An effective double gene knock-in strategy using small-molecule L755507 in the medaka fish (Oryzias latipes).

Homology-directed repair (HDR)-mediated genome editing has become a powerful method for altering chromosomal sequences in a seamless and accurate manner. However, the low efficiency of HDR in most cells hinders the establishment of desired strains harboring accurately modified genomes. To enhance HDR-mediated knock-in events, we explored two approaches, namely low-temperature incubation and chemical compound administration using medaka embryos after microinjection. We validated the performance of each method by calculating the knock-in efficiencies according to the expression area of fluorescent protein in the embryos. The in vivo assay indicated that the reduction in temperature did not promote HDR events, whereas among the nine compounds screened, the small molecule L755507 could enhance the HDR-mediated targeted integration of reporter cassettes. Additionally, the L755507-based approach allowed for the simultaneous integration of two different DNA fragments into the two targeted loci, that is, double knock-in. Our established knock-in system combining L755507, donor plasmids, and the CRISPR/Cas9 nickase system can reduce the workload for genetically modified strain generation, thus accelerating studies on the molecular mechanisms of biological phenomena.