Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9
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New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER T2 upstream of the endogenous ATG of otx2, we utilized this gene’s native promoter and enhancer elements to perfectly match CreER T2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER T2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.
KeywordsCRISPR/Cas9 CreERT2 Knock-in Zebrafish Cre-loxp recombination
We are thankful to the Chen and Wente labs for providing plasmids to generate Cas9 and sgRNA mRNA (via addgene), Daniela Zoeller for help with cloning the bait plasmid with CreERT2, Dilce Gozuyasli for heat shock experiments, past and present members of the Brand lab for discussions and Vasuprada Iyengar for language and content editing. We thank Marika Fischer, Jitka Michling, Claudia Meyer and Daniela Mögel for dedicated zebrafish care. The Light Microscopy Facility, a core facility of BIOTEC/CRTD at the Technische Universität Dresden, supported this work.
G.K was supported by post-doctoral fellowships from Swedish research council (Vetenskapsrådet) and an EMBO long-term fellowship (ALTF 350-2011). This work was also supported by an ERC advanced grant (Zf-BrainReg) and project grants of the German Research Foundation (Deutsche Forschungsgemeinschaft, project number BR 1746/6-1 and BR 1746/3) to M.B.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Brand M, Granato M, Nüsslein-Volhard C (2002) Keeping and raising zebrafish. In: Nüsslein-Volhard C, Dahm R (eds) Zebrafish: a practical approach. Oxford University Press, Oxford, pp 7–37Google Scholar
- Chekuru A, Kuscha V, Hans S, Brand M (2017) Ligand-controlled site-specific recombination in zebrafish. In site-specific recombinases (Springer), pp. 87–97Google Scholar
- Martinez-Barbera JP, Signore M, Boyl PP, Puelles E, Acampora D, Gogoi R, Schubert F, Lumsden A, Simeone A (2001) Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2. Development 128:4789–4800PubMedGoogle Scholar
- Ramachandran, R., Reifler, A., Wan, J., and Goldman, D. (2012). Application of Cre-loxP recombination for lineage tracing of adult zebrafish retinal stem cells. Retinal Development: Methods and Protocols, 884 129–140Google Scholar
- Westerfield, M. (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edition. University of Oregon Press, EugeneGoogle Scholar