Cancer is one of the major leading causes of death worldwide. Many therapies and strategies have been applied and are still being discovered in the treatment of this cruel disease. Among them, one such novel genetic study found an RNA domain-containing endonuclease-based genome engineering technology, called the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9), as a powerful technique for the treatment of cancer cells.
Advantages of genome editing CRISPR technology
Apart from other treatment options, genome editing technology has been proposed in cancer treatment that can target any gene in the affected area and create knock-in and knock-out alterations.
This technology has brought significant improvement in diagnosis and treatment of the solid tumours, such as breast, lung, liver, etc. cancers where treatment via gene therapies show little to slow progress when compared to non-solid tumours like leukaemia.
Traditionally, two different genome techniques, such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) have been used for DNA repair by targeting the gene of interest. The efficiency of these techniques depends on the specificity and affinity of nucleases.
These techniques have been used for targeting various oncogenes like for human papillomavirus (HPV), however, they exerted off-target effects and are also it is less efficient, and a time taking process. Thus, the need for developing a highly efficient and accurate technique was born to compensate the negatives of the old techniques.
Structure & Mechanism of CRISPR/Cas 9
CRISPR/Cas9 System consists of three main components:
- Cas9 protein with DNA endonuclease activity
- Single guided RNA (sgRNA)
- tracrRNA (which makes attraction with Cas9)
The mechanism of action of CRISPR/Cas 9 consists of total 7 steps. CRISPR/Cas9 is used widely by scientists to edit the genes to treat deadly diseases like cancer. The technology itself consists of two parts: single-guided RNA (sgRNA) and Cas9 protein.
- Step 1: Cas9 is an enzyme that cuts off a specific part of infected DNA. This acts as target DNA.
- Step 2: sgRNA and Cas9 together seek out the target DNA downstream to a specific protospacer adjacent motif (PAM).
- Step 3: Cas9-sgRNA complex recognized the PAM of the effected genome and bound to target DNA.
- Step 4-5: Cas9 unzipped the double-strand of target DNA at 3 or 4 nucleotides upstream of the PAM sequence and guided RNA matches the complementary sequence. If the complementary sequence matched, the Cas9 uses molecular scissors to cut the affected part of the target DNA and insert the healthy or desired part of DNA.
- Step 6: The double-strand break (DSB) is repaired by enzymes in two ways, either by homologous recombination (HR) or non-homologous end-joining (NHEJ).
- Step 7: Patient no longer affected by cancer.
Application of CRISPR in the treatment of tumours
CRISPR/Cas9 editing tool provides specificity, efficiency and accuracy in genome editing. CRISPR/Cas9 technology targets tumour-suppressor genes to inhibit or reduce the tumorigenesis by restoring the activities of tumour-suppressor genes.
Below table shows some examples of invitro and in vivo use of CRISPR/Cas9 in the treatment of different tumours:
|Cancer Type||Target Choice||Cell line/gene||Study type||Vector|
|Breast cancer||TNBC||MDA-MB-231||In vitro||Lentiviral||Western blotting, PCR||Knockout of the CXCR4 or CXCR7 gene||Delay the conversion of the G1/S cycle, inhibit cell proliferation, invasion, and mitigation|
|Lung cancer||PD-1||T-cells||Phase 1 clinical trial||Plasmid||Next-generation sequencing||Knockout the PD-1||Effectively targeted exon 2 of PD-1 gene to|
|Liver cancer||β-catenin||PTEN and p53 (TP53 and Trp53)||In vivo||Plasmid||PCR||Knockout of both alleles||Akt phosphorylation|
|Colorectal cancer||KRAS||SW-480 CRC cells||In vitro||Polymer||Flow cytometry||Knockout of both alleles||Induce apoptosis, suppress cell proliferation and promote tumor cell death|
|Anal cancer||HPV16||293 T||In vivo||AVV||PCR||Knockout oncogenes (E6 and E7)||Inhibit the expression of HPV16 E6 and E7 genes to reduce anal tumour|
|Prostate cancer||ERRα, PGC1α||PC3, DU145, 293FT||In vivo||Lentiviral||Chromatin immunoprecipitation, bioinformatic analysis||Knockout of ERRα||Inhibit MYC levels to suppress metastasis and invasion of lung cancer growth|
TNBC: triple-negative breast cancer; AVV: adeno-associated vector.
Delivery of CRISPR/Cas9
This is the most important step in the genome editing strategy to minimise the off-target effects. The CRISPR components, that consists of sgRNA, ribonucleoproteins, and Cas9 are delivered to target site in two common ways:
- Viral vectors (lentiviral and adenoviral)
- Non-viral delivery system including DNA plasmids, nanoparticles, polymers, and micro-injection
Challenges and Conclusion
Besides Cas9, researchers continue to develop more members of CRISPR system.
Although, the tool produced remarkable results, the impending challenge remains during synthesis of the CRISPR system is to reduce the off-target effects of Cas9 nuclease.
Further recommendations include to improve the optimization of Cas9 and minimize the off-target effects. Also, the in vivo delivery of Cas9 should extend its application to cervix and stomach to locate and treat other cancers.
- CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions. Elsevier Public Health Emergency Collection. CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions – PMC (nih.gov)