Abstract Body (Do not enter title and authors here): Introduction:
Our previous studies have shown that sustained activation of the DNA damage response (DDR) in cardiomyocytes leads to p53/p21 activation and cardiac dysfunction. Although the DDR generally involves molecules in DNA replication and repair pathways, the non-proliferative nature of cardiomyocytes suggests a cardio-specific DDR mechanism. However, our understanding of DDR in cardiomyocytes remains limited. Here, we aim to use CRISPR interference (CRISPRi) knockdown screens to identify genes critically involved in DDR regulation in human cardiomyocytes. We hypothesize that identifying these gene clusters may allow us to develop methods to prevent cardiac dysfunction by suppressing DDR in cardiomyocytes.
Methods and Results:
We established a human iPS cell line stably expressing dCas9-KRAB, which allows CRISPRi-mediated gene knockdown, and differentiated the cells into cardiomyocytes. The resulting human iPS cell-derived cardiomyocytes (hiPSCMs) showed the achievement of approximately 80% knockdown efficiency after gRNA transfection. We stimulated the hiPSCMs with H₂O₂ and quantitatively evaluated the expression levels of the DDR markers γH2AX and p21 by immunostaining using the Operetta^® high content imaging system. The DDR markers showed a significant concentration-dependent increase in response to H₂O₂ administration. For arrayed CRISPRi screening, we constructed a gRNA library targeting 437 DDR-related genes. Using this library, we knocked down each DDR-related gene in hiPSCMs followed by H₂O₂ stimulation. We quantified the expression levels of DDR markers by calculating the fluorescence intensity ratios relative to control after gene knockdown, and standardized them to calculate Z scores for all 437 genes. The screening successfully revealed the differential impact of each gene knockdown on γH2AX and p21 expression. We identified 71 genes that significantly affected their expression (Z-score < -1 or > 1). Mapping these genes to DDR pathways highlighted the differential impact of gene knockdown within the same pathway, and stratified their importance in cardiomyocytes.
Conclusions:
Arrayed CRISPR screening using hiPSCMs revealed differential functional significance of DDR-related genes in cardiomyocytes, identifying 71 genes of particularly significant importance. These findings provide a critical understanding of the cardio-specific DDR pathway and important clues for establishing an appropriate method to suppress DDR in the failing heart.