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

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.