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bioRxiv · 10.1101/2020.07.21.214452

Endogenous zebrafish neural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Almeida, M. P.·Welker, J. M.·Ekker, S. C.·Clark, K. J.·Essner, J. J.·McGrail, M.
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Abstract

The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous gene expression patterns are needed. Here, we apply CRISPR/Cas9 targeting to integrate a 2A-Cre recombinase transgene with 48bp homology arms into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

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Almeida, M. P., Welker, J. M., Ekker, S. C., Clark, K. J., Essner, J. J., McGrail, M.. 2020-07-22. Endogenous zebrafish neural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration. https://doi.org/10.1101/2020.07.21.214452

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