In the workflow of two-step amplification-based CRISPR, pathogens are first rapidly amplified using isothermal amplification techniques. After amplification is completed, the amplified products are manually introduced into the CRISPR reaction system.

The main application examples of amplification-based CRISPR technology include SHERLOCK (Kellner et al., 2019) and DETECTR (Broughton et al., 2020). SHERLOCK focuses on the CRISPR-Cas13 system and achieves sensitivity detection by RPA and transcribing DNA into RNA. The core of this process is the enzymatic activity of CRISPR-Cas13, which recognizes target sequences through crRNA and cleaves them after recognition. A significant advantage of this technology is 100% sensitivity and specificity demonstrated in clinical samples ( Figure 3A ). DETECTR is based on the CRISPR-Cas12 system and is primarily used for DNA detection. It combines RPA with CRISPR-Cas12 to achieve detection of DNA ( Figure 3B ). In two-step amplification-based CRISPR detection, the need to manually transfer amplified products into the CRISPR system not only increases operational complexity but also raises the risk of aerosol contamination.

To address these issues, researchers have explored various strategies to integrate amplification and CRISPR reactions into a single process. Currently, the main solutions include physically separating and optimizing reaction conditions to enabling one-pot reactions.

Firstly, the physically separation can be achieved by modifying the reaction tube design. For example, a double-layer reaction tube was designed, where the inner and outer layers are connected by a thin tube (Hu et al., 2022a). The inner layer is used for RPA and after completion, the amplified products are transferred to the outer layer containing the CRISPR reaction system through centrifugation, thus enabling one-pot detection. Additionally, the CRISPR reaction system was placed on the cap of the reaction tube (Sun et al., 2021). After amplification, the two systems are mixed by centrifugation. This optimized method can detect SARS-CoV-2 within 50 minutes, with a detection limit of 1 copy/μL. Secondly, physical separation can also be achieved by adding dense reagents (such as sucrose, mineral oil, and glycerol). A dynamic aqueous multiphase reaction system (DAMR) that exploits sucrose-density differences to compartmentalize two otherwise incompatible reactions in the same tube was developed (Yin et al., 2020). Paraffin was used to separate the RAA and CRISPR-Cas12a systems, ensuring that sufficient RAA amplicons diffuse to activate the CRISPR-Cas12a reaction (Tan et al., 2024). This method achieves a sensitivity of 2.5 × 10 copies/μL ( Figure 3C ).

Researchers have explored various innovative methods to efficiently integrate the two processes. LAMP and Cas12b-both active at 58°C-were combined to enable one-pot CRISPR detection (Gong et al., 2024). It was found that AacCas12b exhibits minimal cis-cleavage but robust trans-cleavage at 30-39°C, based on this, RPA and CRISPR/AacCas12b were integrated in a single tube to create the Rcod system (Lin et al., 2024). Additionally, a one-pot detection method utilizing suboptimal protospacer adjacent motifs (PAMs) for Cas12a, such as VTTV, TCTV, and TTVV, was developed (Lu et al., 2022). Compared to the traditional TTTV PAM, these suboptimal PAMs exhibit lower binding affinity, thereby reducing the cis-cleavage activity of Cas12a. This allows to more effectively accumulate amplified products, providing sufficient substrates for the Cas12a reaction and offering more flexible crRNA design. A photocontrolled one-pot CRISPR workflow was likewise established (Hu et al., 2022b). However, this inevitably entails low sensitivity and mutual interference between amplification and CRISPR. ( Figure 3D ).

In the CRISPR reaction system, crRNA scans and recognizes specific sequences to activate the Cas protein. The more crRNA species present, the more Cas proteins are activated, thereby enhancing the trans-cleavage activity. In amplification-free CRISPR detection strategies, to activate more Cas proteins and improve detection sensitivity, researchers have employed cascade methods, primarily including multi-protease cascade signal amplification and nucleic acid circuit signal amplification.

The multi-protease cascade signal amplification method mainly involves designing multiple crRNAs on the target. The detection limit for single crRNA was found to be around 80.52 pM, whereas for triple or quadruple crRNAs, the detection limit was between 6.92 and 1.25 pM. The detection limit for multiple crRNAs was 6 to 64 times higher than that for single crRNA (Zeng et al., 2022). Four crRNAs were designed on the target sequence, achieving a detection sensitivity of 0.16 pM (Yang et al., 2024). Similarly, a CRISPR-Cas12a system with multiple crRNAs was utilized to detect microsporidians (Zhang et al., 2024), which not only saved time but also reduced the risk of aerosol contamination, offering a new approach for detecting other pathogens.

Nucleic acid circuit signal amplification involves specially designed nucleic acid fragments to activate a secondary CRISPR reaction. A U-rich hairpin (URH) structure was be designed to replace the reporter molecule, with the loop part rich in uracil preferred for Cas13a cleavage and the stem part containing complementary bases to ensure the stability of the URH structure. In the presence of the target, the CRISPR system is activated, the URH structure is cleaved, and the released stem part serves as a target for secondary cleavage. Without amplification, this method can detect 100 copies/μL of SARS-CoV-2 within 5 minutes (Zeng et al., 2024). However, the method requires operation at low temperatures and is prone to false-positive results. Additionally, the DSAC detection system was developed by introducing circular nucleic acid sequences into the CRISPR reaction system (Zhou et al., 2024c). These sequences are cleaved into double strands by the trans-cleavage activity of Cas12a, thereby activating the CRISPR system. Furthermore, an amplification-free collateral cleavage-enhanced CRISPR-CasΦ method (TCC) was established, enabling pathogen detection with a sensitivity as low as 1.2 CFU/mL within 40 minutes (Chen et al., 2025a). This approach can also be extended for miRNA detection ( Figure 3A ).

However, Cascade CRISPR also bring many challenges. First, the complexity of crRNA design is increased. Second, nucleic acid circuit signal amplification is prone to false-positive results, which can affect the accuracy of detection.

At present, amplification-free CRISPR technology can also use more sensitive sensor devices to efficiently output weak signals. Among them, graphene field-effect transistors (gFET), electrochemical biosensors (ECL), and surface-enhanced Raman spectroscopy (SERS) are the three main types of sensor devices.

gFET achieve ultrasensitive nucleic acid detection by manipulating the conductivity of graphene. gFET consists of a graphene channel, source and drain electrodes connected to the graphene channel, and a gate electrode (Sun et al., 2024). The Cas13a was utilized in combination with a gFET, successfully enabling the detection of SARS-CoV-2 at the aM level within 30 minutes at 37°C (Li et al., 2022c). In this system, negatively charged RNA reporter molecules (such as PolyU20) are immobilized on the surface of the gFET via a molecular linker 1-pyrenebutyrate succinimide ester (PBASE). Upon activation of CRISPR Cas13a, the reporter molecules are cleaved, leading to a reduction in the electron-doping effect on the graphene surface and a positive shift in the charge neutrality point voltage (VCNP). The quantitative detection of target nucleic acids is achieved by measuring the change in VCNP. Further optimized this technology by designing reporter molecules (RP) with stronger electron-doping effects, significantly enhancing the specificity and sensitivity of detection (Sun et al., 2023). However, the stability of graphene in liquid environments remains a challenge. Although hydrophobic treatments (such as OTS coating) can enhance stability, the long-term stability of graphene in complex clinical samples still needs to be verified.

ECL are commonly used signal output devices in amplification-free CRISPR detection, consisting of an electrode system, an electrolyte solution, and an electrochemical indicator. The electrochemical CRISPR (E-CRISPR) method that uses methylene blue (MB)-modified reporter molecules. In the presence of the target, the trans-cleavage of the reporter molecules releases MB, altering the redox reactions on the electrode surface and generating a current signal. This method successfully detected HPV-16, PB-19, and TGF-β1 with a detection limit at the nanomolar (nM) level (Dai et al., 2019). Additionally, methylene blue (MB) and ferrocene (Fc) were employed as electrochemical indicators to detect the S gene and ORF1ab gene of SARS-CoV-2, utilizing the MB current signal in the negative potential range and the Fc current signal in the positive potential range (Kashefi-Kheyrabadi et al., 2023). Electrochemical biosensors offer sensitivity, rapid response, and ease of operation, making them suitable for rapid diagnosis and large-scale screening.

SERS is a spectroscopic technique based on the enhancement of Raman signals by metal nanostructures. SERS technology was utilized for the detection of SARS-CoV-2 without amplification, achieving a detection limit of 1 fM (Liang et al., 2021). This method involves attaching reporter molecules modified with biotin and thiol groups to streptavidin-coated magnetic beads and silver nanoparticles@4-ATP to form SERS probes. After activation of the CRISPR-Cas12 system, the reporter molecules are cleaved, causing the silver nanoparticles@4-ATP to dissociate and resulting in a decrease in Raman signals, with the signal change being proportional to the viral concentration. In addition to 4-ATP, commonly used Raman reporter molecules include 4-mercaptobenzoic acid (4-MBA). As demonstrated in one study, 4-MBA was conjugated to gold nanoparticles for this purpose (Ma et al., 2023). Furthermore, the operation process was significantly simplified by employing centrifugation or polyether sulfone (PES) membrane filtration for signal detection (Liang et al., 2021).

While gFET, ECL, and SERS technologies offer high sensitivity and specificity for amplification-free CRISPR detection, they differ in cost, stability, and operational complexity. gFET sensors require improvement in stability and remain in the laboratory research and development stage, making accurate estimation of their practical large-scale production costs challenging. ECL sensors strike a favorable balance between cost and performance, with relatively low instrument prices (approximately $5,000–$20,000), yet they still face background interference issues that require further optimization to improve the signal-to-noise ratio. In contrast, despite its outstanding detection performance, the high equipment acquisition cost of SERS technology (ranging from about $50,000 to $200,000 depending on configuration) limits its large-scale application in clinical settings. Future advancements in materials science and nanotechnology will drive the development of more stable, cost-effective, and user-friendly sensor devices, thereby laying a critical foundation for the practical application and widespread adoption of amplification-free CRISPR detection technology. ( Figure 3B ).

Digital droplet CRISPR technology is an emerging method for pathogen detection, utilizing microdroplet techniques to achieve quantitative detection of pathogens at the single-molecule level. The core of digital droplet CRISPR technology lies in the integration of the CRISPR system with droplet digital technology. The basic principle of droplet technology is to use microfluidics to divide sample liquids into micrometer-sized droplets, each of which can independently undergo a reaction. STAMP digital CRISPR-Cas13a technology to achieve quantitative detection of HIV-1 RNA, with a detection limit of 2000 copies/ml (Nouri et al., 2023). This technology uses commercial polycarbonate track-etched (PCTE) membranes to digitally partition the reaction mixture, utilizing its high-density and uniform-sized pores to divide the sample into tiny reaction units. Further proposed by combining the CRISPR system and target nucleic acids with an oil phase (90% isopropyl palmitate and 10% Abil EM 180) to generate polydisperse droplets with diameters of 10-100 μm through simple vertexing, with 90% having diameters less than 50 μm (Xue et al., 2023). This method significantly reduces the difficulty of droplet generation, eliminating the need for complex microfluidic devices and enhancing the convenience and accessibility of detection, making it particularly suitable for POCT and pathogen detection in resource-limited environments. By combining sample micro-segmentation with the CRISPR system, it may become a valuable tool for crisis response as the technology matures.( Figure 3C ).

In brief, CRISPR-based nucleic acid detection has develop from amplification-dependent to amplification-free approaches. The two strategies differ in sensitivity, specificity, limitations, and strengths, as summarized in Table 2 .

Viruses are simple-structured, minuscule pathogens (Taylor and Leiman, 2020). During infection, they spread rapidly and widely, and are prone to mutation, especially RNA viruses, a trait that often affects the efficacy of vaccines and drug treatments. In 2020, the SHERLOCK detection technology was optimized, leading to the establishment of a one-pot CRISPR-Cas12b SARS-CoV-2 detection method known as STOPCovid.v1 (Joung et al., 2020). This method integrates magnetic bead extraction, LAMP and LFA, enabling virus detection within one hour. The sensitivity and specificity were 93.1% and 98.5%, respectively, as tested on 202 positive and 200 negative samples. In 2021, an amplification-free CRISPR-Cas13a SARS-CoV-2 detection method was developed, which enabled direct quantification of viral concentration using a mobile phone microscope (Fozouni et al., 2021). This technique, which employs triple crRNA to directly recognize and cleave reporter molecules, can detect the virus within 30–40 minutes, with a sensitivity of 30 copies/μL. The method established is a rapid POCT technology that could be adapted for portable detection, reducing reliance on specialized laboratories and personnel. SATORI (Iida et al., 2023) is an automated digital droplet CRISPR detection platform that can detect SARS-CoV-2 and influenza A/B viruses in just 9 minutes, with a sensitivity of 6.5 aM after magnetic bead enrichment. Mpox, a zoonotic virus (also known as monkeypox virus), has prompted researchers to develop a CRISPR-Cas12a-based detection system that can simultaneously detect mpox virus and other Ortho poxviruses, highlighting its importance in rapidly responding to outbreaks (Singh et al., 2023). Hepatitis D virus (HDV) can accelerate the progression of chronic hepatitis B virus infection and poses a health burden to patients. A CRISPR detection method based on RT-RAA was established, demonstrating a sensitivity of 10 copies/μL in positive samples. Ebola virus infection causes hemorrhagic fever, a disease with an extremely high mortality rate, for which there is currently no specific treatment. An amplification-free Cas-Roller method, which can detect Ebola virus down to 291 aM within 40 minutes at 37°C (Hang et al., 2022). In summary, the application of CRISPR technology, especially the Cas13a system, is gradually changing the way RNA viruses are detected, providing new solutions for viral detection.

In the detection of DNA viruses, the CRISPR-Cas12a system has emerged as a unique and advantageous technology. HPV is considered a major cause of cervical cancer, and timely detection and typing of HPV are crucial for risk prediction and management. A POCT detection system that can detect six high-risk HPV types within 40 minutes, with a sensitivity of 1 copy/μl (Liu et al., 2024). Additionally, an amplification-free CRISPR platform can detect HPV16 (Yu et al., 2023). HBV is closely related to the occurrence of liver cancer and can be transmitted through mother-to-child, blood, and sexual contact. A CRISPR-Cas12a-mediated SERS detection method was established, enabling the detection of HBV DNA at 0.67 pM within 50 minutes (Du et al., 2023). The application of the CRISPR-Cas12a system in DNA virus detection, especially in the detection of HPV and HBV, offering important insights for the development of future pathogen detection technologies. It is worth noting that when using Cas13 to detect DNA, the amplified products need to be transcribed into RNA by T7 (Tian et al., 2022). In the study, DNA amplified by RPA was reverse transcribed into RNA using T7 prior to reaction with CRISPR-Cas13 (Xu et al., 2024). Separately, a CRISPR-Cas14a-based cascade colorimetric detection method was established, capable of detecting ASFV as low as 5 copies/μL and distinguishing mutant ASFV DNA with a 2-nt difference (X. Zhao et al., 2024).

In summary, the application of CRISPR technology in the detection of viral pathogens has demonstrated its superiority in sensitivity, specificity, and rapid response, providing significant technical support for clinical testing and public health (Shokoohi et al., 2025b). With continuous technological advancements and optimizations, future CRISPR detection platforms are expected to detection more viruses.