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

项目摘要 使用CRISPR-Cas9技术进行的全基因组筛选彻底改变了 宿主-病原体相互作用，导致鉴定了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，这种强大的方法还没有 在昆虫细胞中使用，以揭示昆虫传播的病原体所需的宿主因子， 负责一长串的传染病，如疟疾，登革热，西尼罗河，寨卡， 莱姆病这在很大程度上是由于技术障碍：无法有效地递送基因组- 宽的向导RNA文库导入昆虫细胞。我们最近克服了这个障碍，开发了一个 在培养的果蝇细胞中进行全基因组CRISPR-Cas9筛选方法。使用这种方法， 通过利用诺伯特·佩里蒙博士实验室在昆虫模型方面的专业知识， 在董的细菌毒素实验室，我们进行了广泛的初步研究，导致 鉴定已知的主要细菌毒素家族成员的潜在昆虫受体 证明了我们无偏见的全基因组筛选的能力和可行性 approach.在这些成功的基础上，在目标1中，我们将侧重于进一步发展， 用主要Tc毒素家族成员进行全基因组筛选以建立 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。我们 进一步建议扩大我们的方法，建立第一个全基因组CRISPR-Cas9 在蚊子细胞中筛选Aim 2的方法和工具，然后利用这种方法来识别 两种新型细菌毒素的关键宿主因素，对蚊子显示出选择性毒性，但对蚊子没有毒性。 果蝇细胞。我们的提案的成功将揭示受体和关键的宿主细胞因子 并建立可推广的方法和基本工具， 在昆虫细胞中全基因组水平上研究病原体和毒素， 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.