Center for Reproductive Biology, School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
Biol Reprod. 2019 Jul 1;101(1):177-187. doi: 10.1093/biolre/ioz075.
Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.
基因编辑技术，如 CRISPR-Cas9，在哺乳动物胚胎中有重要的应用，可用于生成新的生物医学研究动物模型和具有增强生产特性的家畜品系。然而，缺乏将基因编辑试剂有效引入各种物种的受精卵的方法，以及在小鼠中进行胚胎手术转移的需要，一直是广泛应用的技术障碍。在这里，我们描述了克服这些限制的方法，用于小鼠、牛和猪的胚胎。我们使用 Nanos2 基因的突变作为读出，优化了电穿孔参数，使用预组装的 sgRNA-Cas9 RNP，无需溶解透明带，从而实现了高效的 INDEL 编辑。此外，我们优化了培养条件，以支持三种物种的受精卵从受精卵到多细胞阶段的成熟，从而生成可用于产生基因编辑动物的胚胎。此外，对于小鼠，我们设计了一种非手术胚胎转移方法，其效率可与传统的手术方法相媲美。总的来说，这些研究的结果提供了简化的基于 CRISPR-Cas9 的基因编辑管道，适用于多种哺乳动物物种。
