Niu Yiyuan, Zhao Xiaoe, Zhou Jiankui, Li Yan, Huang Yu, Cai Bei, Liu Yutai, Ding Qiang, Zhou Shiwei, Zhao Jin, Zhou Guangxian, Ma Baohua, Huang Xingxu, Wang Xiaolong, Chen Yulin
College of Animal Science and Technology, Northwest A&F University, #22, Xinong Road, Yangling 712100, China.
College of Veterinary Medicine, Northwest A&F University, #22, Xinong Road, Yangling 712100, China.
Reprod Fertil Dev. 2018 Jan;30(2):307-312. doi: 10.1071/RD17068.
The recent emergence of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has attracted significant attention for its potential to improve traits of agricultural importance. However, most applications in livestock species to date have depended on aberrant DNA repair to generate frameshifting indels. Whether this genomic engineering technique involving homology-dependent repair (HDR) can be used to introduce defined point mutations has been less explored. Previously, we reported a G→A point mutation (g.231A>G, p.Val397Ile) in the growth differentiation factor 9 (GDF9) gene that has a large effect on the litter size of cashmere goats. In the present study we report that by co-injecting synthesised RNAs and single-stranded oligo deoxynucleotide (ssODN) donor sequences into goat zygotes, we successfully introduced defined point mutations resulting in single amino acid substitutions in the proteins as expected. The efficiency of this precise single-nucleotide substitution in newborn kids was as high as 24% (4/17), indicating that ssODN-directed HDR via zygote injection is efficient at introducing point mutations in the goat genome. The findings of the present study further highlight the complex genome modifications facilitated by the CRISPR/Cas9 system, which is able to introduce defined point mutations. This represents a significant development for the improvement of reproduction traits in goats, as well as for validating the roles of specific nucleotides in functional genetic elements in large animals.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas）9系统最近的出现，因其改善重要农业性状的潜力而备受关注。然而，迄今为止在牲畜物种中的大多数应用都依赖异常DNA修复来产生移码插入缺失。这种涉及同源依赖性修复（HDR）的基因组工程技术是否可用于引入特定的点突变，此前较少有人探索。此前，我们报道了生长分化因子9（GDF9）基因中的一个G→A点突变（g.231A>G，p.Val397Ile），该突变对绒山羊的产羔数有很大影响。在本研究中，我们报告称，通过将合成RNA和单链寡脱氧核苷酸（ssODN）供体序列共同注射到山羊受精卵中，我们成功引入了特定的点突变，如预期那样导致蛋白质中单个氨基酸的替换。新生羔羊中这种精确单核苷酸替换的效率高达24%（4/17），表明通过受精卵注射的ssODN介导的HDR在山羊基因组中引入点突变是有效的。本研究结果进一步突出了CRISPR/Cas9系统促进的复杂基因组修饰，该系统能够引入特定的点突变。这对于改善山羊繁殖性状以及验证大型动物功能基因元件中特定核苷酸的作用而言，是一项重大进展。
