CRISPR/Cas-mediated gene editing using purified protein in Drosophila suzukii

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is an invasive pest of stone fruits and berries currently without effective control management. The sterile insect technique (SIT) is an environmentally friendly and effective pest control method that releases sterile males to mate with wild females resulting in the suppression or eradication of targeted pest populations. New molecular technologies and genome editing methods are paving the way to bring this technology to invasive species like D. suzukii. The CRISPR/Cas technology is emerging as a versatile option for efficient tailored gene manipulation. Components of this system, the guide RNA (gRNA) and Cas9 protein, can be delivered into the organism in the form of DNA, RNA, or protein. We report CRISPR/Cas-mediated site-specific white gene editing using purified Cas9 protein delivered directly into D. suzukii embryos. Mutant flies with 13, 3, and 1 nt deletions within the cutting s ite of the Cas9 protein were retrieved. Moreover, the two independent G1 deletion events (13 and 3 nt) derived from the same G0 female. Our results show that the recombinant Cas9 protein could be a method of choice for generating heritable gene modifications in D. suzukii.