DHFR Gene Editing for Methotrexate-Resistance

Abstract

The DHFR (Dihydrofolate reductase) gene plays an important role in producing enzymes necessary for DNA synthesis, DNA repair, and cellular replication. Due to uncontrolled cell division in cancer patients, methotrexate is administered to the patients and it inhibits the DHFR enzyme. This inhibition causes disruption in DNA synthesis, thereby slowing down the uncontrolled cell division of cancer patients. There is no information found on introducing mutations into the DHFR gene to achieve resistance to methotrexate. My experiment determines whether changing leucine to tyrosine at position 22 in DHFR will result in methotrexate resistance. The gene edit was conducted via the use of CRISPR-cas9 and a donor molecule containing the tyrosine 22 mutation. The incubation of 3T3 cells in methotrexate showed that the cells containing the DHFR gene edit from transfection via the PCR donor molecule 3 and PUC-57 circular plasmid had higher resistance to a selective methotrexate concentration compared to the negative control due to colony growth. Analysis from the DNA sequencing confirmed that the 3T3 cells with higher levels of resistance to methotrexate contained the tyrosine 22 mutation. Also, the PUC-57 plasmid generated a larger number of methotrexate-resistant colonies than the PCR donor molecule 3 transfection. Thus, these results can potentially be used in clinical trials to provide methotrexate resistance to cancer patients via a bone marrow transplant with genetically modified stem cells. This will allow patients to receive higher concentrations of the drug for a potent treatment.