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Generation of stable cln3 mutant lines in zebrafish using CRISPR/Cas9. (A) Pipeline for the generation of cln3 homozygous mutants. Cas9 protein was co-injected with cln3 and slc45a2 sgRNAs into one-cell embryos. Chimeric larvae showing a pronounced albino phenotype were raised to adulthood (P0) and outcrossed with nacre fish for creating the F1 generation. F1 adult fish were screened for indel mutations in the cln3 gene. Founders with a predicted early stop codon were incrossed to create the F2 generation comprising potentially wild-type (+/+), heterozygous (+/−), and homozygous progeny (−/−). (B)cln3 sgRNA was injected into freshly fertilized embryos, and genomic DNA was extracted 24 hpf. Agarose gel analysis confirmed the high efficiency of cln3 sgRNA-Cas9 to induce indel mutations at the target site. (C) Left panel: dorsal view of a 4 dpf uninjected control wild-type and a chimeric larva. The albino phenotype can be evaluated in the eye (red arrowhead). Right panel: chimeric male (upper part) and female (lower part) P0 adults. (D) Schematic illustration of the cln3 gene showing the two primer pairs used for qPCR analysis. cln3 expression levels in MUT larvae relative to the expression levels in WT at 5 dpf. All expression levels were normalized to either the rpl13α or the ef1α reference genes. (E) Carriers were incrossed and raised to adulthood. Genotyping of F2 adult fish showed that 46% and 30% of the progeny were homozygous mutants for the nonsense and exon 4 deletion mutation, respectively. (F, G) Body length and eye area of 6 dpf larvae (n = 10 per genotype). (H) Survival curve of HET1 and MUT1 zebrafish (n = 50 per genotype).
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