Team:NTU-Singapore/Description
Cancer Diagnostic
Our initial project aim was to develop a cancer diagnostic assay, specifically colon cancer as colon cancer is one of the most common cancers in Singapore. During the 2014 - 2018 period, the National Cancer Center Singapore stated that 16.9% of males diagnosed with cancer suffered from colorectal cancer while 13.3% of females diagnosed with cancer suffered from colorectal cancer[1]. This alarming statistic made our team interested in developing a diagnostic assay for colorectal cancer as early detection can save the patient’s life.
One occurring metastatic colon cancer (mCRC) is caused by the mutation on the BRAF gene, in which the amino acid Valine (V) is changed to Glutamic Acid (E) at the 600th position in the gene. By using the CRISPR-Cas12a system, we aim to detect this mutation on the BRAF gene that causes mCRC. Cas12a is a single RNA-guided endonuclease that creates a double strand break (DSB) at the target DNA whose sequences are complementary to the guide RNA (gRNA) sequences of the CRISPR-Cas12a. The Cas12a homolog that we will be using is LbCas12a as it was found to have CRISPR-Cas12a high sensitivity with mutations. By designing a gRNA complementary to the mutated BRAF V600E gene, the LbCas12a will be able to detect the mutation of the BRAF gene.
One occurring metastatic colon cancer (mCRC) is caused by the mutation on the BRAF gene, in which the amino acid Valine (V) is changed to Glutamic Acid (E) at the 600th position in the gene. By using the CRISPR-Cas12a system, we aim to detect this mutation on the BRAF gene that causes mCRC. Cas12a is a single RNA-guided endonuclease that creates a double strand break (DSB) at the target DNA whose sequences are complementary to the guide RNA (gRNA) sequences of the CRISPR-Cas12a. The Cas12a homolog that we will be using is LbCas12a as it was found to have CRISPR-Cas12a high sensitivity with mutations. By designing a gRNA complementary to the mutated BRAF V600E gene, the LbCas12a will be able to detect the mutation of the BRAF gene.
How COVID-19 Affected Our Project
As the COVID-19 pandemic hit Singapore, our team felt that this virus is a more urgent matter as it is extremely contagious. To curb the spread of the virus, we shifted the aim of the project to develop a COVID-19 diagnostic assay instead. We realised that CRISPR-Cas enzymes are not efficient in detecting multiple variants as more than one Cas enzyme is required in the single reaction. We decided to design a probe that removes this restriction on multiplexing. By using Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) together with a probe, we are able to detect the presence of the virus in an RNA sample by measuring the fluorescence emitted by the sample.
Our end goal: Diagnostic Assay
After optimising the reaction in the biological lab, we intend to develop a portable diagnostic assay that can be operated by everyone. We plan to build a circuit that will heat the sample at 65°C and have a LED to stimulate fluorescence emission. With our diagnostic assay, we hope that it will help in early detection of the virus and allow patients to be isolated in order to prevent the spread, flattening the curve. Patients will also be able to undergo treatment earlier which will allow them to recover without any health complications.
Reference
- “Cancer statistics In Singapore,” Cancer Statistics. [Online]. Available: https://www.nccs.com.sg/patient-care/cancer-types/cancer-statistics. [Accessed: 21-Oct-2021].