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

基本信息

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
项目类别：
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

项目摘要

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，仍然是全球的主要负担。如今，绝大多数寄生虫的基因组尚未得到表征，这极大地阻碍了我们的研究。开发新的抗疟药、疗法和疫苗的能力对于性配子体来说尤其如此。寄生虫的阶段，跟随人类宿主的症状阶段并传播到目前的药物疗法不容易清除蚊子的配子体，甚至导致传播的发生。迄今为止，在治疗感染者后，研究多种疟原虫基因的方法很少。曾经，特别是在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。使用 Cas9 碱基编辑，可以实现特定的 C 到 T 突变。仅使用 gRNA 引入早期终止该系统非常高效，可用于敲除。为了筛选配子体发育所必需的基因，我收集了多个基因。生成了基于转录组预测对性阶段至关重要的 250 个基因的列表丰度、蛋白质组丰度和进化保守。表达且高度保守的基因对于配子体阶段至关重要，并且会产生适应性为了测试这一点，我将使用这种新颖的编辑系统进行基因筛选。配子体荧光标记物的分选和测序将识别荧光寄生虫。 gRNA 含量不足，代表配子体的必要性，我们的遗传筛选中排名前五的将是。然后单独敲除并进行表型分析，以检查蚊子的形态和损伤该提案的研究结果将有助于确定新的药物和疫苗目标，从而有所帮助。防止疟疾传播。