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Kok Gan, C. Cas12-Based CRISPR-Dx. Encyclopedia. Available online: (accessed on 01 December 2023).
Kok Gan C. Cas12-Based CRISPR-Dx. Encyclopedia. Available at: Accessed December 01, 2023.
Kok Gan, Chan. "Cas12-Based CRISPR-Dx" Encyclopedia, (accessed December 01, 2023).
Kok Gan, C.(2021, December 18). Cas12-Based CRISPR-Dx. In Encyclopedia.
Kok Gan, Chan. "Cas12-Based CRISPR-Dx." Encyclopedia. Web. 18 December, 2021.
Cas12-Based CRISPR-Dx

Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing.

CRISPR cas Assays

1. Two-Pot Assays

Given that rRT-PCR is deemed as the standard diagnostic test for the confirmation of COVID-19, Huang et al. [1] demonstrated the ease of coupling RT-PCR with a CRISPR-Cas12a assay, termed specific enhancer for detection of PCR-amplified nucleic acids (SENA), to improve the sensitivity and specificity of the technique for SARS-CoV-2 detection [1]. Following the completion of the RT-PCR reaction, the SENA reaction is set up by adding the amplicons to the SENA reagent (LbCas12a, crRNAs, and FQ reporter) and the increase in fluorescent signal due to the cleavage of the FQ reporter is then measured with a fluorescence reader. By using a mixture of crRNAs targeting the Orf1ab and N genes, the authors found that the mix-SENA was more sensitive than detecting each of the targets alone and showed that the limit of detection (LoD) of mix-SENA (1.6 copies/reaction) was lower than that of rRT-PCR (4.0 copies/reaction). As such, mix-SENA may play a role in resolving samples with ambiguous rRT-PCR results that are associated with high cycle threshold (Ct) value “grey zones”. Mix-SENA was also able to identify two false positives and four false negative results by rRT-PCR as corroborated by next-generation sequencing results when evaluated with 295 clinical specimens. The potential application of mix-SENA as an indicator of viral clearance was also demonstrated with samples from three COVID-19 recovering patients, whereby rRT-PCR-negative samples were found to be positive by mix-SENA, highlighting the risk of patients being discharged prior to complete viral clearance [1].
A particular clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins12 (Cas12) system detection system may also be developed to be compatible with both non-isothermal- and isothermal-based amplification techniques. For example, the CRISPR-based fluorescent diagnosis system for COVID-19 (COVID-19 CRISPR-FDS) developed by Huang et al. [2] could be used to detect RT-PCR- or RT-RPA-amplified N and Orf1ab genes without changes in the detection limit of the test [3]. Furthermore, the LoD of the COVID-19 CRISPR-FDS (2 copies/test) was reported to be comparable to that of rRT-PCR (5 copies/test). Based on the analysis of 29 nasal swab specimens from suspected COVID-19 cases, CRISPR-FDS showed complete concordance with the state laboratory-generated rRT-PCR positive samples (100% PPA), but not with rRT-PCR negative samples (71.4% NPA). The authors could not conclude whether the three discordant samples represented false positive CRISPR-FDS or false negative rRT-PCR results due to the lack of information and further testing. The large discrepancy between the rRT-PCR results of the 29 nasal swab specimens generated by a hospital laboratory and the state laboratory in the study further emphasizes the need for diagnostic tests that are not only rapid and sensitive, but also robust in detecting SARS-CoV-2 positive samples [2].
In terms of target amplification, isothermal amplification-based CRISPR-Cas assay is the preferred approach for COVID-19 diagnosis with DNA endonuclease-targeted CRISPR trans reporter (DETECTR) being a typical representative of the Cas12-based detection schemes. Notably, the SARS-CoV-2 DETECTR Assay and the SARS-CoV-2 DETECTR Reagent Kit are the first and only CRISPR-Cas12-based diagnostic tests to receive an emergency use authorization (EUA) from the United States Food and Drug Administration (FDA) in July and August 2020, respectively [4]. The assay consists of two monoplex reactions and is designed to amplify the target N gene and internal control RNase P separately. RNA extraction is a prerequisite, and the RNA extract serves as a template for the 30-min RT-LAMP reaction at 62 °C followed by a 15-min Cas12 assay at 37 °C. A real-time thermocycler is required for fluorescence measurement and a cut-off value of 500,000 relative fluorescent units is used to interpret positive/negative result for the target and control. The SARS-CoV-2 RNA DETECTR Assay [5] and SARS-CoV-2 DETECTR Reagent Kit [6] share the same performance characteristics (LoD = 20 copies/µL; PPA = 95%; NPA = 100%), but the test is only authorized to be conducted in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories that meet the requirements to perform high complexity tests. Despite similar personnel and instrument requirements, the SARS-CoV-2 DETECTR Assay was six- to twenty-fold less sensitive than the FDA-EUA approved CDC 2019 novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel (1–3.16 copies/µL) [7].
In the RT-LAMP-DETECTR assay developed by Broughton et al. [8], the E gene was used to detect three SARS-like CoVs (SL-CoVs), including SARS-CoV-2, bat SL-CoV, and SARS-CoV, whereas the N gene was used to detect SARS-CoV-2 specifically. The target genes, along with an internal control RNase P, were amplified individually in separate tubes prior to CRISPR-Cas12a detection [8]. Other than Broughton et al. [8], many researchers have also sought to demonstrate the feasibility of using either an LFD or a fluorescence reader for CRISPR-Cas result interpretation without compromising the test performance [9][10]. This flexibility provides the test operator with the option of using either one of the devices based on the resources that are available as well as the expected throughput and turnaround time in a particular setting. Other researchers opted for naked eye detection of fluorescence emission of positive samples under blue or UV light, such as in the RT-RPA-coupled CRISPR-Cas12a assays developed by Wang et al. [11] and Mayuramart et al. [12]. Although results interpretation based on naked eye detection is advantageous for POC and resource-limited settings, the risk of operator bias cannot be avoided due to the subjective nature of visual inspection.
Other than LFD and fluorescence reader, a low-cost, three-dimensional (3D)-printed, smartphone-based device for fluorescence imaging has been described for the analysis of RT-RPA-DETECTR results [13]. The highly portable device consists of a sample tray with a capacity of eight PCR tubes, a smartphone holder, and an imaging compartment. Images captured with the phone camera are then sent to the cloud where a machine learning algorithm will be used for result interpretation. The LoD of this RT-RPA-DETECTR assay (6.25 copies/µL) was comparable to the RT-LAMP-DETECTR assay (10 copies/µL) developed by Broughtonet al. [8], but higher than that of rRT-PCR (1–3.2 copies/μL) [8][13][7]. The PPA and NPA of the RT-LAMP-DETECTR assay relative to rRT-PCR (n = 82; PPA = 95%; NPA = 100%) [8] were also better than the RT-RPA-DETECTR assay (n = 115; PPA = 87%; NPA = 92%) [13]. Nevertheless, the PPA and NPA of this RT-RPA-DETECTR assay improved to 97% and 93%, respectively, when clinical samples were restricted to those with high viral loads (Ct < 33; n = 96), indicating that the developed model required further fine-tuning on clinical samples with low viral loads [13].

2. One-Pot Assays

Post-amplification analysis that requires the opening and closing of the amplification reaction tube, particularly the isothermal amplification reaction tube, increases the risk of aerosol contamination that can lead to false positive results. Therefore, the development of a closed-tube format for CRISPR-Dx would be ideal to minimize the risk of carry-over and cross contaminations. The RT-All-In-One Dual CRISPR-Cas12a (RT-AIOD-CRISPR) assay is one of such examples whereby RT-RPA and CRISPR-Cas12a reagents are added in a single tube and incubated at 37 °C for 40 min before the result is visualized under blue or UV light [14]. Unlike the DETECTR assay in which the collateral activity of Cas12 is induced by the binding of crRNA to dsDNA in a PAM-dependent manner, RT-AIOD-CRISPR relies on crRNAs that are designed without PAM so that the binding of crRNAs to single-stranded amplicon during RPA will still induce the collateral cleavage activity of Cas12. In addition to achieving a LoD of 5 target RNA copies and complete concordance with rRT-PCR results when tested with 28 clinical samples, the authors also showed that the RT-AIOD-CRISPR assay could be performed with a hand warmer and positive results could be observed in as little as 20 min [14].
Contrary to the strategy used by Ding et al. [14], other researchers sought to avoid the cis-cleavage activity of Cas12 during the amplification process physically by separating the CRISPR-Cas reaction mixture from the amplification reaction mixture within the confine of a single tube. This is typically achieved by placing the CRISPR-Cas reaction mixture within the lid of the tube while the amplification reaction mixture is placed at the bottom of the tube with or without a layer of mineral oil [15][16][17][18][19]. Upon completion of the amplification process, the solution is either mixed by inverting the tube manually or subjecting the tube to a brief spin. Due to the use of RT-LAMP as the amplification method, the assay protocol developed by Chen et al. [15], Wanget al. [16], and Pang et al. [17] required different incubation temperatures for amplification and Cas12, assay whereas the RT-RPA-based OR-DETECTR assay developed by Sun et al. [18] only requires a single incubation temperature. Result are then interpreted based on visual inspection under blue/UV light or via a fluorescence read-out. The reported LoD for these one-pot assays ranged from 2.5 copies/µL to 45 copies/µL and achieved 97%–100% concordance with rRT-PCR results when tested with clinical specimens (n = 14–100) [16][17][18]. Like Samacoits et al. [13], Chen et al. [15] also capitalized on 3D printing technology to fabricate a portable instrument for fluorescence imaging with a smartphone camera, but result interpretation was based on visual inspection instead of a cloud-based analysis and the LoD attained was 20 copies/reaction [15].
As RT-LAMP-based CRISPR-Cas12a detection requires different incubation temperatures, this drawback can be overcome by substituting Cas12 with a thermostable ortholog such as the Cas12b from Alicyclobacillus acidiphilus (AapCas12b) and Alicyclobacillus acidoterrestris (AacCas12b). Unlike LbCas12a, which operates at an optimal temperature of 37 °C, AapCas12b is able to function at temperatures up to 65 °C [20], making it compatible with RT-LAMP to create CRISPR-Cas12b-based one-pot assays that only require a single incubation temperature. For example, the in vitro specific CRISPR-based assay for nucleic acids detection (iSCAN) developed by Ali et al. [21] began as a two-pot assay in which RT-LAMP (62 °C, 30 min) and Cas12a assay (37 °C, 10 min) were performed in separate tubes [21]. To further simplify the assay protocol, the team proceeded to develop a one-pot iSCAN by replacing LbCas12a with the thermophilic variant AapCas12b. When the RT-LAMP and Cas12b reagents were added together, lower amplification efficiency was achieved as compared to the two-pot format. This was attributed to the cleavage of target amplicon by the activated Cas12b during the amplification process. Hence, the CRISPR-Cas12b reagent mixture was placed on the tube wall near the top of the tube to allow the RT-LAMP reaction (62 °C, 30 min) to proceed to completion. The tube was then subjected to a brief spin followed by the Cas12b assay (62 °C, 15 min) and detection. The one-pot and two-pot iSCAN exhibited the same LoD (10 copies/reaction) and were two-fold higher than that of rRT-PCR (5 copies/reaction). Evaluation with 24 clinical specimens revealed that the PPA and NPA of the one-pot and two-pot iSCAN using fluorescent-based detection were the same. Specifically, the PPA of iSCAN was dependent on the target gene (N gene, 85.7%; E gene, 38.1%), whereas NPA was 100% for both formats [21].
In the SHERLOCK Testing in One Pot (STOP) SARS-CoV-2 (STOPCovid.v2) assay [20], a 10-min magnetic bead-based RNA extraction was first performed and, by retaining only the RNA-bound magnetic beads in the tube under a magnetic field, the same tube was used for the RT-LAMP and Cas12 assay by adding the STOPCovid.v2 reaction mixture to the beads. The tube was then incubated at 60 °C in a real-time thermocycler for 1–2 h with fluorescence measurements taken 1 h prior to LFD-based detection. Compared to the LoD of rRT-PCR (1000 copies/mL), the LoD of STOPCovid.v2 (33–83 copies/mL) was found to be 12–30 times lower. Evaluation of the STOPCovid.v2 with 402 clinical samples yielded a PPA of 93.1% and an NPA of 98.5% [20]. Guo et al. [19] coupled RT-RAA with a CRISPR-Cas12b-mediated DNA detection (CDetection) to develop a CRISPR-assisted detection (CASdetec) platform [19]. Due to the drastic decrease in sensitivity when RT-RAA and CDetection were concurrently executed within a single tube, Guo and colleagues separated the RT-RAA (42 °C, 30 min) and CDetection (42 °C, 30 min) reaction mixtures by placing the CDetection reagents within the lid of the tube. A brief spin was sufficient to bring the CDetection reagents down after RT-RAA was completed. Measurement of the fluorescence emission with a fluorescence reader resulted in a LoD of 1 × 104 copies/mL of SARS-CoV-2 pseudovirus. Despite the apparent advantage of using AapCas12b due to its thermostable nature, the longer sgRNA required as compared to the crRNA of LbCas12a may increase the risk sporadic collateral activity arising from the overlapping between the sgRNA and LAMP primers [17].

3. Other Assay Formats

The work of Ramachandran et al. [22], Park et al. [23], and Ning et al. [24] highlights the use of a chip-based approach that consumes less reagents than conventional devices. Ramachandran et al. [22] used an electrokinetic microfluidic technique called isotachophoresis (ITP) to automate the RNA extraction process and to control the Cas assay within an in-house built microfluidic chip through the application of an electric field. The major disadvantage of the present ITP-CRISPR design for SARS-CoV-2 detection is the off-chip steps whereby sample lysis and RT-LAMP remain as tube-based procedures. The ITP-CRISPR in its current design also requires laboratory-based instruments (such as a source meter and camera-mounted inverted epifluorescence microscope) and the LoD obtained (10 copies/µL) was similar to that achieved with LFD in other CRISPR assays [8][10][18]. Nonetheless, the sample-to-result time of ITP-CRISPR (~35 min), which is inclusive of the RNA extraction step, is still shorter than RNA extraction-free CRISPR-based assays (50–75 min) [25][26][27]. Furthermore, ITP-CRISPR is amenable to automation, miniaturization, and integration of different analytical operations. The development of its associated detection systems into hand-held devices would make the platform applicable for POC use.
Park et al. [23] took a different approach and utilized a commercially available chip (QuantStudio 3D Digital PCR 20K Chip, Thermo Fisher Scientific) to develop a digital CRISPR-Cas-based assay called digitization-enhanced CRISPR/Cas-associated one-pot virus detection (deCOViD) [23]. Both ITP-CRISPR and deCOViD are designed for monoplex detection of target gene with a single sample loaded per chip, but unlike ITP-CRISPR [22], the only off-chip step in deCOViD is the sample processing step in which either RNA extract or heat-inactivated SARS-CoV-2 is obtained. Once the template is added to the mixture of reagents for RT-RPA and Cas12a assay, a specialized chip loader will be used to partition the mixture into 20,000 nanoscale reaction wells that are etched on the chip. Subsequently, deCOViD required only a single incubation step at 42 °C for 30 min, which is carried out in a custom-assembled miniature heater, before fluorescence intensity is measured under a fluorescence microscope [23]. A five- to ten-fold increase in sensitivity was observed when the LoD of deCOViD (20 GE/µL heat-inactivated SARS-CoV-2; 1 GE/µL) was compared to that of RT-RPA-CRISPR detection with a real-time thermocycler (100 GE/µL heat-inactivated SARS-CoV-2; 10 GE/µL RNA). Although deCOViD was shown to accelerate qualitative and quantitative detection along with a broad dynamic range and increased sensitivity through digitization, its highly specialized equipment requirement (such as a chip loader and fluorescent microscope) would need to be addressed if the platform were to gain acceptance for more widespread use.
A 15-min sample-to-result, chip-based assay that combines RT-RPA, CRISPR-Cas12a, and a fluorescence detection system (FDS) was recently described by Ning et al. [24], making this proof-of-concept study a breakthrough in CRISPR-Dx for COVID-19. The CRISPR-FDS assay, which is designed to analyze saliva samples following a 5-min lysis step, utilizes a compact, in-house built chip containing five reaction wells that can accommodate the analysis of five assays in parallel with a smartphone-based fluorescence microscope [24]. To conduct the assay, an aliquot of lysed sample is added to the reaction well of a chip that is pre-filled with premixed RPA and CRISPR-Cas solution. The chip only requires a 10-min incubation step at room temperature before it is ready to be inserted into a smartphone-based fluorescence microscope for imaging under blue light. The CRISPR-FDS assay developed by Ning et al. [24] demonstrated good linearity over a broad range of viral concentrations (1–105 copies/µL) with a calculated LoD (0.38 copies/µL) below that of the CDC 2019 novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel (1–3.16 copies/µL). A clinical evaluation with 103 saliva and 103 nasal swab samples also revealed that the performance of CRISPR-FDS in relation to rRT-PCR was similar (PPA = 99%; NPA = 99%) when using either the smartphone-based fluorescence microscope or a plate reader. Furthermore, viral load was also found to be correlated in the 43 saliva samples that were CRISPR-FDS- and rRT-PCR-positive (r = 0.63). Nonetheless, further improvements that include on-chip sample lysis, incorporation of microfluidic channels, and the development of a custom smartphone app for assay regulation and result analysis have been proposed to make the platform more user-friendly for POC testing [24].
Wu et al. [25] demonstrated how a low-cost polypropylene (PP) bag-based approach may be used to facilitate at-home COVID-19 nucleic acid testing [25]. The three-chamber PP bag was designed to be flexible so that mixing could be performed by pressing the chamber with fingers and a foam is also placed in the lid of the PP bag to enable the device to float on water. More importantly, the PP bag allowed simultaneous amplification of the target gene (Orf gene) and an internal control RNase P as well as the subsequent CRISPR-Cas detection to be conducted in a closed environment. To perform the assay, lysis buffer, wash buffer, and RT-LAMP reagents are placed into the respective chamber within the PP bag before a layer of oil is added to form an immiscible interface between the different chambers. Magnetic particles followed by a sample is then added into the lysis chamber and the solution is mixed before the PP bag is incubated at 65 °C for 10 min in a milk warmer. An external magnet is used to transfer the RNA-bound magnetic particles between chambers. Adsorption of the RNA on magnetic particles allows the nucleic acids to be transferred to the wash chamber and finally to the amplification chamber where RT-LAMP takes place at 65 °C for 30 min. The CRISPR-Cas reagent mixture for the target gene is only added at the end of the amplification process and a further incubation at 37 °C for 10 min is required prior to the visualization of fluorescence emission with a portable UV lamp in a dark room. A visible fluorescent signal indicates that the sample is positive, but a negative sample must be verified with a second round of CRISPR-Cas assay for RNase P to rule out a false negative result [25]. The LoD obtained (20 copies/reaction) was the same as that achieved in the tube format with a real-time thermocycler as well as that of the visual-based, one-pot RT-LAMP-CRISPR assay developed by Chen et al. [15].

4. RNA Extraction-Free Protocols

Development of CRISPR-Dx with the capability to detect SARS-CoV-2 from unpurified clinical specimens is of particular interest to researchers due to the reduction in sample-to-result time, the number of liquid-handling steps, and reliance on laboratory-based equipment (such as a centrifuge). Manual RNA extraction involves multiple liquid-handling steps and hence, is tedious and time-consuming particularly when dealing with a large number of samples. Automated RNA extraction instruments provide a walk-away solution and free up personnel for other tasks, but such instruments are expensive, making them useful and cost effective if used in large laboratories with high-throughput testing. For example, the Qiagen EZ1 DSP Virus Kit (Qiagen, Hilden, Germany) that was validated for RNA extraction in the FDA-EUA approved SARS-CoV-2 DETECTR Reagent Kit, has an estimated time per run of 40 min on the EZI Advanced XL instrument (Qiagen, Hilden, Germany). Therefore, it would be ideal, particularly for POC testing, if the RNA extraction could be substituted with a simple specimen processing method such as a 5-min heat lysis step at 80 °C, as described by Xiong et al. [26], that was shown to be capable of liberating SARS-CoV-2 genomic RNA for RT-RPA [26]. Based on visual inspection of the LFD, analytical sensitivity of the RT-RPA-CRISPR-Cas12 assay (1 copy/μL) was 10–50 times more sensitive than RT-RPA alone (10-50 copies/μL) [26]. Similarly, Garcia-Venzor et al. [27] showed that a 25-min heat lysis protocol (42 °C for 20 min; 64 °C for 5 min) with a lysis buffer could be used to generate RNA template for a RT-LAMP-CRISPR-Cas12a assay and measurement of fluorescence with a real-time thermocycler resulted in a LoD of 16 copies/µL [27]. Other RNA extraction-free methods that have been described include a 5-min lysis step at 37 °C [24], a 5-min proteinase K and heat treatment at 95 °C [28], a 30-min heat inactivation step at 65 °C [23], and a 10-min magnetic bead-based purification step [20]. These simplified protocols highlight the potential use of unextracted samples for isothermal amplification techniques to achieve significant reduction in the total assay time of CRISPR-Cas12a-based diagnostics.

5. Sensitivity and Specificity Enhancement Strategies

In an attempt to enhance the sensitivity and specificity of CRISPR-Cas assays as well as to minimize mutational escape, several researchers have chosen to use multiple guide RNAs that bind to different regions of the target gene [9][11][12][14][28][29]. Moreover, the activation of more Cas12a protein per unit time by multiple crRNAs would also lead to greater signal amplification as compared to that of single crRNA, leading to higher sensitivity [29]. In the RT-LAMP-DETECTR assay developed by Brandsma et al. [9], two guide RNAs were used to target different regions of the N gene; the analytical sensitivity of the resulting assay was found to be 10–100 times more sensitive than rRT-PCR when tested with 10-fold dilutions of RNA extract from four COVID-19 patients [9]. Cross-reactivity was not observed with four other human CoVs, suggesting an analytical specificity of 100%. The authors also demonstrated that Cas12 ribonucleoprotein (RNP) was incapable of detecting SARS-CoV-2 RNA following RT even in samples with high viral load (Ct < 20), whereas the combination of RT-LAMP and Cas12 resulted in an assay that exhibited higher sensitivity than either of the methods alone. Evaluation of this RT-LAMP-DETECTR assay with a large cohort of COVID-19 patients (n = 378) from three hospitals showed a 94.9% concordance with rRT-PCR results [9].
Another strategy that may lead to the universal enhancement of CRISPR-Cas12a system is to use engineered crRNA [28][30][31]. By combining the CRISPR-enhanced analysis of nucleic acids with CrRNA extensions (ENHANCE) technology with lateral flow-based detection of the RT-LAMP-amplified N gene, Nguyen et al. [30] showed that the band intensity obtained with engineered crRNA containing a 7-mer AT-rich 3′-overhang was stronger due to the augmentation of Cas12a-mediated collateral cleavage activity as compared to that of un-engineered crRNA [30]. In the S gene-targeting variant nucleotide guard (VaNGuard) assay developed by Ooi et al. [28], the authors not only capitalized on the enhancement brought about by using engineered crRNAs, but also that of an engineered variant of AsCas12a (enAsCas12a) [28]. Compared to LbCas12a, the enAsCas12a was found to be active over a wide range of temperatures (37 °C to 65 °C) and exhibited a higher mismatch tolerance that makes it robust against viral genome mutations. Ooi and colleagues also explored the effect of different crRNA modifications on the collateral activity of enAsCas12a, including 3′- or 5′-extended crRNAs, crRNAs bearing 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro-ribonucleotides, and phosphorothioate linkages as well as DNA–RNA hybrid crRNAs. Using unmodified crRNAs as the benchmark, the DNA–RNA hybrid crRNAs were able to increase the rate of enAsCas12a detection reaction at 60 °C while suppressing collateral activity in the absence of the target sequence to negligible levels [28]. In another study, Ma et al. [29] discovered that manganese ion could increase the sensitivity of Cas12a detection up to 13-fold after screening a series of divalent cations [29]. The manganese-enhanced Cas12a (MeCas12a) system was then developed to detect the RT-RAA-amplified E gene of SARS-CoV-2, but an additional step of desalting the amplicon was required prior to the MeCas12a assay. The MeCas12a assay exhibited a LoD of 5 RNA copies and perfect agreement with rRT-PCR results when evaluated with 24 clinical nasopharyngeal specimens [29].


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