In a recent study posted to Research Square*, researchers developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic technique for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) ribonucleic acid (RNA).
Although quantitative polymerase chain reaction (qPCR) tests are robust and considered the gold standard for detecting nucleic acids, it requires trained personnel, sophisticated equipment, efficient specimen handling, and long turnaround times.
While this may be acceptable for many other diagnostic applications, the coronavirus disease 2019 (COVID-19) pandemic has revealed a pressing need for developing diagnostics that are easily distributed, simple to perform, and rapid. While these factors are addressed by rapid antigen tests (RATs) and isothermal amplification methods, several limitations exist related to their sensitivity, specificity, or versatility. CRISPR RNA-guided diagnostics are nascent technologies that can address the current limitations.
The study and findings
In the present study, researchers developed a CRISPR-based diagnostic for sequence-specific capture and concentration of SARS-CoV-2 RNA from heterogeneous samples.
Type III CRISPR RNA-guided complexes such as Cmr and Csm bind to and cleave complementary single-stranded (ss) RNA. A mutant (D34A), ribonuclease (RNase)-dead type III-A CRISPR complex (Csm3) from Thermus thermophilus (TtCsmCsm3-D34A) was mixed and incubated with radioactively labeled (32Phosphorous or 32P) target and non-target RNAs to increase sensitivity and test whether it could concentrate sequence-specific RNAs. They observed that most (76±5.8%) of the 32P-labeled target RNA was captured by the complex, while the non-target RNA was concentrated in the supernatant. They also found that the type III CRISPR-based RNA concentration increased the levels of cyclic oligoadenylates, cA3 and cA4.
Further, another type III CRISPR system (TtCsm6), activated by cA4, was previously repurposed for real-time fluorescent readout for detecting viral RNA. It was speculated that the increased cA4 levels mediated by Csm-based RNA enrichment could boost the activity of TtCsm6, thereby improving the sensitivity. To this end, the nucleocapsid (N) gene RNA (target) of SARS-CoV-2 was titrated into the total RNA (non-target) isolated from HEK 293T cells for TtCsmCsm3-D34A-based concentration of target RNA and subsequently transferred to a reaction mix of TtCsm6 and fluorescent RNA reporter. The Csm-based enrichment of RNA increased the assay sensitivity by 100-folds, demonstrating that type III-A CRISPR complexes could capture sequence-specific RNAs, increase cyclic nucleotide levels, and improve the sensitivity of RNA detection.
The CRISPR-associated Rossman fold (CARF) domains in some Csm family proteins form homodimers and bind to cA4 or cA6, activating the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain. Still, in some Csm6 proteins, the CARF domain degrades the cyclic nucleotide inactivating the nuclease.
To overcome this limitation, they explored CARF-nucleases that do not degrade cA4. The team tested the nuclease activity of CRISPR ancillary nuclease 1 (Can1) from T. thermophilus (TtCan1) against plasmid deoxy-RNA (DNA) in the presence of five cyclic oligoadenylates and observed robust degradation with cA3 and manganese ions (Mn+2). Further investigation revealed that TtCan1 was a double-stranded (ds) deoxyribonuclease (DNase) without any sequence specificity when activated by cA3 and ssRNase if activated by cA4. Similarly, Can2 from Archaeoglobi archaeon JdFR-42 (AaCan2) was found to be a dsDNase in the presence of cA3 and Mn+2 and ssRNase in the presence of cA4 and Mn+2 or magnesium ions (Mg+2).
Next, the authors noted that both TtCan1 and AaCan2 cleaved the same synthetic RNA, but TtCan1-mediated cleavage required higher levels of cA4 and produced a weaker fluorescent signal than AaCan2. It was found that AaCan2 induced a similar fluorescent signal as TtCsm6 when activated by a 20-fold lesser amount of cA4. Therefore, coupling AaCan2 with the TtCsmCsm3-D34A increased the sensitivity of RNA detection.
Finally, nasopharyngeal swabs from 17 SARS-CoV-2-positive and six SARS-CoV-2-negative individuals were tested to see whether the TtCsm complex could capture the total extracted RNA. Samples with high levels of viral RNA, i.e., cycle threshold (Ct) > 17, were positive with the TtCsm-AaCan2 reaction. A 100-fold increase in sensitivity was observed when the Csm-based RNA captured method complemented it. Moreover, it was investigated whether the TtCsm complex could directly capture RNA from swabs without RNA extraction. The TtCsm complex detected RNA when swab samples were treated with Triton X-100 and egtazic acid (EGTA) as lysis buffers in a TtCsm6-based fluorometric assay. Lastly, this was tested with the TtCsm-AaCan2 assay, which detected 5 x 104 RNA copies per microliter.
To summarize, the researchers developed a technique to capture and concentrate sequence-specific RNA from unprocessed samples using the RNase-dead TtCsmCsm3-D34A complex. However, the sensitivity of this assay remains comparable to RAT and could not match that of qPCR diagnostic tests. More research is needed to apply the type III CRISPR systems to eliminate the RNA extraction step, optimize lysis buffers, and develop novel next-generation readout methods to boost sensitivity and shorten the time-to-result of the assay.
Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Source: News Medical