Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens
Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens
复制标题
DOI：
10.1096/fj.202001065rrr
复制
发表时间：
2020-10-08
期刊：
影响因子：
4.8
通讯作者：
Han, Jae Yong
中科院分区：
文献类型：
--
作者：
Lee, Kyung Youn;Lee, Hong Jo;Han, Jae Yong
碱基编辑技术能够生成精确基因组修饰的动物模型。在这项研究中，我们将碱基编辑应用于鸡，这是农业，营养和研究领域的重要家畜动物，通过原始生殖细胞（PGC）介导的种系传递。使用这种方法，我们成功地产生了两个基因组修饰的鸡株，其编码卵转铁蛋白（TF）和肌肉生长抑制素（MSTs）的基因中含有突变;然而，只有55.5%和35.7%的基因组修饰的鸡分别在TF和MSTs中具有所需的碱基替换。为了解释低碱基编辑活性，我们进行了分子分析，以比较PGC和鸡成纤维细胞系DF-1之间的DNA修复途径。结果显示，相对于DF-1细胞，PGC中碱基切除修复（BER）相关基因显着升高。随后的功能研究证实，编辑活性可以通过调节尿嘧啶N-糖基化酶（UNG）的表达来调节，尿嘧啶N-糖基化酶是BER途径的上游基因。总的来说，我们的研究结果表明，鸡PGC独特的DNA修复特性导致基因组修饰过程中的编辑活性较低，然而，BER功能的调节可以促进具有所需基因型的基因组修饰生物体的产生。
Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions inTFandMSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.