Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins

Area of Science:

• Genomics
• Molecular Biology
• Microbiology

Background:

• Forward genetic screens using pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation are vital for functional genomics.
• CRISPR screens investigate genotype-phenotype relationships, enabling study of cellular responses to biological agents like bacterial toxins.
• Investigating toxin mechanisms is challenging due to the diverse effects of various toxins.

Purpose of the Study:

• To provide a detailed protocol for a genome-wide pooled CRISPR perturbation screen specifically designed for studying bacterial toxins.
• To outline technical considerations, experimental steps, and analysis methods for effective toxin-related genetic screening.
• To facilitate the discovery of host factors critical for cellular intoxication pathways.

Main Methods:

• Genome-wide pooled CRISPR perturbation screen protocol.
• Introduction of gain-of-function or loss-of-function mutations into cell populations.
• Analysis of perturbation-associated genotypes and screening for specific phenotypes.
• Detailed steps covering library construction, screen execution, and result validation.

Main Results:

• The protocol enables the investigation of bacterial toxin effects on host cells.
• Identifies host factors such as receptors, trafficking/translocation factors, and substrates involved in intoxication.
• Applicable to a wide range of bacterial toxins.

Conclusions:

• The developed CRISPR screening protocol is a robust tool for dissecting bacterial toxin-host interactions.
• It is expected to reveal crucial host factors mediating cellular responses to toxins.
• The protocol is adaptable and accessible, requiring only basic biological knowledge.