With the rise of CRISPR technologies and the innovative use of T cells in personalized medicine, we have seen a huge boost in both the therapeutic potential of and interest in modified T cells. Genetic screens have become an incredibly valuable tool for discovering and validating new biological insights, drug targets, and understanding how drugs can either resist or become sensitive to treatments. Since the first successful demonstration of a lentiviral approach for pooled functional genomic screening in T cells¹, there has been a rise in advanced pooled CRISPR screening technologies in T cells. These advancements have significantly enhanced our ability to identify optimal genetic targets and develop more effective adoptive T cell therapies².

Advantages of pooled screening

Pooled screening offers the benefits of lower costs and a broader scope, making it a quick and reliable method for identifying potential new drug targets. At Revvity, our scientists have successfully completed several pooled lentiviral CRISPR-Cas9 knockout screens in primary T cells. From extensive development, our customers have reaped the benefits of our screening platform, which uses freshly isolated and stimulated primary T cells. These cells are first transduced with a lentiviral sgRNA library before introducing Cas9 mRNA.

In our validation screen (Figure 1), we conducted a drug-gene interaction analysis by exposing an edited pool of primary T cells to prolonged treatment with a sub-lethal dose of a specific small molecule inhibitor³. We took multiple time points and used T cells from various donors, allowing us to analyze response kinetics and evaluate donor-specific effects. To validate our platform, we monitored the T cells' response rate to a control set of sgRNAs and compared the performance of the primary T cell screen directly with a Jurkat cell line analysis.

Our screen successfully identified new genes that are crucial for the survival of primary T cells, as well as genes that influence how T cells respond to the small molecule inhibitor treatment. Because the drug we used had a known mechanism of action, these findings directly supported the specificity, sensitivity, and overall quality of our platform.