A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish

Ablain et al. (2015). Dev Cell DOI: 10.1016/j.devcel.2015.01.032 Citations: 380

CRISPR knockouts in zebrafish required whole-embryo injection, giving mosaic disruption. A Tol2 vector expressing Cas9 from a tissue-specific promoter restricted editing spatially. Stable F1 fish reached ~70% mutant alleles at the target locus.

Key findings

  • Ablain et al. developed a modular Tol2-integratable CRISPR/Cas9 vector for tissue-specific gene disruption in zebrafish. The vector incorporated a zebrafish U6-3 promoter for gRNA expression and a Gateway-compatible tissue-specific promoter cassette driving codon-optimized Cas9, with a cmlc2:GFP heart-specific marker enabling identification of stable transgenic embryos.

  • CRISPR/Cas9 targeting of the urod gene using erythrocyte-specific gata1 promoter-driven Cas9 yielded red fluorescent erythrocytes in approximately 70% of injected zebrafish embryos at 30 hours post fertilization, recapitulating the yquem mutant phenotype. Deep sequencing confirmed that more than 80% of detected mutations induced frameshifts, demonstrating efficient tissue-specific gene disruption.

  • Stable F1 transgenic zebrafish generated by Tol2-mediated integration of the tissue-specific CRISPR vector displayed highly penetrant erythrocyte fluorescence, confirming germline transmission of disrupted urod alleles. The modular vector design requires only substitution of the tissue-specific promoter and 20-nucleotide seed sequence, enabling rapid adaptation to any target gene and tissue in zebrafish.

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Abstract

Highlights d Urod inactivation using CRISPR yields a fluorescent phenotype in zebrafish embryos d A CRISPR-based vector system enables tissue-specific gene inactivation in zebrafish d The targeting vector can be easily modified for any gene and tissue-specific promoter Authors Julien Ablain, Ellen M. Durand, ..., Yi Zhou, Leonard I. Zon Correspondence zon@enders.tch.harvard.edu In Brief Ablain et al. describe a vector system for spatial control of gene disruption in zebrafish. Based on the CRISPR technology, this modular vector, which only requires a tissue-specific promoter and a gene-specific target sequence, greatly expands the range of loss-of- function studies in vivo. Ablain et al., 2015, Developmental Cell 32 , 756–764 March 23, 2015 ª 2015 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2015.01.032 Developmental Cell Resource A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish Julien Ablain, 1 Ellen M. Durand, 1 Song Yang, 1 Yi Zhou, 1,2 and Leonard I. Zon 1,2,3, * 1 Stem Cell Program and Division of Hematology/Oncology, Boston Children’s Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA 2 Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA 3 Howard Hughes Medical Institute, Boston, MA 02115, USA *Correspondence: zon@enders.tch.harvard.edu http://dx.doi.org/10.1016/j.devcel.2015.01.032 SUMMARY CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In ze- brafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebra- fish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, spe- cifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish em- bryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very pene- trant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.