Spatiotemporal Control of Genetic and Epigenetic Editing through Covalent Tethering of CRISPR Nanoparticles to Zwitterionic Microgels

CRISPR gene editing offers unprecedented genomic and transcriptomic control, positioning it as a powerful tool for cell therapies. Non-viral CRISPR delivery avoids the immunogenicity, genomic integration, and packaging limits of viral vectors, but local or systemic injections of non-viral nanoparticles suffers from transient action and poor biodistribution. Alternatively, biomaterial-based delivery improves nanoparticle localization and sustains delivery, yet current approaches rely on non-specific adsorption of nanoparticles to scaffolds, risking aggregation, destabilization, and unreliable release kinetics. This work establishes a novel strategy to covalently tether nanoparticles to biomaterial substrates through SnoopTag and SpyTag bioconjugation systems for spatiotemporal control of non-viral CRISPR delivery. The RALA cell-penetrating peptide, an efficient and low-cytotoxicity CRISPR delivery system, was functionalized with SnoopTag without altering nanoparticle formation or transfection capacity. SnoopTag-functionalized nanoparticles were then covalently tethered to SpyTag-decorated zwitterionic microgels via a SnoopCatcher-SpyCatcher fusion protein. This platform achieves sustained RALA-mRNA nanoparticle delivery and prolonged CRISPR activation in human mesenchymal stem cells seeded within CRISPR-loaded microgel scaffolds. This work establishes a defined biorthogonal conjugation system that tethers non-viral nanoparticles to biomaterial platforms for sustained and localized CRISPR gene editing.