Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket <i>Gryllus bimaculatus</i>

Studies of traditional model organisms such as the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly understood organisms into viable laboratory organisms for functional genetic study. To this end, we present a method to induce targeted genome knockout and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germline transmission of induced mutations is comparable with that reported for other well-studied laboratory organisms, and knock-ins targeting introns yield viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knockout method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.