A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions

Area of Science:

• Genetics
• Molecular Biology
• Systems Biology

Background:

• Epistasis analysis is crucial for understanding cellular pathways.
• CRISPR-Cas9 genetic screens offer genomic-scale insights but face challenges like Cas9 competition and limited temporal resolution.
• Assessing epistasis with essential genes and understanding kinetic impacts on viability are difficult with conventional methods.

Purpose of the Study:

• To develop and present a high-throughput, flow-based approach for studying genetic interactions.
• To overcome limitations of existing CRISPR screens, including Cas9 competition and temporal resolution.
• To enable robust epistasis analysis with both essential and non-essential genes.

Main Methods:

• Utilized two distinct Cas9 orthologs to mitigate competition issues.
• Implemented a flow-based system for monitoring cell viability.
• Collected and analyzed viability data at multiple time points post-perturbation.

Main Results:

• Successfully mitigated Cas9 competition and recombination challenges.
• Enabled high-resolution temporal analysis of genetic interaction impacts on cell viability.
• Facilitated the assessment of epistasis involving essential genes.

Conclusions:

• The developed flow-based CRISPR approach significantly enhances the study of genetic interactions.
• This method provides a powerful tool for dissecting cellular pathways and gene functions with improved accuracy and temporal detail.
• It offers a scalable solution for comprehensive genetic interaction mapping.