In a recent study, researchers developed a new variant of the Cas12f1 gene-editing tool, referred to as Cas12f1_v8, aimed at improving gene editing efficiency. This advancement is significant for applications in gene therapy and biotechnology.
The team initiated their work by constructing an initial plasmid for the advanced Cas12f1 variant, dCasMINI-VPR, which was engineered to activate gene expression efficiently in mammalian cells. The study further optimized the sgRNA (single guide RNA) for Cas12f1, leading to a notable 867-fold increase in gene editing efficiency.
Subsequent experiments demonstrated that the codon optimization of Cas12f1 increased gene editing efficiency across multiple human genes. The transfection efficiency of the plasmid in HEK293T cells reached 96%, confirming the effectiveness of the delivery method.
The researchers also explored the stability of sgRNA by introducing a stable hairpin sequence, resulting in improved gene editing efficiency. Notably, the introduction of mutations in the DNA-binding pocket of Cas12f1 enhanced its interaction with sgRNA, further boosting editing capabilities.
In addition to optimizing Cas12f1, the study investigated the potential of this variant for cancer therapy. The enhanced Cas12f1 (eCas12f1) demonstrated significant gene editing activity in targeting critical cancer genes, such as PLK1, leading to a remarkable reduction in cancer cell survival rates.
Furthermore, the study established the utility of eCas12f1 for base editing by fusing it with deaminases, achieving efficient A-to-G conversions. The researchers also constructed an activator and inhibitor version of eCas12f1, confirming its versatility in regulating gene expression.
Overall, the development of eCas12f1 positions it as a promising tool for precision gene editing, with implications for cancer therapy and other genetic engineering applications.