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.

疟疾的致病药物疟原虫仍然是全球的主要负担。如今，寄生虫的绝大多数基因组尚未被描述。开发新的抗疟药，疗法和疫苗的能力。寄生虫的阶段，遵循人类宿主的症状阶段，并传播到蚊子不容易被当前的药物治疗清除在治疗受感染的个体之后。曾经，特别是对于血液阶段的阶段。使用CAS9基础编辑，特定的C到to-to-to 仅使用AgRNA引入早期终止。以合并格式的多个基因。产生了一个基于转录组的250个基因的列表，这对于性阶段至关重要丰度，蛋白质组学丰度和进化保守。表达且高度保守的基因对于配子细胞阶段至关重要，并且会产生健身沉默时的成本。荧光细胞的荧光标记。不足的grnas，抑制配子的必要性。然后被单独淘汰并表现出来，以检查蚊子上的形态和障碍提议的调查结果将有助于识别新药和疫苗目标防止疟疾的传播。