Cas12a multi-target rapid detection method and kit based on multiple crRNA arrays

Abstract

The invention relates to a Cas12a multi-target rapid detection method and a Cas12a multi-target rapid detection kit based on multiple crRNA arrays. The detection method comprises the following steps: designing and synthesizing a crRNA array containing at least two crRNA units, wherein the crRNA units are directly connected or connected through a variable-length connecting sequence; the crRNA array and Cas12a protein are combined to form a compound; the complex is reacted with a sample containing the target nucleic acid and detected by a fluorescent or colorimetric signal. The method not only can realize rapid and parallel detection of different pathogens, but also can identify single base mutation, and has the advantages of high throughput, low cost, portability and the like. The application covers food safety, environmental monitoring and clinical pathogen on-site rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and biological detection technology, and particularly relates to a rapid detection method and kit for Cas12a multi-target based on multiple crRNA arrays. Background Technology

[0002] In recent years, the demand for rapid detection of foodborne pathogens and clinical pathogens has been increasing. Existing detection methods mainly include amplification technologies such as PCR, real-time quantitative PCR, and LAMP. Although these methods have high sensitivity, they suffer from problems such as strong equipment dependence, limited detection throughput, and poor field applicability.

[0003] Cas12a is an RNA-guided endonuclease with programmable target recognition capabilities. Upon binding to a target, it triggers non-specific single-stranded DNA cleavage activity, thereby amplifying the signal. Most existing Cas12a molecular detection methods use a single crRNA for target recognition, thus only allowing the detection of a single target within a single reaction system. This limits the simultaneous detection of multiple targets in a single reaction, restricting its application in multi-pathogen detection and on-site testing.

[0004] In food safety testing, pathogen surveillance, and clinical multiplex diagnostics, it is often necessary to simultaneously detect multiple target sequences within a limited time and reaction system. Existing multiplex detection strategies mainly rely on the parallel detection of multiple single-reaction systems or spatial separation, which has problems such as operational complexity, high cost, and signal inhomogeneity.

[0005] Reference 1 and the invention patent (application number 201880089710.2, publication number CN111836903A) disclose a method for simultaneously detecting four target molecules by using Cas enzymes (Cas12a, Cas13a and Cas13b) with different cleavage preferences and their guide RNA, in combination with RNA or ssDNA probes with different fluorescent labels. Reference 1: GOOTENBERG JS, ABUDAYYEH OO, KELLNER MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science (New York, NY), 2018, 360(6387):439-44. The above method is a high-level strategy for multi-target detection. This technology utilizes multiple CRISPR-Cas enzymes with different cleavage preferences (e.g., Cas12a, Cas13a, and Cas13b) targeting DNA and RNA targets respectively, and is paired with RNA or ssDNA reporter probes with different fluorescent labels, thereby achieving simultaneous detection of multiple targets in a single reaction system by distinguishing them by fluorescence wavelength. However, it suffers from drawbacks such as overly complex detection systems and high difficulty in design and application. Cas13 can only recognize RNA targets transcribed in vitro via T7, and the establishment of the reaction system is cumbersome and complex; the detection results are presented by scanning four fluorescence signals simultaneously, which is highly dependent on instruments and difficult to apply in POCT. Significant limitations exist in terms of practicality, cost, and scalability. The core strategy employs a "multi-enzyme, multi-channel" approach, using multiple Cas enzymes (such as Cas12a, Cas13a, Cas13b) and multiple fluorescent reporter probes, distinguished through spectral channels. Regarding system complexity and cost, it suffers from extreme complexity and high cost. It requires the simultaneous expression, purification, and optimization of multiple Cas enzymes, resulting in high enzyme costs. Multiple reporter probes with different fluorescent groups need to be synthesized, leading to complex design and synthesis, and high probe costs. Expensive equipment (such as real-time fluorescence PCR instruments or more complex detection systems) capable of multicolor fluorescence detection and spectrometry is required, resulting in high equipment costs. In terms of scalability, it suffers from limited scalability; the number of distinguishable targets is limited by the number of available Cas enzymes (cutting bias) and the number of resolvable fluorescent channels, with a theoretical upper limit. Each additional channel requires the introduction of a new enzyme and a new reporter probe, exponentially increasing the development difficulty. In terms of applicable scenarios, it is more suitable for complex multi-target molecular typing in well-equipped central laboratories. In terms of technical barriers, it requires profound knowledge of protein engineering and multicolor fluorescence optics, and the system optimization is extremely complex, resulting in a high technical threshold.

[0006] The invention patent (application number 201910673246.8, publication number CN112301101A) discloses a CRISPR multi-target detection method and its kit, which requires the independent design and synthesis of a structurally complex "guide RNA-reporter nucleic acid co-probe" for each target. This probe couples recognition and reporting functions into a single molecule. The design is complex, and the synthesis cost is extremely high. Each co-probe requires complex and specific chemical modification and synthesis, resulting in a cumbersome process. For n targets, n different complex probes need to be synthesized, and the cost increases linearly with the number of targets. The system is complex, containing multiple composite probes with different structures and properties, which may lead to high background signal, poor stability, and difficulty in optimization due to probe interactions. Scalability is poor; for each new target, a completely new and structurally complex co-probe must be designed and synthesized, making expansion cumbersome and costly.

[0007] Therefore, there is an urgent need to develop a new method that is simple, efficient, scalable, and capable of multi-target identification and detection in a single reaction system to meet the practical needs of food safety and clinical diagnosis. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides a rapid detection method and kit for multiple targets of Cas12a based on multiple crRNA arrays. The present invention, by designing a tandem crRNA array, enables parallel detection of multiple targets in a single system, and has advantages such as high specificity and strong mutation recognition capability.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0010] This invention provides a Cas12a detection method based on multiplex crRNA arrays, comprising the following steps:

[0011] (1) Design and synthesize a crRNA array containing at least two or more crRNA units, wherein the crRNA units are directly connected or connected by a variable-length linker sequence;

[0012] (2) The crRNA array is bound to the Cas12a protein to form a complex; in the presence of a reporter probe, the complex is reacted with a sample containing the target nucleic acid and detected by fluorescence or colorimetric signal.

[0013] The beneficial effects of adopting the above technical solution include: achieving simultaneous detection of multiple targets and improving detection throughput through innovative crRNA array structure; achieving signal equalization and high sensitivity by adjusting the ratio of crRNA to Cas12a; identifying single-base mutations and improving detection specificity; and being suitable for rapid on-site detection when combined with portable detection platforms. The above method is applicable to portable on-site detection devices.

[0014] Furthermore, the crRNA array is a crRNA array with ≥2 targets. The crRNA array described in this invention can be a dual-target array (DcrRNA), an eight-target array (OcrRNA), or other arrays with different numbers of targets.

[0015] Furthermore, the linker sequence is composed of nucleotides. The length of the linker sequence can be, but is not limited to, 0–60 nucleotides.

[0016] Furthermore, the molar ratio of Cas12a to crRNA can be (50-800):600.

[0017] Furthermore, the Cas12a protein is FnCas12a, LbCas12a, AsCas12a, or a modified variant thereof.

[0018] This invention significantly improves the detection sensitivity and specificity of the system by optimizing the structure of the multiplex crRNA array, the Cas12a / RNA ratio, and the reaction conditions.

[0019] The present invention also provides a kit for implementing the above method, comprising one or more of Cas12a protein, multiplex crRNA array, reporter probe and reaction buffer.

[0020] Furthermore, the reporting probe can be a fluorescence quenching probe or a colorimetric probe.

[0021] Furthermore, it may also include a reading module for portable detection.

[0022] The kit provided by this invention can identify single-base mutations, enabling simultaneous detection of multiple targets and improving detection throughput. It offers advantages such as signal balance, high sensitivity, and good specificity. Furthermore, it can be integrated with portable detection platforms, making it suitable for rapid on-site detection. The kit is applicable to portable on-site detection devices.

[0023] The present invention provides the application of the above method in any one or more of (1) to (6); (1) multi-target detection; (2) identification of single base mutations in target nucleic acids; (3) food safety detection; (4) simultaneous detection of specific gene sequences of multiple foodborne pathogens; (5) clinical pathogen detection; (6) environmental sample monitoring.

[0024] The present invention provides the application of the above-mentioned kit in any one or more of (1) to (6); (1) multi-target detection; (2) identification of single base mutations in target nucleic acids;

[0025] (3) Food safety testing; (4) Simultaneous detection of specific gene sequences of multiple foodborne pathogens; (5) Clinical pathogen detection; (6) Environmental sample monitoring.

[0026] The pathogenic bacteria may be, but are not limited to, one or more of the following: Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, Salmonella enteritidis typhimurium serotype, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Aspergillus flavus, and Bacillus cereus vomiting type.

[0027] The target genes are not limited to one or more of the following: gyrB gene, 16SrRNA gene, rfbE gene, hlyA gene, Sdf gene, fimY gene, sed gene, lolB gene, tlh gene, aflR gene, cesB gene, escV gene, stx2 gene, and ipaH gene. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the method of the present invention.

[0029] Figure 2 The effect of different concentrations of Cas12a on the detection results of the single crRNA system.

[0030] Figure 3 These are experimental results validating the single crRNA fluorescence system.

[0031] Figure 4 This is a schematic diagram of the structure of a dual-target crRNA array (DcrRNA-ES).

[0032] Figure 5 To compare the detection results of the tandem crRNA array detection system and the single crRNA detection system.

[0033] Figure 6 The effects of linker sequence length (A), linker sequence (B), and the evaluation of the effects of DcrRNA-ES and DcrRNA-SE on the detection response (C).

[0034] Figure 7 Experimental results for crRNA concentration optimization (A) and reporter probe concentration optimization (B).

[0035] Figure 8 Experimental results for optimizing pre-incubation time (A) and reaction temperature (B).

[0036] Figure 9 For specificity assessment.

[0037] Figure 10 The results are from the experiments used to assess sensitivity.

[0038] Figure 11 This is a sequence diagram of an OcrRNA array.

[0039] Figure 12 Visualization of OcrRNA for each single target.

[0040] Figure 13 Signal uniformity analysis of OcrRNA arrays under different Cas12a concentrations.

[0041] Figure 14 Performance of the Cas12a system based on OcrRNA arrays in single-target and mixed-target detection.

[0042] Figure 15 Detection of 12 targets based on multiple crRNA arrays and verification of the universality of different Cas12a proteins. Detailed Implementation

[0043] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0044] like Figure 1 As shown, this invention provides a multi-target nucleic acid detection method based on Cas12a and crRNA arrays, comprising the following steps:

[0045] (1) Design and transcribe in vitro to obtain a tandem crRNA array containing at least two crRNAs;

[0046] (2) The crRNA array is combined with the Cas12a protein to form a complex;

[0047] (3) The complex is mixed with the sample to be tested, which can be a single target sample, a dual target sample or n target samples (n>2), and a fluorescent reporter probe is added to achieve the identification and cleavage of the target.

[0048] (4) Under suitable reaction conditions, if the sample contains a target nucleic acid that matches the crRNA array, a fluorescent signal will be generated. The detection results can also be visualized under fluorescent conditions.

[0049] Specifically, the entire reaction procedure may include the following steps: adding diluted buffer, Cas12a enzyme, RNA, and fluorescent probe; mixing appropriately under non-freezing conditions (e.g., room temperature 20-38℃); and pre-incubating to allow the Cas12a enzyme and RNA to form a complex. Adding 1 μL of target (target concentration can be set according to the experiment), the reaction temperature is 30-45℃ (preferably 39℃), and the reaction time can be 40 min to 1 h. As time increases, the fluorescence intensity increases, which can be detected using real-time instruments or observed visually after blue light irradiation. The target can be an amplicon of the target microbial nucleic acid, and amplification can be isothermal amplification such as PCR, RPA, or LAMP.

[0050] Cas12a enzymes include, but are not limited to, one or more of the following proteins: AsCas12a, BbCas12a, BoCas12a, FnCas12a, LbaCas12a, HkCas12a, OsCas12a, and their mutants. The present invention preferably uses FnCas12a, AsCas12a, or LbCas12a proteins. Cas12a enzymes can be obtained by purchase or purified through prokaryotic expression. For prokaryotic expression and purification methods, please refer to the following reference: Wang D, Ma D, Han J, et al. CRISPR RNA array-guided multisite cleavage for gene disruption by Cas9 and Cpf1[J]. ChemBioChem, 2018, 19(20):2195-2205.

[0051] Reaction buffers include, but are not limited to, NEBuffer™ 2.1, NEBuffer™ 3.1, NEBuffer™ CutSmart, or commercially available Cas reaction buffers purchased from New England Biolabs (NEB). Buffers in the reaction system help maintain high activity of the Cas12 enzyme and enhance its trans-cleavage activity after activation during the reaction.

[0052] The fluorescent probe can be a fluorescent single-stranded DNA (ssDNA) reporter probe labeled with a fluorescent group carboxyfluorescein (FAM) and a quenching group black hole quencher (BHQ); the quenching group black hole quencher includes one or more of black hole quencher 1 (BHQ-1), black hole quencher 2 (BHQ-2) or black hole quencher 3 (BHQ-3).

[0053] The present invention provides a kit that may include Cs12a protein, multiplex crRNA array, fluorescent probe and buffer.

[0054] This invention boasts advantages such as high throughput, high sensitivity, good specificity, wide applicability, and short detection time. Through crRNA array design, it can simultaneously detect ≥2 targets. Experiments using spacer sequences revealed that the signal intensity is highest in spacerless structures, ensuring high detection sensitivity. It can distinguish between single-base mutants and wild-type targets, making it suitable for drug resistance and mutation monitoring. Furthermore, it can be directly used for rapid screening of complex food and clinical samples, demonstrating excellent practicality and industrialization potential. In on-site testing of mixed samples of foodborne pathogens, results can be obtained within 40 minutes.

[0055] The sequences involved in the embodiments are shown in Tables 1 and 2.

[0056] Table 1 shows the target sequences and M13 primers involved in the examples.

[0057]

[0058]

[0059]

[0060] Table 2 shows the single crRNA sequences and tandem crRNA arrays involved in the examples.

[0061]

[0062]

[0063]

[0064] Note: General backbone sequences are underlined, while random base linkers are shown in bold.

[0065] In this example, the fluorescent probe was 5′-FAM-TTATT-BHQ1-3′, which was synthesized by General Biotech (Anhui) Co., Ltd.

[0066] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels.

[0067] Unless otherwise specified, the DNA or RNA and primers involved in the examples can be synthesized by General Biotech (Anhui) Co., Ltd.

[0068] The following is a description through specific embodiments.

[0069] Example 1

[0070] A single crRNA detection system was established, and the effect of different Cas12a concentrations on the detection results was investigated.

[0071] The single crRNA detection reaction system was prepared using standard buffer solutions. The reaction system included Cas12a 50-500 nM, crRNA-S 600 nM, and fluorescent probe 500 nM. All of the above concentrations were the final concentrations of the components in the reaction solution.

[0072] In actual operation, taking a 30μL reaction system as an example, add 3μL of standard buffer, 1μL of Cas12a, 1μL of crRNA-S, 1.5μL of fluorescent probe, and make up to 30μL with water.

[0073] Mix the above components thoroughly and pre-incubate at room temperature for 10 min. Add 1 μL of 40 ng / μL target gene amplicon. Monitor fluorescence kinetics changes for 40 min using a CFX96 real-time PCR system (Bio-Rad Laboratories, California) at 39°C. Experimental data are expressed as the mean ± standard error of three independent experiments, with two technical replicates for each experiment. Student's t test was used for pairwise comparisons between groups, and Kruskal-Wallis test combined with Dunn's post-hoc test was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0074] Another template-free control (NTC) was set up, in which an equal amount of ddH2O was used to replace the target gene amplicon, and the same applies below.

[0075] The standard buffer consisted of 10 mM Tris-HCl (pH 8.0), 0.1 mM CaCl2, and 20 mM MgCl2, with 1 U RNase inhibitor (Thermo Fisher Scientific, Beijing, China) added. The solvent was water (such as RNase-free water or DEPC water). Cas12a was FnCas12a. crRNA-S could be obtained through whole-gene synthesis or in vitro transcription. The preparation method of the target gene amplicon included the following steps: A specific fragment (300 bp, Target-S, Table 1) targeting the Staphylococcus aureus 16S rRNA gene was used as the target DNA. This fragment was synthesized in the plasmid backbone pUC57-GW-Kan (purchased from Suzhou Genewise Biotechnology Co., Ltd.). Using this plasmid as a template and M13f and M13r as primers, the target gene amplicon was obtained by PCR amplification. Amplification system: M13f (20μM) 0.5μL, M13r (20μM) 0.5μL, 2×HiFi polymerase mix 25μL, plasmid template (10ng / μL) 1μL, ddH2O 23μL. Amplification program: 94℃, 2min; 94℃, 30s, 60℃, 30s, 72℃, 15s, 30 cycles; 72℃, 5min, 14℃∞. The fragment size of the amplified products was confirmed by agarose gel electrophoresis.

[0076] Test results as follows Figure 2 As shown, the single crRNA system reaches its optimal concentration at 100 nM Cas12a, while the signal decreases at 300–500 nM.

[0077] Example 2

[0078] The Cas12a concentration was set to 100 nM, and all other parameters were the same as in Example 1. This study verified whether crRNA-E could produce fluorescent signals against the gyrB gene target (Target-E) of *E. coli* O157:H7 and the 16S rRNA gene (Target-S) of *Staphylococcus aureus*. crRNA-E can be obtained through whole-gene synthesis or in vitro transcription.

[0079] The Cas12a concentration was set to 100 nM, and all other parameters were the same as in Example 1. This study verified whether crRNA-S could produce fluorescent signals against the gyrB gene target (Target-E) of Escherichia coli O157:H7 and the 16S rRNA gene (Target-S) of Staphylococcus aureus.

[0080] Test results as follows Figure 3As shown, in the single crRNA system, crRNA-E produces a fluorescent signal against the gyrB gene target (Target-E) of *E. coli* O157:H7 within 40 min, but does not produce a fluorescent signal against the 16S rRNA gene (Target-S) of *Staphylococcus aureus*. crRNA-S produces a fluorescent signal against the 16S rRNA gene (Target-S) of *Staphylococcus aureus*, but does not produce a fluorescent signal against the gyrB gene target (Target-E) of *E. coli* O157:H7. This demonstrates that both crRNA-E and crRNA-S designed in this study have high specificity and can be used to detect *E. coli* O157:H7 and *Staphylococcus aureus*, respectively, and that the established single crRNA fluorescence system is feasible.

[0081] Example 3

[0082] Establish a dual crRNA array-mediated Cas12a detection system and optimize Cas12a concentration.

[0083] A schematic diagram of the dual-target crRNA array (DcrRNA-ES) structure is shown below. Figure 4 As shown in Table 2, the DcrRNA-ES sequence contains crRNA-E targeting E. coli O157:H7 and crRNA-S targeting Staphylococcus aureus. crRNA-E targets the gyrB gene of E. coli O157:H7, and crRNA-S targets the 16S rRNA gene of Staphylococcus aureus. The preparation method of DcrRNA-ES can include the following steps: PCR amplification of the DcrRNA-ES DNA fragment; verification of the PCR product by agarose gel electrophoresis; purification using the Oligo Clean & Concentrator purification kit (Zymo Research, Beijing, China); and subsequent in vitro transcription using the TranscriptAid T7 high-efficiency transcription kit (Thermo Fisher Scientific, Beijing, China). The transcribed RNA is purified again using the same purification kit, quantified using a Nano-300 ultra-micro spectrophotometer (Ausun Technology, Hangzhou, China), and stored at -80℃.

[0084] The target amplicon was the amplification product of Target-S. In the reaction system of Example 1, crRNA-S was replaced with DcrRNA-ES, and all other steps were the same as in Example 1. The detection results of the dual crRNA array detection system and the single crRNA detection system were compared, and the results are as follows: Figure 5As shown, the single crRNA system reached its optimal concentration at 100 nM Cas12a, while the signal decreased at 300–500 nM, indicating that the single crRNA system was inhibited under high enzyme conditions. In contrast, the DcrRNA system showed the strongest fluorescence at 300 nM FnCas12a and maintained high activity at 500 nM, indicating that the DcrRNA system had improved tolerance to higher concentrations of Cas12a.

[0085] Example 4

[0086] (1) The effect of spacer sequence length on the detection results of the dual crRNA array detection system

[0087] For DcrRNA, a double crRNA array was constructed with no spacer (L0) between crRNA-E and crRNA-S or with a 10–60 nt spacer sequence added. The RNA sequences are shown in Table 2, and the RNA was obtained by in vitro transcription.

[0088] The Cas12a concentration was set at 100 nM, the target amplicon was the amplification product of Target-S or Target-E, and the DcrRNAs were DcrRNA-ES(L0), DcrRNA-ES(L10), DcrRNA-ES(L20), DcrRNA-ES(L30), and DcrRNA-ES(60), respectively. The other detection reaction system and methods were as described in Example 3.

[0089] The effect of crRNA linker sequence length, such as Figure 6 As shown in Figure A, this structural variable may affect crRNA folding, accessibility, and Cas12a assembly. This invention constructed four variants, separating crRNA-E and crRNA-S with random RNA linker sequences of 10, 20, 30, or 60 nucleotides, respectively. Fluorescence detection using the corresponding plasmid targets revealed significant positional asymmetry: the activity of the first (5′) crRNA-E decreased with increasing linker sequence length, while the activity of the second (3′) crRNA-S remained stable with only slight decay. The variant without the linker sequence (0-nt) produced the strongest and most balanced signal, representing the optimal conformation.

[0090] (2) The effect of the ligation sequence on the detection results of the dual crRNA array detection system

[0091] For DcrRNA-ES(L20), two constructs containing different 20nt random ligation sequences were set up, namely DcrRNA-ES(L20a) and DcrRNA-ES(L20b). The RNA sequences are shown in Table 2, and they were obtained by in vitro transcription.

[0092] The Cas12a concentration was set at 100 nM, the target amplicons were the amplification products of Target-S or Target-E, and the DcrRNAs were DcrRNA-ES(L20), DcrRNA-ES(L20a), and DcrRNA-ES(L20b), respectively. The remaining detection reaction system and methods were as described in Example 3.

[0093] Test results as follows Figure 6 As shown in Figure B, it can be seen that all three 20-nt variants produce similar fluorescence signals for the dual targets, indicating that the linker sequence does not affect performance.

[0094] (3) Evaluate the effects of DcrRNA-ES and DcrRNA-SE on the detection response.

[0095] The sequence of DcrRNA-SE is shown in Table 2. The linking order of crRNA-S and crRNA-E in DcrRNA-SE is the reverse of the linking order of crRNA-E and crRNA-S in DcrRNA-ES.

[0096] A dual-array reaction was performed using DcrRNA-ES and DcrRNA-SE, with the Cas12a concentration set to 100 nM. The detection system and method were as described in Example 3. The results were compared with conventional detection using single crRNA or an equimolar mixture of crRNA or its mixed crRNA pool. The mixed crRNA pool consisted of a mixture of crRNA-S and crRNA-E, with both crRNA-S and crRNA-E having a final concentration of 600 nM.

[0097] like Figure 6 As shown in Figure C, the detection results indicate that the fluorescence signals of all detection modes are comparable when the crRNA is located at the array leader position. Taking DcrRNA-SE as an example, when crRNA-S is located at the array leader position, its fluorescence signal value is consistent with that of the mixed crRNA pool and the single crRNA-S detection system. For both DcrRNA-SE and DcrRNA-ES, the dual-array mode produces a stronger signal than the single crRNA or mixed format. These results suggest that the dual-crRNA array is equivalent to the conventional method in detecting the first (5′) crRNA while significantly enhancing the activity of the second (3′) crRNA.

[0098] Example 5

[0099] (1) Optimization of crRNA concentration

[0100] Referring to the detection method in Example 3, based on Example 3, Cas12a was fixed at 100 nM, the fluorescent probe at 500 nM, and the target amplicon at Target-S. The effects of 200 nM, 400 nM, 600 nM, and 800 nM DcrRNA-ES on the detection results were tested respectively.

[0101] like Figure 7 As shown in Figure A, this invention systematically evaluated the impact of crRNA concentration on the performance of the Cas12a detection system and found that the dual crRNA array (DcrRNA-ES system) has significant advantages over the single crRNA system. Experiments showed that the single crRNA system reached its peak fluorescence signal at a crRNA concentration of 600 nM, while exhibiting an inhibitory effect at 800 nM, consistent with the negative impact of high-concentration crRNA on ribonucleoprotein complex activity in existing technologies. In contrast, the DcrRNA-ES system showed a stronger signal response across all tested concentration ranges, with the most significant improvement at low concentrations.

[0102] (2) Report probe concentration optimization

[0103] Referring to the detection method in Example 3, based on Example 3, the concentration of Cas12a was fixed at 100 nM, the concentration of DcrRNA-ES at 600 nM, and the target amplicon was Target-S. The effects of 200 nM, 300 nM, 400 nM and 500 nM fluorescent probes (ss DNA reporter) on the detection results were tested respectively.

[0104] like Figure 7 As shown in Figure B, increasing the fluorescent probe concentration linearly enhances the fluorescence signal of both the dual-crRNA array (DcrRNA-ES system) and the single-crRNA system, without signal saturation or background increase in the absence of a template control. Notably, the DcrRNA-ES system outperforms the single-crRNA system under all fluorescent probe concentration conditions. These results confirm that the dual-crRNA array maintains high efficiency even at low input levels, providing crucial technical support for the development of low-cost, multiplex molecular diagnostic platforms.

[0105] Example 6

[0106] (1) Investigate the effect of pre-incubation time on the reaction

[0107] Based on Example 3, the Cas12a concentration was fixed at 100 nM and the DcrRNA-ES concentration at 600 nM, with all other parameters remaining the same as in Example 3. The standard buffer, Cas12a, DcrRNA-ES, and fluorescent probe were mixed and pre-incubated at room temperature for 0 min, 5 min, 10 min, and 20 min, respectively. 1 μL of 40 ng / μL target amplicon Target-S was added, and fluorescence kinetics were monitored for 40 min at 37°C using a CFX96 real-time PCR system.

[0108] The effect of different pre-incubation times on detection performance, such as Figure 8 As shown in Figure A, pre-incubation promotes the formation of the Cas12a-crRNA complex in the dual-crRNA array, thereby improving target cleavage efficiency. Comparing the DcrRNA-ES system and the single-crRNA system under the same conditions, it can be seen that in the single-crRNA system, the fluorescence signal output is largely unaffected by the pre-incubation time; while in the DcrRNA-ES system, a brief pre-incubation of only 5 minutes is sufficient to significantly enhance the signal intensity compared to the single-crRNA system. These findings reveal the key advantage of the DcrRNA-ES system: its ability to rapidly form an active Cas12a-DcrRNA complex, thus enhancing detection performance.

[0109] (2) Investigating the effect of reaction temperature on detection

[0110] Based on Example 3, the Cas12a concentration was fixed at 100 nM and the DcrRNA-ES concentration at 600 nM, with all other parameters remaining the same as in Example 3. The standard buffer, Cas12a, DcrRNA-ES, and fluorescent probe were mixed and pre-incubated at room temperature for 10 min. 1 μL of 40 ng / μL target amplicon Target-S was added. Fluorescence kinetics were monitored for 40 min at 35℃, 37℃, 39℃, and 41℃ using a CFX96 real-time PCR system.

[0111] Comparing the DcrRNA-ES system and the single crRNA system under the same conditions, such as Figure 8 As shown in Figure B, it can be seen that the signal intensity of both systems gradually increases within this range as the temperature rises. Notably, in the 35-39℃ range, the DcrRNA-ES system exhibits significantly higher fluorescence intensity than the single crRNA system. This result indicates that under more easily achievable field-ready moderate temperature conditions (<41℃), the DcrRNA-ES system has higher Cas12a-crRNA complex formation efficiency and / or target cleavage activity.

[0112] Example 7

[0113] The Cas12a concentration was fixed at 100 nM, and the mixture was pre-incubated at room temperature for 10 min. The target assay was then added at a concentration of 2 nM. Figure 10 The target S is shown to have single-base mutants and multi-point mutants of the protospacer neighbor motif (PAM) and protospacer. Fluorescence kinetics were monitored using a CFX96 real-time PCR system at 39°C for 0-40 min. All other parameters were the same as in Example 3.

[0114] like Figure 9 As shown, single-base mutants and multi-point mutants of target S, PAM and protospacer, were constructed. Figure 9 In the diagram, red text indicates mutated bases, A represents the sequence of target S, B shows the detection results of DcrRNA-ES and single crRNA systems for different single-base mutants of PAM targeting the target S sequence, and C shows the detection results of DcrRNA-ES and single crRNA systems for different single / multi-base mutants of protospacer targeting the target S sequence. It can be seen that mutations at key PAM sites (-4, -6, -7, -9) significantly reduce the detection signal; mutations in the seed region are sensitive, while distant mutations have high tolerance; three or more mutations completely block detection.

[0115] This invention can distinguish between single-base mutants and wild-type targets, making it suitable for drug resistance and mutation monitoring.

[0116] Example 8

[0117] Sensitivity assessment: Using the amplification products of Target-S or Target-E as target amplicon, the DcrRNA-ES system and the single crRNA system were used for detection.

[0118] For the DcrRNA-ES system detection, based on Example 3, the Cas12a concentration was set to 100 nM, the DcrRNA-ES concentration was set to 600 nM, and the final concentrations of the target amplicon in the reaction system were (0.3-8.5 nM), with the rest being the same as in Example 3.

[0119] For single crRNA system detection, crRNA-E and crRNA-S were used instead of DcrRNA-ES, and the rest of the methods were the same as those for DcrRNA-ES system detection.

[0120] Test results as follows Figure 10 As shown, both systems exhibit a fluorescence signal that increases proportionally with the target concentration, and show a linear response (R0) in the low concentration range (<2 nM). 2>0.99). These results confirm that the Cas12a / DcrRNA system can achieve precise quantitative differentiation of target abundance while maintaining high specificity comparable to single crRNA systems. Furthermore, within the range of 0–2 nM, the fluorescence of the DcrRNA system is linearly correlated with the target concentration (R0). 2 >0.99), achieving high-precision quantification.

[0121] Example 9: Multi-target detection mediated by an octet crRNA array

[0122] like Figure 11 As shown in Table 2, eight target OcrRNA arrays were designed and constructed, corresponding to Perosamine synthetase homolog rfbE (S83460.1) of E. coli O157:H7, Listeriolysin O hlyA (AF253320.1) of Listeria monocytogenes, Salmonella difference fragments Sdf (AF370707.1) of Salmonella enterica serovar Enteritidis, Fimbriae Y fimY (M90677.1) of S. enterica serovar Typhimurium, Staphylococcal entererotoxin D variant v4 sed (KX168622.1) of Staphylococcus aureus, Outer membranelipoprotein lolB (GQ403683.1) of Vibrio cholerae, and Thermolabile hemolysin of V. parahaemolyticus. tlh (MH047289.1), Aflatoxin regulator aflR (AF441435.2) of Aspergillusflavus, etc. (Table 1).

[0123] Visual detection of each single target was performed using either crRNA or OcrRNA. The reaction system included: standard buffer, 100 nM FnCas12a, 600 nM crRNA or OcrRNA, and 500 nM fluorescent probe (5′-FAM-TTATT-BHQ1-3′). After mixing, the components were pre-incubated at room temperature for 10 min. 1 μL of target amplicons (i.e., unpurified, quantified, and diluted using the ZYMO kit, and directly used for detection after amplification) were added. The target amplicons were Target-1 to Target-8 (Table 1). Fluorescence was then monitored using a CFX96 real-time PCR system (Bio-Rad Laboratories, California) at 39°C. Fluorescence values ​​were recorded after 60 min of reaction and observed under 470 nm blue light. A template-free control (No Template Control) was also included.

[0124] The above standard buffer solution includes: 10 mM Tris-HCl (pH 8.0), 0.1 mM CaCl2 and 20 mM MgCl2, with 1 U RNase inhibitor added (Thermo Fisher Scientific, Beijing, China), and water as the solvent.

[0125] Test results as follows Figure 12 As shown, "-" indicates NTC, meaning No Template Control; "+" indicates a positive result, meaning the sample contains the target. Figure 12 It can be seen that the OcrRNA system can detect eight targets simultaneously in a single reaction, and the signals can be observed with the naked eye.

[0126] Example 10

[0127] Based on Example 9, the concentrations of FnCas12a were adjusted to 100 nM, 400 nM, and 800 nM, and fluorescence kinetics were monitored for 60 min using a CFX96 real-time PCR system (Bio-Rad Laboratories, California) at 39°C. Experimental data are expressed as the mean ± standard error of three independent experiments, with two technical replicates for each experiment. Student's t-test was used for pairwise comparisons between groups, and Kruskal-Wallis test combined with Dunn's post-hoc test was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. All other aspects were the same as in Example 9.

[0128] Experimental results are as follows Figure 13 As shown, "-" indicates NTC, meaning No Template Control; "+" indicates a positive result, meaning the sample contains the target. From Figure 13It can be seen that OcrRNA can generate a signal. The OcrRNA signal at both ends of the array (i.e., positions 1, 2, 7 and 8 in the order of 5′ to 3′, located in the non-middle positions on both sides) is slightly weak, but it can be restored to the single crRNA level when the Cas12a concentration is increased to 800 nM.

[0129] Example 11

[0130] (1) Based on Example 10, the concentration of FnCas12a was set to 100 nM, and Target-1 to Target-8 were detected using crRNA-1 to crRNA-8 and OcrRNA, respectively. All other aspects were the same as in Example 10.

[0131] Test results as follows Figure 14 As shown in Figure A, it can be seen that OcrRNA can simultaneously trigger the fluorescence output of eight targets in a single reaction system. The signals of each target are consistent with the corresponding single crRNA control, proving that the system can achieve parallel detection of eight pathogen genes.

[0132] (2) Prepare simulated samples of multiple targets. Following the method described in (1) above, test simulated samples of multiple targets. Take target amplicon of the same molar concentration and volume, mix them as follows to prepare simulated samples of multiple targets, and take 1 μL of the mixture for testing.

[0133] The simulated samples for multiple targets are as follows: T1+T2 represents Target-1 amplicon + Target-2 amplicon. T1+T5 represents Target-1 amplicon + Target-5 amplicon. T1+T2+T8 represents Target-1 amplicon + Target-2 amplicon + Target-8 amplicon. T2+T4+T6 represents Target-2 amplicon + Target-4 amplicon + Target-6 amplicon. T3+T6+T7+T8 represents Target-3 amplicon + Target-6 amplicon + Target-7 amplicon + Target-8 amplicon. T1+T3+T5+T7 represents Target-1 amplicon + Target-3 amplicon + Target-5 amplicon + Target-7 amplicon. T1+T3+T6+T8 represents Target-1 amplicon + Target-3 amplicon + Target-6 amplicon + Target-8 amplicon. T2+T3+T4+T5+T6 represents Target-2 amplicon + Target-3 amplicon + Target-4 amplicon + Target-5 amplicon + Target-6 amplicon. Mix of All 8 Targets represents Target-1 amplicon + Target-2 amplicon + Target-3 amplicon + Target-4 amplicon + Target-5 amplicon + Target-6 amplicon + Target-7 amplicon + Target-8 amplicon.

[0134] Test results as follows Figure 14 As shown in Figure B, in mixed target (2–8 types) samples, the OcrRNA system can accurately output all positive signals without cross-reactivity. This demonstrates that the array strategy of this invention has scalability and high-throughput advantages.

[0135] In summary, when multiple pathogen targets are added to a mixed sample, the detection system of this invention can simultaneously output positive signals, and the different targets do not interfere with each other. It can be used for multi-target detection of complex samples, and the method of this invention is suitable for detection applications of food safety or clinical samples.

[0136] Example 12

[0137] This embodiment expands the detection system to 12 targets and evaluates the universality of this strategy for multiple Cas12a proteins such as LbCas12a and AsCas12a.

[0138] This demonstrates the scalability and universality of the Cas12a multi-target detection strategy based on multiple crRNA arrays provided by the invention.

[0139] crRNA array design and synthesis: Twelve corresponding crRNA units were designed based on the specific gene sequences of 12 common foodborne pathogens (e.g., Salmonella, Listeria, Escherichia coli O157:H7, Staphylococcus aureus, etc.). Each crRNA unit was tandemly linked with a previously optimized 0-nt linker sequence (crRNA-1 to crRNA-12) in a 5′ to 3′ direction to synthesize a single 12-fold crRNA array transcript. The sequences of the 12-fold tandem crRNA array are shown in Table 2.

[0140] Cas12a protein: Prokaryotic expression of purified FnCas12a, LbCas12a and AsCas12a proteins, expressed using the same method as FnCas12a.

[0141] Target nucleic acids: Short DNA fragments (~300bp, containing PAM sequences and crRNA recognition regions, Table 1) of the target genes of the above 12 pathogenic bacteria were synthesized, and amplicon for each target was obtained by PCR amplification. A negative control group without target addition was also set up (i.e., the target was replaced with an equal amount of water).

[0142] The single-stranded DNA fluorescent probe, 5′-FAM-TTATT-BHQ1-3′, was synthesized by General Biotechnology (Anhui) Co., Ltd.

[0143] The standard buffer consists of 10 mM Tris-HCl (pH 8.0), 0.1 mM CaCl2 and 20 mM MgCl2, with the addition of 1URNase inhibitor (Thermo Fisher Scientific, Beijing, China), and the solvent is water (such as RNase-free water or DEPC water).

[0144] Detection Reaction: The fluorescence-based Cas12a detection reaction was prepared using standard buffer and performed in a 30 μL system containing Cas12a protein, 600 nM of 12-replenish crRNA transcripts, and 200 nM of single-stranded DNA fluorescent probe. When Cas12a was selected from AsCas12a, the final concentration of AsCas12a in the reaction system was 80 nM. When Cas12a was selected from LbCas12a, the final concentration of LbCas12a in the reaction system was 100 nM. When Cas12a was selected from FnCas12a, the final concentration of FnCas12a in the reaction system was 800 nM.

[0145] The above components were mixed and pre-incubated at room temperature for 10 min. 1 μL of target amplicon (i.e., unpurified, quantified, and diluted using the ZYMO kit, 1 μL directly used for detection after amplification) was added. The target amplicones were Target-1 to Target-12, as shown in Table 1. Fluorescence was then monitored using a CFX96 real-time PCR system (Bio-Rad Laboratories, California) at 39°C. The fluorescence value was recorded after 1 h of reaction and observed under 470 nm blue light.

[0146] Experimental results are as follows Figure 15 As shown, when using FnCas12a protein, the addition of 12 single targets can induce strong fluorescence signals compared with the negative control (NC), indicating that each crRNA unit in the multiplex crRNA array can independently and effectively guide FnCas12a to cleave its corresponding target and activate its trans-cleavage activity.

[0147] The performance of FnCas12a, LbCas12a, and AsCas12a in the same system was compared, and the experimental results are as follows: Figure 15 As shown, both LbCas12a and AsCas12a produce a rapid fluorescence response similar to that of FnCas12a. Meanwhile, their respective negative control groups showed no significant signal. This result fully demonstrates that the "multiplex crRNA array" strategy proposed in this invention is not limited to a specific Cas12a protein, but is applicable to a variety of Cas12a homologous proteins, including FnCas12a, LbCas12a, and AsCas12a, and has broad versatility.

[0148] This invention proposes a single Cas12a strategy based on multiplex crRNA arrays, also known as a "single-enzyme, multi-target" strategy. It uses a single type of Cas12a protein and a multiplex crRNA array, along with a single, universal reporter molecule, and interprets the results through reaction kinetics or endpoint signal intensity. This strategy achieves extreme simplification and low cost, eliminating reliance on complex multi-enzyme systems and multicolor fluorescence detection equipment. The system is simple and extremely low-cost, requiring only one Cas12a protein and a single, universal, and inexpensive ssDNA-fluorescence-quenched reporter molecule. It can be performed on a single-channel fluorescence detector or even a portable blue light transilluminator (colorimetric method), making the equipment barrier very low. This significantly reduces the overall cost of reagents and detection equipment, making this technology feasible for rapid on-site testing, primary healthcare institutions, and resource-scarce areas. This invention has excellent scalability; theoretically, the maximum number of crRNA units that can be tandemly linked in a single crRNA array is very high (capable of detecting >12 targets). Adding a new target only requires adding the corresponding sequence to the array DNA template, with negligible cost increase. This invention achieves near-linear, low-cost scalability, making it particularly suitable for scenarios requiring high-throughput screening (such as multi-site pathogen typing and multi-gene SNP detection). Designed specifically for rapid on-site detection and portable applications, the system is simple and stable, with low equipment requirements and rapid response, making it ideal for on-site food safety sampling, rapid pathogen detection in primary hospitals, and home testing. It precisely addresses the pain point of existing high-end technologies being unable to be applied on-site, filling a market gap. The main technical challenge of this invention lies in the sequence design and optimization of the crRNA array, which is relatively low-barrier and easy for professionals in the field to master and implement. This lowers the technical implementation threshold, facilitating rapid technology promotion and industrialization. This invention takes a different approach, providing a disruptive strategy based on "single enzyme multifunctionality." By cleverly designing multiple crRNA arrays, it maximizes the multi-target detection capability of the single, inexpensive Cas12a protein. The core advantage of this strategy lies in its extreme simplicity—it abandons complex multi-enzyme systems and multicolor fluorescent probes, instead using an optimized, modular crRNA array to guide a single Cas protein to complete all tasks. Therefore, this invention is not a simple improvement on existing technologies, but rather provides a completely new technological path that is more closely aligned with practical and commercial needs. It successfully maintains the high sensitivity and specificity of CRISPR technology while revolutionarily reducing costs, simplifying operation, and achieving outstanding scalability, making it possible to develop truly "high-throughput, low-cost, and portable" molecular diagnostic products, representing a clear technological advancement and commercial breakthrough in this field.

[0149] Compared to the existing technology CN201910673246.8, this invention uses a simple linear multiplex crRNA array to guide the Cas12a protein, paired with a universal, single fluorescent reporter molecule (such as ssDNA-FQ). This achieves decoupling in principle, separating target recognition (crRNA array) from signal output (universal reporter molecule), resulting in a simpler and more robust system. This invention is simple to design and has extremely low synthesis costs. The crRNA array can be obtained through a single in vitro transcription, a mature and inexpensive method. The ligation sequence can be standardized, lowering the design threshold. The universal reporter molecule can be synthesized on a large scale at low cost. This significantly reduces development and production costs, making it particularly suitable for applications requiring the detection of a large number of targets (e.g., more than 10), solving the fundamental problem of existing technologies being difficult to commercialize due to excessive cost. The system of this invention is simple: it contains only one Cas12a protein, one crRNA array, and one universal reporter molecule. The components are single, resulting in low background signal, system stability, and ease of optimization and standardization. This improves the reliability and reproducibility of detection, making it easier to develop into a stable and reliable reagent kit. This invention boasts exceptional scalability: by simply replacing or adding new crRNA unit sequences to the DNA template within a validated array backbone, detection throughput can be easily expanded. This high degree of modularity and scalability enables the rapid development of product series for different detection panels. In summary, while existing technology CN201910673246.8 achieves multi-target detection, it relies on costly and complex dedicated matching probes, exhibiting significant disadvantages in commercialization cost, system stability, scalability, and universality, severely hindering its practical application, particularly in rapid on-site detection and low-cost screening scenarios. This invention fundamentally overcomes these shortcomings by introducing an innovative decoupled design of "multiple crRNA array + universal reporter molecule." This invention not only achieves lower cost and higher scalability but also provides a more stable and universal detection platform, representing a significant technological advancement in this field and laying a solid foundation for developing truly high-throughput, low-cost, and portable molecular diagnostic kits.

Claims

1. A Cas12a multi-target rapid detection method based on a multiple crRNA array, characterized by, The method comprises the following steps: (1) designing and synthesizing a crRNA array comprising two or more crRNA units, which are directly connected or connected by variable length connection sequences; (2) forming a complex of the crRNA array and a Cas12a protein; (3) contacting the complex with target nucleic acids in a sample to be tested, and reacting in the presence of a reporter probe; (4) detecting the results by fluorescence or colorimetric signal output of the reporter probe to simultaneously identify one or more target nucleic acid sequences.

2. The method of claim 1, wherein, The crRNA array is a crRNA array with a target number of ≥2.

3. The method of claim 1 or 2, wherein, The connection sequence consists of nucleotides.

4. The method of claim 1 or 2, wherein, The molar ratio of Cas12a to crRNA is (50-800):

600.

5. The method of claim 1 or 2, wherein, The Cas12a protein is FnCas12a, LbCas12a, AsCas12a or a modified variant thereof.

6. A kit for carrying out the method according to any one of claims 1 to 5, characterized in that, The kit comprises one or more of a Cas12a protein, a nucleic acid molecule containing a double-target or multi-target crRNA array, a reporter probe and a reaction buffer.

7. The kit of claim 6, wherein The reporter probe is a fluorescence quenching probe or a colorimetric probe.

8. The kit according to claim 6 or 7, characterized in that It also includes a reading module for portable detection.

9. Use of the method of any one of claims 1-5 in any one or more of (1) to (6); (1) multi-target detection; (2) identifying single base mutations in target nucleic acids; (3) food safety detection; (4) detecting specific gene sequences of multiple foodborne pathogens; (5) clinical pathogen detection; (6) environmental sample monitoring.

10. Use of the kit of any one of claims 6-8 in any one or more of (1) to (6); (1) multi-target detection; (2) identifying single base mutations in target nucleic acids; (3) food safety detection; (4) detecting specific gene sequences of multiple foodborne pathogens; (5) clinical pathogen detection; (6) environmental sample monitoring.

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