157 Production of β-lactoglobulin gene knockout blastocyst stage embryos of Indian water buffalo using CRISPR and somatic cell nuclear transfer technology

In many tropical countries, buffalo milk holds greater value than cow milk due to its superior nutritional and economic benefits. In India, buffaloes are the primary dairy animals, contributing a significant 45% to the nation’s total milk production. However, despite advances in breeding strategies, nutritional management, and quantitative genetics to improve milk yield, there have been limited achievements in altering milk composition. The emergence of biotechnology, particularly genome editing tools such as CRISPR, now offers the potential to create new value-added products, such as designer hypoallergenic milk, to enhance human health. While gene editing technology has been previously applied to produce BLG-free cows and goats (Wei et al. 2018 Sci. Rep. 8, 1–13; Zhou et al. 2017 PloS One 12, e0186056), no such attempts had been made in buffaloes. Therefore, this study aimed to utilise the CRISPR/Cas9 system to target the BLG gene in buffalo fibroblasts and generate BLG-gene edited cloned embryos. Three sgRNAs were designed against the BLG locus, and their editing efficiency was assessed through Sanger sequencing, T7E assay, TIDE, and ICE analysis. The most efficient sgRNA was chosen to create a clonal population of edited cells. Fifteen single-cell clones were established and screened using TA cloning and Sanger sequencing methods, out of which seven were found to have BLG-gene disruption. These edited single-cell clones exhibited various types of mutations, including bi-allelic heterozygous, bi-allelic homozygous, and mono-allelic gene disruptions, each involving distinct nucleotide deletions and insertions. The edited single-cell clones were then used as nuclear donors for somatic cell nuclear transfer applications. Although the embryonic developmental rates varied among different cell clones, with embryo production rates ranging from 30%–60% for edited cells and 45% for non-edited control cells, the total cell number and apoptotic index were comparable between the two groups. Embryo developmental data indicated that CRISPR-based genetic modifications did not adversely affect the cloned embryonic developmental competence. The study is currently making efforts to establish pregnancies from BLG-gene edited cloned embryos. The creation of BLG gene disrupted cloned embryos represents a valuable resource for future production of genome-edited buffaloes, offering the potential to provide hypoallergenic milk and encourage the wider consumption of buffalo milk among individuals with milk allergies.