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

Area of Science:

• Developmental Biology
• Genomics
• Stem Cell Biology

Background:

• Long non-coding RNAs (lncRNAs) play roles in development, but most are functionally uncharacterized.
• Understanding lncRNA function is critical for deciphering developmental processes.

Purpose of the Study:

• To develop novel human embryonic stem cell (hESC) lines for functional lncRNA screening during early human development.
• To identify functional lncRNAs essential for differentiation into all three germ layers.
• To establish a methodology for discovering and characterizing novel developmental regulators.

Main Methods:

• Generation of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) hESC lines.
• Genome-wide CRISPRi screening targeting lncRNA loci during endoderm differentiation.
• Development of a clustering approach to infer lncRNA mechanisms of action.
• Validation of identified functional lncRNAs, including FOXD3-AS1.

Main Results:

• Dozens of lncRNA loci were found to be essential for proper endoderm differentiation.
• Most differentially expressed lncRNAs were not functionally required, highlighting the need for functional screening.
• FOXD3-AS1 was identified and validated as a critical lncRNA for maintaining pluripotency and facilitating differentiation.
• A robust methodology for lncRNA discovery in developmental contexts was established.

Conclusions:

• The developed hESC lines and screening methodology enable efficient functional characterization of lncRNAs during early development.
• Functional screening is superior to expression analysis for identifying critical developmental regulators.
• FOXD3-AS1 is a key regulator of pluripotency and differentiation, underscoring the importance of lncRNAs in these processes.