Abstract 18968: A Crispr Interference Platform in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes for the Characterization of Gene Loss-of-function in Cellular Electrophysiology
Introduction: The recent development of CRISPR interference (CRISPRi) shows highly efficient and specific gene silencing in different cell types including human induced pluripotent stem cells (iPSC). Our goal was to develop to develop a high throughput platform for functional validation of loss-of-function gene mutations in a cellular electrophysiological phenotype by combining human iPSC technology with CRISPRi and a genetically encoded voltage sensing fluorescent indicator, Arclight.
Methods and results: We first established a human iPSC colony stably and constitutively expressing dCas9-KRAB as our CRISPRi platform. We designed single-chimeric guide RNAs (sgRNAs) targeting around the transcription start sites of KCNH2, KCNQ1 and KCNJ2. These sgRNAs were subcloned into a lentiviral vector containing the U6 promoter and mCherry fluorescent protein under CMV promoter. The CRISPRi iPSC were differentiated into cardiomyocytes (CMs) and transduced with a lentiviral vector carrying Arclight for monitoring cellular action potential duration (APD), and were further infected with lentiviruses carrying different sgRNAs. After 9 days of transduction with lentivirus carrying sgRNAs, Arclight fluorescence was recorded in polyclonal cardiomyocytes populations and APD at 80% repolarization (APD80) was analyzed. mRNA was then extracted and the expression of KCNH2, KCNQ1 and KCNJ2 was determined by qPCR. A sgRNA that targets KCNH2 in polyclonal iPSC-CM resulted in 46% downregulation of KCNH2 mRNA and a significant prolongation of APD80 (+15%; 309±12 ms vs. 354±12 ms, p<0.05) serving as proof-of-concept. sgRNAs targeting KCNQ1 or KCNJ2 caused only 20% or 40% downregulation of their mRNA levels in polyclonal iPSC-CMs, and resulted in 6% or 13% prolongation of APD80, respectively.
Conclusion: Our findings suggest that CRISPRi mediated knockdown of LQT associated genes in human iPSC derived cardiomyocytes can recapitulate their physiological cellular phenotype. Combination of CRISPRi and iPSC technologies potentially provides a powerful cellular platform to evaluate gene function in cellular electrophysiology.
Author Disclosures: L. Xiao: None. S. Clauss: None. E. Bailey: None. P. Ellinor: None. D. Milan: None.
- © 2016 by American Heart Association, Inc.