Rapid, point-of-care molecular diagnostics with Cas13

Abstract

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/μL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.

Figure 1.. Schematic of point-of-care DISCoVER platform for molecular diagnostics.

Patient samples, such as saliva, are collected and heat-inactivated in direct lysis buffer, followed by loading onto a single-use, gravity-driven microfluidic cartridge. The cartridge is then inserted into a companion instrument which automatically runs and the DISCoVER assay in a closed system to minimize reaction contamination. In Step 1, an initial rLAMP (RNA transcription following LAMP) reaction employs two mechanisms for amplification of target nucleic acids. Cas13 enzymes are programmed with a guide RNA to specifically recognize the desired RNA molecules over non-specifically amplified products. Subsequent activation of Cas13 ribonuclease activity, in Step (2), results in cleavage of reporter molecules for saturated signal within 5 minutes of CRISPR detection. In the left half of the cartridge, guide RNAs targeting SARS-CoV-2 enable rapid and selective detection of attomolar concentrations of virus. The mirrored half of the cartridge is used for an internal process control, enabling a negative test result by ensuring the presence of adequate patient sample. By exploiting template switching and CRISPR programmability, the point of care DISCoVER system can contribute to increased surveillance of diverse pathogens.

Figure 2.. Direct nucleic acid detection with CRISPR-Cas enzymes and LAMP.

A. Cas13 and Cas12 detection kinetics at varying activator concentrations. Values are mean ± SD with n = 3. B. Cas13 and Cas12 time to half-maximum fluorescence. †, Time to half maximum fluorescence was too rapid for reliable detection. ††, Time to half maximum fluorescence could not be determined within the 120 min assay runtime. Values are mean ± SD with n = 3. C. Schematic of SARS-CoV-2 genome sequence, with LAMP primer set locations indicated. D. Representative fluorescence plots of LAMP amplification of 100 copies/ μL of synthetic SARS-CoV-2 RNA or NTC. NTC, no-template control. Shaded regions denote mean ± SD with n = 3. E. Time-to-threshold of 9 screened LAMP primer sets, targeting synthetic SARS-CoV-2 RNA or NTC. Replicates that did not amplify are represented at 0 minutes. Error bars represent SD of amplified replicates. F. Limit of detection assay of LAMP using N Set 1 primer set. Replicates that did not amplify are represented at 0 minutes. Error bars represent SD of amplified technical replicates.

Figure 3.. Development of RNA transcription following LAMP (rLAMP) for two layers of nucleic acid amplification.

A. Schematic of rLAMP mechanism for exponential DNA amplification using F3/B3 and FIP/BIP primers, resulting in higher-order inverted repeat structures. Red arrows indicate location of T7 promoter sequence, inserted in the mBIP primer. Upon T7 transcription, the resulting RNA contains one or more copies of the Cas13 target sequence (orange). B. Schematic of the location of different T7 promoter locations on the rLAMP dumbbell structure and loop primers. C. rLAMP time to threshold of 6 distinct T7 promoter insertions. Replicates that did not amplify are represented at 0 minutes. Error bars represent SD of amplified technical replicates. D. Denaturing PAGE gels of mBIP rLAMP products to verify successful T7-mediated transcription. AfeI cleaves in the crRNA target region of templated products, which is expected to result in a single major transcribed species. E. Kinetics of T7 transcription and Cas13 detection on mBIP rLAMP products. RNP, ribonucleoprotein. Values are mean ± SD with n = 3. F. Cas13 detection of 8 technical replicates of mBIP rLAMP amplification on genomic RNA, depicted as fold-change over NTC at different reaction end-points.

Figure 4.. Development of DISCoVER for extraction-free detection of saliva and validation on patient samples.

A. Direct saliva lysis conditions were tested for compatibility with the DISCoVER workflow. Replicates that did not amplify are represented at 0 minutes. Error bars represent SD of amplified technical replicates. IA: inactivation reagent; QE: QuickExtract. B. Schematic of contrived saliva sample generation, quantification via qRT-PCR, and detection via DISCoVER to determine analytical sensitivity. C. Fold-change in DISCoVER signal relative to NTC on SARS-CoV-2 positive saliva samples at 5 minutes of Cas13 detection. D. Fold-change in DISCoVER signal relative to NTC on 30 negative saliva samples collected before November 2019, at 5 minutes of Cas13 detection. E. Schematic of rLAMP multiplexing with SARS-CoV-2 (N Gene) and human internal control (RNase P) primer sets. F. DISCoVER signal of SARS-CoV-2 positive saliva samples after multiplexed rLAMP. Values are mean ± SD with n = 3. G. Fold-change in DISCoVER signal relative to NTC on 30 negative clinical nasal samples at 5 minutes of Cas13 detection. H. Fold-change in DISCoVER signal relative to NTC on 33 positive clinical nasal samples at 5 minutes of Cas13 detection.

Figure 5.. Implementation of a point of care, microfluidic-driven diagnostic platform

(A) Illustration of the final instrument in which the cartridge is inserted (left). Picture of the entire microfluidic gravity-driven cartridge (inset). On right, images of the sample metering and rLAMP reaction chamber (1), rLAMP metering and Cas13 mixing chamber (2), and the detection chamber (3). (B) Front (left) and rear (right) illustration of the instrument components, that include detection system, heaters, air-driven valves and mechanical valves. (C) Schematic of the cartridge function. The reaction can be separated in four steps: 1. Saliva metering; 2. amplification reaction; 3. post-amplification metering + Cas13 mix; 4. Cas13 reaction in detection chambers. Reagents stored on the cartridge are separated via a proprietary hydrophobic solution to avoid premature initiation of the reactions. After the LAMP reaction, the sample can be split into two reactions: The left part of the cartridge will expose the sample to N gene crRNA (step 3a) while the right side of the cartridge will act as internal control with only RNase P crRNA (step 3b). Saliva samples only went through step 3a while clinical samples went through step 3a and step 3b. (D) Heat map depicting the fold-change in DISCoVER signal on negative and positive saliva samples relative to the no-template control (NTC). (E) Heat map depicting the fold-change in SARS-CoV-2 RNA positive and negative clinical samples from nasal swabs, relative to NTC. (F) Fluorescent images of detection chamber for NTC cartridge (no viral sample), negative and positive clinical sample (with qRT-PCR Ct ranging from 16 to 21). The left detection chamber is specific for N gene detection whereas the right chamber is specific for RNase P. (G) Graph depicting raw fluorescence over time for both detection chambers (blue for RNase P and red for N gene) in NTC (left), negative (middle) and positive (right, Ct 16) samples.