Genetically Targeted Antifungal Therapeutics: A CRISPR-dCas9--RNAi Hybrid Framework with Dual-Gene Targeting, Evolutionary Escape Modeling, and In Vitro Validation Protocol

This study introduces an advanced framework for Genetically Targeted Antifungal Therapeutics (GTAT), incorporating dual-gene silencing of \textit{ERG11} and \textit{FKS1}, probabilistic modeling of evolutionary escape, and a comprehensive \textit{in vitro} validation protocol. Although the core model relies on synthetic mRNA data for demonstration, this limitation is explicitly acknowledged, with recommendations for RT-qPCR quantification in \textit{Candida albicans} and cytotoxicity assessments in HepG2 cells to confirm specificity and safety. The framework further models nuclease degradation kinetics, macrophage clearance, and potential immune activation by chitosan-lipid nanoparticles (CLNPs). Dual-gene knockdown reduces the evolutionary escape probability from $2.4 \times 10^{-3}$ (single-target) to $5.8 \times 10^{-6}$ (dual-target). All assertions are designed to be falsifiable, reproducible, and firmly rooted in established genomic, pharmacological, and immunological principles.