Results
1. Extracellular Targeted Cleavage Of mecA Gene
CRISPR/Cas9 system is a powerful gene-editing tool. It employs single-guide RNA to direct Cas9 nuclease to a specific sequence, where Cas9 nuclease introduces a double-strand break in the target DNA. In our design, the CRISPR/Cas9 system functions as a molecular scissor that targets and cleaves the mecA gene of methicillin-resistant staphylococcus aureus (MRSA). Given the potential biosafety concerns associated with the mecA gene under intracellular conditions, we aimed to evaluate the feasibility of using the CRISPR/Cas9 system for targeted cleavage of the mecA gene in extracellular environments.
To evaluate the efficacy of the CRISPR/Cas9 system, we first amplified the mecA gene from the plasmid by polymerase chain reaction and subsequently incubated the amplified mecA gene with sgRNA-ΔmecA/Cas9 complex. A control group with sgRNA-ΔEGFP/Cas9 complex was included to verify the specificity of the CRISPR/Cas9 system. Two additional control groups, one containing only sgRNA-ΔmecA and the other containing only Cas9 protein, were used to confirm that the sgRNA-ΔmecA/Cas9 complex functions as an integrated unit. A negative control group with only Cas9 nuclease reaction buffer and the mecA gene was also established. DNA bands of each group were visualised by 1% agarose gel electrophoresis analysis. The image was analysed using Photoshop and Microsoft PowerPoint software. Details of the gene cleavage experiment can be found in the protocol (1 Extracellular targeted cleavage of mecA gene).
In the experimental group treated with sgRNA-ΔmecA/Cas9 complex, two DNA bands were observed at approximately 1300bp and 700bp, corresponding to the targeted cleavage site of the sgRNA-ΔmecA/Cas9 complex (Figure 1). In contrast, each control group only showcased a single DNA band at around 2000bp, corresponding to the full-length of the mecA gene (Figure 1). These results demonstrated the feasibility of the sgRNA-ΔmecA/Cas9 complex for targeted cleavage of the mecA gene in extracellular environments.
Figure 1. 1% agarose gel electrophoresis analysis of the mecA gene treated with different components. A. The mecA gene was treated with the Cas9 nuclease reaction buffer. B. The mecA gene was treated with only the Cas9 protein. C. The mecA gene was treated with only sgRNA-ΔmecA. D. The mecA gene was treated with sgRNA-ΔEGFP/Cas9 complex. E. The mecA gene was treated with sgRNA-ΔmecA/Cas9 complex.
2. Extracellular Targeted Cleavage Of lacZ Gene
Since the CRISPR/Cas9 system will be delivered into cells via DNA origami and the final experimental setting is intracellular, it is essential to choose a safer model than MRSA. Given that lacZ cleavage can be easily monitored by the colour of E.coli MG1655 colonies, we selected lacZ as a safer alternative to mecA for intracellular experiments. Therefore, the feasibility of the CRISPR/Cas9 system for targeted cleavage of the lacZ gene also required verification. This step, however, involved more than just replicating the last experiment. In our design, DNA origami served as a delivery platform for the sgRNA/Cas9 complex. To load the sgRNA-ΔlacZ onto DNA origami, the 3' terminal extended sgRNA-ΔlacZ (sgRNAL) was generated by in vitro transcription from a pre-designed double-stranded DNA template. The sgRNAL was designed to hybridise with the complementary staple strands of DNA origami. A critical aspect of this design was to evaluate whether the introduction of the linker influenced the function of the sgRNAL/Cas9 complex.
To evaluate the efficacy of the sgRNAL/Cas9 complex, we first amplified the lacZ gene from E. coli MG1655. After purifying, we incubated the lacZ gene with the sgRNAL/Cas9 complex, serving as the experimental group. The lacZ gene with the Cas9 nuclease reaction buffer served as the control group. DNA bands of each group were analysed by 1% agarose gel electrophoresis. The image was analysed using Photoshop and Microsoft PowerPoint software. Details of the gene cleavage experiment can be found in the protocol (2 Extracellular targeted cleavage of lacZ gene).
The expected outcome was that the sgRNAL/Cas9 complex would generate two cleavage fragments of approximately 1330 bp and 670 bp. In the experimental group, two distinct DNA bands were observed at the anticipated positions, around 1300 bp and 700 bp, thereby confirming successful cleavage at the lacZ target site (Figure 2). In contrast, the control group showed a single DNA band around 2000bp, corresponding to the full-length of the lacZ gene (Figure 2). These results indicated that the sgRNAL/Cas9 complex effectively cleaved the lacZ gene, confirming its gene-editing capability.
Figure 2. 1% agarose gel electrophoresis analysis of the lacZ gene treated with different components. A. The lacZ gene was treated with sgRNAL/Cas9 complex. B. The lacZ gene was treated with buffer.
Protocol
1. Extracellular targeted cleavage of mecA gene (click me to open the protocol!)
2. Extracellular targeted cleavage of lacZ gene (click me to open the protocol!)
