Generation of functional Na_V1.5 current by endogenous transcriptional activation of SCN5A

Abstract

The sodium channel Na_V1.5, which is encoded by the SCN5A gene, underlies the fast upstroke of cardiac action potential and thus plays a crucial role in cardiac electrophysiology, but the mechanism governing the regulation of Na_V1.5 has not been fully elucidated. The newly developed clustered regularly interspaced short palindromic repeats (CRSPR)/Cas9 transcription factors offer a powerful and precise approach for modulating gene expression. We investigated the potential of this new tool for activating stringently silenced SCN5A in human cells. We first selected the most efficient single guide RNA (sgRNA) targeting upstream transcription start sites to induce effective expression of SCN5A mRNA. We observed significant transcriptional activation of endogenous SCN5A, with the highly effective activity of sgRNA targeting the human SCN5A promoter. The optimized dCas-VP64/sgRNA enhanced the endogenous SCN5A transcription up to 20-fold in human HEK293T cells and ultimately generated the Na_V1.5 protein. Interestingly, multiple transcript variants of SCN5A were generated by endogenous transcriptional activation. Functionally, the Na_V1.5 current produced by endogenous activation exhibited a similar electrophysiological property to that produced by ectopic overexpression of Na_V1.5. The results of our study suggest that Cas9-mediated transcriptional activation is a useful tool for modulating gene expression and conducting electrophysiological studies in human cells.

Keywords:

• CRSPR/Cas9
• SCN5A
• transcriptional activation
• Na_V1.5 current

Acknowledgements

We are thankful to Jiahe Xu for supporting us for draft and figure preparation.

Authors’ contributions

Rui Shi and Liang Xu designed the study. Liang Xu designed, performed the experiments, and Rui Shi analyzed the results. Rui Shi and Liang Xu drafted the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was funded by the National Key Research and Development Program (2018YFC1312504 to Liang Xu).