Abstract
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.
Highlights
IntroductionThe advent of deep sequencing technology has revealed the extensive and widespread nature of transcription across the human genome, with ~80% being transcribed at some point during
We found that 8,190 long non-coding RNAs (lncRNAs) genes corresponding to 12,611 lncRNA transcripts were expressed at 0.1 tpm in at least one of the three lineages (Fig 2D and S3B Fig in S1 Appendix; S1 Table)
Among the 1117 single guide RNAs (sgRNAs) that overlapped between our screen and CRiNCL, we found that sgRNA drop out correlated between the two screens (S5F Fig in S1 Appendix)
Summary
The advent of deep sequencing technology has revealed the extensive and widespread nature of transcription across the human genome, with ~80% being transcribed at some point during. Endoderm differentiation CRISPR interference screen targeting long non-coding RNA loci available at link: https://github.com/kmattioli/ 2019__lncRNA_CRISPRi. All processed data are available within the manuscript’s Supporting information files
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