Rapid and robust validation of pooled CRISPR knockout screens using CelFi

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Pooled CRISPR screens are essential for unbiased gene function discovery and identifying therapeutic targets.
• Validating hits from these screens is crucial but often requires robust and rapid methods.
• Current validation approaches may lack the precision to confirm observed genetic effects.

Purpose of the Study:

• To develop a simple and robust CRISPR-based method for validating hits from pooled CRISPR screens.
• To introduce the Cellular Fitness (CelFi) assay for assessing the impact of genetic perturbations on cell fitness.
• To provide a tool for rapid verification of putative gene function discoveries.

Main Methods:

• Developed a CRISPR-based assay (CelFi) to monitor indel profiles at target loci over time.
• The assay assesses the effect of genetic perturbations on cell fitness by tracking changes in indel frequency.
• Correlates indel profile alterations with selective growth advantages or disadvantages in individual cells.

Main Results:

• The CelFi assay effectively elucidates cellular vulnerabilities by monitoring genetic changes.
• It successfully verifies hits identified from pooled CRISPR knockout screens.
• Demonstrated utility in evaluating gene dependencies and testing hypotheses irrespective of experimental variables.

Conclusions:

• The CelFi assay offers a novel and efficient approach for hit validation in CRISPR screening studies.
• This method provides a quantitative measure of cellular fitness changes linked to specific genetic perturbations.
• CelFi enhances the reliability of gene function discovery and therapeutic target identification through precise validation.