Fig. 5.

Effects of trim33 knockdown on neutrophil migration in Tg(mpx-cas9) zebrafish embryos. (A) Representative fluorescent images of mCherry-labelled neutrophil at caudal fin 3.5 h post-wounding in 3 dpf Tg(mpx-Cas9) embryos injected with two trim33 gRNAs, compared to uninjected transgenic embryos (WT), demonstrating the impaired migratory neutrophil response to caudal fin injury in gRNA-microinjected crispant embryos compared to WT. (B) Quantification of neutrophil response. (C) Sanger sequencing chromatogram of independent experiment demonstrating on-target Cas9 activity in FACS-purified mCherry-positive neutrophils. (D) Manhattan plots from NGS of the same neutrophil DNA preparation as in C, displaying cumulative distribution of aligned deleted alleles at the target locus. (E) WT reference sequence compared to five predominant variants (Var 1-5), representing 43.6% on-target gene editing in neutrophils. (F) Predicted amino acid sequences of variants 1-5 with nonsense mutations. Red arrows indicate the sequencing direction; red asterisks mark sequence heterogeneity; green vertical dashed lines indicate cropped areas of the chromatogram; PAM, protospacer adjacent motif highlighted in red boxes and font; red dots, deletion; purple font, truncated protein; black dots, sequence continues as WT. Unpaired two-tailed Student’s t-test (P=0.001) of pooled data from two independent experiments indicated by different colours. Scale bar: 100 µm.