Direct and sensitive detection of SARS-CoV-2 through a Cas-13a-based assay

Last Wednesday the CRISPR/Cas-based genome editing technique was awarded with the Nobel Prize in Chemistry 2020. Although surrounded by some controversy in the field of gene editing, the excellent research and applications that Emmanuelle Charpentier and Jennifer Doudna have developed by adapting the CRISPR/Cas system to basic and translational research have been rewarded. At the end of September, Doudna together with Daniel A. Fletcher and Melanie Ott published a preprint in medRxiv [1] in which they took CRISPR/Cas technology one step further in the diagnosis of SARS-CoV-2.

The quantitative reverse transcription polymerase chain reaction (RT-qPCR) is well established and widely used as the gold-standard diagnostic method for SARS-CoV-2 infection, with an analytical limit of detection (LOD) of 1 viral copy/µl [2]. Recent modeling of viral dynamics suggests that frequent testing with a fast turn-around time is required to break the current pandemic, and that an LOD of 100 copies/µl would be sufficient for screening [3]. It has been shown that when the viral load drops below 1,000 copies/µl, few infectious particles are detected and the risk of transmission is low [4, 5]. The high sensitivity of RT-qPCR may pick up RNAs freed by infected and dead cells after infectious particles have waned, therefore identifying individuals that are SARS-CoV-2 RNA-positives but no longer infectious. 

Schematic of two different RNPs binding to different locations of the same SARS-CoV-2 RNA, leading to cleavage of the RNA reporter and increased fluorescence. Fozouni, P. et al. Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone. medRxiv 2020.09.28.20201947; doi: 10.1101/2020.09.28.20201947

The need for a rapid, widespread test able to identify infectious individuals led to the development of viral RNA detection systems based on CRISPR technology. In their recent preprint, Doudna et al. adapted the Cas13a nuclease, which targets single-stranded (ss)RNA substrates when complexed with a CRISPR RNA (crRNA) containing a programmable spacer sequence to form a ribonucleoprotein complex (RNP). When the RNP binds to its target ssRNA sequence, the Cas13a becomes active and cleavages any surrounding ssRNA in a process called collateral activity. Target RNA binding and subsequent Cas13a cleavage activity can therefore be detected with a fluorophore-quencher pair linked by an ssRNA, which will fluoresce after cleavage by the Cas13a. 

Current CRISPR diagnostic strategies rely on pre-amplification of target RNA for subsequent detection by the Cas protein, thus achieving high sensitivity. As we previously explain in a MolecularCloud article about the SHERLOCK diagnostic strategy, RNA sensing entails the conversion of RNA to DNA by RT, DNA-based amplification, and transcription back to RNA for detection by the Cas13. This method was recently adapted for SARS-CoV-2 detection [6], in a procedure that takes approximately an hour to complete and that can be read out with paper based lateral flow strips. 

The new CRISPR-Cas13a-based diagnostic method has been developed for the direct detection of SARS-CoV-2 RNA, therefore not requiring pre-amplification of the viral genome for detection and thus offering a promising option for rapid, point-of-care testing. There are several key features of this technique that make it stand out from previous detection systems. The combination of several crRNAs targeting different sequences of SARS-CoV-2 RNA can be used to detect viral target RNA in the attomolar range or as few as ~30 copies/µl. Combining three crRNA in the same assay, the method is able to detect SARS-CoV-2 viral RNA of ~100 copies/µl within 30 minutes. Furthermore, multiple crRNAs targeting different parts of the genome also safeguards against a potential loss of detection due to naturally occurring viral mutations. 

Another advantage is the ability of the diagnostic platform to directly translate the fluorescent signal into viral loads. By avoiding pre-amplification and employing direct detection, the reaction rate directly correlates with viral copy number and may be used for quantification. Therefore, the patient’s course of infection can be monitored and one can determine if the infection is increasing or decreasing, because in symptomatic cases viral loads follow the course of the infection [5]. Monitoring viral loads quantitatively will allow the estimation of the infection stage and will help predict infectivity, recovery and return from quarantine in real time. 

Schematic of mobile phone-based microscope for fluorescence detection. Fozouni, P. et al. Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone. medRxiv 2020.09.28.20201947; doi: 10.1101/2020.09.28.20201947.

To perform quantitative RNA measurements, the diagnostic method incorporates a mobile phone camera and a compact device that includes a low-cost laser illumination and collection optics. This device was an order of magnitude more sensitive than a plate reader due to reduced measurement noise and the ability to collect more time points, enabling ~100 copies/µl sensitivity in 30 minutes and accurate diagnosis of a set of patient samples in 5 minutes, also making the assay portable. The choice of a mobile phone as the basis of the detection system was motivated by the high sensitivity of current mobile phone cameras, the simplicity of integrating a mobile phone for detection, their robustness and cost-effectiveness, and the fact that they are widely available. 

One major advantage of CRISPR diagnostics is that they can be highly specific. Doudna et al. confirmed the specificity of their method by testing it against a set of other respiratory viruses such as alphacoronavirus HCoV-NL63, betacoronavirus HCoV-OC43, Middle East respiratory syndrome coronavirus (MERS-CoV), H1N1 Influenza A and Influenza B or with RNA extracted from primary human airway organoids, detecting no signal above RNP background for any of the viral or human RNAs tested. 

If scaled up, this diagnostic method could fulfill the need for a test that provides rapid results and is available to be administered frequently. Nevertheless, we need to take into account that this is a pre-print article and must be validated in a peer-reviewed journal before being proposed for clinical application. This is another huge step for the development of CRISPR-based diagnostic methods, and I am sure that we will see the development of other direct-detection strategies with multiple Cas proteins in the near future.

References

1. Fozouni, P., et al., Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone. 2020: p. 2020.09.28.20201947.

2. Vogels, C.B.F., et al., Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat Microbiol, 2020. 5(10): p. 1299-1305.

3. Larremore, D.B., et al., Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance. 2020: p. 2020.06.22.20136309.

4. La Scola, B., et al., Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 2020. 39(6): p. 1059-1061.

5. Wolfel, R., et al., Virological assessment of hospitalized patients with COVID-2019. Nature, 2020. 581(7809): p. 465-469.

6. Joung, J., et al., Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv : the preprint server for health sciences, 2020: p. 2020.05.04.20091231.

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