Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9

Xuemei Zhang,Ning Zhang,Lei Chen,Xuejiao Li,Liqin Wang,Jiapeng Lin,Juncheng Huang,Bing Han,Mayila Mayila,Sangang He,Wenrong Li,Xinrong Peng,Mingjun Liu,Yangsheng Wu,Chenxi Liu

doi:10.1038/s41598-017-08636-0

Abstract

Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D9 and/or D5 preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D9/D5 mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.

Highlights

Animal coat color as one of the major breed characteristics is an important production and economy trait
A 5 bp deletion of exon 2 which resulted in a frame shift followed by a premature stop at 63 amino acids was recognized as a recessive mutation for black coat color[13]
The knowledge of the role and mechanism of ASIP gene in determination of coat color pattern predominantly relys on spontaneous mutations in farm animals

Summary

Introduction

Animal coat color as one of the major breed characteristics is an important production and economy trait. The knowledge of the role and mechanism of ASIP gene in determination of coat color pattern predominantly relys on spontaneous mutations in farm animals. Precise modification of ASIP gene by genome manipulation offers a powerful means to comprehensively recognize the function of ASIP gene and get insight into the mechanism of coat color formation and alteration. In attempt to get colorful fine wool sheep and furtherly understand the role and molecular mechanisms of ASIP in formation and regulation of coat color, hereby we applied CRISPR/Cas[9] gene editing approach to disrupt sheep ASIP gene under the guidance of sgRNA. We speculated that the variation of coat color phenotypes in targeted sheep was likely attributable to comprehensive factors of Cas9-mediated modifications, duplications and other spontaneous mutations. It offers technical approaches to change coat color and provides evidence and animal models to study the function and mechanism of ASIP gene in formation of coat color pattern in farm animals

Methods

Results

Conclusion