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Related Concept Videos

CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells
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Tailoring Magnetite-Nanoparticle-Based Nanocarriers for Gene Delivery: Exploiting CRISPRa Potential in Reducing

David Arango1, Javier Cifuentes1, Paola Ruiz Puentes1

  • 1Department of Biomedical Engineering, Universidad de Los Andes, Bogotá 111711, Colombia.

Nanomaterials (Basel, Switzerland)
|June 10, 2023
PubMed
Summary

This study introduces novel iron oxide nanoparticle (ION) gene delivery vehicles that release therapeutic nucleic acids (tDNA) under reducing conditions. These biocompatible nanocarriers efficiently deliver CRISPR activation sequences, successfully overexpressing the pink1 gene by 130-fold.

Keywords:
CRISPRacell deliverydisulfide bondgene deliverymagnetite nanoparticlesnanoconjugatespink1

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Area of Science:

  • Biotechnology and Nanomedicine
  • Gene Therapy
  • Materials Science

Background:

  • Gene delivery offers an alternative to conventional treatments but faces challenges with degradation and cell penetration.
  • Nanostructured vehicles, particularly iron oxide nanoparticles (IONs) like magnetite nanoparticles (MNPs), show promise due to their properties.
  • Effective gene delivery requires vehicles that protect cargo and facilitate cellular uptake and release.

Purpose of the Study:

  • To develop and characterize an ION-based nanocarrier for controlled release of linearized nucleic acids (tDNA) under reducing conditions.
  • To demonstrate the potential of this nanocarrier for gene therapy applications, specifically for gene overexpression.
  • To evaluate the biocompatibility, cellular uptake, and functional gene delivery efficiency of the developed nanocarrier.

Main Methods:

  • Functionalization of magnetite nanoparticles (MNPs) with polyethylene glycol (PEG), 3-[(2-aminoethyl)dithio]propionic acid (AEDP), and an OmpA translocating protein.
  • Conjugation of a thiol-modified CRISPR activation (CRISPRa) sequence to the MNP carrier via a disulfide bond for triggered release.
  • Physicochemical characterization (TGA, FTIR), biocompatibility assays (hemocompatibility, platelet aggregation, cytocompatibility), and functional testing (RT-qPCR).

Main Results:

  • Successful synthesis and functionalization of MNP-based nanocarriers confirmed by TGA and FTIR.
  • Demonstrated remarkable biocompatibility with primary human astrocytes, rodent astrocytes, and human fibroblast cells.
  • Achieved efficient cargo penetration, cellular uptake, endosomal escape, and a 130-fold overexpression of the pink1 gene via CRISPRa delivery.

Conclusions:

  • The developed ION-based nanocarrier is a versatile and promising platform for gene delivery, capable of releasing nucleic acid cargo under reducing conditions.
  • This MNP-based system exhibits excellent biocompatibility and efficient gene delivery, with potential applications in gene therapy.
  • Represents the first MNP-based nanocarrier for nucleic acid delivery triggered by reducing conditions, accommodating sequences up to 8.2 kb.