Cancer is the major cause of morbidity and mortality worldwide according to statistics. It has been reported that more than 14 million people in the world suffer from cancer, and 8.2 million died from cancer in 2012 (1). Numerous complicated causes of cancer make it difficult to be cured. Our project aims to provide a sensitive and effective therapy for cancer that can avoid the side-effects of current therapy. Our project focuses on melanoma, the most serious type of skin cancer with high potential for metastasis and a low survival rate (2, 3), which results in 59800 deaths around the world in 2015. Many studies have shown that BRAF is one of the most common mutant genes in Melanoma. The BRAF mutation incidence rate reaches up to 40% - 70% in malignant melanomas (4).
Nowadays, the major treatments to cancer are surgery, radiation therapy and chemotherapy, all of which are very harmful to human body. In order to upgrade current treatments, we, the SFLS_iGEMers, designed a synthetic device based on the CRISPR/Cas9 system that can only express in melanoma cells containing BRAF V600E mutation. Our device can discriminate between the oncogenic mutant and wild-type BRAF alleles and eliminate the carcinogenic mutant BRAF allele with high accuracy. Thus we can achieve efficient, specific and personalized melanoma treatment.
BRAF, one of the most important proto-oncogenes in human, is a serine or threonine kinase from the RAF family and has the strongest kinase activity in the RAF family. The most common mutation in BRAF is V600E, which is caused by the substitution of adenine (A) for thymine (T) and lead to the change of amino acid from valine to glutamate in the protein sequence. Mutant BRAF will continually activate MEK (mitogen-activated extracellular signal-regulated kinase) and ERK (extracelluar signal regulated kinase), resulting in activation of MAPK (mitogen-activated protein kinase) pathway and leading to the cell cycle being out of control, which promotes tumorigenesis (4). Despite V600E, other BRAF mutation sites have also been reported with low mutation rates. Consequently, we choose BRAF V600E mutation as our target.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) is a unique prokaryotic defense system against foreign genetic elements which is now utilized as a genome editing technology that allows permanent modification of genes. Compared with other genome editing methods such as ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nucleases), CRISPR may be a better choice for its simplicity, high success rate and efficiency in genome targeting (5). In our project, we designed a single-guide RNA (sgRNA) that targets the mutant BRAF oncogene to meet our goal. By simply changing the sequence of the sgRNA, we can target any specific oncogene sequence and achieve personalized therapy for cancer.
However, the CRISPR/Cas9 system has limitations, the most serious one of which is off-target. Off-target means the system may knockout genes in normal cells besides the target sequence and cause serious side-effect. Because of the simple principle of the CRISPR/Cas9 system, it has a lower specificity in cleavage of the targeted sequence compared to ZFN and TALEN. The extensive genome-wide scanning of mutations at all sites with similar homology is necessary, but it will be considered as a heavy workload.
Recently, it has been reported that a long non-coding RNA (lncRNA) named SAMMSON (Survival Associated Mitochondrial Melanoma Specific Oncogenic Non-Coding RNA), a target of the lineage-specific transcription factor SOX10, was found in more than 90% human melanoma cells. It is proved to be required for melanoma growth and survival (6).
In order to reduce the harm of off-target, we designed a synthetic device, which specifically binds to SAMMSON and allowed the CRISPR/Cas9 system to be expressed only in melanoma cells.