背景:掠食性花虫Oriusstrigicollis是针对小型节肢动物的有价值的生物防治剂;但是,它的有效性可能会有所不同,尤其是当由于猎物/害虫密度低而无法建立种群时。提高O.strigicollis作为生物防治剂的功效的一种有希望的方法是通过基因编辑。然而,当雌性在植物组织中产卵时,在这个物种中,传统的胚胎注射方法具有挑战性。
结果:在这项研究中,我们旨在使用直接亲本CRISPR(DIPA-CRISPR)开发一种有效且实用的O.strigicollis基因编辑技术。雌虫在不同的出苗后阶段接受了Cas9核糖核蛋白注射,随后使用PCR和异源双链迁移率测定对其后代(G0)进行基因分型。我们针对犬尿氨酸3-单加氧酶基因(朱砂),昆虫全色素生物合成的关键。通过实验优化,我们实现了52%的峰值基因编辑效率,即,52%的G0后代在出苗后1天注射时携带基因编辑的等位基因。值得注意的是,一些基因编辑的G0成年人表现出红眼马赛克表型,与黑眼野生型相反。交叉实验证实了在随后的世代(G1)中引入的突变的遗传性,能够建立一条明亮的红色眼睛的朱砂击倒线。
结论:我们证明了我们为O.strigicollis定制的DIPA-CRISPR基因编辑方法是有效和实用的。我们的发现强调了DIPA-CRISPR作为O.strigicollis基因工程工具的效力,并建议了更广泛的应用来增强其他生物防治剂。©2024化学工业学会。
BACKGROUND: The predatory flower bug Orius strigicollis serves as a valuable biocontrol agent against small arthropods; however, its effectiveness can vary, especially when population establishment fails due to low prey/pest densities. A promising approach to improve the efficacy of O. strigicollis as a biocontrol agent is through gene editing. However, as females lay their eggs in plant tissue, the conventional embryo injection approach is challenging in this species.
RESULTS: In this study, we aimed to develop an efficient and practical gene editing technique for O. strigicollis using direct parental CRISPR (DIPA-CRISPR). Female bugs at various postemergence stages received Cas9 ribonucleoprotein injections, with subsequent genotyping of their offspring (G0) using PCR and a heteroduplex mobility assay. We targeted the kynurenine 3-monooxygenase gene (cinnabar), pivotal for insect ommochrome pigment biosynthesis. Through experimental optimization, we achieved a peak gene editing efficiency of 52%, i.e., 52% of G0 progeny carried gene-edited alleles when injecting 1 day postemergence. Notably, some gene-edited G0 adults exhibited a red-eye mosaic phenotype, in contrast to the black-eyed wild type. Crossing experiments confirmed the heritability of the introduced mutations in the subsequent generation (G1), enabling the establishment of a cinnabar-knockout line with bright red eyes.
CONCLUSIONS: We demonstrate that our DIPA-CRISPR gene editing method tailored for O. strigicollis is efficient and practical. Our findings highlight the potency of DIPA-CRISPR as a tool for O. strigicollis genetic engineering and suggest broader applications for enhancing other biocontrol agents. © 2024 Society of Chemical Industry.