Exploring the virulence gene interactome withCRISPR/dCas9 in the human malaria parasite

Mutually exclusive expression of thevarmultigene family is key to immune evasion and pathogenesis inPlasmodium falciparum, but few factors have been shown to play a direct role. We adapted aCRISPR-based proteomics approach to identify novel factors associated withvargenes in their natural chromatin context. Catalytically inactive Cas9 (dCas9) was targeted tovargene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins,DNAhelicases, and chromatin remodelers. Functional characterization ofPfISWI, an evolutionarily divergent putative chromatin remodeler enriched at thevargene promoter, revealed a role in transcriptional activation. Proteomics ofPfISWIidentified several proteins enriched at thevargene promoter such as acetyl-CoA synthetase, a putativeMORCprotein, and an ApiAP2 transcription factor. These findings validate theCRISPR/dCas9 proteomics method and define a newvargene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.