Using a novel gene knock-in technique, effective insertion of an exogenous gene was demonstrated in human cells and in animal models, including silkworms and frogs. This strategy universally enables gene knock-in not only in cultured cells, but also in various organisms. This technique will enhance the usefulness of genome editing techniques in a variety of cells and organisms, especially in those in which gene knock-in is hindered by low homologous recombination efficiency.