Chromosomal translocations that result in the generation of oncogenic fusion-genes are a hallmark of lymphoblastic leukaemia. Current approaches to study these aberrations are largely limited to enforced overexpression of the oncogenic fusion transcript. While this has been fruitful for understanding of how these lesions drive cancer, they cannot capture the physiological significance of fusion products arising at endogenous gene loci.
Here we demonstrate that CRISPR technology can be leveraged to generate large chromosomal translocations by simultaneously targeting two loci, allowing a directed chromosomal translocation with a defined breakpoint. Further, we show that this process can be enhanced by co-delivery of a homology-directed-repair (HDR) template. For this, we have developed a HDR template containing a fluorescent marker (dsRed) under the control of a CMV promoter. Homology arms for the two target loci (on different chromosomes) can be rapidly amplified from target cells and cloned into this plasmid. The final HDR template, is then provided to pro-B cells with Cas9 and gRNA complexes. Using Snx2-Abl1 as a proof-of-principle target, we show that despite successful generation of the chromosomal translocation, expression of the Snx2-Abl1 transcript from it’s endogenous locus was not sufficient for cell transformation. Strikingly, amplification of the CRISPR-induced fusion, followed by enforced overexpression in pro-B cells revealed that overexpression of this transcript was now sufficient to drive transformation and downstream signaling, consistent with our interpretation that expression of the endogenous fusion alone was not sufficient for transformation. We further explore the role of a ‘second-hit’ in co-operation with fusion oncogenes, and perform large-scale transcriptomic and methylome analysis of leukemia patient samples to understand how expression of the 5’ fusions partners may be altered in disease. Critically, this work, provides a unique approach to functionally characterize the chromosomal aberrations that are identified in leukaemia. In addition, while CRISPR is commonly utilized to knock-out or modify single-genes, here we exploit CRISPR technology and HDR in an ingenious manner, to coax the fusion of two chromosomes.