5220 - Impact of CCL4 knockout using CRISPR Cas-9 technology on colorectal tumor progression

Background

The Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 system (CRISPR/Cas9 system), is the defense mechanism in Streptococcus pyogenes against viruses, now it is widely used by researchers as a precise gene editing tool that allows a targeted genomic editing with the least possible off targets. Colorectal cancer (CRC) has recently become a devastating disease with mortality rate exceeded 9% in 2018, where the survival rate worsens once it metastasizes to vital organs such as the liver or lungs. Chemokines are one of the key players that promote cancer cell metastasis in some types of cancers, including colorectal cancer, where increasing evidence shows that CRC accumulates invasive and metastatic capacities through tumor microenvironment. In the light of this, we aimed at understanding the tumor microenvironment to achieve a comprehensive therapy, aiming in preventing early metastasis.

Methods

Microarray was done by our research group on early metastasized CRC cells to liver in a group of rats with CRC. In silico sgRNA CRISPR designing tools were used to design a sgRNA targeting C-C motif chemokine ligand 4 (CCL4) gene. The insert was ligated into a Cas9 expressing plasmid and transformed into E.coli. The Plasmid was then extracted and sequenced. CRC cells were transfected with the cloned plasmid. Functional assays were performed.

Results

Microarray results showed that CCL4 was highly expressed in early metastasized CRC cells to liver that gives a hypothesis that it has a role in cancer progression. Sequencing results confirmed a successful sgRNA insertion into the Cas9 plasmid. In addition, knocking out the expression of CCL4 in CRC cells significantly inhibited the cellular metabolic activity compared to cells transfected with empty plasmid. Furthermore, the ability of CRC cells to proliferate and to form colonies was dramatically decreased in response to CCL4 knockout in comparison to that transfected with empty plasmid.

Conclusions

CCL4 has oncogenic effect on CRC by its positive effect on cell proliferation properties and metabolic activity. The results indicate that CCL4 may act as a promising target for further studies to reveal its downstream impact on CRC progression.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Hassan Adwan, German University in Cairo, Cairo, Egypt.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.