Characterization of P. falciparum gametocyte-essential genes using a novel genetic screen

恶性疟原虫是疟疾的病原体，它仍然是全球的主要负担。连 今天，这种寄生虫的绝大多数基因组还没有确定特征。这大大阻碍了我们的 开发新的抗疟疾药物、治疗方法和疫苗的能力。有性配子体尤其如此。 寄生虫的阶段，它遵循人类宿主的症状阶段，并传播给 蚊子。配子细胞不容易被目前的药物方案清除，甚至可以发生传播 在治疗了一个感染者之后。到目前为止，研究多个疟原虫基因的方法还很少。 一次，尤其是对于血液阶段以外的阶段。我一直致力于开发一种新的基因编辑系统 这既是可访问的，也是高度可扩展的。使用Cas9碱基编辑，可以使特定的C-T突变 只使用一个gRNA就可以引入早期终止。这个系统效率很高，可以用来击倒 多个基因以池的形式存在。为了筛选配子体发育所必需的基因，我已经 根据转录本生成了一份包含250个基因的清单，这些基因被预测为有性阶段所必需的 丰度、蛋白质组丰度和进化守恒。我假设这些丰富的 表达和高度保守的基因对配子体阶段是必不可少的，并将导致适合性 静默时要付出代价。为了测试这一点，我将使用这个新颖的编辑系统执行基因筛选，使用 配子体的荧光标记。荧光寄生虫的分类和测序将识别 GRNA含量不足，代表配子体的重要性。我们基因筛查的前五名将 然后分别敲除蚊子并进行表型鉴定，观察蚊子的形态和损伤情况 变速箱。该提案的发现将有助于确定新的药物和疫苗靶点，这可能会有所帮助 防止疟疾的传播。

Plasmodium falciparum, the causative agent of malaria, continues to be a major global burden. Even today, the vast majority of the parasite’s genome has yet to be characterized. This has greatly hindered our ability to develop new antimalarials, therapies, and vaccines. This is particularly true of the sexual gametocyte stage of the parasite, which follows the symptomatic stages in the human host and is transmitted to the mosquito. Gametocytes are not easily cleared by current drug regimens, allowing transmission to occur even after treating an infected individual. To date, there have been few ways to study multiple Plasmodium genes at once, particularly for stages outside of the blood stage. I have worked to develop a new gene editing system that is both accessible and highly scalable. Using Cas9 base-editing, specific C-to-T mutations can be made to introduce early terminations using only a gRNA. This system is highly efficient and can be used to knock out multiple genes in a pooled format. In order to screen for genes essential to gametocyte development, I have generated a list of 250 genes predicted to be essential for the sexual stage based on transcriptomic abundance, proteomic abundance, and evolutionary conservation. I hypothesize that these abundantly expressed and highly conserved genes will be essential to the gametocyte stage, and will incur a fitness cost when silenced. In order to test this, I will perform a genetic screen with this novel editing system using a fluorescent marker for gametocytes. Sorting and sequencing of fluorescent parasites will identify underabundant gRNAs, representative of gametocyte essentiality. The top five hits from our genetic screen will then be individually knocked out and phenotyped to examine the morphology and impairment on mosquito transmission. The findings of this proposal will aid in identifying new drug and vaccine targets that could help prevent the spread of malaria.