Cell panel screens play nicely with CRISPR screens: an integrated approach to drug target discovery.

Getting a long list of potential genes that influence the response to your new therapeutic is one sign of a successful CRISPR screen. Still, the hard work has only just begun. How do you prioritize this list of targets that could have an impact on the clinic? Bioinformatics and data mining can help, but other screens can also add value, including large cell panel screens with the same therapeutic of interest.

This integrated approach, combining Revvity's cell panel screening and functional genomic screening services, offers a powerful solution for drug development programs seeking to identify and validate disease-relevant targets.

A flexible CRISPRko library to fit any workflow

Revvity scientists have developed a whole-genome CRISPR knockout library that enables the use of four, six, or eight guides, depending on the biological question being addressed and the modality being used for the screen. For example, eight guides could be used to find genes that, when knocked out, result in sensitivity to a new drug in cancer cell lines in culture (a dropout screen), whereas a four-guide library might be best placed for in vivo CDX-based CRISPR screens where there is a need to keep the library small. This flexibility enables researchers to tailor their screening approach to meet specific project requirements, striking a balance between depth of coverage and practical considerations.

A proof-of-principle screen

To test the utility of this new CRISPRko library, Revvity's preclinical services team conducted a whole genome drug-gene interaction screen in the HT29 cancer cell line using the PARP inhibitor, olaparib. As reported in The CRISPR Journal (Blanck, M. et al. 2020), the screen successfully identified genes that, when knocked out, either increased or decreased sensitivity to the drug compared with DMSO-treated control cells. This demonstrated the library's effectiveness in identifying potential resistance mechanisms and synthetic lethal interactions.

Triaging hits with cell panel screening

So far, so good, but how best to triage this list? Large cell panel screens, which utilize many cancer cell lines representing various cancer types, can also be informative in identifying sensitivity and resistance mechanisms. Revvity scientists tested olaparib and an additional PARP inhibitor (talazoparib) across a panel of more than 300 cancer cell lines. Using these data, along with drug response and CRISPR screen data from the DepMap portal, a set of genes influencing sensitivity and resistance to these drugs was also compiled.

Substantial "Hit" overlap with complementary methods

The genes influencing sensitivity and resistance to olaparib and talazoparib in the cell panel screen had substantial overlap with the genes identified in the CRISPR screen. Genes involved in DNA damage response, cell survival, and cell division pathways were heavily involved. A combination drug screen using clinical-grade compounds was used to initially validate some of the genes whose loss appears to increase sensitivity to olaparib. Drug-mediated inhibition of proteins involved in DNA damage, such as ATM, ATR, CHK1, and CHK2, as well as proteins involved in cell survival, such as BCL-XL, and proteins involved in cell division, such as WEE1, all increased sensitivity to olaparib.

Integrated platform offers more disease-relevant information

Assessing overall concordance between Revvity's CRISPRko screen in HT29 cells and similar CRISPRko screens published in other cell types using olaparib shows that agreement between CRISPR screens carried out in different labs is substantially greater than that observed in RNA interference screens. However, differences between cell lines are still evident, indicating that robust conclusions from drug-gene interaction screens are best made from screens conducted across diverse genetic and lineage backgrounds to better describe the effects of the drug-gene interaction.

Conclusion

This integrated approach can be achieved with Revvity's fully integrated, pooled CRISPRko and high-throughput cell panel screening platform. The orthogonal approach of cell panel screening provides rapid initial validation of selective hits in high throughput, enabling targets to be considered in terms of their tractability in the clinic. These data also illustrate how CRISPR screens and high-throughput cell panel screens can be used to rapidly validate new, disease-relevant genetic interactions.