Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic. Here we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring. Considering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.