Comparison of efficacy and functional consequences of siRNA and CRISPRi gene modulation with different effector domains in post-differentiated human iPSC-CMs.

Abstract

Current models for cardiotoxicity screening are limited in their ability to only evaluate hERG inhibition and related prediction of QT prolongation and torsadoegnic potential. More comprehensive cardiotoxicity assays are being developed using in vitro platforms with human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), which are further aided by experimental and computational approaches. CRISPRi gene perturbation is a powerful source for human functional genomics in the cardiac field as it can link genes to electrophysiological phenotype. We developed a high-throughput system involving all-optical cardiac electrophysiology in 96-well format and CRISPRi gene modulation in post-differentiated iPSC-CMs. Single guide RNAs (sgRNAs) were tested targeting around the transcription start sites of KCNH2, an ion channel responsible for the repolarization of the cardiac action potential. Optogenetic pacing and spectrally-compatible voltage and calcium sensors were used to obtain functional measurements for voltage and calcium responses. The efficacy and functional consequences of gene modulation by siRNA and CRISPRi with two effector domains (dCas9-KRAB and dCas9-Zim3) were compared. We found that adenoviral transduction with a recently developed dCas9-Zim3, induced the most robust APD prolongation (+20%, p<0.0001) in the hiPSC-CMs upon KCNH2 knockdown when compared to using siRNA (+11%, p=0.0002) and transfection of an inducible-dCas9-KRAB (+<10%, p=0.02). Knockdown of hERG in our system yielded mild but specific functional changes which can be combined with computational approaches using dimension reduction approaches to help visualize and quantify electrophysiological changes in the heart. Usage of this platform for CRISPRi mediated knockdown of diseases-associated genes in pre-differentiated cardiomyocytes can improve the assessment of gene function in cellular cardiac electrophysiology.