The African turquoise killifish Nothobranchius furzeri (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the duration of experiments, and we think that these advances will accelerate aging and developmental studies using N. furzeri.

The African turquoise killifish (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the experimental duration and we think that these will accelerate aging and developmental studies using N. furzeri.