In this project, we use the Nobel prize-awarded methodology Crispr/Cas9 to extend our understanding of NK cell cytotoxicity. Using specific guide-RNAs (gRNA) we can knock out a specific gene of interest from Cas9-expressing malignant cells. In CRISPR sceens, we expose malignant cells to a lentiviral library of gRNAs targeting many different genes. After exposure to NK cells, knockouts conferring resistance to NK cells will be enriched, while knockouts sensitizing the target cells to NK cells will be depleted. Using NGS (next-gen sequencing), we can identify resistor and sensitizer genes.
A challenge with a CRISPR screen approach in an NK cell context is that the NK cytotoxic responses may result from several different activating receptors acting in concert to kill a target cell. As the experimental conditions needed to generate firm enrichment of survival mutants are usually harsh, a gene knock-out that confers a partial protection may be undetected as other activating signals are sufficient to kill the target cell. Alternatively, the killing may be highly dependent on one receptor-ligand pair, which will prevent any other deletion to confer resistance to NK cells.
To address these issues, we take advantage of our experience in NK cell assays and our access to unique reagents to define major receptor-ligand pairs involved in target cell killing. With the help of these results we can genetically engineer target cells with specific gRNAs to skew the cytotoxicity in different directions to address different research questions.
Participating group members: PhD student Linnea Kristenson, MSC student Chiara Badami, MSc student Mohammed Nehmé