GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Genome-wide CRISPR screens are vital for discovering genes influencing cell fitness.
• Loss-of-function screens face challenges due to limited dynamic range in detecting decreased sgRNA sequences.

Purpose of the Study:

• To introduce Guide-Only control CRISPR (GO-CRISPR), an advanced loss-of-function screening workflow.
• To present the companion software, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS).
• To enhance the discovery of essential genes in difficult biological contexts.

Main Methods:

• Development of the GO-CRISPR workflow incorporating guide-only controls.
• Application of GO-CRISPR in a 3D spheroid model of dormant high-grade serous ovarian cancer.
• Utilizing the TRACS software for analyzing pooled sgRNA library quality and guide-only controls.

Main Results:

• GO-CRISPR demonstrated superior performance compared to standard screening methods.
• TRACS successfully identified novel molecular pathways in tumor dormancy.
• The workflow proved effective in growth-arrested cells, overcoming limitations of other analysis packages.

Conclusions:

• GO-CRISPR and TRACS significantly improve the discovery of essential genes in challenging biological scenarios.
• This integrated approach enhances the reliability and scope of loss-of-function CRISPR screens.
• The methodology is particularly valuable for studying non-proliferative or dormant cell states.