One-step Generation of Zebrafish Carrying a Conditional Knockout-Knockin Visible Switch via CRISPR/Cas9-Mediated Intron Targeting

SUMMARY The zebrafish has been becoming a popular vertebrate animal model in biomedical research. However, it is still challenging for making conditional gene knockout (CKO) zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2 zCKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2 zCKOIS was switched to a non-functional CKO allele hey2 zCKOIS-inv associated with TagRFP expression, enabling to visualize CKO alleles. Thus, the simplification of the design, and the visibility and combination of both CKO and KI alleles’ engineering make our zCKOIS strategy an applicable CKO approach for zebrafish.