CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor‐Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster‐derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR‐applied genetic manipulation in this and other rising model organisms.

中文翻译：

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通过卵巢上的CRISPR-Cas9核糖核蛋白的卵巢诱变纳森氏球菌的生殖。

CRISPR / Cas9基因编辑是一项功能强大的技术，可用于研究正在崛起的模型生物的遗传学，例如宝石黄蜂Nasonia vitripennis。然而，当前涉及CRISPR试剂的胚胎显微注射的方法具有挑战性。将Cas9核糖核蛋白递送到雌性卵巢中是另一种选择，只有少数昆虫，例如蚊子，粉虱和甲虫才进行了研究。在这里，我们通过在成年的核糖核蛋白注入Cas9开发了种系基因编辑一个简单的协议N. vitripennis使用任一REMOT控制（货物的受体介导的转导子房）或BAPC（支化两亲性肽胶囊）子房递送方法的女性。对于ReMOT控制，我们使用了果蝇（Drosophila melanogaster）衍生的肽“ P2C”与EGFP融合以可视化卵巢递送，并与Cas9蛋白融合以使用皂素作为内体逃逸试剂对朱砂基因进行基因编辑。对于BAPC，我们优化了蛋白质，sgRNA和转染试剂的浓度。我们展示了通过P2C肽和BAPC将蛋白货物如EGFP和Cas9传递到发育中的卵母细胞中。另外，证明了体细胞和种系基因编辑。这种方法将大大促进在这种以及其他新兴模型生物中应用CRISPR技术的遗传操作。