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Truong, D.J.J. ; Armbrust, N. ; Geilenkeuser, J. ; Lederer, E.-M. ; Santl, T. ; Beyer, M. ; Ittermann, S. ; Steinmaßl, E. ; Dyka, M. ; Raffl, G. ; Phlairaharn, T. ; Greisle, T. ; Živanić, M. ; Grosch, M. ; Drukker, M. ; Westmeyer, G.G.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Nat. Cell Biol. 24, 1666-1676 (2022)

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Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

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