Establishing CRISPR cell screening for the deer tick Ixodes scapularis, a vector of Lyme and other diseases

建立针对鹿蜱肩突硬蜱（莱姆病和其他疾病的载体）的 CRISPR 细胞筛选

• 批准号: 10428801
• 负责人: STEPHANIE E MOHR
• 金额: $ 26.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428801
• 关键词: Anaplasma phagocytophilum; Anopheles Genus; Arthropod Vectors; Arthropods; Babesia microti; Biological Assay; Biology; Black-legged Tick; Borrelia; Borrelia burgdorferi; CRISPR screen; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell Survival; Cells; Cellular Assay; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communities; Complement; Complex; Culicidae; Cultured Cells; Data; Databases; Detection; Development; Disease; Disease Vectors; Drosophila genus; Face; Flavivirus; Fostering; Funding; Genes; Genetic Techniques; Genetic Transcription; Genetic study; Genome; Geographic Locations; Guide RNA; Human; Immune; Immune signaling; Immune system; Immunity; Immunology procedure; Individual; Insecta; Intervention; Ixodes; Knock-out; Knowledge; Lead; Lentivirus; Letters; Libraries; Life Cycle Stages; Light; Lyme Disease; Mammalian Cell; Mediating; Mentors; Methods; Microbe; Modification; Molecular Genetics; Parasites; Pathway interactions; Pesticides; Physiology; Plasmids; Powassan virus; Prevention; Research; Research Design; Research Personnel; Resources; Rickettsia; Scientist; System; Technology; Testing; Tick-Borne Diseases; Ticks; Vector-transmitted infectious disease; Work; bacterial vector; base; cell type; design; emerging human pathogen; extracellular; gene function; genetic approach; genome wide screen; genome-wide; human disease; immune function; in vivo; insight; knock-down; lentiviral integration; microorganism interaction; next generation; novel; novel strategies; pathogen; pathogenic microbe; pathogenic virus; preventive intervention; promoter; recombinase-mediated cassette exchange; reference genome; resistance gene; reverse genetics; screening; tick-borne pathogen; tool; vector; vector control; vector mosquito; vector tick

Project Summary/Abstract Prevention of tick-borne illnesses would be significantly aided by the availability of new strategies for uncovering the functions of tick genes. Ixodes scapularis is an arthropod vector of the Lyme disease spirochete Borrelia burgdorferi and other emerging human pathogens, including the rickettsial agent Anaplasma phagocytophilum, the flavivirus Powassan, and the parasite Babesia microti. Unfortunately, genetic studies of ticks are significantly hindered by practical barriers, including the approximately two-year life cycle of I. scapularis. An alternative approach is to perform CRISPR screening in cultured cells, which in other systems is an established robust and large-scale approach to uncovering new information about cellular activities and pathways. We have successfully developed a method for genome-wide CRISPR screening in insect cells that is extensible to ticks. In this R21 application, we will establish a genome-wide pooled CRISPR cell screening platform for I. scapularis cultured cells as a scientific resource for the community. Tick cell culture has been extensively used to investigate microbial interactions and can be used to predict the complex physiology of I. scapularis without the challenges associated with the long-life cycle of ticks. Specifically, we will use our combined knowledge of I. scapularis cultured cells and CRISPR cell screening to: (i) design single guide RNAs (sgRNAs) for CRISPR modification based on the reference genome sequence and other genome sequences of this species; (ii) identify appropriate U6 promoters for sgRNA expression in I. scapularis cells; (iii) modify cells for screening, such as by making the competent for recombination mediated cassette exchange (RMCE) and by introducing Cas9; and (iv) test and optimize the efficiency of CRISPR-based knockout strategies in these cells. Concurrent with this work, we will develop cell-based assays appropriate for pooled-format screens that interrogate tick immune signaling via the immune defense (IMD) and JAK/STAT pathways. Notably, the type of large-scale and unbiased approach we propose will provide an important complement to reverse genetic in vivo analyses. Establishment of CRISPR screening in I. scapularis cells will propel the field of tick biology forward at a rapid pace, provide novel insights into relationships between arthropod vectors and the microbes they host, foster collaborations between investigators with distinct expertise, and offer new opportunities for mentoring the next generation of scientists.

项目总结/摘要 预防蜱传疾病将大大有助于新的战略， 揭示蜱虫基因的功能肩突硬蜱是莱姆病螺旋体的节肢动物传播媒介 伯氏疏螺旋体和其他新出现的人类病原体，包括立克次体无形体 嗜吞噬细胞菌、黄病毒Powassan和寄生虫小Babalyprotimicroti。不幸的是， 蜱的传播受到实际障碍的严重阻碍，包括I的约两年生命周期。 肩胛肌另一种方法是在培养的细胞中进行CRISPR筛选，这在其他系统中是可行的。 一种已建立的强大和大规模的方法来揭示有关细胞活动的新信息， 途径。我们已经成功开发了一种在昆虫细胞中进行全基因组CRISPR筛选的方法， 可扩展到蜱虫。在这次R21申请中，我们将建立一个全基因组合并的CRISPR细胞筛选， 平台为I。肩胛骨培养细胞作为社会科学资源。蜱细胞培养已经 广泛用于研究微生物的相互作用，并可用于预测复杂的生理I。 肩胛骨没有与蜱虫的长生命周期相关的挑战。具体而言，我们将使用我们的 结合我的知识。肩胛肌培养的细胞和CRISPR细胞筛选，以：（i）设计单向导RNA 基于参考基因组序列和其他基因组序列， （ii）鉴定合适的U6启动子用于在I.肩胛肌细胞;（iii）修饰 用于筛选的细胞，例如通过使其能够进行重组介导的盒交换（RMCE）， 以及通过引入Cas9;以及（iv）测试和优化基于CRISPR的敲除策略在 这些细胞。在这项工作的同时，我们将开发适合于合并格式筛选的基于细胞的测定方法 通过免疫防御（IMD）和JAK/STAT途径询问蜱免疫信号。特别是 我们提出的一种大规模和无偏见的方法将为逆转 体内遗传分析。CRISPR筛选在I.肩胛肌细胞将推动蜱虫的领域 生物学的快速发展，提供了新的见解之间的关系节肢动物载体和 它们宿主的微生物，促进具有不同专业知识的研究人员之间的合作，并提供新的 培养下一代科学家的机会。