Diagnosing COVID-19 with CRISPR and cellphones
A novel way of detecting the novel coronavirus disease (COVID-19) uses the clustered regularly interspaced short palindromic repeats (CRISPR) technology to sidestep the need for RNA amplification and to shorten test turnaround times.
Inserting RNA sequences complementary to the N and E regions of the SARS-CoV-2 genome in between the CRISPR repeats creates a highly specific targeting mechanism. The present diagnostic system couples this with the functionality of the Cas13 protein, a naturally occurring component of bacterial immunity.
When the CRISPR RNA (crRNA) binds to its opposite sequence, Cas13 is activated, unleashing a large-scale RNAse activity, breaking apart any surrounding single-stranded RNAs (ssRNA). The current assay also uses an ssRNA-linked fluorophore-quencher detection system that is triggered by Cas13 activity.
“This assay, unlike previous CRISPR diagnostics, does not require preamplification of the viral genome for detection. By directly detecting the viral RNA without additional manipulations, the test yields quantitative RNA measurements rather than simply a positive or negative result,” the researchers said.
Quantitative and accurate
In developing the novel CRISPR assay, choosing the best Cas13 and crRNAs was crucial to maximize the assay accuracy without needing to amplify the SARS-CoV-2 RNA beforehand. Ten initial crRNA candidates were able to detect target RNA at a concentration of 480 fM, displaying reactivity signals stronger than the no-target controls.
Of these crRNAs, two (crRNAs 2 and 4) were chosen to proceed with the subsequent experiments and were complexed with a Cas13 homologue from Leptotrichia buccalis. When both systems were tested individually on serial dilutions of the target RNA, signals were detected at RNA concentrations as low as around 1,000 copies/µL, beyond which no difference could be observed relative to controls. [Cell 2020;doi:10.1016/j.cell.2020.12.001]
Moreover, the observed rate of Cas13 activity scaled proportionally with the set concentration of target RNA, confirming that the assay was quantitative.
Two crRNAs were the combined, each targeting a different site on the viral genome, to boost the sensitivity of the assay.
“In theory, a single target RNA could activate multiple Cas13a ribonucleoprotein complexes (RNPs) if each RNP is directed to different regions of the same viral target RNA, effectively doubling the active enzyme concentration,” the researchers said. “Targeting multiple sites is especially beneficial in cases where target RNA is the limiting reagent,” as in instances where amplification is absent.”
The theory played out well in practice: When crRNAs 2 and 4 were combined, the slope of detection jumped from 213±1.6 and 159±1.7 AU/min, respectively, to 383±3.0 AU/min combined. When the assay was performed on SARS-CoV-2 RNA from infected cell cultures, the limit of detection improved to 270 viral copies/µL.
The combination of crRNAs likewise remained strongly specific, displaying no signal above the control when tested on other respiratory viruses, including alpha- and betacoronaviruses, and the Middle East respiratory syndrome coronavirus, as well as influenza.
Rapid and reliable
Before testing the assay out on patient samples, the researchers added crRNA 21 to improve performance and to help screen out potential noise from the swab samples.
Five RNA samples from de-identified nasal swabs were used for the analysis. All source patients were positively diagnosed with COVID-19, and clinical analysis yielded cycle threshold values of 14.37–22.13, indicating copy numbers ranging from 2.08×107–1.27×105 copies/µL.
The CRISPR assay successfully identified all test samples as positive for viral RNA, with calculated copy numbers ranging from 3.2×105–1.65×103 copies/µL. When tested on samples who had been determined as COVID-19-negative, the current assay returned no signal above the control.
Notably, a mobile phone-based microscopy device could also be used to detect the fluorescence signal with good accuracy.
Testing on five RNA samples from COVID-19-positive patients, the researchers saw that the mobile phone microscope could identify all swabs as positive within the first 5 minutes of measurement. This showed that “the device can provide a very fast turnaround time of results for patients with clinically relevant viral loads.”
“This result highlights the inherent trade-off between sensitivity and time in the Cas13a direct detection assay,” they said. “High viral loads can be detected very rapidly because their high signals can be quickly determined to be above the control, and low viral loads can be detected at longer times once their signal can be distinguished above the control.”