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: 10686381
• 负责人: STEPHANIE E MOHR
• 金额: $ 20.51万
• 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/10686381
• 关键词: Anaplasma phagocytophilum; Anopheles Genus; Applied Genetics; 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; Lentivirus; Letters; Libraries; Life Cycle Stages; 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; Resistance; Resources; Rickettsia; Scientist; System; Technology; Testing; Tick-Borne Diseases; Ticks; Vector-transmitted infectious disease; Work; bacterial vector; 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; 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.

项目摘要/摘要 预防硬虱传播的疾病将大大有助于制定新的防治策略 揭开扁虱基因的功能。肩部硬蜱是莱姆病螺旋体的一种节肢动物媒介 伯氏疏螺旋体和其他新出现的人类病原体，包括立克次体病原体无浆体 吞噬细胞、黄病毒鲍瓦桑和寄生虫巴贝斯虫。不幸的是，基因研究表明 扁虱受到实际障碍的严重阻碍，包括I。 肩胛骨。另一种方法是在培养细胞中进行CRISPR筛选，这在其他系统中是 已建立的强大且大规模的方法来发现有关细胞活动和 小路。我们已经成功地开发了一种在昆虫细胞中进行全基因组CRISPR筛选的方法 是可扩展到Tick的。在R21的应用中，我们将建立全基因组池CRISPR细胞筛选 为肩胛鱼培养细胞提供平台，为社会提供科学资源。扁虱细胞培养已经被 广泛用于研究微生物相互作用，并可用于预测I。 没有与扁虱长生命周期相关的挑战。具体来说，我们将使用我们的 结合肩胛肌培养细胞和CRISPR细胞筛选的知识：(I)设计单引导RNA (SgRNAs)，用于基于参考基因组序列和其他基因组序列的CRISPR修饰 该物种；(Ii)确定合适的U6启动子，用于在肩周炎细胞中表达sgRNA；(Iii)修饰 用于筛选的细胞，例如通过使其具有重组介导盒交换(RMCE)的能力 通过引入Cas9；以及(Iv)测试和优化基于CRISPR的淘汰赛策略在 这些细胞。在这项工作的同时，我们将开发适合于池格式屏幕的基于细胞的分析 这是通过免疫防御(IMD)和JAK/STAT通路询问扁虱免疫信号。值得注意的是， 我们提出的一种大规模和不偏不倚的方法将为扭转 体内遗传分析。建立肩胛肌细胞CRISPR筛查方法将推动硬蜱领域的发展 生物学的快速发展，为节肢动物媒介之间的关系提供了新的见解 它们是微生物的宿主，促进了具有独特专业知识的研究人员之间的合作，并提供了新的 指导下一代科学家的机会。