Efficient genome editing in the olive fruit fly, Bactrocera oleae

Abstract

The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well‐known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early‐stage embryos. Successful CRISPR‐induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR‐based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology‐based pest control strategies.

Stably inherited transfer of the bacterial symbiont Candidatus Erwinia dacicola from wild olive fruit flies Bactrocera oleae to a laboratory strain

“…This study, together with recent developments both in genomic resources (Estes et al ., 2018 b ; Bayega et al ., 2020) and genome modification technology (Koidou et al ., 2020) may facilitate research to better understand the molecular basis of the olive fruit fly – Erwinia symbiosis (and possibly in other insect systems) in a systemic manner and utilize acquired knowledge towards the development of innovative control strategies that go beyond traditional approaches. The development of possible alternative efficient ‘dysbiosis’ approaches to control olive fly (Sinno et al ., 2020) may prove a valid alternative, given the current and forthcoming strict EU regulations to reduce the use of neurotoxic insecticides.…”

First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera)

“…The CRISPR/Cas9 system is a straightforward and effective genome‐editing method with tremendous potential for genetic modification and investigating gene function in species beyond model insects (Ashok et al, 2023; Choo et al, 2018; Wang et al, 2017). Several Tephritid genomes have been edited using this method (Bai et al, 2019; Koidou et al, 2020; Li & Handler, 2019; Meccariello et al, 2017; Paulo et al, 2022; Sim et al, 2019; Zhao et al, 2019). The current investigation concentrated on the role of the white gene in B. dorsalis .…”

“…Hence, it is necessary to do in‐depth research on the pest, particularly at the molecular level, using the recently developed technology. Of late, genetic biocontrol achieved through gene drive mediated by CRISPR/Cas9 is gaining momentum for designing an effective area‐wide pest management strategy for several pests such as Aedes aegypti (Li et al, 2017), Anopheles gambiae (Galizi et al, 2016; Hammond et al, 2016), Drosophila suzukii (Li and Scott, 2016), Ceratitis capitata (Meccariello et al, 2017), Zeugodacus cucurbitae (Paulo et al, 2022), Bactrocera tryoni (Choo et al, 2018), B. oleae (Koidou et al, 2020) that include B. dorsalis (Bai et al, 2019; Zhao et al, 2019). The above approach, called the precision‐guided sterile insect technique (pgSIT), has been successfully demonstrated in Drosophila melanogaster (Kandul et al, 2019), which is yet to be validated in pests of agricultural importance.…”

“…Unlike in plants, one cannot use invasive method for edit characterization as individual flies carry unique edits due to the random nature of the double‐stranded DNA break repair mechanism and also limited by the low success rate of microinjection (Chaverra‐Rodriguez et al, 2020; Heu et al, 2022; Koidou et al, 2020). In addition to the above, maiming test animals are not permitted to determine the type of genomic edits in the treated individuals.…”

“…Rendering sequencing is the ultimate method to determine the nature of the edit/s (Bi et al, 2022; Fu et al, 2022; Zhang et al, 2015). In this regard, it is relatively easy to identify the genomic edits in phenotypic genes, such as white , that play a vital role in eye color pigmentation, where the edited flies bear white eyes as compared to the wild eye color (Bai et al, 2019; Chaverra‐Rodriguez et al, 2020; Choo et al, 2018; Khan et al, 2017; Koidou et al, 2020, Heu et al, 2022). But the same is not possible for the non‐phenotypic, functional genes mentioned above, requiring 2–3 months of wait time to know whether or not editing has happened.…”