Abstract Body: Background: The direct conversion of human fibroblasts into induced cardiomyocytes (iCMs) holds great promise for generating mature cardiomyocytes for clinical applications. However, the process remains inefficient, and the underlying reprogramming mechanisms are not well understood. Given the crucial role of epigenetic regulation in defining and maintaining cellular identities, we aim to systematically identify key epigenetic barriers using large-scale CRISPR-based screening tools to enhance reprogramming efficiency and generate mature human cardiomyocytes.
Result: We developed a high-throughput CRISPR-based loss-of-function screening pipeline to systematically identify key epigenetic barriers that restrict direct cardiac reprogramming. A custom knockout sgRNA library was designed to target epigenetic regulators, consisting of ~8,000 sgRNAs, including seven sgRNAs per gene and non-targeting controls. To enable the screen, we generated Cas9-expressing human fibroblast cells and confirmed robust Cas9 nuclease activity using a functional assay. We then conducted the first pooled direct cardiac reprogramming screen over a 14-day period. sgRNA-infected cells were indicated by GFP expression, while iCMs at different stages were distinguished based on cardiac troponin T (cTnT) levels. Using fluorescence-activated cell sorting (FACS), we isolated GFP⁺/cTnT^-, GFP⁺/cTnT^low, GFP⁺/cTnT^medium, GFP⁺/cTnT^high cell populations. For each group, genomic DNA was extracted, sequencing libraries were prepared, and raw reads from each sample were analyzed to assess sgRNA representation. We identified several sgRNAs that were specifically enriched in GFP⁺/cTnT^high cells compared to other populations, demonstrating the effectiveness of our strategy and uncovering novel epigenetic factors that can be targeted for facilitating iCM generation.
Conclusion: We established a CRISPR knockout loss-of-function screening platform specifically adapted to human iCM reprogramming, providing an unbiased approach to systematically identify epigenetic barriers that influence cell fate determination. Removing the potential barriers may enhance reprogramming efficiency and facilitate strategies to improve iCM generation and maturation.