Fig 1.
Egg microinjection site and embryo appearance.
(A) Illustration depicting an Amphiprion ocellaris egg (< 1-hour post-fertilisation) with demarcated injection site at the animal pole. (B) Brightfield micrograph of a live A. ocellaris egg injected by a microneedle with released fluid marked by red-fluorescent dye. (C) Wild-type A. ocellaris embryo at 64–88 hours post-fertilisation with formed eyes and pigmentation.
Fig 2.
Targeted gene regions and in-vitro cutting assay.
Sites and sequences targeted by sgRNA designed for the (A) RH2B and (B) tyr genes in Amphiprion ocellaris. Expanded regions show the target sequence (underlined in green) and ‘NGG’ PAM (underlined in black) for each sgRNA. For Exon 4 of RH2B, the Lys296 chromophore binding site (coloured blue) is also depicted down-stream of target sequence 1. Gel images to the right of each gene illustration depict DNA fragments size when amplicons that contained target (C) RH2B and (D) tyr gene regions were incubated (in-vitro) with (+) or without (-) Cas9 protein and sgRNA. Dotted boxes highlight cut DNA fragments.
Table 1.
Clutch survival and mutation rates for RH2B targeted injection rounds.
Survival rates of embryos injected with sgRNA/Cas9 (treatment) or sgRNA-only (positive control) targeting RH2B, and non-injected (negative control) embryos at time of collection (64–88 hpf), number of genotyped embryos, and mutation rate per clutch and target sequence. Note: Clutches 8–11 (sgRNA 4) were injected with a lower concentration of sgRNA/Cas9.
Table 2.
Clutch survival and mutation rates for tyr targeted injection rounds.
Survival rates of embryos injected with sgRNA/Cas9 or sgRNA-only targeting tyr, and non-injected embryos at time of collection (64–88 hpf), number of genotyped embryos, and mutation rate per clutch and target sequence.
Fig 3.
Genotype analysis of RH2B mutant Amphiprion ocellaris embryos.
Subcloned sequences and next generation shotgun amplicon sequences (NGS) belonging to A. ocellaris embryos (clutch 3, sgRNA RH2B 1; clutch 12, sgRNA tyr 1) with mutations at targeted sequences (underlined) located on (A) Exon 4 of the RH2B opsin gene, and (B) Exon 2 of the tyr gene. Wildtype (WT) sequences are included for reference. Mutations included deletions (dashes), substitutions (green), and insertions (blue). Sequence labels on the left-side indicate mutant embryo and allele no., while numbers on the right-side indicate the base pair change (Δbp), proportion of each allele out of the total number of cloned sequences for each embryo, or the percentage (%) of reads out of total reads for NGS. (C) Number of frameshift and in-frame mutations per RH2B mutant embryo. (D) Micrographs of tyr mutant A. ocellaris embryos exhibiting full knockout (tyr-M1 and -M2) and partial knockout (tyr-M3 and -M4) phenotypes, and a wildtype embryo for comparison. (E) Number of frameshift and in-frame mutations per tyr mutant embryo.
Fig 4.
Genotype analysis of four-month-old RH2B mutant Amphiprion ocellaris.
(A) Subcloned sequences belonging to A. ocellaris juveniles (clutch 11, sgRNA RH2B 4) with mutations at targeted sequences (underlined) located on Exon 1 of the RH2B opsin gene. Wildtype (WT) sequence is included for reference. Mutations included deletions (dashes), substitutions (green), and insertions (blue). Sequence labels on the left-side indicate mutant fish and allele no., while numbers on the right-side indicate the base pair change (Δbp) and the proportion of each allele out of the total number of cloned sequences for each fish. (B) Images of the RH2B mutant A. ocellaris juveniles. (C) Number of frameshift and in-frame mutations per RH2B mutant fish.