Zhou Xiaoyang, Wang Lulu, Du Yinan, Xie Fei, Li Liang, Liu Yu, Liu Chuanhong, Wang Shiqiang, Zhang Shibing, Huang Xingxu, Wang Yong, Wei Hong
Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Hum Mutat. 2016 Jan;37(1):110-8. doi: 10.1002/humu.22913. Epub 2015 Oct 23.
Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.
对模式动物进行精确的基因突变对于人类突变的功能研究具有很高的价值。成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）诱导的同源定向修复（HDR）通常用于精确的基因突变，但与非同源末端连接（NHEJ）相比，其效率相对较低，受到一定限制。尽管抑制NHEJ已被证明可增强HDR衍生的突变，但在本研究中，我们在不抑制NHEJ的情况下，首先通过使用单链寡聚DNA（ssODN）作为模板，在受精卵中利用CRISPR/Cas9诱导的HDR产生了携带精确直系同源人类突变（Sox10 c.A325>T）的基因修饰猪，效率高达80%，这表明猪受精卵相对于NHEJ表现出较高的HDR活性，并且非常适合通过CIRSPR/Cas9诱导的HDR进行基因突变。此外，我们发现较高浓度的ssODN显著降低了猪受精卵中HDR衍生的突变，这表明在受精卵中，对于最佳的HDR衍生突变，可能存在一种平衡，即HDR模板的过度可及性与HDR相对于NHEJ的活性之间的平衡，而这种平衡似乎与ssODN浓度呈负相关。此外，HDR衍生的突变以及NHEJ产生的突变广泛整合到包括奠基猪性腺在内的各种组织中，未检测到脱靶现象，这表明在受精卵中通过CRISPR/Cas9诱导的HDR是猪精确基因突变的可靠方法。
