Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis.

The Philadelphia (Ph) chromosome was the first translocation identified in leukemia. It is supposed to be generated by aberrant ligation between two DNA double-strand breaks (DSBs) at the BCR gene located on chromosome 9q34 and the ABL1 gene located on chromosome 22q11. Thus, mimicking the initiation process of translocation, we induced CRISPR/Cas9-mediated DSBs simultaneously at the breakpoints of the BCR and ABL1 genes in a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent human leukemia cell line. After transfection of two single guide RNAs (sgRNAs) targeting intron 13 of the BCR gene and intron 1 of the ABL1 gene, a factor-independent subline was obtained. In the subline, p210 BCR::ABL1 and its reciprocal ABL1::BCR fusions were generated as a result of balanced translocation corresponding to the Ph chromosome. Another set of sgRNAs targeting intron 1 of the BCR gene and intron 1 of the ABL1 gene induced a factor-independent subline expressing p190 BCR::ABL1. Both p210 and p190 BCR::ABL1 induced factor-independent growth by constitutively activating intracellular signaling pathways for transcriptional regulation of cell cycle progression and cell survival that are usually regulated by GM-CSF. These observations suggested that simultaneous DSBs at the BCR and ABL1 gene breakpoints are initiation events for oncogenesis in Ph+ leukemia. (200/200 words).