FIGURE

Fig. 3

ID
ZDB-FIG-130430-20
Publication
Chang et al., 2013 - Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos
Other Figures
All Figure Page
Back to All Figure Page
Fig. 3

Cas9/gRNA induces indels in the gata5 and gata4 loci in zebrafish. (A) Cartoon showing the position of the gRNA-targeting site and its sequence in the gata5 locus in zebrafish. (B) SSA recombination assay showing luciferase activity with and without cleavage of the target site by the Cas9/gRNA system in 293T cells. The luciferase activity increased by 16-fold in the group with gRNA (Cas9 + gRNA) compared with the control group (Cas9 + empty vector). Error bars indicate SD, n = 3. **P < 0.01. (C) Representative SURVEYOR assay showing the efficiency of Cas9-mediated cleavage, 26.4% in a single embryo at 50 hpf. (D) Representative Sanger sequencing results of the PCR amplicons of 8 individual embryos at 50 hpf, showing indels induced by Cas9/gRNA in the targeted gata5 locus. Twenty to thirty clones were sequenced for each embryo. Table summarizes the frequencies of site-specific indels, ranging from 50% (11/20) to 90% (18/20), in 8 individual embryos tested by Sanger sequencing. TGG (blue) is the PAM sequence. Deletion is represented by a dashed line and insertion is highlighted in red. (E) Two small hearts were formed in a Cas9/gRNA-induced mutant (right), which phenocopied that in the fautm236a genetic mutant, whereas a single heart formed in control embryos at 60 hpf. The heart was stained with phalloidin. * indicates two small hearts. Scale bar, 100 μm. (F) Representative Sanger sequencing results of the PCR amplicons of 8 individual embryos at 50 hpf, showing indels induced by Cas9/gRNA in the targeted gata4 locus as in D. AGG (blue) is the PAM sequence. AGG in 2 out of 16 sequencing results was deleted.

Expression Data

Expression Detail
Antibody Labeling
Phenotype Data
Fish:
Knockdown Reagent:
Observed In:
Stage: Pec-fin

Phenotype Detail
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Cell Res.