Genome- wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome- wide CRISPR loss- of- function screen to identify genes important for (interferon gamma) IFN-gamma -dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF- 1. We then performed an ISG- targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-gamma -mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213- mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-gamma. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell- autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.