CRISPR-Based Assays for Effective Detection of SARS-CoV-2: History
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Contributor:
Pornchai Kaewsapsak
,
Naphat Chantaravisoot
,
Pattaraporn Nimsamer
,
Oraphan Mayuramart
,
Suwanan Mankhong
,
Sunchai Payungporn

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. 

  • CRISPR
  • SARS-CoV-2
  • COVID-19

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spread worldwide and led to an outbreak. Until 18 July 2022, more than 567 million people have been infected, and 6 million people died due to viral infection or its complications [1]. SARS-CoV-2 is an RNA virus that relies on its unique RNA polymerase enzyme to replicate its RNA in the human host. This process can be erroneous, leading to high mutation rates in RNA viruses. In September 2020, the Alpha variant (B1.1.7) occurred, followed by the spread of the Beta variant. In late 2020, the Gamma variant (P.1) had spread, and the Delta (B.1.617.2) variant had taken over afterward. Now, Omicron variants (both BA.4 and BA.5) are dominating worldwide during 2022 [2]. The mutations in spike proteins in SARS-CoV-2 for different variants helped the virus evade host immunity. For example, the D614G mutation observed in all strains enhances viral replication, infectivity, and virion stability [3].
To prevent the spread of SARS-CoV-2, molecular diagnostic techniques have been used to detect and subsequently quarantine the infected cases. The standard detection technique is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Briefly, RT-qPCR works by converting SARS-CoV-2 RNA to cDNA by reverse transcriptase and then amplifying cDNA by DNA polymerase. This technique has high sensitivity and specificity, but it takes time (more than 2 h) and requires an expensive thermocycler. Hence, there are developments in other techniques, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Both LAMP and RPA are isothermal amplification; hence, they are faster and do not require a thermocycler. However, they are prone to false positives because of non-specific amplification. Many studies have incorporated the CRISPR-Cas system to detect the amplification of the target genes. This incorporation improves the accuracy and specificity comparable to RT-qPCR [4][5].

2. CRISPR-Cas Detection System

CRISPR-Cas system is a bacterial defense mechanism against phage. Cas nuclease can recognize and hydrolyze DNA or RNA targets using CRISPR RNA (crRNA), which has a complementary sequence to the target. This system has been applied for many applications, including gene knock-in and knock-out [6]. However, some of the Cas proteins such as Cas12a, Cas12b, and Cas13 have collateral nuclease activity, meaning that once these Cas proteins recognize the target, they can cleave nucleotide non-specifically. For Cas12a and Cas12b, the Cas protein together with its crRNA recognize double-stranded DNA (dsDNA) target and have the trans-cleavage activity for single-stranded DNA (ssDNA) [7][8]. For Cas13, the Cas protein, along with its crRNA, can hydrolyze the RNA target and later cleave single-stranded RNA (ssRNA) non-specifically [4]. This collateral activity has been utilized for nucleic acid detection.
For CRISPR detection, the major difference between Cas12 and Cas13 is that Cas12 only detects DNA targets with protospacer adjacent motif (PAM) sequence, while Cas13 can detect RNA targets without PAM restriction. To detect SARS-CoV-2, the CRISPR Cas12-based system needs to convert viral RNA to cDNA, followed by DNA amplification and detection by Cas12 protein. In contrast, the CRISPR Cas13-based system needs to turn RNA into cDNA for amplification with the T7 promoter. The resulting DNA was then in vitro transcribed to generate RNA for Cas13 detection. This additional in vitro transcription (IVT) could introduce more complications such as misincorporation from T7 RNA polymerase (about 0.005% error rate) [9].
In general, the limit of detection for the CRISPR-Cas system alone is in picomolar to femtomolar ranges, which is not sensitive enough to detect a few copies of targets in the attomolar range [4][10][11]. Hence, CRISPR-Cas has been combined with nucleic amplification techniques, including PCR, RPA, and LAMP, lowering the detection limit to ~10 copies per µL.

3. Mismatches in the Amplification Step and CRISPR-Cas Detection Step

Three major DNA amplification techniques have been used in combination with CRISPR-based assays. The first one is PCR. This approach is conventional, where the amplification consists of three main steps: denaturation at 95 °C, annealing of primers at 50–60 °C, and extension at 68–72 °C. The second one is LAMP. LAMP uses 4–8 primers binding to distinct regions. With the special design of the assay, DNA polymerase would amplify DNA products with a loop-like structure, enhancing its further amplification of numerous repeat sequences of the target. This reaction occurs at a single temperature of around 60–65 °C. The third one is recombinase-based assay, which is commercialized under recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA), or enzymatic recombinase amplification (ERA). In this technique, the recombinase pairs the primers to the complementary sequence in the DNA target. The resulting displaced strand is secluded by single-stranded DNA-binding protein (SSB), followed by primer extension and DNA amplification by a strand displacing DNA polymerase. This process can happen isothermally at 37–42 °C.
Since SARS-CoV-2 has different mutations across the variants, the primers must be designed carefully to avoid mismatches. PCR has higher primer specificity than LAMP or RPA due to higher temperature during amplification [12]. However, the position of mismatches on the primer could also affect DNA amplification differently. The mismatches within the 3′ ends of primers are typically more detrimental than the internal mismatches [13][14].
In the CRISPR-Cas detection step, mutations of the virus can affect the binding of crRNA, resulting in compromising collateral activity. However, there is no generalizable rule to predict the effect of the mismatch positions on the trans-cleavage activity. Ooi et al. had shown that Cas12a from Acidaminococcus spp. with E174R/S542R/K548R (enAsCas12a) can tolerate mutation better than wild-type AsCas12a and Lachnospiraceae bacterium Cas12a (LbCas12a) [15]. In addition, the study also presented that the mismatches at positions 7–9 and 19 of crRNA have the most detrimental effect, while positions 1 and 17 have less effect. For Cas13, there is no systematic study on how the mismatch affects its collateral activity. However, Abudayyeh et al. reported that a single mismatch in gRNA only minimally affects Leptotrichia shahii Cas13a’s knockdown ability, while double mismatches in any positions in gRNA reduce knockdown efficiency dramatically [16]. For Cas13d, Wessels et al. demonstrated that the knockdown is most affected by mismatches in the seed region of gRNA between nucleotides 15–21 [17].

4. Mismatches in Published CRISPR-Cas Detection System

Firstly, the search was conducted electronically on PubMed from January 2020 to December 2021. The search strategy included keywords within titles or abstracts “((SARS-CoV-2) OR (severe acute respiratory syndrome coronavirus 2) OR (COVID-19) AND (CRISPR))”. Search results were imported into Covidence (https://www.covidence.org/ (accessed on 20 December 2021) for systematic research management. The abstracts of 258 candidate publications were evaluated by three researchers focusing on the research related to the molecular techniques (amplification-free or PCR or LAMP or RPA) combined with a CRISPR-based assay for SARS-CoV-2 detection. Research and irrelevant studies were excluded during the evaluation process. Finally, the primer and crRNA sequences were retrieved from the full texts of 53 candidate research. Genome sequences of SARS-CoV-2, including Alpha, Beta, Gamma, Delta, and Omicron variants, were multiple aligned and then analyzed for primer and crRNA binding sites.
For the compilation, 33% (4/12) of PCR primer pairs have mismatches for different variants, while there are 21% (7/33) and 54% (26/48) of LAMP and RPA/RAA primers containing mismatches, respectively. However, in the PCR-based assay, only one of the crRNAs (1/12) has mismatches with the Omicron variant. For LAMP-based assay, 6% (2/34) of the crRNAs have mismatches with Alpha, Beta, and Omicron variants. For RPA/RAA-based assay, 23% (11/48) of the crRNAs have mismatches with Alpha, Gamma, Delta, and Omicron variants.
Considering which viral genes have the most mismatches in amplification primers, researchers found that the S gene has the most mismatches (82%), while the E and N genes have 50% and 39% mismatches, respectively. This result agrees that the S gene has the highest variation among the genes in the SARS-CoV-2 genome. Strain-wise, the primer pairs have the lowest mismatches for gamma strain (11%), while mismatches in other strains (alpha, beta, delta, and Omicron) are almost double (18%, 21%, 19%, and 22%, respectively).
Next, researchers examined CRISPR-Cas reactions, and researchers found that 15% of the CRISPR-Cas reactions have mismatches between the viral genome and crRNA. Dividing by type of Cas (Cas12 versus Cas13), crRNAs in Cas12 has slightly lower mismatch percentages than crRNA in Cas13 (14% versus 20%, respectively). Strain-wise, crRNAs have the most mismatches with Delta and Omicron (7%), while Beta, Gamma, and Alpha have 2%, 2%, and 4% mismatches, respectively. Considering the target genes, the S gene has the most mismatches with crRNAs (25%), while E, ORF1ab, and N genes have mismatches at 0%, 15%, and 17%, respectively. This suggests that crRNAs of the E gene are robust for SARS-CoV-2 detection. Hence, the E gene could have potential use as a universal detection gene, while the S gene could be beneficial for designing assays to differentiate viral strains.

This entry is adapted from the peer-reviewed paper 10.3390/pathogens11090968

References

  1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Available online: https://covid19.who.int/ (accessed on 18 July 2022).
  2. World Health Organization. Tracking SARS-CoV-2 Variants. Available online: https://www.who.int/activities/tracking-SARS-CoV-2-variants (accessed on 18 July 2022).
  3. Plante, J.A.; Liu, Y.; Liu, J.; Xia, H.; Johnson, B.A.; Lokugamage, K.G.; Zhang, X.; Muruato, A.E.; Zou, J.; Fontes-Garfias, C.R.; et al. Spike Mutation D614G Alters SARS-CoV-2 Fitness. Nature 2021, 592, 116–121.
  4. Gootenberg, J.S.; Abudayyeh, O.O.; Lee, J.W.; Essletzbichler, P.; Dy, A.J.; Joung, J.; Verdine, V.; Donghia, N.; Daringer, N.M.; Freije, C.A.; et al. Nucleic Acid Detection with CRISPR-Cas13a/C2c2. Science 2017, 356, 438–442.
  5. Chen, J.S.; Ma, E.; Harrington, L.B.; da Costa, M.; Tian, X.; Palefsky, J.M.; Doudna, J.A. CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity. Science 2018, 360, 436–439.
  6. Cong, L.; Ran, F.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.; Wu, X.; Jiang, W.; Marraffini, L.; et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339, 819–823.
  7. Li, S.-Y.; Cheng, Q.-X.; Wang, J.-M.; Li, X.-Y.; Zhang, Z.-L.; Gao, S.; Cao, R.-B.; Zhao, G.-P.; Wang, J. CRISPR-Cas12a-Assisted Nucleic Acid Detection. Cell Discov. 2018, 4, 20.
  8. Teng, F.; Guo, L.; Cui, T.; Wang, X.-G.; Xu, K.; Gao, Q.; Zhou, Q.; Li, W. CDetection: CRISPR-Cas12b-Based DNA Detection with Sub-Attomolar Sensitivity and Single-Base Specificity. Genome Biol. 2019, 20, 132.
  9. Huang, J.; Brieba, L.G.; Sousa, R. Misincorporation by Wild-Type and Mutant T7 RNA Polymerases: Identification of Interactions That Reduce Misincorporation Rates by Stabilizing the Catalytically Incompetent Open Conformation. Biochemistry 2000, 39, 11571–11580.
  10. Ramachandran, A.; Santiago, J.G. CRISPR Enzyme Kinetics for Molecular Diagnostics. Anal. Chem. 2021, 93, 7456–7464.
  11. Santiago-Frangos, A.; Hall, L.N.; Nemudraia, A.; Nemudryi, A.; Krishna, P.; Wiegand, T.; Wilkinson, R.A.; Snyder, D.T.; Hedges, J.F.; Cicha, C.; et al. Intrinsic Signal Amplification by Type III CRISPR-Cas Systems Provides a Sequence-Specific SARS-CoV-2 Diagnostic. Cell Rep. Med. 2021, 2, 100319.
  12. Zou, Y.; Mason, M.G.; Botella, J.R. Evaluation and Improvement of Isothermal Amplification Methods for Point-of-Need Plant Disease Diagnostics. PLoS ONE 2020, 15, e0235216.
  13. Ayyadevara, S.; Thaden, J.J.; Shmookler Reis, R.J. Discrimination of Primer 3’-Nucleotide Mismatch by Taq DNA Polymerase during Polymerase Chain Reaction. Anal. Biochem. 2000, 284, 11–18.
  14. Kwok, S.; Kellogg, D.E.; McKinney, N.; Spasic, D.; Goda, L.; Levenson, C.; Sninsky, J.J. Effects of Primer-Template Mismatches on the Polymerase Chain Reaction: Human Immunodeficiency Virus Type 1 Model Studies. Nucleic Acids Res. 1990, 18, 999–1005.
  15. Ooi, K.H.; Liu, M.M.; Tay, J.W.D.; Teo, S.Y.; Kaewsapsak, P.; Jin, S.; Lee, C.K.; Hou, J.; Maurer-Stroh, S.; Lin, W.; et al. An Engineered CRISPR-Cas12a Variant and DNA-RNA Hybrid Guides Enable Robust and Rapid COVID-19 Testing. Nat. Commun. 2021, 12, 1739.
  16. Abudayyeh, O.O.; Gootenberg, J.S.; Konermann, S.; Joung, J.; Slaymaker, I.M.; Cox, D.B.T.; Shmakov, S.; Makarova, K.S.; Semenova, E.; Minakhin, L.; et al. C2c2 Is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector. Science 2016, 353, aaf5573.
  17. Wessels, H.-H.; Méndez-Mancilla, A.; Guo, X.; Legut, M.; Daniloski, Z.; Sanjana, N.E. Massively Parallel Cas13 Screens Reveal Principles for Guide RNA Design. Nat. Biotechnol. 2020, 38, 722–727.
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