Progress in applying CRISPR in pathogen detection

Heating/chemical pyrolysis methods

In 2018, Gootenberg et al.’s (2018) team proposed a scheme called heating unextracted samples to eliminate nucleases technology (HUDSON), which utilizes heating and chemical reduction to lyse virus particles and inactivate high concentrations of RNase, thereby eliminating the nucleic acid extraction step in the detection process (Fig. 2A) (Myhrvold et al., 2018). HUDSON is applicable to a variety of clinical samples (including urine, whole blood, plasma, serum, and saliva) and can destroy viral particles and inactivate RNase within 10 min without dilution or purification and without affecting subsequent amplification or detection. The protocol has been widely applied to SHERLOCK, DETECTR, Streamline Highlights of Infections to Navigate Epidemics (SHINE), and other major detection platforms (Table 2) (Aquino-Jarquin, 2019; Arizti-Sanz et al., 2020; Myhrvold et al., 2018). However, HUDSON cannot achieve good detection results when facing relatively high concentrations of nuclease. Therefore, the SHERLOCK Rapid Parasite Extraction Protocol (S-PREP) was proposed for high-specificity parasite detection without the need for nucleic acid extraction (Lee et al., 2020). In the S-PREP scheme, Chelex-100 (with paired iminodiacetate ions) was first used as a chelating agent that can bind polyvalent metal ions. Because a nuclease requires metal ions as cofactors, the chelating agent can inhibit its activity. In this case, the enzyme can then be heated at 95 °C for 10 min to inactivate the nuclease. It can inactivate a high level of nuclease without nucleic acid extraction, and its detection specificity for Plasmodium species reaches 100%. In addition, to eliminate the equipment needed for sample heating, the sample was treated with FastAmp lysis reagent supplemented with 5% RNase inhibitor in SHINEv.2, and the RNase activity was reduced to 85% at room temperature (Arizti-Sanz et al., 2022). Compared with SHINE, SHINEv.2 eliminates the need for heating steps and reagent refrigeration, enabling equipment-free SARS-CoV-2 detection with a sensitivity of 200 copies/µL.

Although the heating/chemical pyrolysis method is compatible and has been widely verified in various pathogen tests, the lack of nucleic acid purification and concentration steps may compromise the ability of CRISPR to detect microtargets in complex matrix samples.

Magnetic bead purification method

To further enhance the sensitivity of extraction-free CRISPR/Cas detection for trace pathogen nucleic acids, it is necessary to employ simple and efficient methods for separating and purifying cell lysates from complex matrices. Nanoscale micromagnetic beads possess unique surface characteristics and can be rapidly immobilized in the target region to facilitate the separation and purification of nucleic acids released into sample lysates (Fig. 2B) (Chen et al., 2021). For example, in CRISPR-mediated DNA-FISH, Ni-NTA magnetic beads were used to pull out the cell lysate sample directly, eliminating the need for gene purification and the subsequent nucleic acid extraction step (Guk et al., 2017). In addition, in the SHERLOCK One-Pot testing (STOP), COVID-19 uses magnetic bead adsorption to concentrate the SARS-CoV-2 RNA genome into a STOPCovid reaction mixture, thus eliminating the ethanol washing and RNA elution steps and shortening the sample extraction time to 15 min with the least manual operation time (Table 2) (Joung et al., 2020).

Magnetic bead purification can quickly concentrate and purify the target from large-volume samples without tedious operation steps, thereby improving the sensitivity of detection and increasing its applicability, which further promotes the field deployment of CRISPR/Cas detection. However, magnetic bead purification carries the risk of bead residue and is dependent on cost and equipment, with significant consumable costs in high-throughput applications.

Others

In addition to nucleic acid purificatio using micromagnetic beads, nucleic acid isolation membranes formed by nanoscale or microscale fibers can also be employed for efficient nucleic acid purification and concentration from many samples at a lower cost than the magnetic bead purification method (Fig. 2C) (Rodriguez et al., 2015). The principle involves rapid and efficient separation of nucleic acids from lysed samples in high-salt solutions via electrostatic interactions and hydrogen bonding. The nucleic acids adsorbed on these membranes bind stably via simple washing, allowing for the removal of pollutants, and can be amplified in situ without elution. For example, with the minimum-instrument SHERLOCK (miSHERLOCK), SARS-CoV-2 RNA was isolated from two mL of saliva using gravity and capillary action through a four mm polyether sulfone (PES) membrane (Table 2) (De Puig et al., 2021). The simplicity of this design enables instrument-free, intuitive liquid handling, achieving significant sample concentrations that enhance the overall signal by a factor of 2 to 20. Further studies have reported that microfluidic technology enables the rapid purification and concentration of nucleic acids in small-volume samples (Fig. 2C) (Obino et al., 2021). This technology can integrate sample preparation, amplification, the CRISPR reaction, and signal readout steps into a microfluidic platform, simplifying detection and potentially improving its analytical and diagnostic performance (Ramachandran et al., 2020).

In addition, the solid-phase extraction and enhanced detection assay, integrated with CRISPR-Cas12a (SPEEDi-CRISPR), utilizes the sequence recognition of CRISPR/Cas12a for nucleic acid extraction for the first time, integrating CRISPR/Cas enrichment and detection capabilities into a single platform (Fig. 2D) (Tian, Yan & Zeng, 2024). Cas12a-crRNA RNPs are first bound to the surface of magnetic beads, and the target DNA in the sample is captured on the beads through sequence recognition by the RNP, activating the trans-cleavage activity of Cas12a and generating a signal through the cleavage of the ssDNA reporter. The platform is capable of detecting human papillomavirus 18 (HPV-18) at concentrations as low as 2.3 fM in 100 min and 4.7 fM in 60 min. The simplicity and compatibility of its equipment facilitate POC testing.

Signal conversion strategy

Electrochemical technology is an ultrasensitive detection technology that can convert the extremely weak signal produced by CRISPR/Cas trans-cleavage into a detectable signal, enabling low-cost, rapid and simple ultrasensitive detection. Dai et al. (2019) combined an electrochemical biosensor with the CRISPR/Cas system for the first time to develop a CRISPR/Cas12a-based electrochemical biosensor (called E-CRISPR). Methylene blue (MB)-modified ssDNA was paired on the electrode surface. When the target nucleic acid is present, complementary Cas12a-crRNA binds to the target DNA and activates its trans-cleavage activity. The ssDNA-MB was cleaved, and the MB was released, resulting in electron transfer that allowed the target to be detected directly by monitoring the change in electrochemical current. The limit of detection (LOD) of human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19) by this platform reach picolar levels. The same team then proposed a CRISPR/Cas system-enhanced electrochemical DNA sensor (E-DNA), which can induce a conformational change in the surface signal probe (containing the electrochemical tag MB) upon binding to the target, resulting in a change in the electron transfer rate of the electrochemical tag (Xu et al., 2020). The platform utilizes hairpin DNA as a signal probe, which causes the MB to move closer to the surface of the gold electrode, thereby increasing the current response. Cis-cleavage mediated by Cas9 or Cas12a resulted in the separation of MB from the probe after recognition of the PAM and target sequence, leading to a change in the electron transfer rate of MB. Consequently, the detection sensitivity of PB-19 reached the fM level. Another study combined silver metallization with Cas12a to propose a new silver-enhanced E-CRISPR biosensor (E-Si-CRISPR) (Suea-Ngam, Howes & De Mello, 2021). The ssDNA fixed on the electrode is cleaved by the Cas12a:crRNA:target DNA ternary complex, followed by silver metallization, and the degree of silver deposition is proportional to the amount of remaining ssDNA. The final electrochemical signal can be read via square wave voltammetry (SWV). Using this platform, the sensitivity of the MRSA mecA gene was improved to the fM level.

Graphene field-effect transistors (gFETs), as sensitive and advanced electronic devices, can achieve hypersensitive signal conversion and amplification. A field-effect transistor (FET) is a three-terminal electronic device with a gate that can regulate the source–drain current through the semiconductor channel by applying an external electric field to the gate (Liang et al., 2020). Graphene is a low-cost zero-gap material with extremely high electron carrier mobility and has been used for high-current-gain FETs. Bruch, Urban & Dincer (2019) proposed a biosensor based on CRISPR/dCas9-gFETs. The detection principle involves fixing the target-specific dCas9-crRNA on the surface of gFETs and regulating the electrical characteristics of gFETs through specific binding between the target and the dCas9-crRNA complex for sensor signal transduction. The platform can produce an electrical output with a LOD of 1.7 fM within 15 min. Later, Li et al. (2022a) developed a biosensor based on CRISPR/Cas13a-gFET, which fixes a negatively charged RNA reporter (PolyU20) on gFET. When the target is present, the trans-cleavage activity of Cas13a is activated, the ssRNA reporter is shed, and a decrease in the number of electron carriers in the graphene channel is observed. Compared with that of CRISPR/dCas9-gFET, the multi-turnover trans-cleavage of Cas13a leads to high-speed signal generation, which can detect SARS-CoV-2 and respiratory syncytial virus (RSV) genomes as quickly as 1 aM.

Signal enhancement strategy

The key factor in ultrasensitive detection is to maximize the detection signal intensity. Gold nanoparticles, as highly sensitive nanomaterials, can achieve amplification-free detection by enhancing the signal readout. Choi et al. (2021a) utilized ssDNA-functionalized AuNPs and CRISPR/Cas12a-mediated trans-cleavage to achieve target amplification-free detection, employing the principle of metal-enhanced fluorescence. The target DNA sequence activates the CRISPR/Cas12a complex, which subsequently degrades the ssDNA between the AuNPs and the fluorophores, resulting in a colour change from purple to reddish purple. The system can achieve high-sensitivity detection within 30 min. In addition, Fu et al. (2021) designed a spherical nucleic acid (SNA) reporter using gold nanoparticles and developed a stable and sensitive CRISPR assay. An SNA reporter was formed by immobilizing fluorescently labelled ssDNA on the gold nanoparticle (AuNP) core. Due to the high quenching efficiency of AuNPs for fluorescent chromophores, the platform enables high signal-to-noise ratio DNA detection at the femtomolar (fM) level. In addition, nanoenzymes are nanomaterials with enzyme-like catalytic activity that play a crucial role in enhancing the sensitivity of CRISPR-based detection, enabling both visual and rapid CRISPR detection (Zhang et al., 2024b). Labeling recognition molecules (such as antibodies and DNA) with nanoenzymes instead of natural enzymes can enhance signal readout, resulting in highly sensitive colorimetric or ratio fluorescence sensors (Chi, Wang & Gu, 2023; Wu et al., 2024a).

Surface-enhanced Raman scattering (SERS), a signal enhancement technique, can manipulate the surface plasmon effect on a metal surface and further enhance the Raman vibration signal of the target, allowing for the detection of low-concentration biomolecules with extremely high sensitivity (Dai et al., 2021). Su et al. (2023) combined the CRISPR/dCas9 system with SERS detection and enzyme-catalyzed reactions to develop a CRISPR-SERS assay. The CRISPR/dCas9/sgRNA complex immobilized on magnetic beads facilitates DNA enrichment and separation. Horseradish peroxidase (HRP) was introduced into biotinized target DNA to catalyze the substrate TMB (3,3′,5,5′-tetramethylbenzidine) to produce a blue–green oxidation product (oxTMB), which can provide colorimetry and SERS signal output, and the SERS spectrum of oxTMB was measured by gold nanostars with a silica shell to achieve double amplification. This platform can rapidly detect the ng level of HPV-DNA without the need for preamplification.

In addition, the sensitivity of SERS-based detection can be further enhanced by utilizing the surface plasmon effect of precious gold nanoparticles. Choi et al. (2021b) developed an amplification-free CRISPR/Cas12a-SERS sensitive detection platform based on Raman probe-functionalized gold nanoparticles (RauNPs). In this platform, ssDNA-RauNP probes are immobilized on graphene oxide (GO)/triangular gold nanoflower (TANF) arrays to enhance the SERS signal through GO-TANF arrays. In the presence of target viral DNA, the CRISPR/Cas12a complex was activated to cleave ssDNA between RauNPs and GO-TANFs. The platform can detect hepatitis B virus (HBV), HPV-16 and HPV-18 with a sensitivity of 1 aM within 20 min. Du et al. (2023) combined gold nanoparticles (AuNPs) with magnetic spheres as Raman probe molecules and proposed a CRISPR/Cas12a-based surface-enhanced Raman spectroscopy assay. The SERS signal was enhanced by the AuNPs modified with the Raman reporter molecule 4-ATP. AuNPs were attached to the magnetic spheres via ssDNA to achieve the maximum SERS effect and a differential Raman spectral signal. The target gene could activate the Cas12a/crRNA complex to cleave ssDNA attached to the AuNPs and magnetic spheres. Therefore, the system can detect HBV DNA rapidly and sensitively within 50 min.

Target/signal enrichment strategy

Using ultralocal or closed microvolume reactors (such as droplet microfluidics, microchamber arrays, and micromagnetic beads) to highly aggregate local, low-concentration targets or signals can achieve single-molecule sensitivity detection. For example, Tian et al. (2021) proposed a CRISPR/Cas13a assay based on the droplet microfluidic restriction effect for single-molecule RNA diagnosis without amplification or reverse transcription. The platform utilizes droplet microfluidics to confine the target RNA-triggered Cas13a catalytic system in a cell-sized reactor, thereby simultaneously increasing the local concentrations of both the target and the reporter. Compared to batch Cas13a detection, the sensitivity is improved by more than 10,000 times, enabling absolute digital quantification of single-molecule RNA. The same research group further proposed a double-crRNA droplet-based CRISPR/Cas12a assay for amplification-free and absolute quantification of DNA at the single-molecule level (Yue et al., 2021). The platform employed a dual crRNA strategy to enhance reaction efficiency and performed Cas12a reactions in pL water-oil droplets, which can directly detect 17.5 copies/µL of African swine fever virus (ASFV).

However, the droplet has unstable performance, which may affect detection. Compared with the droplet method, the microchamber array has a more uniform reaction volume, and its fL volume results in a better concentration effect (Cohen et al., 2020). Shinoda et al. (2021) combined microchamber array technology with CRISPR/Cas13a to develop a CRISPR-based amplification-free digital RNA detection platform (SATORI). Similarly, a microchamber array can increase the local molecular concentration and accelerate detection. The device contains more than 1 ×10⁶ through-holes and can observe a large number of chemical reactions in parallel at the single-molecule level, enabling the detection of SARS-CoV-2 in as little as 5 min with a sensitivity of 10 fM. By combining multiple crRNAs, the sensitivity can be increased to 5 fM. However, some manual processing procedures may introduce human errors, which may lead to reduced detection accuracy of SATORI. Therefore, a fully automated platform for SATORI (opn-SATORI) is proposed to avoid complex solution exchange steps (Shinoda et al., 2022). When the optimal Cas13a enzyme and magnetic bead technology are utilized, the limit of detection (LoD) of opn-SATORI for SARS-CoV-2 genomic RNA is less than 6.5 aM, with a sensitivity comparable to that of RT-qPCR. Recently, Wang et al. (2023) combined magnetic bead capture with a microchamber array to develop a CRISPR/Cas13a-based single-molecule digital detection method for nucleic acids. The target nucleic acid fragments were captured and enriched with magnetic beads. The target-induced Cas13a cleavage reaction was dispersed and confined to a million single-fL micropores, thereby increasing the local signal intensity and enabling single-molecule detection. The LOD of the method for H1N1 virus and SARS-CoV-2 is 2 aM.

In addition, a study reported the use of a CRISPR/Cas-driven single micromotor (Cas-DSM). The micromotor is used as a universal microreactor for CRISPR/Cas reactions and as a unique readout platform to enrich all the generated fluorescence signals in a very small location (Chen et al., 2023a). By fixing the fluorescently labeled ssDNA or RNA on the magnetic micromotor, the trans-cleavage capability of the CRISPR/Cas system is activated when the target DNA/RNA is present, and the FQ-ssDNA/ssRNA reporter on the surface of the micromotor is cleaved, resulting in the separation of the fluoro group (FAM) from the quenching group (BHQ1), leaving only FAMs on the micromotor surface, thus illuminating the microreactor under laser light. The platform utilizes highly enriched signals to detect nucleic acid targets at the single-molecule level directly, eliminating the need for amplification steps.

Cascade signal amplification strategy

Although the above methods enable sensitive detection of pathogens without amplification, detection is dependent on other sensitive sensing systems, which require the discovery of biosensors integrated with CRISPR/Cas systems. The CRISPR/Cas cascade signal amplification strategy (including Cas-mediated and crRNA-mediated cascade amplification) can achieve direct detection of pathogens without combining with other sensing systems and has great application prospects for amplification-free CRISPR/Cas detection.

The Cas protein cascade can improve the sensitivity and specificity of detection and has the potential for amplification-free detection. For example, in SHERLOCKv2, the type III effector protein Csm6, in conjunction with Cas13, can increase the rate of RNA detection by 3.5 times compared with Cas13 alone. Additionally, 4-channel multiplexing is achieved by using three Cas13 enzymes and one Cas12a enzyme (Gootenberg et al., 2018). Researchers subsequently proposed fast integrated nuclease detection in the tandem platform (FIND-IT), which integrates Cas13a and Csm6 and can directly detect pathogen RNA quickly (20 min), with a sensitivity of 30 copies/µl (Liu et al., 2021). Csm6 is a dimeric RNA endonuclease. When the target RNA activates Cas13a-crRNA, Cas13a cuts the ssRNA substrate, and the degraded substrate acts as an activator of Csm6, inducing the Csm6 trans-cleavage RNA reporter probe to achieve signal cascade amplification.

Cascaded signal amplification initiated by crRNA can also enable direct detection of the target sequence. For example, one study developed a method for the direct detection of SARS-CoV-2 via the synergy of crRNA, which was designed to contain more than ten crRNAs and could detect SARS-CoV-2 RNA at concentrations as low as 170 aM within 30 minutes (Fozouni et al., 2021). However, this assay, which combines multiple crRNAs, is only suitable for detecting long-strand nucleic acids and detecting long-strand nucleic acids and has limited ability to detect short-strand nucleic acids. Another study proposed the use of the autocatalytic nucleic acid positive feedback network of the CRISPR/Cas12a system, namely, the CRISPR/Cas-only amplification network (CONAN), to overcome these difficulties (Shi et al., 2021). To achieve target nucleic acid index signal amplification, the platform builds a feedback amplification network by creating a gRNA for target dsDNA (gRNA-T) and a gRNA hybridized with a ssDNA reporter (scgRNA) (Fig. 3A). The self-reporting ability of scgRNA serves to output amplified fluorescent signals and multiple active gRNA molecules in response to each active Cas12a protein with trans-cleavage activity. Each target nucleic acid molecule is converted into an exponentially amplified fluorescence signal in CONAN. Thus, ultrasensitive nucleic acid detection is realized with attomolar sensitivity. Moreover, according to a recent study, full-sized crRNA and split crRNA can amplify cascade signals through their competitive response to Cas12a (Fig. 3B) (Moon & Liu, 2023). The asymmetric trans-cleaving activity of Cas12a is induced by a competitive response between full-sized crRNA and split crRNA. This competitive CRISPR response triggers a conformational reset of the CRISPR enzyme, significantly enhancing the detection sensitivity of CRISPR-Cas12a without requiring additional DNA amplification steps. Furthermore, researchers have demonstrated that Cas12a can be utilized to identify broken RNA/DNA targets, thereby enabling the direct detection of RNA.

The CRISPR/Cas assay with cascaded signal amplification is simple and fast, and it does not require any special instruments or equipment, which is crucial for the realization of POCT and on-site diagnosis. However, its sensitivity is relatively weak, and improving the compatibility of this multienzyme reaction system to achieve simple and sensitive molecular diagnostics remains a challenge.

One-pot detection

One-pot detection integrates nucleic acid amplification and the CRISPR/Cas reaction into a single buffer system, which not only simplifies the operation procedure but also reduces cross-contamination (Fig. 4A). Because heat-resistant Cas12b is compatible with the operating temperature (60 °C∼65 °C) of loop-mediated isothermal amplification (LAMP), one-step detection can be realized. For example, HOLMESv2 (Li et al., 2019), created by combining heat-stable AacCas12b with LAMP, integrates nucleic acid amplification and target detection steps into a single system, enabling one-step detection, simplifying operation procedures and avoiding cross-contamination. In addition, the newly discovered Brevibacillus sp. Cas12b (BrCas12b) was shown to have superior heat resistance. The CRISPR single-pot assay for detecting emerging volatile organic compounds (VOCs) (CRISPR-SPADE), based on BrCas12b, can accurately detect SARS-CoV-2 variants via simple and robust one-pot detection with a sensitivity of 95% (Ct value ≤ 32) and above (Nguyen et al., 2022). Compared with other one-pot methods, the thermal stability of BrCas12b alleviates some limitations of LAMP primer design, allowing for greater flexibility in primer selection. However, simply combining nucleic acid amplification with the CRISPR/Cas system significantly affects detection sensitivity due to the competitive reaction between Cas-mediated cis-cleavage and target amplification. During target amplification, the target and primer are likely to be degraded by Cas-mediated cis-cleavage, which affects amplification efficiency (Ali et al., 2020; Nguyen et al., 2022).

Purposefully limiting the cis-cleavage rate of the Cas protein is a feasible strategy to improve the performance of one-pot assays. Therefore, to avoid the influence of cis-cleavage on target amplification, one study addressed this issue by employing an all-in-one double CRISPR/Cas12a (AIOD-CRISPR) detection method (Ding et al., 2020). The platform employs two PAM motif-free gRNAs that allow Cas12a-gRNA complexes (RNPs) to bind to amplification targets and induce Cas12a trans-cleavage activity to cleave quenched ssDNA probes but does not induce cis-cleavage activity, thereby preventing cleavage of the bound amplification target and attenuation of the amplification reaction (Fig. 4B). In addition, the suboptimal PAM of Cas12a (sPAMC)-based test, which was developed using gRNA targeting suboptimal PAM sequences, can also effectively inhibit cis-cleavage of Cas proteins and improve the sensitivity of one-pot assays (Lu et al., 2022). Cas12a reactions based on suboptimal PAM exhibit weaker and slower cis-cleavage activity than reactions involving standard PAM, thus effectively slowing the degradation of targets and primers by the CRISPR/Cas system during nucleic acid amplification, allowing the amplification to accumulate sufficient products for subsequent CRISPR/Cas detection (Fig. 4C). However, the Cas-mediated cis-cleavage effect of the above methods still exists.

A light-controlled one-pot assay can better regulate crRNA-guided Cas enzyme activity in both spatial and temporal dimensions, effectively addressing the influence of cis-cleavage on target amplification efficiency. For example, one study designed a photocleavable protective RNA (p-RNA) to block the activity of crRNA (Fig. 4D) (Hu et al., 2022). After preamplification, p-RNA was degraded and isolated from the crRNA under light irradiation to restore Cas12a activity. This new one-pot detection system separates target amplification from CRISPR detection in space and time, thus eliminating the influence of CRISPR/Cas cleavage on the amplification reaction and achieving a sensitivity comparable to that of PCR. However, this method requires the synthesis of customized crRNA and additional human intervention, which hinders its simplification and automated detection. Recently, a study proposed an in situ one-pot assay that only achieved a sensitive one-pot assay by utilizing the slow association kinetics between the target-specific sgRNA and Cas12a to limit the cis exonuclease activity of Cas12 (Fig. 4E) (Lesinski et al., 2024). In the early stages of the reaction, RNP concentrations are limited by relatively slow kinetics, thereby circumventing the disadvantages associated with early-reaction cis exonuclease activity and retaining the signal transduction benefits of high RNP concentrations. However, owing to the overall decrease in RNP concentration, the trans-cleavage of the ssDNA reporter is reduced, which affects the final detection sensitivity.

Microfluidic integrated detection

Microfluidic chips typically integrate miniaturized flow channels, valves, reaction chambers and detectors, enabling the integration of nucleic acid extraction, amplification, and CRISPR/Cas detection on a minimal chip (Li et al., 2023c). Once a drop of sample is added to the microfluidic chip, it automatically flows through all the components in the chip and eventually generates signals, eliminating the need for manual pipetting and enabling fast, high-throughput, multichannel, and digital detection (Fig. 4F) (Chen et al., 2023b). Lu et al. (2023) combined digital microfluidic technology with CRISPR/Cas12a to develop a fluorescence readout platform for detecting Staphylococcus aureus (S. aureus). Staphylococcus aureus cells were enriched from the raw materials via immunomagnetic beads. The enriched magnetic beads were collected by centrifugation, and the super-serum containing S. aureus cells was added to the digital microfluidic chip for further analysis. Cell lysis, RPA amplification, and Cas12a trans-cleavage occur automatically in sequence on the digital microfluidic chip. Due to its small volume and automation capabilities, the platform can complete detection in 55 min with a LOD of 32 CFU/mL. Li et al. (2022c) developed a simple, sensitive, and instrument-free CRISPR-based method for diagnosing SARS-CoV-2 using an independent microfluidic system. The platform utilizes a low-cost, portable hand warmer to culture microfluidic chips, maintaining optimal temperatures for RPA amplification and trans-cleavage. The microfluidic chip integrates isothermal amplification, CRISPR cleavage, and lateral chromatography detection in a closed microfluidic platform, enabling pollution-free visual inspection that can detect up to 100 copies of SARS-CoV-2 RNA.

In addition, Zhang et al. (2024a) designed a multiplex, portable, centrifugal microfluidic detection system that integrates magnetic bead-based nucleic acid extraction, RPA amplification, and CRISPR-Cas13a detection in a single user step, with an overall turnaround time of 45 min. All reagents are preloaded onto the chip and can be automatically released. By utilizing a multichannel chip, it can simultaneously detect 10 infectious viruses with a LOD of 1 copy per reaction. The self-contained reaction system, designed using a microfluidic chip, can effectively prevent contamination and potentially achieve the goal of being pipette-free. Additionally, well-designed microchannels can distribute a single sample across multiple reaction chambers to achieve multiple detections (Ackerman et al., 2020; Shang et al., 2024). However, the complex preparation of microfluidic chips limits their widespread use, which requires further commercialization and the integration of instruments and chips to facilitate POC testing.

Ultrasensitive lateral flow chromatography system

The lateral flow assay (LFA) is a portable, easy-to-operate, and cost-effective solution for resource-poor areas and POCT. The principle is that the DNA probe sequence complementary to the reporter is fixed on the test line of the lateral flow rod to capture the reporter. When the target is present, the reporter is cleaved, resulting in the generation of a light signal on the test line. Due to their unique advantages of simplicity and portability, LFAs, such as SHERLOCK v2 (Table 2) (Gootenberg et al., 2018) and STOPCovid (Joung et al., 2020), have been widely utilized in CRISPR assays; however, their sensitivity is generally low. Therefore, to improve the sensitivity of CRISPR/Cas-LFA-based assays, Zhou et al. (2022) utilized quantum dot-enhanced fluorescence signals to develop a fluorescence-enhanced LFB based on CRISPR/Cas12a, known as CRISPR/Cas-recombinase-assisted amplification-based LFB (CRA-LFB), for detecting Staphylococcus aureus. QDs are fluorescent semiconductor nanocrystals with high brightness, wide excitation and strong light stability (Deng et al., 2017). On this platform, streptavidin (SA) modified with quantum dots (QDs) is embedded on the T-line, allowing for the generation of a large number of specific amplification products through recombinant enzyme-assisted amplification (RAA). The trans-cleavage activity of Cas12a is then activated to degrade the biotinylated probes. The degraded probe cannot be captured by the QD-SA group on the T-line, resulting in no signal accumulation. In contrast, a significant fluorescence signal is observed on the C-line, and the target concentration is negatively correlated with the fluorescence intensity on the T-line. The limit of detection for Staphylococcus aureus DNA was 75 aM.

Similarly, Zhong et al. (2023) utilized the fluorescence enhancement characteristics of QDs to develop a CRISPR/Cas12a-based QD nanobead (QDNB)-labelled lateral flow assay (CQ-LFA) for detecting varicella zoster virus (VZV) with results that were completely consistent with those of q-PCR. The platform introduced the QDNB-anti-FAM antibody conjugate into the Cas12a cleavage system. When the target DNA was present, the cleavage activity of Cas12a was triggered to degrade the biotin-ssDNA-FAM probe, and the QDNBs leaked out of the streptavidin-embedded T-line, resulting in no or a weak fluorescence signal on the T-line. Instead, in the absence of a target, the reporter is intact and bound to the T-line, where a fluorescent signal is visible.

Smartphone readout system

Compared to the naked eye, smartphones are capable of converting the signals generated by CRISPR/Cas cleavage into accurate digital data, displaying more sensitive and precise readings. They offer the advantages of low cost, wide availability, and high portability. In addition, smartphones also have great potential and broad application prospects in statistics, summary and query data. Fozouni et al. (2021) designed a smartphone-based fluorescence microscope and reaction chamber to quantify the fluorescence signal produced by Cas13a, enabling direct detection of SARS-CoV-2. Detection integrated with a mobile phone-based reader device is expected to achieve rapid, low-cost, quantitative, immediate screening of SARS-CoV-2. Yin et al. (2021) developed a G-quadruplex-based CRISPR/Cas12a bioassay with smartphone readings for pathogen detection. On this platform, ssDNA is designed with a guanine-rich sequence, and the addition of K⁺ forms a stable G-quadruplex DNase. DNase can catalyze the TMB-H2O2 reaction in the presence of heme, resulting in changes in absorbance and color at 454 nm, which can be easily distinguished by the naked eye and smartphones using a color selecting app. The LOD of this strategy for Salmonella was 1 colony-forming unit (CFU) per millilitre (mL). In addition, Song et al., (2022) reported a colorimetric DNA enzyme reaction triggered by LAMP and CRISPR/Cas9, and developed a machine learning (ML)-based smartphone application to check the diagnostic results of SARS-CoV-2 and variant strains periodically. The CRISPR/Cas9 system eliminates false-positive signals generated by LAMP, thereby improving the accuracy of detection. Smartphones achieve end-user-friendly detection. The system can detect SARS-CoV-2 and its variants quickly and accurately within 1 h in the field, with sensitivity reaching the attomolar level.

Innovative small readout system

The combination of various innovative small readout strategies, such as fluorometers, thermometers, and glucose meters, with the CRISPR/Cas system, can achieve portable and sensitive POC diagnosis of pathogens, offering the advantages of low cost and user-friendliness. He et al. (2020) designed and developed a fluorescence-based POC system for detecting African swine fever virus (ASFV). The POC system consists of a disposable filter element that can hold up to 80 individual samples and a custom fluorometer. In the presence of ASFV DNA, the Cas12a/crRNA complex was activated and degraded the fluorescent ssDNA reporter. The system can detect ASFV at concentrations as low as 1 pM within 2 h without nucleic acid amplification. Liu et al. (2022) utilized a handheld thermometer as a readout tool to develop a CRISPR/Cas12a-based photothermal platform for detecting citrus-related Alternaria genes. The AuNPs modified with HRP were linked to MB through ssDNA. HRP can oxidize TMB to form oxTMB, which not only appears blue but also exhibits a strong photothermal effect driven by a near-infrared laser. A laser can irradiate it, producing a significant temperature increase, and the changes can be easily recorded with a handheld thermometer. The platform exhibits high sensitivity for semiquantitative detection, with a LOD of 1.5 pM. Recently, Li et al. (2023b) combined a microfluidic platform with glucose biosensing technology to develop a bioinspired CRISPR-mediated cascade reaction biosensor (CRISPR-MCR) for HIV point-of-care diagnosis. HIV nucleic acid can be digitally detected by a personal blood glucose meter at the endpoint, similar to reading glucose levels in the blood. The detection sensitivities of HIV DNA and HIV RNA via the platform are 43 copies and 200 copies per test, respectively, without the requirement for expensive instruments.

The portable readout strategy enables accurate and sensitive signal readout with minimal instrumentation, demonstrating great potential for POC detection. However, most strategies still require complex sample extraction, preamplification and pipetting steps, making the overall strategy unsuitable for field point-of-care diagnosis. In future work, an extraction-free and amplification-free integrated reaction strategy will be combined with a portable readout to achieve real-time, on-the-spot diagnosis.