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

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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.