Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency Haswell, Jeffrey R. Peinado Fernández, Paola Medina Vico, Pedro Pablo This work was funded by the US National Institutes of Health (NIH) grant P01GM099117, and by the National Science Foundation Graduate Research Fellowship under the grant DGE1144152 (J.R.H. and K.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage. 2022-03-09T07:40:16Z 2022-03-09T07:40:16Z 2021-11-03 journal article Haswell JR... [et al.] (2021) Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency. PLoS ONE 16(11): e0252848. [https://doi.org/10.1371/journal.pone.0252848] http://hdl.handle.net/10481/73240 10.1371/journal.pone.0252848 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España Public Library of Science