SHERLOCK: CRISPR-Cas13 based diagnostic test to detect COVID-19 (Part 6)
Welcome to the 6th part of the 12-part series on CRISPR-Cas system based diagnosis of COVID-19.
On 6th May 2020, Massachusetts-based biotech firm Sherlock Biosciences with its “Sherlock CRISPR diagnostic kit,” became the first company that received the FDA approval for the CRISPR-based COVID-19 diagnostic test. SHERLOCK is an acronym for “Specific High-sensitivity Enzymatic Reporter unLOCKing. This kit uses the Cas13 enzyme, which targets the RNA. For the diagnosis, a guide RNA (gRNA) is designed that recognizes a specific RNA sequence found in the SARS-CoV-2 genome. Cas13 and the gRNA then form a complex and search for the sequence match in the RNA of the SARS-CoV-2. When the gRNA binds to the programmed sequence of the target RNA, the Cas13 enzyme cuts the target SARS-CoV-2 RNA. After cleaving the target RNA, Cas13 doesn’t get inactivated; instead, HEPN motifs of Cas13 get further activated to cut the surrounding unrelated single-stranded RNA reporter molecules that may be nearby in the reaction solution.
In the test, the cleavage of these single-stranded RNA reporter molecules resulting from the collateral activity of the Cas13 enzyme is determined.
Mechanism of CRISPR-Cas13 based COVID-19 diagnostic test
Diagnosing COVID-19 requires taking a sample from the patient. Sample taken is usually a nasopharyngeal or oral pharyngeal swab. To execute CRISPR-based diagnosis, the viral RNA is extracted from the patient’s sample. The extracted RNA may contain other viral, bacterial, or patient’s own RNA as well. Thus, to increase the test’s sensitivity, the SARS-CoV-2 RNA is amplified with the Reverse Transcription- Recombinase Polymerase Amplification process, abbreviated as RT-RPA.
In the first step, the reverse transcriptase enzyme converts the SARS-CoV-2 RNA into complementary DNA, referred to as cDNA. The cDNA is then amplified by the RPA process using primers specific to the SARS-CoV-2 genes, for example, S gene, Orf1ab gene, N gene, etc (Fig 1).
Recombinase Polymerase Amplification (RPA) process
The Recombinase Polymerase Amplification is an isothermal nucleic acid amplification. Unlike polymerase chain reaction PCR, the RPA reaction occurs at a single temperature, so there’s no need for a thermocycler, making the RPA an excellent candidate for developing low-cost, rapid, point-of-care diagnosis (POC). And is ideally suited to fields and other settings with minimal resources for diagnosing infectious diseases like COVID-19, food contaminations, etc. RPA is as specific as PCR amplification but is much, much faster. Results are typically generated within 3–10 minutes.
The RPA process occurs by three enzymes: recombinase, single-stranded DNA binding proteins (SSB), and strand displacing polymerase (Fig 2).
· In traditional PCR, the denaturation step in which double-stranded template DNA is separated into two single strands is performed at 94°C. But in RPA, this step is substituted by the two enzymes: recombinase and single-stranded DNA binding proteins. The recombinase enzymes form complexes with the oligonucleotide primers, then these complexes scan the ds viral cDNA target, searching for the homologous sequences. And once the homologous sequences are found, the recombinase primer complexes invade the dsDNA, causing the separation of DNA strands.
· When the primers are paired to their complementary sequences, the single-stranded DNA binding proteins bind to the exposed DNA strand to stabilize it. The local separation of these DNA strands forms a D-loop structure.
· Finally, the strand displacing DNA polymerase enzyme extends the primer, eventually generating the amplicons from the original strands of template DNA. These newly generated DNA strands are then used for another round of RPA for exponential viral cDNA amplification.
Typically, the RPA reactions are executed at a single temperature ranging from 37°C-42°C. At optimal temperature, the reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 3–10 minutes. No other sample manipulation, such as thermal or chemical melting, is required to initiate amplification.
Cas13 collateral activity
After the amplification of SARS-CoV-2 cDNA is done, it is then transcribed into RNA because the kit uses detection by Cas-13 enzyme, which targets the RNA. This amplified RNA is then mixed with the Cas13 enzyme, guide RNA, and single-stranded RNA reporter molecules in a reaction tube. The guide RNA matches a specific RNA sequence found in the SARS-CoV-2 genome.
Each of the single-stranded RNA reporter molecules is coupled with the fluorescent FAM molecule at one end and a biotin molecule at the other end. When the reporter molecules are intact, biotin acts as a quencher, which suppresses the fluorescence emitted by the FAM molecule (Fig 3).
Imagine, if the patient sample contains the SARS-CoV-2 virus, then the Cas13 cleaves the viral RNA and shows collateral activity. Because of collateral activity, Cas13 cleaves the surrounding FAM-biotinylated ssRNA reporter molecules, leading to the separation of the FAM molecules from the biotin molecules (Fig 4a). When excited with light, the freed fluorescent FAM molecules produce a quantifiable fluorescence that can be detected by a detector, indicating the presence of SARS-CoV-2 RNA in the sample (Fig 4b).
Lateral Flow Assay
Visualization of Cas13 activity can also be achieved using lateral flow strips designed to capture labeled nucleic acids. The interpretation of lateral flow strips is very intuitive and easy. Similar to a pregnancy test, the strip changes color if the virus is detected (Fig 5).
Typically, lateral flow test strips are composed of a sample pad, a conjugate pad, a testing area, and an absorbent pad (Fig 6).
· The sample pad is where the test strip receives samples in the form of liquid drops.
· The absorbent pad generates a suction force, pulling the sample from the sample pad towards the conjugate pad and then to the test area.
· The conjugate pad is covered with a large number of colloidal gold particles, conjugated with anti-FAM antibodies.
· The testing area is marked with the letters T and C. T stands for the test area, and C stands for the control area. The generation of colored lines on T or C areas indicates whether the result is positive or negative. In the control area, the streptavidin is immobilized, and in the test area, the antibodies specific for the anti-FAM antibodies are immobilized.
Negative Test: Take the scenario where the sample is negative, which means SARS-CoV-2 is not present in the patient’s sample. In this case, the Cas13 enzyme remains inactive, and thus no collateral cleavage of the FAM-biotinylated ssRNA reporter molecules occurs. When the sample containing the intact reporter molecule is loaded on the sample pad at one end of the strip, it flows to the conjugate pad. The conjugate pad is covered with a large number of colloidal gold particles, conjugated with anti-FAM antibodies. Thus, on reaching the conjugate pad, the intact reporter molecules by their FAM labels get attached to the anti-FAM antibodies conjugated to the gold particles.
The colloidal gold molecules are then pulled into the control © area by the suction force generated by the absorbent pad. When the gold particles reach the C area, the attached intact reporter molecules accumulate at the C area by the interaction of the biotin labels with immobilized streptavidin. The interaction leads to the appearance of a visible colored line in the C area (Fig 7). The bound gold particles are unable to move further into the test area. Thus, a single-colored line appears in the C area on the strip, indicating that the test is negative.
Positive Test: On the other hand, if the patient sample contains SARS-CoV-2, then the Cas13 cleaves the viral RNA and shows collateral activity. Because of the collateral activity, Cas13 cleaves the FAM-biotinylated ssRNA reporter molecules, leading to the separation of biotin molecules and FAM molecules. Therefore, when the sample containing the cleaved reporter molecules is loaded on the sample pad at one end of the strip, it flows to the conjugate pad, where the separated FAM labels get attached to the anti-FAM antibodies conjugated to the gold particles.
The bound colloidal gold molecules and unbound biotin label molecules are then pulled into the control area by the suction force generated by the absorbent pad. When they reach the C area, the biotin labels bind to the C area by interacting with the immobilized streptavidin. The interaction leads to colored line formation in the C area (Fig 8). On the other hand, the FAM label bound colloidal particles are not captured on the C area. Therefore, they continue to move to the next location, the test (T) area of the lateral flow strip containing the anti-rabbit antibody, which binds anti-FAM antibodies. This generates a second visible colored line in the T area.
Thus, the appearance of 2 lines in the Test (T) and Control (C) area indicates a positive test; in other words, the patient is infected with the SARS-CoV-2. On the other hand, the appearance of a single colored line in the C area of the lateral flow strip indicates a negative test; in other words, the patient is not infected with SARS-CoV-2 (Fig 9).
The Sherlock CRISPR-Cas13 based diagnostic test produces results within an hour.
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