CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

Figure 1. Recombinase polymerase amplification (RPA) and nucleic acid detection by lateral flow assay. (A) Recombinase polymerase amplification. Recombinase proteins complex with primers and facilitate strand displacement and binding to homologous target sequence(s) without requiring temperature alterations (i.e., cycling). The DNA polymerase initiates synthesis from the primers and amplifies target DNA exponentially. (B–E) Analyte detection by lateral flow assay (LFA). (B) Reagent components for detection via LFA (Au-NP = gold nanoparticle, Ab = antibody, FITC = fluorescein isothiocyanate). (C) Embedded in the flow assay device conjugate pad are gold nanoparticles decorated with rabbit anti-FITC antibodies. The test and assay control bands are coated with a biotin ligand or anti-rabbit antibodies, respectively. A red arrow indicates the direction of sample flow through the conjugation pad. In the absence of a molecule that is labeled by both FITC and biotin, the Au-NPs flow to and accumulate at the assay control band, where they are bound by anti-rabbit antibodies. Dual FITC:biotin-labeled substrates are bound first by the anti-FITC Au-NPs and then accumulate at the test band via capture…

Figure 2. Biotinylated ‘dead’ Cas9 (bdCas9) and PAM-rich competitor soak DNA allows for the detection of target nucleic acids. (A) SARS-CoV-2 genome and detection strategy. The viral genes are shown as well as the D614G and N501Y amino acid mutations in the S gene and the L84S alteration in ORF8a. The locations of the WHO and CDC genes analyzed in their respective diagnostic assays are indicated with red arrows. The sequence in the ORF8a gene targeted for CRISPR/Cas9 sgRNA detection in the present study is shown with the L84S polymorphic nucleotide at position 28144 (indicated in red) and the CRISPR/Cas9 protospacer adjacent motif (highlighted in green). (B, C) Dual-labeled PCR primer strategy results in false positives. Amplification was performed with a FITC-labeled forward and biotinylated reverse primer in the absence (−) or presence (+) of a template, and products were analyzed via LFA (C). ’Blank’ refers to an LFA test strip loaded with no amplicon. (D, E) Uncoupling the FITC and biotin labeling of DNA using a FITC primer and bdCas9 allows for nucleic acid detection. Amplification was performed with a FITC-labeled forward and unlabeled reverse primer. The amplicon was then…

Figure 3. Optimization of rapid SARS-Co-V2 nucleic acid detection. (A) Simultaneous RPA and bdCas9 detection results in false positives. Room temperature RPA of a SARS-CoV-2 template was performed in the presence of bdCas9 plus 10 or 20 µM competitor (i.e., PAM) soak DNA with a COVID-19 or mismatched sgRNA and analyzed by LFA. (B) Sequential RPA and Cas9 detection. RPA was performed at room temperature followed by amplicon incubation with bdCas9. RPA products were incubated with the indicated concentrations of competitor PAM soak DNA for 20, 40, or 60 min with either a COVID-19 (C19) or mismatched (MM) irrelevant sgRNA, followed by LFA. Blue arrows show the assay control band and black arrows indicate a test band. Images are representative of three independent experiments. LFA-labeled RPA in (A) represents a no DNA template control.

Figure 4. Fluorescence-based CRISPR/Cas9 nuclease detection of SARS-CoV-2 DNA. (A) Design of a CRISPR/Cas9 nuclease fluorescence detection assay. SARS-CoV-2 DNA amplicons were mixed with DNA probes containing a 5′ fluorescein fluorophore and 3′ Iowa black quencher. The probe and amplicons were denatured and renatured, resulting in a heteroduplex of amplicon:probe DNA that was incubated with CRISPR/Cas9 nuclease complexed with COVID-19, or control sgRNAs. Cas9 nucleolytic activity uncouples FAM from the quencher, resulting in a fluorescent signal that can be measured with a fluorometer. (B) CRISPR/Cas9 nuclease fluorescent signal detection of SARS-CoV-2 DNA. A time course was performed to evaluate fluorescence signal intensity generated by Cas9 nuclease conjugated with a COVID-19 sgRNA, two separate unmatched control sgRNAs, or hybridized PCR:probe duplexes (DNA/probe) alone. Shown is the mean relative fluorescent value (RFU) and standard deviation from four independent experiments. p-value (one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test) of **** <0.0001 is shown with asterisks.

Figure 5. Multiplex CRISPR/Cas9 fluorescence detection of viral respiratory pathogens. (A) CRISPR/Cas9 shows high specificity in distinguishing respiratory pathogen targets. Individual DNA amplicons and fluorescent probes for SARS-CoV-2 (FAM-labeled), influenza A (Flu A; TxRed®-labeled) or B (Flu B; Yakima Yellow®-labeled), or respiratory syncytial virus (RSV; TAMRA-labeled) were annealed and interrogated with Cas9 and sgRNAs for each virus DNA. The DNA target amplicons are indicated at the top of each graph and the sgRNAs are labeled vertically on the y-axis. The heat maps represent the mean fluorescent values from three independent experiments performed in duplicate. The horizontal color bars at the top of each heat map are the values normalized to the highest fluorescent signal obtained. (B) Fluorescence values are plotted as the mean of the three independent experiments performed in duplicate from (A). Statistical evaluation of these data was done using one-way ANOVA and Tukey’s multiple comparison to show fluorescence signal above other analytes. *, ***, *** indicates p = <0.05, p = <0.001, and p = <0.0001 respectively, for the appropriate matched sgRNA versus the highest…

Figure 6. SARS-CoV-2 genomic RNA detection by LFA and fluorescence. (A) Viral RNA was diluted serially from 1:10 to 1:10,000 and reverse transcribed and analyzed by qRT-PCR using the CDC N1 and N2 primer:probe sets. Shown are triplicate samples of the mean ± standard deviation (SD) cycle threshold (Ct). (B) Viral cDNA was amplified as in (A) using ORF8a primers and analyzed by agarose gel electrophoresis. Mw = molecular weight and NTC = no template control. (C) Fluorescence detection of SARS-CoV-2 DNA targets. PCR products from serially diluted viral cDNA was hybridized with a COVID-19 probe and incubated with a Cas9:control sgRNA or COVID sgRNA and analyzed for fluorescence generation under isothermal (37 °C) conditions for one hour. Heat maps are shown of the mean of four experiments with the dilution series indicated at the top. Raw fluorescence value scales are shown at the top and the y-axis is the sample identification of DNA:probe alone (ODN) and DNA:probe hybrids interrogated with a control or COVID sgRNA. The x-axis represents the thirty second time points at which fluorescence was measured over the course of one hour (121 total measurements). (D) Copy number limit of detection…