RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Abstract

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has attracted public attention. The gold standard for diagnosing COVID-19 is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, RT-qPCR can only be performed in centralized laboratories due to the requirement for advanced laboratory equipment and qualified workers. In the last decade, clustered regularly interspaced short palindromic repeats (CRISPR) technology has shown considerable promise in the development of rapid, highly sensitive, and specific molecular diagnostic methods that do not require complicated instrumentation. During the current COVID-19 pandemic, there has been growing interest in using CRISPR-based diagnostic techniques to develop rapid and accurate assays for detecting SARS-CoV-2. In this work, we review and summarize reverse-transcription loop-mediated isothermal amplification (RT-LAMP) CRISPR-based diagnostic techniques for detecting SARS-CoV-2.

Keywords: COVID-19; CRISPR; Cas12; Cas13; RT-LAMP; SARS-CoV-2.

Figure 1

Schematic illustration showing a two-pot reaction for the detection of SARS-CoV-2. The viral RNAs are amplified via RT-LAMP in the first pot. The amplicons are then used for the CRISPR-Cas12/13-based detection, followed by the visualization of the result through colorimetry or a fluorescence assay in the second pot.

Figure 2

Schematic illustration shows the one-pot reaction for detecting SARS-CoV-2. The viral RNA amplification via RT-LAMP, CRISPR-Cas12/13-based detection, and visualization of the results through the fluorescence assay, all performed in a single tube. Alternatively, the tube can be opened for a lateral flow assay.

Figure 3

Schematic diagram of RT-LAMP CRISPR-Cas12a/b-based detection of SARS-CoV-2. Samples (nasopharyngeal swabs) were collected from symptomatic and asymptomatic individuals, and viral RNAs were extracted. With the RT-LAMP step, viral RNAs are first converted into cDNAs, which are subsequently amplified. The amplicons were targeted in CRISPR-Cas-based detection, and the results of the tests were visualized via colorimetry or a fluorescence assay.

Figure 4

Schematic diagram of RT-LAMP CRISPR-Cas13-based detection of SARS-CoV-2. Samples (nasopharyngeal swabs) were collected from symptomatic and asymptomatic individuals, and viral RNAs were extracted. With the RT-LAMP step, viral RNAs are first converted into cDNAs, which are subsequently amplified. An additional step was required for Cas13-based detection: T7 transcription to convert DNA amplicons to RNA amplicons, which were then targeted in Cas13-based detection; the results of the test were visualized via colorimetry or a fluorescence assay.

Figure 5

Schematic diagram depicted the trans-cleavage activity of Cas13 enzyme. In the presence of SAR-CoV-2 virus, the RNA amplicon after RT-LAMP and T7 transcription was complemented to pre-designed crRNA (ribonucleoprotein complex, Cas13 + crRNA + amplicon). As a result, the Cas13 enzyme was activated to cleave the reporter probes, and the lateral flow assay (IFA) showed both a control (C) and a test (T) line, indicating a positive COVID-19 result. In absence of SAR-CoV-2 virus, there was no amplification of target site and binding to crRNA. Thus, Cas13 enzyme remain inactivated, and the reporter probes were not cleaved. The LFA showed only control line, indicating negative COVID-19 result. The un-cleaved reporter molecules are captured at the first detection line (control line), whereas indiscriminate Cas13 cleavage activity generates a signal at the second detection line (test line).