Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR‐based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl‐CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene‐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.

中文翻译：

---

探索人类疟疾寄生虫中毒力基因与 CRISPR/dCas9 的相互作用。


var多基因家族的相互排斥表达是恶性疟原虫免疫逃避和发病机制的关键，但很少有因素被证明发挥直接作用。我们采用基于 CRISPR 的蛋白质组学方法来识别与var基因在其天然染色质环境中相关的新因子。催化失活的 Cas9（“dCas9”）针对var基因调控元件，进行免疫沉淀，并进行质谱分析。丰富了已知和新的因子，包括结构蛋白、DNA 解旋酶和染色质重塑剂。 Pf ISWI（一种在var基因启动子处富集的进化上不同的假定染色质重塑剂）的功能特征揭示了其在转录激活中的作用。 Pf ISWI 的蛋白质组学鉴定出几种在var基因启动子处富集的蛋白质，例如乙酰辅酶 A 合成酶、一种推定的 MORC 蛋白质和 ApiAP2 转录因子。这些发现验证了 CRISPR/dCas9 蛋白质组学方法，并定义了一种新的var基因相关染色质复合物。这项研究建立了一种工具，用于对未改变的基因组位点进行靶向染色质纯化，并鉴定了可能参与人类疟原虫毒力基因的转录控制和/或染色质组织的新型染色质相关因子。