Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae

Abstract

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo‐we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene‐specific single‐guide RNAs. Gene disruption of Bo‐we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo‐we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype

“…Use of double sgRNA simultaneously for embryo microinjection cleaves the target gene at multiple sites more effectively compared to the use of sgRNA individually (Chen et al, 2014; Dong et al, 2019; Zhang & Reed, 2017). Embryonic microinjection of double sgRNA was successful in generating a mutation of the white gene in B. dorsalis (Bai et al, 2019), B. tryoni (Choo et al, 2018), B. oleae (Meccariello et al, 2020). Meccariello et al (2017) reported embryonic microinjection of RNPs (Cas9+sgRNA) by CRISPR‐Cas9 to disrupt the white gene in the C. capitata .…”

“…CRISPR/Cas9 based white gene mutagenesis efficiency in G 0 adults in B. dorsalis was 2.13% and 3.7% (Bai et al, 2019), 0.92% and 5.36% (Zhao et al, 2019), and 0.05% (Sim et al, 2019), 0% in B. tryoni (Choo et al, 2018), 0.1% in B. oleae (Meccariello et al, 2020), 0.2% in Anastrepha ludens (Sim et al, 2019), in C. capitata was 0.11% and 0.6% (Sim et al, 2019), and 1.2%, 0.6%, 0% (Meccariello et al, 2017), 3.1% in Z. cucurbitae (Paulo et al, 2022). In our study, we report mutagenesis efficiency of 1.12% in Z. cucurbitae .…”

“…Embryonic microinjection of double sgRNA was successful in generating a mutation of the white gene in B. dorsalis (Bai et al, 2019), B. tryoni (Choo et al, 2018), B. oleae (Meccariello et al, 2020). Meccariello et al (2017) reported embryonic microinjection of RNPs (Cas9+sgRNA) by CRISPR-Cas9 to disrupt the white gene in the C. capitata.…”

“…In our study, we report mutagenesis efficiency of 1.12% in Z. cucurbitae. Low mutagenesis efficiency associated with embryonic microinjection in tephritid fruit flies might be due to technical effects of microinjection procedure (Meccariello et al, 2017(Meccariello et al, , 2020. Mutagenesis efficiency should be calculated by the number of mutant flies generated from the total number of embryos injected.…”

CRISPR-Cas9-Based Technology and Its Relevance to Gene Editing in Parkinson’s Disease

“…Injection of RNP into embryos of various organisms has been successful so far, including mice [193], zebrafish [194], rabbit [195], reef-building corals [196], and axolotl [197], olive fruit fly [198], and spider mite [199]. Contrary to this, embryo microinjection may result in inevitable cell damage, and it necessitates highly skilled manipulation and sophisticated tools, both of which are difficult to implement in non-specialist facilities.…”