Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization

Area of Science:

• Cell Biology
• Genetics
• Molecular Biology

Background:

• Pooled-library CRISPR screening is a high-throughput method for genetic discovery.
• Current pooled screening methods are limited to phenotypes not requiring imaging.
• Complex cellular phenotypes like morphology and molecular organization necessitate imaging-based readouts.

Purpose of the Study:

• To develop an all-imaging-based pooled-library CRISPR screening approach.
• To enable high-throughput identification of genetic regulators for complex cellular phenotypes.
• To investigate the regulation of RNA localization in nuclear compartments.

Main Methods:

• Combined high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification.
• Utilized a lentiviral delivery system for codelivery of sgRNAs and barcodes.
• Employed Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) for accurate sgRNA identification.

Main Results:

• Successfully screened 162 sgRNAs targeting 54 RNA-binding proteins.
• Identified novel regulatory factors influencing RNA localization to nuclear compartments.
• Uncovered both positive and negative regulators of MALAT1 lncRNA localization to nuclear speckles.

Conclusions:

• The developed imaging-based CRISPR screen effectively analyzes complex cellular phenotypes.
• This method advances the study of genetic regulation of RNA localization.
• Revealed dynamic regulation of long noncoding RNA localization within subcellular compartments.