The primary purpose of the CRISPR/Cas system is to identify target nucleic acids via crRNA and to initiate the nucleic acid cleavage activity of Cas proteins, which includes both the precise cleavage of target nucleic acids and the collateral cleavage activity of non-specific reporter molecules (Kaminski et al., 2021). The Cas12a protein can non-specifically cleave single-stranded DNA (ssDNA) (Chen et al., 2018). Using this attribute, ssDNA can be altered on the surface of nanozymes to function as a “molecular switch.” After target recognition, CRISPR/Cas12a cleaves ssDNA on nanozymes’ surfaces, altering their surface charge, dispersibility, or active-site exposure and thus modulating their catalytic activity (Liu et al., 2015; Zeng et al., 2019). For instance, the MnO₂ nanozyme-mediated CRISPR/Cas12a colorimetric approach delineated by Wu et al. (2022), wherein the activation of Cas12a triggers the cleavage of ssDNA affixed to the MnO₂ nanozyme surface, reactivating the nanozyme-catalyzed chromogenic reaction for the detection of SARS-CoV-2 (Wu et al., 2022). Furthermore, Tao et al. (2025) showed a MoS₂ quantum-dot system in which Cas12a-regulated DNA cleavage on MoS₂ QDs alters the enzyme-mimicking properties, facilitating sensitive colorimetric detection of viral DNA targets (Tao et al., 2025). Collectively, these examples highlight a common design logic: CRISPR/Cas trans-cleavage acts as the “gate” that rewires nanozyme surface chemistry into a measurable catalytic signal, providing a compact and modular coupling strategy.

The improved catalytic activity of nanozymes allows multi-tier amplification of the CRISPR/Cas system recognition signal, addressing the issue of limited signal intensity in the conventional CRISPR/Cas system (Broto et al., 2022; Shamsabadi et al., 2024). This process generally combines CRISPR-mediated target identification with the catalytic activity of nanozymes, creating a sequential “recognition-cleavage-catalytic amplification” response (Babaei et al., 2025). In a standard design framework, crRNA recognition initiates Cas trans-cleavage to cleave various nucleic-acid linkers, releasing numerous nanozymes. Each nanozyme catalyzes multiple substrate molecules to provide enhanced optical or electrochemical outputs (Chen et al., 2023; Xu et al., 2023; Luo et al., 2024).

For example, Zhang et al. (2023) created a nanozyme-mediated amplification-free CRISPR system. It employs the collateral cleavage activity initiated by Cas12a following target recognition to cleave the DNA linker that connects nanozymes to magnetic beads, releasing the nanozymes from the magnetic bead surface. The released nanozymes facilitate the chromogenic process. A single Cas12a can cleave numerous DNA linkers to release multiple nanozymes, with each nanozyme capable of catalyzing several substrate molecules. This results in multi-level signal amplification, enabling the visual detection of target nucleic acids without the need for nucleic acid amplification steps, reducing the detection time to 30 min (Zhang et al., 2023). Mechanistically, this coupling mode emphasizes reporter multiplicity and catalytic turnover rather than solely changing the intrinsic activity of a single nanozyme particle.

Beyond activity gating and catalytic amplification, because of the assembly characteristics of nanomaterials, the CRISPR/Cas system and nanozymes can be systematically organized into functional complexes, which would increase their synergistic effect and strengthen the specificity and stability of detection (Liu S. et al., 2024; Sui et al., 2025; Zhang X. et al., 2025). In practice, this coupling mode often relies on programmable co-localization (e.g., DNA-guided assembly) or confined microenvironments (e.g., nanostructured scaffolds/nanoflowers) to increase effective local concentrations, suppress nonspecific interactions, and stabilize catalytic activity under challenging conditions.

Jiang et al. (2024) proposed a dCas9-mediated dual-signal platform that facilitates target-dependent assembly of Au–Pt nanozyme units, thus simultaneously amplifying catalytic and SERS responses, which enables high-specificity detection, including single-base discrimination (Jiang et al., 2024). Moreover, Qiu et al. (2024) developed a self-assembled bifunctional nanoflower-based CRISPR/Cas platform for dual-readout detection of Salmonella enterica, demonstrating that confined microenvironments and co-encapsulation can increase assay stability and efficacy in complex matrices (Qiu et al., 2024). Overall, this coupling mode highlights that assembly is not merely a packaging step. It can be a functional design lever that improves robustness, reduces interference, and supports integrated readouts, which is particularly relevant for practical deployment.

DNA-target detection includes bacterial genomic markers, viral sequences, and clinically significant biomarkers (Niemz et al., 2011; Kellner et al., 2019; Broughton et al., 2020). Nanozyme-mediated CRISPR assays are particularly beneficial in this field. Collateral-cleavage CRISPR nucleases, such as Cas12a and Cas14a, convert sequence recognition into collateral cleavage, whereas nanozymes transform these molecular events into enhanced colorimetric, electrochemical, or electrochemiluminescent (ECL) outputs (Chen et al., 2018; Harrington et al., 2018; Huang et al., 2019; Li C. et al., 2023; Arshad et al., 2024).

Li et al. (2023) presented a CRISPR Cas14a electrochemical biosensor, employing PtPd@PCN-224 nanozymes as catalytic signal tags to enhance the hydrogen peroxide reduction current, demonstrating a characteristic electrochemical format that promotes catalytic signal amplification (Figure 1A). In the presence of target DNA, activated Cas14a initiates trans-cleavage of phosphorylated ssDNA at the electrode interface, inhibiting the assembly of PtPd@PCN-224 on the electrode through Zr-O-P coordination and resulting in a signal-off reduction in current, thus facilitating ultrasensitive detection of pathogenic bacterial DNA (Li C. et al., 2023).

Arshad et al. (2024) proposed an electrochemical transduction approach by creating a CeO₂ nanozyme-mediated RPA-CRISPR/Cas12a dual-mode biosensor targeting the invA gene of Salmonella (Figure 1B). This strategy involves the initial amplification of the invA sequence via RPA, which then activates the CRISPR/Cas12a-crRNA complex to cleave a FAM-quencher ssDNA reporter, producing a fluorescent signal for sequence-specific identification. The technology employs a CeO₂ nanozyme-mediated colorimetric readout for orthogonal validation. The technology utilizes the inherent oxidase- and peroxidase-like properties of CeO₂ in the presence of H₂O₂ to produce a target-dependent chromatic transition, providing reliable dual-signal confirmation. This assay attained a minimal detection threshold of 10 CFU/mL and exhibited strong anti-interference characteristics in complex food matrices, including milk and chicken (Arshad et al., 2024).

RNA-target detection focuses on viral genomes and regulatory non-coding RNAs (Abudayyeh et al., 2016; Barnes et al., 2020; Yang and Patel, 2024). Relative to DNA targets, RNA analytes are generally rarer, more prone to degradation, and frequently considerably smaller, particularly miRNAs (Garneau et al., 2007; Dong et al., 2013; Jet et al., 2021). These attributes require robust signal transduction and efficient background suppression (Dong et al., 2013; Chen F. et al., 2022). For this, nanozymes provide high-turnover catalytic amplification and adaptable readout modalities, enhancing the programmability of CRISPR systems (Thamilselvan and Kim, 2024; Zhang L. et al., 2025).

Wu et al. (2022) presented a MnO₂ nanozyme-assisted CRISPR/Cas12a platform for the fast detection of the SARS-CoV-2 ORF1ab gene in RNA viruses. The method initially transforms viral RNA templates into double-stranded DNA (dsDNA) intermediates by reverse transcription-recombinase polymerase amplification (RT-RPA). Subsequently, the Cas12a-crRNA complex selectively identifies this DNA, initiating trans-cleavage activity that cleaves ssDNA linkers modified on the surface of magnetic beads, therefore releasing the immobilized MnO₂ into the supernatant. The released MnO₂ facilitates TMB color formation through its peroxidase-like activity. This method produces visually discernible outcomes in 45 min, indicating great potential for point-of-care epidemiological surveillance (Wu et al., 2022).

In addition to infections, non-coding RNAs function as essential biomarkers in cancer, although their detection is complicated by their low quantity. Broto et al. (2022) devised the CrisprZyme approach, which combines Cas13/gRNA-mediated RNA detection with an NLISA-type colorimetric output facilitated by extremely efficient Pt@Au nanozymes (Figure 1C). This technique utilizes Cas13 and gRNA recognition to initiate collateral cleavage of a tagged reporter RNA, while the remaining intact reporter binds streptavidin-functionalized Pt@Au nanozymes via biotin contact, catalyzing the oxidation of a chromogenic substrate for quantitative analysis. The authors showed significant applicability across microRNAs, long non-coding RNAs, and circular RNAs in complex samples, including oncological tissue biopsy analysis utilizing circ-AURKA to differentiate between prostate cancer subtypes (Broto et al., 2022).

To improve sensitivity, Shen et al. (2022) integrated miRNA-initiated rolling circle amplification (RCA) with Cas12a trans-cleavage and a hybridization chain reaction circuit, introducing a nanozyme cascade readout that combines beta cyclodextrin-modified gold nanoparticles with an Fe-based MOF nanozyme NH₂-MIL-88B to promote dual photoelectrochemical and colorimetric outputs (Figure 1D). This multi-amplification system attained sub-femtomolar analytical sensitivity, with detection limits of 0.3 fM for photoelectrochemistry and 0.5 fM for colorimetry, facilitating accurate miRNA differentiation in complex conditions (Shen et al., 2022).

Collectively, the above examples illustrate that nanozyme-assisted CRISPR nucleic-acid assays are evolving from proof-of-concept signal conversion toward designs that explicitly optimize matrix tolerance, detection confirmation, and operational convenience. Therefore, A new platforms can be guided by measurable engineering criteria, including time to result, number of manual handling steps, susceptibility to background in real matrices, and the degree to which the readout is quantitative and portable. This analysis helps clarify why specific architectures are selected for distinct nucleic-acid use cases.

Protein biomarkers are essential for clinical screening and disease management; however, their detection by CRISPR systems is limited by the inability of the Cas-crRNA combination to directly recognize them (Tang et al., 2022; Hartl et al., 2023; Carrasco-Zanini et al., 2024). Therefore, the majority of CRISPR-based protein assays depend on an aptamer or antibody to transform a protein-binding event into the release or production of an activator DNA that can initiate the trans-cleavage of the CRISPR/Cas system (Li et al., 2021; Tang et al., 2022; Paialunga et al., 2025). The nanozyme component functions as the signal output and amplification module, converting CRISPR-mediated nucleic acid cleavage into a visual or electrochemical alteration (Mu et al., 2023).

Mu et al. (2023) established a visual biosensor for carcinoembryonic antigen (CEA) by integrating rolling circle amplification (RCA), CRISPR/Cas12a, and peroxidase-mimicking DNA–Ag/Pt nanoclusters (NCs) (Figure 2A). The design involves CEA binding to an aptamer complex, which releases a primer strand that promotes padlock probe circularization and initiates RCA, resulting in the production of long repetitive DNA products comprising Cas12a activator sequences; a subsequent nicking step increases the quantity of activator to activate Cas12a. The activated Cas12a subsequently cleaves the DNA scaffold on the Ag/Pt nanoclusters, thus inhibiting the peroxidase-mimetic activity and reducing the UV–vis signal. The assay, employing cascade amplification from RCA and CRISPR, achieved a linear range of 2.5 pg/mL to 2.0 ng/mL, with a detection limit of 0.94 pg/mL, and was verified with serum samples.

The identification of small compounds, including mycotoxins, metabolites, and medication residues, is crucial for guaranteeing food safety and enhancing clinical monitoring (Li et al., 2018). Nanozyme-assisted CRISPR platforms address the inherent limitations of small-molecule detection, such as low molecular weight and the absence of multiple binding epitopes, by combining aptamer-based affinity ligands with the trans-cleavage functionality of Cas proteins, converting small-molecule binding events into substantial signals (Niu et al., 2021; Li et al., 2022; Zhu and Zhao, 2023; Liu M. et al., 2024; Zhu et al., 2025).

Wu et al. (2024) integrated a nanozyme–CRISPR cascade into an electrochemical, centrifugal microfluidic system for the detection of ochratoxin A (OTA). Pd@PCN-222 nanozyme catalyzes the reduction of H₂O₂, producing a reduction current. In the presence of OTA, competitive binding between OTA and the aptamer strand releases cDNA, which subsequently binds to crRNA and activates Cas12a trans-cleavage; the activated Cas12a then cleaves ssDNA connecting Pd@PCN-222 and the electrode, resulting in a reduction of Pd@PCN-222 at the electrode interface and decreasing the H₂O₂ reduction peak current, facilitating preamplification-free detection with a reported detection limit of 1.21 pg mL⁻¹. The on-chip incubation and trans-cleavage processes were programmable, allowing on-site workflow integration (Wu et al., 2024).

Esmaelpourfarkhani et al. (2024) established a CRISPR-Cas12a colorimetric aptasensor for the detection of Aflatoxin M1 (AFM1) in a complementary solution-based colorimetric format by manipulating the quasi-oxidase activity of flower-like MnO₂ nanozymes through a CRISPR/Cas12a system (Figure 2B). In the absence of AFM1, CRISPR/Cas12a is activated and randomly cleaves the ssDNA, restoring the elevated oxidase-like activity of the MnO₂ nanoflowers and generating a pronounced absorbance signal due to enhanced TMB oxidation. In the presence of AFM1, the CRISPR/Cas12a module remains inactive, resulting in the retention of ssDNA on the nanozyme surface, which inhibits nanozyme activity, restricting TMB oxidation and yielding a low absorbance. The detection limit is as low as 0.05 ng/mL, and the detection outcomes in milk samples correspond with those obtained using high-performance liquid chromatography (Esmaelpourfarkhani et al., 2024).

Heavy metal ions (e.g., Pb²⁺ and Hg²⁺) are persistent, non-degradable contaminants that can bioaccumulate and demonstrate significant toxicity at low concentrations, resulting in multi-organ damage. Therefore, there is an urgent need to develop rapid and sensitive detection technologies (Balali-Mood et al., 2021; Pan et al., 2024; Yang et al., 2025). To address the restricted signal transduction and vulnerability to background interference frequently observed in ion-triggered experiments, the integration of the CRISPR/Cas system with a nanozyme readout system improves the amplification of catalytic signals (Xu et al., 2023; Meng et al., 2025).

Xu et al. (2023) developed a DNAzyme-activated, MnO₂ nanozyme-mediated CRISPR/Cas12a colorimetric protocol for the detection of Pb²⁺ without preamplification (Figure 2C). Pb²⁺ is specifically identified by the GR-5 DNAzyme, which cleaves it at the “rA” site, resulting in the release of a short trigger DNA (tDNA) that subsequently activates the CRISPR/Cas12a system. Activated Cas12a cleaves the ssDNA linker connecting MnO₂ nanorods to magnetic beads (ssDNA-MnO₂-MBs), resulting in the cleavage of MnO₂ and a reduced TMB oxidation signal. The assay demonstrated a linear range of 0.8–2,500 nM, with a stated limit of detection (LOD) of 0.54 nM. Furthermore, specificity can be improved by dual recognition, but sensitivity is increased by MnO₂ serving as a stable catalytic signal probe that amplifies color output without target preamplification.

Overall, extending nanozyme-assisted CRISPR diagnostic technology to non-nucleic acid targets shifts the central challenge toward reliable molecular transduction. Future platform development is likely to benefit from transducer designs that minimize background, and tolerate complex sample matrices, as well as integrated devices that reduce manual steps while preserving the catalytic gain of nanozyme reporters.