LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Sci Rep. 2020 Sep 24;10(1):15587. doi: 10.1038/s41598-020-72040-4.
The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
新技术在马属动物基因编辑中的应用可能会产生改良的运动型个体。在这里，我们旨在使用 CRISPR/Cas9 敲除肌肉生长抑制素基因 (MSTN)，该基因是肌肉生长的负调控因子，并首次在马属动物中生成编辑胚胎。我们用 1、2 或 5μg 的 2 种不同的靶向 MSTN 第一外显子的 gRNA/Cas9 质粒对马胎儿成纤维细胞进行电穿孔。我们发现，增加质粒浓度可以提高突变效率。gRNA1 的平均效率为 63.6%（22 个编辑克隆细胞系中的 14 个），gRNA2 的效率为 96.2%（26 个编辑克隆细胞系中的 25 个）。选择了 3 个克隆细胞系通过体细胞核移植来生成胚胎：一个是单等位基因编辑，一个是双等位基因杂合编辑，一个是双等位基因纯合编辑，每个情况下都生成了编辑的囊胚。对胚胎中的 MSTN 编辑和脱靶情况进行了分析。总之，CRISPR/Cas9 被证明是一种高效的方法，可以以剂量依赖的方式在马属动物基因组中进行编辑，具有高度的特异性。通过适应这项技术，可以产生具有运动优势的等位基因，并可以开发出精确的育种计划。
