Base editing screens map mutations affecting IFNγ signalling in cancer

Abstract

IFN{gamma}signalling underpins host responses to infection, inflammation and anti-tumour immunity. Mutations in the IFN{gamma}signalling pathway cause immunological disorders, haematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer, however the function of most clinically observed variants remain unknown. Here, we systematically investigate the genetic determinants of IFN{gamma}response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations with clinical precedence, including causal variants in haematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumour organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumour-reactive T cells. By classifying > 300 missense variants altering IFN{gamma} pathway activity, we demonstrate the utility of base editing for mutagenesis at scale, and generate a resource to inform genetic diagnosis.