Hijacking and recording intracellular RNAs in human cells using eukaryotic reprogrammed tracrRNAs

Recording intracellular RNA expression events is critical for understanding cell states and biological processes, but no method without responsive promoters is available in mammalian cells. Here, we determined the sequence formula of eukaryotic reprogrammed tracrRNA, named euRptr, which can hijack intracellular RNAs to make a complex with CRISPR-associated tools. By leveraging the programmability of euRptr, we devised the system “CHEETAH” to directly capture and record intracellular RNAs in human cells. We validated that CHEETAH induces self-editing by hijacking endogenous mRNAs, that CHEETAH selectively records different viral RNA fragments on a DNA reporter using base editors with a shared-euRptr, and that CHEETAH enables multiplexed, time-resolved recording of viral RNAs on a DNA Tape using prime editors, providing a new avenue for investigating intracellular RNA in eukaryotes.