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Dev Cell. 2015 Mar 23;32(6):756-64. doi: 10.1016/j.devcel.2015.01.032. Epub 2015 Mar 5.

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

Author information

1
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.
2
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
3
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address: zon@enders.tch.harvard.edu.

Abstract

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 zebrafish, 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 zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant 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.

PMID:
25752963
PMCID:
PMC4379706
DOI:
10.1016/j.devcel.2015.01.032
[Indexed for MEDLINE]
Free PMC Article

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