Precise Gene Knock‐In Tools with Minimized Risk of DSBs: A Trend for Gene Manipulation

Abstract Gene knock‐in refers to the insertion of exogenous functional genes into a target genome to achieve continuous expression. Currently, most knock‐in tools are based on site‐directed nucleases, which can induce double‐strand breaks (DSBs) at the target, following which the designed donors carrying functional genes can be inserted via the endogenous gene repair pathway. The size of donor genes is limited by the characteristics of gene repair, and the DSBs induce risks like genotoxicity. New generation tools, such as prime editing, transposase, and integrase, can insert larger gene fragments while minimizing or eliminating the risk of DSBs, opening new avenues in the development of animal models and gene therapy. However, the elimination of off‐target events and the production of delivery carriers with precise requirements remain challenging, restricting the application of the current knock‐in treatments to mainly in vitro settings. Here, a comprehensive review of the knock‐in tools that do not/minimally rely on DSBs and use other mechanisms is provided. Moreover, the challenges and recent advances of in vivo knock‐in treatments in terms of the therapeutic process is discussed. Collectively, the new generation of DSBs‐minimizing and large‐fragment knock‐in tools has revolutionized the field of gene editing, from basic research to clinical treatment.