1. Overview
Our project established a DNA origami-based gene editing system to selectively disrupt antibiotic resistance genes. This system is designed as a multilevel assembly in which DNA origami serves as a delivery vehicle for gene editing constituents, with other functional motifs offering membrane-permeabilizing and targeting capabilities.
To construct our DNA origami-based gene editing system, our experiments are divided into three modules, namely the gene cleavage module, assembly module, and functional validation module.
1.1 Gene cleavage module
For the gene cleavage module of our design, the CRISPR/Cas9 system functions as a programmable gene editing system to cleave specific antibiotic resistance genes. In vitro assay, we confirmed the feasibility of sgRNA-ΔmecA/Cas9 complex for extracellular targeted cleavage of mecA gene. To mitigate the potential biosafety concerns associated with mecA gene in intracellular conditions, we replaced mecA gene with lacZ gene in subsequent experiments. The successful extracellular cleavage of lacZ gene confirmed that the sgRNAL/Cas9 complex could retain its cleavage activity with the incorporation of a linker.
1.2 Assembly module
For the assembly module of our design, atomic force microscopy (AFM) was used to characterize the basic plane of DNA origami, revealing the well-defined nanostructure with the expected dimensions. The sgRNAL/Cas9 complex was then loaded onto the basic plane of DNA origami, and fluorescent modification of both components facilitated the validation of successful loading.
To endow the membrane-permeabilizing capability with the gene editing system, G-quadruplex (G4) strands were incorporated onto the origami. The subsequent loading of hemin onto G4 motifs generated G4/hemin DNAzymes with peroxidase-mimicking activity. Their catalytic activity was confirmed by the ABTS assay, and the NPN uptake assay was employed to further validate the membrane-permeabilizing capabilities of G4/hemin DNAzymes.
The targeting specificity was introduced by incorporating DNA aptamers into the DNA origami. The NPN uptake assay verified the effectiveness of DNA aptamers, and laser scanning microscopy (LSM) revealed a substantial increase in the specific attachment to bacterial cells after aptamer incorporation.
1.3 Functional validation module
For the functional validation module of our design, LSM exhibited successful intracellular uptake of our DNA origami. The blue/white selection was then conducted to verify the overall validation of the well-assembled DNA origami. The white colonies in the experimental group demonstrated the successful gene disruption by our DNA origami-based gene editing system.
To conclude, through the above experiments, our lab provides a proof-of-concept for the specific elimination of antibiotic resistance genes by combining the DNA origami nanostructure with the gene editing system, demonstrating an antibacterial platform with great promise.
1.4 DNA origami constructs and abbreviations
The following list outlines all the DNA origami constructs used in our experiment, along with their corresponding abbreviations.
| Experiments | DNA origami abbreviations | Meanings |
|---|---|---|
| - | DO | M13 + S-x |
| Verification of the DNA origami structure by AFM | DOPAM | M13 + S-x + S-PAM-cap-x + PAM-rich |
| Loading of FITC-sgRNAL/Cas9 complex | Cy5-labeled DOPAMRFITCC | M13 + S-x + S-PAM-cap-x + F-cap-x + PAM-rich + F-H + FITC-sgRNAL/Cas9 |
| Cy5-labeled DOPAM | M13 + S-x + S-PAM-cap-x + F-cap-x + PAM-rich + F-H | |
| Dimerization | DOPAMG | M13 + S-x + S-G4-x + S-PAM-cap-x + PAM-rich |
| Loading of hemin | DOPAMGH | M13 + S-x + S-G4-x + S-PAM-cap-x + PAM-rich + hemin |
| ABTS assay of G4/hemin DNAzymes | DOPAMGH | M13 + S-x + S-G4-x + S-PAM-cap-x + PAM-rich + hemin |
| NPN uptake assay | DOPAMGH | M13 + S-x + S-G4-x + S-PAM-cap-x + PAM-rich + hemin |
| DOAPAMGH | M13 + S-x + S-G4-x + S-PAM-cap-x + Apt-cap-x + PAM-rich + aptamer + hemin | |
| Targeting validation by LSM | Cy5-labeled DOPAM | M13 + S-x + S-PAM-cap-x + F-cap-x + PAM-rich + F-H |
| Cy5-labeled DOAPAM | M13 + S-x + S-PAM-cap-x + Apt-cap-x + F-cap-x + PAM-rich + aptamer + F-H | |
| Internalization validation by LSM | Cy5-labeled DOPAMGH | M13 + S-x + S-G4-x+ S-PAM-cap-x + F-cap-x + PAM-rich + F-H + hemin |
| Cy5-labeled DOAPAMGH | M13 + S-x + S-PAM-cap-x + Apt-cap-x + F-cap-x + PAM-rich + aptamer + F-H + hemin | |
| Blue/white selection | DOAPAMRCGH | M13 + S-x + S-G4-x + + S-PAM-cap-x + Apt-cap-x + PAM-rich + aptamer + sgRNAL/Cas9 |
The mind map of our wet experiment
