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

项目摘要 使用CRISPR-Cas9技术的全基因组筛查已经彻底改变了对 宿主与病原体的相互作用，导致鉴定出许多关键的宿主细胞因子 人类病原体和细菌毒素的作用。然而，这一强大的方法尚未奏效 在昆虫细胞中被用来揭示昆虫传播的病原体所需的宿主因子，这些因子是 对一系列传染病负责，如疟疾、登革热、西尼罗河、寨卡病毒和 莱姆病。这在很大程度上是由于一个技术障碍：无法有效地传递基因组- 广泛引导RNA文库进入昆虫细胞。我们最近克服了这一障碍，开发了一种 果蝇培养细胞全基因组CRISPR-Cas9筛选方法。使用这种方法， 通过利用诺伯特·佩里蒙博士的实验室在昆虫模型方面的专业知识和Min博士的专业知识 董的实验室中的细菌毒素，我们进行了广泛的初步研究，导致 已知主要细菌毒素家族成员潜在昆虫受体的鉴定 作为TC毒素，展示了我们无偏全基因组筛查的力量和可行性 接近。在这些成功的基础上，在目标1中，我们将专注于进一步的发展和 与主要的TC毒素家族成员进行全基因组筛查以建立机制的验证 了解毒素-受体相互作用及其在体内和体外发病机制中的作用。我们 进一步建议扩大我们的方法，以建立第一个全基因组CRISPR-CAS9 在AIM2中筛选蚊子细胞的方法和工具，然后利用该方法进行鉴定 两种对蚊子有选择性毒性但不具有选择性的新型细菌毒素的关键宿主因素 果蝇细胞。我们的计划的成功将揭示受体和关键的宿主细胞因子 对于重要的细菌毒素，并建立通用的方法和必要的工具 在全基因组水平上调查昆虫细胞中与传播有关的病原体和毒素 人类传染病。

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.