Past Issues

2019: Volume 2, Issue 3

CRISPR/Cas9-based Editing of CDK4, p107, and TGFβ1 in Human Breast and Lung Cancer Cells

Hussein Sabit1*, Doha Alaa2, Nourhan Jamal2, Sodfa Tarek2, Magda Fouda2, Bassant Maher2, Abdel-Bary Prince3, Shimaa E Abdel-Ghany2, Emre Cevik1, Huseyin Tombuoglu1, Osama A M Said2, Abdulrahman Sabry4, Afnan F Al-Muhanna5, Mokhtar El-Zawahri2,6

1Department of Genetics, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Saudi Arabia
2College of Biotechnology, Misr University for Science and Technology, Egypt
3College of Vet. Medicine, Cairo University, Egypt
4GIS Research Center, Egypt
5 Breast Imaging Division, KFHU, Imam Abdulrahman Bin Faisal University, Saudi Arabia
6Center for Research and Development, Misr University for Science and Technology, Egypt

Corresponding author: Hussein Sabit, Department of Genetics, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Saudi Arabia.

Received: May 16, 2019
Published: June 26, 2019

ABSTRACT

The CRISPR/Cas9 system is considered one of the most controversial yet powerful genome-editing tool with high potentiality and wide-angled array of biomedical applications. Cancer is considered one of the serious health-threat diseases that could be controlled, and even, cured by CRISPR/Cas9. In the present study, three cancer-related genes (CDK4, p107, and TGFβ1) were targeted in breast cancer cells (MCF-7) and lung cancer cells (A549) with CRISPR/Cas9 cassettes. Cells were transfected with these crRNAs to knockout CDK and TGFβ1, and to activate p107 in a dose-optimized manner. Viable cell count was measured using Trypan blue assay. Semi-quantitative PCR was also performed to detect the knockout and activation of the genes under study. Two-dimensional polyacrylamide gel electrophoresis was carried out to ensure the up/downregulation of the studied genes in both MCF-7 and A549.  Transfecting MCF-7 and A549 has resulted in a significant reduction in the cell count (p>0.005). Cell viability was also measured using MTT assay, and the results showed a significant decrease in the overall viability of cells after being challenged with CRISPR/Cas9. Semi-quantitative PCR was performed against specific primers to ensure the elimination/activation of the target genes. Data indicated a downregulation of CDK4 and TGFβ1 and upregulation of p107. Two-dimensional protein gel electrophoreses was also conducted to indicate the level of up/down regulation of the specific proteins encoded by these genes. Data indicated a partial presence of CDK4 and p107 and a complete absence of TGFβ1 protein. This study might indicate the efficiency of editing tumor-related genes within the genome of malignant cells. However, further confirmative studied are needed to validate the use of CRISPR Knockout (KO)/Activation (AC) in controlling cancer in vitro.

Keywords: CRISPR; Knockout; CDK4; p107; TGFβ1; Lung; Breast; Cancer.

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