Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx

Juan J. Montero, Riccardo Trozzo, Maya Sugden, Rupert Öllinger, Alexander Belka, Ekaterina Zhigalova, Paul Waetzig, Thomas Engleitner, Marc Schmidt-Supprian, Dieter Saur, Roland Rad

Research output: Contribution to journalArticlepeer-review

Abstract

Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.

Original languageEnglish
Pages (from-to)584-596
Number of pages13
JournalNature Methods
Volume21
Issue number4
DOIs
StatePublished - Apr 2024
Externally publishedYes

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