Part:BBa_K5382150:Experience

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Applications of BBa_K5382150

We conducted cellular genome editing experiments to assess the efficiency of a targeted delivery system. Initially, OMVs loaded with Cas9 RNPs were introduced into HeLa cells to target the PRDX4 gene and evaluate Indel editing efficacy. Subsequently, CL7-fused transmembrane peptides and/or monoclonal antibodies were applied to these cells in order to determine their cellular targeting effectiveness and gene editing capabilities. By employing the T7 endonuclease I (T7E1) assay, we confirmed that the engineered OMV-Cas9 RNP complex exhibits genome editing activity comparable to that of commercial lipid-based transfection agents, as depicted in Figure 1.

Figure. 1 The assessment of genome-editing efficiency in HeLa cells using the T7E1 method.

Our findings suggest that OMVs containing Cas9 RNPs have a gene editing efficiency comparable to commercial lipid transfection reagents, achieving a target gene cleavage rate of around 40%. In vitro cell targeting assays highlight the significant gene editing capabilities of our engineered self-assembling system incorporating Cas9 RNPs. Compared to traditional methods for preparing Cas9 RNPs, our system offers notable advantages including the elimination of separate crRNA synthesis and the facilitation of efficient and cost-effective production of stable Cas9 RNP complexes on a large scale through a simplified purification protocol. These results imply that our system is suitable for a wider range of applications.

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