CRISPR-KO screen identifies Dmap1 as a regulator of chemically-induced reprogramming and differentiation of cardiac progenitors.

Direct in vivo reprogramming of cardiac fibroblasts (CF) into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells (EC) and cardiomyocytes (CM) using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells results in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle (SM). Deployment of a genome-wide KO screen with CRISPR-gRNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5 and enhanced the retention of self-renewal, although further differentiation is inhibited due to sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. STATEMENT OF SIGNIFICANCE: This study demonstrates the chemically-induced conversion of mouse cardiac fibroblasts into progenitors using TGF-β/Alk5 inhibition coupled with hypoxia. Utilizing this protocol, we generated two progenitor populations of differing potency; an initial population that could give rise to endothelial and cardiomyocyte lineages, and a second population that is more lineage restricted toward myofibroblast and smooth muscle. In characterizing the biology behind this reprogramming using a CRISPR-KO based genome-wide screen, we aim to drive the development of novel therapeutics that can improve cardiac function by promoting the in-situ induction of cardiac progenitors. © AlphaMed Press 2019.