ERP145837 PRJEB60776 5abd8915-cc92-44e1-bcb2-632e8bd83443 Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx Although lncRNAs dominate the transcriptome, their functions are largely unexplored. lncRNA characteristics, such as extensive overlap with coding and regulatory sequence restrict 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 towards 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 targeting 24,171 lncRNA-families. 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. undefined Although lncRNAs dominate the transcriptome, their functions are largely unexplored. lncRNA characteristics, such as extensive overlap with coding and regulatory sequence restrict 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 towards 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 targeting 24,171 lncRNA-families. 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. ENA-FIRST-PUBLIC 2023-12-01 ENA-LAST-UPDATE 2023-12-01