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

Abstract

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 (ΔΨ_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.

Keywords:

• Skin fibroblast
• heat shock
• ATP1A1 gene
• knockout
• CRISPR/Cas9

Acknowledgments

We duly acknowledge the financial support from the Science and Engineering Research Board (SERB) and Indian Council of Agricultural Research-National Fellow Scheme.

Ethical approval

The experimental procedure for collecting the ear pinna samples was approved by Institutional (National Bureau of Animal Genetic Resources) Animal Ethical committee.

Author contributions

U.K.S. and M.M.: conceptualization. U.K.S., A.S., and M.S.: methodology. U.K.S. and A.S.: analysis. M.S. and U.K.S.: resources. U.K.S., M.M., and A.S.: manuscript writing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the data supporting these findings are present within the manuscript.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This research project was funded by DST-SERB and ICAR-National Fellow Scheme.