Substrate dna for crispr-cas12a system, and application and universal gene mutation detection method thereof

Abstract

The application belongs to the technical field of gene detection, and particularly relates to a substrate DNA for a CRISPR-Cas12a system, application of the substrate DNA and a universal gene mutation detection method. The substrate DNA comprises a complementary auxiliary strand and a target strand, the length of the auxiliary strand is less than that of the target strand, so that a sticky end is formed at the 5' end of the target strand, the end of the sticky end close to the auxiliary strand comprises a starting region complementary to the gRNA, and the starting region comprises 1-8 nucleotides. Through the foregoing arrangement, the CRISPR-Cas12a system has unique PAM independence for the novel substrate of the application. The novel structure and unique property expand the sequence universality of the CRISPR-Cas12a system from isolated ssDNA to partial dsDNA, and have hypersensitivity to base mismatches in the substrate DNA, so that the specificity of single-base difference recognition is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to substrate DNA for the CRISPR-Cas12a system, its applications, and a universal gene mutation detection method. Background Technology

[0002] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system provides bacteria with a prokaryotic adaptive immune system that uses RNA-guided nucleases to target and degrade foreign nucleic acids. CRISPR-Cas12a belongs to the second family of Cas enzymes. The Cas12a nuclease (also known as Cpf1 nuclease) recognizes target DNA on the opposite strand of the pre-interstitial motif (PAM) that is complementary to the spacer region of crRNA (also known as gRNA). Upon recognition, it creates staggered double-strand breaks in the RuvC and Nuc domains. This process involves two types of cleavage: cis-cleavage, where crRNA pairs with the target DNA (single-stranded or double-stranded DNA), activating the cleavage activity of Cas12a; and trans-cleavage, where activated Cas12a can also non-specifically cleave any single-stranded DNA (ssDNA) in the surrounding solution. Based on this characteristic, CRISPR-Cas12a, as a next-generation gene editing technology, has shown great promise in the fields of nucleic acid detection and biosensing. The trans-cleavage activity of the Cas12a nuclease provides a novel signal amplification mode. After the Cas12a / gRNA complex binds to the target DNA, the Cas12a nuclease activates its trans-cleavage activity, thereby continuously cleaving abundant fluorescent ssDNA probes in solution to achieve signal amplification. This method offers advantages such as low cost, ease of operation, simplicity, speed, and high signal-to-noise ratio.

[0003] The powerful signal amplification capabilities of the CRISPR-Cas12a system result in extremely high sensitivity for target DNA recognition. However, in addition to sensitivity, universality and specificity are also crucial for the CRISPR-Cas12a system. Sensitivity refers to the minimum number of targets that can be recognized. In practical applications, target DNA sequences may vary; therefore, the capabilities of the CRISPR-Cas12a system should be generalized to any sequence of interest. Furthermore, target DNA is not isolated but is immersed in a large number of interfering sequences; therefore, the specificity of the CRISPR-Cas12a system is equally important for distinguishing target DNA from interfering sequences. However, the working mechanism of the CRISPR-Cas12a system described above indicates that its universality is limited to target ssDNA; for target dsDNA, it needs to recognize PAM sequences to bind to the target dsDNA. Therefore, in CRISPR-Cas12a-based detection systems, researchers typically need to set the target strand to ssDNA; if the target strand must be dsDNA, it needs to be digested into single strands through enzymatic methods. This pervasive limitation not only sacrifices targeting range or procedural convenience but also introduces additional challenges to the specificity of the CRISPR-Cas12a system. Generally, an important criterion for evaluating the specificity of a detection system is its ability to distinguish interfering strands with a single base difference. Although CRISPR-Cas12a can identify single-base mismatches in dsDNA by finely adjusting mismatch sites within the seed domain (i.e., the 1-6 base sequences following the PAM sequence), the discrimination factor (DF) is not ideal, only 2 to 5 times. In the fields of nucleic acid detection and biosensing, detecting disease-related gene mutations has enormous clinical significance and a wide range of applications, especially the ability to distinguish single-base mismatches, which is crucial for detecting point mutations, particularly those related to cancer. However, the abundance of point mutations is very low (0.01%-10%) in tumor fluid and early tumor tissue biopsies. Therefore, this places high demands on the ability of gene detection systems to distinguish single-base mismatches. However, current research shows that CRISPR-Cas12a's ability to recognize single-base mismatches in target DNA is far from meeting the detection needs. In conclusion, there is an urgent need for CRISPR-Cas12a-based detection systems to expand their versatility and / or significantly improve their specificity while maintaining their high sensitivity. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a substrate DNA for the CRISPR-Cas12a system, and provides the application of this substrate DNA in the fields of gene editing or gene detection, as well as a universal gene mutation detection method based on this substrate DNA, so as to solve or at least partially solve the technical problems of poor universality and / or low specificity of existing CRISPR-Cas12a-based detection systems.

[0005] A first aspect of the present invention provides a substrate DNA for a CRISPR-Cas12a system, comprising a complementary auxiliary strand and a target strand, wherein the length of the auxiliary strand is shorter than the length of the target strand such that the 5' end of the target strand forms a sticky end, wherein the sticky end near one end of the auxiliary strand contains a start region that is complementary to gRNA, the start region comprising 1-8 nt of nucleotides.

[0006] Furthermore, the start region comprises 3-7 nt of nucleotides.

[0007] Furthermore, the start region comprises 5 nt of nucleotides.

[0008] Furthermore, the target chain also includes a branch migration domain comprising 12-19 nt of nucleotides, and the 5' end of the branch migration domain is connected to the 3' end of the start region, forming a complete gRNA binding region.

[0009] Furthermore, the sum of the bases in the branch migration domain and the starting region is 20 nt.

[0010] A second aspect of the invention provides the application of substrate DNA for the CRISPR-Cas12a system as described above in the fields of gene editing or gene detection.

[0011] Furthermore, the gene detection field is the field of gene mutation detection.

[0012] A third aspect of the present invention provides a universal method for detecting gene mutations, comprising the following steps:

[0013] S1. Prepare substrate DNA for the CRISPR-Cas12a system as described above, wherein the substrate DNA includes wild-type target strand and / or mutant target strand;

[0014] S2. The substrate DNA is added to the CRISPR-Cas12a system and incubated at low temperature. Then, a single-stranded DNA fluorescent probe is added to form the reaction system to be tested, and the fluorescence intensity is recorded at room temperature. In the CRISPR-Cas12a system, the gRNA forms a one-base mismatch with the mutant target strand and a two-base mismatch with the wild-type target strand.

[0015] Further, in step S1, the method for preparing the substrate DNA for the CRISPR-Cas12a system includes the following steps:

[0016] S11. Amplify the target gene containing the target chain in the sample to be tested;

[0017] S12. Add an auxiliary strand to the target gene solution, denature at high temperature to allow the target strand and the auxiliary strand to bind complementaryly, and then anneal at low temperature to obtain substrate DNA; wherein the molar concentration of the auxiliary strand is greater than the molar concentration of the target gene.

[0018] Further, in step S12, the molar concentration ratio of the auxiliary strand to the molar concentration of the target gene is 10:1.

[0019] Furthermore, in step S2, the base mismatch between the gRNA and the mutant target strand is located at nucleotides 1-19 of the branch migration domain.

[0020] Furthermore, the base mismatch between the gRNA and the mutant target strand is located at nucleotides 1-10 of the branch migration domain; more preferably at nucleotide 7 of the branch migration domain.

[0021] The advantages and positive effects of this invention are as follows:

[0022] 1. Through the aforementioned settings, the CRISPR-Cas12a system has unique PAM independence for the novel substrates of this invention. This novel structure and unique properties extend the sequence universality of the CRISPR-Cas12a system from isolated ssDNA to some dsDNA, and it is hypersensitive to base mismatches within the substrate DNA, which greatly improves its specificity for recognizing single base differences.

[0023] 2. This invention is based on the CRISPR-Cas12a system's hypersensitivity to base mismatches in substrate DNA. By setting a gRNA targeting a known mutation and ensuring that the gRNA has one base mismatch with the known mutated nucleotide sequence and two base mismatches with the wild-type target gene, a significant difference in fluorescence signal will appear when the sample to be tested has or does not contain the mutant target chain. By comparing the difference in fluorescence signal, the presence or absence of the mutant target chain can be determined. The detection limit for genomic DNA is 0.05% to 0.01%, which has great advantages in the detection of multiple gene mutations and has broad prospects for clinical applications. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0025] Figure 1 This is a schematic diagram illustrating the principle of the CRISPR-Cas12a system recognizing substrate DNA in an embodiment of the present invention.

[0026] Figure 2 This is a diagram showing the cleavage efficiency of the CRISPR-Cas12a system after recognizing substrate DNA in an embodiment of the present invention.

[0027] Figure 3 This is a graph showing the effect of the start region length on the cleavage efficiency of the CRISPR-Cas12a system after recognizing substrate DNA in an embodiment of the present invention.

[0028] Figure 4 This is a graph showing the cleavage efficiency of the CRISPR-Cas12a system in recognizing seven random substrate DNAs in an embodiment of the present invention.

[0029] Figure 5 This is a schematic diagram illustrating the principle of single-base mismatch position counting in an embodiment of the present invention;

[0030] Figure 6 This is a diagram showing the effect of the start region length on the specificity of the CRISPR-Cas12a system in this embodiment of the invention;

[0031] Figure 7 This is a diagram showing the effect of single-base mismatch positions on the specificity of the CRISPR-Cas12a system in the embodiments of the present invention;

[0032] Figure 8 This is a schematic diagram illustrating the design principle of substrate DNA and gRNA base mismatch in embodiments of the present invention;

[0033] Figure 9 This is a diagram showing the ability of the CRISPR-Cas12a system to distinguish between single and double base mismatches in substrate DNA in an embodiment of the present invention.

[0034] Figure 10 This refers to the detection limit of the CRISPR-Cas12a system for single base mismatches of substrate DNA in this embodiment of the invention.

[0035] Figure 11 This is a schematic diagram illustrating the principle of complementary binding between the auxiliary strand and the target strand to form substrate DNA in this invention.

[0036] Figure 12 This is a diagram illustrating the effect of the CRISPR-Cas12a system in distinguishing single-base mismatches of substrate DNA with specific point mutations in an embodiment of the present invention.

[0037] Figure 13This is the detection limit of the CRISPR-Cas12a system for single base mismatches in substrate DNA with specific point mutations in this embodiment of the invention;

[0038] Figure 14 This is a diagram illustrating the effect of the CRISPR-Cas12a system in distinguishing single-base mismatches of plasmid substrates with specific point mutations in an embodiment of the present invention.

[0039] Figure 15 This refers to the detection limit of the CRISPR-Cas12a system for single base mismatches of plasmid substrates with specific point mutations in the embodiments of the present invention;

[0040] Figure 16 This is a flowchart illustrating the gene mutation detection method in an embodiment of the present invention.

[0041] Figure 17 This is a diagram showing the discrimination effect of the CRISPR-Cas12a system on the EGFR T790M plasmid substrate in an embodiment of the present invention;

[0042] Figure 18 This represents the detection limit of the CRISPR-Cas12a system for plasmid substrates with specific point mutations in this embodiment of the invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained commercially.

[0044] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0045] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0046] This invention provides a substrate DNA for a CRISPR-Cas12a system, comprising a complementary helper strand (also called a helper strand) and a target strand. The length of the helper strand is shorter than the length of the target strand so that the 5' end of the target strand forms a sticky end. The sticky end near the end of the helper strand contains a start region that is complementary to gRNA. The start region comprises 1-8 nt of nucleotides, i.e., the start region is sequence complementary to the gRNA, for initiating further invasion of the double-stranded DNA (dsDNA) of the substrate by the gRNA.

[0047] In the context of this invention, the CRISPR-Cas12a system includes a Cas12a nuclease and a gRNA responsible for DNA targeting. The gRNA is used to target the target strand and bind to the target strand through complementary base pairing, thereby anchoring the Cas12a nuclease to the substrate DNA and activating the activity of the Cas12a nuclease.

[0048] This invention achieves its goal by making the length of the auxiliary strand shorter than the length of the target strand. This allows for the formation of a prominent end structure, or sticky end, upstream of the target strand (i.e., at the 5' end) after the two strands bind via complementary base pairing. This results in dsDNA with a sticky-end region, thus yielding substrate DNA suitable for the CRISPR-Cas12a system. See also... Figure 1 This invention incorporates an initiation region in the sticky end of the substrate DNA that is complementary to the gRNA in the CRISPR-Cas12a system. This initiation region is immediately adjacent to the region where the target strand and the helper strand bind (i.e., the double-stranded DNA (dsDNA) structural region of the substrate DNA, with the 3' end of the sticky end connected to the double-stranded DNA structural region of the target strand). Thus, when the gRNA binds to the target strand, the gRNA first recognizes and binds to the initiation region. Then, the gRNA is initiated to further invade the dsDNA region of the substrate. After invasion, the gRNA and the substrate DNA activating the Cas12a nuclease form a triple-lobe structure. Subsequently, the Cas12a nuclease breaks the substrate DNA on both strands.

[0049] For ease of description, the dsDNA region on the target strand that binds complementary to the gRNA is called the branching migration domain. Figure 1 (As shown by the red line in the middle), and the 5' end of the branch migration domain is connected to the 3' end of the initiation region, forming a complete gRNA binding region to anchor gRNA and Cas12a nuclease to the substrate DNA.

[0050] Through the aforementioned configuration, this invention enables gRNA in the CRISPR-Cas12a system to rapidly bind to the sticky-terminated dsDNA (hereinafter referred to as PAM) even without the presence of a PAM sequence. - SE + The gRNA is placed on dsDNA and rapidly activates the Cas12a nuclease, enabling it to recognize and cleave the dsDNA region. Experiments have verified that the gRNA recognizes PAM. - SE + The efficiency of dsDNA is comparable to that of classic substrates (crude-terminated dsDNA containing PAM sequences). In other words, the CRISPR-Cas12a system exhibits unique PAM independence for the novel substrates of this invention. This novel structure and unique properties extend the sequence universality of the CRISPR-Cas12a system from isolated ssDNA to partial dsDNA. Moreover, it is hypersensitive to base mismatches within the substrate DNA. When there is one or more base mismatches between the gRNA and the target strand of the substrate DNA, it will adversely affect the cleavage activity of Cas12a, resulting in a significant difference in fluorescence signal compared to a perfectly matched (no base mismatch) case. This will greatly improve the specificity for recognizing single-base differences, which is extremely beneficial for the application of the CRISPR-Cas12a system.

[0051] In-depth research into the impact of start region length on the recognition efficiency and specificity of the CRISPR-Cas12a system revealed that recognition efficiency gradually increases with the increase of the start region, reaching saturation at 5 nt. Thereafter, recognition efficiency decreases with further increases in the start region length. Furthermore, when the start region length is in the range of 3-7 nt, the CRISPR-Cas12a system exhibits improved performance in PAM recognition. - SE + dsDNA exhibits higher specificity than classical substrates. This is because the CRISPR-Cas12a system is specific to the PAM of this invention. - SE + Base mismatches within the branching migration domain of dsDNA exhibit hypersensitivity; when a base of gRNA is artificially altered to match that of PAM... - SE + The target strand of dsDNA forms a single base mismatch, which interacts with the interfering PAM. - SE + When dsDNA forms two consecutive single-base mismatches, the CRISPR-Cas12a system only recognizes a single-base mismatch PAM. - SE +In dsDNA, when two consecutive single-base mismatches occur, the Cas12a nuclease is essentially inactive, resulting in a greater difference in fluorescence signal rates between the two conditions, allowing for clear differentiation and rapid identification of single-base mismatches. Preferably, the start region comprises 3-7 nt nucleotides, more preferably, it comprises 5 nt nucleotides.

[0052] Optionally, the branch migration domain comprises 12-19 nt of nucleotides.

[0053] Optionally, the sum of the bases in the branch migration domain and the starting region is 20 nt.

[0054] Another embodiment of the present invention provides an application of substrate DNA for the CRISPR-Cas12a system in the fields of gene editing or gene detection.

[0055] For example, when applied in the field of gene detection, such as for the detection of various pathogenic microorganisms, the target gene is prepared into substrate DNA as described above. Then, a CRISPR-Cas12a system targeting the target gene and a non-specific single-stranded DNA fluorescent probe are added. The single-stranded DNA fluorescent probe uses a dual-labeled fluorescent probe with a fluorescent substance (e.g., FAM) at the 5' end and a quencher substance (e.g., BHQ1) at the 3' end. When the probe is intact, the fluorescent substance at the 5' end is inhibited by the quencher substance at the 3' end and cannot emit fluorescence. However, when the dual-labeled fluorescent probe is decomposed, the fluorescent substance at the 5' end is released and emits fluorescence. Therefore, once the target gene is detected, the CRISPR-Cas12a system will initiate and activate the trans-cleavage activity of the Cas12a nuclease. Simultaneously, the fluorescent probe is degraded, releasing a fluorescent signal. By detecting the presence and intensity of the fluorescent signal, it is possible to determine whether the target gene is present in the sample, achieving rapid and highly sensitive detection.

[0056] The advantages of the substrate DNA used in the CRISPR-Cas12a system in gene editing or gene detection compared to existing technologies are the same as those of the substrate DNA used in the CRISPR-Cas12a system compared to existing technologies, and will not be repeated here.

[0057] More importantly, this invention is based on the hypersensitivity of the CRISPR-Cas12a system to the aforementioned substrate DNA base mismatches. Specifically, the trans-cleavage activity of the CRISPR-Cas12a system decreases in gradients when the gRNA and target strand are perfectly matched, when there is one base mismatch, and when there is more than one base mismatch. In particular, the difference in enzyme activity is very significant in the latter two cases, thus enabling its application in the field of gene mutation detection.

[0058] Another embodiment of the present invention provides a universal gene mutation detection method, comprising the following steps:

[0059] S1. Prepare substrate DNA for the CRISPR-Cas12a system as described above, wherein the substrate DNA includes wild-type target strand and / or mutant target strand;

[0060] S2. The substrate DNA is added to the CRISPR-Cas12a system and incubated at low temperature. Then, a single-stranded DNA fluorescent probe is added to form the reaction system to be tested, and the fluorescence intensity is recorded at room temperature. In the CRISPR-Cas12a system, the gRNA forms a one-base mismatch with the mutant target strand and a two-base mismatch with the wild-type target strand.

[0061] In this embodiment, a gRNA targeting a known mutation is configured, ensuring that the gRNA has a one-base mismatch with the known mutated nucleotide sequence and a two-base mismatch with the wild-type target gene. Thus, when the sample to be tested contains the mutant target strand, the gRNA specifically binds to the mutant target strand, activating the cleavage activity of the Cas12a nuclease. The activated Cas12a nuclease can cleave the single-stranded DNA fluorescent probe and release the fluorescent group from the probe, generating a strong fluorescent signal. Conversely, when there is no mutant target strand, the cleavage activity of the Cas12a nuclease is not activated or only minimally activated, resulting in a significantly reduced fluorescent signal. By comparing the differences in fluorescence signals, the presence of the mutant target strand can be determined. In other words, statistical analysis is performed based on the rate of fluorescence signal generation with and without the target gene; a p-value < 0.05 is considered significant, indicating the presence of a single-base mismatch, i.e., a point mutation in the sample to be tested.

[0062] In step S1, the method for preparing the substrate DNA for the CRISPR-Cas12a system includes the following steps:

[0063] S11. Amplify the target gene containing the target chain in the sample to be tested;

[0064] S12. Add an auxiliary strand to the target gene solution, denature at high temperature to allow the target strand and the auxiliary strand to bind complementaryly, and then anneal at low temperature to obtain substrate DNA; wherein the molar concentration of the auxiliary strand is greater than the molar concentration of the target gene.

[0065] In step S11, the method for amplifying the target gene containing the target chain in the sample to be tested includes, but is not limited to, conventional PCR technology, recombinase polymerase amplification (RPA), rolling circle amplification (RCA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP). Any method that can obtain the target gene is acceptable, and this invention does not limit the method.

[0066] In step S12, the molar concentration ratio of the helper strand to the target gene is 10:1. The helper strand, due to its high concentration, is able to hybridize with its target strand.

[0067] In step S2, both high-temperature denaturation and low-temperature annealing are conventional methods in the art, and the present invention does not limit them. For example, the conditions for high-temperature denaturation are 95°C for 10 min, and the conditions for low-temperature annealing are 0°C for 5 min.

[0068] To further improve specificity, preferably, the base mismatch between the gRNA and the mutant target strand is located at nucleotides 1-19 of the branch migration domain. Exemplarily, it can be located at nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or other nucleotide positions. Preferably, it is located at nucleotides 1-9 of the branch migration domain, more preferably at nucleotide 7.

[0069] In single-stranded DNA fluorescent probes, the fluorescent group and its quenching group should effectively overlap to achieve high-efficiency FRET. Optionally, the fluorescent group and its quenching group should be selected from at least one of the following groups: FAM and TAMRA, FAM and BHQ1, and TET and BHQ2. The length of single-stranded DNA fluorescent probes is generally 8-15 nucleotide residues.

[0070] Example 1: Preparation of Substrate DNA and Study on its Properties

[0071] A method for preparing substrate DNA for the CRISPR-Cas12a system includes the following steps:

[0072] S11. Amplify the target gene containing the target chain in the sample to be tested using RPA technology. Specific operations include: […]. Add the target chain to the RPA reaction tube of the kit, then add 2 μM of the corresponding amplification primer (initial concentration is 10 μM), then cap the tube, centrifuge at 1000 rpm, and then react at 37℃ for 10 min to obtain the target gene solution containing the target chain, and adjust the initial concentration to 100 nM.

[0073] S12. Take 10 μL of the target gene solution containing the target strand from step S11, add 10 times the amount of Helper strand (concentration 10 μM), denature at 95°C for 10 min to allow the target strand and helper strand to bind complementaryly, then anneal on ice (0°C) for 5 min to obtain substrate DNA (i.e., PAM). - SE + dsDNA).

[0074] The target chain was artificially synthesized and adjusted according to the experimental purpose, and then diluted with TE buffer to form a liquid with a concentration of 0.5 μM.

[0075] like Figure 1 As shown, PAM - SE + The sticky end region of dsDNA is an elongated overhang, within which is designed an initiation region complementary to the short domain of gRNA, thereby initiating further invasion of the substrate dsDNA by the gRNA. Following the initiation region is a branching migration domain, which, together with the initiation region, constitutes the gRNA-binding region. Upon gRNA invasion, the gRNA forms a triple-lobed structure with the substrate DNA activating Cas12a, subsequently breaking PAM on both strands. - SE + dsDNA.

[0076] To delve deeper into PAM - SE + Based on the properties of dsDNA, the following detection system was constructed:

[0077] 1) Single-stranded DNA fluorescent probe solution: Dilute the single-stranded DNA fluorescent probe powder with TE buffer to prepare a liquid, and then use a Nanodrop instrument to determine the concentration to be 5 μM.

[0078] 2) Preparation of the CRISPR-Cas12a system: Dilute Cas12a (Cpf1) nuclease at a concentration of 2000 pmol 100-fold with ultrapure water and set aside. Then prepare 25 μL of Cas12a / gRNA mixed reaction solution: 2.8 μL of 2.1 μM buffer, 2.2 μL of Cas12a nuclease buffer, 0.94 μL of gRNA (1 μM), and 19.06 μL of ultrapure water and set aside.

[0079] 3) Detection reaction system: 5 μL 2.1 buffer, 5 μL Cas12a / gRNA mixed reaction solution, 20 μL PAM - SE + dsDNA solution, 2 μL MgSO4 (10 mM), 2 μL single-stranded DNA fluorescent probe solution, 16 μL ultrapure water.

[0080] The detection method includes the following steps:

[0081] S1. Prepare the substrate DNA solution. The sequence of the substrate DNA should be adjusted according to experimental requirements.

[0082] S2. Add the substrate DNA solution to the Cas12a / gRNA mixed reaction solution, incubate on ice for 10 min, then add the single-stranded DNA fluorescent probe solution to form the reaction system to be tested, and record the fluorescence generation rate at room temperature.

[0083] By comparing ssDNA, blunt-terminated dsDNA containing PAM sequences, and the derivation pattern of ssDNA (ssDNA / blocker) under different reaction systems (except for the substrate DNA, all other conditions were the same as the detection system described above), PAM... - SE + The efficiency of dsDNA in activating CRISPR-Cas12a when used as substrate DNA is shown in the experimental results. Figure 2 The vertical axis represents the normalized fluorescence intensity, and the horizontal axis represents the detection time. Figure 2 The experimental results show that the PAM of the present invention - SE + dsDNA successfully activated CRISPR-Cas12a, and its cleavage reaction plateaued within 10 minutes, indicating that CRISPR-Cas12a is effective against PAM. - SE + The recognition efficiency of dsDNA is comparable to that of dsDNA substrates containing PAM.

[0084] Subsequently, the impact of the starting region length (1nt, 2nt, 3nt, 4nt, 5nt, and 8nt, respectively) on recognition efficiency was further investigated. Figure 3 As shown, recognition efficiency gradually increases with the expansion of the start region, reaching a near maximum at 5 nt. Notably, the substrate DNA is quite long; therefore, gRNA cannot displace the short strand of dsDNA, indicating that CRISPR-Cas12a cannot recognize PAM via the ssDNA-derived pattern (ssDNA / blocker). - SE + Instead of identifying dsDNA, it recognizes it as a true double-stranded substrate.

[0085] To further investigate the effects of CRISPR-Cas12a on different PAMs - SE + Based on the common recognition characteristics of dsDNA, this invention randomly synthesized 7 PAMs. - SE + Table 1 shows the dsDNA and its corresponding perfectly complementary gRNA sequences. Taking gRNA-1 as an example, the naming rules for the following sequences are as follows: G1-B indicates the strand recognized by CRISPR-Cas12a, the number 1 indicates the strand number, G1-PAM... + -T (forms blunt-terminated dsDNA with G1-B), G1-SE+ -T (forming sticky-terminated dsDNA with G1-B) indicates complementarity with the strand recognized by CRISPR-Cas12a, using these 7 randomly designed PAMs. - SE + Using dsDNA as the substrate and blunt-terminated dsDNA containing the PAM sequence as the control, the experimental results are shown in [Figure number missing]. Figure 4 Where the vertical axis represents PAM - SE + The ratio of the efficiency of dsDNA in activating CRISPR-Cas12a to the efficiency of blunt-terminated dsDNA containing the PAM sequence in activating CRISPR-Cas12a is shown on the x-axis, representing different gRNAs (corresponding to different substrate DNAs). Blue bars represent the efficiency of blunt-terminated dsDNA containing the PAM sequence in activating CRISPR-Cas12a, and red bars represent the efficiency of PAM activation. - SE + The efficiency of dsDNA in activating CRISPR-Cas12a, with the smaller numbers representing the ratio of the latter to the former. This can be seen in the seven randomly designed PAMs. - SE + The activation efficiency of dsDNA in the CRISPR-Cas12a system is quite fast, indicating that the activation of CRISPR-Cas12a by PAM-SE+dsDNA is universal, no longer dependent on the PAM sequence, and is comparable to the activation efficiency of the CRISPR-Cas12a system by dsDNA containing PAM.

[0086] Table 1 gRNA-1 to 7 and their corresponding substrate DNA sequences

[0087]

[0088]

[0089] Note: Underlined regions indicate branch migration regions, shading indicates starting regions, and the direction of all sequences is from 5' to 3'.

[0090] Based on the above data, it can be concluded that this invention discovers a novel substrate DNA suitable for the CRISPR-Cas12a system—dsDNA with sticky ends—which possesses unique PAM independence. Typically, CRISPR-Cas12a requires a PAM sequence to recognize double strands; otherwise, it cannot recognize them. Therefore, the activation mechanism of CRISPR-Cas12a in dsDNA is limited by PAM. This novel structure and unique property of the present invention extends the sequence universality of the CRISPR-Cas12a system from isolated ssDNA to partial dsDNA, which is extremely beneficial for the understanding and application of the CRISPR-Cas12a system.

[0091] To investigate the effect of the CRISPR-Cas12a system on the PAM of this invention - SE + To demonstrate the specificity of dsDNA, using gRNA-1 as a model, eight groups of PAMs with initial lengths ranging from 1 nt to 8 nt were synthesized. - SE + dsDNA, for each PAM group - SE + dsDNA was designed to employ a fully complementary binding target type (target strands with perfectly complementary pairing) and a single-base-mismatch interference type (target strands with a single base mismatch). The interference type forms a base mismatch with gRNA-1 at position 9 (counting from the 5' end to the 3' end, starting from the start region; for example, when the start region length is 1, position 9 is the 8th position in the branching migration domain; when the start region length is 3, position 9 is the 6th position in the branching migration domain, and so on). Figure 5 An example of the counting rule when the starting region length is 5nt is given; experimental results are shown below. Figure 6 Where the vertical axis represents the effect of CRISPR-Cas12a on PAM - SE + The ability of dsDNA to distinguish single-base mismatches is defined by the resolution (DF), which is the ratio of the fluorescence rate of a perfectly matched target strand to the fluorescence rate of a target strand with a single-base mismatch. The horizontal axis represents the mismatch site. As shown in Figure 6, when the length of the start region is in the range of 3-7 nt, CRISPR-Cas12a effectively targets PAM. - SE + dsDNA exhibits higher specificity than classical substrates, with a maximum DF of 8.28 at a start region length of 5 nt.

[0092] The start region was fixed at 5nt, and the mismatch position was adjusted from 1 to 15 (counting from the 5' end to the 3' end, starting from the start region), using gRNA-1, gRNA-2, and gRNA-3 models respectively. Figure 7 The experimental results show that CRISPR-Cas12a has minimal discrimination ability for base mismatches located in the start domain (positions 1 and 3); however, for mismatches located in the branching migration domain (positions 6, 9, 12, and 15, corresponding to amino acids 1, 4, 7, and 10 of the branching migration domain), its specificity significantly increases from 8.2 to 11.3 (median 8.6), nearly 4 times higher than that of classical substrates. Overall, CRISPR Cas12a shows good discrimination ability for PAM. - SE + Mismatches within the branching migration domain of dsDNA are highly sensitive.

[0093] To further enhance the specificity of the CRISPR-Cas12a system, such as Figure 8 As shown, a single base of the gRNA is altered to make it bind to the target PAM. - SE + dsDNA forms a base mismatch, interfering with PAM - SE + The dsDNA exhibited two consecutive single-base mismatches, the corresponding sequence information of which is shown in Table 2. The results are as follows: Figure 9 As shown, the horizontal axis represents the location of mismatched bases, which also corresponds to the interference PAM. - SE + The position of the wavy line in the dsDNA sequence. As can be seen from the figure, DF (discrimination factor) significantly increases, reaching 30.9-631.5 (median 44); where DF is the ratio of the fluorescence rate generated by a single base mismatch to the fluorescence rate generated by two base mismatches. Subsequently, interference PAM was used. - SE + dsDNA to dilute targeted PAM - SE + dsDNA was used to prepare a series of mixed samples at a concentration of 0.5 μM, with target strand ranges of 100%, 10%, 1%, 0.1%, and 0%, such as... Figure 10 As shown, the detection limit for low-abundance target chains was reduced to 0.5%-0.1%.

[0094] Table 2 gRNA-8 to 19 and their corresponding substrate DNA sequences

[0095]

[0096]

[0097]

[0098] Note: In gRNA sequences, underlines (straight lines) indicate the target sequence, and underlines (wavy lines) indicate additional single-base mismatches. In other sequences, underlines (straight lines) represent branching migration domains, shading represents initiation regions, and underlines (wavy lines) indicate single-base mismatches. Sequences with wavy lines are interfering PAMs. - SE + dsDNA, sequences without wavy lines indicating mismatch are targeting PAM. - SE + dsDNA.

[0099] Based on the above data, it can be seen that the CRISPR-Cas12a system is highly sensitive to base mismatches in the substrate DNA of this invention, and the effect on nuclease activity varies completely depending on the location and number of mismatches, thus enabling its application in the field of gene detection.

[0100] Example 2: Study on gene mutation detection

[0101] Gene mutation detection methods include the following steps:

[0102] S1, Preparation of PAM - SE + dsDNA solution, PAM - SE + dsDNA includes wild-type target strands and / or mutant target strands;

[0103] S2, PAM - SE + dsDNA solution was added to the Cas12a / gRNA mixed reaction solution and incubated on ice for 10 min. Then, single-stranded DNA fluorescent probe solution was added to form the reaction system to be tested, and the fluorescence generation rate was recorded at room temperature.

[0104] Utilizing all the properties of the newly discovered substrate DNA, a promising detection modality is proposed: such as Figure 11 As shown, ssDNA is used as the target, and a helper chain is introduced. The helper chain is designed to be complementary to the wild-type ssDNA target chain (WT) and to have single-base mismatches with the mutant ssDNA target chain. The sequence and length of the helper chain are adjusted to form a PAM with either MT or WT. - SE + dsDNA. Subsequently, a "two mismatches, one mismatch" strategy was adopted, where gRNA forms a two-base mismatch with WT and only a one-base mismatch with MT. Using gRNA-1, gRNA-2, and gRNA-3 as models, the single-base mismatch sites were located at positions 6 and 12, with a start region length of 5 nt. Results are as follows... Figure 12 As shown, the vertical axis represents the discrimination index, and the horizontal axis represents the position of the mismatched bases. It can be seen that DF increased to 155-1411 (median 207). Subsequently, the WT target chain was used to dilute the MT target chain, preparing a series of mixed samples at a concentration of 0.5 μM, so that the mutation abundance ranged from 100%, 10%, 1%, 0.1%, 0.05%, 0.01%, 0.001%, to 0%, as shown below. Figure 13 As shown, the detection limit for low-abundance target chains was reduced to 0.0005%. This represents an extremely high level of specificity and functionality, far exceeding the analytical capabilities of current CRISPR-Cas12a-based systems.

[0105] All the above experiments were performed on clinically irrelevant random strands. To evaluate the generality and clinical potential of this method, the system was applied to six clinically significant point mutations: EGFR T790M, EGFR S768I, TP53R248W, PARP1, BARF V600E, and PIK3CA E545D, and the corresponding gRNA, MT target strand, WT target strand, and helper strand were synthesized (sequences are shown in Table 3). Figure 14 As shown, the vertical axis represents the resolution (DF), which is the ratio of the fluorescence rate produced by the MT target chain to that produced by the WT target chain. The horizontal axis represents the point mutation type, with the DF range for the tested mutations from 154 to 446 (median 207). A series of mixed samples at a concentration of 0.5 μM were prepared by diluting the MT target chain using the WT target chain, resulting in mutation abundances ranging from 100%, 10%, 1%, 0.1%, 0.05%, 0.01%, 0.001%, to 0%. Figure 15 As shown, the vertical axis represents fluorescence intensity, and the horizontal axis represents detection time. The detection limit for low-abundance point mutations is 0.01% to 0.005%. The performance matches the results of the random chain described above, demonstrating the versatility, specificity, and clinical potential of the detection method of this invention.

[0106] Table 3. gRNA and substrate DNA sequences corresponding to typical point mutations.

[0107]

[0108]

[0109] In clinical practice, the CRISPR-Cas12a system is typically coupled with the preceding PCR amplification process. The PCR product, whether from traditional hot PCR or the subsequently developed isothermal PCR, is blunt-terminated dsDNA. Therefore, converting the blunt-terminated dsDNA product into ssDNA is a complex process. For a long time, a simple and robust method for converting dsDNA into ssDNA has been a key requirement in the field of mutation detection, especially in post-PCR methods. In this invention, based on another novel characteristic of the CRISPR-Cas12a system—recognition and activation at low temperatures (approximately 0°C) and coupling with unique DNA hybridization kinetics during denaturation and rapid annealing—a very convenient conversion strategy for double-stranded DNA substrates in the CRISPR-Cas12a system has been developed. The workflow is as follows: Figure 16As shown. After conventional hot PCR or isothermal RPA, the double-stranded PCR product and a 10-fold excess of Helper strand were heated to 95°C and incubated for 10 min. Immediately afterwards, the reaction tube was placed on ice and incubated for 5 min, allowing the Helper strand to hybridize with its target strand due to its high concentration. Next, the CRISPR Cas12a system was added and incubated on ice for 10 min. Finally, a single-stranded DNA fluorescent probe was added, and the reaction system was immediately placed in a microplate reader for fluorescence measurement at 37°C. For example, using EGFR T790M as the target mutation, mutant and wild-type plasmids and primers were synthesized. After RPA amplification, the following was performed... Figure 16 The workflow shown is for genetic testing, and the results are available in [link to documentation]. Figure 17 The vertical axis represents the discrimination index, and the horizontal axis represents the CRISPR-Cas12a system incubated on ice for 10, 5, and 0 min, corresponding to groups 1, 2, and 3, respectively. The mutant plasmid produced a strong fluorescent signal, with a DF reaching 257, demonstrating that the system of this invention is compatible with PCR amplification using a simple heating and annealing procedure.

[0110] Subsequently, the aforementioned method was used to detect clinically significant low-abundance point mutations in plasmid samples. Mutant plasmids and wild-type plasmids, including EGFR T790M, EGFR S768I, TP53 R248W, PARP1, BARF V600E, and PIK3CA E545D, as well as primers for other mutations, were synthesized. The mutant plasmids were diluted using wild-type plasmids to achieve mutation abundance ranges of 100%, 10%, 1%, 0.1%, 0.05%, 0.01%, 0.001%, and 0%. Figure 18 As shown in the figure, the vertical axis represents fluorescence intensity and the horizontal axis represents detection time. Figure af corresponds to the point mutations of EGFR T790M, EGFR S768I, TP53 R248W, PARP1, BARFV600E and PIK3CA E545D, respectively. The detection limit for the six tested targeted mutations is 0.05%-0.01%, which proves the function of the detection method of the present invention on genomic samples.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A substrate DNA for the CRISPR-Cas12a system, comprising a complementary auxiliary strand and a target strand, characterized in that, The length of the auxiliary strand is less than the length of the target strand so that the 5' end of the target strand forms a sticky end, and the sticky end near the end of the auxiliary strand contains a start region that binds complementary to the gRNA, the start region comprising 3-7 nt nucleotides; The target strand also includes a branch migration domain comprising 12-19 nt of nucleotides, and the 5' end of the branch migration domain is connected to the 3' end of the start region, the branch migration domain and the start region forming a complete gRNA binding region.

2. The substrate DNA for the CRISPR-Cas12a system according to claim 1, characterized in that, The start region comprises 5 nt of nucleotides.

3. The substrate DNA for the CRISPR-Cas12a system according to claim 1, characterized in that, The sum of the bases in the branch migration domain and the starting region is 20 nt.

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