Lien vers Pubmed [PMID] – 28698275
mBio. 2017 8(4)
relies on monoallelic expression of 1 of 60 virulence genes for antigenic variation and host immune evasion. Each gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless gene in a natural, marker-free chromosomal context. Deletion of the intron resulted in an upregulation of transcription of the gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the gene in trophozoites but did result in increased rates of gene switching in some mutant clones. Transcriptional repression of the intronless gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the gene family. is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the gene intron in transcriptional regulation of monoallelic expression.