1. Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs
- Author
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Dong-Jiunn Jeffery Truong, Niklas Armbrust, Julian Geilenkeuser, Eva-Maria Lederer, Tobias Heinrich Santl, Maren Beyer, Sebastian Ittermann, Emily Steinmaßl, Mariya Dyka, Gerald Raffl, Teeradon Phlairaharn, Tobias Greisle, Milica Živanić, Markus Grosch, Micha Drukker, Gil Gregor Westmeyer, Universitat Politècnica de Catalunya. Doctorat en Enginyeria Biomèdica, Universitat Politècnica de Catalunya. BBT - Grup de recerca en Biomaterials, Biomecànica i Enginyeria de Teixits, Helmholtz Zentrum München. Institute for Synthetic Biomedicine, and Technische Universität München
- Subjects
Base Sequence ,Enginyeria biomèdica [Àrees temàtiques de la UPC] ,RNA ,RNA, Long Noncoding ,Cell Biology ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Introns - Abstract
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.
- Published
- 2021