An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLG^{c.61C > T}), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLG^{c.61C > T} mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLG^{c.61C > T} single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.