Germline mutagenesis ofNasonia vitripennisthrough ovarian delivery ofCRISPR-Cas9ribonucleoprotein
Germline mutagenesis ofNasonia vitripennisthrough ovarian delivery ofCRISPR-Cas9ribonucleoprotein
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DOI：
10.1111/imb.12663
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发表时间：
2020-08-19
影响因子：
2.6
通讯作者：
Akbari, O. S.
中科院分区：
文献类型：
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作者：
Chaverra-Rodriguez, D.;Dalla Benetta, E.;Akbari, O. S.
CRISPR/Cas9基因编辑是一种强大的技术，可以研究新兴模式生物的遗传学，例如宝石黄蜂Nasponia vitripennis。然而，目前涉及胚胎显微注射CRISPR试剂的方法具有挑战性。将Cas9核糖核蛋白递送到雌性卵巢中是一种替代方案，仅在少数昆虫中进行了探索，如蚊子，粉虱和甲虫。在这里，我们开发了一种简单的方案，通过在adultN中注射Cas9核糖核蛋白来进行种系基因编辑。使用ReMOT对照（受体介导的卵巢转运的货物）或BAPC（分支两亲肽胶囊）作为卵巢递送方法对透明质酸拟雌虫进行了研究。对于ReMOT对照，我们使用与EGFP融合的果蝇衍生肽“P2C”来可视化卵巢递送，并使用皂苷作为内体逃逸试剂与Cas9蛋白融合以用于对cinnabar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。我们证明了通过P2C肽和BAPC将蛋白质货物如EGFP和Cas9递送到发育中的卵母细胞中。此外，还展示了体细胞和生殖系基因编辑。这种方法将极大地促进CRISPR应用于这种和其他新兴模式生物的遗传操作。
CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel waspNasonia 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 adultN. vitripennisfemales 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 theDrosophila melanogaster-derived peptide 'P2C' fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of thecinnabargene 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.