From nuclease-based gene knock-in to prime editing – promising technologies of precision gene engineering

Gene knock-in can be defined as the introduction of precisely determined modifications, insertions, or replacements to the genome, which enables the generation of reporter cells, disease modeling and correction, humanization of animal cells and organisms, and so on. To date, gene knock-in systems have reached the fourth stage; i.e., the first, second, and third stages depend on unconstrained homologous recombination (HR)-mediated strategy, genome editing-assisted HR, and genome editing with various DNA double-strand break (DSB) repair pathways such as non-homologous end-joining and microhomology-mediated end-joining, respectively. Finally, in the fourth stage, DSB-free precision gene editors such as base editor and prime editor became available. These diversified strategies open up a new era of intentional editing of the genome, widely contributing to the functional genomics study.