Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan.
Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan.
Cells. 2020 Feb 26;9(3):546. doi: 10.3390/cells9030546.
Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4-1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4-1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called "sequential i-GONAD (si-GONAD)."
通过输卵管核酸递送（i-GONAD）进行改良的基因组编辑是一种能够在怀孕女性输卵管内的胚胎（受精卵）中诱导基因组变化的技术。i-GONAD 通过玻璃微管在解剖显微镜下将包含基因组编辑成分的溶液注入输卵管内，然后使用镊子型电极进行体内电穿孔。i-GONAD 不涉及胚胎的离体处理（受精卵的分离、受精卵的微注射或电穿孔以及处理后的胚胎的卵转移到受体女性的输卵管），这是体外受精卵基因组编辑所必需的。i-GONAD 可实现插入缺失、敲入（KI）约 1kb 感兴趣的序列以及靶标基因座的大片段缺失。i-GONAD 通常在妊娠第 0.7 天进行，相当于受精卵晚期。在该技术的初始发展阶段，我们在第 1.4-1.5 天（相当于 2 细胞期）进行 i-GONAD。理论上，这意味着至少要进行两次 GONAD 步骤（第 0.7 天和第 1.4-1.5 天）。如果这在实践中得到证明，它将为各种聚类规则间隔短回文重复（CRISPR）/ Caspase 9（Cas9）为基础的基因操作提供更多选择。例如，通过同时转染两种向导 RNA 和 Cas9 蛋白，在靶位点诱导两个独立的插入缺失通常很困难，因为它们彼此非常接近。然而，当在不同的日子重复进行 i-GONAD 时，可能可以在靶位点依次诱导插入缺失。此外，当在不同的日子重复进行 i-GONAD 时，同时引入两个突变的lox 位点（Cre 重组酶结合的位点）以产生 floxed 等位基因可能是可行的，因为它经常导致两个 gRNA 靶位点之间的序列缺失。然而，当在不同的日子重复进行 i-GONAD 时，可能可以进行差异 KI 的lox 位点。在这项研究中，我们进行了原理验证实验，以证明我们提出的称为“顺序 i-GONAD（si-GONAD）”的方法的可行性。
