High Throughput SHERLOCK CRISPR SARS-CoV-2 Test
Abstract:
The ability to control the spread of COVID-19 has been hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions. Efforts to increase testing capacity have been adversely impacted by supply chain challenges due to dependencies on a limited set of reagents, consumables, and instrumentation. Here, we present a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive and specific detection of SARS-CoV-2, with the potential for up to 5,000 patient samples per day without thermal cycling instrumentation, and with minimal operator hands-on time. The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a specimen with minimal sample treatment. The assay is implemented in a 384-well format that is compatible with automated liquid handling instrumentation and provides results in less than one hour. Assay performance was evaluated with 105 (60 negative, 45 positive) SARS-CoV-2 specimens tested using FDA emergency use authorized assays (Hologic Panther, Roche Cobas and Perkin Elmer). The high throughput SHERLOCK SARS-CoV-2 assay was 100% concordant with the reference methods, correctly detecting all positive and negative samples.
Introduction:
A significant increase in available SARS-CoV-2 testing has been recognized as a critical requirement to end the COVID pandemic 1 . Most current tests rely on PCR-based amplification and detection of viral RNA, and require expensive, complex and sensitive equipment with highly trained laboratory personnel to operate it 2 . As such, the ability to quickly scale up the volume of testing required to meet demand has been challenging and, in many cases, leads to large delays in results being returned to the patient 3 . Isothermal amplification of viral targets has greatly reduced the complexity of equipment required to amplify viral targets, however off target amplification leading to false positives is a problem when using these methods alone 4–9 . Methods combining the flexibility and simplicity of an isothermal amplification with a high level of specificity are needed.
In recent years, CRISPR-based diagnostics have emerged as a programmable method for rapid, sensitive, and specific detection of nucleic acids 10–12. CRISPR-based diagnostics utilize the specific recognition of a target nucleic acid sequence by a guide RNA/Cas protein complex, which activates collateral nuclease activity of the Cas12 or Cas13 protein complex 12–16. This collateral activity can be converted into various readouts, including lateral flow or fluorescence. Utilizing a highly active Cas13a protein from L. wadei (LwaCas13a) combined with isothermal amplification, the SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) platform was developed as a low-cost CRISPR-based diagnostic that enables detection of DNA or RNA with single-nucleotide specificity 15,16. We have further enhanced the robustness and performance of this method by incorporating a highly sensitive LAMP-based amplification of the target viral RNA.
In May of this year, FDA issued the first Emergency Use Authorization (EUA) for a CRISPR diagnostic test when it granted an EUA for the SHERLOCK CRISPR SARS-CoV-2 kit. The SHERLOCK CRISPR SARS-CoV-2 kit (SHERLOCK kit) is capable of detecting the presence of a target nucleic acid in approximately 1 hour with a Limit of Detection (LoD) of 6.75 copies per microliter of VTM (viral transport medium)17. The SHERLOCK assay was authorized for the detection of SARS-CoV-2 nucleic acid in upper respiratory tissue samples including nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, nasopharyngeal wash/aspirate or nasal aspirate and bronchoalveolar lavage specimens collected from individuals suspected of COVID19 by their healthcare provider. It was recently reported in an independent clinical evaluation that the SHERLOCK assay was 100% concordant to RT-PCR in the detection of SARS-CoV-2 in clinical nasopharyngeal samples.
Loop-mediated isothermal amplification (LAMP) has been extensively studied as a molecular diagnostic amplification method for various viruses including SARS-CoV2 19,20. LAMP based amplification methods are attractive for diagnostics because LAMP has been reported to be more tolerant than PCR to endogenous inhibitors present in biological samples 21,22. However, traditional LAMP-based detection methods suffer from poor specificity and are challenging to multiplex 5–9 . Our SHERLOCK-based methods overcome this limitation with sequence specific Cas13 based amplicon detection.
Here we present several advancements that were developed to improve the simplicity and throughput of the CRISPR diagnostic method implemented in the SHERLOCK kit. These include combining two independent SARS-CoV-2 targets, Nucleocapsid (N) and Open Reading Frame (ORF), in a single reaction, simplifying the sample preparation, and implementing the assay in a 384-well format with minimal liquid handling steps to increase throughput and improve compatibility with automated processes. Additionally, we evaluated multiple extraction methods and demonstrated that a simple heat and proteinase K treatment is sufficient to allow direct sample (swab in saline, saliva) addition to a SHERLOCK reaction while maintaining high sensitivity (2 copies/uL) and specificity.
Results:
SHERLOCK High-throughput Method with 96-well RNA Extraction
Here, we developed a workflow based on the SHERLOCK CRISPR SARS-CoV-2 test that increases throughput, simplifies sample preparation, and combines dual target SARS-CoV-2 amplification and detection into a single reaction (Figure 1). Extracted gRNA samples are added to a LAMP reaction master mix, with primers specific to the target, in a 384-well deep well, fluorescence-compatible plate. The LAMP reactions are then topped with 20 µl of molecularbiology grade mineral oil to prevent condensation and reduce the risk of contamination. The LAMP reaction occurs on any plate heater capable of maintaining 61ºC. The time of the LAMP reaction depends on the template, and is approximately 30 minutes for samples extracted with a nucleic acid extraction kit. After allowing the plate to cool to room temperature, the LAMP reaction plate is moved to a dead air/ post-amplification area and the plate seal is removed. This plate seal can either be disposable or a silicone seal compatible with automated plate handlers. The Cas detection mix (10 µl) containing the Cas enzyme as well as the guide RNA specific for the amplified target is added to each well. The plate is then read on a fluorescence plate reader over 10 minutes at 37ºC. With this method a single operator can process 190 samples in 70 minutes (excluding extraction).
To demonstrate the robustness of the SHERLOCK High Throughput method, we evaluated several bead-based RNA extraction kits on pooled negative NP swab matrix spiked with Zeptometrix NATtrol SARS-CoV-2 viral particles at decreasing concentrations. We tested three commercially available kits: MagMAX Viral/Pathogen Isolation kit, MagMAX Viral RNA isolation kit and the Zymo Quick-DNA/RNA Viral MegBead kit (Supp Table 2A). All three kits had comparable LoDs, however the workflow of the MagMAX Viral RNA was most conducive to our manual protocol and we tested additional concentrations. With this workflow, our LoD was 2cp/µl (20/20 ,100%) (Fig 2A). We also compared the sensitivity of three fluorescent plate readers (Biotek Neo2, Tecan MPlex and Fluoroskan Microplate Fluorometer) to expand accessibility of our assay. All three plate readers had similar LoD of viral genomic RNA spiked into the reaction (4cp/µl for the Tecan, 2cp/µl for the Biotek and Fluoroskan instruments) (Supplemental Table 2B). To determine cross reactivity and competitive inhibition, we tested the high-throughput workflow with a panel of genetically related organisms as well as common high-priority organisms circulating in the area. Data shown in Supplemental Table 2C, D shows 100% specificity towards SARS-CoV-2. To test our clinical sensitivity, we tested 30 positive and 30 negative NP samples, previously tested by molecular diagnostic methods in CLIA labs and confirmed in house using a modified CDC EUA protocol (Supplemental Table 2E), extracted using the MagMAX Viral RNA isolation kit, with a 30-minute LAMP reaction. Our clinical evaluation resulted in a PPA (Positive predicted agreement) of 100% (30/30) and a NPA (Negative predicted agreement) of 100% (30/30) for the SHERLOCK high throughput workflow (Figure 2 B-D). These data support that the SHERLOCK high-throughput workflow for the detection of SARS-CoV-2 in upper respiratory specimens is sensitive, specific and improves the overall turnaround time as compared to Sherlock’s EUA kit.
SHERLOCK High-throughput Direct method
In addition to increasing the throughput of our assay, we were interested in developing a workflow that allowed for use of samples that have not gone through a full RNA extraction process, as there have been consistent shortages of RNA extraction materials 23. The SHERLOCK Direct workflow begins with a simplified sample treatment where 2 µl of proteinase K is added to 18 µl of each sample. The samples are then heated for 6 minutes at 65ºC followed by 98ºC for 3 minutes to heat-kill the proteinase K enzyme, followed by cooling to 4- 10ºC. These samples can be heated in a 96 or 384-well PCR plate on a heat block. Samples can then be added directly to the LAMP reaction as detailed above, with the amplification time extended to 40 minutes.
With an automated implementation parallel processing four 384 well plates, this SHERLOCK Direct method can process 9216 samples per day (Supplemental Table 1A,B).
Saline has become a commonly used storage solution for nasal swabs 24 with high levels of accuracy and stability. We tested the compatibility of our high-throughput workflow with NP swabs in saline with the SHERLOCK Direct workflow. First, we established the LoD of our workflow with saline spiked with SARS-CoV-2 inactivated particles from 100cp/ µl to 0.01 cp/µl. Our LoD for contrived saline samples was 10cp/µl (22/23, 95.7%) (Figure 3A). We then tested the clinical applicability of our SHERLOCK Direct method on clinically collected NP swabs stored in 0.9% saline, 20 positive and 25 negative samples. All samples were purchased from a biobank after having been tested and confirmed COVID positive or negative by an outside CLIA lab. To ensure sample integrity, we also tested the material using an in-house developed protocol modeled on the CDC EUA protocol, i.e., extraction using the Qiagen RNA kit and RTPCR using the CDC primers targeting the N gene of SARS-CoV-2 and the internal control of RnaseP (Supplemental table 3A). The PPA for the SHERLOCK Direct high throughput method in saline was 100% (25/25) and the NPA was 100% (20/20, Figure 3 B-D), while reducing the time to result and cost associated with sample extraction. We also tested saliva with our SHERLOCK Direct method. We were able to show high analytical sensitivity, 5cp/µl in pooled saliva (Supplemental Table 3B).
Discussion:
Most of the population will not have access to a COVID-19 vaccine for many months, therefore testing remains crucial for controlling the spread of the virus 25. The most recent report on average time to results issued by The COVID States Project is 2.7 days with 42% of people waiting at least 3 days 3 , which is too long for reliable contact tracing. Critical to increasing testing capacity is high throughput molecular testing that is not affected by supply chain limitations. Here we demonstrated a high-throughput method for detecting SARS-CoV2 down to 2 copies/µL using a high throughput magnetic bead-based purification of patient samples, or 10 copies/µL direct from patient samples. We also verified key improvements to the SHERLOCK CRISPR SARS-CoV-2 EUA protocol that resulted in an increased number of samples processed and a decreased time to result. With this high throughput method, 96 samples can be tested manually in 100 minutes.
Overall, the improvements demonstrated here are: i) a simplified workflow with decreased liquid handling steps, ii) transition to a 384 well plate format beginning at the sample prep step or LAMP step, and iii) removal of the need for RNA purification of patient samples. Additionally, we demonstrated that this method is compatible with multiple plate readers and magnetic bead RNA isolation kits. Most importantly this high-throughput SHERLOCK CRISPR SARS-CoV-2 test shows 100% specificity and 100% sensitivity.
The modifications to the SHERLOCK CRISPR SARS-CoV-2 EUA protocol demonstrated here result in the development of a more user friendly, faster, inexpensive and robust method for detecting SARS-CoV-2 direct from patient samples. This test can be run on common lab equipment different from what is used for RT-qPCR assays, allowing for individual labs that are currently using COVID-19 RT-qPCR tests to increase their testing capacities. Implementation of this test can increase testing capacity that may enable more efficient and reliable contact tracing and decrease the spread of COVID-19.
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