Genome-wide CRISPR-Cas9 screens in insect cells to characterize insecticidal toxins

在昆虫细胞中进行全基因组 CRISPR-Cas9 筛选以表征杀虫毒素

基本信息

批准号：
10646295
负责人：
Min Dong
金额：
$ 79.49万
依托单位：
BOSTON 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：
2022-06-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646295
关键词：
Bacterial Toxins Bacteriophages CRISPR screen CRISPR/Cas technology Cell Line Cell model Cells Communicable Diseases Communities Culicidae Dengue Development Drosophila genus Drosophila melanogaster Family Family member Future Genetic Recombination Goals Guide RNA Human In Vitro Insecta Insecticides Integration Host Factors Knock-out Knowledge Lentivirus Libraries Life Cycle Stages Lyme Disease Malaria Mammalian Cell Mediating Methods Modeling Molecular Mucins Nematoda Pathogenesis Pesticides Plasmids Proteins RNA library Role Series Site Small Interfering RNA Specificity System Targeted Toxins Technology Toxic effect Toxin Validation Virulence Factors West Nile Xenorhabdus luminescens ZIKV disease design experimental study fly genome wide screen genome-wide human disease human pathogen in vivo knock-down lentivirally transduced member mosquitocidal novel pathogen pathogenic bacteria prototype receptor recombinase-mediated cassette exchange success tool transmission process vector

项目摘要

Project Summary Genome-wide screens using CRISPR-Cas9 technology have revolutionized studies of host-pathogen interactions, leading to identification of many key host cellular factors required for the actions of human pathogens and bacterial toxins. However, this powerful approach has yet to be utilized in insect cells to uncover host factors required for insect-borne pathogens, which are responsible for a long list of infectious diseases such as malaria, Dengue, West Nile, Zika, and Lyme diseases. This is largely due to a technical barrier: the inability to efficiently deliver genome- wide guide RNA library into insect cells. We recently overcame this barrier and developed a genome-wide CRISPR-Cas9 screening method in cultured Drosophila cells. Using this method, and by leveraging the expertise of Dr. Norbert Perrimon’s lab in insect models and of Dr. Min Dong’s lab in bacterial toxins, we carried out extensive preliminary studies, leading to the identification of a potential insect receptor for a member of the major bacterial toxin family known as Tc toxins, demonstrating the power and feasibility of our unbiased genome-wide screen approach. Building on these successes, in Aim 1 we will focus on further development and validation of genome-wide screens with major Tc toxin family members to establish a mechanistic understanding of toxin-receptor interactions and their role in pathogenesis in vitro and in vivo. We further propose to expand our approach to establish the first genome-wide CRISPR-Cas9 screening method and tools in mosquito cells in Aim2, and then utilize this approach to identify key host factors for two novel bacterial toxins that showed selective toxicity on mosquito but not Drosophila cells. The success of our proposal will uncover receptors and key host cellular factors for important bacterial toxins and establish generalizable methods and essential tools for investigating pathogens and toxins at genome-wide scale in insect cells relevant to transmitting human infectious diseases.

项目概要使用 CRISPR-Cas9 技术的全基因组筛选彻底改变了宿主-病原体相互作用，导致鉴定所需的许多关键宿主细胞因子然而，这种强大的方法尚未有效抑制人类病原体和细菌毒素的作用。可在昆虫细胞中发现昆虫传播病原体所需的宿主因子，这些因子是疟疾、登革热、西尼罗河、寨卡等一长串传染病的罪魁祸首这主要是由于技术障碍：无法有效地传递基因组。我们最近克服了这一障碍并开发了一种将广泛引导RNA文库引入昆虫细胞的方法。使用这种方法，在培养的果蝇细胞中进行全基因组 CRISPR-Cas9 筛选。并利用 Norbert Perrimon 博士的昆虫模型实验室和 Min 博士的专业知识董的细菌毒素实验室，我们进行了广泛的初步研究，得出了鉴定已知主要细菌毒素家族成员的潜在昆虫受体作为 Tc 毒素，展示了我们公正的全基因组筛选的力量和可行性在这些成功的基础上，我们将在目标 1 中重点关注进一步的发展和发展。对主要 Tc 毒素家族成员的全基因组筛选进行验证，以建立机制了解毒素-受体相互作用及其在体外和体内发病机制中的作用。进一步建议扩展我们的方法来建立第一个全基因组 CRISPR-Cas9 Aim2中的蚊子细胞筛选方法和工具，然后利用该方法来识别两种新型细菌毒素的关键宿主因素，对蚊子表现出选择性毒性，但没有我们的提案的成功将揭示受体和关键的宿主细胞因子。找出重要的细菌毒素，并建立通用的方法和基本工具在昆虫细胞的全基因组范围内研究与传播相关的病原体和毒素人类传染病。