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

Systematic identification of factors that regulate human embryonic stem cell (hESC) differentiation can provide valuable insights into early development and disease. However, effective modulation of gene expression during differentiation has proven technically challenging. Here, we report the first described hESC lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers. We subsequently performed an unbiased, genome-wide CRISPRi screen targeting long non-coding RNAs during endoderm differentiation. From our screen, we identified known factors, as well as novel lncRNAs required for differentiation. Surprisingly, lncRNA expression was not predictive of enrichment scores, supporting the necessity for functional screening instead of relying on gene expression analysis. Further downstream analysis of two hits, RP11-222K16.2 and FOXD3-AS1, revealed two lncRNAs essential 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. Overall design: We performed two experiments: (1) RNA-sequencing of three lineages (undifferentiated hESCs, endoderm, and mesoderm) and (2) a CRISPR interference screen aimed at assessing lncRNA loci in endoderm differentiation. For the RNA-seq, we provide the raw fastqs for each of the 2 replicates as well as the GENCODE v25 transcript abundances (as determined by kallisto). For the CRISPRi screen, we provide the raw fastqs for three populations: Day Zero input, FOXA2/SOX17 double positive endoderm cells, and FOXA2/SOX17 double negative undifferentiated cells, as well as the corresponding sgRNA counts for each population.