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Updated: Jul 2, 2025
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Published on: May 30, 2025
Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx
Juan J Montero1,2, Riccardo Trozzo3,4, Maya Sugden3,4
1Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, Munich, Germany. jj.montero@tum.de.
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.
Insights
Researchers developed a genome-scale screening method using Cas13d/CasRx to explore the functions of long noncoding RNAs (lncRNAs). This RNA-targeting approach overcomes limitations, enabling a broader understanding of lncRNA biology in health and disease.
Area of Science:
- Genomics
- Molecular Biology
- RNA Biology
Background:
- Long noncoding RNAs (lncRNAs) are abundant but functionally understudied.
- DNA-directed perturbation methods are limited for lncRNA interrogation due to sequence overlap.
Purpose of the Study:
- To develop a genome-scale screening platform for functional lncRNA exploration.
- To overcome limitations of existing methods for studying the lncRNAome.
- To enable pan-cancer applicability and integrate molecular/phenotypic data for lncRNA function inference.
Main Methods:
- Developed a genome-scale lncRNA transcriptome screening system utilizing Cas13d/CasRx for RNA targeting.
- Created a size-reduced, multiplexed guide RNA library (Albarossa) targeting 24,171 lncRNA genes.
- Incorporated target prioritization based on expression, conservation, and tissue specificity for scalable throughput.
Main Results:
- Demonstrated that RNA targeting expands the explorable lncRNAome space.
- Successfully applied the screening platform across multiple cancer entities.
- Identified numerous context-specific and common essential lncRNAs.
Conclusions:
- The developed RNA-targeting screening platform is effective for systematic lncRNA exploration.
- This approach addresses challenges of transcriptome size and tissue specificity.
- The findings pave the way for a deeper understanding of lncRNA biology in health and disease.
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