Editing of and subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock .

A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit () gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle (). The knockout cell lines of Na/K- and (heat shock factor-1, as a positive control) genes were developed by CRISPR/Cas9 method and the gene-editing was confirmed by the genomic cleavage detection assay. The two knockout cell lines ( and ) and wild-type fibroblasts were exposed to heat shock at 42 °C and different cellular parameters ., apoptosis, proliferation, mitochondrial membrane potential (Δ), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that heat shock given to knockout fibroblast cells of both and genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in knockout cells as compared to knockout cells. Taken together, these results indicated that the gene plays a critical role as under heat stress and helps cells to cope with heat shock.