Table_1_A Novel Pale-Yellow Coat Color of Rabbits Generated viaMC1R Mutation With CRISPR/Cas9 System.docx

Coat color is of great importance in animal breed characteristics; it is not only a significant productive trait but also an indispensable economic trait, especially in the rabbit industry. In the present study, the relationship between melanocortin 1 receptor (MC1R) genotypes and coat color phenotypes was observed in five rabbit breeds with popular coat colors that are present in China. These breeds comprised the Lianshan black rabbit (BR), Fujian yellow rabbit (YR), New Zealand white rabbit (WR), Gray Giant rabbit (GR), and Checkered Giant rabbit (CR), which were firstly determined, and the results showed that GR had an E allele; WR, CR, and BR had a 6-bp in-frame deletion (c.281_286del6, ED allele); and YR had a 30-bp deletion (c.304_333del30 E allele). To explore the feasibility of obtaining a novel rabbit coat color through the mutation of MC1R with the CRISPR/Cas9 system, two single-guide RNAs (sgRNAs) were designed for the MC1R gene, and the editing efficiency was confirmed by injection of rabbits’ zygotes. Unlike the donor rabbits whose coat color was originally black, two novel pale-yellow-coated rabbits were generated in the founders. A total of six novel MC1R gene deletions were identified in the two founder rabbits, in which the longest deletion was more than 700 bp. The histological hematoxylin-and-eosin (H&E) staining results indicated that eumelanin amounts were absent in hair follicles of MC1R-knockout (KO) rabbits, when compared with that of donor BR. In addition, the messenger RNA (mRNA) levels of some key downstream genes in the MC1R pathway were all downregulated in MC1R-KO rabbits compared with BR and YR. These results further indicate that loss-of-function MC1R contributed to blocking the synthesis of eumelanin and created a novel pale-yellow coat color in the MC1R-KO rabbits, and gene editing technology may be a useful tool to generate novel phenotypes in rabbit breeding.

被毛颜色在动物品种特征塑造中具有关键价值，其既是重要的生产性状，亦是不可或缺的经济性状，在家兔产业中尤为突出。本研究针对国内主流被毛颜色的5个家兔品种，探究了黑皮质素1受体（melanocortin 1 receptor, MC1R）基因型与被毛颜色表型之间的关联。本次研究的5个品种分别为连山黑兔（BR）、福建黄兔（YR）、新西兰白兔（WR）、巨型灰兔（Gray Giant rabbit, GR）以及花巨兔（Checkered Giant rabbit, CR），首先对其MC1R基因型进行了鉴定。结果显示：GR携带E等位基因；WR、CR与BR存在6 bp的框内缺失（c.281_286del6，ED等位基因）；YR则存在30 bp的缺失（c.304_333del30 E等位基因）。
为探究通过CRISPR/Cas9系统对MC1R基因进行突变以获得新型家兔被毛颜色的可行性，本研究针对MC1R基因设计了两条单向导RNA（single-guide RNAs, sgRNAs），并通过家兔受精卵注射实验验证了编辑效率。与原本被毛为黑色的供体兔不同，本研究在首建个体（founders）中获得了2只具有新型浅黄褐色被毛的个体。在这2只首建兔中，共鉴定出6种新型MC1R基因缺失片段，其中最长缺失片段超过700 bp。组织学苏木精-伊红（hematoxylin-and-eosin, H&E）染色结果显示：与供体连山黑兔相比，MC1R基因敲除（knockout, KO）家兔的毛囊中未检测到真黑素。此外，与连山黑兔和福建黄兔相比，MC1R-KO家兔体内MC1R通路中部分关键下游基因的信使RNA（messenger RNA, mRNA）表达水平均出现下调。
上述结果进一步表明，功能丧失型MC1R可阻断真黑素的合成，从而在MC1R-KO家兔中产生新型浅黄褐色被毛；同时也证实，基因编辑技术可作为一种有效工具，用于家兔育种中的新型表型创制。