Figure 2

Efficient genotyping of 48 hpf transient and stable CRISPR-Cas mutants using genomic DNA derived from the FS protocol (a) Schematics of the zebrafish genomic sequence showing the position of guide-RNAs used to generate the plekhh1 mutant line. (b) PCR result showing the possibility of genotype segregation using the FS-derived gDNA of single embryos from the offspring of plekhh1 mutants outcrossed with WT fish. A fragment of the plekhh1 exon targeted by CRISPR-Cas is amplified. Wild-type (single band, ~ 493 bps) are discernable from heterozygous plekhh1^+/- mutants (double band, ~ 493 and 230 bps, red arrowhead). The asterisk shows not specific bands. The agarose gel shows the result of a single PCR experiment. (c) Survival rate of embryos subjected to FS compared to control individuals from the same clutch, which were not subjected to FS. Log-rank (Mantel Cox) test is used to assess the statistical significance between groups (ns = not significant). Data show the survival rate from a single experiment with n = 40 control embryos non subjected to FS and 24 embryos subjected to FS. (d) Schematics of the zebrafish genomic sequence showing the position of the guide-RNA used to generate the tyr crispant with the base conversion (C > T, here G > A on the complement strand) leading to W273*. (e) Representative phenotypes showing partially (score 1) or severely (score 2) depigmented crispant mutants at 48hpf obtained upon tyr base editing. Reduced melanin content is apparent compared to a not-injected control sibling shown on the left. The images are representative of embryos from a single batch. (f) PCR results showing successful amplification on different crispants of a target fragment surrounding the edited tyr exon using the FS-derived gDNA (upper panel). Amplification of the same target sequence obtained by the gDNA extracted from the remaining WE tissue of the same individuals is shown in the lower panel. The agarose gel shows the result of a single PCR experiment. For both FS and WE two not-injected controls (ctr1 and ctr2) are also shown. (g) Examples of chromatograms resulting from Sanger sequencing of amplicons from WE- and FS- derived gDNA of the same tyr base edited crispants at 48 hpf. Examples of the performance of FS compared to WE for score 1 and score 2 are shown. Visible base conversion (here G > A) leading to a premature STOP codon is indicated by a purple arrow. Percentage of conversion G > A (C > T) and of residual G were calculated by EditR and are shown in green and black, respectively. A representative WT chromatogram from a non-injected sibling is also show on the top as negative control. m = molecular marker (Quick-Load^® 1 kb Plus DNA Ladder, NEB #N0469S), the bands of 0.2 and 0.5 kb are indicated. (h) The bar graph shows the genotype–phenotype correlation obtained by WE or FS-derived genotyping. Incidence (%) of sequences from tyr crispant embryos of different severity obtained by WE or FS exhibiting altered genotype (low or high rate of base conversion) compared to WT controls. N = 2 (controls), 9 for score 1 and 8 for score 2. Two-sided Chi-square’s test in 2 × 2 contingency table (WT vs. altered genotypes) are used to assess statistical significance between the different groups. Adjusted P values are reported (*p < 0.05, **p < 0.01).

Source data are provided as a Source Data file.