CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge)

Culicoidesbiting midges are small blood feeding insects responsible for the transmission of important arthropod-borne viruses (arboviruses) such as bluetongue virus (BTV), Schmallenberg virus (SBV) and epizootic hemorrhagic disease virus (EHDV), which cause major losses to livestock production worldwide.Culicoides sonorensisis the primary vector of BTV in North America and one of the fewCulicoidesspecies to be colonised and reared in artificial conditions. Gene editing technology has been used to explore virus-vector interactions in other vector groups, particularly within mosquitoes. Despite the availability of a reference genome since 2018, to date there have been no reports of gene editing inC. sonorensis. Here, we report the first instance of gene editing inC. sonorensis, achieved by intrathoracic injection of adult females with Cas9 and sgRNAs targeting thewhitegene. We generated heritable mutations in thewhitegene which produced both white eye and red eye phenotypes and went on to establish a homozygous knockout line carrying a single mutation. We observed gene editing efficiencies of up to 12.3%, making this an efficient protocol for genetic manipulation ofCulicoidesbiting midges, opening the door to functional genomics studies and the development of control strategies in these important and understudied disease vectors.