MnO₂ Nanozyme-Mediated CRISPR-Cas12a System for the Detection of SARS-CoV-2

Abstract

The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3′,5,5′-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.

Recent progress on the CRISPR/Cas system in optical biosensors

“…DNA enzymes have the advantages of molecular recognition and catalytic cleavage activity and are more stable than peroxidase.74 Yin et al proposed a peroxidasemimicking G-quadruplex DNAzyme-based colorimetric DNA barcoding (Cricba) platform to detect food authenticity.75 As shown in Fig.3A, in the presence of target genes, the Gquadruplex underwent cleavage by the activation of Cas12a, leading to the inhibition of its peroxidase-like activity to consequently impede the catalysis of TMB. In addition, oxidaselike nanozymes do not rely on the presence of H 2 O 2 and exhibit certain advantages, simplifying and facilitating the use of colorimetric biosensors.Wu et al presented a MnO 2 nanozyme-mediated Cas12a for the colorimetric detection of SARS-CoV-2 8. In this work, the MnO 2 nanozyme exhibited remarkable oxidase-like activity, catalysing TMB from yellow to blue, and was immobilized on magnetic beads through a ssDNA linker (Fig.3B).…”

“…7 It has great potential in molecular diagnosis as it can identify and break down specific nucleic acid sequences by activating the Cas effector protein with the guidance of RNA (gRNA). 8 However, the CRISPR/Cas9 system can also bind to unexpected regions and initiate division, leading to off-target, 9 genotoxic, and carcinogenic effects. 10 To overcome the limitations mentioned above, other types of Cas proteins, such as Cas12, Cas13, and Cas14, have been identified for widespread applications.…”

Recent Advances in Design and Application of Nanomaterials-Based Colorimetric Biosensors for Agri-food Safety Analysis

“…Qualitative detection of glyphosate was achieved by observing the color change of the nanozyme-based system. Similarly, Wu et al proposed a MnO 2 nanozyme-mediated CRISPR-Cas12a system for SARS-CoV-2 detection . MnO 2 nanorods linked to magnetic beads via an ssDNA linker were used as oxidase-mimicking nanozymes.…”

DNA Tile and Invading Stacking Primer-Assisted CRISPR–Cas12a Multiple Amplification System for Entropy-Driven Electrochemical Detection of MicroRNA with Tunable Sensitivity

“…13 Li and colleagues proposed a liposome-based CRISPR−Cas12a strategy for the surface-enhanced Raman scattering and naked-eye detection of DNA fragments with high sensitivity down to the attomolar level. 14 Integrating functional materials, such as nanozymes, 18 MXene, 19 gold nanoparticles, and so on, with CRISPR−Cas12a has promoted the investigation of sensitive detection strategies. Although these methods showed significant biosensing performance, they still suffer from some major disadvantages including high nonspecific backgrounds, complicated probe designs, and timeconsuming processes.…”