Abstract
Approximately 75% of the human genome is transcribed into RNA, yet less than 5% encodes proteins, with the majority producing non-coding RNAs (ncRNAs). Among them, long non-coding RNAs (lncRNAs) represent a major class that exerts broad regulatory influence across cellular processes, disease contexts, and developmental stages. Despite their potential as biomarkers and therapeutic targets, their low sequence conservation, limited abundance, and structural complexity present significant challenges for functional characterization. Traditional RNA interference and CRISPR-Cas9-based methods have offered partial insights but remain limited in efficiency, specificity, and scalability. To address these barriers, Neville E. Sanjana's team developed CaRPool-seq, a transcriptome-scale CRISPR-Cas13 screening platform that directly targets RNA. Applying this approach across diverse human cell lines, they identified 778 essential lncRNAs, including 46 universally required for survival, with distinctive structural features and functional independence from neighboring protein-coding genes. Integration with single-cell transcriptomics revealed their critical roles in cell-cycle regulation, apoptosis, and developmental gene expression, as well as aberrant expression patterns in cancer linked to patient outcomes. This study establishes CRISPR-Cas13 as a precise and scalable strategy for lncRNA functional discovery, expanding opportunities for biomarker identification, therapeutic development, and precision medicine.