ИСТИНА

Chromosomal translocations are major sources of cancer. Specifically, all of lymphoma and almost all of leukemia cases are directly related to chromosomal translocations. Chromosomal translocations arise as a result of an erroneous repair of DNA double strand breaks (DSBs) and they are often cell type-specific. Several factors may affect the occurrence of chromosomal translocations in different cell. These include the transcriptional activity of the genes, the relative distances between the genes in the nuclear space and the chromatin environment. Here we propose to experimentally investigate factors that affect chromosomal translocations by simultaneously inducing several DSBs on non-homologous chromosomes and then comparing the relative rates of chromosomal translocation in different cells. The CRISPR/Cas9 system is used to induce DSBs and observe the resulting chromosomal translocations. The MYC, IGH, RUNX1, RUNX1T1, ETV6 and NTRK3 gene loci were selected for DSB induction as they are known to be involved in chromosomal translocations that are linked to specific types of cancer. Several cell lines of different origins were transfected with Cas9 and gRNAs to target three or four of the selected genes to induce three or four DSBs simultaneously. Cells were collected after 48 hours and the rates of chromosomal translocation are measured by qPCR using translocation-specific primers. Our initial results indicate that translocations involving different loci have different frequency depending on the cell type. For example, in the RUNX1-RUNX1T1-IGH combination, different rates of chromosomal translocations were observed in different cell lines used, with the RUNX1T1-IGH translocation as the most observed in B-cells while the RUNX1-IGH translocation as the most observed in T-cells. To further investigate these observations, we use 3D-FISH to determine the relative distances between the target genes and their respective position in the nuclear space. We are also studying epigenetic marks that can be indicative of a high propensity to form translocations. We plan to extend our study to other translocation partners and to more cell lines with different origin.