Generation of functional NaV1.5 current by endogenous transcriptional activation of SCN5A

• Main Authors: Liang Xu, Rui Shi
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2021-01-01
• Series: Biotechnology & Biotechnological Equipment
• Subjects: crspr/cas9; scn5a; transcriptional activation; nav1.5 current
• Online Access: http://dx.doi.org/10.1080/13102818.2021.1892524

The sodium channel NaV1.5, 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 NaV1.5 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 NaV1.5 protein. Interestingly, multiple transcript variants of SCN5A were generated by endogenous transcriptional activation. Functionally, the NaV1.5 current produced by endogenous activation exhibited a similar electrophysiological property to that produced by ectopic overexpression of NaV1.5. 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.