Induction of t(9;22) (q34;q11) in Bone Marrow-Derived HSPCs Via CRISPR/Cas9 to Reveal the Differences of the Leukemogenic Properties of p210^BCR/ABL1 and p185^BCR/ABL1

The Philadelphia (Ph) chromosome is formed by the reciprocal translocation t(9;22) (q34;q11) and leads to the aberrant fusion of the ABL proto-oncogene 1 (ABL1) gene on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22. The BCR/ABL1 fusion gene encodes the oncogenic protein BCR/ABL1, whose constitutive enhanced kinase activity leads to increased cell proliferation, genomic instability and resistance to cell death. The Ph chromosome is known as the hallmark of chronic myeloid leukemia (CML). In the majority of CML patients, the breakpoint occurs within the major breakpoint region leading to the expression of the BCR/ABL1 transcripts e13a2 and e14a2. Both transcripts encode for the fusion protein p210. Chromosomal breakage within BCR in the minor breakpoint region leads to the BCR/ABL1 transcript e1a2, which encodes for p185 and is associated with inferior patient outcome. Here we report the CRISPR/Cas9-guided generation of bone marrow (BM)-derived cells harboring t(9;22) and expressing either the e13a2 or the e1a2 transcript.

Using the CRISPRscan software, we designed specific sgRNAs cutting in intron 1 of ABL1 and intron 1 or intron 13 of BCR, respectively. By performing in vitro cleavage assays and T7 endonuclease 1 assays we evaluated the cutting efficiency of the sgRNA-guided Cas9 and selected the sgRNAs with the highest efficiency. t(9;22) was induced in BM-derived CD34⁺ hematopoietic stem and progenitor cells (HSPCs) leading to the expression of either e13a2 or e1a2 BCR/ABL1 fusion transcript. The CRISPR/Cas9 system was delivered as a ribonucleoprotein (RNP) via electroporation (Nucleofector™, Lonza Bioscience) which offers a transient transfection. After RNP delivery, t(9;22) can be detected via PCR using specific primer pairs for each breakpoint and fluorescence in situ hybridization (FISH) with an ABL1 dual color break apart probe. We performed Sanger sequencing to validate the fusion of BCR and ABL1. Next, we determined the expression of BCR/ABL1 by RT-PCR and western blot and characterized the immunophenotype of the cells by flow cytometry.

The most promising sgRNAs attained cutting efficiencies up to 70% in in vitro assays and up to 30% in T7 assays. After nucleofection of CD34⁺ HSPCs with two sgRNAs targeting intron 1 of ABL1 and intron 1 or intron 13 of BCR we were able to detect the genomic fusion of BCR and ABL1 via PCR. FISH indicated the translocation affecting one ABL1 locus. Sanger sequencing confirmed the fusion of BCR and ABL1 at the expected breakpoint. By nested RT-PCR and western blot we detected a stable expression of the e1a2 or e13a2 BCR/ABL1 transcript, respectively. As expected, flow cytometry revealed myeloid differentiation of the cells.

In summary, we were able to induce t(9;22) in BM-derived HSPCs. After further characterization and comparison to patient samples, the cells will be used as a patient-like model for CML. This will allow us to gain new insights into the oncogenic pathway of BCR/ABL1 and to compare the leukemogenic properties of p210 and p185 side-by-side. In the future our model can serve as a platform for the improvement of therapeutic approaches.

No relevant conflicts of interest to declare.