PROJECT SUMMARY/ABSTRACT Human cardiac injury, such as a heart attack, leads to irreparable damage and life-long heart complications. Developing translational strategies for inducing heart repair has been limited to laboratory accessible models such as the zebrafish and mouse. Using the zebrafish, which can regenerate their heart after substantial injury, we have previously shown that neural crest-derived cardiomyocytes promote injury-induced proliferation of surrounding cardiomyocytes by re-activating developmental gene networks after injury. Importantly, genetic ablation of neural crest-derived cardiomyocytes leads to a failure of regeneration and a large scar. Now knowing the importance of neural crest-derived cardiomyocytes and re-activating developmental networks, many questions remained unanswered on how these networks are re-deployed after injury and if these networks remain silenced in human hearts after injury. Our current hypothesis is that human neural crest-derived cardiomyocytes are unable to redeploy developmental gene networks after injury and are therefore unable to induce repair mechanisms. Until recently, assessing gene regulatory dynamics in human-derived cardiac tissues was not possible. Now, self-assembling cardiac organoids derived from human pluripotent stem cells have presented a new avenue for exploring cardiac repair in human-derived tissues; however, these cardioid models do not contain cardiomyocytes derived from neural crest. Here, we propose to (i) assess the dynamic chromatin landscapes of the regenerating zebrafish heart using single cell ATAC-seq to unravel critical components necessary for re-activating developmental programs that control cardiac regeneration in the zebrafish, (ii) interrogate the reactivation of developmental programs in a human-derived cardioid model after injury using a multiomics approach, and finally, (iii) use next-generation CRISPR-based functional genomics screens to identify gene circuits responsible for “repair impairment” of human neural crest-derived cardiomyocytes. Ultimately, our goal is to combine gene regulatory network information from zebrafish repair circuits and our human-derived screen to identify optimal targets for potential intervention using any relevant therapeutic modality for driving cardiac repair in vivo post-injury.