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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.

Numerous genomic loci have been implicated in autoimmune disorders, but attempts to identify causal variants, and thereby disease mechanisms, have been hampered by strong linkage disequilibrium; leaving most loci unresolved and the potential of GWAS largely unfulfilled. To overcome this, we developed a massively-parallel reporter assay for use in primary CD4 T-cells, a key effector of many autoimmune diseases, and sought to resolve potential causal variants via their functional effects. This provided testable hypotheses into disease mechanisms, which we illustrate using a multi-disease-associated locus on chromosome 6. By investigating the lead expression-modulating SNP, we uncover an NF-κB-driven regulatory circuit which constrains T-cell activation through the dynamic formation of a super-enhancer that upregulates expression of TNFAIP3 (A20), a key NF-κB inhibitor. In activated T-cells, this circuit is disrupted – and super-enhancer formation prevented – by the risk variant at the lead SNP, thus revealing a molecular mechanism that predisposes to multiple autoimmune diseases.