Alessandro Bertero

Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.