Development of CRISPR/Cas9 gene editing tools for the insect pest sirex noctilio

Sirex noctilio (Hymenoptera: Siricidae) is an invasive pest in South African pine plantations. Current pest management strategies for S. noctilio, such as silvicultural practices or biological control, are not always efficient and gene editing could add to the toolbox of management options. Gene editing has emerged as a new and precision approach for genetic pest control. This approach is facilitated by the discovery of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), with its associated Cas9 nuclease, as a more accessible approach to do gene editing in a wide range of insect pests. Currently, the only feasible option to deliver CRISPR/Cas9 into insects is through microinjection of embryos. However, access to S. noctilio embryos is challenging as they are oviposited into the tree, and if obtained via dissection, will not develop further. In this study, an egg activation protocol was developed for S. noctilio to enable microinjection of large numbers of embryos. In addition, various artificial diets were envaulted with the aim to rear first instar larvae emerging from activated eggs and microinjected embryos. Sirex noctilio eggs from two climatically different regions in South Africa were successfully activated for the first time. Microinjected embryos developed abnormally and had a very low survival rate, and DNA sequencing results revealed that no microinjected embryo was genetically modified. The optimal artificial diet to rear first instar S. noctilio larvae was a modified Anoplophora glabripennis (Coleoptera: Cerambycidae) diet. The results in this study, from rearing to gene editing, are extendable to other insect species with similar characteristics and may enable progress towards their gene editing. Most importantly, this study has unlocked new research opportunities in S. noctilio that may help to develop additional tools for pest management.