As described in the previous article, detection of SARS-CoV-2 genetic material can be done by both RT-PCR and CRISPR. Although the detection system using CRISPR to detect the SARS-CoV-2 is not yet available in the market, a comparison between the two CRISPR platforms (DETECTR from Mammoth Biosciences and SHERLOCK from Sherlock Biosciences) and the RT-PCR (represented by a protocol developed by CDC from the US), made by the Mammoth Biosciences, let us know more about the advantages of using CRISPR in virus detection.1
Basically, when comparing the CRISPR platform and RT-PCR, CRISPR has the advantages of shorter running time, no expensive/ special equipment needed, no highly skilled technical staff needed, and thus lower costs in general to run the test. However, the sensitivity of the CRISPR platform is not as good as that of RT-PCR. The lowest limit for detection by CRISPR is in the range of 10 to 70 copies/ul of virus in the sample, while only 3.6 to 10 copies/ul of virus in the sample is already enough to be detected by RT-PCR.
Neither of the CRISPR platform for virus RNA detection have yet been approved by the FDA in the US or by any other country, however I believe that this detection system will become more widely used than the RT-PCR in the near future. The CRISPR detection system would be particularly useful in countries whose resources are very limited, which lack instruments and technical staff to run RT-PCR.
However, one thing that we do need to be aware of is the possibility of the off-target effect generated from the two exonucleases, Cas12a and Cas13a, used in the CRISPR detection system. No study on this has been published so far. The off-target effect of Cas9 in the gene editing system generates unexpected functions of a gene and may result in genomic instability.2,3 The off-target effect generated from the activity of Cas12a/Cas13a in the CRISPR detection system can result in an incorrect recognition of the nucleotide target, and generates a false positive result. A thorough study of the incident rate of the off-target effect in Cas12a/Cas13a system, and finding out a mechanism to decrease the off-target effect, would give us more assurance of the usability of CRISPR in genetic material detection.
References
1. “A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR” 2nd March, 2020. https://mammoth.bio/wp-content/uploads/2020/03/Mammoth-Biosciences-A-protocol-for-rapid-detection-of-SARS-CoV-2-using-CRISPR-diagnostics-DETECTR.pdf
2. Yanfang Fu, Jennifer A Foden, Cyd Khayter, et al. “High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells” Nat Biotechnol. 2013 Sep; 31(9):822-826.
3. Seung Woo Cho, Sojung Kim, Yongsub Kim, et al. “Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases” Genome Res. 2014 Jan; 24(1): 132–141.
Coronavirus (3): CRISPR as an alternative to PCR technology in detecting SARS-CoV-2
Until now, RT-PCR is the most commonly used method to detect the nucleic acid strand of SARS-CoV-2. With the use of an automatic machine with a robot arm which pipettes the solution and mixes the liquids on a number of tests simultaneously, in general a real time RT-PCR test can be done in 2 hours.
However, new assays based on CRISPR (clustered regularly interspaced short palindromic repeats) technology, which reduces the assay reaction time to 30 to 60 minutes for nucleic acid detection, are being developed.1,2 Two biotech companies, Sherlock Biosciences (based in Cambridge, Massachusetts) and Mammoth Biosciences (based in California), that exploit CRISPR as diagnostic tools have simplified the tests so that each test can be done in one tube without any specialized or expansive equipment. Only test kit, two simple thermometer heat blocks, the sample, and basic laboratory equipment such as pipettes and pipette tips are needed for a test process.
The CRISPR nucleic acid detection tests developed by the two companies also made use of three other technologies: isothermal nucleic acid chain reaction expansion system, quenched fluorescent RNA reporter system, and the lateral flow assay. Isothermal nucleic acid chain reaction amplifies nucleic acid sequence from the sample for detection, and thus enhances sensitivity of the test to the attomolar level, that is, only around 100,000 copies are needed in the original sample for reliable detection (18 orders of magnitude smaller than a mole). CRISPR-Cas is used to identify a target sequence and to initiate the reporter system, which in turn indicates the presence of target SARS-CoV-2 sequences. Lateral flow assay is used for developing and showing the result on a strip of paper.
As the whole test does not involve extensive sample manipulation and expensive machinery, this makes the test field-deployable and available for point-of-care.
CRISPR-based lateral flow test kit.#
How CRISPR technology works
CRISPR technology was first applied in gene editing to fix genetic defects, to inactivate undesired genes, to insert new genes, or to study gene functions. For gene editing, CRISPR technology involves two components: Cas9 (CRISPR-associated 9) enzyme, which acts as molecular scissors (called endonuclease), and a custom-designed single-strand guide RNA, which has a sequence matching the target sequence of a gene to be edited. Guide RNA directs Cas9 to a target gene. By changing the guide RNA to match the target of interest, Cas9 can be programmed to efficiently identify and cut at a precise site on genomic DNA.
If the broken ends are left joined by the cell’s cellular repair process, a non homologous end joining (NHEJ) repair will take place. However, the NHEJ repair system is prone to mistakes and can lead to extra or missing bases in the joins. This often results in the inactivation of a gene. On the other hand, by introducing a separate sequence of a template DNA to the CRISPR cocktail, a cell can be induced to perform a different cellular DNA repair process called homology directed repair. The repair system use template DNA as blueprint to direct the rebuilding process. By addition of a template sequence, a defective gene is repaired, or a completely new gene is inserted.
Due to the ability of CRISPR to fix DNA errors, the technology has been used in the treatment of diseases due to genetic defects, such as Turner syndrome and sickle-cell anaemia. The following video provides clear explanation on how the CRISPR works and in which fields the CRISPR gene editing have been applied.
How CRISPR lets you edit DNA. By Andrea M. Henle
How can a CRISPR technology be used in the field of diagnosis
The CRISPR system is not only to be harnessed for gene editing, it can also be used in the field of diagnosis. With the efforts made by researchers working on identifying Cas molecules in bacterial science, different Cas endocucleases with different molecular properties have been identified in the past few years. Both Cas12a (Cpf1) and Cas13a (C2c2), which are found to possess both nucleic acid sequence recognition ability and dual cleavage activities, are being used in the detection of viral RNA or bacterial DNA genomes that cause disease. Cas12a recognizes a DNA sequence, while Cas13a recognizes an RNA sequence.
Cas12a and Cas13a perform specific binding and cleavage with the aid of guide RNA, which is complementary to the target sequence. This mechanism is similar to that used by Cas9. However, the two endonucleases have trans- or collateral cutting activity, which is activated upon target binding. This property is not possessed by Cas9. Once being activated by finding the target, the endocucleases will cut the target sequence and also indiscriminately cut other single-stranded nucleic acid sequences in the vicinity. The two distinct cleavage (cutting) activities of the endonucleases are used to leverage nucleic acid detection, as every endonuclease activation can lead to cleavage of thousands of reporter nucleic acids. This results in potent signal amplification.
Both biotech companies have published in detail how tests based on CRISPR technology were developed, and their detection protocols for SARS-CoV-2 have been released to the public. DETECTR (from Mammoth Biosciences) detects N gene and E gene from the SARS-CoV-2 genome, while SHERLOCKv2 (form Sherlock Biosciences) detects both S gene and Orf1ab. Basically, the detection systems from the two biotech companies involve 4 steps:
1. Extraction of nucleic acid from sample.
2. Amplification of nucleic acids from sample using isothermal amplification. Since isothermal amplification uses a single temperature, no expensive specialized instrumentation is needed to adjust temperatures over time, as would be needed if conventional PCR were in use.
3. Cas12a/Cas13a activation upon target recognition, and this mediates indiscriminate cleavage (random cutting) of reporter RNA: if the target sequence is present in the pool of amplified nucleotides, the non-specific cleavage (random cutting) activity of the Cas becomes activated. The nucleic acid reporters with quenched fluorescent molecules will be cleaved (cut), resulting in activation of the fluorophore. The fluorescent signal is thus an indicator to signal whether the target sequence is present in the test sample.
4. Visual colour readout using paper lateral flow strip, which captures the cleaved reporter RNA with labelled ends on specific antibody bands.
DETECTR have a separate step to extract the nucleic acid from a sample, and combined reactions in steps 2 and 3 into single tube. On the other hand, SHERLOCKv2 have steps 1 to 3 done in one tube by using the HUDSON method (Heating Unextracted Diagnostic Samples to Obliterate Nuclease), to release viral or bacterial nucleic acid from clinical specimens and to protect it from degradation. This bypasses the need for nucleic acid extraction.
The DETECTR platform enables detection that is 30 minutes faster than SHARLOCKv2. This is because the time spent on the additional in vitro transcription step, which is required for SHARLOCK platform, is saved.
Sherlock Biosciences have used synthetic SARS-CoV-2 virus RNA fragment for validation of the test. Currently, the company is collaborating with scientists from the Harvard School of Public Health in trialling the SARS-CoV-2 diagnostic test on patients.
#Picture adopted from "Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges, and opportunities. Biosensors and Bioelectronics, Volume 166, 15 October 2020, 112445"
References
1. “Coronavirus detection using CRISPR diagnostics” https://www.synthego.com/blog/crispr-coronavirus-detection
2. “How SARS-CoV-2 tests work and what’s next in COVID-19 Diagnostics. The Scientist, 3 March, 2020. https://www.the-scientist.com/news-opinion/how-sars-cov-2-tests-work-and-whats-next-in-covid-19-diagnostics-67210
Since the outbreak of COVID-19 in Wuhan, China last December, the disease has now spread to more than 70 countries worldwide. While much effort has been put into containing the disease by speeding up the testing for the severe acute respiratory syndrome-coronavirus-2 ( SARS-CoV-2), the news last week about the first test kits distributed by the United States CDC being found unable to produce consistent conclusive results from the negative control, is a bit bizarre .1
COVID-19 real time RT-PCR test kit by CDC of the United States.
This may arise your interest in knowing what test methods have been used by your own country for the newly emerged coronavirus. How do the tests work? How accurate are the test results from the test kits, and how the test results usually been validated and verified?
RT-PCR test for SARS-CoV-2
Until now, real time RT-PCR (reverse transcription polymerase chain reaction) technology has been the only method to detect the virus globally. PCR is an amplification method by which a targeted nucleotide sequence of an organism can be multiplied exponentially. By first converting the RNA sequence of the virus genome into complementary DNA and subsequently amplifying the target sequence using the complementary DNA as template, even a tiny amount of the virus genome in collection sample can be detected.
Overview of Reverse Transcription-Polymerase Chain Reaction
In early January, a genetic sequence of the newly emerged coronavirus was first released by China.2 The analysis of the genome structure revealed that SARS-CoV-2 has 79% identity with the SARS-CoV.3 The whole viral genome contains genes encoding non-structural polyprotein, S (Spike) protein, E (Envelope) protein, M (Membrane) and N (Nucleocapsid) protein (S, E and M proteins together form the viral envelope). Based on the viral genome data, researchers from different countries developed test kits by designing primers, short stretches of DNA, to amplify mainly S, N, and E gene regions of new virus.4 The nucleotide regions on the virus which encode the 3 proteins are less prone to mutation and are therefore usually picked for virus detection in RT-PCR test.
3D medical animation still shot showing 2019 novel Coronavirus Structure. From Scientific Animation: www.scientificanimations.com. Click for full-size image.
How reliable are tests using RT-PCR?
RT-PCR tests have been widely used in diagnosis of other viruses such as mumps, HIV, and influenza, and are normally highly reliable. In order to validate the results of each PCR experiment, negative control and positive control are included in every experiment for sample testing. Simply speaking, the positive signal in positive template control is an indication to show that the experiment works. The clear negative signal in the negative control is an indication of no contamination for the experiment. This is used to validate the positive test result of a sample in the same experiment.
For the new coronavirus, every country uses different criteria to make final diagnosis. These include the clinical observations and epidemiological data. If the RT-PCR result did not match with the clinical observation, in most cases RT-PCR will be repeated. Therefore, the chance of misdiagnosis is lower even the RT-PCR test result is wrong.
What are the possible causes of false-negative from RT-PCR test?
Although controls are used in RT-PCR test experiment to validate the positive results and to prove the experiment is working, false-negative from RT-PCR test is unavoidable. According to experience from China, the false-negative accounts for 3% of RT-PCR tests for COVID-19 patients.5
The false-negative from RT-PCR test may come from technical handling errors such as inappropriate specimen collection, storage, and transport. Viral RNA is very much prone to degradation with higher temperature. Therefore, once the sample is collected, it should be placed in a designated collection tube and be kept and transported at 4°C to 8°C. If the test is not going to be done in 24 hours upon sample collection, it should be kept frozen. In addition, the way the tests are being conducted may also cause problems. A dangle or a good rub could mean a big difference in the amount of virus material being collected.
Moreover, insufficient viral material in the specimen collected can also lead to false-negative results. A patient in the early state of infection will shed much less virus; tests taken at this stage have a higher chance of showing negative results. In addition, if the virus is drawn toward the lower respiratory tract for example, then a test from throat or nose swab may miss the virus.6,7 Samples collected from tracheal aspirate or sputum are alternatives for a highly suspicious patient with negative test result.
However, there is the possibility that the tests are accurate and the patients do not have coronavirus at the time of testing, according to Dr MacDermott of King’s College London. The early signs of coronavirus are very similar to other respiratory viruses. The patients may not be actually infected with the new coronavirus, therefore the test result is negative. But they can became infected and later test positive for the coronavirus.7
What we can learn so far from the countries worldwide in their handling of the outbreak of COVID-19?
Since the outbreak in South Korea in late February, the country has now managed to test more than 10,000 people a day for the newly emerged coronavirus, using kits provided by 4 local biotech companies, with sensitivity rates of over 95%. This powerful, fast testing ability is mostly attributed to its painful experience in handling the outbreak of Middle East Respiratory Syndrome (MERS) in 2015. Since then, the country has set up a system to allow rapid approval of testing kits for viruses that may cause pandemics.8 While the shortage of test kits in Japan and the US has jeopardized the containing of the virus in those countries, the effective collaboration system between the regulator and the local biotech companies in South Korea has provide a good example for countries worldwide in handling the outbreak of new disease.
The number of patients with COVID-19 is now surging in European countries over the last two weeks. Let us hope that these countries do not have bureaucratic processes that prevent them from providing a high capacity of tests that can quickly identify and treat COVID-19 patients.
3. Roujian Lu, Xiang Zhao, Juan Li et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020; 395: 565–74.
5. XingzhiXie, Zheng Zhong, and Wei Zhao et al. Chest CT for Typical 2019-nCoV Pneumonia: Relationship to Negative RT-PCR Testing. Radiology, Published Online:Feb 12 2020. https://doi.org/10.1148/radiol.2020200343
6. “What actually happens during a coronavirus test?” Arman Azad, CNN, 5 March 2020. https://web.archive.org/web/20200306034011/https://edition.cnn.com/2020/03/04/health/coronavirus-test-what-happens-explainer/index.html
7. “Are Coronavirus tests flawed?” James Gallagher. BBC news, 13 February 2020.
8. “Virus Testing Blitz Appears to Keep Korea Death Rate Low”. Heejin Kim, Sohee Kim, and Claire Che. Bloomberg, 4 March 2020.
