High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• CRISPR-Cas9 is widely used for loss-of-function screens, primarily in pooled formats with cell-survival assays.
• Arrayed screening formats, enhanced by synthetic crRNA libraries, enable diverse phenotypic readouts, including high-content morphology-based assays.

Purpose of the Study:

• To perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes involved in cell cycle regulation.
• To identify genes regulating the cell cycle using high-content analysis (HCA) and a novel multiparameter analysis technique.

Main Methods:

• Utilized a cell cycle reporter cell line for an arrayed screen targeting 169 genes with over 600 synthetic crRNAs.
• Employed high-content analysis (HCA) with seven parameters to classify cells and a new analysis technique to identify regulatory genes.
• Conducted comprehensive follow-up experiments, including gene expression analysis, indel confirmation, and orthogonal reagent validation.

Main Results:

• The screen identified genes regulating the cell cycle with high confidence and low off-target effects.
• Most validated hits showed consistent phenotypes across multiple independent crRNAs, indicating robust screening.
• The multiparameter HCA approach successfully identified subtle cell cycle phenotypes.

Conclusions:

• Arrayed screening with synthetic crRNAs and multiparameter HCA is a powerful approach for functional phenotypic screening.
• This methodology enhances the identification of genes with critical roles in cellular processes like the cell cycle.
• The study demonstrates the effectiveness of this advanced screening strategy for discovering novel biological insights.