Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes

文献类型： 外文期刊

第一作者： Pi, Wenhui

作者： Pi, Wenhui;Feng, Guangyu;Liu, Minghui;Nie, Cunxi;Chen, Cheng;Wang, Jingjing;Wang, Limin;Wan, Pengcheng;Liu, Changbin;Zhou, Ping;Liu, Yi

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关键词： electroporation; gene editing; CRISPR/Cas9; sheep zygotes; mutation efficiency; homologous recombination; in vitro fertilization (IVF)

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：4.9； 五年影响因子：5.7 ）

ISSN： 1661-6596

年卷期： 2024 年 25 卷 17 期

页码：

收录情况： SCI

摘要： 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 ACTG1 and MSTN 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 MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

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