Spatiotemporal Control of Genetic and Epigenetic Editing through Covalent Tethering of CRISPR Nanoparticles to Zwitterionic Microgels
- 1Lehigh University
Abstract
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.
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BibTeX
@misc{graham2026spatiotemporal,
title = {{Spatiotemporal Control of Genetic and Epigenetic Editing through Covalent Tethering of CRISPR Nanoparticles to Zwitterionic Microgels}},
author = {J. P. Graham and E. E. Wheeler and K. J. Leach and T. Gonzalez-Fernandez},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.751510},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.751510v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Spatiotemporal Control of Genetic and Epigenetic Editing through Covalent Tethering of CRISPR Nanoparticles to Zwitterionic Microgels
AU - J. P. Graham
AU - E. E. Wheeler
AU - K. J. Leach
AU - T. Gonzalez-Fernandez
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.751510
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.751510v1
AB - 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.
ER -