Zebrafish models of SRP component loss-of-function effects on neutrophil abundance. (A) Schematic of global CRISPR/Cas9 srpra knockdown in zebrafish by microinjection of srpra gRNA and Cas9 into 1-cell embryos. (B) Impaired survival of global srpra crispant embryos at 3 days post fertilization (dpf) compared with control noninjected siblings (n = 3 independent experiments; total embryos in each experiment [n control/n crispant]: 179/127, 195/184, and 133/113). Unpaired 2-tailed t test. (C) Representative dysmorphic surviving srpra Tg(mpx:EGFP) crispant embryo at 5 dpf. Panels show bright field (i), rhodamine dextran (tracing reagent delivery) (ii), and enhanced GFP (EGFP) (neutrophil reporter gene) images (iii). (D) Sanger sequencing chromatogram confirming on-target srpa gene editing in global crispants. (i) WT sequence from noninjected control. (ii) Sequence from severely deformed dead embryo, showing multiple superimposed heterogeneous traces starting in the vicinity of the guide RNA (gRNA)–targeted PAM site (red line). (E) Schematic of neutrophil-specific CRISPR/Cas9 srpra knockdown in zebrafish by microinjection of synthetic srpra gRNA into 1-cell mpx-cas9 zebrafish embryos. (F) Depleted trunk neutrophil numbers in mpx-cas9 embryos with neutrophil-specific srpra gene knockdown (pooled embryos from n = 2 experiments, indicated by different color symbols). Unpaired 2-tailed t test. (G) Representative images corresponding to the 2 groups in panel F. White box shows area where trunk neutrophil numbers were enumerated. (H) Sanger sequencing chromatogram confirming on-target srpa gene editing in neutrophils of mpx-cas9 neutrophil-lineage crispants. (i) WT reference sequence from noninjected control. (ii) Sequence from DNA prepared from neutrophils of mpx-cas9 neutrophil-lineage crispants, showing sequence heterogeneity starting in the vicinity of the PAM site (red line). (I) Schematic of global srpr19 knockdown in zebrafish by microinjection of srpra splice-blocking morpholino into 1-cell Tg(mpx:EGFP) zebrafish embryos. (J) Trunk neutrophil numbers in srp19-MO–injected morphants scored at 2 dpf (n = 24-53 embryos per group; experiment 1). One-way ANOVA with Dunnett post hoc test. Corresponding embryo survival rates are shown in supplemental Figure 9E. (K) Trunk neutrophil numbers in srp19-MO–injected morphants scored at 3 dpf (n = 24 embryos per group; experiment 2). One-way ANOVA with Dunnett post hoc test. Corresponding embryo survival rates are shown in supplemental Figure 9F. Ctrl, control; FACS, fluorescence-activated cell sorter; MO, morpholino.