SynAuto-X: A Programmable CRISPR-dCas9 Circuit for Precision Inflammation Control in Autoimmune Disorders

Autoimmune diseases represent a class of complex, multifactorial disorders characterized by the immune system’s aberrant recognition of self-antigens as foreign, leading to chronic inflammation, tissue damage, and progressive organ dysfunction. Despite advances in immunosuppressive pharmacology, current therapies remain largely non-specific, systemically cytotoxic, and incapable of achieving long-term immune tolerance. In response to this therapeutic gap, we present SynAuto-X—a synthetic biology platform integrating a programmable CRISPR-dCas9-based gene circuit designed for the conditional silencing of pro-inflammatory genes that drive autoimmune pathology. SynAuto-X employs a modular genetic architecture based on catalytically inactive Cas9 (dCas9) fused to the KRAB (Krüppel-associated box) repressor domain. This fusion enables precise transcriptional repression of key cytokine and co-stimulatory genes (e.g., IL17A, IFNG, TNFA, CD80) through CRISPR interference (CRISPRi), directed by customizable single-guide RNAs (sgRNAs). Gene silencing is driven by inflammationresponsive promoters, such as NF-B or STAT1 elements, allowing context-specific activation of the repression cascade only in immune cells undergoing aberrant activation. The architecture supports single-input singleoutput (SISO) and combinatorial logic designs, enabling multigene targeting, threshold-dependent activation, and Boolean gating of inflammatory response elements. A key innovation within SynAuto-X is the integration of a drug-inducible kill switch for enhanced biosafety. Utilizing a chemically controlled inducible Caspase-9 (iCasp9) domain responsive to the synthetic small molecule AP1903, the system enables externally triggered apoptosis of transduced cells upon detection of adverse immune modulation or unintended off-target effects. The dynamics of this safety module are modeled through coupled differential equations, representing CID-mediated dimerization and apoptotic cascade activation, with tunable parameters for dose-responsive control. Additionally, the full circuit is encoded within a lentiviral vector framework, optimized via Benchling and CHOPCHOP for minimal off-target sgRNA activity, promoter leakiness, and vector immunogenicity. SynAuto-X represents a programmable, safe, and cell-specific alternative to broad-spectrum immunosuppression. By combining precision CRISPR interference, logic-based gene circuit design, and fail-safe therapeutic control, SynAuto-X advances the frontiers of autoimmune treatment toward intelligent, synthetic immune regulation. This work lays the foundation for a new class of gene circuit-based therapeutics capable of rewiring immune responses with the specificity of molecular computation and the safety of on-demand self-destruction.