BackgroundThe CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficiency of targeted gene integration events in medaka.ResultsWe show that longer homology arms (ca. 500 bp) and linearization of circular donor plasmids by cleavage with bait sequences enhances the efficiency of targeted integration of plasmids in embryos. A new bait sequence, BaitD, which we designed and selected by in silico screening, achieved the highest efficiency of the targeted gene integration in vivo. Using this system, donor plasmids integrated precisely at target sites and were efficiently transmitted to progeny. We also report that the genotype of F2 siblings, obtained by mating of individuals harboring two different colors of fluorescent protein genes (e.g. GFP and RFP) in the same locus, can be easily and rapidly determined non-invasively by visual observations alone.ConclusionWe report that the efficiency of targeted gene integration can be enhanced by using donor vectors with longer homologous arms and linearization using a highly active bait system in medaka. These findings may contribute to the establishment of more efficient systems for targeted gene integration in medaka and other fish species.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-017-0071-x) contains supplementary material, which is available to authorized users.

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Genome editing with CRISPR/Cas9 system has become a powerful technology for targeted modification of chromosomal sequences in a wide range of organisms. This system consisting of Cas9 and guide RNA (gRNA) can introduce the double strand DNA breaks (DSBs) into the genomic target sites, consequently activating the DNA repair pathways such as non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Goodarzi & Jeggo, 2013; Sander & Joung, 2014). Although HDR is more ideal than NHEJ for the seamless and specific integration of foreign genes into genomes, generally HDR is not the dominant repair pathway (Han & Huang, 2020; Lieber et al., 2003; Mao et al., 2008). Thus, a majority of DSBs are repaired by error-prone NHEJ rather than the accurate HDR, and this could result in complex and unpredicted genetic mutations, including small insertions and deletions (indels) or imprecise insertion of a donor DNA, into the genomes (

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