 | CAS9_STRP1 from the Protein Data Bank. Structural analysis of SpyCas9 used a full-length structure to generate the computational stability predictions and provided the basis for chain-specific thermodynamic analysis of the native reference.
• Results: The ThermoMN analysis of the unmodified Cas9 enzyme showed that all chains together contributed a total Gibbs free energy (ΔG) of 106.206 kcal·mol⁻¹. The ΔG per chain of the original enzyme was 25.261, 29.109, 27.146, and 28.331 kcal·mol⁻¹ for chains A, B, C, and D, respectively. After mutagenesis, the rationally designed Cas9 variant had ΔG values of 18.793, 20.411, 21.232, and 22.126 kcal·mol⁻¹ for chains A-D, respectively, with a total Gibbs free energy of 82.967 kcal·mol⁻¹. The decrease in ΔG suggests a substantial improvement in overall thermodynamic stability. Notably, while modeled and designed developments imposed mutational adjustments, the mutational design honored the structural determinants of the essential functional domains - suggesting that the engineered Cas9 variant will keep its catalytic potential and gRNA binding capacity, while demonstrating greater thermostability.
• Conclusion: We demonstrate that with the aid of ThermoMN predictions, specific calculated mutagenesis can effectively alter and enhance the thermostability of SpyCas9. The engineered 25 mutations provided measurable and specific findings for decreasing the Gibbs free energy of the protein, and suggests improved thermodynamic stability, while keeping the structure and function of the essential functional domains intact. Our results provide strong rationale for the design of thermostable Cas9 variants, and further extends possibilities for CRISPR-mediated genome editing in thermophilic organisms, bioprocessing with industrial applications, and increased tolerance for thermal activity. The thermodynamic stability of native Cas9 was modeled with ThermoMN, which forecasts chain-specific Gibbs free energy (ΔG) contributions and can estimate protein stability at higher temperatures. The native enzyme yielded ΔG values of 25.261, 29.109, 27.146, and 28.331 kcal·mol⁻¹ for each of the chains A, B, C, and D, respectively, totaling 106.206 kcal·mol⁻¹. These baseline estimates allowed quantification of the rational mutagenesis experiments to follow. In order to engineer thermal stability we used the ThermoMN predictions table and introduced 25 single-point mutations on pathways that would start to add benefit through intramolecular interactions, hydrophobic packing and hydrogen bonding associated with the protein, whilst maintaining catalytic activity and gRNA binding. Further testing of the engineered Cas9 showed decreased ΔG of all time points, with ΔG values of 18.793, 20.411, 21.232, and 22.126 kcal·mol⁻¹ when applied for chains A, B, C, and D respectively, yielding a total of 82.967 kcal·mol. These results represent a significant increase in predicted thermodynamic stability and exhibit the success of the mutational design in enhancing Cas9 thermostability.
• Keywords: CRISPR-Cas9, Thermostability, Protein engineering, Rational mutagenesis, Gibbs free energy