Generation of a Zebrafish Knock-In Model Recapitulating Childhood ETV6::RUNX1-Positive B-Cell Precursor Acute Lymphoblastic Leukemia.

Approximately 25% of children with B-cell precursor acute lymphoblastic leukemia (pB-ALL) harbor the translocation, leading to the fusion gene. This translocation occurs in utero, but the disease is much less common than the prevalence of the fusion in newborns, suggesting that secondary mutations are required for overt leukemia. The role of these secondary mutations remains unclear and may contribute to treatment resistance and disease recurrence. We developed a zebrafish model for leukemia using CRISPR/Cas9 to introduce the human gene into zebrafish intron 5, resulting in fusion gene expression controlled by the endogenous promoter. As seen by GFP fluorescence at a single-cell level, the model correctly expressed the fusion protein in the right places in zebrafish embryos. The fusion expression induced an expansion of the progenitor cell pool and led to a low 2% frequency of leukemia. The introduction of targeted and gene mutations, mimicking secondary mutations, in the line significantly increased the incidence in leukemia. Transcriptomics revealed that the mut leukemias exclusively represented B-lineage disease. This novel zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype.