CRISPR/Cas9-mediated site-specific homologous recombination for single-plasmid gene insertion system design and validation

Background　The CRISPR/Cas9 system has emerged as a cornerstone technology for genome editing; however, it still faces challenges such as inefficient long-fragment integration and transgene silencing. Objective　To design a circular single plasmid CRISPR/Cas9 system to achieve site-specific integration and stable expression of exogenous genes in multiple cell types. Methods　Cas9, sgRNA targeting specific gene loci and homologous recombination donor templates (green fluorescent protein was selected as the reporter gene for verification) were integrated into a single circular plasmid system. The single plasmid was delivered into the cells by Cationic lipid-based transfection method and verified by fluorescence microscopy observation, PCR amplification and sequencing. Results The single-plasmid system achieved precise site-specific insertion of long gene fragments in both human HEK293T and mouse 3T3 cells. Fluorescence microscopy observations revealed that stable GFP expression was maintained even on the 6th day post-transfection. PCR amplification and Sanger sequencing confirmed the accuracy of the insertion sites. Flow cytometry analysis demonstrated that, under drug-free selection conditions, the targeted integration efficiency in 293T cells reached 5.3%. qPCR analysis indicated that the expression of the endogenous ACTB gene remained largely unaffected following insertion (variation range: 0.2–1.3-fold), confirming the safety of this strategy.Conclusion　This study provides a simple and efficient solution for stable integration of long-fragment, which has significant potential in engineered cell construction and in vitro transgenic model development.