A novel nucleic acid detection method based on CRISPR-Cas12

Abstract

The present application relates to the field of nucleic acid detection, and particularly relates to a novel nucleic acid detection method based on CRISPR-Cas12. By introducing chemical modification (such as locked nucleic acid modification), the present application constructs a method for site-specific cleavage of specific phosphodiester bonds in single-stranded nucleic acid by Cas protein containing RuvC domain and a method for detecting target DNA in a sample to be tested, so as to realize site-specific cleavage and trigger signal cascade amplification, and then realize rapid and high-sensitivity detection of nucleic acid.

Description

[0001] This application claims priority to Chinese invention patent application [CN2021116657136], filed on December 31, 2021, entitled "A method for protecting specific phosphodiester bonds in nucleic acids from cleavage by Cas proteins containing RuvC domains", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of nucleic acid detection, and specifically to a novel nucleic acid detection method based on CRISPR-Cas12. Background Technology

[0003] The CRISPR / Cas system is a prokaryotic adaptive immune system found in most bacteria and all archaea. It serves as a defense mechanism for recognizing and combating invading foreign DNA, bacteriophages, and other pathogens. Guided by guide RNA, Cas proteins scan for, recognize, cleave, and degrade invading nucleic acid sequences at specific sites. Based on this function, the CRISPR / Cas system has been developed into a revolutionary gene-editing tool with wide applications in gene delivery, cancer therapy, stem cell engineering, and pharmacology.

[0004] In recent years, research has revealed that the CRISPR / Cas12a system possesses a unique trans-cleavage effect (i.e., trans enzyme activity): when activated by target DNA, the trans enzyme activity of Cas12a can degrade free single-stranded DNA (ssDNA) through random cleavage. This property has been applied to the development of novel gene detection methods that utilize the trans enzyme activity of Cas12a to cleave fluorescent probe ssDNA, thereby indirectly responding to the content of target DNA. However, since the content of target genes in samples is often lower than the detection limit of conventional CRISPR / Cas system-based gene detection methods, a significant fluorescent signal cannot be triggered, making it difficult to detect target genes.

[0005] To achieve ultrasensitive target gene detection, several novel gene detection methods, such as SHERLOCK, DETECTR, and HOLMES, have been developed by combining linear signal amplification techniques based on CRISPR / Cas systems with polymerase-based nucleic acid exponential amplification techniques (e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)). For example, Ding Xiong et al. combined LAMP reactions with Cas12a to simultaneously perform target gene pre-amplification and efficient signal amplification and output within a short time, achieving convenient and efficient target gene detection. Although nucleic acid amplification-based methods can significantly improve detection sensitivity, this process inevitably complicates the actual detection process and increases the uncertainty of the results. For example, false positive results from nucleic acid amplification cannot be eliminated during the detection process, and the accuracy of the results can be severely affected by subsequent Cas12a-mediated signal amplification. In addition, the detection process introduces components such as nucleotide mixtures, target gene amplification primers, and polymerases, making the detection system complex, the detection process less controllable, the time-consuming process relatively long, and the detection cost further increased. The design and detection of specific primers and other early-stage technologies require specialized and skilled technical personnel, which further limits the widespread application of these methods.

[0006] Recently, leveraging the dual functions of Cas proteins—target gene recognition and nucleic acid cleavage—the CRISPR-Cas system has been integrated into the signal amplification cycle catalyzed by target genes, aiming to achieve ultrasensitive target gene detection with a simpler detection process. Since some type V and VI Cas proteins in the CRISPR / Cas system (such as Cas12a, Cas12b, Cas13a, and Cas14a) exhibit trans-cleavage activity against free ssDNA or ssRNA, this non-site-specific and highly efficient cleavage method results in extremely high product turnover rates after ssDNA digestion. Shi Kai et al.'s CONAN method utilizes these characteristics and combines two CRISPR RNA (crRNA) systems to construct an amplification-free target gene detection method based on Cas12a protein autocatalysis. Compared to previously established gene detection methods based on the CRISPR / Cas12a system, this method directly detects dsDNA without requiring additional nucleic acid amplification or specialized instruments, achieving a detection sensitivity of 5 aM (a more than six-order-of-magnitude improvement) and a signal-to-noise ratio exceeding 13 times solely through the CRISPR-Cas system. This method reduces the complexity of nucleic acid detection to some extent and promotes the application of Cas12a-based nucleic acid detection methods in clinical diagnosis. However, this method still has some drawbacks, such as a long detection time (approximately 4 hours); the introduction of a dual crRNA system, which actually increases the complexity of the CRISPR-Cas system; the detection principle and process are not completely simplified; and the over-reliance on changes in the secondary structure of nucleic acid elements to initiate the reaction results in insignificant signal amplification efficiency and a lack of a clear exponential growth pattern in the detection signal, weakening stability and anti-interference capabilities, and significantly increasing costs. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for site-specifically cleaving a specific phosphodiester bond in a single-stranded nucleic acid by a Cas protein containing a RuvC domain, characterized in that the method comprises: chemically modifying nucleotides in a unit to be cleaved of the single-stranded nucleic acid, wherein the single-stranded nucleic acid includes at least one unit to be cleaved, the unit to be cleaved being a continuous nucleotide sequence of 9 nucleotides in length, the chemically modified nucleotides including a first nucleotide, a fourth nucleotide, and a ninth nucleotide in the 5' to 3' direction of the unit to be cleaved, and the phosphodiester bond being a phosphodiester bond between the seventh nucleotide and the eighth nucleotide in the 5' to 3' direction of the unit to be cleaved.

[0008] In some embodiments, the chemical modification includes one or more of the following: methoxy modification, thio modification, locked nucleic acid modification, bridged nucleic acid modification, 2'-fluoroRNA modification, 2'-aminoRNA modification, morpholine nucleic acid modification, ethylene glycol nucleic acid modification, hexitol nucleic acid modification, and threononucleotide modification.

[0009] In some embodiments, the Cas protein containing the RuvC domain includes one or more of Cas12a, Cas12b, and Cas9 proteins.

[0010] In a second aspect, a method for detecting target DNA in a test sample, characterized in that the method comprises: S1 adding the test sample to a detection system, the detection system comprising Cas12 protein, a first guide RNA, a second guide RNA, a first nucleic acid element, a second nucleic acid element, and a buffer; the first guide RNA comprising a targeting sequence capable of specifically binding to the target DNA; the first nucleic acid element comprising a first binding region and a structural region, the first nucleic acid element comprising a hairpin structure; the second nucleic acid element comprising a second binding region and a signal region, the second nucleic acid element being chemically modified according to the above method such that the phosphodiester bond connecting the second binding region and the signal region in the second nucleic acid element is cleaved; the test sample comprising the target DNA sequence; S2 Under the guidance of the first guide RNA, the Cas12 protein specifically binds to the target DNA and cleaves the phosphodiester bond, so that the second nucleic acid element is cleaved into the second binding region and the signal region and generates a detectable signal; S3 The second bonding region combines with the first bonding region to form a combined activation element; S4 Under the guidance of the second guide RNA, the Cas12 protein specifically binds to the combined activation element and cleaves the second nucleic acid element, forming a positive feedback loop system, which again causes the second nucleic acid element to be cleaved into the second binding region and the signal region and generates the detectable signal; S5 detects the detectable signal to obtain the presence and / or content of the target DNA.

[0011] In some embodiments, the nucleotide sequence of the first nucleic acid element includes SEQ ID NO:139; the nucleotide sequence of the second nucleic acid element includes GaaCgcttAtt (SEQ ID NO:99), wherein uppercase letters represent the chemically modified nucleotides and lowercase letters represent wild-type nucleotides; and the nucleotide sequence of the second guide RNA includes SEQ ID NO:1.

[0012] In some embodiments, the ends of the second nucleic acid element are modified with fluorescent groups and / or quenching groups.

[0013] In some implementations, the detectable signal includes a fluorescence signal.

[0014] In some embodiments, the Cas12 protein includes one or more of Cas12a, Cas12i, Cas12h, Cas12c, Cas12f2, and Cas12f3.

[0015] In some implementations, the Cas12a includes one or more of FnCas12a, AsCas12a, LbCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, and BoCas12a.

[0016] In some embodiments, the chemical modification includes one or more of the following: methoxy modification, thio modification, locked nucleic acid modification, bridged nucleic acid modification, 2'-fluoroRNA modification, 2'-aminoRNA modification, morpholine nucleic acid modification, ethylene glycol nucleic acid modification, hexitol nucleic acid modification, and threononucleotide modification.

[0017] In some implementations, the chemical modification is a locked nucleic acid modification.

[0018] In some implementations, the method further includes a step of plotting a standard curve, the step of which includes: adding positive standards of different concentration gradients to the detection system to react and detect the detectable signal, and plotting the standard curve based on the detectable signal values ​​corresponding to the positive standards of different concentration gradients; the positive standards include the target DNA sequence.

[0019] In some implementations, the combined length of the first binding region and the second binding region comprises 24-28 nucleotides.

[0020] In some implementations, the first binding region is 7 nucleotides long and the second binding region is 14 nucleotides long.

[0021] Thirdly, the present invention provides a reagent kit for nucleic acid detection, characterized in that the reagent kit comprises: (a) A first nucleic acid element, the first nucleic acid element comprising a first binding region and a structural region, the first nucleic acid element comprising a hairpin structure; (b) A second nucleic acid element, the second nucleic acid element comprising a second binding region and a signal region, the second nucleic acid element being chemically modified according to the above method such that the phosphodiester bond connecting the second binding region and the signal region in the second nucleic acid element can be cleaved at specific sites by the Cas12 protein; the first binding region and the second binding region can form a combined activation element; (c) A second guide RNA that guides the Cas12 protein to bind specifically to the combined activation element.

[0022] In some embodiments, the kit further includes the Cas12 protein, a first guide RNA, and a buffer; the first guide RNA includes a targeting sequence capable of specifically binding to the target DNA.

[0023] In some embodiments, the chemical modification includes one or more of the following: methoxy modification, thio modification, locked nucleic acid modification, bridged nucleic acid modification, 2'-fluoroRNA modification, 2'-aminoRNA modification, morpholine nucleic acid modification, ethylene glycol nucleic acid modification, hexitol nucleic acid modification, and threononucleotide modification.

[0024] In some implementations, the chemical modification is a locked nucleic acid modification.

[0025] In some embodiments, the Cas12 protein includes one or more of Cas12a, Cas12i, Cas12h, Cas12c, Cas12f2, and Cas12f3.

[0026] In some implementations, the Cas12a includes one or more of FnCas12a, AsCas12a, LbCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, and BoCas12a.

[0027] Beneficial technical effects

[0028] This invention provides a method for site-specifically cleaving specific phosphodiester bonds in single-stranded nucleic acids using Cas proteins containing RuvC domains. This invention achieves site-specific regulation of the enzymatic activity (including trans and cis activities) of Cas proteins containing RuvC domains (e.g., CRISPR-Cas12a) by chemically modifying specific nucleotides in the target nucleic acid. This prevents the cleavage of specific phosphodiester bonds in the target nucleic acid by Cas proteins containing RuvC domains and / or site-specific cleavage by such proteins. Existing technologies often utilize the characteristic of Cas12a protein, after activation by crRNA, to co-cleave ssDNA (i.e., non-target ssDNA, which can also be understood as "ssDNA to which the crRNA sequence has not hybridized") (i.e., "trans activity"), and perform gene detection using methods such as labeled single-stranded DNA detection reagents. The method provided by this invention can achieve site-specific regulation of the trans enzyme activity of Cas protein containing the RuvC domain. When applied to the field of gene detection, it can achieve cascade amplification of signals through site-specific regulation, eliminating the target gene amplification step and saving time and cost.

[0029] This invention also provides a novel method for activating Cas12 proteins (e.g., LbCas12a): by reintroducing another portion of a truncated single-stranded target DNA into the detection system (e.g., 24 nt of wild-type target DNA, the short single-stranded target DNA being 7 nt from 5' to 3', and the other portion being the remaining 17 nt from 5' to 3'), thereby activating the Cas12 protein. Based on this, this invention also develops a novel nucleic acid detection method. By introducing chemical modifications (e.g., locked nucleic acid modification), a CRISPR-Cas12 autocatalytic system based on the combined activation of two target DNA strands is constructed to achieve site-specific cleavage and cascade amplification of trigger signals. Applying the nucleic acid detection method established in this invention to the rapid detection (55 minutes) of breast cancer-related cfDNA achieves an experimental sensitivity of 10 fM and a detection limit of 4.7 fM. Furthermore, based on the excellent nucleic acid detection specificity of this method, the detection of breast cancer is ultimately achieved by detecting cfDNA secreted by different tumor cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0031] Figure 1A shows the structures of wild-type nucleotides, methoxy-modified nucleotides, thio-modified nucleotides, and locked nucleic acid-modified nucleotides;

[0032] Figure 1 B shows the locations of methoxy, thio, and locked nucleic acid modifications in 8 nt ssDNA;

[0033] Figure 2 The activity of Cas12a trans enzyme is shown to act on ssDNA at different thiomodification sites;

[0034] Figure 3 The activity of Cas12a trans enzyme is shown to act on ssDNA at different locked nucleic acid modification sites;

[0035] Figure 4 High-resolution gel banding results are shown after Cas12a trans enzyme activity is applied to ssDNA at different locked nucleic acid modification sites;

[0036] Figure 5 Michaelis constants for Cas12a trans enzyme activity acting on ssDNA modified with four locked nucleic acids;

[0037] Figure 6 A schematic diagram of locking nucleic acid modification to protect ssDNA from being digested by Cas12a enzyme (the red bases are the locking nucleic acid modification bases);

[0038] Figure 7 High-resolution gel banding results of Cas12a trans enzyme activity acting on ssDNA at different locked nucleic acid modification sites are shown.

[0039] Figure 8 High-resolution gel bands of ssDNA at locked nucleic acid modification sites are shown.

[0040] Figure 9 The principle of signal amplification based on CRISPR-Cas12a and probes specifically modified with locked nucleic acids is illustrated in Figure A: the principle of detection signal amplification by binding locked nucleic acid-modified single-stranded circular DNA and the Cas12a system, where gray represents the CRISPR-Cas12a enzyme and red represents crRNA; the black portion of the single-stranded circular DNA represents the specific target sequence of locked nucleic acid-modified Cas12a, and the red portion represents a random sequence; Figure B: the principle of detection signal amplification by binding locked nucleic acid-modified two specific target sequences and the Cas12a system, where gray and red represent the Cas12a enzyme and crRNA, respectively; the black portion of the two specific target sequences represents the specific target sequence of locked nucleic acid-modified Cas12a, and the red portion represents a random sequence.

[0041] Figure 10The diagram illustrates the technical route for gene detection based on CRISPR-Cas12a and probes specifically modified with locked nucleic acids (A is a schematic diagram of the technical route for gene detection in negative samples by combining locked nucleic acid-modified single-stranded circular DNA and the Cas12a system, where gray represents the CRISPR-Cas12a enzyme and red represents crRNA; the black part in the single-stranded circular DNA is the specific target sequence of locked nucleic acid-modified Cas12a, and the red part is the random sequence; when the target gene is not present in the sample or the target gene is mutated, the fluorescence signal hardly increases; B is a technical route for gene detection in positive samples by combining two specific target sequences modified with locked nucleic acids and the Cas12a system, where gray and red represent the Cas12a enzyme and crRNA, respectively; the black part in the two specific target sequences is the specific target sequence modified with locked nucleic acids, and the red part is the random sequence; when the target gene is present in the sample, a cascade amplification reaction is triggered, and a large number of fluorescent probe single-stranded DNAs mediated by the trans enzyme activity of CRISPR-Cas12a are cleaved, and fluorescent groups are released, resulting in a significant increase in fluorescence signal).

[0042] Figure 11 A schematic diagram of the CRISPR-Cas13-based SHERLOCK gene detection method ( Figure 11 A) and a schematic diagram of the DETECTR gene detection method based on Cripr-Cas12a ( Figure 11 B);

[0043] Figure 12 This is a schematic diagram of a CRISPR-Cas12a-based amplification-free gene detection method.

[0044] Figure 13 a shows the effects of different truncated single-stranded target DNAs based on the N system on LbCas12a activation (A. Effect of 3' truncated DNA on LbCas12a activation; B. Effect of 5' truncated DNA on LbCas12a activation).

[0045] Figure 13 b shows the effects of different truncated single-stranded target DNAs based on the E system on LbCas12a activation (A. Effect of 3' truncated DNA on LbCas12a activation; B. Effect of 5' truncated DNA on LbCas12a activation).

[0046] Figure 14The diagram illustrates the ability of two single-stranded target DNA segments, individually and in combination, to activate As-type and Fn-type Cas12a (A. Structures of different combinations of two single-stranded segments used to activate As-type and Fn-type Cas12a; B. Results of activating AsCas12a with two single-stranded target DNA segments, individually and in combination; C. Ratios of the activation effect of the two single-stranded segments combined to the activation effect of a single strand alone and the activation effect of the intact strand (mean ± standard deviation, n = 3); D. The activation effect of two single-stranded target DNA segments, individually and in combination, on Fn-type Cas12a; E. Ratios of the activation effect of the two single-stranded segments combined to the activation effect of a single strand alone and the activation effect of the intact strand (mean ± standard deviation, n = 3).

[0047] Figure 15 The diagram illustrates the principle of Cas12a activation by combining two target DNA segments (A. Schematic diagram of Cas12a activation by complete single-stranded target DNA; B. Schematic diagram of Cas12a activation by combining two single-stranded target DNA segments (red and blue represent the truncated single-stranded target DNA and its missing portion, respectively)).

[0048] Figure 16 The results of different combinations of N-TS two-single-strand target DNAs activating LbCas12a are shown (A. Structures of different N-TS two-single-strand combinations; B. Results of activating Cas12a with the two single-single-strand target DNAs individually and in combination; C. Ratios of the effect of activating Cas12a with the combination of two single-strands to the effect of activating Cas12a with a single strand alone and the effect of activating with the whole N-TS strand).

[0049] Figure 17 The results of different combinations of E-TS two-single-strand target DNA to activate LbCas12a are shown (A. Structures of different E-TS two-single-strand combinations; B. Results of LbCas12a activation after the two single-single-strand target DNAs are activated separately and in combination; C. Ratio of the activation effect of the two-single-strand combination to the activation effect of a single strand to LbCas12a and the activation effect of the whole E-TS strand).

[0050] Figure 18Different Hairpin and ss structures designed based on the combination 2 of two single-stranded target DNA segments from N-TS are shown, along with the results of Cas12a activation by cross-combination of the two structures (A. Schematic diagram of different Hairpin and ss structures; B. Heatmap results of Cas12a activation by cross-combination of Hairpin and ss; C. Time-dependent activation results of Hairpin 0-ss 0 and Hairpin 0-ss 4 combinations; D. Signal-to-noise ratio of Cas12a activation by Hairpin 0-ss 0 and Hairpin 0-ss 4 combinations).

[0051] Figure 19 The results of high-resolution gel analysis of enzyme digestion of unmodified and locked nucleic acid modified probes at different sites are shown (enzyme digestion results of ss4 and LNA-ss4-CA (red letters indicate locked nucleic acid modification sites, black arrows indicate specific enzyme digestion sites)).

[0052] Figure 20 This is a schematic diagram of the detection method based on locked nucleic acid modification and two-stage single-stranded target DNA activation of Cas12a autocatalytic signal amplification according to the present invention.

[0053] Figure 21 The results show the fluorescence response of the nucleic acid detection method and the direct detection method of Cas12a constructed in this invention to different concentrations of SARS-CoV-2 N gene plasmids (A. Fluorescence response of the method constructed in this invention to SARS-CoV-2 N gene plasmids at 8 concentration gradients; B. Fluorescence response of SARS-CoV-2 N gene plasmids at different concentrations directly detected using Cas12a).

[0054] Figure 22 The standard curves for detecting the SARS-CoV-2 N gene plasmid at different time points using the nucleic acid detection method constructed in this invention are shown (A. Response relationship between N gene plasmid concentration and fluorescence intensity at different time points; B. Standard curves for N gene plasmid detection at different time points).

[0055] Figure 23 The target DNA activation of Cas12a autocatalytic signal amplification system by single point mutations and double site mutations in the SARS-CoV-2N gene is shown (A. Single point mutation sequence and fluorescence results after normalization; B. Fluorescence results of normalized single point mutation in the PAM region; C. Double site mutation sequence and fluorescence results after normalization; D. Fluorescence results of normalized double site mutation in the PAM region).

[0056] Figure 24The specificity experimental results of interfering plasmids for six coronaviruses are shown (A. Alignment results of the six interfering plasmids with the target sequence of SARS-CoV-2-N; B. Fluorescence detection results of the six interfering plasmids, the experimental group, and the background group).

[0057] Figure 25 The performance of the CRISPR-Cas12a autocatalytic cycle nucleic acid detection technology for detecting the BRCA-1 gene under different environments is shown (A. Dose-response curve and standard curve for detecting the BRCA-1 gene in PBS buffer; B. Dose-response curve and standard curve for detecting the BRCA-1 gene in 5% human serum (n≥3)).

[0058] Figure 26 The specificity assessment of the CRISPR-Cas12a autocatalytic cycling method for detecting the BRCA-1 gene is shown (n≥3);

[0059] Figure 27 The following diagram illustrates the application of the nucleic acid detection method constructed in this invention to the detection of three tumor cell lines: (A. Schematic diagram of cell line detection (HEK293T and HeLa cell lines are positive cell lines, MCF-7 is a negative cell line); B. Detection of HEK293T cells using the method constructed in this invention; C. Detection of HeLa cells using the method constructed in this invention; D. Detection of MCF-7 cells using the method constructed in this invention; E. Comparison of detection results for the three cell lines (n>6)).

[0060] Figure 28 This diagram illustrates how locked nucleic acid modification protects specific sites in ssDNA from Cas12a cleavage (red bases represent locked nucleic acid modification bases, and dashed lines indicate specific cleavage sites of trans enzyme activity). The present invention found that when locked nucleic acid modification is introduced into ssDNA, modification at one site can completely protect the two adjacent phosphodiester bonds on either side of that site, while modification at two sites with a two-base interval can effectively protect the three middle and two outer phosphodiester bonds from cleavage by Cas12a trans enzyme activity. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0062] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0063] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0064] In this specification, some embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as specifically disclosing all possible subranges and the individual numerical values ​​within that range. For example, a description of the range 1–6 should be considered as specifically disclosing subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0065] Example 1: Effects of different types of modified nucleic acids on Cas12a enzyme activity

[0066] Materials and Methods

[0067] Sequence dissolution: Using ultrapure water (IQ7010, Millipore, USA), the synthesized DNA or RNA sequences were dissolved to a final concentration of 100 μM. After vortexing for approximately 15 seconds using a vortex mixer (MX-S, Cerrojek, USA), the solutions were centrifuged in a desktop centrifuge (D1008E, Cerrojek, USA). Most of the sequences mentioned in this example (Table 1) contain fluorescent probe labels, so all operations were performed under light-protected conditions. For short-term use, the dissolved DNA or RNA sequences were stored at 4°C; for long-term use, they were aliquoted and stored at -20°C (RNA was aliquoted to a final concentration of 20 μM and stored at -20°C to avoid repeated freeze-thaw cycles).

[0068] Table 1: Sequences involved in this embodiment Note: The "Number" column in this table represents the nucleotide sequence number used in this invention. The "SEQ ID NO:" column represents the sequence number of the non-branched nucleotide sequence of not less than 10 nucleotides in the nucleic acid sequence used in this invention in the sequence listing. NA indicates not applicable.

[0069] annealing: The annealing procedure for the short-stranded double-stranded DNA (dsDNA) used in this embodiment is as follows. Unless otherwise specified, the final concentration of annealed dsDNA is generally 10 μM, and the annealing buffer is 1X PBS solution. The annealing equipment used is a conventional PCR instrument (SimpliAmp PCR instrument, Applied Biosystems, USA), with the program set as follows: 95℃-5 min, 65℃-30 min, 50℃-30 min, 37℃-30 min, 25℃-30 min, 4℃-store. After annealing, the product is stored at -20℃ for long-term storage and at 4℃ for short-term storage.

[0070] Assay of the reactivity of Cas12a with different modified probe substrates (using LNA1-LNA5 Cas12a cleavage) (Taking experimental fluorescence testing as an example): The Cas12a enzyme used in this embodiment is the Lb type Crispr-Cas12a enzyme, namely Cpf1. Sample loading procedure (taking one reaction as an example): Mix Cas12a enzyme (NEB) to a final concentration of 50 nM, crRNA (Shanghai Sangon Biotech) to a final concentration of 50 nM, 1X Buffer 2.1 (Cas12a enzyme working buffer), and target strand NT (Shanghai Sangon Biotech) to a final concentration of 2 nM in a PCR tube, and dilute with ultrapure water to a total volume of 16 μL. Incubate the PCR tube in a 37°C metal bath (LS-D202, Waterman, UK) for 5 min, then add 4 μL of locked nucleic acid modified DNA probes (LNA1-LNA5) to a final concentration of 200 nM. Vortex to mix and centrifuge. Sample testing: Transfer the reaction system from the above PCR tube to a white 384-well plate and monitor the fluorescence kinetics using a cell imaging microplate reader (Cytation™ 3, Berten Instruments, USA). The program was set as follows: a) Vibration mode: linear vibration mode, frequency 100 rpm, duration 10 seconds; b) Kinetic process: reading once per minute, total measurement time 1 hour; c) Fluorescence measurement: excitation and emission light 492 nm and 518 nm respectively, gain set to 50, temperature constant at 37℃. The probe determination process for other modifications (such as methoxy and thiosulfate modifications) is the same as described above.

[0071] Procedure for determining the Michaelis constant of Cas12a reaction with locked nucleic acid-modified probe substrates:First, a Cas12 and crRNA complex at a final concentration of 4 nM was mixed with a target strand ssDNA or dsDNA at a final concentration of 40 nM in 2X Buffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 100 μg / mL BSA, pH 7.9) and incubated at 37°C for 30 minutes to obtain a 4 nM activated enzyme solution. Then, LNA1 to LNA5 modified DNA fluorescent reporter probes were added at final concentrations of 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1.25 μM, 2.5 μM, and 5 μM, respectively, to trigger the substrate cleavage reaction. The Cas12a enzyme concentration was maintained at a constant 2 nM in 1X Buffer 2.1 (NEB, MA). Fluorescence assays were performed three times in a microplate reader at 37°C, with fluorescence readings taken every 30 seconds. Background subtraction of the fluorescence signal was calculated by subtracting the fluorescence signal of the control group (without the target chain) from the original fluorescence signal of the experimental group. For each type of fluorescent probe, the initial reaction rate (AU / s) was obtained by linear regression fitting of the first 600 seconds of data at each concentration. The reaction rate was converted from AU / s to nM / s using a calibration curve. The measured reaction rates were fitted to the corresponding fluorescent probe concentration data using GraphPad Prism software (GraphPad, CA, USA) to obtain the Michaelis-Menten equation, and finally, kcat and K were calculated. M .

[0072] Verification of Cas12a's cleavage of locked nucleic acid-modified probes using high-resolution denaturing polyacrylamide gel electrophoresis:Sample preparation: Mix Cas12a enzyme (NEB) to a final concentration of 50 nM, crRNA (Shanghai Sangon Biotech) to a final concentration of 50 nM, 1X Buffer 2.1 (Cas12a enzyme working buffer), and target strand NT (Shanghai Sangon Biotech) to a final concentration of 2 nM, place the mixture in a PCR tube, and dilute with ultrapure water to a total volume of 50 μL. Incubate at 37°C in a metal bath for 5 min, then add 50 μL of locked nucleic acid modified DNA probes (LNA1P-LNA5P) to a final concentration of 500 nM. After mixing, incubate at 37°C in a PCR instrument (SimpliAmp PCR instrument, Applied Biosystems, USA). Take 20 μL of sample at intervals of 0 min, 30 min, 60 min, and 120 min, inactivate at 95°C for 5 min, and then allow to cool naturally at room temperature. Mix in an equal volume of 2X DNA loading buffer (Sangon Biotech) containing urea to prepare the sample for gel electrophoresis. High-resolution denaturing polyacrylamide gel preparation process: 24g of urea (Sangon) was placed in a 50mL BD tube. 5mL of 10X TBE (Sangon) and 25mL of 40% acrylamide / methylenebisacrylamide solution (19:1) (Sangon) were added separately. The tube was placed in a 37℃ water bath until the urea particles were completely dissolved. 166μL of freshly prepared 30% (w / w) APS solution and 20μL of TEMED (Sangon) were added, mixed well, and immediately poured into a 1.5mL gel mold (JY-scz6, Junyi) to form the gel. Gel running process: After removing the urea from the sample wells, 40μL of the prepared sample was added. The running voltage was set to 300V; the electrophoresis buffer was 1X TBE; and the running time was set to 4 hours. The power supply used was a DYY-6D power supply manufactured by Beijing Liuyi Biotechnology Co., Ltd. The photopolymer coating instrument was an Invitrogen iBright 1500 (Thermo Fisher Scientific, USA).

[0073] High-resolution denaturing polyacrylamide gel electrophoresis was used to verify the cleavage of locked nucleic acid-modified TS strands by Cas12a. (Verification experiment of cis enzyme activity):Sample preparation: Dilute Cas12a (final concentration 250 nM), crRNA (final concentration 500 nM), and 1X Buffer 2.1 to 30 μL with ultrapure water. Incubate at 37°C for 5 min. Then add 30 μL of LNA-TS-2, LNA-TS-1, and WT DNA (final concentration 100 nM). Mix well and incubate at 37°C in a PCR instrument (SimpliAmp PCR instrument, Applied Biosystems, USA). Perform incubation at 95°C for 5 min at 5 min and 20 min intervals. Prepare gel electrophoresis samples using 2X urea DNA loading buffer (Sangon Biotech). Control experiment preparation: Follow the steps described above. Dilute the prepared sample system to 10 μL with ultrapure water and incubate directly at 95°C for 5 min to inactivate the DNA. Subsequently, 10 μL of LNA-TS-2, LNA-TS-1, and WT DNA were added to a final concentration of 100 nM. Samples for gel electrophoresis were then prepared using 2X urea DNA loading buffer (Sangon Biotech). The gel electrophoresis instrument was an Invitrogen iBright 1500 (Thermo Fisher Scientific, USA).

[0074] 1.1 Inhibitory effects of different types of modified nucleic acids on the trans enzyme activity of Cas12a

[0075] Following the method described above for "determination of the reactivity of Cas12a with different modified probe substrates," fluorescence kinetic experiments were used to investigate the inhibitory effects of different types of modified nucleic acids on the enzyme activity (especially trans enzyme activity) of Cas12a. The following methods were employed: Figure 1 The three modification types shown in A are methoxy modification, thio modification, and locked nucleic acid modification. In addition, other modification types such as bridging nucleic acid, ethylene glycol nucleic acid, and 2'-fluoroRNA modification can also be selected.

[0076] Methoxy (2'OMe1) modification is a common nucleoside modification in RNA, formed by the methylation of the hydroxyl group at the 2-position of the ribose in the nucleoside. In experiments investigating the transase activity of Cas12a, methoxy-modified nucleotides can be inserted at different sites on an 8-nt ssDNA segment to construct a methoxy-modified probe ssDNA. This example uses ssDNA with all 8 nucleotides modified by methoxy groups (NO. 8 in Table 1) to study the transase activity of Cas12a. Figure 1B). The results showed that when methoxy-modified nucleic acid was used as a substrate, the trans enzyme activity of Cas12a was significantly inhibited: during the 2-hour cleavage reaction, the fluorescence intensity was more than 50-fold different from that of the control group (i.e., the unmodified wild-type probe ssDNA), indicating that full-site methoxy modification can effectively inhibit the trans enzyme activity of Cas12a.

[0077] Phosphorthioate modification involves replacing non-bridging oxygen atoms in the phosphate backbone of a nucleic acid chain with sulfur atoms. Phosphor-modified oligonucleotides primarily prevent nucleic acid degradation by nucleases. To investigate whether phosphor-modified nucleic acids also effectively inhibit Cas12a transase activity, this example used three different phosphorthioate modification methods (as shown in Table 1, NO.14, NO.15, and NO.16): full-site modification, four-site modification near the 5' and 3' ends, and two-site modification near the 5' and 3' ends, respectively. Figure 1 B). For example Figure 2 As shown, full-site modification can clearly inhibit the trans enzyme activity of Cas12a, with a fluorescence intensity nearly 50-fold different from the control group (i.e., the unmodified wild-type probe ssDNA). This indicates that full-site thiolation modification can effectively and significantly inhibit the trans enzyme activity of Cas12a. The other two thiolation modification methods did not show effective inhibition, and their fluorescence results showed no significant difference between each other or between them and the control group. This suggests that the four- or two-site modifications at the 5' and 3' ends have almost no effect on the trans enzyme activity of Cas12a, and this is significantly different from the principle used in common molecular experiments that involves introducing 2-5 thio-modified bases at both ends of primers to prevent degradation by nucleases. This also suggests that the trans enzyme activity of Cas12a differs significantly from that of ordinary nucleases.

[0078] Locked nucleosides (LNAs) are synthetic nucleic acid analogs containing bridging bicyclic glycosyl groups. The addition of a methylene group between the 2'-O- and 4'- positions "locks" the furanose ring to a 3'-internal conformation. LNAs fully comply with the Watson-Crick base pairing rules, and studies have shown that LNA:DNA hybrids exhibit significantly higher annealing temperatures and strong resistance to nucleases compared to their corresponding DNA:DNA hybrids. To investigate whether LNA-modified nucleotide chains inhibit the transase activity of Cas12a, this embodiment employed five different LNA modification methods based on the 8-base length of the probe DNA (i.e., adjacent site modification (LNA1), intermediate site modification (LNA2), intermediate two-base modification (LNA3), intermediate three-base modification (LNA4), and intermediate four-base modification (LNA5)). These five modification methods essentially cover possible adjacent and intermediate modification logics. Figure 1 B).

[0079] First, fluorescence experiments were performed on the five different LNA modifications mentioned above. For example... Figure 3 As shown, it is evident that the trans enzyme activity of Cas12a was inhibited to varying degrees with changes in the location and amount of LNA modification. LNA1 modification completely inhibited Cas12a trans enzyme activity. For LNA2 and LNA3 modifications, their inhibitory effect on Cas12a trans enzyme activity was similar to that of full-site methoxy and thio modifications, achieving highly efficient inhibition. For LNA4 and LNA5 modifications, only a weak inhibitory effect on Cas12a trans enzyme activity was observed, with LNA4 showing a significantly better inhibitory effect than LNA5. Based on these results, it can be concluded that, similar to methoxy and thio modifications, LNA modification inhibits Cas12a trans enzyme activity, and full-site modification can achieve 100% inhibition. Furthermore, it is evident that the five different LNA modifications exhibit a gradient inhibitory effect in the fluorescence experiment results. That is, as the number of LNA modifications increases and the modification position changes (from LNA5 to LNA1), the inhibitory effect on the trans enzyme activity of Cas12a shows a significant positive correlation.

[0080] To more intuitively reflect the cleavage effect of Cas12a on five probes modified with different LNA1-LNA5, high-resolution denaturing polyacrylamide gel electrophoresis was used to further verify the above results. As described in the above method "Verification of Cas12a cleavage of probes modified with locked nucleic acids using high-resolution denaturing polyacrylamide gel electrophoresis", five LNA fluorescent reporter substrates with the same modification were constructed. It is hoped that the size of the reporter substrate fragments before and after Cas12a cleavage can be used to intuitively reflect the cleavage effect of Cas12a.

[0081] Different banding results were obtained in reactions involving different substrate types at different time points. Figure 4 It is evident that no product bands from enzyme digestion were observed in LNA1-3 (this differs from the results of the fluorescence experiment, possibly because the sensitivity of the fluorescence experiment is much higher than that of the polyacrylamide gel, so the weaker fluorescence growth of LNA2 and LNA3 in the fluorescence experiment was not clearly reflected in the product bands on the gel). However, in LNA4 and LNA5, it is clearly visible that as the reaction time increases, the substrate band becomes fainter, while the product band becomes more prominent; and the product band of LNA5 is significantly more prominent than that of LNA4. Furthermore, comparing the product bands of LNA4 and LNA5, it is clear that the product band of LNA5 appears to be significantly higher than that of LNA4 (i.e., the interval between the product bands and the substrate bands of LNA4 and LNA5 is different). This suggests that although LNA5 and LNA4 modifications only exhibit similar weak activity towards the trans enzyme activity of Cas12a, the cleavage sites of Cas12a in LNA5 and LNA4 appear to be different.

[0082] 1.2 Determination of the Michaelis constant of Cas12a response to four locked nucleic acid-modified probe substrates

[0083] To investigate whether different LNA modification methods can yield different results in inhibiting the trans enzyme activity of Cas12a, this example used four different LNA-modified probes as substrates (LNA1 was excluded from the Michaelis constant determination due to its 100% inhibitory effect), and measured the Michaelis constant of Cas12a response to the locked nucleic acid-modified probe substrates.

[0084] This embodiment selected a relatively low concentration of the Cas12a and crRNA complex, and at seven substrate concentrations, the reaction maintained linear growth for the first 10 minutes. Simultaneously, to better reflect the true changes in each group of data, a background group with the same substrate concentration was set up in each reaction. The fluorescence signal value of each experimental group was subtracted from the fluorescence signal value of the background group to obtain the true experimental value for each group, thereby calculating the initial rate for each group. Figure 5As shown, from LNA2 to LNA5, the initial reaction rate increases significantly with increasing substrate concentration, exhibiting a relatively clear Michaelis growth model. Based on the Michaelis function, various parameters of Cas12a for substrates from LNA2 to LNA5 were calculated, including the maximum reaction rate (VL). max ), Michaelis constant (K) m ), transformation number (K) cat and the ratio K cat / K m As shown in Table 2, the Vc of the Cas12a cleavage reaction varies from substrate LNA2 to LNA5. max Significantly increased. K m Used to measure the affinity between an enzyme and its substrate; K m The smaller the value, the lower the substrate concentration required for the enzyme to react, and the greater the affinity between the enzyme and the substrate. The K values ​​for the four substrates LNA2 to LNA5 are... m The differences between the values ​​are not very significant, indicating that the affinity of the Cas12a enzyme for these four substrates is not significantly different. In other words, the large differences in the reaction efficiency of the Cas12a enzyme with these four substrates are not strongly related to the enzyme's affinity for the substrate. By calculating K... cat Values ​​were found to indicate that K values ​​varied from substrates LNA2 to LNA5. cat The values ​​show a clear positive correlation. cat With K m The ratio of K to α is an important parameter for measuring the catalytic efficiency of an enzyme. As can be seen from the data in Table 2, K... cat / K m The values ​​showed a significant positive correlation with the substrates LNA2 to LNA5, indicating that these four modification types significantly affected the cleavage efficiency of Cas12a. Therefore, this embodiment found that the effect of LNA modification on the trans enzyme activity of Cas12a is mainly manifested in its significant impact on the cleavage activity of Cas12a for LNA-modified substrates, while having almost no effect on its recognition and binding ability.

[0085] Table 2: Michaelis constant determination of locked nucleic acid modified substrates LNA2-LNA5

[0086] On the other hand, the reaction results of LNA2-LNA5 show that both the meta modification of LNA2 (one wild-type base in the middle) and the two meta-base modification of LNA3 (two wild-type bases in the middle) have strong inhibitory effects on Cas12a. For LNA4 and LNA5, both substrates contain only two locked nucleic acid modifications. LNA4 contains three wild-type bases and four phosphodiester bonds between the two modification sites, while LNA5 contains four wild-type bases and five phosphodiester bonds between the two modification sites. Neither of these two modification methods significantly inhibits Cas12a. Therefore, this example further hypothesizes that a single LNA modification can effectively protect the adjacent nucleotides, meaning that modifications of two LNA sites can effectively protect the 6nt probe from Cas12a degradation (e.g., ...). Figure 6 (A schematic diagram showing how LNA modification protects ssDNA from being digested by Cas12a).

[0087] 1.3 LNA modification site-specifically regulates the trans enzyme activity of Cas12a

[0088] Having established that LNA can effectively inhibit the trans enzyme activity of Cas12a and the effects of different LNA modifications on the trans enzyme activity of Cas12a, and even preliminarily determined the protective effects of possible LNA modifications on different sites, this embodiment further explores whether different LNA modification sites and amounts can achieve site-specific regulation of the trans enzyme activity of Cas12a.

[0089] Based on LNA3, LNA4, and LNA5 modification methods, different types of probes were constructed to investigate the effects of different LNA modification sites and amounts on the transase activity of Cas12a. As shown in Table 1 (NO.17, NO.18, NO.19, NO.20, NO.21, and NO.22), six sequence types were constructed for verification. All DNA strands were modified with fluorescently labeled FAM at the 5' end, and the length of the fluorescently labeled DNA probe chains was precisely analyzed using high-resolution denaturing polyacrylamide gel electrophoresis to illustrate the site-specific regulation of Cas12a transase activity by LNA. Denaturing polyacrylamide gel electrophoresis was used to analyze the DNA strands after Cas12a treatment; the cleavage sites of the Cas12a enzyme on the DNA strand were determined by analyzing the length of the DNA probe chains. Results are shown below. Figure 7 As shown, the following conclusions can be drawn from the gel graph:

[0090] First, changing the amount of LNA on the probe sequence can alter the cleavage site of Cas12a on the LNA-modified DNA strand. For example... Figure 7As shown in Figure C, in the "Trans enzyme activity WT 7+4" (i.e., the unmodified control group), the substrate band gradually weakened with increasing reaction time, while the product band, compared to the marker band, was significantly smaller than 6 nt. This result is consistent with the literature's conclusion that the trans enzyme activity of Cas12a can cleave ordinary ssDNA to fragments of approximately 4 nt. Furthermore, other DNA strands modified with LNA showed bands significantly longer than approximately 4 nt, such as... Figure 7 A and Figure 7 As shown in B.

[0091] Secondly, changing the position of LNA on the probe sequence can alter the cleavage site of Cas12a on the LNA-modified DNA strand. In this embodiment, three bases were selected from an 11nt DNA strand to modify LNA, resulting in five representative modification methods. The selection of modification sites was based on 1.1. As shown in Table 1 (NO.9, NO.10, NO.11), LNA modification with an interval of two or fewer nucleotides effectively protects those two nucleotides from Cas12a cleavage. However, as shown in Table 1 (NO.12, NO.13), LNA modification with an interval of three or more nucleotides does not effectively protect the DNA from Cas12a enzymatic activity. Utilizing the characteristics described above, five different LNA-modified DNA probe strands were designed: NO.18, NO.19, NO.20, NO.21, and NO.22.

[0092] like Figure 7As shown in Figure A, comparing the results of sequences LNA-5c 7+4 and LNA-5b 7+4 reveals that LNA-5c 7+4, after trans digestion by Cas12a, produces three product bands: 8 nt, 6 nt, and a fragment smaller than 6 nt. With increasing reaction time, the 8 nt fragment band gradually weakens, while the fragment smaller than 6 nt band gradually strengthens. In contrast, LNA-5b 7+4 produces only two product bands: 7 nt and a fragment smaller than 6 nt. Similarly, with increasing reaction time, the 7 nt product band significantly weakens, while the fragment smaller than 6 nt band strengthens, indicating that the 7 nt fragment is further degraded. By analyzing the sequence differences between LNA-5b and LNA-5c, this embodiment deduces that the 8nt and 7nt product sequences are GAACGCTT and GAACGCT, respectively. Furthermore, the further degradation of the 8nt and 7nt products is due to the presence of three unmodified nucleotides (i.e., four phosphodiester bonds) between the two LNA modification sites in the sequence, similar to the fluorescence results of LNA4 described above, consistent with previous conclusions. In addition, compared to LNA-5b, LNA-5c produces 8nt and 6nt fragments, rather than 7nt fragments, also due to the presence of four unmodified bases (i.e., five phosphodiester bonds) between the two LNA sites, similar to the results for LNA5 described above.

[0093] To improve this result, the second LNA modification at the 5' end of the probe was modified by migrating one and two base sites to the 5' end, respectively, resulting in LNA-4c, LNA-4b, and LNA-3b sequences. As expected, none of these three modified products showed further degradation; that is, no bands significantly smaller than 6 nt were observed in the gel electrophoresis (for the LNA-3b sequence, since there are 5 unmodified bases and 6 phosphodiester bonds between the two LNA modifications at the 3' end, the product is clearly concentrated around 5 nt). Comparing the LNA-4b and LNA-4c sequences, it is evident that the 7 nt sequence produced by the LNA-4b substrate shows only very slight degradation with increasing reaction time. This result is similar to the LNA-5c product, where there are 4 unmodified bases and 5 phosphodiester bonds between the two LNA modification sites at the 3' end. Although LNA-4b modification can clearly achieve site-specific cleavage of Cas12a at the 7th phosphodiester bond at the 5' end of the substrate, trace amounts of non-site-specific cleavage products still occur. In summary, by altering the position and number of locked nucleic acids on the probe sequence, site-specific regulation of Cas12a transactivation can be effectively achieved. That is, by introducing locked nucleic acid modifications at specific sites, the random cleavage activity of Cas12a on ssDNA can be converted into site-specific cleavage.

[0094] exist Figure 28 In the diagram shown, taking an 11nt ssDNA segment as an example, when locked nucleic acid modifications are introduced at three specific sites (as indicated by the red 'x' bases in the diagram, from the first locked nucleic acid modified nucleotide to the third locked nucleic acid modified nucleotide, a total of 9nt), the activated Cas12a transase is restricted to a specific cleavage site on this 11nt ssDNA. Figure 28 (As shown by the dashed line between c and d), this results in the 11nt substrate being ultimately cut into two fragments of 8nt and 3nt in size.

[0095] 1.4 LNA modification inhibits Cas12a cis enzyme activity.

[0096] Existing literature reports a possible mechanism by which CRISPR-Cas12a exerts trans enzyme activity: after the complex formed by CRISPR-Cas12a and crRNA recognizes and binds to the target gene, the conformation of Cas12a changes, exposing its RuvC restriction site, which is used to cleave the recognized target strand (TS strand) (called cis enzyme activity); the exposed RuvC restriction site can further achieve random degradation of free ssDNA in the system (called trans enzyme activity). Therefore, the trans enzyme activity of Cas12a may essentially still originate from the RuvC active site, which is also responsible for cis enzyme activity.

[0097] Based on the above theoretical foundation and conclusions, this embodiment hypothesizes that LNA modification can also effectively inhibit the cis enzyme activity of Cas12a. This embodiment, having verified that LNA modification can inhibit and efficiently regulate the trans enzyme activity of Cas12a, explores its inhibitory effect on the cis enzyme activity of Cas12a.

[0098] As shown in Table 1 (NO.29-NO.31), three target sequences (TS) for cis enzyme activity verification were designed. The experimental groups modified the high-frequency sites of cis enzyme digestion (i.e., the six bases preceding the 5' end of the region paired with crRNA) with LNA. The results are as follows: Figure 8As shown, it is evident that neither the LNA-modified TS-1 nor TS-2 groups exhibited the specific fragment bands larger than 30 nt found in the unmodified WT group. Specifically, the LNA-TS-1 group (LNA meta-modification) showed three distinct bands at the potential cis restriction site of 30 to 40 nt, indicating that meta-modification with LNA effectively alters the cis restriction site. In contrast, the LNA-TS-2 group (LNA full-site modification) showed only one distinct band at the potential cis restriction site of 30 to 40 nt, with a size close to 40 nt. This demonstrates that full-site modification with LNA efficiently inhibits the cis restriction site. In this experiment, this manifested as a change in cis restriction site preference; that is, after locking the Cas12a cis enzyme activity site, the ternary complex formed by ssDNA and Cas12a / crRNA has the ability to adjust its conformation, bringing the unmodified portion of the ssDNA strand closer to the Cas12a nuclease's active domain.

[0099] Based on this, the method for regulating trans enzyme activity (and cis enzyme activity) provided by this invention is applicable to CRISPR-Cas series enzymes containing RuvC cleavage sites. Taking Cas12a (i.e., cpf1) as an example, the domains containing RuvC are: 885-940, RuvC-I; 941-957, RuvC-II; 1263-1307, RuvC-III. For Cas12b, the domains containing RuvC are: 519-628, RuvC-I; 785-900, RuvC; 975-993, RuvC-III. For Cas9, the domains containing RuvC are: 1-41, RuvC-I; 435-481, RuvC-II; 650-775, RuvC-III.

[0100] Chemical modifications applicable to the methods provided in this invention include one or more of the following: methoxy modification, thiomodification, locked nucleic acid (LNA) modification, bridged nucleic acid (BNA) modification, 2'-fluoroRNA modification, 2'-aminoRNA modification, morpholine nucleic acid modification, ethylene glycol nucleic acid (GNA) modification, hexitol nucleic acid (HNA) modification, and threonine nucleic acid (TNA) modification. The chemically modified nucleotides (naturally occurring or artificially synthesized nucleotide analogs) can conform to the Watson-Crick base pairs with natural nucleic acids (DNA or RNA). Since these chemical modifications all occur on the pentose units of the nucleotides, the modified pentose units, due to changes in rigidity or increased stability, endow the modified nucleic acid chains with a more significant resistance to nucleases.

[0101] 1.5 Application of site-specific regulation of Cas12a trans enzyme activity in gene detection

[0102] Figure 9 A illustrates a signal amplification strategy mediated by locked nucleic acid-modified circular DNA, which can be simply described as follows: The sequence of the target gene to be detected is designed as a small single-stranded circular DNA. Given that the specific target sequence of CRISPR-Cas12a is 24 nt in length, the length of the small single-stranded circular DNA can be designed to be approximately 30 nt, containing a 24 nt locked nucleic acid-modified specific target sequence and approximately 6 nt of unmodified random sequence. When the target gene is absent, due to the rigidity of the circular DNA itself, the 24 nt target sequence in the circular DNA cannot bind normally to crRNA, and therefore will not activate the transenzyme activity of CRISPR-Cas12a. Therefore, when there is only a large amount of free circular DNA in the system, since the transenzyme activity is not activated, the probe single-stranded DNA modified with the fluorescent quencher group cannot be cleaved, the fluorescent group cannot be exposed, and the fluorescence signal hardly increases. Figure 10 As shown in Figure A, when the target gene is present, a small amount of CRISPR-Cas12a is activated. Its transzyme activity then cleaves approximately 6 nt of random sequences of a large amount of free circular DNA in the system (the target sequence is protected from cleavage due to site-specific modifications of locked nucleic acids). This leads to linearization of the target sequence and binding to crRNA, activating more CRISPR-Cas12a, thus creating a cascade amplification effect. A large amount of fluorescently quenched probe single-stranded DNA is cleaved, resulting in a significant increase in fluorescence signal, such as... Figure 10 As shown in B.

[0103] Furthermore, cascade amplification effects can be achieved using ordinary linear DNA modified with locked nucleic acids, such as... Figure 9 As shown in B, the 24nt target sequence was divided into two segments. A 4-8nt random sequence was added to the 3' side of one single-stranded target DNA segment at its 5' end, and a similar 4-8nt random sequence was added to both the 5' and 3' sides of the other single-stranded target DNA segment at its 3' end. Similarly, locked nucleic acid modification was used to protect only the two target DNA segments at specific sites. When the target gene was absent, due to interference from the random sequence, the two target DNA segments could not bind properly to the crRNA, resulting in the inability to activate the transase activity of CRISPR-Cas12a. The fluorescently quenched probe single-stranded DNA could not be cleaved, the fluorescent group could not be exposed, and the fluorescence signal showed almost no increase. Figure 10As shown in Figure A, when the target gene sequence is present, a small amount of CRISpr-Cas12a is effectively activated. Its trans enzyme activity cleaves the interfering random sequences on the two target sequences at specific sites, generating two complete target sequences. These sequences then bind to crRNA, activating more CRISpr-Cas12a and creating a cascade amplification effect. A large amount of fluorescently quenched probe single-stranded DNA is cleaved, resulting in a significant increase in the fluorescence signal, such as... Figure 10 As shown in B.

[0104] The method for regulating the trans enzyme activity of CRISPR-Cas12a provided by this invention has the following advantages in nucleic acid detection compared with other Cas12a-based nucleic acid detection methods: ① It can achieve amplification-free nucleic acid detection, that is, signal amplification is achieved through site-specific regulation, eliminating the need for... Figure 11 The most common existing method shown is the process of amplifying DNA signals through PCR or isothermal amplification, thereby saving detection time and simplifying the detection procedure. ② and Figure 12 Compared with existing technologies, the Cas12a-based amplification-free nucleic acid detection method shown has the advantage of relying on only one crispr-cas12a and crRNA system to complete the detection, and has higher signal amplification efficiency and shorter detection time (e.g., Figure 12 The existing technology shown involves two CRISPR-Cas12a systems, namely, using two CRISPR-Cas12a-crRNA systems, T1 and T2, for signal amplification. This method can achieve amplification-free gene detection to a certain extent, but the system is too complex and expensive.

[0105] The two signal amplification strategies and gene detection methods mentioned above are just examples of applications based on site-specific regulation of the trans enzyme activity of CRISPR-Cas12a by locked nucleic acid modification. More in vitro gene detection methods can be developed based on the methods provided in this invention.

[0106] The method provided by this invention has specific applications in in vitro detection, such as: military biochemical emergency infection detection, food quarantine, animal quarantine, disease control, and customs inspection; specific applications include clinical respiratory disease detection (influenza, avian influenza, COVID-19, etc.), intestinal infectious disease detection (hand-foot-mouth disease, norovirus, Salmonella), zoonotic and vector-borne infectious disease detection (dengue fever, Zika), blood-borne and sexually transmitted diseases (hepatitis B and C, Ebola), foodborne pathogen detection, meat component detection, and genetically modified food identification. Specific applications in in vivo detection include, for example, gene editing applications based on CRISPR.

[0107] Example 2:

[0108] Materials and Methods

[0109] Table 8. Deoxynucleotide chain sequences related to the N system. Note: Red underlines in this table indicate mutation sites.

[0110] Table 3. Deoxynucleotide chain sequences associated with the E system.

[0111] Table 4. Sequences of nucleic acids related to the P system

[0112] Table 5. Preferred DNA sequences with different structures Note: The probe DNA is modified with 5'FAM and 3'BHQ1 at the 5' and 3' ends, respectively; the probe DNA used for high-resolution gel electrophoresis is only modified with 5'FAM; the red underlined sites in this table are the locked nucleic acid modification sites of the probe DNA.

[0113] Table 6 Core sequences of different interfering plasmids

[0114] Table 7. Relevant sequences for tumor cell detection experiments.

[0115] Cell imaging microplate analyzer detection: (1) Sample loading: Transfer the sample to be tested to a white 384-well plate, with a sample volume of 20 μL in each well; centrifuge for 1 min and then place it on the sample stage of the cell imaging microplate detector for detection. (2) Setting the running program: Set the linear vibration mode, the frequency to 100 r / min, and the time interval to 10 s; set the fluorescence detection interval to 1 min, and the total measurement time to 2 h; set the excitation and emission wavelengths to 492 nm and 518 nm, respectively; set the gain value to 50; and set the temperature to 37 ℃. (3) Detection on the instrument: Run the cell imaging microplate detector monitoring program to analyze the fluorescence results.

[0116] Efficiency assessment of truncated single-stranded target DNA activating Cas12a:To investigate the efficiency of truncated single-stranded target DNA in activating the Cas12a / crRNA system, a series of truncated single-stranded target DNAs targeting different crRNA systems (including the N, E, and P systems, as shown in Tables 8, 3, and 4) were synthesized. The activation effects were evaluated using Lb-type Cas12a enzymes (N and E systems) and Fn-type and As-type Cas12a enzymes (P system). The reaction systems in this experiment were prepared according to Table 9-1.

[0117] Table 9-1. Reaction system configuration for single-stranded target DNA activation of Cas12a

[0118] Add the components listed in the table to RNase-free centrifuge tubes sequentially and mix well. Incubate at 37°C for 5 min, then add 6 μL of single-stranded target DNA of different lengths to a final concentration of 50 nM and mix thoroughly. Immediately add 20 μL of reaction solution to each well of a white 384-well plate, with three replicates for each length of single-stranded target DNA. Then, place the plate in a microplate reader for detection, using the same detection program as described above.

[0119] The combined activation of Cas12a by two single-stranded target DNA segments: In this invention, all experiments involving the combined activation of Cas12a by two single-stranded target DNA segments were prepared according to the reaction system in Table 9-2.

[0120] Table 9-2. Reaction system configuration for activating Cas12a with a combination of two single-stranded target DNA segments.

[0121] Add the components listed in the table to an RNase-free centrifuge tube sequentially and mix well. Incubate at 37°C for 5 min, then add 6 μL of 5' end single-stranded DNA and 6 μL of 3' end single-stranded DNA (final concentration 50 nM). Immediately add 20 μL of reaction solution to a white 384-well plate, setting three replicates for each combination of two single-stranded target DNA. Then, place the plate in a microplate reader for analysis, using the same detection program as above.

[0122] Two-segment single-stranded target DNA activation Cas12a assay with different structures

[0123] The reaction system was prepared as shown in Table 9-2. The mixed solution was incubated at 37°C for 5 min. Then, 6 μL of hairpin DNA and 6 μL of single-stranded DNA with a final concentration of 500 nM were added (as shown in Table 5). Immediately afterwards, 20 μL of reaction solution was added to a white 384-well plate. Samples with different combinations of hairpin DNA and single-stranded DNA were set up in three replicates. The plates were then placed in a cell imaging microplate reader for detection, and the detection program was set up as above.

[0124] High-resolution polyacrylamide gel electrophoresis for detection of Cas12a cleavage lock nucleic acid modified probes

[0125] (1) Sample preparation. The effect of locked nucleic acid modification on Cas12a trans-cut DNA sites and resistance to nuclease cleavage was investigated. The reaction system was prepared according to Table 9-3. The components listed in the table were added to RNase-free centrifuge tubes and mixed thoroughly. The mixed solution was incubated at 37°C for 5 min, and 50 μL of locked nucleic acid modified fluorescent probe strands with a final concentration of 500 nM (as shown in Table 5) were added. After mixing, the solution was incubated at 37°C. Then, 20 μL of the sample was taken at different time points (0 min, 30 min, 60 min, 120 min) and inactivated at 65°C for 10 min. (2) The preparation method of high-resolution polyacrylamide gel was the same as in Example 1. (3) The electrophoresis process of the locked nucleic acid modified substrate samples by Cas12a site-specific enzyme digestion was the same as in Example 1.

[0126] Table 9-3. Reaction system configuration for locked nucleic acid modified probe cleavage experiments

[0127] Using SARS-CoV-2N gene detection to evaluate site-directed cleavage and two-segment single-cell cleavage based on locked nucleic acid modification regulation. Detection performance of gene detection methods based on the autocatalytic signal amplification of Cas12a activated by target DNA.

[0128] (1) Sensitivity evaluation of the gene detection method established in this invention.

[0129] The reaction system for detecting the SARS-CoV-2N gene based on the gene detection method of Cas12a autocatalytic signal amplification was prepared as shown in Table 9-4. The components listed in the table were added to the RNase-free centrifuge tubes in sequence and mixed thoroughly.

[0130] Table 9-4. Reaction system configuration for locked nucleic acid modified probe cleavage experiments

[0131] The mixed solution was incubated at 37°C for 5 min. Nine groups of incubated samples were then added with 6 μL of SARS-CoV-2N gene plasmid at final concentrations of 0 fM, 10 fM, 50 fM, 100 fM, 500 fM, 2 pM, 10 pM, 40 pM, and 100 pM (Table 6). Immediately afterwards, 20 μL of reaction solution was added to each well of a white 384-well plate. Samples containing different concentrations of SARS-CoV-2N gene were placed in triplicate. The plates were then placed in a microplate reader for detection, with the detection program set up as above.

[0132] (2) Specificity analysis of the gene detection method established in this invention.

[0133] The solution was prepared as shown in Table 9-4. The mixed solution was incubated at 37°C for 5 minutes and then set aside.

[0134] The conditions for analyzing the specificity of single-stranded target DNA were set as follows: 6 μL of wild-type single-stranded target DNA with a final concentration of 2 nM, as well as single-site and double-site mutated single-stranded target DNA, were added to each of the 41 groups of incubated samples (as shown in Table 8). Immediately afterwards, 20 μL of reaction solution was added to each well of a white 384-well plate. Samples containing different mutated single-stranded target DNA were placed in three replicates, and then placed in a cell imaging microplate reader for detection. The detection program was set as above.

[0135] The conditions for analyzing plasmid specificity were set as follows: Eight groups of the above-mentioned incubated samples were each given 6 μL of six interfering plasmids (as shown in Table 6, constructed in pUC57 plasmid) at a final concentration of 50 pM: the N gene plasmid of human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome-associated coronavirus Tor2 (SARS-CoV-Tor2), and the E gene plasmid of SARS-CoV-2 (SARS-CoV-2-E). 6 μL of the SARS-CoV-2-N gene plasmid at a final concentration of 500 fM and 6 μL of RNase-free water were added to each plasmid as the experimental group and the control group, respectively. Immediately afterwards, 20 μL of reaction solution was added to each well of a white 384-well plate. Samples containing different interfering plasmids were placed in three replicates, and then the plates were placed in a cell imaging microplate reader for detection. The detection program was set as above.

[0136] BRCA-1 gene detection experiment

[0137] Prepare the reaction system as shown in Table 10. Use a 1.5 mL RNase-free centrifuge tube to add the reagents listed in the table in sequence and mix them by pipetting with a micropipette.

[0138] Table 10 Reaction system configuration for BRCA-1 gene detection experiment

[0139] After short-term separation, the samples were placed in a metal bath and incubated at 37°C for 5 minutes. Six groups of incubated samples were then taken, and 6 μL of BRCA-1 ssDNA (sequences shown in Table 7) at final concentrations of 0 fM, 10 fM, 100 fM, 1 pM, 10 pM, and 100 pM were added respectively. The mixture was vortexed and centrifuged. Immediately afterwards, 20 μL of the mixture was transferred to white 384-well plates using a micropipette. After short-term separation, the plates were placed in a microplate reader for analysis. Each reaction was performed in triplicate. Due to the presence of fluorescent and quenching groups in the reaction system, the entire reaction preparation process was conducted in the dark. In the specificity experiment, nine groups of the above-mentioned incubation samples were taken, and 6 μL of BRCA-2, miRNA-21, miRNA-let7a, miRNA-141, miRNA-let7d, miRNA-7e, and miRNA-122 with a final concentration of 10 pM and 6 μL of RNase-free water were added as blanks and 6 μL of BRCA-1 with a final concentration of 100 fM as experimental groups for detection.

[0140] In HEK293T, HELA, and MCF-7 cell assays, well-growing cells were passaged at ratios of 1:6 (HEK293T and MCF-7 cells) and 1:4 (HELA cells), respectively. Cell counts were performed using a cell counter, and the cells were passaged until the cell density reached approximately 5 x 10⁻⁶ cells / cells. 5 / mL, 6x10 5 / mL and 7x10 5 After collecting 7 mL of cell culture medium, immediately freeze at -80°C. Once a sufficient number of cell culture groups (8 groups per concentration per cell) have been collected, thaw the culture medium and extract cfDNA from the culture medium using the Ezup Column-Based Cell-Free DNA Extraction Kit, following the instructions in the kit (elution volume 40 μL).

[0141] Prepare the reaction system according to Table 10. After momentary centrifugation, incubate in a metal bath at 37°C for 5 min. Take 6 μL of cfDNA extraction solution from HEK293T, HELA, and MCF-7 cells of different densities, add 6 μL of the incubated samples, vortex to mix, and centrifuge. Immediately transfer 20 μL of each solution to a white 384-well plate using a micropipette. After momentary centrifugation, place the plate in a cell imaging microplate reader for detection. Each reaction is performed in triplicate. Due to the presence of fluorescent and quenching groups in the reaction system, the entire reaction preparation process must be carried out in the dark.

[0142] In the cell detection experiments, each group of experiments was independently repeated 6 times for statistical significance analysis.

[0143] Statistical analysis The experimental data were statistically analyzed using GraphPad Prism 7.0. The experimental data are expressed as mean ± standard deviation (Mean ± SD). The comparison between multiple groups was performed using analysis of variance, and the comparison between two groups was performed using t-test. P < 0.05 was considered statistically significant. Each experiment was repeated 3 times.

[0144] Activation of CRISPR / Cas12a by different truncated single-stranded target DNAs

[0145] The gene-targeting recognition function of CRISPR / Cas12a depends on two factors: first, the protospacer adjacent motif (PAM) interaction domain (PAM-interacting, PI) of the Cas12a protein recognizes the PAM sequence in the target sequence, namely 5'-TTTV-3' (V = A, C, G); second, the 20-24 nt RNA strand at the 3' end of the guide crRNA pairs complementaryly with the target DNA strand in the target sequence. Previous studies have shown that, guided by crRNA, the CRISPR / Cas12a system can recognize not only double-stranded DNA but also ssDNA, and that the recognition of ssDNA is not strictly dependent on the presence of the PAM sequence.

[0146] This embodiment investigated the effect of different single-stranded target DNA lengths on the activation of Cas12a (the commonly used Lb-type Cas12a enzyme, derived from Lachnospiraceae bacterium), when the crRNA length was fixed at 24 nt. The activation level of Cas12a was characterized using an ssDNA fluorescent probe (the TA base combination reported to have the highest cleavage efficiency was used in this embodiment). As shown in Tables 8 and 3, the different single-stranded target DNA lengths used can be divided into two categories: one where the target DNA is gradually shortened by one nucleotide from the 3' end of the DNA strand, and the other where the target DNA is gradually shortened by one nucleotide from the 5' end of the DNA strand.

[0147] Experimental results are as follows Figure 13 a and Figure 13b shows that as the length of the single-stranded target DNA corresponding to both genes decreases, their ability to activate Cas12a gradually weakens. For the N system (a set of crRNA and single-stranded target DNA (N-TS ssDNA, 24nt) recognition combinations), when shortening starts from the 3' end of the N-TS ssDNA, the truncated ssDNA (removing the first 7 bases) shows no significant difference in activation effect on LbCas12a compared to the intact N-TS ssDNA. When the 8th base is removed, the activation effect of the truncated ssDNA on LbCas12a decreases by about 50%. And when 9 or more bases are removed, the truncated ssDNA loses its ability to activate LbCas12a. Figure 13 (A portion of a). When shortening begins from the 5' end of N-TS ssDNA, the activation effect of the truncated ssDNA on LbCas12a gradually weakens with decreasing base number: when 4 bases are removed, the activation effect of the truncated ssDNA on LbCas12a decreases by 50%; and when more than 4 bases are removed, the truncated ssDNA almost completely loses its ability to activate LbCas12a. Figure 13 (Part B of a). Although both the truncated variants shortened from the 5' end and the truncated variants shortened from the 3' end completely lose their ability to activate Cas12a after being shortened to a certain extent, it can be found that Cas12a has a lower tolerance for the truncated variant ssDNA shortened from the 5' end.

[0148] For the E system (another set of crRNA and single-stranded target DNA (E-TS ssDNA) recognition combinations), we can see results almost identical to those of the N-TS ssDNA truncated variant. Specifically, in the truncated variant shortened from the 3' end, when the 9th base is removed, 50% of the Cas12a activation activity is lost; when shortened by more than 9 bases, the activation activity of the truncated ssDNA is almost completely lost. Figure 13 (part A of b). In the truncated form shortened from the 5' end, when the third base is removed, 50% of the Cas12a activation activity is lost; when shortened by more than three bases, the activation activity of the truncated ssDNA is almost completely lost. Figure 13 (Part B of b).

[0149] This embodiment further designed the P system (a novel combination of crRNA (44nt) and single-stranded target DNA recognition, as shown in Table 4) to analyze the activation effects of different truncated ssDNA on AsCas12a and FnCas12a. Figure 14As shown in A and 14B, the activation of AsCas12a and FnCas12a gradually weakens with the reduction of 5' and 3' bases. Similar to LbCas12a, the 5' bases are also more critical for the activation of FnCas12a and AsCas12a. For FnCas12a, removing more than 5 bases from the 5' end almost completely eliminates the activation of the truncated ssDNA; while for AsCas12a, removing more than 8 bases from the 5' end almost completely eliminates the activation of the truncated ssDNA. Although FnCas12a and AsCas12a seem to have a higher tolerance for 5' truncated ssDNA compared to LbCas12a, considering that P-TS ssDNA in the P system is 29 nt (5 nt more at the 5' end than N-TS ssDNA and E-TS ssDNA), it can be considered that the tolerance of these three types of Cas12a enzymes for 5' truncation is consistent.

[0150] The results above suggest that the activation of Cas12a by truncated single-stranded target DNA may be related only to the characteristics of Cas12a itself, and not to different crRNA systems (including different crRNA lengths (40-44 nt) and different base compositions).

[0151] In summary, single-stranded target DNA can activate different types of Cas12a (including Lb, As, and Fn types), and specific truncated single-stranded target DNA can also effectively activate its transase activity. Furthermore, the presence or absence of the PAM sequence does not affect the activation effect of single-stranded target DNA. It can also be seen that the bases at the 5' end of the single-stranded target DNA appear to be more important for Cas12a activation, and the key site for recognizing and activating Cas12a may be located in a structural region 3-4 nt away from the 5' end.

[0152] Example 3: Efficiency of combined activation of Cas12a by two single-stranded target DNA segments

[0153] In the aforementioned study of the differences in the activation of Cas12a by single-stranded target DNA of different lengths, a series of truncated ssDNAs that completely lost their activating effect were obtained. These truncated ssDNAs have the potential to function as switches, regulating the activity of Cas12a trans-cleavage of probe DNA. To further apply these truncated ssDNAs to construct efficient signal amplification methods, this embodiment uses a complementation experiment to explore the switching effect of truncated ssDNAs on the dual function of Cas12a recognition and cleavage. Figure 15 A and Figure 15As shown in B, the missing portion of the truncated ssDNA was added to the reaction system of the truncated ssDNA that had lost its Cas12a activation function to observe whether Cas12a could be reactivated and the magnitude of its activation effect.

[0154] Based on the above results showing that the N-TS ssDNA completely loses its Cas12a activation function starting from the 5' end after subtracting the 5th base and the 3' end after subtracting the 9th base, the N-TS ssDNA was truncated at positions 6, 7, 8...14 after the 5' end into two pairs of sequences: the 5' y segment and the 3' x segment, totaling 9 pairs of sequences. Figure 16 A) and the activation capacity of each truncated ssDNA for LbCas12a was detected separately when it was present alone and when mixed. Figure 16 As shown in B, neither of the truncated ssDNA sequences alone could effectively activate LbCas12a, consistent with the results of the previous examples. When both the 5' y and 3' x sequences were present simultaneously, the trans enzyme activity of LbCas12a was reactivated efficiently. The activation effect of intact N-TS ssDNA was taken as 100% activation, and the difference in activation between the combined activation of LbCas12a by the two truncated ssDNA sequences and the activation of LbCas12a by the 5' y sequence alone was compared. Figure 16 As shown in Figure C, neither the truncated ssDNA combinations achieved 100% activation compared to the complete N-TS ssDNA, and there were significant differences in the reactivation effects of LbCas12a among the nine truncated ssDNA combinations. Relatively speaking, the activation effect of truncated ssDNA combinations 1-6 was stronger than that of truncated ssDNA combinations 7-9, indicating that the closer the truncation site is to the middle of the N-TS ssDNA, the weaker the activation effect by filling in the missing part.

[0155] To further verify that the cas12a activation effect of truncated ssDNA is independent of different crRNA systems and different types of cas12a enzymes, this example also performed complementation experiments using E-TS ssDNA with LbCas12a enzyme and P-TS ssDNA with AsCas12a and FnCas12a enzymes. In the reaction system of E-TS ssDNA and LbCas12a enzyme, the E-TS ssDNA was truncated into two pairs of sequences, namely the 5' y segment and the 3' x segment, from the 6th, 7th, 8th...14th position after the 5' end. Figure 17 A). For example Figure 17As shown in Figure B, some combinations of two truncated ssDNA segments can still efficiently activate LbCas12a, but only combinations 1, 2, 3, and 8 show significant activation effects on LbCas12a, which differs from the results for N-TS ssDNA. Using activation of LbCas12a with intact E-TS ssDNA as 100% activation, the activation efficiency of the two truncated E-TS ssDNA segments was less than 75%, far less than the activation effect of the two truncated N-TS ssDNA segments. Furthermore, it was found that the location of most cleavage sites had no significant impact on the reactivation efficiency of Cas12a, with a fluorescence intensity difference of approximately 10 times compared to the background fluorescence of individual activations. However, when the 5' end of the DNA strand of both N-TS ssDNA (N gene) and E-TS ssDNA (E gene) was at the 12th base site, the fluorescence intensity of the combination decreased significantly. This indicates a significantly weakened ability to activate Cas12a, suggesting that the 12th base site is likely a key region for Cas12a activation.

[0156] In the reaction system of P-TS ssDNA and AsCas12a (derived from Acidaminococcus sp. BV3L6) and FnCas12a (derived from Francisella novicida U112), the P-TS ssDNA is truncated into two segments, y at the 5' end and x at the 3' end, starting from positions 15, 16, 17...22 or 23 after the 5' end. Figure 14 A). For example Figure 14 B and Figure 14 As shown in C, all eight sequence combinations effectively activated AsCas12a. Starting with the third combination, the difference in AsCas12a activation between the truncated ssDNA (y = 17 nt) and the truncated combination gradually decreased because the 5'y truncated ssDNA itself has a certain Cas12a activation effect. Similar results were observed in the system where the truncated P-TS ssDNA activated FnCas12a. Figure 14 D and Figure 14 E).

[0157] The mechanism of CRISPR / Cas12a recognition and cleavage of target DNA reveals that crRNA interacts with the WED, RuvC, and REC2 domains of the Cas12a protein to form a stable Cas12-crRNA binary complex. Subsequently, the cyclic lysine helical loop (LKL) region in the PI domain promotes complementary pairing between the target strand (TS) and crRNA in the target double-stranded DNA. The generation of the crRNA-TS DNA heteroduplex triggers a conformational rearrangement of Cas12a. This allosteric change exposes the DNase catalytic site in the RuvC domain, thereby activating cis and trans enzyme activities. Given that CRISPR / Cas12a recognition and activation primarily depend on the complementary pairing of crRNA and the target sequence mediated by the PI domain of the Cas12a protein, leading to the subsequent conformational rearrangement... Therefore, it is speculated that the two combined single-stranded target DNA segments in this invention can effectively activate Cas12a because: First, the Cas12a protein has a high tolerance for recognizing single-stranded target DNA and activating cis and trans enzyme activities (as can be seen from the fact that truncated ssDNA can effectively activate various types of Cas12a). Second, when truncated ssDNA alone cannot activate Cas12a, but when a partial truncated ssDNA and the missing portion coexist, Cas12a can be reactivated. This indicates that when a segment of the single-stranded target DNA pairs complementaryly with crRNA, the resulting crRNA-TS DNA does not complete normal extension, and therefore is insufficient to cause Cas12a to undergo conformational rearrangement and expose the RuvC restriction domain. However, when the missing portion is also present in the system, it pairs complementaryly with the unpaired crRNA, allowing the crRNA to restore its normal secondary structure. This normal secondary structure of crRNA further triggers the conformational rearrangement of Cas12a, ultimately producing trans enzyme activity.

[0158] Based on the above results, it can be inferred that the characteristic of this combination of two single-stranded target DNAs to effectively activate Cas12a may not be related to different crRNA systems or different types of Cas12a enzymes, but is a common activation mode that exists in all types of Cas12a enzymes.

[0159] In summary, when ssDNA is used as the target strand of the Cas12a protein, it can be truncated into two truncated ssDNA segments at a specific site. When only one segment is present, Cas12a cannot be activated; however, when both truncated ssDNA segments are present simultaneously, Cas12a can be reactivated efficiently. This feature provides a basis for subsequent research in this invention, namely, controlling the activation process of Cas12a by introducing one of the two single-stranded target DNA segments as a switch. Furthermore, based on the dual function of Cas12a—gene recognition and nucleic acid cleavage—this new feature can be applied to multiple fields, such as the development of new biological signal amplification methods and the research of "biological circuits" with molecular logic gating functions. In addition, the discovery of this co-activation of Cas12a by two ssDNA segments also provides new insights into the target sequence recognition of CRISPR series proteins, the subunit conformational changes involved in the Cas12a activation process, and the final nuclease activity generation process.

[0160] Example 4: The Activation Effect of Different Dual-Segment Target DNA Combinations on CRISPR / Cas12a

[0161] To better utilize the features discovered in the above embodiments, a combination 2 (with a high signal-to-noise ratio and relatively low background signal in system N) is selected. Figure 17 C, consisting of two ssDNA segments (7 nt at the 5' end and 17 nt at the 3' end), serves as two nucleic acid elements in the signal amplification method. The aim is to utilize these two special nucleic acid elements in combination 2 to combine the dual functions of Cas12a—nucleic acid recognition and enzyme cleavage—to produce an autocatalytic activation effect. That is, Cas12a first recognizes and then activates, and this activation promotes further recognition, creating a positive feedback loop that ultimately amplifies the signal. In this method, when trace amounts of the target gene are present, a very small amount of crRNA and the Cas12a complex are activated. This small amount of activated Cas12a, due to the specially designed nucleic acid elements in its trans-enzyme cleavage system, generates more "target strands" that can reactivate Cas12a, thus creating a cascade amplification effect. This leads to the rapid amplification of the trace target gene signal until sufficient fluorescence is produced for detection. Such trace amounts of the target gene are undetectable by conventional methods (such as DETECTR) due to excessively low fluorescence yield.

[0162] To construct this special nucleic acid element, the two ssDNA segments of combination 2 in N-TS were first structurally modified to verify their feasibility as elements controlling Cas12a recognition and activation. To prevent the 17nt single strand at the 3' end from being degraded by the transzyme activity of activated Cas12a in the reaction system, this invention designed it as a special hairpin structure, while the 7nt at the 5' end retained its single-stranded structure (as shown in Table 5).

[0163] To verify the feasibility of controlling the activation of Cas12a using these two sequences, a single T base, two TA bases, three TAT bases, and four TATT bases were introduced at the breaks in the two sequences, respectively, thus constructing sequences including Hairpin0-4 and ss0-4. Figure 18 A). Then, through pairwise cross-combinations, a total of 25 specific activation combinations were formed, and the activation effects of these combinations were examined. For example... Figure 18 As shown in Figure B, when Hairpin 0 is combined with ss 0-4 respectively, the activation effect is strongest when combined with ss 0, and the activation effect gradually weakens with the increase of the number of bases introduced at the 3' end of ss 0. Similarly, when ss 0 is combined with Hairpin 0-4 respectively, the activation effect is strongest when combined with Hairpin 0, and the activation effect gradually weakens with the increase of the number of bases introduced at the 3' end of Hairpin 0. Other combinations of Hairpin chains with ss chains do not have a significant activation effect on Cas12a. Figure 18 C represents the fluorescence detection results after Cas12a activation by the combination of Hairpin 0 and ss 0, the combination of Hairpin 0 and ss 4, and the complete N-TS. It can be seen that when the 7nt linear strand in the Hairpin 0 and ss 4 combination changes from the ss 4 structure to ss 0, it can rapidly activate Cas12a to produce trans enzyme activity within a short time. This result indicates that in both Hairpin 0 and ss 4 combinations, the transition of the 7nt linear strand from ss 4 to ss 0 can act as a switch-like nucleic acid element to control the rapid activation of Cas12a.

[0164] This embodiment further analyzes the signal-to-noise ratio of Cas12a activation at different time points using the Hairpin 0 and ss 0 combination and the Hairpin 0 and ss 4 combination. For example... Figure 18As shown in Figure D, the effective signal-to-noise ratio (SNR) of Hairpin 0-ss 0 / Hairpin 0-ss 4 reached 16.6-fold at 15 minutes and peaked at 19.3-fold at 30 minutes, with activation levels approaching those of the complete N-TS. These results strongly suggest that the two ssDNA segments in N-TS combination 2, existing as Hairpin 0 and ss 4, have significant potential as key nucleic acid elements for constructing Cas12a-based signal amplification methods.

[0165] The results above show that, in the presence of the Hairpin 0 structure, when the four TTAT bases at the 3' end of ss4 are cleaved to form the ss 0 structure, the trans activity of Cas12a can be efficiently activated. Therefore, the TTAT sequence at the 3' end of ss4 becomes the key switch for Cas12a activation. This invention aims to activate a small amount of Cas12a by the target gene in the constructed signal amplification method; then, the activated Cas12a cleaves the nucleic acid element ss4 through its trans enzyme activity to generate ss 0; subsequently, due to the co-activation of the Hairpin 0 and ss 0 nucleic acid elements in the system, a large amount of Cas12a is activated, generating a positive feedback signal amplification effect, and then achieving qualitative and quantitative detection of the target gene by cleaving the probe DNA to produce fluorescence. However, the trans enzyme activity of Cas12a usually cleaves free ssDNA randomly, and cannot achieve the desired site-specific cleavage of ss4 to generate ss 0 as proposed in this invention.

[0166] Based on Example 1, locked nucleic acid modification was used to site-specifically regulate the activity of Cas12a. As shown in the LNA-ss4-CA sequence in Table 5, locked nucleic acid modification was performed on the straight-chain ss4 at a specific site to regulate the exonuclease activity of Cas12a, achieving site-specific cleavage of the straight-chain ss4, thereby generating the seamlessly cleaved target straight-chain ss0. The results are as follows: Figure 19 As shown, it can be clearly seen that LNA-ss 4-CA, upon cleavage by activated Cas12a, produces a specific 7nt ss 0. Hairpin 0, with a Tm value of 81.5℃, is sufficiently stable at 37℃ and will not be recognized and randomly degraded by the Cas12a exonuclease.

[0167] Example 5: A gene detection method based on site-directed cleavage regulated by locked nucleic acid modification and amplification of the autocatalytic signal of Cas12a activated by two-segment single-stranded target DNA.

[0168] Therefore, this invention constructs a high-efficiency signal amplification method based on Cas12a, such as... Figure 20As shown, the detection system contains two premixed DNA structures: Hairpin 0 and a single-stranded fluorescent probe (LNA-ss 4) modified with locked nucleic acid. When the CRISPR-Cas12a system (inactive Cas12a / crRNA) recognizes the target DNA strand, it activates the trans-activation of Cas12a (active Cas12a), thereby cleaving the fluorescent probe LNA-ss 4. Due to the site-specific modification of the locked nucleic acid, the fluorescent probe LNA-ss 4 is specifically cleaved into ss 0 and emits fluorescence. Hairpin 0 and ss 0 combine to form a complete target DNA strand, which can efficiently activate a new CRISPR-Cas12a system. Each trans-cleavage of Cas12a not only accumulates the fluorescent signal but also activates other Cas12a enzymes, forming a Cas12a-driven cascade amplification effect to amplify the target nucleic acid signal.

[0169] When the target gene is absent from the system, the crRNA-Cas12a complex is inactive, preventing the Hairpin 0 and reporter-labeled LNA-ss 4 dual nucleic acid elements from functioning. This results in a quiescent state with no significant fluorescence signal. However, when trace amounts of the target gene are present, the crRNA-Cas12a complex recognizes the gene and activates its trans enzyme activity. The free reporter-labeled LNA-ss 4 in the system is cleaved by the Cas12a trans enzyme activity, transforming into ss 0. ss 0 then combines with another nucleic acid element, Hairpin 0, to reactivate Cas12a. This positive feedback effect activates a large amount of Cas12a, cleaving more reporter-labeled LNA-ss 4 to accumulate a significant fluorescence signal, thus enabling the detection of trace target genes.

[0170] The nucleic acid detection method of this invention involves two modules—a signal triggering module and a positive feedback signal amplification module. In the signal triggering module ( Figure 20 (Left), trace amounts of the target gene can activate the exonuclease activity of Cas12a; in the subsequent positive feedback signal amplification module ( Figure 20 (Right) The activated Cas12a exonuclease activity of the target gene can cleave the LNA-ss4 element in the module and generate ss0. The combination of ss0 and another element in the module, Hairpin0, reactivates Cas12a. The activated Cas12a continues to cleave the LNA-ss4 element and generate a large number of ss0, forming a positive feedback loop signal amplification, and the trace target gene signal is significantly amplified and read.

[0171] In the positive feedback signal amplification module, LNA-ss4 and Hairpin 0, as two nucleic acid elements, are derived from the combination of two single-stranded target DNA segments (a 7nt single-stranded DNA at the 5' end and a 17nt single-stranded DNA at the 3' end) selected for the N system constructed in the above embodiments. Figure 13 and Figure 18 As shown in the diagram. The reason it is a preferred element is that two single strands alone cannot activate Cas12a; only when both single strands are present simultaneously can they activate Cas12a. Besides the sequence types shown in SEQ ID NO:99 and SEQ ID NO:139, Hairpin 1 and LNA-ss4 can also be used as nucleic acid elements to achieve positive feedback amplification of the signal. When using a combination of two single-stranded target DNA segments from the N system as the preferred core nucleic acid element, structural changes or modifications to the two single-stranded target DNA segments can transform them into nucleic acid elements in a positive feedback signal amplification module. Structural changes to the 17nt 3' end include changing it to a completely double-stranded structure or simultaneously adding additional interfering bases (0-8nt in length, and the type of interfering base is not limited) to the 5' end of the double-stranded structure complementary to the crRNA strand. Structural alterations or modifications to the 7nt at the 5' end include: changing it to a double-stranded structure without modification and adding an additional interfering base (0-8nt in length, type of interfering base is not limited) to the 3' end of the complementary strand to the crRNA; or modifying the single strand using chemical modifications (e.g., locked nucleic acid and other modification types) and adding an additional interfering base (0-8nt in length, type of interfering base is not limited) to the 3' end of the complementary strand to the crRNA. In summary, structural modifications or alterations to the 17nt at the 3' end and the 7nt at the 5' end of the nucleic acid element core prevent Cas12a activation when either the individual nucleic acid element or both modified elements are present simultaneously. That is, Cas12a can only be activated when the two modified elements are specifically cleaved into the 17nt at the 3' end and the 7nt at the 5' end of the core.

[0172] In addition to the Hairpin and ss sequences of the N system selected in this invention (derived from...) Figure 16 Combination 2), such as Figure 16 As shown, combinations 4, 5, 6, 8, and 9 in system N also possess the controllable activation effect of Cas12a required by this invention; that is, the two single strands cannot be activated, but the combination can efficiently activate Cas12a. By modifying the two single strands according to the above structural changes and modifications, nucleic acid elements can also be constructed for use in positive feedback signal amplification modules. Furthermore, in system E of this invention, as... Figure 17As shown, combinations 2, 3, and 8 also possess the controllable activation effect of Cas12a required by this invention. Based on the above structural changes and modifications, nucleic acid elements can also be constructed for use in positive feedback signal amplification modules. And as... Figure 14 As shown, in the P system targeting As and FnCas12a enzymes, Figure 14 Combination 1 and in C Figure 14 Combinations 1, 2, and 3 in E also possess the effect of controllable activation of Cas12a required by this invention, in order to construct nucleic acid elements for use in positive feedback signal amplification modules.

[0173] Example 6: Performance Analysis of the Gene Detection Method Constructed in This Invention

[0174] This embodiment uses the N gene of SARS-CoV-2 as the detection target, designs a set of target genes and crRNA systems and constructs a plasmid of the N gene to verify the feasibility of the constructed gene detection method.

[0175] In this embodiment, the SARS-CoV-2N gene plasmid was diluted to eight gradient concentrations: 10 fM, 50 fM, 100 fM, 500 fM, 2 pM, 10 pM, 40 pM, and 100 pM. The plasmids were then detected using the detection method constructed in this invention and the direct Cas12a detection method, respectively. Figure 21 As shown in Figure A, the detection method constructed in this invention can detect N gene plasmids with concentrations as low as 50 fM within 1 hour, and the fluorescence intensity signals between plasmids of different concentrations are clearly distinguishable, indicating that the cascade signal amplification method constructed in this invention has a clear and resolving response to target genes of different concentrations. The fluorescence signal produced by the 10 fM N gene plasmid cannot be distinguished from the background. This is because the method itself has a strong exponential signal amplification effect, which leads to the amplification of the background signal after a period of accumulation. In contrast, the direct detection method of Cas12a takes nearly 1.5 hours to distinguish the 40 pM N gene plasmid from the background. Figure 21 B) The detection method constructed in this invention shortens the detection time while improving the detection sensitivity of target genes by three orders of magnitude compared with the direct detection method.

[0176] from Figure 21 As shown in Figure A, with the extension of detection time, N gene plasmids with concentrations from 10 pM to 10 fM sequentially enter the exponential growth region of the fluorescence signal. Within this time period, the differentiation effect for quantitative quantification of the target gene concentration is relatively good. Therefore, this embodiment selects detection results at different time points within this period for analysis to further determine the optimal detection time and the shortest detection time for this method. Figure 22As shown in Figure A, at detection time points of 35-60 min, the concentration of the N gene plasmid and the fluorescence value can be well fitted to the standard dose-response curve, indicating that the gene detection method established in this invention can respond rapidly (within 35 min) to different concentrations of the target gene. This is achieved by logarithmically processing the concentration of the N gene plasmid and linearly fitting it with the fluorescence γ signal value. Figure 22 B) It was found that as the detection time increased, the R-value of the linear fit decreased. 2 The value increased from 0.87 (35 min) to 0.96 (50 min), indicating that the response between the N gene plasmid concentration and the fluorescence signal reached its optimal level at 50 min. Analysis... Figure 21 Based on the background fluorescence signal of the blank group in A at 50 min, the detection limit of this method can be calculated to be approximately 25 fM.

[0177] This embodiment further evaluates the specificity of the method. First, a series of single point mutations of the SARS-CoV-2N gene were designed and synthesized. Figure 23 A) and double mutation at two sites ( Figure 23 C) Double-stranded DNA. To better evaluate the ability of this method to distinguish base differences before and after mutation, inter-purine and inter-pyrimidine mutations were selected, namely, mutations between adenine (A) and guanine (G), and mutations between thymine (T) and cytosine (C) (as shown in Table 8). Figure 23 As shown, when Cas12a was activated using 100pM of different single-mutated and double-mutated double-stranded target DNA of the SARS-CoV-2N gene instead of wild-type (WT) target DNA, the activation efficiency was significantly lower than that of WT. The activation efficiency of single mutations varied from 4.2 to 21.1 times, while the activation efficiency of double mutations varied from a minimum of 4.3 to a maximum of 30 times. Furthermore, from... Figure 23 As can be seen from B and 23D, the efficiency decreases more significantly when the mutation occurs in the double-stranded PAM sequence. This is consistent with previous reports, as the PAM region is more critical for the recognition and activation of the crRNA-Cas12a complex for double-stranded target DNA. Therefore, this method demonstrates good specificity for both single and double mutations in the target gene, proving its feasibility for detecting mutant genes.

[0178] To further analyze the specificity of this method in distinguishing different coronaviruses in practical applications, this embodiment selected the N gene plasmids of six known human-infecting coronaviruses and the E gene plasmid of SARS-CoV-2 (SARS-CoV-2-E) as interference to evaluate the method's resistance to common contamination encountered in practical applications. These six coronaviruses include human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome-associated coronavirus Tor2 (SARS-CoV-Tor2). Figure 24 As shown in Figure A, by comparing these six interfering plasmid sequences with the target sequence of SARS-CoV-2-N designed in this study, it is demonstrated that the crRNA designed in this invention has strong specificity. The detection results are as follows: Figure 24 As shown in B, when both 5 pM of the interfering plasmid (a concentration 100 times higher than the SARS-CoV-2-N gene target plasmid) and 50 fM of the SARS-CoV-2-N plasmid are detected simultaneously ( Figure 24 B) It can be seen that the fluorescence value produced by the 50 fM concentration of SARS-CoV-2-N plasmid is much higher than that of other interference groups, and the fluorescence value of the interference groups is at the same level as the background signal. This indicates that the method has high specificity, not only distinguishing between single and double mutations of the target DNA, but also excluding interference from other viral genes present in the environment in potential practical applications. The above experimental results prove that the gene detection method constructed in this invention has advantages such as high sensitivity, rapid detection, and high specificity. Table 11 lists the analytical performance of this method and other Cas12a-based amplification-free nucleic acid detection methods reported in the literature. In this invention, the gene detection method constructed by combining locked nucleic acid modification and two single-stranded target DNA segments can rapidly detect target genes with concentrations as low as 25 fM in only 50 minutes, which has certain advantages in sensitivity and detection time compared with other Cas12a-based electrochemical methods. In addition, the gene detection method constructed in this invention adopts a "one-tube" reaction method, which significantly improves the ease of operation. Furthermore, the nucleic acid element for controlling the Cas12a enzyme activity switch designed in this invention can be applied to various detection strategies based on Cas12a trans enzyme activity, as well as to develop methods suitable for the detection of various target genes, thus further demonstrating the strong versatility of this method.

[0179] Table 11 Comparison of the gene detection method constructed in this invention with existing Cas12a-based amplification-free nucleic acid detection methods. Note: HRP, horseradish peroxidase; MB, methylene blue; Fluo, fluorescence; colorimetry, colorimetric method.

[0180] Example 7: Signal amplification technology based on CRISPR-Cas12a autocatalytic cycle for breast cancer detection

[0181] cfDNA is a short, double-stranded DNA molecule, approximately 50-200 bp in size, originating from processes such as cell metastasis and apoptosis and secreted into body fluids (including blood, saliva, tears, and urine) via exocytosis. During tumor development, the rapid apoptosis and necrosis of tumor cells, or their active release, leads to an increase in cfDNA levels in the blood, and the cfDNA level in the blood of cancer patients is significantly correlated with the stage of tumor development. Therefore, the level of cfDNA in the blood has become a promising biomarker of great interest in the early diagnosis and prognostic monitoring of tumors. Breast cancer gene 1 (BRCA-1) and breast cancer gene 2 (BRCA-2) are two common tumor suppressor genes, with BRCA-1 being a representative cfDNA-related tumor marker that has been extensively studied. These two tumor suppressor genes encode BRCA1 and BRCA2, two DNA repair proteins responsible for repairing chromosomal damage. Although BRCA-1 and BRCA-2 are expressed in various endocrine tissues, their expression levels are higher in the breast and thymus, and lower in the lung, ovary, and spleen. It has been reported that over 400 mutations have been identified in the BRCA-1 and BRCA-2 genes, and a large number of mutations may still remain undiscovered. Compared to individuals carrying normal BRCA-1 and BRCA-2 genes, individuals carrying BRCA mutations often have a significantly increased risk of developing breast cancer. Therefore, accurately assessing BRCA-1 levels in bodily fluids is crucial for the early diagnosis of breast cancer. However, due to the extremely low concentration and short half-life of cfDNA in bodily fluids (for example, in blood, the concentration is typically below 2 μg / mL, with a half-life between 16 minutes and 2.5 hours), the sensitivity, specificity, and timeliness of detection methods are extremely important, with specificity being key to detecting BRCA-1-related mutations in breast cancer.

[0182] In summary, BRCA-1 mutations are significantly associated with the risk of breast cancer. Therefore, methods for breast cancer diagnosis based on BRCA-1 detection must be specific enough to accurately detect at least single-base mutations. In the preceding embodiments of this invention, a nucleic acid detection technology based on the CRISPR-Cas12a autocatalytic cycle was established. The detection results using the SARS-CoV-2 N gene plasmid not only validated the method's femtomolar sensitivity but also demonstrated its ability to complete detection in a short time (less than 1 hour). More importantly, specificity was further evaluated using single and double-base mutations in the SARS-CoV-2 N gene and different coronavirus plasmids. Therefore, the method established in this invention is well-suited for the detection of BRCA-1 in blood.

[0183] First, the detection performance of the normal BRCA-1 gene fragment was characterized using CRISPR-Cas12a autocatalytic cycling nucleic acid detection technology. The principle of BRCA-1 detection is as follows: Figure 25 As shown in Figure A, a specifically designed crRNA recognizes the BRCA-1 gene in the system, thereby activating the trans enzyme activity of Cas12a. The free reporter-labeled LNA-ss4 in the system is cleaved into ss0 by the directional cleavage of Cas12a trans enzyme activity. ss0 combines with another nucleic acid element in the system, Hairpin 0, which reactivates Cas12a. This positive feedback effect leads to the activation of a large number of Cas12a in the system, which cleaves LNA-ss4 to produce a significant fluorescent signal, thus enabling the detection of the target gene. However, when the BRCA-1 gene is absent or a mutant of the BRCA-1 gene is present in the system, it cannot be recognized by the specific crRNA, therefore the downstream circulation system cannot be activated, and no fluorescent signal is produced.

[0184] Characterization results as follows Figure 25 B- Figure 25As shown in Figure C, BRCA-1 was diluted to five concentration gradients using 1xPBS or 5% human serum: 10 fM, 100 fM, 1 pM, 10 pM, and 100 pM. It can be seen that, in different buffer environments, the method constructed in this invention consistently yielded a good response relationship between the fluorescence readout signal and the initial concentration of BRCA-1, with correlations greater than 0.9 in both PBS and serum. This indicates that the method constructed in this invention can achieve femtomolar-level detection of BRCA-1 in complex serum environments. Comparing the detection sensitivity (lower limit of standard curve 50 fM, LOD 25 fM) of the SARS-CoV-2 N gene using the same method, it was found that the sensitivity for BRCA-1 detection can be further improved to a lower limit of standard curve of 10 fM and an LOD of 4.7 fM. This suggests that the sensitivity of this invention varies to some extent due to differences in the activation efficiency of different target genes and crRNAs; higher efficiency leads to higher sensitivity, while lower efficiency affects sensitivity. The detection strategy based on the Cas12a autocatalytic cycle established in this invention is an amplification-free method. Utilizing a relatively simple one-pot detection system, it can rapidly achieve femtomolar level detection sensitivity in a short time (55 minutes for BRCA-1 detection) and is also applicable to complex serum environments, demonstrating promising clinical application prospects.

[0185] To evaluate the specificity of this method for detecting the BRCA-1 gene, seven ssDNAs with expression levels directly associated with breast cancer in the blood were used for testing, including the breast cancer gene 2 BRCA-2, miRNA-21, miRNA-let7a, miRNA-141, miRNA-let7d, miRNA-7e, and miRNA-122. The results are as follows: Figure 26 As shown, when using interfering ssDNA (10 pM) at a concentration 100 times higher than that of the experimental group BRCA-1 (100 fM), the fluorescence readout signal of the interfering group was at the same level as the blank control, far lower than that of the experimental group. Since the source of initiating the CRISPR-Cas12a autocatalytic cycle in this method lies in the specific recognition of BRCA-1 crRNA, this demonstrates that the BRCA-1 gene crRNA designed in this invention has strong recognition specificity, further illustrating that this method possesses sufficient specificity in detecting BRCA-1.

[0186] To evaluate BRCA-1-based detection for breast cancer detection, this invention selected three tumor cell lines: human embryonic kidney cells (HEK293T), cervical cancer cells (HeLa), and breast cancer cells (MCF-7). Using the established BRCA-1 detection method based on the CRISPR-Cas12a autocatalytic cycle, the BRCA-1 secreted by these three cell lines was detected. Figure 27 A). Because BRCA-1 exists in a mutated form in breast cancer cell lines but in a normal form in other cell lines, BRCA-1 detection results can be used to determine different tumor cell types and their corresponding proliferation levels. Results are as follows... Figure 27 As shown, this method can be used to extract cells from HEK293T cells (…). Figure 27 B) and HeLa cells ( Figure 27 BRCA-1 was successfully detected in C), and there was a significant correlation between different numbers of cells and the detected fluorescence signal (P<0.01). However, for MCF-7 cells, such as Figure 27 As shown in D, the fluorescence signal did not increase significantly with the increase in cell number, and the fluorescence signal was also much lower than that of the other two cell types with the same cell number. Figure 27 E). In summary, it can be seen that by using the CRISPR-Cas12a autocatalytic cycle method, different tumor cells can be identified by detecting BRCA-1 released from different tumor cells.

[0187] Summarize:

[0188] This invention systematically investigated the effect of single-stranded target DNA of different lengths on the activation of Cas12a activity when the length of crRNA was fixed. It was found that as the length of the single-stranded target DNA shortened, its activation effect on Cas12a gradually weakened and eventually disappeared. Unexpectedly, it was also found that when another portion of the truncated single-stranded target DNA was reintroduced into the system (e.g., 24 nt of wild-type target DNA, a shorter 5' to 3' 7 nt single-stranded target DNA, and the remaining 5' to 3' 17 nt portion), Cas12a was reactivated. This invention verified the aforementioned two-segment activation characteristics in Lb-type, As-type, and Fn-type Cas12a using three different crRNAs and target DNAs. Based on similar structures, Cas12a proteins such as HkCas12a, OsCas12a, TsCas12a, BbCas12a, and BoCas12a are also within the scope of protection of this invention. Besides the activation of Cas12a by double-stranded single-stranded target DNA, double-stranded double-stranded target DNA can also activate Cas12a. This invention has two key findings: first, it discovers that different lengths of ssDNA have varying activating Cas12atrans enzyme activity; second, it reveals that in experiments with truncated ssDNA, by restoring the missing ssDNA segments, the combination of two single-stranded target DNA segments can reactivate Cas12a.

[0189] The method for activating the Cas12a protein provided by this invention can actually be applied to various Cas12a proteins, including Cas12a, Cas12i, Cas12h, Cas12c, Cas12f2, and Cas12f3. According to the current CRISPR protein classification, Cas12a belongs to a class II system of single-effect Cas proteins, and is further subdivided into a fifth class based on sequence similarity. This fifth class includes a series of Cas12(ak) proteins as well as Cas14a, Cas14b, and Cas14c proteins; among them, except for Cas12g, which is an RNA editing enzyme, the rest are DNA editing enzymes. In this fifth class, proteins with structural similarities to Cas12a include Cas12a, Cas12i, Cas12h, Cas12c, Cas12f2, and Cas12f3. All of the above proteins contain consecutive RuvC I, RuvC II, and RuvC III domains (distinguishing them from cas12e, cas12b, etc.), and do not require trans-activation of crRNA (i.e., tracrRNA) to function. These six cas12 enzymes belong to the same taxonomic subtype and have similar structural compositions; therefore, they possess functions similar to cas12a and are suitable for the activation method and autocatalytic amplification detection method involved in this invention.

[0190] In this invention, locked nucleic acid (LNA) modified double-segment single-stranded target DNA was mainly used to perform controlled activation experiments of Cas12a and the amplification effect was verified. Other chemical modifications suitable for the method of this invention include methoxy modification, thio modification, bridged nucleic acid (BNA) modification, 2'-fluoroRNA modification, 2'-aminoRNA modification, morpholine nucleic acid modification, ethylene glycol nucleic acid (GNA) modification, hexitol nucleic acid (HNA) modification, and threonine nucleic acid (TNA) modification.

[0191] Combining the controllable activation effect of the dual-segment single-stranded target DNA on Cas12a and the site-specific cleavage of Cas12a induced by locked nucleic acid modification, this invention constructs an amplification-free signal amplification method for rapid and highly sensitive nucleic acid detection. Based on the above embodiments, a new gene detection method is constructed based on the site-specific cleavage regulated by locked nucleic acids and the activation of Cas12a by two-segment single-stranded target DNA. In this detection method, the modification of locked nucleic acids causes the activated Cas12a to site-specifically cleave the straight-stranded ss4 to generate ss0, which, after combining with Hairpin0, reactivates Cas12a, forming an autocatalytic effect. Through positive feedback, a cascade amplification effect of the signal is ultimately achieved. This invention further evaluates the analytical performance of this method using the N gene of SARS-CoV-2, finding that the signal cascade amplification method constructed in this invention has an fluorescence signal amplification efficiency nearly 1000 times compared to direct Cas12a detection, with an experimental sensitivity of 50 fM and a detection limit of 25 fM. In subsequent embodiments of this invention, a series of artificially constructed target DNAs with single and double site mutations and N gene plasmids from six common coronavirus-like organisms were used to systematically analyze the specificity of the method. The results showed that the method constructed in this study possesses excellent detection specificity. Furthermore, this invention applied the established gene detection method to the rapid detection of breast cancer-related cfDNA (55 minutes), achieving an experimental sensitivity of 10 fM and a detection limit of 4.7 fM. Based on the excellent specificity of this method, the detection of breast cancer was ultimately achieved by detecting cfDNA secreted by different tumor cells. By applying the method established in this invention to detect cfDNA secreted by different tumor cells, and ultimately achieving rapid detection of the breast cancer cell line MCF-7, a new potential clinical application scheme for the early diagnosis of breast cancer is provided.

[0192] The gene detection method established in this invention demonstrates advantages in terms of detection time, detection efficiency, ease of operation, and specificity in specificity verification and comparison with other Cas12a-based amplification-free nucleic acid detection methods. Furthermore, the method established in this invention, based on the activation of Cas12a through a combination of two target strands to initiate an autocatalytic effect, can not only be used to develop different gene detection methods, but is also applicable to the intrinsic design principles of biological logic circuits.

[0193] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for enabling a Cas protein containing a RuvC domain to cleave specific phosphodiester bonds in a single-stranded nucleic acid at a specific site, characterized in that, The method includes: chemically modifying the nucleotides in the unit to be cleaved of the single-stranded nucleic acid, wherein the single-stranded nucleic acid includes at least one unit to be cleaved, the unit to be cleaved is a continuous nucleotide sequence of 9 nucleotides in length, the chemically modified nucleotides include a first nucleotide, a fourth nucleotide, and a ninth nucleotide in the 5' to 3' direction of the unit to be cleaved, and the phosphodiester bond is a phosphodiester bond between the seventh nucleotide and the eighth nucleotide in the 5' to 3' direction of the unit to be cleaved; the chemical modification is a locked nucleic acid modification; and the Cas protein containing the RuvC domain is a Cas12a protein.

2. The method as described in claim 1, characterized in that, The Cas12a protein includes one or more of LbCas12a, FnCas12a, and AsCas12a.

3. A method for detecting target DNA in a sample for non-disease diagnostic purposes, characterized in that, The method includes: S1. The sample to be tested is added to a detection system, which includes Cas12 protein, a first guide RNA, a second guide RNA, a first nucleic acid element, a second nucleic acid element, and a buffer solution; the first guide RNA includes a targeting sequence capable of specifically binding to the target DNA; the first nucleic acid element includes a first binding region and a structural region, and the first nucleic acid element includes a hairpin structure; the second nucleic acid element includes a second binding region and a signal region, and the second nucleic acid element is chemically modified according to the method described in claim 1, such that the phosphodiester bond connecting the second binding region and the signal region in the second nucleic acid element is cleaved; the sample to be tested includes the target DNA sequence; the Cas12 protein is Cas12a; S2 Under the guidance of the first guide RNA, the Cas12 protein specifically binds to the target DNA and cleaves the phosphodiester bond, so that the second nucleic acid element is cleaved into the second binding region and the signal region and generates a detectable signal; S3 The second bonding region combines with the first bonding region to form a combined activation element; S4 Under the guidance of the second guide RNA, the Cas12 protein specifically binds to the combined activation element and cleaves the second nucleic acid element, forming a positive feedback loop system, which again causes the second nucleic acid element to be cleaved into the second binding region and the signal region and generates the detectable signal; S5 detects the detectable signal to obtain the presence and / or content of the target DNA.

4. The method as described in claim 3, characterized in that, The nucleotide sequence of the first nucleic acid element is SEQ ID NO: 139; the nucleotide sequence of the second nucleic acid element is GaaCgcttAtt, where uppercase letters indicate transnucleotides. The nucleotides chemically modified according to the method of claim 1, wherein lowercase letters represent wild-type nucleotides; the nucleotide sequence of the second guide RNA is SEQ ID NO:

1.

5. The method as described in claim 3, characterized in that, The second nucleic acid element is modified at its end with a fluorescent group and / or a quenching group, and the detectable signal includes a fluorescent signal.

6. The method as described in claim 3, characterized in that, The Cas12a includes one or more of LbCas12a, FnCas12a and AsCas12a.

7. The method as described in claim 3, characterized in that, The method further includes a step of plotting a standard curve, which includes: adding positive standards of different concentration gradients to the detection system for reaction and detecting the detectable signal, and plotting the standard curve based on the detectable signal values ​​corresponding to the positive standards of different concentration gradients; the positive standards include the target DNA sequence.

8. The method as described in claim 3, characterized in that, The combined length of the first binding region and the second binding region is 24-28 nucleotides.

9. A reagent kit for nucleic acid detection, characterized in that, The kit includes: (a) A first nucleic acid element, the first nucleic acid element including a first binding region and a structural region, the first nucleic acid element including a hairpin structure; (b) A second nucleic acid element, the second nucleic acid element comprising a second binding region and a signaling region, the second nucleic acid element being chemically modified according to the method of claim 1 such that the phosphodiester bond connecting the second binding region and the signaling region in the second nucleic acid element can be cleaved at specific sites by the Cas12 protein; the first binding region and the second binding region can form a combined activation element; (c) A second guide RNA, which guides the Cas12 protein to bind specifically to the combined activation element; the Cas12 protein is Cas12a.

10. The kit according to claim 9, characterized in that, The kit also includes the Cas12 protein, a first guide RNA, and a buffer; the first guide RNA includes a targeting sequence capable of specifically binding to the target DNA; the Cas12a includes one or more of LbCas12a, FnCas12a, and AsCas12a.

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