Pig Coat Color Manipulation by MC1R Gene Editing

Abstract

Black coat color in pigs is determined by the dominant E allele at the MC1R locus. Through comparing MC1R gene sequences between recessive e and dominant E^D1 alleles, we identified four missense mutations that could affect MC1R protein function for eumelanin synthesis. With the aim of devising a genetic modification method for pig coat color manipulation, we mutated the e allele in the Duroc breed to the dominant E^D1 allele using CRISPR-mediated homologous recombination for the four mutation substitutions at the MC1R locus. The MC1R-modified Duroc pigs generated using the allele replacement strategy displayed uniform black coat color across the body. A genotyping assay showed that the MC1R-modified Duroc pigs had a heterozygous E^D1/e allele at the MC1R locus; in addition, the pigs remained in the Duroc genetic background. Our work offers a gene editing method for pig coat color manipulation, which could value the culture of new pig varieties meeting the needs of diversified market.

Keywords: CRISPR; MC1R; coat color; gene editing; homologous recombination; pig.

Figure 1

Gene editing strategy at the MC1R locus for coat color switch in Duroc pigs. (A) MC1R sequence alignment among different pig breeds. The four SNPs at AA positions 95, 102, 164, and 243 of the MC1R gene distinct between Duroc breed (e) and Chinese indigenous black breed (E^D1) are shown in red. Landrace and Large White breeds have a specific D124N substitution compared with other breeds, which is the key variation forming the E^P allele. The E allele associated with each MC1R variant is indicated in parentheses. (B) CRISPR system and donor design for SNP substitutions in Duroc MC1R. A guide RNA (gRNA) was designed to recognize the cleavage target in the SNP regions. A 2757-bp DNA sequence was amplified from the MC1R genomic region in Chinese indigenous black pigs as the donor to replace the Duroc MC1R homologous region by HR. The gRNA-recognized sequence in the donor was synonymously mutated to avoid second CRISPR cleavage after HR. The genotyping primers amplifying a 1627-bp sequence covering homology arm and adjacent genome region after HR were used in PCR assay to detect the occurrence of gene replacement. AA, amino acid position.

Figure 2

Blackish Durocs generated by MC1R gene editing. (A) The newborn MC1R-modifed Duroc pigs with the dark black coat color (upper panel). As the control, the newborn wild-type Durocs with the red coat color are shown (lower panel). (B) The cloned MC1R-modified piglets displayed three types of coat color: dark black (a), light black (b), and wild-type red (c). Total substitution in four SNP sites (in one allele or both alleles) resulted in coat color switch in Durocs (a and b). Light black pigs (b) harbored an intact substituted MC1R allele, but its expression was lower than the same substituted allele in the dark black pigs (a). Expression level of the substituted MC1R allele may decide the black color depth. Non-modification or knockout of the MC1R e allele generated the red coat color (c). (C) Sanger sequencing results showed that all four SNPs were substituted with corresponding E^D1 genotypes on at least one MC1R allele in dark black pigs. Among them, nine had the four SNPs substituted on only one allele (dark black genotype 2), and eight had three SNPs substituted on one allele and one SNP substituted on both alleles (dark black genotype 1). AA, amino acid position.

Figure 3

Analysis of MC1R gene of cloned Durocs with light black coat color. (A) Representative image of a cloned Duroc (#8) showing the light black coat color. (B) Quantification of MC1R mRNA level in ear skin among cloned Durocs with three different coat colors (red, dark black, and light black). (C) Sanger sequencing results of genome DNA showed that #27 had the heterozygous E^D1/e alleles, and #8 had the heterozygous E^D1/E⁺ alleles (one allele of MC1R was changed to E^D1 genotype and the other allele had a V164A substitution forming an E⁺ genotype). Sanger sequencing of cDNA showed an equal expression level for both E alleles in light black pigs, but a great higher expression for the E^D1 allele in dark black pigs. Red arrows point to SNP sites varied in E^D1 and e alleles. AA, amino acid position.

Figure 4

Results of admixture analysis for breed tracing of cloned pigs. Four pig breeds were illustrated with different colors. Each bar represents an individual pig and values on the Y axis represent the proportions of ancestry.