Targeted integration of Cas9 to the mitfa locus with GeneWeld knockin system.

(A) Schematic depicting integration site and GeneWeld knock-in cassette. 5’ and 3’ homology arms are designed to target exon 2 of the zebrafish mitfa gene. The knock-in cassette includes p2A followed by Cas9 and BFP driven by the eye-specific promoter gamma-crystallin. (B) Pipeline to produce F1 mitfaCas9 zebrafish. The GeneWeld knock-in vector and gRNAs targeting the mitfa genomic insertion site or specific sites on the knock-in vector (UgRNA) are co-injected into one-cell stage wild-type zebrafish embryos. Embryos are screened for BFP+ eyes marking mosaic integration. These mosaic founder fish are then raised to adulthood and crossed with wild-type fish. The resulting embryos are screened for BFP+ eyes and sequenced to confirm precise integration. (C) Fluorescent in situ hybridization chain reaction (FISH) on 3dpf wild-type and mitfaCas9 embryos treated with 1-phenyl 2-thiourea (PTU). The presence of mitfa and Cas9 RNA was assessed by confocal microscopy at 40x magnification. Maximum intensity projections are shown. n=17 WT embryos and n=16 mitfaCas9 embryos were screened. Scale bars, 100µm. Created with BioRender.com.

Melanocyte lineage-specific knockout of albino using mitfaCas9 fish.

(A) Pipeline to generate F0 and F1 mitfa:GFP;U6:gRNA (MG-gRNA) zebrafish. (B) Adult mitfaCas9 MG-NT and MG-albino F0 fish. (C) Proportion of MG-NT (n=19) and MG-albino (n=25) F0 fish with loss of pigmentation phenotype. (D) Adult mitfaCas9 MG-NT and MG-albino F1 fish. (E) Pigmented area/ mm2 calculated for n=5 fish/ genotype within a defined rectangular region of interest (ROI) encompassing the top and middle melanocyte stripes. Two-sided student’s t-test was used to assess statistical significance, **** p < 0.0001; error bars, SD. (F) 3dpf mitfaCas9 MG-NT and MG-albino F2 fish. Representative embryos are shown for each genotype. (G) Mean grey value of head melanocytes calculated for n=10 embryos/genotype within a defined hexagonal ROI indicated as a red outline in 2F. Two-sided student’s t-test was used to assess statistical significance, **** p < 0.0001. (H) Schematic for CRISPR sequencing protocol. (I) CRISPR-seq results are shown for WT and n=3 independent F1 MG-albino fish. Only GFP negative cells were isolated from WT fish. Results are shown as a fraction of sequences with indels calculated using CRISPRESSO. Created with BioRender.com.

Melanocyte lineage-specific knockout of sox10 using mitfaCas9 fish.

(A) Adult mitfaCas9 MG-NT and MG-sox10 F0 fish. (B) Proportion of MG-NT (n=19) and MG-sox10 (n=16) F0 fish with disrupted stripes phenotype. (C) Adult mitfaCas9 MG-NT and MG-sox10 F1 fish. (D) The average width of melanocyte stripe 1D and xanthophore interstripe X0 was calculated for each fish by averaging 5 stripe/interstripe width measurements. N=5 fish per genotype. Two-sided student’s t-test was used to assess statistical significance, *** p < 0.001; error bars, SD. (E) F1 MG-NT (n=5) and MG-sox10 (n=5) adult fish were treated with epinephrine and melanocytes were counted within a defined rectangular region of interest 3.46mm x 2.54mm encompassing the top and middle melanocyte stripes. Two-sided student’s t-test was used to assess statistical significance, * p < 0.05; error bars, SD. (F) Schematic of neocuproine (neo) experimental setup. Adult mitfaCas9 MG-NT (n= 5) and MG-sox10 (n=5) F1 fish were treated with neocuproine for 24 hours to ablate melanocytes then imaged at day 7,15, and 70 to measure regeneration of melanocytes compared to day 0. (G) Representative images are shown for MG-NT and MG-sox10 fish pre- and post-neocuproine treatment. (H) Quantification of melanocyte regeneration. Fish were treated with epinephrine prior to imaging to enable counting of melanocytes. Two-sided student’s t-test was used to assess statistical significance, ** p < 0.01; error bars, SD. Created with BioRender.com.

Non-autonomous function of tuba1a on melanocytes.

(A) 4dpf zebrafish embryos injected with either NT or tuba1a Alt-R CRISPR Cas9 gRNAs. (B) Percentage of NT or tuba1a Alt-R zebrafish embryos with dispersed melanocyte phenotypes. N=2 independent experiments. (C) Survival percentage is shown for NT and tuba1a Alt-R embryos. Embryos were counted at 24hpf and again at 14dpf to determine survival. (D) 4dpf mitfaCas9 MG-tuba1a F2 embryos (BFP+ eyes) compared to sibling controls with no mitfaCas9 (BFP-eyes). Representative embryos are shown for each genotype. (E) Pigmented area/ mm2 calculated for n=19 embryos/genotype from two independent MG-tuba1a F2 clutches. Two-sided student’s t-test was used to assess statistical significance, ns: no significance. (F) 4dpf Alt-R injected zebrafish embryos imaged before and after epinephrine (epi) treatment. (G) Pigmented area/ mm2 calculated for n=20 embryos/genotype from two independent clutches. Two-sided student’s t-test was used to assess statistical significance, **** p < 0.0001.

Generation of a zebrafish melanoma model in mitfaCas9 fish.

(A) Adult mitfaCas9 MG-NT; mitfa:TdT;U6:NT (MTdt-NT) and MG-ptena; MTdT-ptenb F0 fish. (B) Proportion of MG-NT; MTdT-NT (n=14) and MG-ptena; MTdT-ptenb (n=14) F0 fish with disrupted stripes phenotype. (C) Schematic of zebrafish tumorigenesis assay. Indicated plasmids are injected into one-cell stage embryos from crosses between mitfaCas9 and WT fish. The mitfa:BRAFV600E plasmid includes cardiac-specific cmlc2:GFP. Embryos were sorted for GFP+ hearts and BFP+ eyes and fish were screened every 2 weeks for tumors. (D) Tumor free survival curve. N=2 independent experiments. Tumors were tracked over the course of 30 weeks. Log-rank (Mantel-Cox) test was used to assess statistical significance, *** p < 0.001; **** p < 0.0001; ns: no significance. (E) Histology was performed on one fish from each indicated injection group. Dotted lines indicate site of sectioning. Red chromogen was used for all IHC staining. Scale bars, 50µm. (F) CRISPR-seq results are shown for normal skin dissected from WT fish and tumors dissected from injection conditions 2, 3, and 4. Results are shown as a fraction of sequences with indels calculated using CRISPRESSO. Created with BioRender.com.

Melanoma specific knockout of sox10 reduces tumor burden and induces phenotypic switching.

(A) Schematic of zebrafish tumorigenesis assay. Indicated plasmids are injected into one-cell stage embryos from crosses between mitfaCas9 and WT fish. Embryos were sorted for GFP+ hearts and BFP+ eyes and fish were screened every 2 weeks for tumors. (B) Tumor free survival curve. N=3 independent experiments. Tumors were tracked over the course of 50 weeks. Log-rank (Mantel-Cox) test was used to assess statistical significance, **** p < 0.0001. (C) Histology is shown for one fish from each injection group. Dotted lines indicate site of sectioning. Red chromogen was used for all IHC staining. Scale bars, 50µm. (D) Color thresholding was used on IHC images to calculate the percentage of nuclei that stained positive for sox9. NT-1 n=1222 cells, NT-2 n=1620 cells, NT-3 n=2185 cells, sox10-1 n=970 cells, sox10-2 n=594, sox10-3 n=1046. Cells from n=3 images were analyzed for each tumor. (E) Schematic of scRNA-seq experimental setup from Wouters et al. Patient derived cell lines were treated with siRNAs targeting SOX10 or NTC and scRNA-seq was conducted at 72hrs. (F) UMAP of scRNA-seq dataset for MM057 cell line from Wouters et al. Cells from siSOX10 and siNT conditions are labeled. (G) Normalized expression of Sox10 per cell in UMAP space. (H) Violin plots of normalized expression of Sox10 per cell. Median is shown as dashed red line. Wilcoxon Rank Sum Test was used to assess statistical significance, **** p < 0.0001. (I) Normalized expression of Sox9 per cell in UMAP space. (J) Violin plots of normalized expression of Sox9 per cell. Wilcoxon Rank Sum Test was used to assess statistical significance, **** p < 0.0001. Created with BioRender.com.