Multiplexed mammalian gene control via modular splicing riboswitches

Precise, multiplexed gene regulation in mammalian cells remains constrained by a heavy reliance on heterologous protein regulators, whose associated immunogenicity and strict cargo-size limits impede clinical translation. Here, we introduce SpliceRACER (Splice-based R iboswitch A rray for C ombinatorial E xpression R egulation), a multiplexed RNA switch platform that achieves scalable, orthogonal, and combinatorial control of gene expression in mammalian cells through alternative splicing, eliminating the need for exogenous protein co-expression. SpliceRACER independently orchestrates three orthogonal channels to regulate fluorescent reporter outputs, yielding 3³ = 27 discrete combinatorial expression states, vastly exceeding the capacity of conventional binary gene switches. Furthermore, we engineered three generalizable strategies to effectively suppress the basal leakage inherent to synthetic riboswitches, thereby enabling tight OFF-state control of the potent pyroptotic effector GSDMD-NT for precise cell-death induction. This work offers broad utility in synthetic biology and therapeutic applications.