Programmable CRISPR Protease Circuit for Targeted Cancer Cell Elimination

Abstract Precise targeting of malignant cells remains a central objective in oncology. We developed a programmable intracellular kill-switch that links oncogenic RNA recognition to activation of a lytic effector pathway. The system repurposes a Type III-E CRISPR-associated RNA-activated protease to detect mutation-specific transcripts and trigger cleavage of engineered gasdermin constructs. Activation of the protease liberates the pore-forming gasdermin domain, resulting in membrane permeabilization and cell death. The circuit was assembled and evaluated in Saccharomyces cerevisiae as a eukaryotic model platform. Inducible expression of the full system demonstrated trigger-dependent proteolysis and selective loss of viability. Control strains lacking the target RNA or expressing cleavage-resistant gasdermin variants remained unaffected, confirming mechanistic specificity. These findings establish a modular RNA-responsive cytotoxic framework that operates independently of genome editing and can be retargeted through guide RNA redesign. The work supports further investigation of CRISPR-guided proteases as precision therapeutic tools for transcript-specific cancer targeting.