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
No comments:
Post a Comment