Nanopuncturing‐Enabled Efficient Gene Editing for Isogenic Human iPSC‐Derived Organoid Engineering

Abstract Genetically engineering human induced pluripotent stem cells (hiPSCs) with clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) to construct isogenic organoid models holds great promise for disease modeling. However, genetic editing of iPSCs has been hindered by low transfection efficiency and low CRISPR/Cas9 editing efficiency. To address this, a nanoneedle‐assisted nanopuncturing strategy is employed to facilitate the high‐throughput delivery of CRISPR/Cas9 plasmids into hiPSCs, achieving stable editing efficiency. A meticulously designed silicon nanoneedle array temporarily pierces the cell membrane, allowing the exogenous plasmid to efficiently diffuse into the intracellular region without causing serious damage to the cells. The nanopuncturing operation can be accomplished within 30 min, with a transfection efficiency of 45 ± 4%, nearly five times higher than conventional lipid‐based methods. Additionally, this strategy results in high plasmid expression across most transfected cells, ensuring comprehensive gene knockout effects. By efficiently knocking out the PTPRB and ZNF521 genes, implicated in heart development and liver cancer, respectively, novel isogenic organoid models are constructed without altering their stemness and differentiation potential. This approach offers a novel and efficient method for developing iPSC‐derived organoids and disease models, enabling precise studies of gene function and disease mechanisms.