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

使用新型遗传筛选表征恶性疟原虫配子体必需基因

• 批准号: 10619569
• 负责人: Sean Taylor Windle
• 金额: $ 4.77万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10619569
• 关键词: Alleles; Antimalarials; Back; Biological Assay; Bite; Blood; CRISPR/Cas technology; Candidate Disease Gene; Cessation of life; Consumption; Culicidae; Cytidine Deaminase; Data; Data Set; Development; Epitopes; Erythrocytes; Essential Genes; Fluorescence-Activated Cell Sorting; Foundations; Generations; Genes; Genetic Screening; Guide RNA; Hepatocyte; Human; Immunofluorescence Immunologic; Impairment; Individual; Knock-out; Libraries; Life Cycle Stages; Malaria; Manuals; Morbidity - disease rate; Morphology; Mutation; Nonhomologous DNA End Joining; Nonsense Codon; Oocysts; Parasites; Pathway interactions; Pharmaceutical Preparations; Phenotype; Plant Genes; Plasmids; Plasmodium; Plasmodium falciparum; Play; Process; Proteomics; Random Allocation; Regimen; Reporter; Research; Role; Screening Result; Sexual Development; Sexual Reproduction; Sorting; Sporozoites; System; Technology; Testing; Time; Transfection; Vaccines; Work; asexual; base editing; candidate identification; cost; fitness; gene conservation; gene function; in silico; knockout gene; liver infection; loss of function; malaria infection; malaria transmission-blocking vaccine; mortality; novel; novel therapeutics; novel vaccines; parasite genome; prevent; repaired; symptom treatment; transcriptomics; transmission process; vector mosquito

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.

恶性疟原虫是疟疾的病原体，它仍然是全球的一个主要负担。甚至 时至今日，这种寄生虫的绝大多数基因组仍有待鉴定。这极大地阻碍了我们 开发新的抗疟药、疗法和疫苗的能力。这一点在有性配子体中尤为明显 阶段的寄生虫，其中以下的症状阶段，在人类宿主和传播到 蚊子目前的药物治疗方案不易清除配子细胞，甚至允许发生传播。 在治疗一个感染者之后。到目前为止，研究多种疟原虫基因的方法很少， 一次，特别是对于血液阶段之外的阶段。我致力于开发一种新的基因编辑系统 既可访问又可高度扩展。使用Cas9碱基编辑，可以进行特定的C到T突变， 仅使用gRNA引入早期终止。该系统效率高，可用于敲除 多个基因的混合形式。为了筛选配子母细胞发育所必需的基因， 根据转录组学的研究， 丰度、蛋白质组丰度和进化保守性。我假设这些大量的 表达的和高度保守的基因将是配子体阶段所必需的， 沉默的代价为了测试这一点，我将使用一个新的编辑系统执行一个基因屏幕， 配子体的荧光标记物。荧光寄生虫的分选和测序将识别 gRNA丰度不足，代表配子母细胞的必要性。我们基因筛选的前五名 然后将其单独敲除并进行表型分析，以检查蚊子的形态和损伤 传输这项提案的发现将有助于确定新的药物和疫苗靶点， 防止疟疾的传播。