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

Abstract

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.

Genetically Modified Rabbits for Cardiovascular Research

“…Spontaneous mutations in rabbits can be found accidentally and they can be used in controlling the coat color for commercial purposes such as tyrosinase and the melanocortin 1 receptor (Aigner et al, 2000 ; Xiao et al, 2019 ). However, some spontaneous mutations in rabbits can cause a pronounced phenotype that can mimic human diseases, such as Watanabe heritable hyperlipidemic (WHHL) rabbits (Watanabe, 1980 ), St. Thomas hyperlipidemic rabbits (Laville et al, 1987 ; Seddon et al, 1987 ) and complement 6 deficient rabbits (Rother, 1986 ; Liu et al, 2007a ).…”

“…The transgenic constructs are typically composed of the transgene (either cDNA or genomic DNA) under the control of a tissue-specific promoter such as liver-and macrophage-specific promoter. In addition to the pronuclear microinjection method, other methods such as sperm vector (Wang et al, 2003;Li et al, 2006Li et al, , 2010Shen et al, 2006), ICSI-mediated transgenesis (Li et al, 2010;Zhang et al, 2016), somatic cell nuclear transfer (SCNT) (Li et al, 2009) or chimeric SCNT (Matsuda et al, 2002;Skrzyszowska et al, 2006), lentiviral vectors (Hiripi et al, 2010), transposonmediated transgenesis (Katter et al, 2013;Ivics et al, 2014), and novel genome editing technology (Song J. et al, 2016;Yang et al, 2016;Li et al, 2019) have been reported to produce Tg rabbits. In spite of this, the pronuclear microinjection is still the most common method even though transgene integration rate is low.…”

Analysis of MC1R, MITF, TYR, TYRP1, and MLPH Genes Polymorphism in Four Rabbit Breeds with Different Coat Colors

“…In this study, two indels (c.284-285del and c.292-295del) were found in the TB and SW breeds, and polymorphism distribution of the c.292-295del sites were significantly different between TBs and SWs ( p < 0.05). Previous investigations found that MC1R c.280_285del6 was more frequently presented in black rabbits, c.304_333del30 was recessive red/yellow, and c.[124A;125_130del6] was connected with Japanese brindling coat color [ 6 , 7 , 30 ]. The MITF gene polymorphism is known to be associated with the black and white colors in mice [ 31 ], pigs [ 32 ], cattle [ 33 ], horses [ 34 ], chickens [ 35 ], ducks [ 36 ], llamas [ 37 ], and so on.…”

Rabbits – their domestication and molecular genetics of hair coat development and quality

“…They have shown that the Gray Giant rabbit has an E allele, whereas the New Zealand White rabbit, the Checkered Giant rabbit and the Lianshan Black rabbit have a 6 bp in‐frame deletion (Xiao et al . 2019). The experimental MC1R ko causes the blockage of the synthesis of eumelanin and has created a novel pale‐yellow coat color.…”