One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in 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 hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.