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Digital archive of theses discussed at the University of Pisa

 

Thesis etd-10242023-103807


Thesis type
Tesi di dottorato di ricerca
Author
KONSTANTINIDOU, SOULTANA
URN
etd-10242023-103807
Thesis title
Photo-switchable gold-based nanoformulations of the CRISPR/Cas9 protein for efficient delivery and genome editing
Academic discipline
BIO/11
Course of study
BIOLOGIA
Supervisors
tutor Prof.ssa Raffa, Vittoria
Keywords
  • CRISPR-Cas9
  • gene editing
  • gold nanoparticle
  • light-activation
  • nanotechnology
Graduation session start date
03/07/2023
Availability
Withheld
Release date
03/07/2026
Summary
The CRISPR/Cas9 technology has found extensive application in introducing DNA double-strand breaks (DSBs) across various experimental models. Nevertheless, there remains a pressing need to mitigate unintended DNA cuts, known as off-targets, and enhance the in vivo delivery of the editing complex. In this context, I propose two gold nanoformulations of the Cas9 protein, optimizing its delivery efficiency and enabling light-induced DSBs. In the first approach, Cas9 is conjugated to 12nm spherical gold nanoparticles via affinity binding, exhibiting high cleavage efficiency in vitro and proficient gene editing in zebrafish. Notably, the nanoformulation internalizes in human melanoma cells spontaneously and localizes within the nucleus, effectively carrying out gene editing. The second strategy introduces spatial and temporal control over the activation of the editing complex via near-infrared (NIR) irradiation. Here, dCas9 is linked to a gold nanorod, acting as a heat source, while dCas9 serves as a DNA sensor to target specific areas. Two nanoformulations converge in close proximity to DNA, guided by their respective gRNA. Upon coupling, the resulting plasmonic properties of the dimer facilitate heat generation, reaching temperatures between 100-150°C within an extremely minuscule volume (zeptoliter). To validate the efficacy of this system, it was administered to zebrafish embryos at the 1-cell stage, followed by irradiation with NIR ultrashort pulses. The DNA of the irradiated embryos displayed large fragment deletions within the targeted DNA region. These innovative nanoformulations present promising solutions to address the current limitations of gene editing technologies.
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