Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency

The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4 , which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN , which acts as a putative negative regulator of VRTN , affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.