Crispri Ion Channel Gene Modulation in Human iPSC-Cardiomyocytes

Abstract

Cardiotoxicity is a major limiting factor of drugs going to market. Current means to assess cardiotoxicity are outdated as compounds are only screened for hERG (KCNH2) channel sensitivity. The sole measurement of hERG activity is restrictive as there are multiple modes in which a drug may induce off-target effects and potentiate arrhythmia. Therefore, there is a need for models measuring multiple channel effects on cardiomyocytes. Our objective was to functionally validate loss-of-function gene perturbations in cardiac electrophysiology to potentiate a high-throughput platform for cardiotoxicity studies. We employed CRISPRi, a means for highly specific gene modulation, commercial pre-differentiated human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM), and an all-optical dynamic cardiac electrophysiology system (OptoDyCE) which simultaneously records voltage and calcium in high-throughput (96-well) format. Pre-differentiated cardiomyocytes were transduced with sgRNAs subcloned into a lentiviral vector containing an eGFP fluorescent marker. sgRNAs were designed around the transcription start site of Kir2.1(KCNJ2) and KCNH2. Five days post-transduction, hiPSC-CMs were transfected with a dox-inducible dCas9-KRAB, containing a mCherry fluorescent marker. A near-infrared voltage-sensitive dye, BeRST, was used to monitor action potential duration (APD) and responses were recorded using the OptoDyCE system. sgRNAs targeting KCNJ2, resulted in about a 15-40% decrease in basic cycle length. Additionally, sgRNAs, targeting KCNH2, exhibited about a 10-40% increase in APD at 90% (APD90) in spontaneous recordings and showed about a 5-20% increase in APD90 from hiPSC-CMs subjected to pacing at 0.5Hz. Considering the role of these two K+ channels in modulating heart rate and repolarization properties, these results were within expected outcomes. Our data corroborates the potential of a CRISPRi-based platform for precise gene modulation of ion channels with all-optical cardiac electrophysiology technology for high throughput functional interrogation in already differentiated hiPSC-CMs for human cardiac disease modelling, cell therapies and drug discovery.