Programmable Targeted RNA Degradation via dCas13d-Directed Chaperone-Mediated Autophagy (dCasCMA)

RNA-targeted degradation technologies offer significant promise for treating diseases by selectively disrupting gene expression. However, a robust method to specifically, efficiently, and programmability degrade targeted RNAs in mammalian cells is still in demand. Here, we present a versatile platform, dCas13d-directed chaperone-mediated autophagy (dCasCMA), which integrates the precise targeting capabilities of dCas13/CRISPR with the degradation efficiency of chaperone-mediated autophagy (CMA) to achieve efficient degradation of specific RNAs. By combining dCas13d with a CMA-targeting motif and customizable guide RNA (gRNA), the platform allows for accurate targeting of both exogenous and endogenous RNAs in cells. Moreover, the incorporation of multiplexed gRNA expression arrays enables the simultaneous degradation of multiple RNA targets during viral pathogenesis in live cells and in vivo. Our findings emphasize the platform’s modular design, which enables flexible combinations of dCCTM components with user-defined gRNA sequences. This versatility positions it as a promising tool for developing innovative therapies for various diseases. 293T cells were transfected with constructs expressing either WT Cas13d or dCCTM, each paired with EGFP-targeting gRNAs or non-targeting (NT) gRNA, followed by RNA-Seq analysis.