CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

Grand Challenges in Analytical Science

“…Alternatively, lateral flow assays (LFA) by taking advantages of nucleic acid amplification like isothermal recombinase polymerase amplification (RPA) and specificity of CRISPR-its associated proteins (Cas) have become popular for detection of pathogens. For example, a RPA-CRISPR/Cas9 LFA with a fluorescent detection mode shows sensitive and selective detection of SARS-CoV-2 (Osborn et al, 2021). However, cost, analysis time, false positive and negative results, and complicated operation procedure are concerns before CRISPR/Cas LFA assays with nucleic acid amplification can be ready for POC.…”

CRISPR/Cas13-Based Platforms for a Potential Next-Generation Diagnosis of Colorectal Cancer through Exosomes Micro-RNA Detection: A Review

“…Therefore, Cas12 and Cas13 are not only natural beacon-like reporters, but they also intrinsically amplify the detection signal mediated by their trans-collateral activity. Although Cas9 does not exhibit any trans-collateral activity (see Figure 3 ), there have been some CRISPR/Cas9-based platforms, including lateral flow detection [ 180 ] and fluorescence readouts [ 181 ], which display a good detection performance for the CRISPR-Dx toolbox; however, the implementation may have drawbacks due to logical adjustment of Cas9 to transform it into a functional molecular detector, including less cost-effectiveness due to the requirement of antibodies, fluorescent probes, and extra enzymes, which also leads to complications for setup.…”

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

“…The feasibility of utilizing dCas9 for SARS-CoV-2 detection was explored by both Azhar et al [74] and Osborn et al [75]. Both assays relied on the visual detection of a labeled dCas9-sgRNA-target DNA complex with a LDF but employed different Cas9 orthologs and labeling strategies.…”

“…FELUDA was shown to be capable of detecting 2 ng of SARS-CoV-2 RNA extract and the total assay time from RT-PCR to result visualization with LFD was found to be ~45 min. In the study by Osborn et al [75], synthetic SARS-CoV-2 DNA was initially used to demonstrate the specific recognition of the target sequence by dCas9 [75]. Instead of labeled sgRNA, Osborn et al [75] used biotinylated Streptococcus pyogenes dCas9 and unlabeled sgRNA to bind to FAM-labeled, RPA target amplicon (Orf8a gene) (Figure 3B).…”

“…In the study by Osborn et al [75], synthetic SARS-CoV-2 DNA was initially used to demonstrate the specific recognition of the target sequence by dCas9 [75]. Instead of labeled sgRNA, Osborn et al [75] used biotinylated Streptococcus pyogenes dCas9 and unlabeled sgRNA to bind to FAM-labeled, RPA target amplicon (Orf8a gene) (Figure 3B). The 20-min RPA amplification and dCas9 assay were performed sequentially, as combining the steps in a one-pot assay led to non-specific positive results.…”