A Streamlined Point-of-Care CRISPR Test for Tuberculosis Detection Directly from Sputum

Abstract

Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (10⁵ to 10² CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

Figure 1.. CRISPR-Cas12a assay optimization for detecting Mtb.

A. Schematic of Cas12a/Cas13a based CRISPR reaction, where DNA is amplified using target specific primers, recombinases, strand-displacing polymerase, and ssDNA binding proteins and directly detected by Cas12a or transcribed to RNA with T7 reverse transcription for detection with Cas13a. B. Cas12a detecting synthetic DNA for six different crRNA-primer designs. Amplification was allowed to proceed for 30 minutes before the addition of crRNA and Cas12a. C. Cas12a detecting synthetic DNA with varying concentrations of Cas12a. Molar concentration alone indicates all components present from the start of the reaction. Molar concentration spiked indicates amplification was allowed to proceed for 30 minutes before the addition of crRNA and Cas12a. D. Cas12a detecting synthetic DNA in a one-pot format, as all components are present from the start of the reaction. E. Performance of Cas12a assay for decreasing concentrations of H37Rv genome. F. Specificity panel for Cas12a assay against various NTMs and other bacteria/fungi at 1 ng/μL. In B and D, error bars: SD based on n=3 replicates. In C, E, and F, error bars: SD based on n=2 replicates.

Figure 2.. CRISPR-Cas13a based detection of Mtb.

A. Cas13a detecting synthetic targets for six different crRNA-primer designs against two different targets, IS6110 and IS1081. B. Comparison of Cas12a to Cas13a detection of IS6110 synthetic target. Cas13a is a single-crRNA assay for direct comparison. C. Cas13a detecting H37Rv in single-target detection (IS6110 only) and dual-target detection (IS6110 and IS1081) formats. D. Cross reactivity panel for Cas13a dual detection assay against various NTMs and other pathogens at 0.1 ng/μL. Error bars: SD based on n=3 replicates.

Figure 3.. Validation of Cas13a dual detection assay against clinically relevant samples.

A. Cas13a detecting BCG genomic DNA spiked into sputum:TE matrix and Cas12a detecting internal control. Sp is sputum and gBCG is genomic BCG. Units are genomes/μL. Cas13a reporter is FAM and Cas12a reporter is HEX. B. Comparison of fresh Cas13a (left) or lyophilizer Cas13a (right) assays detecting synthetic internal control (IC) and BCG genome. Synthetic targets were mixed at a concentration of 10⁴ and 10³ copies/μL, respectively. C. Lateral Flow read-out of Cas13a detecting dual targets IS6110+IS1081 (top) and Cas12a detecting internal control (bottom) in BCG spiked into sputum:TE matrix. Appearance of the top line is a positive readout. Faint lines are interpreted as negative. Disappearance of the bottom line is not necessary. D. Range finding experiment demonstrating the dynamic range of fresh Cas13a dual detection assay. Cas13a detecting BCG and Cas12a detecting internal control. BCG culture (left) and BCG spiked into sputum:TE (right). gBCG is genomic BCG and units are genomes/μL. All other units are CFU/mL. E. Testing fresh Cas13a and Cas12a coupled assay against TB-negative clinical sputum samples. Numbers indicate patient number. F. Limit of detection (LoD) for H37Rv. LOD = 69.0 (51.0 – 86.9). G. Limit of detection (LoD) for BCG. LOD = 80.5 (59.4 – 101.6). In A, B, D, and E, error bars: SD based on n=3. C shows a single representative sample. F (N=20) and G (N=21), was plotted using ggplot2 in R.

Figure 4.. TB Detection from Sputum using Fresh SHINE-TB.

A. Schematic showing workflow of patient sample testing. Sputum samples were collected and tested using GeneXpert at CIDEIM in Colombia. Samples were shipped to Rutgers for novel sample processing. Processed samples were used for CRISPR assays and dPCR. B. TB detection in 14 different sputum samples using Cas13a dual detection. For each sample, a line is shown at the mean of 3 technical replicates. Shading: Grey - Controls, Green - correct call, Yellow - unmatched call for GeneXpert vs CRISPR and culture. LoD is shown as a dotted line at 2900 a.u. Triplicates were averaged for determination of positive and negative. Positive sputum is 500 CFU/mL. Plus signs mean positive detection and minus signs mean no detection. C means contamination/not determined. ND means not done or data not collected. C. Tables showing Cas13a dual detection compared to culture (top left), GeneXpert (top right), and dPCR (middle right). Table showing GeneXpert vs Culture (bottom right). Further details in Supplementary Table 4. D. Average Cas13a (FAM) vs Cas12a internal control (HEX) signal for each sample seen in (B). Linear trend was determined for the positive samples with an R squared value of 0.7058.