Fig. 5

Light-induced gene editing in zebrafish. Light-induced gene knockout can be achieved by combining the TAEL system with the CRISPR/Cas9 system. (A) Three promoter construct containing: (1) the heart-specific promoter myl7 in front of GFP to label injected embryos, (2) Cas9 under the control of the C120 promoter and (3) a U6 promoter in front of the guide RNA sequence targeting tyrosinase, a protein essential for pigment formation. TAEL mRNA was co-injected with the construct depicted in A into wild-type single-celled embryos. Embryos were illuminated globally with 465 nm blue light and then scored for disruption in pigment formation at 2 dpf. (B-D) Wild-type, uninjected embryos (B) exhibit normal pigmentation as do injected dark controls (C). Pigment formation is disrupted in injected and illuminated embryos (D, arrow). Approximately 90% of illuminated embryos (n=18) showed various levels of pigment disruption, while 100% of dark control embryos examined (n=11) exhibited pigmentation comparable to wild-type uninjected embryos. (E) Representative CEL I nuclease assay indicates mutagenesis of the tyr locus in illuminated but not dark or uninjected control embryos. Arrows indicate cleavage fragments. Scale bar: 100 µm.