CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes
CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes
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DOI：
10.1016/j.celrep.2019.07.089
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发表时间：
2019-08-27
期刊：
影响因子：
8.8
通讯作者：
Menke, Douglas B.
中科院分区：
文献类型：
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作者：
Rasys, Ashley M.;Park, Sungdae;Menke, Douglas B.
CRISPR-Cas9 介导的基因编辑使得能够直接操纵许多物种的基因功能。然而，爬行动物的生殖生物学给该技术的使用带来了独特的障碍，并且没有爬行动物具有有效的定向诱变方法。在这里，我们证明，显微注射 Anolis sagrei 雌性卵巢内的未成熟卵母细胞能够产生 CRISPR-Cas9 诱导的突变。该方法能够产生具有单等位基因或双等位基因突变的 FO 胚胎和幼体。我们证明这些突变可以通过种系传播以建立转基因蜥蜴品系。现在可以在 Anolis 蜥蜴中进行基因功能的直接测试，Anolis 蜥蜴是研究爬行动物进化和发育的重要模型。
CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate that the microinjection of immature oocytes within the ovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing FO embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.