Activation Of Endogenous Transcription Factors By CRISPRa Mediates Cardiac Reprogramming

Studies of cardiac reprogramming - the conversion of fibroblasts to cardiomyocyte-like cells - have deepened our understanding of cell identity changes, and they hold promise for potential clinical application. At the core of most cardiac reprogramming approaches lie three transcription factors: MEF2C, GATA4, and TBX5 (MGT). Exogenous expression of MGT can convert fibroblasts into cardiomyocyte-like cells, but it is unknown if activation of endogenous MGT can mediate reprogramming, or if these reprogrammed cells would be phenotypically different. In this study, we implemented CRISPR-based gene activation (CRISPRa) for targeted activation of endogenous MGT in mouse and human fibroblasts. Transcriptional activation ranged from 10X-10,000X greater than the non-targeted control, varying by gene and target locus. In combination with miR-133 overexpression, CRISPRa of all three of the MGT factors induced cardiac reprogramming of human fibroblasts. Multiple combinations of guide RNAs were capable of mediating human reprogramming with varying efficiencies. Induced cardiomyocytes appear phenotypically different under immunofluorescence. Finally, we simplified the human CRISPRa platform by combining the guide RNAs into a single, multiplex vector. Cellular and molecular studies of this new reprogramming model may provide mechanistic insights into cell fate conversion.