Method and reagent for directly detecting gene methylation based on Cas12a system and application of method and reagent

Abstract

The invention relates to the technical field of biology, in particular to a method and a reagent for directly detecting gene methylation based on a Cas12a system and application of the method and the reagent. According to the method for directly detecting the gene methylation of the gene in the Cas12a system, disclosed by the invention, a Cas12a protein and specific Multiplex-gRNAs of a target gene are combined in a CRISPR-Cas12a buffer system to form a compound, the target gene is specifically recognized, the trans-cleavage activity of the Cas12a protein is activated, a fluorescence-labeled reporter probe is cleaved to generate a fluorescence signal, and the methylation of the gene is directly detected in the CRISPR-Cas12a buffer system. Qualitative / quantitative analysis of target gene methylation is carried out by detecting the intensity of a fluorescence signal generated by a Cas12a protein trans-cleavage fluorescence labeled reporter probe. The method has the advantages that hydrosulfite pretreatment and amplification are not needed, and the complex process and sensitivity limitation of a traditional methylation detection technology are remarkably broken through. The methylation state of the target DNA can be quickly distinguished, and the result can be read within 30 minutes in the whole detection process. Compared with the prior art, the method disclosed by the invention has the characteristics of simplicity and convenience in operation, no amplification pollution risk, low requirement on sample concentration and the like, and is particularly suitable for rapid detection of methylation sites in cfDNA samples from clinical plasma and auxiliary diagnosis of cancers.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for directly detecting gene methylation based on a Cas12a system, reagents and applications thereof. BACKGROUND

[0002] DNA methylation is a key epigenetic modification that plays a crucial role in gene expression regulation, cell differentiation, and tumorigenesis. Aberrant DNA methylation patterns, particularly hypermethylation in promoter regions or genome-wide hypomethylation, have been widely demonstrated to be closely related to the occurrence of various tumors, and usually precede genetic mutations. Therefore, accurately and sensitively detecting the DNA methylation status of the promoter region of a gene has extremely important clinical significance for early diagnosis, prognosis evaluation, and individualized treatment of tumors. However, the existing mainstream DNA methylation detection methods, such as bisulfite sequencing and various PCR and sequencing methods based on restriction endonucleases, although have made certain progress in sensitivity and coverage, still have many shortcomings. These methods generally have complex operation, long detection period, high cost, and the need for large amounts of DNA samples, which greatly limits their widespread application in clinical large-scale screening and rapid diagnosis.

[0003] In recent years, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system as a new nucleic acid detection tool has attracted widespread attention in the field of molecular diagnosis. Among them, Cas12a, Cas13a, Cas14 and other nucleases can activate their own "trans-cleavage" activity after recognizing specific targets, cutting the quenching group on the fluorescent reporter probe to emit fluorescent signals, thereby realizing signal amplification and improving detection sensitivity. Taking Cas12a as an example, it activates its own single-stranded DNA nuclease activity, i.e. "trans-cleavage" activity, after recognizing the target DNA, which cuts the fluorescent reporter probe and triggers the fluorescent signal, and has been widely used in virus detection, tumor-related mutation site identification, single nucleotide polymorphism analysis, etc. At present, a variety of CRISPR-based detection platforms have been developed, such as SHERLOCK, DETECTR, HOLMES, etc., which have shown significant potential in portable, biosafety and low-cost molecular diagnosis.

[0004] Although existing CRISPR detection platforms have made significant progress in sequence recognition, they still face many challenges in detecting epigenetic modifications such as DNA methylation. Most existing CRISPR-Cas system strategies for detecting methylation rely on chemical pretreatment (such as bisulfite treatment) to convert methylation information into sequence differences, which still has problems such as complex detection process, severe DNA damage, and high non-specific background. Some studies have attempted to combine the CRISPR-Cas system with methylation-sensitive enzymes, bisulfite treatment, and strand displacement amplification to indirectly detect the presence of 5-methylcytosine in DNA. However, these methods have their own drawbacks: the method of using methylation-sensitive restriction endonuclease to treat target DNA and then using Cas12a for detection can achieve single CpG site resolution, but it relies on specific site enzyme digestion recognition, has limited scope of application, and has a complex process (Van Dongen JE et.al, Biosens Bioelectron, 2021); the method of using bisulfite treatment to deaminate non-methylated cytosine to uracil and then combining it with the Cas12a system for detection has high universality, but the pretreatment process involves chemical treatment and harsh reaction conditions, which can lead to DNA degradation and reduced detection sensitivity, limiting its practical application in low-abundance samples such as cfDNA; there is also a method of using restriction endonuclease, isothermal amplification (RPA), and Cas12a system to improve detection sensitivity, but this method is complex, has a complicated process, and relies on multiple enzyme systems and non-specific amplification, increasing the risk of false positives and experimental difficulty.

[0005] In summary, the existing CRISPR-Cas12a methylation detection methods have the following defects: (1) they rely on enzyme digestion or chemical treatment, making it difficult to apply to microcirculating free DNA (cfDNA); (2) the amplification system introduces non-specific signals, affecting specificity and stability; (3) bisulfite treatment can cause cfDNA fragmentation. Therefore, there is an urgent need to develop a new CRISPR detection strategy that does not require tedious pretreatment, can directly distinguish between methylated and non-methylated sequences, has high sensitivity and specificity, and can accurately identify epigenetic modification states, expanding its potential applications in early tumor screening and clinical liquid biopsy. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a method for directly detecting gene methylation based on the Cas12a system, reagents and their applications, which can directly and efficiently distinguish between gene methylation and non-methylation without tedious pretreatment such as enzyme digestion, chemical pretreatment, or amplification reaction, and has the advantages of simple operation, high sensitivity, and good specificity.

[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0008] The present application provides a method for directly detecting gene methylation based on a Cas12a system, which comprises the following steps: S1: incubating Cas12a protein and specific Multiple-gRNAs targeting a target gene in a CRISPR-Cas12a buffer system to form a complex, wherein the Multiple-gRNAs can specifically guide the Cas12a protein to bind to the DNA sequence of the promoter region of the target gene; S2: adding a fluorescently labeled reporter probe to the system of S1 to read the initial background fluorescence, wherein the fluorescently labeled reporter probe comprises a fluorescent group and a quenching group and is connected by a polynucleotide chain; S3: adding a target DNA to the system of S2 to initiate the Cas12a cis-cleavage reaction, activate the trans-cleavage activity of the Cas12a protein, remove the quenching group in the fluorescent probe, and generate a fluorescent signal; and S4: qualitatively and / or quantitatively analyzing the methylation of the target gene according to the intensity of the fluorescent signal.

[0009] Further, the method comprises the following steps:

[0010] S1: incubating Cas12a protein and specific Multiple-gRNAs targeting a target gene in a CRISPR-Cas12a buffer system to form a complex, wherein the Multiple-gRNAs can specifically guide the Cas12a protein to bind to the DNA sequence of the promoter region of the target gene;

[0011] S2: adding a fluorescently labeled reporter probe to the system of S1 to read the initial background fluorescence, wherein the fluorescently labeled reporter probe comprises a fluorescent group and a quenching group and is connected by a polynucleotide chain;

[0012] S3: adding a target DNA to the system of S2 to initiate the Cas12a cis-cleavage reaction, activate the trans-cleavage activity of the Cas12a protein, remove the quenching group in the fluorescent probe, and generate a fluorescent signal;

[0013] S4: qualitatively and / or quantitatively analyzing the methylation of the target gene according to the intensity of the fluorescent signal.

[0014] Preferably, the method is used for directly detecting 5mC methylation sites located in the region distal to the PAM sequence.

[0015] Preferably, the Cas12a protein is FnCas12a, and the optimal detection time is 30 min after the reaction.

[0016] Preferably, the fluorescent group of the fluorescently labeled reporter probe is selected from ROX, Cy3, Cy5, and FAM, and the quenching group is selected from BHQ1, BHQ2, and Dabcyl. In one embodiment, the fluorescently labeled reporter probe is Texas Red-CCCCC-BHQ2.

[0017] In one embodiment, the target gene is CCDC140 gene, the specific Multiple-gRNAs targeting the target gene include gRNA-1 and gRNA-2, wherein the 3' end last thymine deoxyribonucleotide T of gRNA-1 overlaps with the first thymine deoxyribonucleotide T of the PAM sequence of gRNA-2, and the sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0018] The application also provides a reagent for directly detecting gene methylation based on a Cas12a system, which comprises a Cas12a protein, specific Multiple-gRNAs targeting a target gene, a fluorescently labeled reporter probe, and a CRISPR-Cas12a buffer system; wherein the Multiple-gRNAs can specifically guide the Cas12a protein to bind to the DNA sequence in the promoter region of the target gene; the fluorescently labeled reporter probe comprises a fluorescent group and a quenching group, and is connected by a polynucleotide chain.

[0019] In the application, the Cas12a protein in the reagent binds with the specific Multiple-gRNAs to specifically recognize the target gene, activates the transcleavage activity of the Cas12a protein, removes the quenching group in the fluorescently labeled reporter probe to generate a fluorescent signal, and qualitatively / quantitatively analyzes the methylation state of the target gene based on the intensity of the fluorescent signal.

[0020] Preferably, the reagent is used to directly detect the methylation site in the region distal to the PAM sequence in the target gene.

[0021] Preferably, the Cas12a protein is FnCas12a, and the optimal detection time is 30 min after the reaction.

[0022] Preferably, the fluorescent group of the fluorescently labeled reporter probe is selected from ROX, Cy3, Cy5, and FAM, and the quenching group is selected from BHQ1, BHQ2, and Dabcyl. In one embodiment, the fluorescently labeled reporter probe is Texas Red-CCCCC-BHQ2.

[0023] Preferably, the CRISPR-Cas12a buffer system comprises NaCl, Tris-HCl, MgCl2, and recombinant albumin.

[0024] Further preferably, the CRISPR-Cas12a buffer system further comprises a reducing agent DTT and KCl, and in the buffer system, Mg 2+The concentration of the metal ions is 45 mM, and the concentration of DTT is 10 mM. By optimizing the concentration of the metal ions, the discrimination of the DNA methylation state can be enhanced under certain conditions, which is suitable for in vitro synthetic sample detection, and the ion conditions of the clinical sample can be adjusted according to the detection requirements to obtain the best effect.

[0025] In one embodiment, the target gene is a CCDC140 gene, and the specific Multiple-gRNAs targeting the target gene include gRNA-1 and gRNA-2, wherein the 3' end of the last thymine deoxyribonucleotide T of gRNA-1 overlaps with the first thymine deoxyribonucleotide T of the PAM sequence of gRNA-2, and the sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0026] The application also provides the use of the reagent for directly detecting gene methylation in any one of the above embodiments in the detection of gene methylation, which includes the following:

[0027] S1: incubating the Cas12a protein and the specific Multiple-gRNAs targeting the target gene in a CRISPR-Cas12a buffer system to form a complex;

[0028] S2: adding a fluorescently labeled reporter probe to the system of S1 to read the initial background fluorescence;

[0029] S3: adding the DNA to be detected to the system of S2 to start the reaction;

[0030] S4: detecting the fluorescence signal intensity, and qualitatively / quantitatively analyzing the methylation of the target gene according to the fluorescence signal intensity.

[0031] In one embodiment, the application is the use in the detection of the methylation of the CCDC140 gene.

[0032] The application also provides the use of the reagent in any one of the above embodiments in the preparation of a tumor detection reagent or a detection kit.

[0033] The application also provides a pancreatic cancer or lung cancer detection reagent or detection kit, comprising a FnCas12a protein; specific Multiple-gRNAs targeting a target gene; a fluorescently labeled reporter probe; a CRISPR-Cas12a buffer system; wherein the target gene is a CCDC140 gene, the specific Multiple-gRNAs targeting the target gene can specifically guide the Cas12a protein to bind to the DNA sequence in the promoter region of the CCDC140 gene, comprising gRNA-1 and gRNA-2, wherein the 3' end of the last thymine deoxyribonucleotide T of gRNA-1 overlaps with the first thymine deoxyribonucleotide T of the PAM sequence of gRNA-2, and the sequences are shown in SEQ ID No. 1 and SEQ ID No. 2 respectively; the fluorescently labeled reporter probe comprises a fluorescent group and a quenching group, and is connected by a polynucleotide chain, wherein the fluorescent group is selected from ROX, Cy3, Cy5, FAM, and the quenching group is selected from BHQ1, BHQ2 and Dabcyl.

[0034] Preferably, the fluorescently labeled reporter probe is Texas Red-CCCCC-BHQ2.

[0035] The application has the following beneficial effects:

[0036] The application first discloses the rule and mechanism of DNA methylation sequence inhibiting Cas12a transcleavage activity, and based on this experiment, it is verified that the CRISPR-Cas12a system can directly recognize the methylation and non-methylation states in DNA. The system does not need to be pretreated and amplified by bisulfite, thereby significantly simplifying the complex process of traditional DNA methylation detection technology, and greatly improving the detection sensitivity. The method for directly detecting gene methylation based on the Cas12a system provided by the application is to use the methylation modification to inhibit the Cas12a transcleavage activity, to realize the rapid differentiation of the target DNA methylation state and the non-methylation state, and the whole detection process is efficient and fast, and the result reading can be completed in only 30 minutes. Compared with the prior art, the application is more convenient to operate, effectively avoids the pollution that may be generated in the detection process, and significantly increases the detection sensitivity of low-concentration samples. These advantages make the application particularly suitable for the rapid detection of cfDNA methylation from clinical plasma, and provide a powerful tool for the early diagnosis of cancer. Further, the application also provides a special kit, which can fully utilize the sensitivity of the transcleavage activity of Cas12a to the DNA methylation state, to realize the rapid and reliable differentiation of the target DNA methylation state.

[0037] To explore the recognition mechanism, the present application systematically designs target sequences containing continuous methylation sites, single methylation sites and interval methylation modification, and combines single molecule fluorescence resonance energy transfer (FRET) technology and molecular docking simulation, reveals that 5mC modification affects the conformational stability and recognition binding kinetics of Cas12a complex, thereby leading to differences in trans-cleavage efficiency, providing theoretical support for the mechanism of methylation detection.

[0038] In terms of system construction, the present application determines the signal-to-noise ratio and sensitivity of FnCas12a in methylation recognition by screening different Cas12a variants, significantly enhances the distinction between methylated and non-methylated sequences, and improves the detection sensitivity and accuracy.

[0039] The present application further proves the effectiveness and specificity in practical application through clinical sample verification. In the detection of preoperative plasma samples of healthy people and pancreatic cancer and lung cancer patients, the detection system constructed by the present application can stably distinguish between methylated and non-methylated cfDNA, and the fluorescence signal detected in pancreatic cancer and lung cancer samples is significantly lower than that in healthy samples, verifying the feasibility and reliability of the present application in clinical detection, and having wide clinical popularization value.

[0040] In summary, through innovative detection strategy and technical optimization, the present application solves the deficiencies of existing methylation detection methods in sensitivity, specificity, operation simplicity and sample adaptability, provides a new methylation detection method which is efficient, simple and low-cost, brings a major breakthrough for epigenetic research and clinical application, and has significant scientific significance and social value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1Figure 2. The different effects of target 5-methylcytosine (5mC) modification and unmodified double-stranded DNA (dsDNA) on Cas12a transcleavage activity. (A): Schematic diagram of the mechanism of Cas12a-gRNA complex recognizing and cleaving 5mC-modified or unmodified dsDNA, thereby triggering Cas12a transcleavage activity, red cytosine represents 5mC site (diagram drawn using BioRender.com); (B): Schematic diagram of gRNA binding to DNA in “T group” dsDNA, which contains 1-4 5mC modification sites from the PAM distal end to the PAM proximal end, red cytosine represents 5mC site. (C-E): Changes in fluorescence intensity of LbCas12a (C), FnCas12a (D), and AsCas12a (E) at 0, 30, 60, 90, and 120 minutes under the induction of “T group” dsDNA (n=3). (F): Schematic diagram of gRNA binding to DNA in “C group” dsDNA, which contains 1-4 5mC modification sites from the PAM proximal end to the PAM distal end along the target strand, red cytosine represents 5mC site. (G-I): Changes in fluorescence intensity of LbCas12a (G), FnCas12a (H), and AsCas12a (I) at 0, 30, 60, 90, and 120 minutes under the induction of “C group” dsDNA (n=3).

[0042] Figure 2 Figure 3. The effects of single 5-methylcytosine (5mC) site and interval 5mC modification of double-stranded DNA (dsDNA) on Cas12a transcleavage activity. (A): Schematic diagram of different single 5mC site modification of dsDNA, red cytosine represents 5mC site. (B): Schematic diagram of interval 5mC modification of dsDNA, red cytosine represents 5mC site. (C-E): The effects of single 5mC site modification on LbCas12a (C), FnCas12a (D), and AsCas12a (E) transcleavage activity, bar chart shows fluorescence intensity at 30 minutes (n=3). (F-H): The effects of interval 5mC modification on LbCas12a (F), FnCas12a (G), and AsCas12a (H) transcleavage activity, bar chart shows fluorescence intensity at 30 minutes (n=3).

[0043] Figure 3Effects of hemi-methylated double-stranded DNA (dsDNA) on Cas12a trans-cleavage activity. (A) Schematic diagram of gRNA binding to unmethylated, target strand (TS) hemi-methylated, non-target strand (NTS) hemi-methylated, and fully methylated dsDNA, with red cytosine representing 5mC sites. (B-D) Comparison of trans-cleavage activity of LbCas12a (B), FnCas12a (C), and AsCas12a (D) induced by four types of 3CG target dsDNA, including unmethylated, TS hemi-methylated, NTS hemi-methylated, and fully methylated dsDNA (n = 3), with column chart showing fluorescence intensity at 30 minutes.

[0044] Figure 4 Conformational changes and FRET energy transfer properties of Cas12a in recognizing dsDNA with different methylation states. (A) Schematic diagram of binding mechanism. (B-D) Energy transfer from Fd to Fa was evaluated using LbCas12a (E), FnCas12a (F), and AsCas12a (G) on “T group” dsDNA containing 1-4 5mC modification sites (n = 3), with column chart showing Fa fluorescence values measured every 10 minutes for a total duration of 2 hours. (E-G) Energy transfer from Fd to Fa was evaluated using LbCas12a (H), FnCas12a (I), and AsCas12a (J) on “C group” dsDNA containing 1-4 5mC modification sites (n = 3), with column chart showing Fa fluorescence values measured every 10 minutes for a total duration of 2 hours. (H-J) RMSD analysis based on the structures of the three Cas12a variants, with schematic diagram of protein structure showing unmethylated dsDNA in wheat color, methylated dsDNA in light green, target strand (TS) in red, non-target strand (NTS) in blue, and gRNA in magenta. RMSD of LbCas12a (PDB: 5XU9) was

[0045] RMSD of FnCas12a (PDB: 6I1K) was RMSD of AsCas12a (PDB: 5b43) was Enlarged structure showing conformational differences between unmethylated NTS and methylated NTS.

[0046] Figure 5 Trans-cleavage fluorescence intensity heat map of LbCas12a RNP, FnCas12a RNP, and AsCas12a RNP in recognizing double-stranded DNA (dsDNA) with single 5-methylcytosine (5mC) modification at different sites.

[0047] Figure 6 Optimization results of Cas12a buffer system.

[0048] Figure 7 Figure 11. The CpG island and methylation status of CCDC140 promoter. (A): The CpG island in CCDC140 promoter was predicted by EMBOSS CpGplot (https: / / emboss.bioinformatics). (B): The methylation level of CCDC140 promoter in LUAD (n=449), LUSC (n=370) and healthy control (n=68) samples was analyzed by SMART. The beta value showed that the tumor tissue was hypermethylated compared with the normal tissue (http: / / www.bioinfo-zs.com / smartapp / ). (C): The methylation level of CCDC140 promoter in PAAD (n=184) and healthy control (n=10) samples was analyzed by SMART. The beta value showed that the tumor tissue was hypermethylated compared with the normal tissue (http: / / www.bioinfo-zs.com / smartapp / ). (D): The methylation status of CpG sites near CCDC140 promoter was analyzed by MALDI-TOF. Control (n=7), lung cancer (n=9) and pancreatic cancer (n=7) (E): The methylation status of CCDC140 in pancreatic cancer patients and their adjacent tissues was detected by ddPCR.

[0049] Figure 8 Figure 12. The results of cfDNA based on Cas12a detection system to distinguish cancer patients from healthy donors. The fluorescence signal triggered by cfDNA was detected using FnCas12a transcleavage activity assay. The preliminary cohort included 15 healthy donors, 10 pancreatic cancer patients and 9 lung cancer patients, each sample was detected twice (n=2). The fluorescence intensity was measured at 30 minutes of reaction. Orange, purple and blue dots represent healthy donors, pancreatic cancer patients and lung cancer patients, respectively (ns: not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively and explain the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology achieved based on the above description of the present application is covered within the scope of protection intended by the present application. It should be noted that if not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. The experimental reagents in the examples, if not specifically indicated, can be commercially available. The experimental methods in the examples, if not specifically indicated, are conventional methods. Specifically, the experimental reagents and experimental methods used in the present application include the following:

[0051] 1. Experimental reagents

[0052] The Cas12a enzymes used in the present application include Lbcas12a (Cat No: M0653T) purchased from New England BioLabs, AsCas12a (Cat No: Z03502) purchased from Genscript, and FnCas12a (Cat No: LXC002L) purchased from Lixi Biosciences. The buffer used is 10x NEB buffer r2.1 (Cat No: B6002S), which contains 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 and 100 μg / mL recombinant albumin. The inorganic salts MgCl2 (Cat No: M875550) and KCl (Cat No: R885888) used are from Macklin Company, the reducing agent DTT is from Thermo (Cat No: R0861), and all DNase / RNase-free water is provided by Promega Company (Cat No: MC119A).

[0053] 2. Oligonucleotide synthesis and annealing

[0054] DNA and RNA oligonucleotides were synthesized by Sangon Biotech Company, and fluorescently labeled Reporter probes were synthesized by GENCEFE Company. All ssDNA oligonucleotides were prepared to a concentration of 100 μM with nuclease-free water. dsDNA was obtained by annealing complementary TS and NTS strands in equimolar amounts (1:1), and the concentration after annealing was 100 μM. The construction of hemimethylated dsDNA included:

[0055] (1) annealing the methylated TS strand with the unmethylated NTS strand to form TS hemimethylated dsDNA; (2) annealing the unmethylated TS strand with the methylated NTS strand to form NTS hemimethylated dsDNA. All annealing products were stored at -20°C. gRNAs were dissolved in nuclease-free water at a concentration of 20 μM and also stored at -20°C.

[0056] 3. Cas12a trans-cleavage standard reaction system

[0057] The total reaction volume was 100 μL / well, including 25 nM target DNA, 25 nM Cas12a enzyme, 25 nM gRNA, and 150 nM fluorescent probe (Texas Red-CCCCC-BHQ2), and the reaction buffer was 1x NEBuffer r2.1. Cas12a and gRNA were pre-incubated at 26°C for 30 minutes, then the fluorescent probe was added and the initial background fluorescence was read (Ex / Em: 570 / 615 nm), followed by the addition of target DNA to start the reaction. The fluorescence signal was recorded every 10 minutes for 2 hours, and the instrument was BioTek Synergy H1 microplate reader.

[0058] 4. Fluorescence resonance energy transfer (FRET) assay method

[0059] Cy3-labeled gRNA and Cy5-labeled DNA were used for FRET experiment. In the experiment, 25 nM of Cy5-DNA was added to 25 nM of Cy3-gRNA-Cas12a complex, with an excitation wavelength of 520 nm, and the donor fluorescence (565 nm) and acceptor fluorescence (665 nm) were recorded, respectively. The instrument was TECAN Spark multi-functional plate reader.

[0060] 5. Bioinformatics and structural analysis method

[0061] All protein sequences and three-dimensional structure data were from the UniProt database. Protein structure prediction and protein-DNA complex modeling used the AlphaFold3 platform. Structure alignment and root mean square deviation (RMSD) calculation used PyMOL software (version 2.6, Schrödinger LLC).

[0062] 6. FnCas12a method for detecting human cfDNA

[0063] Using BeyoMag TM Cell-free DNA Isolation kit (Cat. No. D0085S) was used to extract cfDNA from 2 ml of plasma, following the instructions. Qubit4 fluorescence instrument was used for quantification. The total reaction system was 20 μL, including 50 nM FnCas12a, 50 nM gRNA-1 and gRNA-2 (equimolar mixture), 2.5 μM fluorescent probe was added after incubation at 26°C for 30 minutes. 2 ng of cfDNA sample (healthy person or cancer patient) was added to each well of a 384-well plate, and incubated at room temperature for 30 minutes, and the fluorescence signal was recorded.

[0064] Example 1 Effect of the number of methylation sites in target DNA on Cas12a trans- cleavage activity

[0065] The effect of the number of 5-methylcytosine (5mC) modifications on Cas12a activity was evaluated. Two strategies were used to construct the experimental system: ① In the “C” group, 5mC modification sites were gradually increased along the target double-stranded DNA (dsDNA) from 5’ to 3’ direction of PAM; ② In the “T” group, 5mC modification sites were gradually increased along the 3’ to 5’ direction. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) complementary to the guide RNA (gRNA) were set in each group. The experimental results showed that Figure 1 A–I), in the three homologous proteins LbCas12a, FnCas12a and AsCas12a, 5mC modification inhibited the trans-cleavage efficiency of the three Cas12a.

[0066] Effect of single methylation site position on Cas12a enzyme trans-cleavage activity

[0067] To clarify the recognition sensitivity of Cas12a to the methylation and non-methylation state of the target DNA, a target DNA containing a single methylation site was designed for systematic verification. The experimental results show that Figure 2 A, C, D, E), among the three Cas12a proteins of different sources (LbCas12a, FnCas12a and AsCas12a), the inhibition of methylation DNA on Cas12a activity is more obvious as the distance between the methylation site and the PAM increases. And in double-stranded DNA, the discrimination of methylation and unmethylation DNA on Cas12a activity is greater. This phenomenon is defined as "edge effect". Further analysis found that this effect is particularly pronounced in FnCas12a, while in LbCas12a and AsCas12a it shows similar but weaker trends. This finding provides an important basis for the design of gRNA sequences and DNA methylation target detection for Cas12a in methylation detection.

[0068] Effect of interval methylation site on Cas12a enzyme trans-cleavage activity

[0069] To explore the effect of the number of interval bases between methylation sites on the trans-cleavage activity of Cas12a, methylation sites with different interval base numbers were designed for verification. Specifically, on the basis of the fixed methylation of cytosine at the 4th position of the target strand, a second methylation site was introduced at a position 3, 7, or 11 bases away from the first, resulting in three groups of methylation sequences with different interval distances. The experimental results show that Figure 2 B, F, G, H), under different interval distances, Cas12a shows a significant "edge effect" in cutting activity on methylated and non-methylated DNA, i.e., as the distance between the methylation site and the PAM sequence increases, the recognition ability of Cas12a for the methylation state increases, and the inhibition of DNA methylation with an interval of 7 nt on Cas12a activity is most obvious, further verifying the important influence of methylation position on the trans-cleavage activity of Cas12a.

[0070] Effect of half-methylation state of target strand (TS) and non-target strand (NTS) in double-stranded DNA on Cas12a trans-cleavage activity

[0071] Two groups of double-stranded DNA (dsDNA) containing 5mC modification were constructed to systematically evaluate the effect of half-methylation state of TS and NTS on Cas12a enzyme cleavage activity Figure 3A). The experimental results show that the trans-cleavage activity of Cas12a is significantly reduced when 5mC modification exists in the PAM distal region, and the inhibition is most significant when the NTS strand is in the hemi-methylation state Figure 3 B, C, D). Through comparative detection of three homologous proteins LbCas12a, FnCas12a and AsCas12a, it is found that FnCas12a performs best in distinguishing different methylation states, with higher sensitivity and discrimination.

[0072] Example 5 AlphaFold3 and FRET verify the mechanism of CRISPR-Cas12a distinguishing methylation and non-methylation DNA

[0073] To clarify the mechanism of 5mC modified DNA leading to the reduction of trans-cleavage efficiency of CRISPR-Cas12a complex, the present application first speculates that this modification may affect the conformational change of Cas12a-RNP. To verify this hypothesis, single molecule fluorescence resonance energy transfer (FRET) technology is used, with Cy3 as the donor and Cy5 as the acceptor, to detect the effect of 5mC modification on the conformation of Cas12a-RNP Figure 4 A). The experimental results Figure 4 B, C, D, E, F, G) show that the energy transfer efficiency of 5mC modified single-stranded DNA is lower than that of unmethylated single-stranded DNA, while the energy transfer signal of 5mC modified double-stranded DNA is enhanced compared with unmethylated double-stranded DNA, and this trend can be observed in various Cas12a variants such as FnCas12a, AsCas12a, etc. Further, the three-dimensional structure of Cas12a-gRNA-target DNA ternary complex is predicted and constructed by AlphaFold3, and structure comparison is performed by PyMOL Figure 4 H, I, J). The RMSD analysis results show that the NTS strand undergoes significant conformational change in the methylation state, which is speculated to be an important structural basis for the decrease in enzyme activity. In summary, methylation in the PAM distal region can significantly interfere with the recognition and activation of Cas12a, providing a theoretical basis and technical support for the detection method of circulating free DNA (cfDNA) methylation based on the CRISPR system, and has wide application prospects in early tumor screening and precise epigenetic diagnosis.

[0074] Example 6 Cas12a protein screening

[0075] The four groups of dsDNA substrates containing a single 5mC modification and the modification sites sequentially located in the PAM adjacent region to the distal region were compared by heat map, and the trans-cleavage fluorescence signal intensity of three Cas12a homologous enzymes (LbCas12a, FnCas12a and AsCas12a) was compared and evaluated. The experimental results show that FnCas12a shows better discrimination ability under different 5mC modification sites, so the present application preferably uses FnCas12a for subsequent detection system optimization and application. Figure 5 )。

[0076] Example 7 Optimization of CRISPR-cas12a buffer system

[0077] The CRISPR-cas12a buffer system is optimized by adjusting the concentrations of Mg 2+ , K + and DTT in the reaction system to improve the discrimination ability of the CRISPR-Cas12a system for synthetic double-stranded DNA methylation and non-methylation. Figure 6 The specific operation is as follows: after mixing the synthetic dsDNA with Cas12a protein and corresponding gRNA, Mg 2+ (10, 25, 35 or 45mM), K + (15, 45 or 65mM) and DTT (5, 10 or 20mM) are added to the reaction system for system optimization. At low concentration of Mg 2+ , the slight structure or chemical difference of dsDNA caused by methylation modification is difficult to be amplified and recognized by Cas12a, while when the concentration of Mg 2+ is 45mM (84%) and the concentration of DTT is 10mM (70%), the Cas12a enzyme activity is significantly enhanced, and the fluorescence difference between methylated and non-methylated dsDNA is higher than that of the control group, achieving a better discrimination effect. + K plays an auxiliary role in maintaining the ionic strength and environmental stability of the system, DTT prevents the oxidation and cross-linking of cysteine residues of Cas12a, maintains the correct folding and active state of the enzyme, and thus ensures the stability and signal reliability of the entire detection system.

[0078] Example 8 Clinical sample verification (pancreatic cancer, lung cancer and healthy human plasma cfDNA)

[0079] (1) Multiple-gRNAs design

[0080] According to existing research, the double gRNA (Multiple-gRNA) strategy can produce a synergistic effect when targeting the same gene, significantly enhancing the trans-cleavage rate and detection sensitivity of Cas12a compared to single gRNA (Morales-Moreno MD et.al, CRISPR J, 2023). Based on this advantage, the present application selects the CCDC140 gene as the detection target gene according to the previous results, and selects two groups of highly specific target sites in the gene sequence of the CCDC140 gene by time-of-flight mass spectrometry (Table 1, Table 2, Figure 7 ), and then designs a Multiple-gRNA system suitable for cfDNA detection (Table 3), aiming to achieve high sensitivity and high specificity detection of targeted methylated DNA in clinical samples.

[0081] Table 1 CCDC140 methylation detection primer table

[0082]

[0083] Table 2 Sequence information of target gene CCDC140

[0084]

[0085] Table 3 Specific Multiple-gRNAs information targeting the target gene CCDC140

[0086]

[0087] (2) Clinical sample verification (pancreatic cancer, lung cancer and healthy human plasma cfDNA)

[0088] The clinical sample information is shown in Table 4.

[0089] Table 4 Sample information

[0090]

[0091] The collection and processing of pancreatic cancer / lung cancer patient plasma and healthy human plasma samples were carried out according to the standardized operation procedure.

[0092] The specific steps are as follows: 10 mL of peripheral venous blood was collected from the patient in a fasting state, and the blood collection container was a sterile vacuum blood collection tube containing EDTA anticoagulant. Within 1 hour after blood collection, the collected whole blood was centrifuged at 2000 x g for 10 minutes (4°C) to separate the plasma. The supernatant plasma was transferred to a new tube and centrifuged again at 12,000 x g for 10 minutes (4°C) to remove cell debris and residual cells.

[0093] In this study, the cell-free plasma obtained was subjected to cfDNA extraction by magnetic bead method, and the specific operation procedure was as follows:

[0094] Sample lysis: Take 4 mL of plasma sample, add 4 mL of lysis solution and 400 μL of proteinase K, mix well, and incubate at 56°C for 10 minutes to fully lyse the sample. After incubation, cool the reaction system to room temperature.

[0095] Magnetic bead binding: Add 4 mL of anhydrous ethanol and 50 μL of pre-mixed BeyoMag TM Magnetic bead suspension. Note that the magnetic beads must be fully mixed and shaken before use to ensure their binding efficiency. After gentle mixing, stand at room temperature for 5 minutes, and mix gently 1-2 times during this period. Then, place the centrifuge tube in the magnetic stand, and carefully remove the supernatant after the magnetic beads are fully aggregated.

[0096] Washing and purification: Add 1 mL of washing solution I, gently shake to disperse the magnetic beads evenly, gently invert 2 times, and then place the centrifuge tube in the magnetic stand again. After the magnetic beads are aggregated, discard the residual liquid. Add 1.2 mL of washing solution II, and repeat the above operation to further purify the magnetic bead-bound cfDNA.

[0097] Drying treatment: After washing, you can choose to stand the centrifuge tube at room temperature for 5-10 minutes, or place it in a 37°C air dryer for 5 minutes to ensure that the residual ethanol is completely volatilized.

[0098] cfDNA elution: Add 30 μL of elution solution, shake to fully suspend the magnetic beads, and incubate at room temperature for 3-5 minutes. During this period, you can gently shake the centrifuge tube to promote mixing. After the magnetic beads are re-aggregated, transfer the supernatant to a new centrifuge tube. If you want to improve the yield of cfDNA, you can repeat the elution 1-2 times. The obtained cfDNA product can be stored at -80°C. The extracted cfDNA is quantified using the Qubit fluorometer from Thermo.

[0099] Trans -cleavage experiment was performed using the Cas12a standard reaction system. The total reaction volume was 20 μL / well, including 50 nM FnCas12a and 50 nM two kinds of gRNA, incubated at 26°C for 30 minutes, and then 2.5 μM ssDNA reporter (TexasRed-CCCCC-BHQ2) was added. The prepared reaction solution was added to a 384-well plate, and the mixture was deposited at the bottom of the well by centrifugation at 1200 rpm for 1 minute at 4°C. A total of 2 ng of cfDNA sample (derived from healthy individuals, pancreatic cancer or lung cancer patients) was added to the reaction system to activate the Cas12a trans -cleavage activity. After 30 minutes of reaction at 26°C, the fluorescence intensity was measured using a BioTek Synergy H1 microplate reader. The experimental results are as follows Figure 8As shown, the Cas12a-based detection system can effectively distinguish the methylation status of cfDNA of pancreatic cancer and lung cancer cancer patients from healthy donors.

[0100] The above merely provides the preferred embodiment of the present application and not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. A reagent for direct detection of gene methylation based on the Cas12a system, characterized in that, The reagents include: Cas12a protein; Specific multiple-gRNAs targeting specific genes; Fluorescently labeled reporter probes; CRISPR-Cas12a buffer; The multiple-gRNAs specifically guide the Cas12a protein to bind to the DNA sequence of the target gene promoter region; the fluorescently labeled reporter probe includes a fluorescent group and a quencher group, which are linked by a polynucleotide chain.

2. The reagent according to claim 1, characterized in that, The reagent is used to directly detect 5mC methylation sites located in the distal region of the PAM sequence.

3. The reagent according to claim 1, characterized in that, The Cas12a protein is FnCas12a, and the optimal detection time is 30 minutes after the reaction.

4. The reagent according to claim 1, characterized in that, The fluorescently labeled reporter probe has a fluorescent group selected from ROX, Cy3, Cy5, and FAM, and a quenching group selected from BHQ1, BHQ2, and Dabcyl.

5. The reagent according to claim 1, characterized in that, The CRISPR-Cas12a buffer system contains NaCl, Tris-HCl, MgCl2, and recombinant albumin.

6. The reagent according to claim 5, characterized in that, The CRISPR-Cas12a buffer system also contains the reducing agents DTT and KCl. In the buffer system, Mg... 2+ The concentration was 45 mM, and the DTT concentration was 10 mM.

7. The reagent according to claim 1, characterized in that, The target gene is the CCDC140 gene, and the specific multiple-gRNAs targeting the target gene include gRNA-1 and gRNA-2. The last thymine deoxynucleotide T at the 3' end of gRNA-1 overlaps with the first thymine deoxynucleotide T of the PAM sequence of gRNA-2, and their sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

8. A method for directly detecting gene methylation based on the Cas12a system, characterized in that: The method includes the direct detection of gene methylation using the reagents described in any one of claims 1 to 7, comprising the following steps: S1: The Cas12a protein and specific multiple-gRNAs targeting the target gene are incubated in a CRISPR-Cas12a buffer system to form a complex. S2: Add a fluorescently labeled reporter probe to the system of S1 and read the initial background fluorescence; S3: Add the DNA to be tested to the system of S2 to initiate the trans-cutting reaction; S4: Detect the fluorescence signal intensity, and perform qualitative and / or quantitative analysis of the methylation of the target gene based on the fluorescence signal intensity.

9. The use of the reagent according to any one of claims 1 to 7 in the preparation of tumor detection reagents or detection kits.

10. A reagent or kit for detecting pancreatic cancer or lung cancer, characterized in that, include: FnCas12a protein; Specific multiple-gRNAs that target specific genes; Fluorescently labeled reporter probes; CRISPR-Cas12a buffer system; The target gene is the CCDC140 gene. Specific multiple-gRNAs targeting the target gene specifically guide the Cas12a protein to bind to the CCDC140 gene promoter region DNA sequence, including gRNA-1 and gRNA-2. The last thymine deoxynucleotide T at the 3' end of gRNA-1 overlaps with the first thymine deoxynucleotide T in the PAM sequence of gRNA-2, as shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The fluorescently labeled reporter probe includes a fluorescent group and a quencher group linked by a polynucleotide chain. The fluorescent group is selected from ROX, Cy3, Cy5, and FAM, and the quencher group is selected from BHQ1, BHQ2, and Dabcyl.

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