Protocol for genome-wide CRISPR knockout screens of bacterial cytotoxins in HeLa cells

CRISPR screening is a powerful tool to identify host factors for pathogenic agents including viruses and bacterial toxins. Here, we present a protocol to conduct a genome-scale CRISPR screen on HeLa cells for host factors involved in the toxin action of Clostridioides difficile TcdB4. We describe in detail how to prepare the library, set up the screen, obtain the gene sequences, and analyze the results. This protocol can also be modified for other genome-scale libraries, cell lines, and cytotoxins.For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).

Automated summary

The article introduces a protocol for conducting a genome-scale CRISPR screen on HeLa cells to identify host factors involved in the action of Clostridioides difficile TcdB4 toxin. The protocol provides detailed instructions on preparing the library, setting up the screen, obtaining gene sequences, and analyzing the results. This protocol can be adapted for other genome-scale libraries, cell lines, and cytotoxins as well.