gRNAs targeting on 4 genes (smc1a, tarbp1, vps13c, ccdc129) were injected into the one-cell embryos of zebrafish, and mutation events were detected by using restriction enzymes (RE) at following embryogenesis stages: 64-cell (2 hpf), 256-cell (2.5 hpf), 1k-cell (3 hpf), dome (4.33 hpf), bud (10 hpf), 24 hpf and 48 hpf, a 48 hpf control was included. (A) PCR products of each Cas9 target were treated by specific RE. (B) Fluorescence intensity was transferred to mutation rates by ImageJ. Blue, red, purple and grey line represent mutation rate of gRNAs targeting on smc1a, tarbp1, vps13c and ccdc129, respectively. X-axis was arranged according to hpf time course of zebrafish embryo development. Error bars were calculated from replicates of gRNAs targeting on each gene.