Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, 890-8544, Japan.
Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, 259-1193, Japan.
Arch Pharm Res. 2018 Sep;41(9):898-910. doi: 10.1007/s12272-018-1053-z. Epub 2018 Jul 4.
The discovery of sequence specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 has revolutionized genome editing. The CRISPR/Cas9 system has particularly emerged as a highly simple and efficient approach towards generating genome-edited animal models of most of the experimental species. The limitation of these novel genome editing tools is that, till date, they depend on traditional pronuclear injection (PI)-based transgenic technologies developed over the last three decades. PI requires expensive micromanipulator systems and the equipment operators must possess a high level of skill. Therefore, since the establishment of PI-based transgenesis, various research groups worldwide have attempted to develop alternative and simple gene delivery methods. However, owing to the failure of chromosomal integration of the transgene, none of these methods gained the level of confidence as that by the PI method in order to be adapted as a routine approach. The recently developed genome editing systems do not require complicated techniques. Therefore, presently, attention is being focused on non-PI-based gene delivery into germ cells for simple and rapid production of genetically engineered animals. For example, a few reports during the previous 1-2 years demonstrated the use of electroporation (EP) in isolated zygotes that helped to overcome the absolute dependency on PI techniques. Recently, another breakthrough technology called genome editing via oviductal nucleic acids delivery (GONAD) that directly delivers nucleic acids into zygotes within the oviducts in situ was developed. This technology completely relieves the bottlenecks of animal transgenesis as it does not require PI and ex vivo handling of embryos. This review discusses in detail the in vivo gene delivery methods targeted towards female reproductive tissues as these methods that have been developed over the past 2-3 decades can now be re-evaluated for their suitability to deliver the CRISPR/Cas9 components to produce transgenic animals. This review also provides an overview of the latest advances in CRISPR-enabled delivery technologies that have caused paradigm shifts in animal transgenesis methodologies.
诸如 ZFNs、TALENs 和 CRISPR/Cas9 等序列特异性核酸酶的发现彻底改变了基因组编辑。CRISPR/Cas9 系统尤其成为生成大多数实验物种的基因组编辑动物模型的高度简单和高效的方法。这些新型基因组编辑工具的局限性在于，迄今为止，它们仍然依赖于过去三十年发展起来的传统核注射 (PI) 为基础的转基因技术。PI 需要昂贵的微操作器系统，并且操作人员必须具备高水平的技能。因此，自从建立了基于 PI 的转基因技术以来，世界各地的各种研究小组都试图开发替代的简单基因传递方法。然而，由于转基因的染色体整合失败，这些方法都没有像 PI 方法那样获得足够的信心，无法被采用为常规方法。最近开发的基因组编辑系统不需要复杂的技术。因此，目前，人们的注意力集中在非 PI 基的基因传递到生殖细胞上，以简单快速地生产基因工程动物。例如，在过去 1-2 年中，有几份报告表明在分离的受精卵中使用电穿孔 (EP) 有助于克服对 PI 技术的绝对依赖。最近，另一种突破性技术称为通过输卵管核酸传递进行基因组编辑 (GONAD)，该技术可直接将核酸递送到输卵管内的受精卵中。该技术完全消除了动物转基因的瓶颈，因为它不需要 PI 和胚胎的体外处理。这篇综述详细讨论了针对女性生殖组织的体内基因传递方法，因为这些在过去 2-3 十年中开发的方法现在可以重新评估其是否适合将 CRISPR/Cas9 组件递送到产生转基因动物。这篇综述还概述了 CRISPR 使能的传递技术的最新进展，这些进展导致了动物转基因方法的范式转变。
