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

基本信息

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
项目类别：
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

项目摘要

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/摘要通过新策略的可用性，预防壁虱传播疾病将得到明显的帮助揭示壁虱基因的功能。 ixodes肩cap骨是莱姆病螺旋体的节肢动物载体 Borrelia Burgdorferi和其他新兴的人类病原体，包括立克剂Anaplasma 吞噬细胞，黄病毒Powassan和寄生虫microti。不幸的是，实际障碍极大地阻碍了壁虱。包括I的大约两年生命周期。肩cap骨。另一种方法是在培养的细胞中进行CRISPR筛查，在其他系统中一种既定的强大而大规模的方法，用于揭示有关蜂窝活动和的新信息途径。我们成功地开发了一种在昆虫细胞中筛查全基因组CRISPR筛查的方法滴答可扩展。在此R21应用程序中，我们将建立一个全基因组的CRISPR细胞筛选 I.肩cap培养细胞的平台作为社区的科学资源。 tick细胞培养已经广泛用于研究微生物相互作用的方法，可用于预测I的复杂生理。肩cap骨没有与tick虫的长寿周期相关的挑战。具体来说，我们将使用我们的 I.肩capularis培养的细胞和CRISPR细胞筛选的结合知识为：（i）设计单引导RNA （SGRNA）基于参考基因组序列和其他基因组序列进行CRISPR修饰这个物种；（ii）识别出适当的U6启动子，用于肩cap骨细胞中的SGRNA表达；（iii）修改用于筛选的细胞，例如通过使重组介导的盒式盒式交换（RMCE）胜任并引入CAS9；（iv）测试和优化基于CRISPR的淘汰策略的效率这些细胞。与这项工作同时，我们将开发适合合并格式屏幕的基于单元的测定通过免疫防御（IMD）和JAK/STAT途径询问tick免疫信号传导。值得注意的是我们提出的大规模和公正方法的类型将为反向提供重要的补充遗传体内分析。在I.肩cap骨细胞中建立CRISPR筛选将推动tick虫领域生物学以快速的速度向前发展，提供有关节肢动物载体与的关系的新见解他们举办的微生物，促进具有独特专业知识的调查员之间的合作，并提供新的指导下一代科学家的机会。