College of Animal Science and Technology, Shihezi University, Shihezi 832003, China.
State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
Int J Mol Sci. 2024 Aug 23;25(17):9145. doi: 10.3390/ijms25179145.
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the and genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of -modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
电穿孔法用于递送 CRISPR/Cas9 系统组件，使哺乳动物受精卵中的基因编辑变得高效，从而促进了基因组编辑动物的发展。在这项研究中，我们专注于对绵羊的 和 基因进行靶向研究，揭示了电穿孔过程中的一个门限现象，即在绵羊体外受精（IVF）受精卵中存在电压耐受现象。我们研究了各种接近 40V 的穿孔电压和脉冲持续时间，以对绵羊受精卵进行电穿孔。研究得出结论，更强的电场需要更短的脉冲持续时间，才能实现高基因突变率和合理囊胚发育的最佳条件。本研究还评估了 Cas9/sgRNA 核糖核蛋白复合物（Cas9 RNPs）的质量及其对绵羊早期胚胎基因组编辑效率的影响。研究强调，在电穿孔之前，Cas9 蛋白与单链向导 RNA（sgRNA）的预复合对于实现高突变率至关重要。通过对绵羊 IVF 受精卵使用合适的电穿孔参数，可以显著提高突变率和杂合子比率。通过电穿孔将 Cas9 RNPs 和单链寡脱氧核苷酸（ssODNs）递送至受精卵，靶向 （肌肉生长抑制素）基因，实现了 26%的敲入效率。通过将 Cas9 RNPs 电穿孔递送至 IVF 受精卵，成功生成了 基因修饰的羔羊。
