Optimizing CRISPR/Cas9 technologies to develop disease model systems

The CRISPR/Cas9 system is widely used as state of the art genetic engineering to create specific disease models in vitro. We used this system to mimic leukemic chromosomal translocations like KMT2A-AFF1 t(4;11) and KMT2A-MLLT4 t(6;11) in K562 cells and HSPCs. It turned out that many aspects have to be considered regarding the transfection parameters and culture conditions. Interestingly, the K562 cell line was not only suitable to check sgRNA efficiency, we were also able to induce both chromosomal translocations, the t(4;11) and t(6;11), in K562 cells with CRISPR/Cas9. The K562 cells are undifferentiated blast cells growing in suspension and dividing rapidly caused by a bcr-abl fusion. Due to the high cell proliferation, the NHEJ repair mechanism appeared as preferential repair method after double strand break induction. Is the proliferation status of HSPCs also mandatory to induce chromosomal translocations or is it just donor dependent? Future perspectives will be discussed.

Publication History

Article published online:
12 May 2023