Marco Vincenzo Russo, Scientific Manager, Memorial Sloan Kettering Cancer Center, explained that the Gene Editing and Screening Core Facility offers various services, including CRISPR-based gene editing, generation of knockout and knock-in cell lines, and pooled and arrayed library screenings.
Russo’s research focuses on a project related to the identification of drivers of resistance and sensitizers to PRMT5 inhibition in lymphoma. He decided to investigate this topic because this protein is highly expressed in primary lymphoma and is also a predictor of full outcomes. Many PRMT5 inhibitors have been tested in cancer patients, but the mechanism of action of the inhibitors remains unclear.
The Memorial Sloan Kettering Cancer Center has a partnership with GSK, which provided them with a compound called GSK 591, a selective and potent PRMT5 inhibitor. Combining their knowledge of PRMT5 with their whole genome library screening, they found that PRMT5 presents an attractive target in lymphoma, and by knocking down PRMT5, Russo was able to validate this data about the importance of this factor for lymphoma cell growth.
The goal of the screening is to identify the mechanism of resistance to this PRMT5 inhibition. One question that frequently crops up is whether a two-vector system or a vector system is more suitable for the screening and cell line generation. Russo’s case study used a two-vector system. The study used CRISPR pooled screens to identify key factors like P53 and MUSASHI2.
The study found that PRMT5 is essential for lymphoma cell growth, and its inhibition by GSK 591 is mediated by P53. Meanwhile, MUSASHI 2 was identified as a top driver of resistance to PRMT5 inhibitors.
Russo also stressed the importance of Guide RNA design. He used a tool called Guidescan to design his own guide that would satisfy the following three conditions: fewer off-target effects, high cutting efficiency, and good specificity. He commented that he prefers to deliver the guides via electroporation or transfection, depending on the application. To conclude, Russo stated that the team plans to develop combination therapies targeting PRMT5 and MUSASHI 2, as well as drugs that interfere with specific pathways like BCL2.
