Representing Type V systems, Cas12a exhibits unique post-cleavage trans-cleavage activity, non-specifically cleaving single-stranded DNA (ssDNA) reporters upon target engagement. This property enables ultra-sensitive DNA detection (e.g., DNA viruses, SNP genotyping). Cas12a recognizes T-rich PAM sequences (5’-TTTN-3’), expanding targeting scope, and generates sticky ends post-cleavage, facilitating downstream cloning applications (Nouri et al., 2021).

As a Type VI effector, Cas13a uniquely targets RNA rather than DNA. Similar to Cas12a, it activates collateral RNA cleavage upon target binding, making it ideal for RNA virus detection (e.g., SARS-CoV-2, dengue, Zika). Cas13a requires a protospacer flanking site (PFS) and its trans-cleavage activity underpins high-sensitivity RNA detection platforms (Gootenberg et al., 2017).

While Cas9 revolutionized genome editing, its lack of trans-cleavage activity limits direct diagnostic utility. Researchers circumvent this by coupling catalytically inactive dCas9 with signal output modules (e.g., fluorescent reporters) or using it for target enrichment, thereby enhancing detection sensitivity (Xin et al., 2022).

Cas14 represents a class of exceptionally compact RNA-guided nucleases (400–700 amino acids), with most Cas14 proteins being nearly half the size of the smallest Cas9 or Cas12 nucleases. Despite their small size, Cas14 proteins are capable of targeting and cleaving single-stranded DNA (ssDNA) without stringent sequence constraints. Furthermore, target recognition by Cas14 triggers collateral cleavage of ssDNA molecules, an activity that enables applications such as high-precision SNP genotyping (Cas14-DETECTR) (Nguyen et al., 2022).

To overcome limitations of native systems—such as narrow target range, stringent assay conditions, and insufficient sensitivity or specificity—protein engineering and directed evolution have yielded CRISPR-Cas variants that advance diagnostic performance.

AapCas12b is a thermostable variant derived from Alicyclobacillus acidiphilus that maintains high activity at 60–65 °C. Elevated-temperature reactions not only accelerate the assay (to within 5–20 minutes) but also reduce nonspecific amplification (e.g., primer-dimer formation), thereby enhancing the signal-to-noise ratio and sensitivity. Platforms based on AapCas12b have been successfully deployed for rapid screening of pathogens like SARS-CoV-2 (Ali et al., 2022).

By leveraging a catalytically dead Cas9 (dCas9) mutant with retained DNA-binding capability, methods such as Bio-SCAN have been developed. This approach enables precise detection of the SARS-CoV-2 genome without requiring additional reporters, probes, enhancers, or complex readout devices. Incorporating variant-specific sgRNAs further allows efficient discrimination between SARS-CoV-2 variants (e.g., Alpha, Beta, and Delta) (Zhang et al., 2021).

These engineered variants are driving diagnostics toward higher sensitivity, specificity, ease of use, and broader application scenarios.

Developed by Zhang’s group in 2017, this platform utilizes Cas13a/crRNA complexes to recognize specific RNA targets, activating collateral RNA cleavage of fluorescently labeled reporters. The workflow integrates reverse transcription-recombinase polymerase amplification (RT-RPA) or loop-mediated isothermal amplification (LAMP) for target pre-amplification, achieving aM sensitivity (Gootenberg et al., 2017). SHERLOCKv2 (2018) introduced multiplexing capabilities through orthogonal Cas13 variants (Cas13a/b/c) and Csm6 nucleases, coupled with lateral flow assay readout, significantly enhancing field applicability (Gootenberg et al., 2018).

Pioneered by Doudna’s team in 2018, this platform employs Cas12a’s trans-cleavage activity on single-stranded DNA (ssDNA) reporters following target dsDNA recognition (Chen et al., 2018). Combined with RPA isothermal amplification, DETECTR completes detection within 30–60 minutes at aM sensitivity. Clinical validation demonstrated 100% concordance with PCR for HPV16/18 genotyping, establishing its diagnostic accuracy (Zhao et al., 2023).

HOLMES (HOur Low-cost Multipurpose Highly Efficient System) was developed by the Shanghai Institute of Life Sciences, the first-generation HOLMES utilized Cas12a for viral DNA detection and genotyping. HOLMESv2 introduced Cas12b, maintaining activity across 37-60 °C and improving thermal stability. This PCR-CRISPR hybrid system achieves 10 aM sensitivity through synergistic amplification and detection (Li et al., 2019). The operational characteristics, clinical validation status, and a critical appraisal of the strengths and limitations of SHERLOCK, DETECTR, and HOLMES are systematically consolidated in Table 2. This synthesis provides a foundational framework for evaluating their suitability in various diagnostic scenarios discussed in the following sections.

Furthermore, continuous innovation has led to the emergence of derived platforms such as the Cas14-based DETECTR-CAS14, designed specifically for detecting single-stranded DNA viruses and single-nucleotide polymorphisms (SNPs) (Harrington et al., 2018), and the CRISPR-Dx platform, capable of simultaneous detection of both DNA and RNA pathogens (Yoshimi et al., 2022). These ongoing developments enrich the CRISPR diagnostic toolkit, offering expanded options for pathogen detection, genotyping, and mutation analysis.

The most widely used and sensitive method relies on fluorescently labeled reporter molecules. Upon target activation, Cas proteins (e.g., Cas12/13) cleave fluorescence-quenched (FQ) reporter probes, releasing fluorescent signals. These signals are quantifiable via real-time qPCR instruments, microplate readers, or portable fluorometers. Platform-specific examples include SHERLOCK’s RNA reporters (e.g., 6-FAM/UU/3IABkFQ) and DETECTR’s ssDNA reporters (e.g., 6-FAM-TTATT-3BHQ) (Gootenberg et al., 2017). While offering quantitative capabilities and high-throughput potential, this method requires sophisticated equipment, limiting point-of-care utility.

This approach advances field deployment by using biotin/FAM-labeled ssDNA reporters. Cas-mediated cleavage releases FAM-tagged fragments that bind anti-FAM antibodies on test lines (T-line), while biotinylated moieties interact with streptavidin on control lines (C-line). Integrated into SHERLOCKv2 and DETECTR platforms, this method enables visual interpretation within 30 minutes using paper-based strips, achieving fM-level sensitivity (Myhrvold et al., 2018). Though less sensitive than fluorescence methods, its portability and simplicity make it ideal for resource-limited settings.

Other emerging readout modalities include electrochemical sensors and colorimetric assays. Electrochemical sensors integrate CRISPR reactions with electrode systems, detecting target nucleic acids by measuring changes in current, voltage, or impedance. For example, when an ssDNA reporter is immobilized on an electrode surface, activation of Cas12 cleaves the reporter, altering the electron transfer properties of the electrode and producing a measurable electrical signal. This method offers high sensitivity, ease of miniaturization, and system integrability (Dai et al., 2019). Colorimetric methods rely on visual color changes mediated by nanomaterials (e.g., gold nanoparticles) or enzymatic reactions (e.g., horseradish peroxidase, HRP). For instance, activated Cas13 can cleave an HRP-conjugated RNA reporter, modulating color intensity (Kellner et al., 2019). These emerging approaches are driving CRISPR diagnostics toward higher sensitivity, lower cost, and greater portability.

Leveraging the Cas12a system, researchers have developed multiple innovative strategies for human immunodeficiency virus (HIV) detection. These include a CRISPR-Cas12a-PCHA method that activates palindromic-catalytic hairpin assembly for multiplex signal amplification, an amplification-free HIV DNA assay using gold nanoparticles (AuNPs) for dual signal enhancement, and a gel separation-based “one-pot” detection system (Zhao et al., 2022; Zhou et al., 2022; Uno et al., 2023). These approaches enable ultrasensitive detection down to the aM level and facilitate visual diagnosis within 30 minutes. Furthermore, an innovative strategy that converts CRISPR-mediated signals into glucometer readings shows promise for at-home point-of-care HIV testing (Li et al., 2023). Simultaneously, the Cas13a system enables RNA-targeted point-of-care screening via lateral flow strips, while an amplification-free digital CRISPR platform (STAMP-dCRISPR) provides precise viral load quantification comparable to RT-PCR, offering powerful tools for early HIV diagnosis and therapeutic monitoring (Nouri et al., 2023).

The integration of CRISPR-Cas12a/b systems with isothermal amplification has led to efficient detection platforms for hepatitis viruses that belong to RNA viruses. For hepatitis C virus (HCV), the engineered thermostable Cas12b system SPLENDID enables detection within 20 minutes at 67 °C (Nguyen et al., 2023). A single-tube visual platform reduces detection time to 15 minutes with 100 copies/μL sensitivity, while a lateral flow strip-based method achieves 10 copies/μL sensitivity in 30 minutes (Wang et al., 2022; Wei et al., 2024). For hepatitis D virus (HDV), the CRISPR-Cas13a system demonstrates superior performance with an 81.9% positive detection rate in clinical samples, significantly outperforming conventional molecular assays (Tian et al., 2023b). For hepatitis E virus (HEV), CRISPR-Cas13a-based methods achieve detection limits of 12.5 IU/mL and 200 IU/mL for fluorescence and lateral flow readouts, respectively, with a single-tube assay shortening detection time to 35 minutes while maintaining accuracy (Li et al., 2023).

Multiple CRISPR-based approaches have been developed for SARS-CoV-2 detection. Wei et al. employed entropy-driven amplification combined with CRISPR/Cas13a system, achieving 7.39 aM sensitivity via electrochemiluminescence signaling (Wei et al., 2023). Ma et al. developed a CRISPR/Cas12a-based assay integrated with surface-enhanced Raman spectroscopy (SERS), enabling amplification-free detection within 45 minutes with 200 copies/mL sensitivity in a single tube (Ma et al., 2023). Yang et al. integrated CRISPR/Cas13a with catalytic hairpin assembly, attaining 84 aM detection limit within 35 minutes while effectively discriminating SARS-CoV-2 from related viruses (Yang et al., 2023).

Furthermore, The Cas13a-based assay enables direct detection of Ebola virus RNA without amplification, achieving 175 copies/μL detection limit and completing workflow within 40 minutes at 37 °C without pre-amplification or centrifugation (Hang et al., 2022). Additionally, the Naval Medical University established a single-pot RT-RPA-CRISPR/Cas12a system targeting conserved regions across dengue virus serotypes, obtaining an impressive detection limit of 9.17 copies/μL (Zhang et al., 2025).

DNA virus detection primarily utilizes Cas12 proteins (e.g., Cas12a/b) through trans-cleavage of ssDNA reporters. For instance, a Cas12a/QDNB-based lateral flow assay (CQ-LFA) targeting the varicella-zoster virus ORF31 gene demonstrated a limit of detection (LOD) of 0.2 copies/μL, showing 100% concordance with qPCR results across 86 clinical vesicular samples without requiring nucleic acid extraction (Zhong et al., 2023). In CRISPR/Cas12a-based HPV-16 detection, a microfluidic fluorescence assay integrated with a transition-state molecular switch employs a dual screening mechanism to effectively suppress non-specific adsorption, achieving low-background and highly specific detection while distinguishing various HPV subtypes and base mismatches, with LODs of 7.64 pM (mean fluorescence intensity) and 9.91 fM (pixel count) (Sui et al., 2025).

The Cas13 protein can also be applied to DNA virus detection. One study reported a test strip method based on RAA-CRISPR/Cas13a for detecting HBV DNA in patients with low-level viremia, demonstrating a sensitivity of 10¹ copies/μL and 100% specificity (Tian et al., 2023a). Zhang X et al. established a novel CRISPR-Cas13-based method for cccDNA detection by integrating sample pretreatment, amplification, and detection steps. Following rolling circle amplification (RCA) and nested PCR, the assay achieved detection of HBV cccDNA at a sensitivity as low as 1 copy/μL through fluorescence readout (Zhang et al., 2022).

Tuberculosis (TB) remains one of the deadliest infectious diseases globally, with multidrug-resistant TB (MDR-TB) posing substantial diagnostic challenges. Although the traditional phenotypic drug susceptibility testing (pDST) is the “gold standard”, it is time-consuming and delays treatment initiation. Researchers at Shenzhen Third People’s Hospital developed the CRISPR-Cas14a MTB RIF/INH platform for direct detection of rifampicin (RIF)- and isoniazid (INH)-resistant genetic mutations (e.g., rpoB, katG, and inhA genes) in clinical MTB isolates and sputum samples. This platform employs specific gRNAs to distinguish wild-type from mutant sites, integrating multiplex PCR amplification and CRISPR trans-cleavage reactions (Xiao et al., 2025). Beyond Cas14a, Cas12a and Cas13a systems have also been applied for MTB and drug-resistance gene detection, exhibiting high sensitivity and specificity (Ai et al., 2019; Sam et al., 2021).

Methicillin-resistant Staphylococcus aureus (MRSA), a major pathogen in hospital- and community-acquired infections, derives resistance primarily from the mecA gene. Professor Wei Cheng’s team at the First Affiliated Hospital of Chongqing Medical University developed a CRISPR/Cas12a-based method combined with cross-priming amplification (CPA), achieving detection within 30 minutes with a LOD of 5 CFU/mL. Lateral flow strip visualization enables field deployment (Wu et al., 2023). Another study established an RPA-CRISPR Cas13a assay for rapid MRSA resistance gene detection (Gu et al., 2025).

For Salmonella, a common foodborne pathogen, the EXPAR-CRISPR/Cas12a method targets the yfiR gene, achieving a 10 fM LOD on synthetic DNA and successfully identifying Salmonella-containing genomic DNA extracts (threshold: 1 pg/μL) within 1 hour. This approach offers speed, affordability, and high specificity (Lin et al., 2025). A centrifuge-driven microfluidic chip integrated with RPA-CRISPR enabled visual Salmonella detection with 1×10² CFU/mL sensitivity while preventing aerosol contamination (Chen et al., 2025).

Neisseria gonorrhoeae antibiotic resistance is a growing concern, necessitating rapid diagnostics for sexually transmitted infection control. A team at the First Affiliated Hospital of Chongqing Medical University developed an RPA-CRISPR/Cas12a system for equipment-free N. gonorrhoeae detection within 1 hour, demonstrating 100% concordance with traditional culture methods (Tu et al., 2023).

In bacterial pathogen detection, an RAA-CRISPR/Cas13a assay targeting Brucella based on the BCSP31 gene achieved LOD of 100 copies/μL and 10¹ copies/μL for fluorescence and lateral flow readouts, respectively, both with 100% specificity (Liu et al., 2025). For Haemophilus influenzae (HI), the EFORCA platform integrated rapid lysis with RAA and CRISPR technologies, enabling extraction-free single-tube detection of bacterial suspensions at 50 CFU/mL within 30 minutes (Cao et al., 2025).

In antimicrobial resistance detection, Professor Feng Ren’s team established a CRISPR/Cas13a system for precise identification of carbapenem-resistant Klebsiella pneumoniae (CRKP), where the PCR-CRISPR method achieved an LOD of 1 copy/μL for KP, blaKPC, and blaNDM genes, while the RAA-CRISPR method reached 10¹ copies/μl (Feng et al., 2025).

For Mycoplasma pneumoniae, the novel TRACER technology enabled seamless integration of RPA amplification and CRISPR-Cas12b detection in a single tube through precise temperature control, achieving a detection sensitivity of 1 copy/μL (Zeng et al., 2025).

Furthermore, CRISPR has been extended to fungal detection. An ERA-CRISPR/Cas12a system targeting the ITS2 region of Candida albicans was optimized into a one-pot temperature-controlled assay, completing detection within 30 minutes with an LOD of 100 ag/µL. Its high specificity and closed-tube operation effectively prevent cross-reactivity and aerosol contamination (van Dongen et al., 2020).

These findings collectively demonstrate the broad clinical application prospects of CRISPR technology in pathogen diagnostics, particularly highlighting its unique advantages in enabling rapid, highly sensitive, and user-friendly point-of-care testing.

When coupled with isothermal amplification technologies like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), CRISPR assays achieve aM sensitivity, detecting even single-copy viral nucleic acids. For example, SHERLOCKv2 demonstrated a LOD of 1 copy/μL for Zika and dengue viruses, matching or surpassing traditional gold-standard methods like PCR (Gootenberg et al., 2017).

Guided by crRNA, CRISPR-Cas systems exhibit unparalleled specificity, enabling discrimination of single-nucleotide polymorphisms (SNPs). This facilitates precise differentiation of closely related pathogen species, subtypes, or drug-resistant mutations—such as distinguishing all 144 possible combinations of H1-H16 and N1-N9 influenza subtypes (Ackerman et al., 2020).

Traditional qPCR requires hours and specialized thermal cyclers. In contrast, CRISPR diagnostics integrated with isothermal amplification reduce total workflow time (from nucleic acid release to result interpretation) to 30–60 minutes. Simplified protocols minimize hands-on steps and reduce reliance on trained personnel.

A hallmark advantage, CRISPR reactions operate at constant temperatures with results readable via portable fluorometers or naked-eye lateral flow dipsticks (LFD). This enables POCT in resource-limited settings like clinics, airports, and outbreak sites (Kellner et al., 2019). Recent platforms such as hippo-CORDS, PddCas, and CLIPON further advance viral POCT applications (Ackerman et al., 2020; Tang et al., 2022; Xue et al., 2023).

Efficient, rapid, and low-cost extraction of high-quality nucleic acids from complex clinical samples (e.g., sputum, blood, nasal swabs) remains a major bottleneck in achieving fully integrated “sample-to-result” systems. Inhibitors present in samples (such as hemoglobin and mucin) can significantly impair amplification efficiency and CRISPR reaction performance.

Although CRISPR systems themselves are highly specific, the preceding isothermal amplification step requires carefully designed primers to avoid non-specific amplification. Similarly, crRNAs must be meticulously optimized to maximize on-target specificity and cleavage efficiency while minimizing off-target effects and non-specific signals. This imposes high demands on bioinformatics tools and design strategies (Chen et al., 2020).

The activity of Cas enzymes is highly dependent on reaction conditions such as temperature, pH, and ion concentration (e.g., Mg²⁺). Developing Cas variants that maintain high activity and stability across a broad range of conditions, as well as producing lyophilized reagents suitable for storage and transport at ambient temperatures, is essential to ensure reliability and broad applicability (Teng et al., 2019).

The current cost of recombinant Cas enzymes and synthetic crRNAs remains relatively high. Achieving large-scale production and application will require optimization of protein expression and purification processes, development of efficient lyophilization formulations, and miniaturization and cost reduction of detection devices.

Ensuring the reliability of CRISPR diagnostics necessitates building a comprehensive system for quality control, spanning from manufacturing to performance evaluation. On one hand, lot-to-lot consistency of the core Cas enzyme reagents is critical for maintaining stable assay performance. Manufacturing must implement standardized controls for each batch’s activity, purity, and contaminants to minimize variations in sensitivity or background signal caused by reagent variability, thereby ensuring long-term comparability of patient results. On the other hand, the field urgently requires standardized reference materials, such as accredited pseudoviral particles or synthetic biological samples with precisely defined concentrations. Such universal “yardsticks” enable objective comparison and independent verification of performance across different platforms, provide a basis for regulatory assessment and inter-laboratory result alignment, and ultimately empower clinicians to select and trust diagnostic products based on harmonized criteria.

As in vitro diagnostic (IVD) devices, CRISPR-based tests must undergo stringent regulatory review (e.g., by the FDA, CE, or NMPA) (Mustafa and Makhawi, 2021). The clinical translation of CRISPR-based diagnostics follows distinct regulatory pathways, the choice of which determines a product’s intended use and evidentiary standard. Emergency Use Authorization (EUA) facilitates rapid deployment during public health emergencies, relying on relatively limited clinical data and intended for temporary screening purposes. In contrast, obtaining full in vitro diagnostic (IVD) approval requires evidence from large-scale prospective clinical studies, along with the establishment of a complete quality system and post-market surveillance. This pathway represents the ultimate goal for a product to be used as a routine diagnostic tool guiding long-term clinical decision-making. Clinicians must understand this hierarchical nature of regulatory approvals to critically evaluate the strength of evidence and the appropriate context for applying results from different products.