Genomic targeting by recombinase-mediated cassette exchange in the spotted wing drosophila, Drosophila suzukii.

Abstract

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon-mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase-mediated cassette exchange (RMCE) system for D.suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon-mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D.suzukii, and quantitative PCR determined that polyubiquitin-regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology-directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.