“Gene Scissors” in COVID-19 Diagnostics: Next Generation Tests Will Deliver the Result in 30 Minutes

OLYMPUS DIGITAL CAMERAWith the aggressive spread of Coronavirus around the globe, existing controls have proved to be ineffective. It takes some time to obtain the results of the polymerase chain reaction (PCR) tests and to detect SARS-CoV-2 infection, and the tests are then sometimes inaccurate. It is difficult and dangerous for personnel to take samples from the nasopharynx of potentially infected persons. In later stages of the disease, it is difficult to detect the virus in the upper respiratory tract.

Therefore, the world is in a rush to find alternative technologies to improve rapid PCR testing. One such technology is CRISPR endonucleases – the “gene scissors”. Using this and other innovative technologies Lithuanian scientists at the Life Sciences Centre of Vilnius University and other scientific institutions of the country are developing COVID-19 tests suitable for examining saliva samples.

Current tests are not perfect

Today, COVID-19 disease is tested using the so-called gold standard quantitative reverse- transcription polymerase chain reaction (RT-PCR) test. It detects at least two SARS-CoV-2 gene targets in the tested sample. The sequence of the virus is determined in a sample during the laboratory RT-PCR test and the quality of the sampling is assessed. Using in vitro diagnostic (IVD) kits, high diagnostic sensitivity and specificity – at 95% and 99% respectively – is achieved. The analytical sensitivity is also high – from 5 to 100 virus copies per microliter of the specific sample for analysis is sufficient for testing. Emergency hospitals cannot do without rapid RT-PCR tests. This test requires a 10- to 100-fold increase in viral RNA, but the sensitivity and specificity of the method is sufficiently high. The method works in a closed system and requires certain equipment which is not always available.

Rather than examining genetic virus material, antigen tests examine virus proteins which cannot be amplified prior to detection. As a result, the analytical sensitivity of the method is also different. The antigen test requires thousands more viruses in the sample. This test is only effective in the active phase of infection and is not suitable for the diagnosis of COVID-19 without confirmation of the findings using RT-PCR. As a result, it is not recommended as the main testing method, but is more suitable for mass testing in high-risk zones (e.g. airports).

Antibody tests look for specific proteins made in response to infection. IgM and IgA classes of antibodies indicate active infection and IgG antibodies indicate a long-term immune response. Unfortunately, antibody tests can only be used in instances in which viral testing is delayed and their informative value is low. Antibody tests allow detection of lower respiratory SARS-CoV-2 infection characteristic in subsequent stages of the disease, when the nasopharyngeal sample is no longer informative, but is best suited to confirm infection which has remained asymptomatic.

Next generation tests are super rapid

The variety of tests does not solve the main challenge of the pandemic, which is to quickly and reliably identify cases of infection to stop spreading of the disease and to provide the necessary treatment. It is clear that a rapid technological breakthrough in COVID-19 diagnosis is required during the pandemic.

Next generation tests for use in the home, hospital or nursing settings are being developed in the laboratories. No additional personnel are required to collect the sample or evaluate the result. You simply carry the diagnostic devices in your pocket, need a small amount of test sample, a few reagents, and the results can be obtained in 30 minutes and are simply displayed on the screen of the device itself or on a mobile phone. Usually easily accessible biological fluids or other samples, such as saliva, blood, urine, oral mucous, are used for such testing.

“Gene scissors” in COVID-19 diagnostics

Alternative technologies are being developed both in Lithuania and elsewhere for the development of rapid PCR tests. One of these technologies is the adaptation of “gene scissors” – the CRISPR endonucleases. The original CRISPR-Cas system is a bacterial molecular mechanism designed to protect against viruses. The system not only destroys viral nucleic acid, but also builds the acquired immunity, so the bacterium will no longer be infected with the same virus a second time.

CRISPR sequences were accidently identified more than three decades ago, but exploration of the system became more widespread in recent years. It was adapted for treating diseases by means of genetic editing. Over the past five years, HIV was nipped out of the genome in mice and the genome in disease-carrying mosquitoes was suppressed with the aim of preventing the spread of malaria; treatment of muscle dystrophy, cystic fibrosis, Huntington’s disease, cancer and many other diseases have been explored. This year, the interest in CRISPR-Cas reached its highest point when the system was first used to edit the genome directly in the human body, and was crowned with the Nobel Prize for its broad and significant application.

The CRISRR-Cas system operates on the principle of endonuclease selectivity – the activity of the enzyme depends on the sequence in the complex, which is complementary to the target fragment. In case of a match, a cut in the reporter sequence is made thereby releasing the molecule causing the emission of a colour signal or any other reaction demonstrating a positive result. It is not surprising that this technology has also been applied to identify COVID-19.

Prototypes for rapid COVID-19 detection

CRISPR-Cas9 is the oldest used system that cuts its target. In case of COVID-19 testing, where the result is positive a specific cut would be made and the result would be displayed as two bands. However, this method is not optimal because of the need for specific equipment to amplify virus RNA and to display the result.

The Cas13 protein recognizes RNA, so it can be used for SARS-CoV-2 detection directly, however the amount of virus RNA may be insufficient unless it is amplified for COVID-19 testing. In order to increase the sensitivity of the test, the virus RNA would be converted into DNA and then amplified by alternative PCR techniques that do not require specific equipment and save time (LAMP, RPA, etc.) and the result will be visible using a paper strip or similar detection systems. These technologies were developed prior to the COVID-19 pandemic for sensitive and rapid detection of other viral infections. Several CRISPR-based prototypes for rapid COVID-19 detection have already been developed – HUDSON, SHERLOCK, STOP, DETECTR, iSCAN, etc. Currently, such tests can analyse one sample at a time, which may take up to 30 minutes, and the diagnostic sensitivity and specificity of the tests is high.

Next generation sequencing is irreplaceable

Sequencing, a detailed identification of the gene sequence, is currently widely used for disease diagnosis. The next generation sequencing (NGS) is very common today and allows a full genome analysis to be performed. Unlike PCR or CRISPR-based tests, sequencing allows a new strain of the virus to be detected immediately. Diagnostic tests directed at the target may, unfortunately, give a false negative response if the target sequence changes, and therefore allows a new strain of virus to spread among the population. Viruses are one of the fastest mutating forms of life in the world, so in order to monitor the evolution of the virus and select specific sequences for accurate diagnostic methods, the sequencing technology remains irreplaceable.

Using NGS, SARS-CoV-2-induced zoonosis, evolution, and virulence have been extensively researched. A larger-scale NGS involves sequencing of human immune cells, which may also be performed in individual cells. Such research may help clarify immune response mechanisms, find the most informative biomarkers for diagnosis and targets for treatment. For example, patients with COVID-19 suffer from a reduction in the total number of white blood cells. The NGS data indicate that when the disease develops, the p53 protein response pathway and depletion of lymphocytes (apoptosis) are activated, which explains the patient-specific cell deficiency.

Complex testing to obtain valuable information

Antigen tests are cheap and rapid, because viral proteins, and also RNA, are detected in early phases of infection. Proteins are simply detected in the liquid biopsy samples, i.e. nasopharyngeal swabs or saliva, where the virus remains alive. At present, antigen tests are provided in small pregnancy test type containers. The sensitivity of antigen tests, like that of antibody tests, is several times lower than that of PCR tests, because the tested proteins are not amplified. The specificity is lower too, because the tests are prone to false positive results. The possibility of combined tests – to use one sample for viral RNA and viral proteins – would significantly increase the sensitivity and specificity of tests, reduce false positive and false negative test results, and help to better identify cases without symptoms.

Antibody tests are usually performed using blood samples, but other body fluids such as saliva may also be used. Such tests are not inferior to routine serological tests, are non-invasive, and do not require specialized personnel to take the sample. However, the immune system reaches far broader and deeper than the antiviral antibodies, for example, a distinctive secretory immune response is characteristic of coronavirus infection. It has been established that stage 3 of COVID-19 disease – hyperinflammation – is caused by the cytokine storm (IL-6, IL-1β, IL-10, TNF, etc.) caused by Th-17 cells. Where the immune response component is tested in addition to the RNA assay, the diagnosis can be verified and the course of the disease can be predicted. With higher levels of cytokines secreted by Th-17 cells a patient could be hospitalized to prevent the lethal outcome of the disease. A complex testing of viral antigens, antibodies and specific components of the immune system could provide valuable information not only about the infection, but also about the response of the organism to it.

A mask signalling that you have a disease

How about a protective mask that would signal that you are sick before you feel unwell? This is not fantasy, but an electrochemical system on paper, which detects hydrogen peroxide in the exhaled breath. To detect COVID-19, antibodies and colour signals could be placed on the mask to inform about the infection before you know about it.

Jennifer Anne Doudna, one of this year’s Nobel Prize winners in Chemistry, together with her colleagues, is developing a CRISPR-based coronavirus test with a read-out in a smartphone. The optical lenses of smartphone cameras are capable of detecting fluorescence generated by the test and display the result on the smartphone screen. The test is RNA amplification-free, the virus detection limit is 100 copies of viral genome per microliter and read-out in 30 minutes.

We can apply the saying that perfection lies in simplicity to the latest COVID-19 detection methods. Three in four people with COVID-19 experience partial or complete loss of sense of smell, so people who do not experience other symptoms can perform the u-Smell-it company’s test. In addition, specially trained dogs can detect a COVID-19 patient – this test method is used at Helsinki Airport.

It is said that what goes around comes around. The CRISPR system, a naturally occurring system that microbes use to fend off viruses, due to science achievements can be used for COVID-19 control. Who knows, maybe by uniting forces, scientists will adapt the CRISPR technology in the near future to quickly build the acquired immunity and effectively treat COVID-19.

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