A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (NIH) grant R01 EB012637. The authors thank support of the NRI microscopy center, the Olympus confocal microscope was funded by the NIH grant 1S10RR022585-01A1. The authors thank A. Mikhailovsky for helpful conversations and aid of the UCSB Optical Characterization Facility. The ultrafast laser system was funded by DURIP ARO grant 66886LSRIP. The authors would like to thank S. Merkel for aid with flow cytometry studies. The authors would like to thank C. Woodcock for his aid and discussions on the purification of Cre Recombinase. The authors would also like to thank G. Braun, A. Pallaoro, and X. Huang for helpful discussions.
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- gene editing
- near-infrared light endosome escape
- protein delivery
- spatiotemporal control