Huang Yu, Ding Yige, Liu Yao, Zhou Shiwei, Ding Qiang, Yan Hailong, Ma Baohua, Zhao Xiaoe, Wang Xiaolong, Chen Yulin
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; and Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Reprod Fertil Dev. 2019 Aug;31(9):1533-1537. doi: 10.1071/RD18485.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL-1 Cas9 mRNA and 25ngμL-1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.
成簇规律间隔短回文重复序列（CRISPR）/Cas9系统是一种生产基因编辑动物的有效方法。我们通过向受精卵注射Cas9信使核糖核酸（mRNA）和单向导RNA（sgRNA）混合物，成功培育出了基因修饰的山羊和绵羊。然而，显微注射的递送系统在很大程度上参照的是为小鼠建立的方法；对于大型动物的培育而言，迫切需要优化注射条件。在此，我们设计了一项研究来优化山羊的Cas9 mRNA和sgRNA递送系统。通过比较四种用于sgRNA设计的计算工具并在山羊成纤维细胞中验证靶向效率，我们提出了一种选择具有更高靶向效率且脱靶突变可忽略不计的理想sgRNAs的方案。我们进一步评估了注射Cas9:sgRNA（sg8）的山羊受精卵中的编辑效率，发现注射50ngμL-1的Cas9 mRNA和25ngμL-1的sgRNA可提高编辑效率。我们的结果为通过受精卵注射方法优化大型动物基因组高效编辑的注射条件提供了参考方案。
