Integrative GWAS and CRISPR Functional Validation of Gene Variant Structure of AtHKT1; 1 in KCl

Understanding ion transport mechanisms in plants is crucial for enhancing stress tolerance and crop yields. The Arabidopsis thaliana High-Affinity Potassium Transporter 1; 1 (AtHKT1; 1) is vital for maintaining sodium and potassium balance under saline stress. However, the structural and functional effects of genetic variants in AtHKT1; 1, especially in potassium chloride (KCl) environments, are not fully understood. This research combiness Genome-Wide Association Studies (GWAS) with CRISPR-based functional validation to examine AtHKT1; 1 gene variants, focusing on the protein structure with PDB ID: 8W9O. The study first used GWAS and SNP discovery to find significant genetic variations linked to ion transport and salt tolerance. Candidate SNPs were selected based on their statistical significance and potential biological roles. Structural analysis of the protein 8W9O involved PDB and MMDB resources, with validation through ERRAT and visualization/mutation mapping in PyMOL. InterProScan was used to identify conserved functional motifs. To validate SNP effects, CRISPR guide RNAs were designed with E-CRISP and CHOPCHOP, targeting key gene regions for precise editing. This integrated approach linked genetic variations to structural changes and their potential impact on ion binding and transport, especially under KCl conditions. Results showed certain SNPs cause conformational shifts in key transmembrane regions of AtHKT1; 1, possibly influencing ion selectivity and transport efficiency. Structural validation confirmed the accuracy of the modeled variants, and domain analysis revealed disruptions in conserved functional areas. CRISPR strategies proved feasible for precise gene editing to test these functional hypotheses. Overall, this study offers a comprehensive framework that combines GWAS, structural bioinformatics, and CRISPR technology to explore how genetic variants affect AtHKT1; 1 function. These insights improve understanding of ion transport regulation in saline environments and support the development of genetically modified crops with enhanced salt tolerance.