Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5-43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.
Wang et al. (2026) studied this question.