Introduction: Colorectal cancer is the third leading cause of cancer death in the world and the second leading cause of cancer death in the United States. CRISPR/Cas9 is a powerful tool in genome manipulation for therapeutic purposes. Using CRISPR, it is possible to correct genome errors and turn genes on or off in cells and organisms. When DNA suffers a double strand failure, there are two pathways HDR and NHEJ to repair DNA. NHEJ pathway is used to delete the defective gene and HDR is used to replace it with a healthy allele. Commonly mutated genes involved in colorectal cancer are APC, Tp53, KRAS and SMSD10. The main obstacle in the drug treatment of CRC is drug resistance.
Methods: New technologies:
CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies utilize nuclease-deactivated Cas9 (dCas9) that binds to the target genomic region with the same efficiency as Cas9 but cannot generate a DSB and instead results in RNA-directed transcriptional control of the target region.
Bace editing is a novel genome editing method that can make and transversion and transition mutations at the single-base level without double-stranded DNA breaks, donor templates, or undesirable effects of NHEJ and HDR mechanisms. Prime editing is a ‘search and replace’ tool that can do any intended changes, including all 12 possible base-to-base conversions, insertions, and deletions without requiring DSBs or donor DNA templates.
Applications of CRISPR/Cas9 in CRC:
CRISPR/Cas9 can be used as a powerful tool to elucidate the precise function of mutations that underlie the development of CRC. It is also used to investigate the natural course of CRC progression and to elucidate the sequence of mutations that contribute to tumorigenesis. With CRISPR Cas9, this versatile system can facilitate efficient genome editing, enabling the simultaneous insertion and deletion of multiple genes. The evolution of CRC is greatly influenced by the accumulation of gene mutations. However, the specific function of the genes and the impact of these genomic changes are still unclear. Traditional gene editing tools have many limitations, especially the efficiency of gene editing. Compared to other tools, CRISPR/Cas9-mediated genome editing is simple and effective. Also, precise genome editing can be done using sgRNA of the CRISPR/Cas9 system. These tools have been used in CRC cell lines, mouse models, and human-derived organoid models, as well as CRISPR/Cas9-based gene screening and gene therapy.
The metastatic site of CRC can provide suitable conditions for targeted gene therapy because these sites are almost limited to the intestinal cavity, liver or abdomen compared to other cancers such as breast cancer and lung cancer.
We can use the CRISPR/ Cas9 library to screen functional genes in colon cancer cells, identify genes that direct the development of tumors, and shed light on the initiation and progression of cancer. Mass genomic screening is a powerful tool for detecting mutated genes that can reveal phenotypic changes following drug treatment or other stimuli, and thus lead to the identification of new targets for cancer therapy.
According to existing studies, the CRISPR / Cas9 gene editing system has been widely used in early cancer research. Also, concurrent with the rapid development of Cas9-based biotechnology, a number of Cas9-based clinical trials may point to extracellular somatic cell editing and future use in patients. It has been achieved in inhibiting the growth and progression of colon cancer .
CRISPR / Cas9 engineering:
CRISPR / Cas9 engineering that improves fidelity and specificity, which are mainly divided into three categories: Cas9 engineering; sgRNA modification; SaCas9 modification.
A novel approach in CRC:
Multiple pieces of evidence have shown that a high tumor mutation burden indicates an effective immunotherapy response, and immune checkpoint inhibitors (ICIs) can effectively treat metastatic colorectal cancer (mCRC) with low microsatellite instability and deficient mismatch repair. However, current ICIs are still ineffective for pMMR CRC or MSI-H CRC (known as pMMRMSI-H tumors).
Results: The results of studies investigating many genes involved in CRC show that CRISPR technology is effective in the treatment of colorectal cancer.
Conclusion: CRISPR/Cas9 gene editing technology provides a new method for both targeted therapy and gene therapy. However, due to the off targeting, and limited application of the CRISPR/Cas9 gene editing technology to cell lines and organoid models, further research is needed for gene therapy of CRC patients.