Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing
Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing
复制标题
DOI：
10.1016/j.cub.2021.06.056
复制
发表时间：
2021-09-13
期刊：
影响因子：
9.2
通讯作者：
Furuta, Yasuhide
中科院分区：
文献类型：
--
作者：
Kiyonari, Hiroshi;Kaneko, Mari;Furuta, Yasuhide
有袋类动物是现存的三个哺乳动物亚类之一，具有其他哺乳动物所不具有的非常独特的特征。最值得注意的是，在相对较短的妊娠期后出生的新生儿的大部分发育发生在外部环境中。在有袋类动物中，灰色短尾负鼠（Monodelphis arctica;以下简称负鼠）是少数几种建立的实验室模型之一。由于许多生物学上独特的特征和实验上的优势，负鼠被用作有袋动物生物学基础研究的原型物种。（1，2）然而，负鼠体内基因功能的研究，因此一般有袋动物，远远落后于真兽目哺乳动物，由于缺乏可靠的手段来操纵它们的基因组。在这项研究中，我们描述了通过生殖生物学和胚胎操作中的精细方法相结合成功产生基因组编辑负鼠。我们利用了负鼠在体型和繁殖特征上与流行的啮齿动物模型（如小鼠和大鼠）的相似性。首先，我们建立了一个易于处理的生殖技术管道，从诱导排卵，定时交配，受精卵收集到胚胎移植到假孕雌性，这保证了一个操纵负鼠受精卵的重要平台。此外，我们成功地证明了通过使用CRISPR/Cas9基因组编辑显微注射原核期合子，在Tyr基因座处产生基因敲除负鼠，沿着编辑的等位基因的种系传递到F1代。这项研究为将哺乳动物反向遗传学扩展到元兽亚类提供了一个重要的基础。
Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.(1,2) However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.