CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.
CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.
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DOI：
10.1128/mbio.00729-17
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发表时间：
2017-07-11
期刊：
影响因子：
6.4
通讯作者：
Scherf A
中科院分区：
文献类型：
--
作者：
Bryant JM;Regnault C;Scheidig-Benatar C;Baumgarten S;Guizetti J;Scherf A
恶性疟原虫依赖于60个变种毒力基因中的1个的单等位基因表达来进行抗原变异和宿主免疫逃避。每个var基因都含有一个保守的内含子，在以前的研究中，内含子通过几种表观遗传机制参与了转录的激活和抑制，包括与var启动子的相互作用，长非编码RNA（lncRNA）的产生以及抑制性核周位点的定位。然而，功能研究主要依赖于人工表达构建体。使用最近开发的恶性疟原虫成簇规则间隔短回文重复序列（CRISPR）/Cas9系统，我们直接删除了var 2csa恶性疟原虫3D7_1200600（Pf3D7_1200600）内源性内含子，从而在天然、无标记的染色体环境中产生无内含子var基因。var 2csa内含子的缺失导致在环阶段的寄生虫的var 2csa基因的转录上调，并随后在晚期寄生虫的PfEMP 1蛋白的表达。内含子缺失并不影响滋养体中var基因的正常时间调节和随后的转录沉默，但确实导致了一些突变克隆中var基因转换速率的增加。无内含子var 2csa基因的转录抑制可以通过长期培养或用CD 36受体淘选来实现，之后用硫酸软骨素A（CSA）淘选可以重新激活。这些数据表明，var 2csa内含子是不需要的沉默或激活在环阶段的寄生虫，但指出一个微妙的作用，在调节开关的var基因家族。恶性疟原虫是疟疾寄生虫中毒性最强的一种，在感染者中造成高发病率和死亡率。慢性感染依赖于称为抗原变异的免疫逃避机制，而抗原变异又依赖于~60个变异基因中的1个的单等位基因表达。了解抗原变异和单等位基因表达的转录调控对于开发药物和/或疫苗是重要的。var基因家族编码修饰受感染红细胞的抗原性表面蛋白。直到最近，研究恶性疟原虫中调节单等位基因表达的潜在遗传元件是困难的，大多数研究依赖于人工系统，如附加型报告基因。我们的研究是第一个使用CRISPR/Cas9基因组编辑以内源性，无附加体的方式对var基因的重要，保守的遗传元件-内含子进行功能研究。我们的研究结果阐明了var基因内含子在单等位基因表达的转录调控中的作用。
Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var 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 var 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 P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa 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 var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa 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 var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. Plasmodium falciparum 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 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum 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 var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.