CRISPR based diagnostic tests use smartphone cameras to detect SARS-CoV-2 (Part 12)
Welcome to the 12th part of the 12-part series on CRISPR-Cas system based diagnosis of COVID-19.
Scientists at Gladstone Institutes and UC Berkeley have developed a CRISPR-based COVID-19 diagnostic test that can provide the test results with a smartphone’s help in 15–30 minutes. The test eliminates the need to convert RNA to DNA steps and then amplify the DNA segments, thus reducing the time needed to complete the analysis. The diagnostic test utilizes three guide RNAs CRISPR-Cas13 protein complexes, which bind and cleave viral RNA. CRISPR Cas13 proteins are “programmed” to recognize three different regions of SARS-CoV-2 viral RNA and then combined with reporter molecules that become fluorescent when cleaved. Cas13 proteins get activated upon binding of guide RNAs to SARS-CoV-2 RNA, and the activated Cas13 proteins then start to cleave the reporter molecules, leading to separation of fluorescent label from the quencher (Fig 1).
The mobile phone camera converted into a fluorescent microscope can detect the fluorescence emitted by the cleaved reporter molecules. Higher the fluorescence detected, higher is the number of virus particles in the sample and vice versa.
Besides rapid testing, the high sensitivity of mobile phone cameras, along with the connectivity, GPS, and data-processing capabilities of smartphone, has the potential to make the CRISPR-based test portable and an attractive tool for point-of-care disease (POC) diagnosis in low-resource regions.
Additionally, the test can detect tiny amounts of virus, in under 30 minutes, and much larger concentrations of the virus, such as those in highly contagious people, in under 5 minutes.
How is a smartphone converted into a fluorescent microscope?
Fluorescence microscope is a type of microscope that works on the principle of fluorescence. A substance is said to be fluorescent when it absorbs the energy of invisible shorter wavelength radiation (such as UV light) and emits longer wavelength radiation of visible light (such as green or red light).
Higher energy and shorter wavelength of lights (UV rays or blue light) generated from mercury lamp passes through the excitation filter (Fig 2). The excitation filter allows only the short wavelength of light to pass through it and removes the non-specific wavelengths of light. The filtered light is then reflected by the dichroic filter and falls on the sample which is fluorophore-labeled. The fluorochrome absorbs shorter wavelength rays and emits rays of longer wavelength (lower energy) that passes through the emission filter. The emission filter blocks any residual excitation light and passes the desired longer emission wavelengths to the detector. Thus, the microscope forms glowing images of the fluorochrome-labeled reporter molecules against a dark background.
The smartphone is converted into a fluorescent microscope by placing a lens between the sample of interest and the cell-phone camera unit (Fig 3). The placed lens collects the fluorescent signal. Simple LEDs can be used to excite the fluorescent reporter molecules while a
simple plastic filter rejects the scattered excitation light, thereby creating a dark field background necessary for viewing fluorescent images.
Everyday the scientists are exploring new ways to develop more efficient, more sensitive, more accurate POC diagnostic kits and treatment methods to combat the SARS-CoV-2 pandemic.
Moreover, the CRISPR-based diagnostic kits that we have discussed in Parts 6–12 are not just for diagnosing SARS-CoV-2, but they can also be used to detect other infectious diseases. All needed is to design guide RNA specific to the genome of the organism that is required to be detected.
If you liked this article and want to know more about SARS-CoV-2 and how it can be detected with the CRISPR-Cas system, follow the below links:
