CRISPR-Based Approaches for Efficient and Accurate Detection of SARS-CoV-2

Wancun Zhang, PhD; Kangbo Liu, MD; Pin Zhang, MD; Weyland Cheng, PhD; Linfei Li, MD; Fan Zhang, MD; Zhidan Yu, PhD; Lifeng Li, PhD; Xianwei Zhang, PhD

Disclosures

Lab Med. 2021;52(2):116-121. 

In This Article

Visualization and Portable Onsite Detection of SARS-CoV-2

Reducing the global infectivity of SARS-CoV-2 requires efficient and accurate nucleic acid diagnostic tools. However, the typical detection time for screening and diagnosing patients with suspected SARS-CoV-2 has been >24 hours, given the need to ship specimens overnight to designated laboratories. In addition, testing typically relies on expensive equipment and well-trained personnel, all of which is not conducive to the rapid control of the epidemic.[25–27] In such a backdrop, any development toward ultrasensitive, cheaper, and portable diagnostic tests for the assessment of suspected infection, regardless of the presence of qualified personnel or sophisticated equipment for virus detection, could help advance the diagnosis of COVID-19.

Isothermal amplification methods, such as recombinase polymerase amplification[28] and loop-mediated isothermal amplification (LAMP),[29] have been developed as attractive alternatives to the conventional PCR method because of their simplicity, rapidity, and low cost. However, there is still a challenge to develop these methods into a reliable point-of-care diagnostic for clinical applications because of nonspecific signals.[30,31] Notably, whereas CRISPR is a biotechnological technique well-known for its use in gene editing, it has been recently used for the in vitro detection of nucleic acids, thereby emerging as a powerful and precise tool for molecular diagnosis.[32–34]

Lucia et al[26] developed a Cas12-based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. More important, the Cas12-based diagnostic tool can provide visualization of the results. Ding et al[35] developed the All-In-One Dual Cas12a (AIOD-CRISPR) assay for simple, rapid, ultrasensitive, one-step approach for visual detection of SARS-CoV-2. In the AIOD-CRISPR assay, a crRNA pair is introduced to initiate dual Cas12a detection, improving the detection of SARS-CoV-2 nucleic acids (DNA and RNA) with a sensitivity of few copies. Therefore, the AIOD-CRISPR assay has potential for the development of next-generation point-of-care molecular diagnostics.

Joung et al developed a simple chemical test that is suitable for point-of-care use in detecting SARS-CoV-2 in 1 hour, called STOPCovid (SHERLOCK Testing in One Pot). This simplified test, STOPCovid, provides a sensitivity comparable to RT-PCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns results in 70 minutes. Using fluorescence readout, the test returns results in 40 minutes. Moreover, in their study, 12 positive and 5 negative results from nasopharyngeal swabs were detected by STOPCovid and by RT-PCR, meaning that STOPCovid and RT-PCR test results were consistent with each other. Thus, STOPCovid can significantly aid "test-trace-isolate" efforts, especially in low-resource settings, which is critical for long-term public health safety and for effectively reopening society.[27] Therefore, the CRISPR-based approach is critical for virus detection in regions that lack resources to use the currently available methods.

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