Cas9 Stable Cell Line - HEK293T

Especially in leukemia, characteristic fusion genes created by translocation following a chromosomal break are frequently seen in cancer. To disrupt the BCR-ABL fusion gene, the researchers investigate CRISPR-Cas9/dual-sgRNA targeting of the BCR and cABL introns. Changes in BCR-ABL expression in CML cells are achieved using lentiviruses expressing Cas9-GFP/dual-BA-sgRNA. The effects, which include cell proliferation, clonogenicity, apoptosis, and downstream signaling, are assessed at the genomic and protein levels. Validated in CML mice models and patient-derived cells, effective disruption results in BCR-ABL tyrosine kinase pathway blockage, decreasing cell proliferation and eliciting apoptosis.

Figure 1. The researchers used Cas9-GFP/dual-BA-sgRNA to break the BCR-ABL fusion gene in CML cells. Dual sgRNAs targeted the BCR and cABL introns, respectively. The Cas9, GFP, and sgRNA co-expression vector was transfected into K562 cells. GFP+ cells were sorted for the targeted disruption efficiency assay, which confirmed genomic disruption with PCR and lowered BCR-ABL fusion protein levels with western blot analysis. The control empty vector Cas9 was co-transfected into HEK293T cells along with auxiliary plasmids pSPAX2 and pMD2G to generate the virus, serving as the negative control virus (NC). (Zeng J, et al., 2024)