Cellular signal-responsive m5C editing by an inducible split CRISPR platform

RNA 5-methylcytidine (m⁵C) modification plays an essential role in regulating RNA metabolism and functions in cellular processes. Tools achieve temporal and transcript-specific m⁵C editing for functional studies are still limited. Furthermore, methods enabling m⁵C editing, triggered by specific cellular signals, can contribute to the understanding of m⁵C functions under specific physiological conditions but still lacking. Here, we present a temporally and conditionally controlled m⁵C writing platform engineered through integrating abscisic acid (ABA)-mediated chemically induced proximity with split-dCas13b-NSUN2/NSUN6 technology. This system enables the writing of m⁵C by reconstituting the split dCas13b-based m⁵C editing complex at the guide RNA (gRNA)-targeted RNA transcript sites under the control of the inducer ABA. The deposition of m⁵C is inducible, reversible and selective. The deposited m⁵C is biologically active and influence the stability of endogenous mRNA transcripts. Moreover, by incorporating ABA prodrugs, the m⁵C writing can be triggered by signals associated with distinct physiological or disease conditions (e.g., tumor microenvironment and senescence). This conditional m⁵C editing strategy provides a new programmable tool for studying m⁵C biology in context dependent manners and expands the repertoire of RNA modification editing technologies.