Evaluation Of Geminin Fused Crispr/cas9 System In Targeted Genome Editing

Abstract

In a diverse range of cellular kinds and organisms, genome editing involves making changes to the genomic DNA at a specific location. The CRISPR/Cas system has become the preferred approach for accurate genome editing because of its straightforwardness, cost-efficiency, and exceptional performance. This system causes a DNA double-strand break (DSB) when doing gene editing. The cell can fix the DSB in two ways: the HDR route, active during the S and G2 phases, and the NHEJ route, active throughout the entire cell cycle, introduces random indels. The NHEJ pathway presents this system with challenges such as off-target effects and on-target modifications. The Cas9-Geminin fusion construct was engineered beforehand is expected to increase the HDR rates over NHEJ to enhance gene editing efficiency. In our project, we assessed the efficacy of this complex in boosting genome editing efficiency compared to the traditional Cas9 via the HDR pathway in NIH/3T3 cells, emphasizing precise DNA repair using homologous sequences. Further validation of the inserted area using PCR genotyping will be required and comparison of Cas9- Geminin and non-fused Cas9 protein expression levels will provide deeper insights into the translational efficiency and cellular effects of the construct, opening the pathways for future studied in gene editing.