Fig. 1

Fig. 1

a Two gRNAs (a, b) targeting slc45a2 exon 1 in zebrafish (top). Experimental setup to analyze CRISPR-Cas9, SpG, and SpRY-mediated mutations in zebrafish by injecting one-cell-stage embryos (bottom). b Phenotypes obtained after the injection of the mRNA–gRNA duplex targeting slc45a2 (albino) showing different levels of mosaicism (albino-like (alb-like), severe, mild) compared to the WT. Lateral views (scale bar, 1 mm) and insets of the eyes (scale bar, 0.2 mm) of 48 h post-fertilization (hpf) embryos are shown. c Percentage of albino- phenotypes (panel b) in embryos 48 hpf. (n) total number of injected embryos. The results were obtained from at least two independent experiments. d Phenotypic evaluation at different concentrations of gRNAs and mRNAs. Stacked bar plots show the percentage of the phenotypes described in panel b. The results were obtained from at least two independent experiments. e gRNAs were complexed with purified proteins to form RNPs for in vitro and in vivo testing in C. elegans by microinjection⁶⁵. f Sequences of gRNAs targeting dpy-10 with distinct complementarity. RNP combinations comprised of each gRNA and WT SpCas9, SpG, or SpRY were tested in vitro. Top bands show uncleaved PCR product. Lower bands show cleaved products. g The dpy-10 matched and +5 gRNAs were tested for in vivo activity in C. elegans by injecting a single gonad arm. Each dot represents the editing efficiency in each P₀ that produced at least 100 F₁s. The results were obtained from two independent experiments, with both conditions carried out in parallel injections (Student’s t test p value). h Schematic representation of in vivo experiments in C. elegans using a gtbp-1::wrmScarlet reporter by screening for loss of fluorescence. dpy-10 gRNA was used for co-CRISPR. i An anti-wrmScarlet gRNA with NGG PAM was complexed with 1.3 µM of SpCas9, SpG, or SpRY to compare their in vivo efficiencies. In a separate experiment, the SpG-anti-wrmScarlet (NGG) RNP was injected at 8.0 µM. Each dot represents the editing efficiency in each P₀ that produced at least five Dpy or Rol F₁s. (One-way ANOVA followed by Tukey’s test for multiple comparisons p values).