A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection

Abstract

Background Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. Methods A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans -cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab ( O ) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. Findings The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix- FCratio ) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix- FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix- FCratio were determined as 1.145 for positive and 1.068 for negative. Interpretation SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. Funding Detailed funding information is available at the end of the manuscript.

A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics

“…Similar to fluorescent readouts, it was frequently encountered with “grey zone” issue, where the Ct value was too “vague” to judge a sample especially for the ones with fairly low viral loads. This led to false negatives or false positives ( Huang et al, 2020 ). Our method, in this regard, balanced the sensitivity, accuracy and POC detection potentials.…”

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics

“…The typical workflow of a CRISPR-Dx for COVID-19 consists of RNA extraction, reverse transcription (RT), target amplification, Cas assay, and collateral cleavage activity detection as shown in Figure 2A. RNA extraction is firstly carried out to lyse and purify the RNA genome of SARS-CoV-2 from clinical specimens, such as nasopharyngeal swab [35][36][37][38][39] nasal swab [40], oropharyngeal swab [14,41], saliva [42,43], bronchoalveolar lavage [35,39] and sputum [35]. The viral RNA is then converted into complementary DNA through RT followed by a DNA-based amplification technique in a one-step or a two-step approach to generate a large amount of target DNA prior to the Cas assay and collateral cleavage activity detection.…”

“…Given that rRT-PCR is deemed as the standard diagnostic test for the confirmation of COVID-19, Huang et al [41] demonstrated the ease of coupling RT-PCR with a CRISPR-Cas12a assay, termed specific enhancer for detection of PCR-amplified nucleic acids (SENA), to improve the sensitivity and specificity of the technique for SARS-CoV-2 detection [41]. Following the completion of the RT-PCR reaction, the SENA reaction is set up by adding the amplicons to the SENA reagent (LbCas12a, crRNAs, and FQ reporter) and the increase in fluorescent signal due to the cleavage of the FQ reporter is then measured with a fluorescence reader.…”

Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP

“…The nonspecific trans-cleavage activity of Cas12 or Cas13 nuclease can be activated after the recognition of specific target nucleic acids, and thus to cleave the reporter probe [ 13 , 15 ]. Most of these methods used fluorescent probes, which need a special instrument to monitor fluorescent signal [ 16 , 17 ].…”