A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish.

Zebrafish husbandry and maintenance

All zebrafish were maintained in a temperature-controlled (28.5C), light-controlled (14h on/10h off) room as per standard conditions. Embryos were raised in temperature-controlled (28.5°C) incubators in E3 medium. AB and WIK strains were obtained from the ZIRC zebrafish stock center (Eugene, OR); TE was a generous gift from Patrick Müller (MPI Tübingen); roy orbison was originally obtained from the Dowling laboratory (Harvard Medical School); tra b6/b6 ;nac w2/w2 from the Gilmour laboratory (EMBL Heidelberg). Positional cloning of roy orbison Positional cloning was performed as previously described ( Zhou and Zon, 2011 ). Briefly, adult roy orbison mutants were bred to homozygous WIK fish to obtain heterozygous progeny. S from this group were then incrossed, and the embryonic offspring screened at 3–4dpf to look for defects in eye iridophores. This revealed Mendelian segregation of the roy orbison phenotype, with ~25% of offspring having severely reduced or absent iridophores. A total mapping panel of 1672 fish were then used for low-resolution linkage analysis, which placed the defect on roy on linkage group 20. We then ped medium and high resolution mapping using previously described SNP markers along chromosome 20, as shown in Figure 2 . For the fine-resolution mapping between markers z13672 and z28453, custom designed PCR primers based on the Zv9 zebrafish build were used, which led to few recombinants between markers zp6 and zp10, with only 8 genes in that interval. Based on this small number of genes, we chose a candidate approach and examined expression by ISH and qRT-PCR, revealing the gene mpv17 to be the most likely candidate in this region. cDNA cloning of mpv17 3 AB adults and and 3 roy orbison adults were tail-clipped, and RNA isolated using Trizol and the RNEasy kits. This was followed by reverse transcription using SuperScript III with both oligo- dT and random hexamers. The entire mpv17 ORF was PCR-amplified, and cloned into the TOPO/TA cloning vector. 4–5 colonies from each TOPO reaction was mini-prepped and subject to Sanger sequencing. The sequence traces were compared to the reference genome sequence. mRNA rescue The full-length mpv17 cDNA from the AB strain was subcloned into the pCS2+ vector. Capped mRNA was transcribed using the mMessage machine kit per manufacturer’s protocol. mRNA was resuspended in water and microinjected into 1-cell roy embryos at 100ng/μl. Embryos were scored for rescue of eye iridophores at 4–7 dpf. Morpholino phenocopy An ATG translation-blocking morpholino was designed by GeneTools using the AB reference sequence. The morpholino was resuspended in water, and microinjected into 1-cell AB embryos at doses ranging from 0.02mM to 4mM. Embryos were scored for loss of eye iridophores at 4–7 dpf. Array CGH Genomic DNA was isolated from the skin and kidney of 3 AB and 3 roy adults. This DNA was hybridized to Nimblegen zebrafish DNA arrays, as previously described ( Chen et al., 2013 ; Freeman et al., 2009 ). This array has both autosomes in linkage groups 1–25, as well as mitochondrial probes. After normalization for probe intensities, a log2-fold change of skin versus kidney for each individual fish was obtained. The values across each of the three fish for each genotype was averaged.

Statistics

Iridophore counts between groups were compared using unpaired t-tests with a significance level of 0.05. The array CGH data was compared by averaging of replicates and comparison by t-test for mitochondrial chromosome (chrM) DNA signal. sgRNA synthesis and genotyping The mpv17 sgRNA was designed using CHOPCHOP ( Labun et al., 2016 ; Montague et al., 2014 ) ( http://chopchop.cbu.uib.no/index.php ). sgRNA sequences are (PAM underlined): sgRNA[ mpv17 ccB ]: 5 ′ -GATGGCCAAACACCCATGGAAGG-3 ′, sgRNA[ mpv17 ccC ]: 5 ′ -GGTGCGTCTCGCGTTGTGATTGG-3 ′ sgRNA synthesis using oligo-based, cloning-free template generation and in vitro transcription ( Bassett et al., 2013 ; Burger et al., 2016 ). Primers for oligo based sgRNA synthesis were: sgRNA T7 fwd : 5 ′ - GAAATTAATACGACTCACTATA-N20-GTTTTAGAGCTAGAAATAGC-3 ′, where N20 indicates the sgRNA target sequence without PAM and the invariant sgRNA rev : 5 ′ -AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3 ′ (PAGE-purified, Sigma Aldrich). Primer extension was performed using Phusion polymerase (Thermo Scientific), followed by column purification and in vitro transcription using T7 RNA polymerase (Roche). sgRNAs were assembled with Cas9-EGFP into ribonucleopeotein complexes (RNP) solubilized with 300 mM KCL as described and were injected into 1-cell stage embryos of the TE wildtype strain. F1 animals were genotyped and alleles recovered by amplifying the locus using PCR primers ccB forward : 5 ′ -TAACCGTTTGTCATAATGTGGC-3 ′, ccB reverse 5 ′ - CTAAACAAACTGCTGCTTAGGGAG-3, ccC forward 5 ′ -AATAGGGAGTGAATGGGG-3 ′ and ccC reverse 5 ′ -GTGGCCAGCAAAATGTAAA-3 ′; resulting PCR products were sub-cloned into pJet and sequenced from DNA column-free isolated off colony PCR of individual clones, as established before. The panel plots describing the mpv17 alleles were generated using the CrispRVariantsLite software ( Lindsay et al., 2016 ) ( http://imlspenticton.uzh.ch:3838/CrispRVariantsLite/ ) from clonal Sanger sequencing data of PCR clones from individual embryos. Crispant and complementation phenotype assessment Injected clutches were sorted for Cas9-EGFP-positive embryos 3 hours post-injection to quality-control injections and mortality was assessed at 24 hpf. Phenotypes were assessed at 3 dpf by stereomicroscopy. Statistical analysis and graphical representation were performed with GraphPad Prism 7. Embryos for imaging were raised in E3 and treated with 3% MS-222 (pH = 7) during screening and imaging. Image acquisition of mpv17 ccB crispants and control embryos was performed with a Leica M205 FA stereomicroscope equipped with a 1x magnification lens at 100x magnification. Iridophores were visualized by illuminating embryos from above with a swan neck light source attached to the base of the stereomicroscope. Images were processed with ImageJ 1.51h and Adobe Photoshop CS6.

Supplementary Material 1 Supplemental Figure 1: Targeting details for sgRNA ccB and allele spectrum in mpv17ccB crispants. Schematic of mpv17 locus with coding sequence (CDS) and total mRNA-coding region including introns, sgRNA ccB indicated as red triangle. Panel plot shows allele spectrum and mutagenesis efficiency in four individual mpv17 ccB crispant embryos. Genomic reference sequence depicted on top, black vertical bar indicates Cas9 cleavage site, blue box depicts PAM. Mutant sequences reveal re-occurring alleles in crispants; note absence of wildtype sequences, suggesting high mutagenesis efficiency for Cas9 RNPs with sgRNA ccB. 2 Supplemental Figure 2: Mitochondrial DNA (mtDNA) signal from array CGH of skin:kidney from AB shows a severe loss of mtDNA only from the roy orbison skin, consistent with the protein leading to a tissue specific defect in mtDNA maintenance (*=p