Fig. 4

(A) Diagram of plasmid pZα-Cas9. The Cas9-P2A-tRFP cassette is driven by zebrafish muscle-specific α-actin promoter. The fusion protein Cas9-P2A-tRFP expressed in muscle cells is digested into Cas9, which is bound by transferred sgRNA, resulting in silencing the target gene in muscle cells. (B) Diagram of plasmid pZα-Pgk1. Pgk1 is specifically overexpressed in muscle cells. (C–G) Injection of different materials, as indicated, into embryos from transgenic line Tg(mnx:GFP) and observation of fluorescent signals expressed in embryos at 30-hpf. GFP-labeled motor neurons observed under confocal microscopy. (C’–G’) Location of RFP-labeled muscle cells in which Cas9 and/or Pgk1 is overexpressed. (C’’–G’’) Two fluorescent signals were merged. Numbers shown in the lower right corner were the number of phenotypes out of total examined embryos. (C–C”) Untreated embryos served as the control group. (D–D”) Injection of pZα-Cas9. NOM was not affected. (E–E”) Injection pZα-Cas9 combined with pgk1 sgRNA. The length of NOM became shorter (white arrows). (F–F”) Injection of pZα-Cas9 combined with pgam2 sgRNA (served as negative control). The NOM was not affected. (G–G”) Injection of pZα-Pgk1. The NOM became increasingly ectopic toward the muscle cells in which Pgk1 was overexpressed (white arrowheads).