Editing of HSF-1 and Na/K-ATPase α1 subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock in vitro .

A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit ( ATP1A1 ) gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle ( Bos indicus ). The knockout cell lines of Na/K- ATP1A1 and HSF-1 (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 ( ATP1A1 and HSF-1 ) and wild-type fibroblasts were exposed to heat shock at 42 °C in vitro and different cellular parameters viz ., apoptosis, proliferation, mitochondrial membrane potential (Δ Ψ m ), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that in vitro heat shock given to knockout fibroblast cells of both ATP1A1 and HSF-1 genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in HSF-1 knockout cells as compared to ATP1A1 knockout cells. Taken together, these results indicated that the ATP1A1 gene plays a critical role as HSF-1 under heat stress and helps cells to cope with heat shock.