National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA; Division of Animal Science, University of Missouri, Columbia, MO, USA.
National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA; USDA-ARS, Plant Genetics Unit, Columbia, MO, USA.
Reprod Toxicol. 2021 Oct;105:44-52. doi: 10.1016/j.reprotox.2021.08.002. Epub 2021 Aug 15.
Pigs have become an important model for agricultural and biomedical purposes. The advent of genomic engineering tools, such as the CRISPR/Cas9 system, has facilitated the production of livestock models with desired modifications. However, precise site-specific modifications in pigs through the homology-directed repair (HDR) pathway remains a challenge. In mammalian embryos, the use of small molecules to inhibit non-homologous end joining (NHEJ) or to improve HDR have been tested, but little is known about their toxicity. The compound RS-1 stimulates the activity of the RAD51 protein, which plays a key role in the HDR mechanism, demonstrating enhancement of HDR events in rabbit and bovine zygotes. Thus, in this study, we evaluated the dosage and temporal effects of RS-1 on porcine embryo development and viability. Additionally, we assessed the effects of its vehicle, DMSO, during embryo in vitro culture. Transient exposure to 7.5 μM of RS-1 did not adversely affect early embryo development and was compatible with subsequent development to term. Additionally, low concentrations of its vehicle, DMSO, did not show any toxicity to in vitro produced embryos. The transient use of RS-1 at 7.5 μM during in vitro culture seems to be the best protocol of choice to reduce the potentially toxic effects of RS-1 while attempting to improve HDR in the pig. Direct injection of the CRISPR/Cas9 system, combined with strategies to increase the frequency of targeted modifications via HDR, have become an important tool to simplify and accelerate the production of genetically modified livestock models.
猪已成为农业和生物医学目的的重要模型。基因组工程工具（如 CRISPR/Cas9 系统）的出现促进了具有所需修饰的家畜模型的生产。然而，通过同源定向修复（HDR）途径在猪中进行精确的定点修饰仍然是一个挑战。在哺乳动物胚胎中，已经测试了使用小分子抑制非同源末端连接（NHEJ）或提高 HDR 的方法，但对其毒性知之甚少。化合物 RS-1 可刺激 RAD51 蛋白的活性，RAD51 蛋白在 HDR 机制中起关键作用，可增强兔和牛受精卵的 HDR 事件。因此，在这项研究中，我们评估了 RS-1 的剂量和时间对猪胚胎发育和活力的影响。此外，我们还评估了其载体 DMSO 在胚胎体外培养中的影响。短暂暴露于 7.5 μM 的 RS-1 不会对早期胚胎发育产生不利影响，并且与随后的足月发育兼容。此外，其载体 DMSO 的低浓度对体外产生的胚胎没有任何毒性。在体外培养过程中短暂使用 7.5 μM 的 RS-1 似乎是减少 RS-1 潜在毒性影响的最佳选择方案，同时试图提高猪中的 HDR。CRISPR/Cas9 系统的直接注射，结合通过 HDR 增加靶向修饰频率的策略，已成为简化和加速基因修饰家畜模型生产的重要工具。
