Generation of the ETV6::RUNX1 (E::R) knock-in line. (a) A schematic illustrating the integration of the gene-breaking cassette into intron 5 of the etv6 gene using the GeneWeld technique. The process required four reagents: (1) Cas9 mRNA, (2) genomic sgRNAs, (3) universal sgRNAs, and (4) a donor plasmid containing the gene-breaking cassette flanked by 48 bp homology arms and by universal sgRNA target sites. In vivo, Cas9-induced a targeted double-strand break (DSB) in the genome and generated two targeted DSBs in the donor plasmid, liberating the cassette along with the short homology arms. Subsequently, integration of the cassette followed. Red arrowheads: Cas9 nuclease-induced DSBs; pink and turquoise rectangles: homology arms; UgRNA: universal sgRNA target sites. (b) Schematic of the E::R zebrafish model showing the integrated gene-breaking cassette and the UAS:GFP transgene. E::R fusion protein and GAL4-VP16 protein expression are driven by the endogenous etv6 promoter. GAL4-VP16 binds to UAS enhancer sequences, leading to GFP expression. SA: splice acceptor; TE: ocean pout antifreeze gene transcriptional termination and polyadenylation sequence. (c) Sequence of the in-frame splicing of the etv6 and RUNX1 transcripts. Primers used in the RT-PCR are either complementary to a region in exon 5 of etv6 or the inserted human RUNX1 cDNA, as indicated by arrows on the schematic of the etv6 locus (Table S5). (d) Lateral view of a 2 day old E::R zebrafish larva with two images: (d(I)) GFP fluorescence in the caudal hematopoietic tissue and (d(II)) a merged image of GFP fluorescence with transmitted light, providing the structural context. Scale bar: 50 µm.

Flow cytometry analysis of major blood cell lineages in the whole kidney marrow of the ETV6::RUNX1 (E::R) zebrafish based on light scatter characteristics. (a) Flow cytometry plots representative of the UAS:GFP and E::R zebrafish lines are shown. (b) Scatter plots representing the percentage of cells in precursor, myeloid, and lymphoid fractions for the E::R (n = 10) and UAS:GFP zebrafish lines (n = 10). The statistical significance was represented as ‘ns’ for no statistical significance, * p < 0.05, and *** p < 0.001. All quantifications are presented as mean ± s.e.m.

CRISPR/Cas9 targeting of the pax5 gene and features of leukemic E::R;pax5mut fish. (a) Structure of the zebrafish Pax5 protein has five conserved functional domains: paired (PD), octapeptide (OP), homeo (HD), transactivation (TAD), and inhibitory (ID) domains. Brackets indicate the boundaries of the eleven encoding exons. Red arrowheads mark the Cas9 cut positions in exon 3 and exon 5, and the respective sequences of the sgRNA target sites are shown. (b) Phenotypic and histological analysis of E::R;pax5mut zebrafish. Images depict lateral views of representative control UAS:GFP zebrafish (b(I)) and leukemic zebrafish (b(II)). The E::R;pax5mut leukemic zebrafish developed subcutaneous bleedings in the ventral body region (highlighted by a red frame in b(II)). An inset provides a close-up view of the bleeding (marked with red asterisks) (b(II)). Top views of the entire kidneys of UAS:GFP and E::R;pax5mut leukemic zebrafish are shown (b(III–IV)). The kidney of the leukemic zebrafish exhibited enlargement along its entire length (b(IV)). Giemsa staining of a peripheral blood smear from the UAS:GFP fish (b(V)) revealed normal nucleated erythrocytes, while Giemsa staining of the leukemic blood smear (b(VI)) showed the presence of clusters of lymphoblasts. Hematoxylin and Eosin staining of the sagittal paraffin sections of tissues from UAS:GFP fish (b(VII,IX,XI,XIII)) and leukemic fish (b(VIII,X,XII,XIV)) highlighted the presence of lymphoblasts in the kidney marrow (b(VIII)), muscle tissue (b(X), arrows), epidermis (b(XII), arrows), and liver (b(XIV), arrows). Scale bars: (b(V,VI)): 10 µm; (b(VII–XIV)): 100 µm. (c) Flow cytometry plot (right) showing cell populations separated by their light scatter characteristics in the whole kidney marrow of leukemic E::R;pax5mut fish, along with the UAS:GFP control plot (left). The predominant cell population in the leukemic fish exhibited light scatter characteristics similar to the precursor cell fraction in the control group. (d) Western blot analysis was performed using protein extracted from kidney marrow cells of UAS:GFP fish, non-leukemic E::R knock-in fish, and 6 out of 10 leukemic E::R;pax5mut fish (#4–#9). Primary antibodies targeting human RUNX1 and β-Actin were used. E::R protein was detected in all tumor samples but not in the samples from the UAS:GFP control or non-leukemic E::R knock-in fish. The uncropped blots are shown in Supplementary Materials.

Generation of cdkn2a/b^+/− zebrafish using the CRISPR/Cas9 genome editing and features of leukemic E::R;cdkn2a/b^+/− fish. (a) Genomic structure of the cdkn2a/b gene. Red arrowheads mark the Cas9 cut positions in exon 2, and the sequences of the three sgRNA target sites are shown. The cdkn2a/b^+/− zebrafish line with a premature stop codon after 210 nucleotides was established. (b) Phenotypic and histological characteristics of leukemic E::R;cdkn2a/b^+/− zebrafish. Images depict lateral views of representative control UAS:GFP zebrafish (b(I)) and leukemic zebrafish (b(II)). The E::R;cdkn2a/b^+/− leukemic zebrafish developed subcutaneous bleeding in the ventral body region (highlighted by a red frame in (b(II))). An inset provides a close-up view of the bleeding (marked with a red asterisk) (b(II)). Giemsa staining of a peripheral blood smear from the UAS:GFP fish (b(III)) revealed normal nucleated erythrocytes, while Giemsa staining of a leukemic blood smear (b(IV)) showed a significant presence of lymphoblasts. Hematoxylin and Eosin staining of sagittal paraffin sections of tissues from the UAS:GFP fish (b(V,VII)) and leukemic fish (b(VI,VIII)) revealed the presence of large deposits of lymphoblasts in the kidney marrow (b(VI)) and epidermis (b(VIII), arrows) of leukemic fish. The spleen was enlarged in the leukemic E::R;cdkn2a/b^+/− fish (b(X)) compared to the UAS:GFP control fish (b(IX)). Scale bars: (b(III,IV)): 10 µm; (b(V–VIII)): 100 µm; (b(IX,X)): 1 cm.

Heatmap visualization of the significantly differentially expressed B and T lineage-associated genes between the B-ALL and E::R;pax5mut versus T-ALL zebrafish transcriptomes and their expression across the different zebrafish leukemia types (adjusted p-value ≤ 0.05). B-ALL and E::R;pax5mut zebrafish leukemias expressed several B-lineage-associated genes at significantly higher levels compared to zebrafish with T-ALL, while the expression difference was the opposite for multiple T lineage-associated genes.