Efficient knockout of the albino gene using the SpRY protein. In A the albino locus is schematically shown, exons are in grey, introns are shown as gaps, not to scale. The coding sequence is in dark grey and the different target sites in exons 1, 3 and 6 are indicated, the position of the alb^b4 mutation in exon 6 is marked by a red asterisk. The different categories for the evaluation of the knockout efficiency are shown in B-F′. Lateral views (B-F) and dorsal views (B′-F′) of larvae 3 dpf are shown, scale bar: 2 mm. Category 1, no knockout (none) (B), (B′), category 2 weak (C), (C′), category 3 moderate (D), (D′), category 4 good (E), (E′) and category 5 very good/complete knockout (F), (F′). In G the results for the six target sites tested are shown. For detailed results see Table S1.

Homology directed repair of the albino^b4 mutation using the Cas9 variant SpRY. In A the partial sequence of exon 6 of the albino gene is depicted, coding sequence in capital letters, intron sequence in small letters. The target sites U3, U4, U5 and U6* are indicated by arrows; the mutation in alb^b4 leading to a premature stop codon is shown in red and marked with an asterisk. In B the oligonucleotides used as donors for HDR are shown. The two base pairs that are altered are shown in red; note the 5′ and 3′ ends of the oligonucleotides indicating the DNA strand they correspond to. The different categories for the evaluation of the repair efficiency are shown in C-F′. Lateral views (C-F) and dorsal views (C′-F′) of larvae 3 dpf are shown, scale bar: 2 mm. Category 1, no repair (none) (C), (C′), category 2 weak (D), (D′), category 3 moderate (E), (E′) and category 4 good repair (F), (F′). In G the results for the six tested donor oligonucleotides are shown. For detailed results see Table S2.

Improvement of knockout and repair efficiencies by adding an aNLS. In A the efficiencies of knocking out the function of the albino gene are compared between Cas9 and aNLS-Cas9 and SpRY and aNLS-SpRY. Whereas in the case of Cas9 the addition of an aNLS leads to a significantly higher efficiency, 93.5% in the best two categories compared to 55.1%, this is not the case for SpRY. Detailed results see Table S3. Purified aNLS-Cas9 performs as well as commercially available Cas9 protein (Alt-R™ S.p. Cas9 V3, IDT) when tested for the knockout of the albino gene (B). In addition, the repair efficiency for aNLS-SpRY is significantly higher compared to SpRY, with 19% versus 9.1% in the category ‘good’ (C). For detailed results see Tables S4 and S5.

Allele exchange in the obelix gene. In A the start of the coding sequence for obe is shown. The CRISPR target site is indicated by an arrow, the two point mutations introduced by HDR are shown in red and green, respectively, the recognition site for SalI that is generated by the HDR is underlined. The oligonucleotide used as donor DNA is also depicted. In B sequences from wild-type fish, and fish heterozygous or homozygous for the engineered allele are shown, the two positions that were engineered are boxed. There are no discernible differences between wild-type siblings (C) and fish heterozygous for the engineered allele (kcnj13^ten/+) (E), nor between fish that carry a kcnj13 loss-of-function allele over a wild-type allele (kcnj13^t24ui/+) (F) or over the engineered allele (kcnj13^t24ui/ kcnj13^ten) (G). For comparison, a fish homozygous mutant for a loss-of-function allele of obe (kcnj13^t24ui/kcnj13^t24ui) is shown in D, scale bar: 1 cm.