ETTAS: a modular aptamer-recruited platform for programmable translational activation

Precise enhancement of endogenous protein synthesis offers a reversible therapeutic strategy without permanent genomic modification. However, existing Cas13-mediated translational activation systems are limited by modest potency and restricted modular expandability. Here, we developed the Enhanced Targeted Translational Activation System (ETTAS), a modular RNA-guided platform that combines dCas13a, the SINEB2 translational activation element, and an independently recruitable aptamer-mediated auxiliary module. Systematic ortholog screening identified dCas13a as the most effective scaffold for SINEB2-mediated translational activation, whereas direct tandem duplication of SINEB2 elements impaired rather than enhanced activity. To overcome this architectural limitation, we used aptamer-mediated recruitment to spatially separate target recognition from auxiliary activation. A binding-validated, non-interfering dCas13a-binding aptamer enabled construction of a dual-module system in which an aptamer-recruited SINEB2 element enhanced translation without altering target mRNA abundance or stability. Compared with the previously reported dCasRx-SINEB2 system, ETTAS produced stronger reporter activation, stronger endogenous induction of P53 and PTEN, and greater antiproliferative and pro-apoptotic effects in bladder cancer cells. Proteomic analyses showed selective target protein upregulation with limited global perturbation. In vivo, dual-AAV delivery of ETTAS activated endogenous P53 and suppressed tumor growth. ETTAS establishes a programmable framework for modular post-transcriptional upregulation of endogenous proteins.