A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.
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.
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.
Julien
J
Ablain
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.
Ellen M
EM
Durand
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.
Song
S
Yang
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.
Yi
Y
Zhou
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.
Leonard I
LI
Zon
D029503:Anemia, Diamond-Blackfan / Q000235:genetics; D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004195:Disease Models, Animal; D004912:Erythrocytes / Q000166:cytology; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D016159:Tumor Suppressor Protein p53; D029961:Zebrafish Proteins; D000097573:Red Fluorescent Protein; C571228:tp53 protein, zebrafish