Abstract 15721: CRISPR Activates Heterochromatic Cardiac Genes in Fibroblasts to Initiate Lineage Conversion Into Cardiac Progenitors

Abstract

Introduction: Our previous studies have demonstrated that CRISPR activation (CRISPRa) of endogenous genes was sufficient to convert fibroblasts into induced cardiovascular progenitor cells (iCPCs) for myocardial regenerative therapy. However, reprogramming efficiency remains a challenge and little has been elucidated about the epigenetic mechanisms of cellular reprogramming, which will be important for methodological improvements or refinements. Hypothesis: CRISPRa complex can alter the chromatin accessibility and activity of heterochromatic cardiac genes in fibroblasts, directly initiating cardiac reprogramming. Methods: A fibroblast-specific CRISPRa mice model (Pdgfra-cre/ERT; ROSA-dCas9-SunTag) was generated. SgRNAs targeting the promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5 genes were transduced through an AAV system in cardiac fibroblast isolated from transgenic mice. iCPCs were confirmed via immunostaining of CPC markers and cardiac electrophysiological assays. iCPCs were collected for RNA-seq, ATAC-seq, and ChIP-seq after reprogramming induction to reveal the epigenetic landscape modulated by CRISPRa. Results: The CRISPRa system was activated in ~80% of fibroblasts after tamoxifen induction and AAV-sgRNA transfection. The reprogrammed iCPCs were characterized by immunostaining of proliferative markers (Ki67 and Pcna), progenitor surface markers (Flk1 and Ssea1), and markers of cardiac tri-lineage cells after induced differentiation. RNA-seq data showed the upregulation of genes associated with cell cycling (Cdkn1b, Aurkb, etc.) and heart development (Myh6, Irx4, etc.) in CRISPRa-induced cells. ATAC-seq showed increased chromatin accessibility at the loci of cardiogenic genes targeted by CRISPRa in fibroblasts. Furthermore, ChIP-seq of Gata4, Nkx2.5, and Tbx5 associated DNA shows that these factors can bind to their own promoter regions in fibroblasts after CRISPRa induction. Conclusions: CRISPRa complexes open the chromatin of the targeted genes in fibroblasts and establish an endogenous autoregulatory loop to ensure stable reprogramming into cardiac progenitors.