Genetic manipulation of anIxodes scapulariscell line

Abstract

AbstractAlthough genetic manipulation is one of the hallmarks in model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the blacklegged tickIxodes scapularis,an arthropod that serves as a vector of several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology dependent recombination. Targeting the E3 ubiquitin ligase X-linked inhibitor of apoptosis (xiap) and its substratep47led to alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agentAnaplasma phagocytophiluminI. scapularisISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticksin vivoand aid in circumventing the long-life cycle ofI. scapularis,of which limits efficient and scalable molecular genetic screens.ImportanceGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology dependent recombination. We ablated the expression ofxiapandp47, two signaling molecules present in the immune deficiency (IMD) pathway ofI. scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agentA. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tickIxodes scapularis.