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CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish

Abstract

Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.

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Figure 1: A diagram of CRISPR/Cas9-mediated conversion of an eGFP-transgenic line into an eGFPbait-E2A-KalTA4 transgenic line.
Figure 2
Figure 3: Two different strategies for targeting endogenous exons.
Figure 4: Analysis of a stably converted transgenic line.
Figure 5: Representative results from three eGFP-transgenic lines with examples of mosaic RFP expression in the former eGFP expression domain.
Figure 6: A representative gel of in vitro–transcribed mRNA encoding Cas9 nuclease and the noncapped eGFP sgRNA.

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Acknowledgements

Special thanks to C. Wyart and M. Kapsimali for the Tg(olig2:eGFP), Tg(nkx2.2a:meGFP) and Tg(CLGY786) lines. We thank J. Wittbrodt for scientific discussion and support, and C. Giovannangeli and members of the Del Bene laboratory for general discussion and comments. We thank the members of the Developmental Biology Curie imaging facility (PICT-IBiSA@BDD, UMR 3215/U934) for their help and advice with confocal microscopy. Del Bene laboratory 'Neural Circuits Development' is part of the Laboratoire d'Excellence (LABEX) entitled DEEP (ANR -11-LABX-0044). T.O.A. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. This work has been supported by an ATIP/AVENIR program starting grant (F.D.B.), by ERC-StG no. 311159 (F.D.B.), by ANR TEFOR (J.-P.C.), and by CNRS, INSERM, Institut Curie and the Muséum National d'Histoire Naturelle.

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T.O.A., J.-P.C. and F.D.B. contributed to the protocol development; T.O.A. and K.D. built donor constructs, and carried out the PCR diagnosis and Southern blotting; T.O.A. performed microinjections and confocal imaging; T.O.A. and F.D.B. wrote the manuscript with inputs from J.-P.C. and K.D.

Corresponding authors

Correspondence to Thomas O Auer or Filippo Del Bene.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Plasmid map of the donor plasmid.

The corresponding Genbank sequence can be found in Supplementary Note 1.

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Auer, T., Duroure, K., Concordet, JP. et al. CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat Protoc 9, 2823–2840 (2014). https://doi.org/10.1038/nprot.2014.187

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