4.1.1 Fluorescence instrument reading

Huang et al. [47] developed CAS-EXPAR, a novel nucleic acid amplification technique that combines Cas9-specific cleavage with EXPAR constant temperature rapid amplification. The CRISPR/Cas9 system recognizes, binds and cleaves the target DNA through PAM to generate specific fragments. The specific fragments are then used as primers to amplify the reaction performed cyclically to generate numerous DNA replicates, which are detected using a real-time fluorescence monitoring system. Compared with conventional nucleic acid amplification techniques, CAS-EXPAR does not require exogenous primers and avoids non-specific amplification of the target. SHERLOCK is a nucleic acid detection system developed by Gootenberg et al. [48] based on Cas13, which can independently detect RNA viruses and mRNA and combine amplification techniques and DNA transcription for DNA detection. This technology can be used to detect viral loads and distinguish different strains. Combined with HUDSON technology [49], direct detection of whole blood, plasma, serum, urine or saliva samples can be achieved. Subsequently, Gootenberg et al. [50] built a quantitative, highly sensitive and multiplexable nucleic acid detection platform, SHERLOCK v2, based on SHERLOCK, which is based on four proteins: LwaCas13a, PsmCas13b, CcaCas13b and AsCas12a. SHERLOCKv2 is limited by the type of Cas protein and its cleavage propensity, which hampers the simultaneous detection of dozens or even more target nucleic acids. In contrast, the CARMEN [51] microwell-array system combines Cas13 and different crRNAs to achieve high-throughput, miniaturized, scalable and self-organizing simultaneous detection of hundreds of pathogens. CARMEN has a wide range of uses for distinguishing viral species, strains and SNPs and can be rapidly expanded to perform high-throughput detection of novel pathogens, which is of great benefit to patients and public health. CARMEN is the first scalable high-throughput detection platform based on the CRISPR/Cas system, which will undoubtedly push the CRISPR/Cas system to new heights in nucleic acid detection.

4.1.2 Smartphone reading

Doudna et al. [52] reported developing an amplification-free CRISPR-Cas13a assay to detect SARS-CoV-2 from nasal swab RNA that can be read using a mobile phone microscope. This research team combined Cas13a with various crRNAs to generate different RNPs that act in series on the same RNA target region and achieved 100 copies/μL sensitivity in under 30 min of measurement time. Subsequently, the number of RNPs was increased to eight and concatenated with Csm6 to achieve higher sensitivity and a faster detection time of RNA targets without amplification [53]. Ning et al. [54] developed an RT-RPA CRISPR-fluorescence detection system assay to sensitively quantify SARS-CoV-2 present in saliva without RNA isolation and adapted this assay to a smartphone-read chip format. The built-in smartphone camera app was used to focus on samples and capture images, removing the need for mechanical focusing and lowering the weight and cost of the device while improving its optical stability and usability.

4.1.3 Visual observation

Wang et al. [55] developed a CHP system for the high-fidelity and sensitive detection of dsDNA by combining the CRISPR/Cas9 system with two isothermal amplification techniques. Cas9/gRNA can specifically recognize and cleave dsDNA, leading to NTS exposure. Exposed NTS binds to the hairpin probe to initiate strand displacement amplification, and the amplification product triggers subsequent rolling circle amplification to amplify long ssDNA. Long ssDNA can be used to open the fluorescent hairpin probe and complete the reporting of target dsDNA. Wang et al. [56] developed a one-pot visual reverse transcription (RT)-LAMP-CRISPR method for ultrasensitive visual detection of SARS-CoV-2, which simplified operations and avoided contamination. The RT-LAMP reagents were incubated at the base of the tube. The CRISPR/Cas12a reaction reagents were added to the lid, which was added to the reaction system by an external force after the completion of the RT-LAMP reaction. In the presence of target DNA, the fluorescent probes are trans-cleaved by Cas12 to generate fluorescence, and the fluorescent signal is visible under blue light.

4.2.1 Immobilized electrochemical biosensing systems

Researchers [57-61] covalently modified the electrochemical indicator with agents such as methylene blue (MB) to one end of the DNA or RNA to form an electrochemical probe and fixed the other end of the probe to the electrode surface using the difference in electrochemical signals generated by the physical distance between MB and the electrode surface to report whether the target DNA exists in the reaction system. In contrast, Bruch et al. [62] fixed streptavidin (SA) on the surface of the carrier to link biotin-RNA-fluorescein and then linked anti-fluorescein antibody-glucose oxidase via fluorescein. In the presence of the target RNA, activated Cas13a trans-cleaved the biotin-RNA-fluorescein, releasing glucose oxidase. After cleaning the microchannel, a certain concentration of glucose solution was injected, and the RNA content was determined by measuring the current density generated by H₂O₂, the oxidation product of glucose. In addition, DNA/RNA electrical signals without electrochemical indicators or glucose oxidase were directly detected. Li et al. [63] modified Au nanoparticles (AuNPs) on the surface of reduced graphene oxide (RGO) and fixed thiolated reporter RNA (reRNA) on the surface of AuNPs for reporting target RNA. The pre-incubated Cas13a/crRNA/RNA ternary complex was then placed onto the surface of the RGO-EFT chip. Using the trans-cleavage activity of Cas13a to cleave the reRNA immobilized on the chip induced p-doping with a positive shift of the Dirac point, with the displacement reflecting the target RNA concentration. Hajian et al. [64] combined catalytically inactivated Cas9 with gRNA and fixed this complex on a graphene field effect transistor. The change in graphene conductivity triggered by the specific binding of these two molecules to target dsDNA reported the presence of the target dsDNA. The label-free nucleic acid detection chip can be used with a handheld reader, and detection can be completed within 15 min with a sensitivity of 1.7 fM.

4.2.2 Non-immobilized electrochemical biosensing systems

Researchers [65, 66] modified electrochemical indicators such as MB at the end of DNA or RNA to make electrochemical probes, which are free in the system. They used the differences in signals of the electrochemical probes caused by steric hindrance and diffusion coefficient changes to report the presence or absence of target DNA in the system. Liu et al. [65] used the trans-cleavage activity of Cas12a on ssDNA combined with the diffusivity difference and electrostatic repulsion of the ssDNA-MB probe on a negatively charged indium-tin-oxide (ITO) electrode to prepare a simple, non-immobilized and homogeneous electrochemical biosensor for detecting human HPV-16. Specifically, the ssDNA-MB probe cannot freely diffuse to the surface of the negatively charged ITO electrode because of the strong electrostatic repulsion, giving rise to a weak electrochemical signal. The presence of target DNA activates the trans-cleavage activity of the CRISPR/Cas12a system, which cleaves the negatively charged ssDNA-MB probe to release MB. Because of higher diffusivity and less electrostatic repulsion, the smaller MB diffuses more readily to the surface of the ITO electrode to enhance the electrochemical response. Li et al. [66] added a pulsed electric field on this basis to increase the distance between the negatively charged ssDNA-MB probe and the electrode surface, reducing the background and enhancing the signal-to-noise ratio. With the help of pulsed electric fields, the detection limit of the reaction system without the assistance of amplification techniques is 1 pM.

4.3.1 Lateral flow chromatography

Lateral flow chromatography based on chromogenic substances such as colloidal gold or latex microspheres has the advantages of simple operation, rapid reaction time and low cost. Researchers have combined lateral flow chromatography with CRISPR/Cas systems to achieve better detection performance and have established various nucleic acid detection systems based on a chromogenic response. Researchers [67-71] combined the amplification technology with the CRISPR/Cas9 system to realize the convenient detection of target DNA based on lateral flow chromatography strips. Initially, target DNA is labeled with biotin, fluorescein isothiocyanate, digoxin or carboxyfluorescein at the end of the amplification primer and then combined with the Cas9/gRNA complex or the Cas9/gRNA complex containing modified groups to form a ternary complex. AuNPs conjugates bind to the target DNA, gRNA or modifier group in the ternary complex to form a quaternary complex, which is then captured by SA, complementary ssDNA or a secondary antibody fixed to the test line, thereby reporting the target DNA by the appearance of a band. Wang et al. [69] incubated biotin-labeled target dsDNA amplification products with Cas9/gRNA to form ternary complexes and placed them on a sample pad. After lateral flow, the stem-loop structure of gRNA in the ternary complexes hybridized with the AuNPs-DNA probes to form quaternary complexes. The quaternary complexes continued to flow to the test line and were captured by fixed SA, while the remaining AuNPs-DNA probes continued to flow to the control line and were captured by the capture probe. Continuous accumulation of AuNPs increases the intensity of the test and control lines, and the presence or absence of the target DNA can be judged by the color indicator. On this basis, Xiong et al. [70] added digoxin-labeled amplification primers to achieve a simultaneous dual-gene diagnosis.

The unique trans-cleavage activity of the CRISPR/Cas12 and Cas13 systems facilitates more novel system construction methods. Mukama et al. [72] placed AuNPs-SA on the conjugate pad to conjugate biotin-ssDNA or biotin in the reaction product and generated AuNP reporter complexes. Single-strand capture probes complementary to ssDNA were fixed at the test line to capture the AuNPs-SA-biotin-ssDNA reporter complexes via base complementation. Biotinylated rabbit polyclonal antibodies immobilized on the control line were used to capture excess AuNPs reporter complexes by binding to SA. The presence of Pseudomonas aeruginosa DNA in the system caused the CRISPR/Cas12a/dsDNA ternary complex to trans-cleave biotin-ssDNA, and the single-stranded capture probe no longer captured the AuNPs-SA-biotin reporter complexes, thus reporting the target DNA as a non-detectable test line.

4.3.2 Homogeneous chromogenic systems

Homogeneous chromogenic systems are simpler than lateral flow chromatography systems. Researchers used the transition of aggregation or dispersion between AuNPs and CRISPR/Cas systems to establish more simple chromogenic response nucleic acid detection platforms. Yuan et al. [73] developed a chromogenic foodborne pathogen detection system based on the trans-cleavage activity of the CRISPR/Cas12a and Cas13a systems and the distance-dependent optical properties of AuNPs-DNA probes. In this strategy, the AuNPs-DNA probes are connected by ssDNA or ssRNA to cause aggregation of the AuNPs-DNA probes, and the solution color is magenta. After low-speed centrifugation, the probe settles to the bottom, and the solution is colorless. When the target nucleic acid is present in the system, the activated Cas12a or Cas13a cleave ssDNA or ssRNA, causing dispersion of the AuNPs-DNA probes and the solution color changes from magenta to red. Moreover, the dispersed AuNPs-DNA probes are less affected by centrifugation, and the solution remains red after centrifugation. On this basis, Zhou et al. [74] greatly improved the signal-to-noise ratio of the detection system by improving the DNA hybridization buffer and were able to detect 6.4 × 10² CFU/mL of Staphylococcus aureus ST398 within 50 min.

4.3.3 Catalytic chromogenic systems

Researchers have combined a catalytic chromogen and the CRISPR/Cas system to achieve chromogenic detection using the catalytic effect of enzymes or enzyme mimics on substrates to report target nucleic acids. Chen et al. [75] proposed a sequence-specific, homogeneous and label-free chromogenic technique for detecting Vibrio parahaemolyticus. The target DNA amplification product activates the trans-cleavage activity of the CRISPR/Cas12a system, allowing this system to arbitrarily cleave G-quadruplexes (G4s) and inhibit its peroxidase mimic activity. The intact G4s conjugate hemin to catalyze ABTS²⁻ to ABTS, and the solution color changes from colorless to green. When the G4s structure loses the activity of the peroxidase mimic, the catalytic process no longer occurs, and the solution is colorless. Based on this principle, Yin et al. [76] replaced the catalytic substrate of G4s with 3,3′,5,5′-tetramethylbenzidine (TMB). The combination of G4s and hemin chloride catalyzes TMB to produce oxTMB under the action of K⁺, and the solution changed from colorless to blue, enabling the rapid detection of Salmonella SPP.

In addition to the nucleic acid sequences with an enzyme mimic activity, proteases produced by translation can participate in catalytic color development. Collins et al. [77] combined isothermal nucleic acid amplification technology with the toehold switch sensor and the CRISPR/Cas9 system to develop a chromogenic system that distinguishes Zika genotypes with single-base resolution. For the American strain, the CRISPR/Cas9 system cleaves the amplified product to yield short DNA by recognizing the PAM. The short RNA produced by transcription cannot activate the sensor H toehold switch, preventing LacZ production. For the African strain, the CRISPR/Cas9 system cannot effectively recognize the PAM and does not cleave the DNA. The complete RNA produced by transcription activates the sensor H toehold switch to produce the LacZ enzyme, which converts the yellow substance on the paper platform to the purple substance. Thus, the genotype of the Zika virus can be discerned only by the color change on the paper platform. Compared with other detection strategies, the chromogenic response-based CRISPR/Cas12 sensing system can fully use the high sensitivity and specificity of the CRISPR/Cas12 system to convert target nucleic acids into visual signals for output, broadening the application scope.