INTERNATIONAL CONGRESS OF
CancerGenetics
Design and Computational Analysis of CRISPR-Based Base Editors: Predicting Efficiency and Protein Stability for Precision Gene Editing
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Mohammad Mehdi Sadehsani,1,* Bahare Shadafza,2 Ali Gholizad,3 Eiliya Panahi,4 Ilin Dadashi,5
1. Department of Cellular and Molecular Biology, Islamic Azad University of Sari, Iran
2. Department of Cellular and Molecular Biology, Islamic Azad University Tehran Medical Branch, Tehran, 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 of Sari, Iran
- Introduction: CRISPR-based base editors allow for precise single-nucleotide changes, without generating double-strand breaks, but their application relies on proper gRNA targeting and a stable and active, functional, fusion protein. The purpose of this study was to use computational approaches to assess the structural, thermodynamic, and physicochemical characteristics of an SpCas9-sgRNA base editor (PBD: 6VPC, Chain A). Using SWISS-MODEL and ThermoMN, we evaluated the domain orientation, thermal stability, solubility, and predicted half-life in a variety of cellular parameters. The protein exhibited a very stable Instability index (37.36) and demonstrate a very high aliphatic index (89.76), and hydrophilic characteristics (GRAVY = -0.585). These findings provide an indication that the base editor is suitable for strong expressivity and functional performance. Our extinction coefficients showed very minimal contribution from disulfide bonds, while our predicted in vivo half-life indicated potential rapid degradation potential for the base editor in various cellular environments. This workflow represents a streamlined computational approach to selecting and optimizing base editors and may serve to inform gRNA targeting practices and the protein engineering process in order to maximize editing efficiency and base editor structural stability.
- Methods: For this analysis, we selected a representative CRISPR-based base editor for an extensive computational analysis of its structure and stability levels. The details are as follows. The protein of interest was the SpCas9-sgRNA complex from Streptococcus pyogenes that is associated with PDB, entry ID 6VPC (Chain A). This complex represents the Cas9 nuclease in association with a single-guide RNA (sgRNA), which plays a central role in the functionality of CRISPR-associated genome editing systems. The high-resolution structure, obtained from the Protein Data Bank, permitted an accurate analysis of the protein's three-dimensional alignment of each domain orientation and active-site accessibility. The familiar assessment of some structural characteristics of the protein was provided through the variable modeling of SWISS-MODEL, which produces information on various aspects of secondary and tertiary structure, domain order, and possible functional regions of the protein. After the assessment of structure, we proceeded with an extensive stability analysis through the use of ThermoMN. ThermoMN is a computational tool for predicting the thermodynamic stability and the majority of relevant physicochemical properties. Several aspects of the protein were calculated, such as the molecular formula of protein (C₇₀₈₀H₁₁₂₈₂N₁₉₃₂O₂₀₈₈S₂₃) and the total number of atoms in the protein (22,405), in addition to several other stable and useful indicators of stability and solubility (Instability Index, aliphatic index and GRAVY). The Instability Index of 37.36 determined the protein to be stable and a high aliphatic index (89.76) indicated strong thermal stability over an extensible temperature range. The negative GRAVY value (-0.585) determined that the protein is generally hydrophilic and can be expected to be soluble in aqueous environments. Also evaluated were extinction coefficients at 280 nm where the oxidized and reduced states were nearly equal which indicated that absorbance in this region of the spectrum was not significantly influenced by the presence of disulfide bonds. The predicted half-life in vitro was 1.3 hours (mammalian reticulocytes) and 3 minutes in vivo (yeast and E. coli) where the reason for the low predicted half-life in vivo was likely due to an N-terminal lysine residue that directed it to cytoplasmic degradation via ubiquitination pathways. By combining structural modeling from SWISS-MODEL with stability predictions from ThermoMN, we were able to evaluate the protein's physical and thermodynamic properties as well as to quantitatively assess overall molecular stability. This workflow can be seen as a standard protocol to assess base editor stability, solubility and suitability for practical gene-editing applications.
- Results: The SpCas9-sgRNA complex investigated in this research exhibited promising structural and stability attributes. The Instability Index of 37.36 categorized the protein as stable, corroborated by maintaining functional folding when shifting from test tube to physiological conditions. The thermal stability exhibited a high aliphatic index of 89.76, indicative of resilience to temperature variation effects. Hydropathy yielded a GRAVY index of -0.585, indicating a largely hydrophilic nature of the protein, so likely soluble in aqueous environments, which favors cellular expression. The extinction coefficient determination showed insignificant differences (as a factor of 120,575 calculated with, and 120,450 in the fully reduced form) between states reinforced the immaterial contribution of disulfide bonds to either absorbance and folding stability. Predictive half-life determinations exposed that the in vitro stability is acceptable by mammalian reticulocytes (1.3 hours, overall), while the in vivo half-life is much shorter (3 minutes E. coli and yeast), perhaps as a result of the N-terminal lysine residue, which serves a role in ubiquitination pathways. Although substantial work remains to conduct protein engineering for the purpose of increasing in vivo stability, which would increase base editor efficacy, these results suggest inherent stability in the Cas9-sgRNA complex.
- Conclusion: Our computational analyses reveal that the SpCas9-sgRNA base editor demonstrates favorable thermodynamic and structural features, supporting its use in genome-editing applications. Having favorable thermal stability, solubility, and a stable folding signature reinforces that the protein possesses favorable function capabilities under physiological conditions. However, short in vivo half-lives may suggest targeted modifications to enhance cellular persistence and overall editing efficiencies, such as N-terminal stabilization or optimal fusion domains. This study combined structural modelling with thermodynamic and physicochemical considerations providing a pathway to rationale design and optimization of CRISPR-based base editors, which would decrease experimental screening and accelerate precise gene-editing strategies.
- Keywords: Base Editors, Computational Analysis, Protein Stability, CRISPR-Cas9
