Abstract
Background and objective: Modeling of human physiologic traits using pluripotent stem cell derived cardiomyocytes has been hampered in part by variability between cardiomyocytes derived from different stem cell lines. While the advent of genome editing affords unprecedented opportunities to evaluate gene function, the variation between cell lines creates challenges in distinguishing signal from noise. We recently developed a genetically encoded voltage sensing fluorescent protein, Arclight, that enables serial recording of cellular electrical activity. Using an inducible CRISPR-CAS9 nuclease we have developed a method to knockout genes of interest in differentiated cardiomyocytes. The use of the Arclight voltage reporter allows monitoring of the loss of gene function over time. This real-time knockout method is demonstrated for the KCNH2 gene and compared to shRNA mediated knockdown. Methods and Results: Human iPSCs that were genetically engineered to contain a doxycycline (DOX) inducible Cas9 allele were differentiated into cardiomyocytes and transduced with lentiviral vector carrying the genetically encoded fluorescent voltage indicator, A242-Arclight. These human Cas9 iPSC-CMs expressing Arclight were further transduced with lentiviral vector containing a guide RNA (gRNA) targeting KCNH2 to achieve knockout upon DOX treatment. Arclight fluorescence was repeatedly recorded in these cardiomyocytes at day 0, 3, 5, 7, and 9 after DOX treatment. APDs at 90% repolarization (APD90) were analyzed and compared to APD90 from CMs without DOX treatment. APD90 in DOX treated iPSC-CMs gradually prolonged from day 3 to day 5 and remained relatively stable between days 5 and 9. At day 7, APD90 was significantly prolonged in DOX treated iPSC-CMS (321 ± 21 ms) compared to untreated cardiomyocytes (231 ±15 ms). RNAi-mediated knockdown of KCNH2 using short hairpin RNA (shRNA) on the iPSC-CMs expressing Arclight did not significantly alter APD90 throughout the study time course. Conclusion: In this in vitro human cell model, Crispr/Cas9 knockout of KCNH2 results in APD prolongation in iPSC-CMs in a real-time manner, which provides a powerful cellular platform to evaluate gene function in cellular electrophysiology.
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