Abstract Summary Although gene editing using the CRISPR/Cas9 system enables the rapid generation of knockout mice, constructing an optimal gene disruption strategy is still labourious. Here, we propose KOnezumi, a simple and user-friendly web application, for use in automating the design of knockout strategies for multiple genes. Users only need to input gene symbols, and then KOnezumi returns target exons, gRNA candidates to delete the target exons, genotyping PCR primers, nucleotide sequences of the target exons and coding sequences of expected deletion products. KOnezumi enables users to easily and rapidly apply a rational strategy to accelerate the generation of KO mice using the CRISPR/Cas9 system. Availability and implementation This web application is freely available at http://www.md.tsukuba.ac.jp/LabAnimalResCNT/KOanimals/konezumi.html. Supplementary information Supplementary data are available at Bioinformatics online.