Genome editing has been found to be a valuable tool for lightening the hair and coat colour to better adapt dairy cattle to rapidly changing climatic conditions.
These are the findings by researchers from the AgResearch Centre in New Zealand. High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat colour, black absorbs more solar radiation translating into radiative heat gain which is a contributing factor to heat stress in cattle, negatively affecting their production levels, fertility and welfare.
The genome editing targeted the pre-melanosomal protein 17 (PMEL) gene. PMEL is a membrane protein that is exclusively expressed in pigmented cells where it is involved in the maturation of melanosomes, and pigment deposition and polymerisation in these organelles. Mutations in PMEL are associated with black pigment dilution in several species but are found in different parts of the protein.
In their study, the researchers stated that, in different species, PMEL mutations were shown to be responsible for colour dilution effects. For example, they said a deletion of a leucine codon in the signal peptide (p.Leu18del) of PMEL segregates in Highland, Galloway and Tuli cattle and has been proposed as a causative mutation for coat colour effects. “While the role of this variant has not been functionally confirmed, its proposed coat colour impacts make it an excellent candidate for introgression into Holstein Friesian dairy cattle to reduce radiative heat gain and improve overall heat tolerance,” they said.
In this study, the researchers used the gRNA/Cas9-mediated introgression of the naturally occurring mutation p.Leu18del in the PMEL gene, known from Galloway and Highland cattle, into Holstein Friesian cattle. Using gRNA/Cas9-mediated editing, they introduced a 3 base pair deletion in the PMEL gene proposed as the causative variant responsible for the semi-dominant colour dilution phenotype seen in Galloway and Highland cattle.
Calves homozygous for the PMEL mutation displayed distinct colour dilution to a silvery grey of the otherwise black coat markings in the control calves. They used a cloning method to create embryos set to be genetically modified and applied the gRNA/Cas9-mediation, the CRISPR tool, to mutate the PMEL gene. After 7 days in vitro culture, single embryos were non-surgically transferred to synchronised recipient cows for development.
To determine the phenotypic impact of the PMEL mutation, they used somatic cell nuclear transfer to generate calves with donor cells from the biallelic cell clone CC14 and the parental WT cell line BEF2. Following transfer of reconstructed embryos, a total of 7 pregnancies were detected at day 37 of gestation. For each genotype, one pregnancy failed, resulting in the birth of 2 PMEL mutant calves and 3 control calves. Once the 2 calves were born, the team confirmed that what should have been black markings were silvery grey – and white areas remained unaffected.
Calves generated from cells homozygous for the edited mutation revealed a strong colour dilution effect. Instead of the characteristic black and white coat colour patterning of control calves generated from unedited parental cells, the edited calves displayed a novel pattern of grey and white markings and absence of any black areas. This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat colour in cattle.