CRISPR-Based Tissue Engineering Strategies

Abstract

CRISPR-Cas genome editing has opened unprecedented possibilities for tissue engineering by enabling precise genetic programming of stem cells to enhance differentiation, survival, and functional integration within engineered constructs. This study applied three CRISPR strategies -- CRISPR-Cas9 gene knockout, CRISPR activation (CRISPRa/dCas9-VPR), and base editing (ABE8e) -- to genetically engineer human iPSC-derived mesenchymal stem cells (iPSC-MSCs) for enhanced osteogenic, chondrogenic, and vascular tissue engineering. For osteogenesis, CRISPRa-mediated upregulation of BMP2 (4.8-fold) and RUNX2 (3.2-fold) in iPSC-MSCs produced 2.4-fold higher mineral deposition versus unedited controls at day 21 in 3D-printed PCL/HA scaffolds. For chondrogenesis, Cas9 knockout of MMP13 (matrix metalloproteinase-13) -- the primary collagen II degradation enzyme -- reduced collagen II turnover by 68.4% and increased cartilage matrix retention 1.84-fold in pellet culture. For vascularisation, ABE8e base editing of the HIF1A oxygen-sensing domain (P402A mutation) stabilised HIF-1alpha under normoxia, producing constitutive VEGF secretion (3.8-fold increase) and 2.2-fold higher microvessel density in subcutaneous implants at 4 weeks. Off-target analysis by GUIDE-seq confirmed < 0.1% indel frequency at the top 10 predicted off-target sites for all three strategies. These results establish CRISPR-engineered stem cells as a versatile platform for programming tissue-specific regenerative functions into engineered constructs.