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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.

项目总结/文摘