Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Abstract

Systematic identification of regulatory factors that control human embryonic stem cell (hESC) differentiation can provide valuable insights into early development and disease. The discovery of the CRISPR/Cas gene editing system and its subsequent development in mammalian cells has revolutionized functional genomic studies. However, despite these advances, effective modulation of gene expression during multi-lineage differentiation of stem cells has proven technically challenging. Here, we report the first described hESC lines to repress (via CRISPR interference) or activate (via CRISPR activation) transcription during differentiation into all three germ layers. We subsequently performed an unbiased, genome-wide, FACS-based CRISPRi screen targeting long non-coding RNAs (lncRNAs) during definitive endoderm differentiation. Our screen yielded dozens of hits, including both known and novel lncRNA loci required for proper differentiation. Analysis of common features revealed that hits were more conserved, closer to enhancers, and significantly enriched for endoderm-specific GWAS SNPs compared to non-hits. Surprisingly, expression was not predictive of enrichment scores, supporting the need for functional screening instead of relying solely on gene expression analysis. Further downstream analysis of two hits, RP11-222K16.2 and FOXD3-AS1, revealed two lncRNA loci required for differentiation and pluripotency, respectively. Taken together, the cell lines and screen methodology described herein can be adapted to discover novel regulators of differentiation into any lineage.