CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors

Abstract

Direct in vivo reprogramming of cardiac fibroblasts 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 and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome‐wide knockout screen with clustered regularly interspaced short palindromic repeats‐guide RNA 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 because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958–972

CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy

“… 41 Therefore, a genome-wide screen such as using CRISPR tools is necessary to identify essential genes that control the cell fate of fibroblasts during the reprogramming process. 59 , 60 Other experimental variations such as starting cell sources and culture conditions could contribute to the functional variability of CPCs induced by different reprogramming approaches. Embryonic or neonatal cardiac fibroblasts are considered to be more easily reprogrammed into cardiac lineage as compared with adult or aging cells.…”

CRISPR screening in cardiovascular research

“…A genome-wide CRISPR knockout screen was initially deployed to identify genetic regulators that determine the reprogramming efficiency of murine cardiac fibroblast into cardiac progenitors by a chemically induced approach. Loss of DNA methyltransferase 1-associated protein 1 (Damp1) was shown to enhance Nkx2-5-positive cardiac progenitor conversion and affect further differentiation of these progenitor cells ( Yu et al, 2019 ). Another study created CRISPR-mediated mutant library for more than 6,000 genes in human embryonic stem cells and allow these cells to differentiate into MESP1-positive cardiac mesoderm or ISL1-positive cardiac progenitor cells.…”

Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems

“…CRISPR-based screens have already demonstrated their efficiency in the reprogramming field ( Shifrut et al, 2018 ; Yang et al, 2019 ; Yu et al, 2019 ). Liu Y. et al (2018) went further: after the identification of murine neuronal-fate regulators, they tested whether pairwise activation was more efficient and if it was, for which factors.…”