Engineering Mitochondrial Biogenesis in iPSC-CMs: CRISPR-Guided Approaches for Advanced Cardiomyocyte Development

Human iPSC-derived cardiomyocytes (iPSC-CMs) exhibit fetal-like mitochondrial networks and limited oxidative metabolism, constraining their translational utility. The key bottleneck is mitochondrial immaturity, resulting from blunted PGC-1α-<i>NRF1/2-TFAM</i> axis activation and insufficient nuclear-mitochondrial coordination, rather than sarcomeric or electrophysiological immaturity alone. This review synthesizes genome-guided interventions (CRISPRa and mtDNA editing) and complementary environmental strategies-including metabolic substrate switching, electromechanical stimulation, and extracellular vesicle (EV)-mediated mitochondrial transfer-to drive mitochondrial biogenesis and maturation in iPSC-CMs. We systematically reviewed studies (2005-2025) targeting (1) key regulators of mitochondrial biogenesis (PGC-1α, <i>NRF1/2</i>, <i>TFAM</i>), (2) CRISPR-based transcriptional activators/repressors and mtDNA editors (DdCBE, mitoTALENs), and (3) maturation approaches such as metabolic conditioning, electromechanical stimulation, 3D tissue culture, and EV-mediated mitochondrial transfer. CRISPRa-mediated activation of PGC-1α, <i>NRF1</i>, and <i>GATA4</i>, combined with mtDNA base editors, enhances mitochondrial mass and OXPHOS function, while integration with environmental maturation strategies further promotes adult-like phenotypes. Integrative approaches that combine genome-guided interventions (CRISPRa, mtDNA editing) with environmental maturation cues yield the most adult-like iPSC-CM phenotypes reported to date. CRISPR-guided mitochondrial biogenesis thus represents a frontier for producing metabolically competent, structurally mature iPSC-CMs for disease modeling and therapy. Remaining translational challenges include efficient mitochondrial delivery, metabolic homeostasis, and multi-omics validation. We propose standardized workflows to couple nuclear and mitochondrial editing with maturation strategies.