A DNA detection kit based on CRISPR / Cas9 system and exonuclease lambda Exo and application thereof

Abstract

The application discloses a DNA detection kit based on a CRISPR / Cas9 system and exonuclease lambda Exo and application thereof, and belongs to the technical field of molecular biology and gene detection, wherein the kit comprises Cas9, Spacer RNA, Scaffold RNA, lambda Exo, pDNA and a single-stranded nucleic acid reporter molecule; in detection, the pDNA guides the lambda Exo to open the double strand at a specific site of a dsDNA target to form an R-Loop structure, and then the assembled SCas9 RNP recognizes the target sequence and activates the trans-cleavage activity to cut the reporter molecule to generate a signal. The strategy activates the trans-cleavage activity of the Cas9, and simultaneously eliminates the inherent cis-cleavage activity, thereby not only eliminating the dependence of the traditional CRISPR system on a PAM sequence, but also enabling the nucleic acid detection to be performed in the same tube as an isothermal amplification system, so that the false positive risk is reduced.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and gene detection technology, specifically to a DNA detection kit based on the CRISPR / Cas9 system and the exonuclease λExo, and its applications. Background Technology

[0002] In the existing technology, CRISPR-based nucleic acid detection platforms are mainly divided into two categories: one is based on Cas12 and Cas13 systems (such as DETECTR and SHERLOCK), and the other is based on Cas9 systems.

[0003] Cas12a (such as LbCas12a or AsCas12a) belongs to the Type VA system and naturally possesses non-specific single-stranded DNA cleavage activity (i.e., trans cleavage activity) activated after target binding, thus it is widely used in nucleic acid detection. However, the Cas12a system has two inherent limitations: (1) the recognition of double-stranded DNA (dsDNA) targets strictly depends on the T-rich PAM sequence (usually 5'-TTTN-3' or 5'-TTN-3'), which limits the range of detectable targets; (2) after recognizing the target, Cas12a will cis-cleave the target double-stranded DNA, resulting in the degradation of the target DNA. This makes it difficult to achieve a true "one-tube" reaction with amplification technologies that require the preservation of target integrity (such as RPA). The current conventional two-step reaction detection is subject to aerosol contamination, which can lead to false positives. Therefore, achieving a true "one-tube" reaction is of great significance.

[0004] Traditional Cas9 (such as SpyCas9) belongs to the Type II system and has long been considered to have only cis-cleavage activity for genome editing. Although recent studies have reported that the tracrRNA:crRNA-Cas9 complex has trans-cleavage activity (Chen J, Chen Y, Huang L, Lin X, Chen H, Xiang W, Liu L. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat Biotechnol. 2025 Apr;43(4):558-568.), the inventors found in-depth research that the system has significant defects: (1) its recognition of ssDNA substrates is highly sequence-specific, and it can only recognize specific engineered sequences (such as T01), and has no response to clinically relevant sequences (such as HPV16, monkeypox virus B6R, JAK2, etc.); (2) its recognition of dsDNA is limited to short fragments with a length of no more than 20 bp, and requires nanomolar substrate concentrations, which are far from meeting the needs of clinical detection. Therefore, when the Cas9 system is used for nucleic acid detection, it not only has the same inherent limitations as the Cas12a system, but also has strict limitations on dsDNA substrate recognition, length, concentration, etc. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention aims to develop a novel Cas9 detection system that does not rely on PAM sequences, has high sensitivity to dsDNA, and is compatible with amplification technology, so as to promote the widespread application of Cas9 system in nucleic acid detection.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a DNA detection kit based on the CRISPR / Cas9 system and the exonuclease λExo, comprising at least the following reagents: The Cas9 protein possesses both cis- and trans-cleavage activities. Spacer RNA contains sequences complementary to the target DNA, which are used to specifically recognize the target; Scaffold RNA binds to the Cas9 protein to form the Cas9-Scaffold complex and can activate the trans-cleavage activity of the Cas9 protein in the presence of Spacer RNA; The λ exonuclease (λExo) and its guide DNA (pDNA) are used to guide λExo to specifically bind to and unwind the dsDNA double strand, forming an R-loop structure, thereby exposing the target dsDNA target region as the target single-stranded region. A single-stranded nucleic acid reporter molecule, which is non-specifically cleaved when the trans-cleavage activity of the Cas9 protein is activated.

[0007] It is understood that in the above kit, scaffold RNA and spacer RNA are derived from the splitting of the single-stranded guide RNA (sgRNA) of the complete Cas9 protein. Therefore, the scaffold RNA sequence is determined by the Cas9 protein, meaning that different scaffold RNA sequences are used for different Cas9 proteins. This invention constructs a novel Split gRNA-Cas9 system (hereinafter referred to as the SCas9 system) based on the split guide RNA (split gRNA), and unexpectedly discovered that this treatment causes the detection system to lose cis-cleavage activity but retains highly efficient trans-cleavage activity.

[0008] Preferably, in the above DNA detection kit, the Cas9 protein is derived from Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes The Cas9 protein (hereinafter referred to as SpyCas9) has the amino acid sequence shown in SEQ ID NO.2. In practical applications, the Cas9 protein used can be either a natural protein or an artificially synthesized recombinant protein.

[0009] In the aforementioned DNA detection kit, when the Cas9 protein is SpyCas9, the Scaffold RNA can be the sequence shown in SEQ ID NO.3 or a functional variant thereof. This functional variant should retain the ability to bind to the Cas9 protein and activate Cas9 trans-cleavage activity in the presence of SpacerRNA. In some preferred embodiments of the present invention, the Scaffold RNA is a functional variant with the sequence shown in SEQ ID NO.4. Truncation of this variant compared to the full-length sequence shown in SEQ ID NO.3 significantly improves the detection effect.

[0010] In the aforementioned DNA detection kit, the function of λExo and its pDNA after binding is to target and unwind dsDNA at specific sites, rather than eliminating dsDNA in the traditional way. Furthermore, experiments in this invention demonstrate that the design location of the pDNA is extremely important; when the pDNA is designed to target the non-target strand or target strand upstream of the target dsDNA region, the detection signal can be maximized. This invention innovatively introduces λExo and its pDNA into the SCas9 system, forming a local R-loop structure in the target dsDNA region, enabling Cas9 to recognize and bind to the target without relying on the PAM sequence.

[0011] Preferably, in the above-mentioned DNA detection kit, the single-stranded nucleic acid reporter molecule is an ssDNA probe labeled with a fluorescent group and a quencher group at both ends, respectively; more preferably, the length of the ssDNA probe is not less than 13 nt. When the single-stranded nucleic acid reporter molecule is the above-mentioned ssDNA probe, the ssDNA probe generates a fluorescent signal after being trans-cleaved by the Cas9 protein. Thus, the presence of target DNA in the sample can be determined by whether or not a fluorescent signal is generated, and quantitative analysis can be performed based on the difference in fluorescence signal intensity.

[0012] In the aforementioned DNA detection kits, the Spacer RNA and pDNA need to be specifically designed based on the different detection targets. For example, in some embodiments of the present invention, the target DNA is the human JAK2 gene, the Spacer RNA sequence is shown in SEQ ID NO. 5, and the pDNA sequence may be as shown in SEQ ID NO. 6, SEQ ID NO. 9, or SEQ ID NO. 11. In other embodiments of the present invention, the target DNA is the HPV16 genome, the Spacer RNA sequence is shown in SEQ ID NO. 12, and the pDNA sequence is shown in SEQ ID NO. 13. The term "complementarity" means that the Spacer RNA / pDNA can pair and bind to a specific region (i.e., the target sequence) on the target DNA according to base pairing rules. It is understood that complementarity does not require a 100% match between the Spacer RNA / pDNA and the target sequence; a certain degree of mismatch is permissible as long as it does not significantly reduce the detection effect.

[0013] Preferably, the DNA detection kit further includes primers for specifically amplifying the target DNA, including but not limited to conventional PCR amplification primers and isothermal amplification primers; more preferably, isothermal amplification primers, such as recombinase polymerase amplification (RPA) primers, are used. Experiments of this invention show that introducing such primers helps to further improve detection sensitivity.

[0014] Secondly, utilizing the aforementioned DNA detection kit based on the CRISPR / Cas9 system and the exonuclease λExo, this invention further provides a DNA detection method, comprising the following steps: Assemble the splitting CRISPR / Cas9 ribonucleoprotein complex (hereinafter referred to as SCas9 RNP): After mixing and incubating Cas9 protein with Scaffold RNA to form a Cas9-Scaffold complex, Spacer RNA was added, and after incubation, SCas9 RNP was obtained. Constructing and detecting the reaction system: Establish a reaction system containing the dsDNA sample to be tested, pDNA, λExo, SCas9 RNP, and single-stranded nucleic acid reporter molecules; incubate the reaction system at a suitable temperature and detect the cleavage of the single-stranded nucleic acid reporter molecules to perform qualitative and quantitative analysis of the target DNA in the dsDNA sample to be tested.

[0015] In the aforementioned DNA detection method, when the single-stranded nucleic acid reporter molecule is an ssDNA probe labeled with fluorescent and quenching groups at both ends, qualitative and quantitative analysis can be performed by detecting the fluorescence signal after incubation. It is understood that if the dsDNA sample to be tested contains the target DNA sequence, λExo unwinds the dsDNA under the guidance of pDNA, and the SCas9 RNP recognizes the target and activates its trans-cleavage activity, cleaving the reporter probe and generating a fluorescence signal; if there is no target sequence, no fluorescence signal is generated.

[0016] Preferably, in the above-described DNA detection method, the reaction system further contains isothermal amplification primers and enzymes used for specific amplification of the target DNA. For example, in some embodiments of the present invention, the reaction system simultaneously contains RPA primers for specific amplification and recombinases, single-stranded DNA binding proteins, and strand displacement DNA polymerases used for isothermal amplification. Because the SCas9 system established by the present invention has no cis-cutting activity and will not degrade the amplification products, true one-tube detection can be achieved.

[0017] In the above DNA detection methods, the reaction system should also contain other reagents to support the reaction, including but not limited to buffer solutions and divalent metal cations (such as Mg). 2+ (etc.), dNTPs, etc.

[0018] In the above DNA detection methods, the reaction temperature of the reaction system only needs to be able to maintain the activity of Cas9 protein and λExo simultaneously. In the reaction system containing isothermal amplification primers and the enzymes used, the activity temperature of the enzymes used for isothermal amplification should also be taken into account.

[0019] Thirdly, the DNA detection kit and DNA detection method provided by this invention can be specifically applied to scenarios such as DNA pathogen detection, gene mutation detection, and genetic marker detection. For example, in some embodiments of this invention, they have been successfully used for the detection of pathogens such as human papillomavirus (HPV) and monkeypox virus, as well as for the detection of the human JAK2 gene.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Elimination of PAM sequence restriction: Through the assisted unwinding effect of λExo, the SCas9 system can identify targets without recognizing specific PAM sequences on the target DNA, greatly expanding the range of detectable targets and the flexibility of site selection.

[0021] (2) High sensitivity: By designing the split sgRNA, the SCas9 system itself has highly efficient trans-cutting activity; further combined with RPA isothermal amplification technology, it can realize the detection of attomorlar (aM) dsDNA.

[0022] (3) Compatible with one-tube reaction: The sgRNA splitting strategy unexpectedly eliminates the cis-cleavage activity of Cas9, allowing the system to be carried out simultaneously with amplification technologies such as RPA in the same reaction tube without the need for tube opening and transfer, which greatly reduces the risk of aerosol contamination and reduces false positive results.

[0023] (4) Flexible design and low cost: The design of split sgRNA allows Spacer RNA to be replaced like a "plug-in" according to different detection targets, without the need to synthesize complete sgRNA, which reduces the cost and design complexity of the detection system.

[0024] (5) Good versatility: The system can detect different target DNAs by adjusting the Spacer RNA sequence and pDNA sequence, demonstrating good platform versatility. Attached Figure Description

[0025] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0026] Figure 1 This is a diagram illustrating the working principle of the SCas9 system in this invention. Figure a shows the splitting of traditional sgRNA into Scaffold RNA and Spacer RNA, while Figure b illustrates the principle of the SCas9 system for ssDNA and RNA detection.

[0027] Figure 2 This is a schematic diagram of the ExSCas9 system used for dsDNA detection in this invention.

[0028] Figure 3 Figure 2 shows the effect of different pDNAs on the detection efficiency of the ExSCas9 system in Example 2 of the present invention. Figure a shows the positional relationship between different pDNAs and the target region, and Figure b shows the fluorescence intensity of different pDNAs.

[0029] Figure 4 The figure shows the detection results of the ExSCas9 system under different pDNA combinations in Example 2 of the present invention. In the figure, NTC represents the template-free control, and F1, R1 and R3 are abbreviations for F-PDNA1, R-PDNA1 and R-PDNA3, respectively.

[0030] Figure 5 The figure shows the sensitivity test results of the ExSCas9 system combined with RPA amplification in Example 3 of this invention. NTC in the figure represents no template control.

[0031] Figure 6 The above figures show the detection results of the ExSCas9 system combined with RPA amplification on clinical samples in Embodiment 4 of the present invention. Figure a is the experimental flowchart, and Figures b and c are the detection results of 24 clinical samples. Figure 7 This document presents a comparison of the detection performance of the SCas9 system and the traditional Cas9 system (i.e., the tracrRNA:crRNA-Cas9 system) in Example 5 of this invention. Figure a shows the response of the two Cas9 systems to different ssDNA activators, Figure b shows the comparison of the cleavage efficiency of the two Cas9 systems to the T01 activator, Figure c shows the cis-cleavage activity detection of the three Cas9 systems, Figure d shows the comparison of the trans-cleavage activity of the SCas9 system to dsDNA and ssDNA substrates with the same target sequence, and Figure e shows the structural simulation analysis of the two Cas9 systems. Detailed Implementation

[0032] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0034] Existing CRISPR / Cas9 systems are primarily used for genome editing. Recent studies have reported that the Cas9 protein exhibits trans-cleavage activity under specific conditions, indicating its potential application in nucleic acid detection. However, using the Cas9 protein for nucleic acid detection faces at least the following limitations: ① dependence on the PAM sequence; ② the presence of cis-cleavage activity makes it difficult to achieve a true "one-tube" reaction; ③ strict limitations on the recognition of dsDNA substrates; ④ the need for high substrate concentrations (nanomolar levels). To address these limitations in using the Cas9 protein for nucleic acid detection, this invention utilizes the synergistic effect of split gRNA and λExo to develop a highly sensitive dsDNA detection system (i.e., the ExSCas9 system) that is not limited by the PAM sequence and can achieve a true "one-tube" reaction.

[0035] The working principle of the ExSCas9 system provided by this invention is as follows: First refer to Figure 1 This invention constructs a novel SCas9 system by splitting a complete sgRNA into independent scaffold RNA and spacer RNA; this SCas9 system loses cis-cleavage activity but retains highly efficient trans-cleavage activity. Further, as... Figure 2 As shown, this invention introduces λExo based on the SCas9 system. Using 5' phosphorylated pDNA, λExo is guided to unwind the double strand at a specific site on the dsDNA target, enabling SCas9 to recognize and bind to the target without relying on the PAM sequence, activating its trans-cleavage activity, and then cleaving single-stranded nucleic acid reporter molecules (such as ssDNA probes) to achieve signal output. In the ExSCas9 system of this invention, λExo is not used for non-specific degradation of dsDNA to convert it into ssDNA, but rather as a precise localization auxiliary unwinding tool. The key advantages of this precise mechanism are: (1) Target integrity preservation: λExo only unwinds locally without degrading the target, keeping the target dsDNA intact during detection, providing the possibility for subsequent amplification or verification; (2) Minimized steric hindrance: The pDNA design has been optimized and screened to avoid potential spatial competition from multiple pDNAs used simultaneously; (3) Compatibility with amplification technology: Target integrity preservation allows RPA isothermal amplification to be performed in the same reaction tube as detection, achieving one-tube detection. The EXSCas9 detection system of this invention not only eliminates the PAM limitation, but also, because SCas9 has no cis-cleavage activity, is perfectly compatible with RPA isothermal amplification technology, achieving high-sensitivity, low-contamination-risk "one-tube" dsDNA detection.

[0036] Based on the ExSCas9 system, this invention provides a dsDNA detection kit, which includes at least: Cas9 protein, Spacer RNA, Scaffold RNA, λExo, pDNA, and ssDNA probe.

[0037] Furthermore, in a preferred embodiment, the Cas9 protein is SpyCas9, and the sequence of the Scaffold RNA is shown in SEQ ID NO.4.

[0038] This invention also provides a dsDNA detection method, which includes the following steps: constructing a reaction system containing Cas9 protein, Spacer RNA, Scaffold RNA, λExo, pDNA and ssDNA probe, adding the dsDNA sample to be tested, incubating at a constant temperature and detecting changes in fluorescence signal.

[0039] Furthermore, in a preferred embodiment, the reaction system also includes RPA primers for specific isothermal amplification of the target dsDNA. Combining the ExSCas9 system with RPA technology can effectively improve detection sensitivity.

[0040] In the above detection methods, Cas9 protein can be incubated with Scaffold RNA and Spacer RNA to generate SCas9 RNP, and then the SCas9 RNP can be used to construct the reaction system; alternatively, Cas9 protein, Spacer RNA, Scaffold RNA, λExo, pDNA, and ssDNA probe can be added to the reaction system in one batch.

[0041] The SCas9 system and ExSCas9 system developed in this invention eliminate PAM sequence restrictions and can be widely used for ssDNA, RNA and dsDNA detection. They also have advantages such as high sensitivity and compatibility with one-tube reactions. In this invention, they have been successfully used in scenarios such as human gene detection and detection of pathogenic microorganisms such as HPV16.

[0042] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0043] Example 1 This example provides a dsDNA detection kit and detection method based on the ExSCas9 system, wherein the detection kit includes at least the following components: (1) SpyCas9 protein.

[0044] The SpyCas9 protein used in this example was artificially synthesized. Its preparation method was as follows: the nucleotide sequence encoding the SpyCas9 protein was codon-optimized using *E. coli*, and the optimized sequence is shown in SEQ ID NO.1; the sequence shown in SEQ ID NO.1 was cloned into the pCold II vector and transformed into BL21(DE3) *E. coli* competent cells. After IPTG induction, the protein was purified by Ni-NTA affinity chromatography and gel filtration chromatography, and finally stored in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, and 10% glycerol.

[0045] (2) Spacer RNA and Scaffold RNA.

[0046] Spacer RNA is designed based on the target sequence of the dsDNA to be tested.

[0047] Scaffold RNA, sequence shown in SEQ ID NO.4.

[0048] (3) λExo and its pDNA.

[0049] pDNA is 5' phosphorylated ssDNA, and is designed based on the sequence of the non-target strand or target strand upstream of the target region of the dsDNA to be tested.

[0050] (4) ssDNA probe.

[0051] The ssDNA probe is labeled with a fluorescent group and a quencher group at both ends, respectively. Specifically, a probe such as 5' FAM-TTTTTTTTTTTTTT-BHQ 3' can be used.

[0052] Based on the above-mentioned test kit, the detection method includes the following steps: 1) Assembly of the SCas9 RNP complex.

[0053] The purified SpyCas9 protein was mixed with Scaffold A at a molar ratio of 1:1.5 (SpyCas9 protein: Scaffold A) in reaction buffer (containing 50 mM KAc, 20 mM Tris-HCl, 10 mM MgAc, and 100 μg / mL BSA, pH 7.9) and incubated at 37°C for 5 minutes to form the Cas9-Scaffold subcomplex. Subsequently, Spacer RNA was added at a molar ratio of 1:1.5:1.5 (SpyCas9 protein: Scaffold A: Spacer RNA), and incubation was continued at 37°C for 5 minutes to obtain the complete SCas9 RNP complex.

[0054] 2) Construct the reaction system and detect it.

[0055] A reaction system containing the dsDNA sample to be tested, pDNA, λExo, SCas9 RNP complex and ssDNA probe was established, incubated at 37°C and the fluorescence signal was monitored in real time using a real-time fluorescence quantitative PCR instrument.

[0056] Example 2 Based on the detection kit and detection method in Example 1, this example uses synthetic human... JAK2 The ExSCas9 system was validated using gene dsDNA fragments as test samples, specifically including the following steps: (1) Spacer RNA design.

[0057] Regarding the personnel used in this example JAK2 The specific sequence of the corresponding Spacer RNA for the gene dsDNA fragment is: 5'-AAUUAUGGAGUAUGUGUCUG-3' (SEQ ID NO.5).

[0058] (2) pDNA design.

[0059] like Figure 3 As shown in Figure a, based on the sequence of the dsDNA fragment to be tested, multiple 5' phosphorylated pDNAs were designed upstream and downstream of the target region, including F-PDNA1, F-PDNA2, and F-PDNA3 of the target strand and R-PDNA1, R-PDNA2, and R-PDNA3 of the non-target strand. The sequence information of each pDNA is shown in Table 1.

[0060] Table 1

[0061] (3) Detection.

[0062] The 20 μL reaction system contained 500 nM SpyCas9, 750 nM Scaffold A, 750 nM SpacerRNA, 500 nM FAM-BHQ1-labeled ssDNA probe, 10 U λExo, and different pDNAs (final concentration 10 nM), followed by the addition of 10 pM of [unclear - likely a specific ingredient or ingredient]. JAK2 After dsDNA targeting, the fluorescence signal was monitored in real time at 37°C using a real-time fluorescence PCR instrument.

[0063] Detection results under different pDNA conditions, such as Figure 3 As shown in b, the use of non-target strand R-PDNA1, R-PDNA3, and target strand F-PDNA1 all effectively activated the detection signal, with F-PDNA1 producing the strongest signal. This indicates that the selection of the pDNA binding site has a significant impact on detection efficiency. Furthermore, using multiple pDNAs simultaneously did not further enhance the signal. Figure 4 This suggests the possible presence of steric hindrance.

[0064] (4) Sensitivity test.

[0065] Based on the reaction conditions in step (3) and the optimal pDNA (i.e., F-pDNA1), further serial dilutions were performed. JAK2 The dsDNA target was detected to test the sensitivity of the ExSCas9 system of this invention.

[0066] The test results showed that the detection limit of the ExSCas9 system for unamplified dsDNA was approximately 10 pM.

[0067] Example 3 Using the person in Example 2 as an example JAK2 Using dsDNA fragments as the test sample, this example demonstrates a strategy for constructing a one-tube ultrasensitive dsDNA detection system by combining the ExSCas9 system with RPA technology, specifically including the following: (1) RPA primer design.

[0068] Targeting people JAK2 We designed and synthesized RPA primers for specific amplification of the gene dsDNA fragment. The specific sequences are as follows: JAK2-F: 5'-TTCCTTAGTCTTTCTTTGAAGCAGCAAG-3' (SEQ ID NO. 12); JAK2-R: 5'-TGATCCTGAAACTGAATTTTCTATAT-3' (SEQ ID NO. 13).

[0069] (2) Establishment and detection of a single-tube reaction system.

[0070] In the same test tube, add RPA basic reaction reagent (TwistAmp Basic, containing recombinase, single-chain binding protein, polymerase, etc.), 14 mM MgAc, JAK2-F / R (0.4 μM each), and 10 ng JAK2 The reaction mixture consisted of a dsDNA template and the optimized ExSCas9 detection components from Example 2 (i.e., 500 nM Cas9, 750 nM Scaffold A, 750 nM Spacer, 1 μM ssDNA reporter probe, 10 nM F-PDNA1, and 10 U λExo), totaling 30 μL. The reaction tubes were incubated at 37°C for 60 minutes, with fluorescence signal monitoring performed simultaneously.

[0071] (3) Sensitivity test.

[0072] Based on the reaction system and reaction conditions in step (2), change the system... JAK The concentration of 2 dsDNA templates was used to test sensitivity, and the results showed that the RPA / ExSCas9 one-tube detection system achieved ultrasensitive detection of dsDNA. Figure 5 The detection limit (LoD) is as low as 1 aM, which is about 10^7 times higher than that of ExSCas9 alone. This demonstrates the advantage of the SCas9 system having no cis-cleavage activity, which makes it perfectly compatible with and synergistic with RPA amplification to achieve exponential signal amplification.

[0073] Example 4 Using HPV16 as the pathogen to be tested, this case validated the detection efficacy of the ExSCas9 system in actual clinical samples; the experimental procedure is as follows: Figure 6 As shown in a, the specific steps include: (1) Clinical sample processing.

[0074] Twenty-five human vaginal secretion samples were collected, some of which were confirmed to be HPV16 positive or negative by qPCR.

[0075] Total DNA was extracted from the sample using a commercially available genomic DNA extraction kit.

[0076] (2) RPA / ExSCas9 detection.

[0077] First, spacer RNA, pDNA, and RPA primers targeting the HPV16 genome were designed and synthesized, as follows: Spacer RNA: 5'-GGCCACAAUAAUGGCAUUUGU-3' (SEQ ID NO. 14).

[0078] pDNA: 5'-GATGCCCAAATATTYAATAAACCTTATTGGTTACAACGAGCACAG-3' (SEQ ID NO. 15).

[0079] HPV16-F: 5'-GGTTACCTCTGATGCCCAAA-3' (SEQ ID NO. 16); HPV16-R: 5'-TCATATTCGCTCCCATGTCG-3' (SEQ ID NO. 17).

[0080] Then, following Example 3, a one-tube reaction system was prepared, using DNA extract (approximately 10 ng) from each sample as a template. A negative control (without template) and a positive control (containing HPV16 plasmid) were also included. After reacting at 37°C for approximately 35 minutes, the fluorescence signal of each sample was read. Each sample was tested in triplicate.

[0081] (3) Results analysis.

[0082] The results are as follows Figure 6 As shown in figures b and c, the detection results of RPA / ExSCas9 are highly consistent with the clinical diagnostic results of qPCR, accurately distinguishing between HPV16 positive and negative samples. This indicates that the ExSCas9 detection system has good specificity and accuracy in complex biological sample matrices.

[0083] Example 5 This example compares the detection performance of the SCas9 system of this invention with that of the tracrRNA:crRNA-Cas9 system.

[0084] First, the reaction systems for the SCas9 system and the tracrRNA:crRNA-Cas9 system are as follows: The SCas9 reaction system (10 μL) contains: 500 nM SpyCas9 protein, 750 nM Scaffold RNA, 5 nM Spacer RNA, a final concentration of 0.02% Triton X-100, 500 nM FAM-BHQ1-labeled ssDNA probe, and 1×CutSmart buffer (NEB).

[0085] The tracrRNA:crRNA-Cas9 reaction system (10 μL) contains: 500 nM SpyCas9 protein, 750 nM tracrRNA:crRNA, a final concentration of 0.02% Triton X-100, 500 nM FAM-BHQ1-labeled ssDNA probe, and 1×CutSmart buffer (NEB).

[0086] The same ssDNA reporter probe was used in both reaction systems described above, and different added ssDNA activators (T01, HPV16, monkeypox B6R, JAK2) were detected. Incubation at 37°C for 60 minutes was performed, and fluorescence signals were monitored. Results are as follows: Figure 7 As shown in figure a, the SCas9 system produced significant fluorescence signals for all tested activators, while the tracrRNA:crRNA-Cas9 system only responded to the T01 activator.

[0087] Then, denaturing PAGE analysis was performed on the T01 activator ( Figure 7 (b) The results showed that the SCas9 system completely cleaved the reporter probe within 5 minutes, while the tracrRNA:crRNA-Cas9 system only partially cleaved it after 60 minutes. Quantitative analysis showed that the SCas9 activity was about 20 times that of the latter.

[0088] Subsequently, the cis-cutting activity of these three systems was studied. Figure 7 c), the results showed that the SCas9 system almost lost its cis-cleavage activity against dsDNA, while the tracrRNA:crRNA-Cas9 system was consistent with the traditional Cas9 system and its cis-cleavage activity was not affected. Finally, its trans-cleavage activity was tested using dsDNA. Figure 7 d) The study found that as the length of dsDNA gradually increases, the detection capability of both systems gradually weakens.

[0089] Systematic comparative studies have revealed the following essential differences between the tracrRNA:crRNA-Cas9 system and the SCas9 system: 1) Differences in substrate adaptability: such as Figure 7 As shown in a, the tracrRNA:crRNA-Cas9 system can only be activated by the specific engineered sequence T01, and has no response to clinically relevant ssDNA sequences such as HPV16, monkeypox B6R, and JAK2; while the SCas9 system shows high activation efficiency for all tested ssDNA substrates.

[0090] 2) Differences in cutting efficiency: such as Figure 7 The results of denaturing PAGE showed that, in the presence of T01 ssDNA substrate, the trans-cleavage activity of the SCas9 system was about 20 times higher than that of the tracrRNA:crRNA-Cas9 system.

[0091] 3) Differences in dsDNA recognition capabilities: The tracrRNA:crRNA-Cas9 system strictly limits the recognition of dsDNA to within 20bp and depends on the PAM sequence; while this invention, by introducing the λ Exo auxiliary strategy, achieves PAM-free detection of dsDNA of any length.

[0092] 4) Structural differences: Structural simulation using AlphaFold 3.0 ( Figure 7 e) It was found that in the SCas9 system, splitting the gRNA leads to the HNH domain locking into a rigid, compact conformation, and the solvent-accessible surface area of ​​the RuvC domain increases by 32%. This conformational change is the structural basis for its loss of cis-cleavage activity but retention of highly efficient trans-cleavage activity. The tracrRNA:crRNA-Cas9 system, however, retains some cis-cleavage conformation, resulting in its function being intermediate between that of traditional sgRNA-Cas9 and SCas9.

[0093] In summary, the detection system (ExSCas9) based on SCas9 and λExo constructed in this invention successfully solves the problems of traditional Cas9 systems relying on PAM sequences and difficulty in detecting long-chain dsDNA. Through seamless compatibility with RPA isothermal amplification technology, it achieves aM-level ultrasensitive detection of dsDNA targets and has been successfully validated in clinical samples. This system provides the field of molecular diagnostics with a novel, efficient, flexible, and low-contamination-risk nucleic acid detection tool.

[0094] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A DNA detection kit based on the CRISPR / Cas9 system and the exonuclease λExo, characterized in that, At least the following reagents are included: Cas9 protein has trans-cleavage activity; Spacer RNA contains a sequence complementary to the target DNA; Scaffold RNA, which binds to the Cas9 protein and can activate the trans-cleavage activity of the Cas9 protein in the presence of the Spacer RNA; λExo and its pDNA, wherein the pDNA is 5' phosphorylated ssDNA and contains a sequence complementary to the non-target strand or target strand upstream of the dsDNA target region, and the pDNA guides λExo to bind and unwind the dsDNA double strand; A single-stranded nucleic acid reporter molecule that can be trans-cleaved by the Cas9 protein.

2. The DNA detection kit according to claim 1, characterized in that, The Cas9 protein in question is SpyCas9.

3. The DNA detection kit according to claim 2, characterized in that, The sequence of the Scaffold RNA is shown in SEQ ID NO.

4.

4. The DNA detection kit according to claim 1, characterized in that, The single-stranded nucleic acid reporter molecule is an ssDNA probe with fluorescent and quenching groups labeled at both ends, respectively, and the length of the ssDNA probe is not less than 13 nt.

5. The DNA detection kit according to claim 1, characterized in that, It also includes primers for specifically amplifying the target DNA.

6. The DNA detection kit according to claim 1, characterized in that, The DNA detection kit is used for HPV16 detection, and the sequence of the Spacer RNA is shown in SEQ ID NO.14, and the pDNA sequence is shown in SEQ ID NO.

15.

7. The DNA detection kit according to claim 1, characterized in that, It also includes PRA primers with sequences as shown in SEQ ID NO.16-17.

8. A DNA detection method for non-disease diagnostic purposes, characterized in that, The detection is performed using the kit described in claim 1, and includes the following steps: S1. Incubate Cas9 protein with Scaffold RNA and Spacer RNA to obtain SCas9 RNP; S2. Construct a reaction system containing the dsDNA sample to be tested, pDNA, λExo, SCas9 RNP and single-stranded nucleic acid reporter molecules, and detect the cleavage of single-stranded nucleic acid reporter molecules after isothermal reaction.

9. The DNA detection method according to claim 8, characterized in that, The reaction system also contains primers for specific isothermal amplification of the dsDNA sample to be tested.

10. The DNA detection method according to claim 8, characterized in that, The molar ratio of the Cas9 protein to Scaffold RNA and Spacer RNA is 1:1.5:1.5.

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