Background
The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics.
Methods
We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability.
Results
Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature.
Conclusions
CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.
The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and STOPCovid have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 μL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.
Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has the potential to transform diagnostics due to its programmability. However, many of the CRISPR-based detection methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. A complete one-pot detection reaction using alternative Cas effector endonucleases has been proposed to overcome these challenges. Yet, current approaches using Alicyclobacillus acidiphilus Cas12b (AapCas12b) are limited by its thermal instability at optimum reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction temperatures. Herein, we demonstrate that a novel Cas12b from Brevibacillus sp. SYP-B805 (referred to as BrCas12b) has robust trans-cleavage activity at ideal RT-LAMP conditions. A competitive profiling study of BrCas12b against Cas12b homologs from other bacteria genera underscores the potential of BrCas12b in the development of new diagnostics. As a proof-of-concept, we incorporated BrCas12b into an RT-LAMP-mediated one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs) to enable rapid, differential detection of SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) in 205 clinical samples. Notably, a BrCas12b detection signal was observed within 1-3 minutes of amplification, achieving an overall 98.1% specificity, 91.2% accuracy, and 88.1% sensitivity within 30 minutes. Significantly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, we combined the lyophilized one-pot reagents with a portable multiplexing device capable of interpreting fluorescence signals at a fraction of the cost of a qPCR system. With relaxed design requirements, one-pot detection, and simple instrumentation, this assay has the capability to advance future diagnostics.
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