Bubble type single-stranded blocking nucleic acid for CRISPR system and application thereof

Abstract

The invention belongs to the technical field of gene detection. The invention provides a bubble-type single-stranded blocking nucleic acid BB for a CRISPR system, application of the bubble-type single-stranded blocking nucleic acid BB, and a universal gene mutation detection and nucleic acid modification detection method. The bubble type single-chain blocking nucleic acid BB and the CRISPR system based on the BB do not depend on PAM sites, are good in universality and have high sensitivity to single base mutation or nucleic acid modification. According to the bubble type single-stranded blocking nucleic acid BB, the recognition capability of a CRISPR system on single base mutation or nucleic acid modification in a nucleic acid substrate is improved.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and particularly to the application of a bubble-type single-strand blocking nucleic acid in the CRISPR system, as well as a method for universal gene mutation detection and nucleic acid modification detection. Background Art

[0002] The trans-cleavage property of the CRISPR system allows Cas nucleases such as Cas12a to non-specifically cleave surrounding ssDNA after binding to the target DNA, thus becoming a sensitive molecular detection tool.

[0003] However, the PAM site dependence limits the selection of its target sequences, restricts its universality, and affects the convenience and wide coverage of the application of the CRISPR system.

[0004] On the other hand, in clinical detection, identifying specific SNPs can be used to correct pathogenic mutations for disease prevention and treatment purposes, which poses high requirements for the SNP resolution ability of detection technologies. However, the recognition ability of the CRISPR system for two consecutive base mutations in the 6 bases after the PAM sequence of the ssDNA target is 1 / 1000 of that in the process of dsDNA target recognition and activation, and its SNP resolution ability far fails to meet the needs of clinical detection.

[0005] Therefore, a detection technology that can ensure the convenience and wide coverage of the CRISPR system for ssDNA detection and at the same time improve its SNP resolution ability is urgently needed to be developed. Summary of the Invention

[0006] The present invention provides a bubble-type single-strand blocking nucleic acid BB for the CRISPR system, as well as a universal gene mutation or nucleic acid modification detection method based on the BB, and provides the application of the method in the fields of gene editing, gene detection, or nucleic acid modification detection.

[0007] In the first aspect of the present invention, there is provided a bubble-type single-strand blocking nucleic acid BB for detecting a target nucleic acid, characterized in that the bubble-type single-strand blocking nucleic acid BB comprises a switch region, a stable region, and a bubble region;

[0008] Wherein, the switch region comprises a nucleic acid sequence complementary to the target nucleic acid to-be-detected fragment, and base mutations and / or modifications of the target nucleic acid to-be-detected fragment cause a transition between the single-stranded state and the double-stranded state of the switch region;

[0009] The stable region forms a stable double strand with the target nucleic acid;

[0010] The bubble region is located between the switch region and the stable region, and the bubble region comprises suspended single strands that are not complementary to the target nucleic acid.

[0011] In another preferred example, the detected target nucleic acid includes: base mutations and / or modifications of the detected target nucleic acid.

[0012] In another preferred example, the target nucleic acid fragment to be detected refers to a nucleic acid fragment containing the base mutation and / or modification site to be detected.

[0013] In another preferred example, the base mutation and / or modification includes: base mutation, base modification, and / or binding of the base modification to its specific recognition protein or compound.

[0014] In another preferred example, the single-stranded state serves as a substrate for the CRISPR protein, activating the CRISPR protein, thereby generating a signal; the double-stranded state cannot serve as a substrate for the CRISPR protein, cannot activate the CRISPR protein, and cannot generate a signal.

[0015] In another preferred example, the target nucleic acid includes: single-stranded target nucleic acid or double-stranded target nucleic acid.

[0016] In another preferred example, the target nucleic acid includes: DNA or RNA.

[0017] In another preferred example, the target nucleic acid includes: wild-type target nucleic acid or mutant target nucleic acid.

[0018] In another preferred example, the target nucleic acid includes: wild-type target nucleic acid or modified nucleic acid.

[0019] In another preferred example, the mutant target nucleic acid includes a target nucleic acid with single-base mutation or multi-base mutation.

[0020] In another preferred example, the modified nucleic acid includes a target nucleic acid with single-base modification or multi-base modification.

[0021] In another preferred example, the modification includes: methylation modification, hydroxymethylation modification, carbonylmethylation modification, thiolation modification, or a combination thereof.

[0022] In another preferred example, the modification includes: 5-methylcytidine (5mC), 5-hydroxymethylcytidine (5hmC), 5-formylcytidine (5fC), 5-carboxylcytidine (5caC), or N6-methyladenosine (6mA) modification.

[0023] In another preferred example, the modification includes: N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), N7-methylguanosine (m7G), N4-acetylcytidine (ac4C), pseudouridine (Ψ), or hypoxanthine modification.

[0024] In another preferred example, there is a specific binding protein for the modified nucleic acid, and the specific binding protein includes: an SRA protein domain specifically binding to 5-methylcytidine (5mC)-modified DNA, an SBD protein domain specifically binding to phosphorothioate-modified DNA, a YTH domain specifically binding to N6-methyladenosine (m6A)-modified RNA, an ALYREF domain specifically binding to 5-methylcytidine (m5C)-modified RNA, or a CBC domain specifically binding to N7-methylguanosine (m7G)-modified RNA.

[0025] In another preferred example, the structure of the bubble-type single-strand blocking nucleic acid BB includes: DNA, RNA, 2'-fluoro-modified nucleic acid (2'-FNA), 2'-O-methyl-modified nucleic acid (2'-OMe), 2'-O-methoxyethyl-modified nucleic acid (2'-O-MOE, 2-O-(2-methoxyethyl)), locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino nucleic acid (PMO).

[0026] In another preferred example, the base mutation is a single-base mutation.

[0027] In another preferred example, the base modification is a single-base modification.

[0028] In another preferred example, when the switch region of BB is completely complementary to the target sequence to be tested, BB and the target nucleic acid are in a double-stranded state, the target sequence cannot be used as a substrate for the CRISPR protein, the CRISPR protein cannot be activated, and no signal is generated;

[0029] When the switch region of BB is not completely complementary to the target sequence to be tested, BB and the target nucleic acid are in a single-stranded state, and the target sequence serves as a substrate for the CRISPR protein, activating the CRISPR protein and thus generating a signal.

[0030] In another preferred example, the bubble-type single-strand blocking nucleic acid BB contains a structure shown in Formula I or Formula II:

[0031] 5'-Z1-Z2-Z3-3' (I)

[0032] 5'-Z3-Z2-Z1-3' (II)

[0033] Wherein, Z1 is the switch region, and the switch region responds to the target nucleic acid fragment to be tested;

[0034] Z2 is the bubble region, and the bubble region contains suspended single strands;

[0035] Z3 is the stable region, and the stable region exists in the form of a double strand.

[0036] In another preferred example, the target nucleic acid comprises a structure shown in Formula III or Formula IV:

[0037] 5'-PAM region - spacer region - random sequence region - 3' (III)

[0038] 5’-random sequence region - spacer region - PAM region - 3’ (IV)

[0039] Among them, in the target nucleic acid comprising the structure shown in Formula III, the base numbering of the PAM region, spacer region or random sequence region starts from the 5' end, and the base number of the first base is at 1 nt, and the base numbering increases towards the 3' end;

[0040] In the target nucleic acid comprising the structure shown in Formula IV, the base numbering of the PAM region, spacer region or random sequence region starts from the 3' end, and the base number of the first base is at 1 nt, and the base numbering increases towards the 5' end.

[0041] In another preferred example, for Cas12-class and Cas13-class proteins, the structure of the bubble-type single-strand blocking nucleic acid BB is as shown in Formula I, and the structure of the target nucleic acid is as shown in Formula IV;

[0042] For Cas9-class proteins, the structure of the bubble-type single-strand blocking nucleic acid BB is as shown in Formula II, and the structure of the target nucleic acid is as shown in Formula III.

[0043] In another preferred example, the bubble-type single-strand blocking nucleic acid BB comprises a structure shown in Formula I or Formula II:

[0044] 5'-Z0-Z1-Z2-Z3-3' (I)

[0045] 5'-Z3-Z2-Z1-Z0-3' (II)

[0046] Among them, Z0 is the tail chain, and the tail chain is not complementary to the target nucleic acid,

[0047] Z1 is the switch region, and the switch region responds to the target nucleic acid fragment to be detected;

[0048] Z2 is the bubble region, and the bubble region contains suspended single strands;

[0049] Z3 is the stable region, and the stable region exists in the form of a double strand.

[0050] In another preferred example, the PAM region of the target nucleic acid does not contain a PAM sequence.

[0051] In another preferred example, the molar concentration ratio of the bubble-type single-stranded blocking nucleic acid BB to the target nucleic acid is 2:1 - 50:1, preferably 10:1 - 30:1, and most preferably 20:1.

[0052] In another preferred example, the length of the spacer region is 13 - 28 nt.

[0053] In another preferred example, the length of the spacer region is 18 nt.

[0054] In another preferred example, the spacer region contains base mutations and / or modification sites of the target sequence.

[0055] In another preferred example, the base mutation and / or modification site is located at the 1st nt, 2nd nt, 3rd nt, 4th nt, 5th nt, 6th nt of the spacer region, or a combination thereof.

[0056] In another preferred example, the single base mutation and / or modification corresponds to the 1st nt, 2nd nt, 3rd nt, 4th nt, 5th nt, or 6th nt of the spacer region of the target nucleic acid.

[0057] In another preferred example, the site of the single base mutation and / or modification is located at the 1st nt, 2nd nt, 3rd nt, or 4th nt of the spacer region of the target nucleic acid.

[0058] In another preferred example, the site of the single base mutation and / or modification of the target nucleic acid is located at the 4th nt of the spacer region of the target nucleic acid.

[0059] In another preferred example, the length of the switch region is 2 - 12 nt.

[0060] In another preferred example, the length of the switch region is 6 - 7 nt.

[0061] In another preferred example, the switch region is complementary to the 1st nt - (4 - 6th nt) of the spacer region of the target nucleic acid, preferably, complementary to the 1st nt - 6th nt of the spacer region of the single-stranded target nucleic acid.

[0062] In another preferred example, the switch region is complementary to the last base of the PAM region of the target nucleic acid.

[0063] In another preferred example, the total length of the switch region and the bubble region is 4 - 24 nt.

[0064] In another preferred example, the bubble region contains a suspension sequence that is not complementary to the target nucleic acid.

[0065] In another preferred example, the length of the suspension sequence is 2 - 22 nt.

[0066] In another preferred example, the bubble region contains a suspension sequence that is not complementary to the 7nt - 8nt position of the spacer region of the target nucleic acid.

[0067] In another preferred example, the length of the suspension sequence is 2nt.

[0068] In another preferred example, the length of the stable region is 6 - 65nt.

[0069] In another preferred example, the stable region is complementary to the (5 - 15nt position) of the spacer region of the target nucleic acid - the (1 - 50nt position) of the random sequence region.

[0070] In another preferred example, the length of the stable region is 23nt.

[0071] In another preferred example, the stable region is complementary to the 9nt position of the spacer region of the target nucleic acid - the 13nt position of the random sequence region.

[0072] In another preferred example, the switch region is complementary to the (last base of the PAM region) - the 6nt position of the spacer region of the target nucleic acid; the bubble region is not complementary to the 7nt - 8nt position of the spacer region of the target nucleic acid; the stable region is complementary to the 9nt position of the spacer region of the target nucleic acid - the 13nt position of the random sequence region.

[0073] In another preferred example, the length of the tail chain is 1 - 40nt.

[0074] In another preferred example, the length of the tail chain is 17nt.

[0075] In another preferred example, the switch region of BB can extend to the PAM region of the target nucleic acid.

[0076] In another preferred example, the spacer region does not contain target sequence base mutations and / or modification sites, and the single - base mutations and / or modifications occur in the sequence part of the target sequence that is complementary to the BB switch region.

[0077] In another preferred example, when the switch region of BB is completely complementary to the target nucleic acid to - be - detected fragment, BB blocks the target nucleic acid, and the target nucleic acid cannot serve as a substrate for the CRISPR protein, cannot activate the CRISPR protein, and cannot generate a signal;

[0078] When the switch region of BB is not completely complementary to the target nucleic acid to - be - detected fragment, BB releases the target nucleic acid, and the target nucleic acid serves as a substrate for the CRISPR protein, activates the CRISPR protein, and thus generates a signal.

[0079] In another preferred embodiment, the target nucleic acid fragment to be detected is the sequence corresponding to the switch region in the target nucleic acid.

[0080] In the second aspect of the present invention, a reagent combination is provided, which comprises: the bubble-type single-stranded blocking nucleic acid BB described in the first aspect of the present invention, and a crRNA complementary to the spacer region sequence of the target nucleic acid.

[0081] In another preferred embodiment, the crRNA is completely complementary to the spacer region of the target nucleic acid.

[0082] In the third aspect of the present invention, a CRISPR detection system for detecting a target nucleic acid is provided, which comprises:

[0083] the bubble-type single-stranded blocking nucleic acid BB described in the first aspect of the present invention;

[0084] a CRISPR protein;

[0085] a crRNA; and

[0086] an optional target nucleic acid.

[0087] In another preferred embodiment, the PAM region, spacer region and random sequence region are divided according to the gene mutation and / or modification site of the target nucleic acid, so that the gene mutation and / or modification site of the target nucleic acid is located between 1 nt and 6 nt of the spacer region.

[0088] In another preferred embodiment, the crRNA is complementary to the spacer region sequence of the optionally target nucleic acid.

[0089] In another preferred embodiment, when the switch region in BB is completely complementary to the sequence corresponding to the switch region in the target nucleic acid, the target nucleic acid cannot bind to the crRNA, the CRISPR protein is not activated, and the target nucleic acid cannot be cleaved by the CRISPR protein;

[0090] When the switch region is not completely complementary to the sequence corresponding to the switch region in the target nucleic acid, the target nucleic acid binds to the crRNA, the CRISPR protein is activated, and the target nucleic acid is cleaved by the CRISPR protein.

[0091] In another preferred embodiment, the CRISPR detection system further comprises a reporter molecule.

[0092] In another preferred embodiment, when the target sequence cannot be cleaved by the CRISPR protein, the trans-cleavage activity of the CRISPR protein is not activated, and the reporter molecule does not produce a signal; when the target sequence is cleaved by the CRISPR protein, the reporter molecule produces a signal.

[0093] In another preferred example, the signal is a fluorescence signal.

[0094] In another preferred example, the CRISPR protein includes: CRISPR / Cas12 type, CRISPR / Cas13 type, CRISPR / Cas9, TnpB type, IscB type, IsrB type.

[0095] In another preferred example, the CRISPR detection system detects the target nucleic acid independently of the PAM sequence of the target nucleic acid.

[0096] In the fourth aspect of the present invention, there is provided the use of the bubble-type blocking nucleic acid BB described in the first aspect of the present invention, the reagent combination described in the second aspect of the present invention, or the CRISPR detection system described in the third aspect of the present invention for preparing a kit for detecting a target nucleic acid.

[0097] In the fifth aspect of the present invention, there is provided a kit for detecting a target nucleic acid, which kit contains the bubble-type blocking nucleic acid BB described in the first aspect of the present invention, the reagent combination described in the second aspect of the present invention, or the CRISPR detection system described in the third aspect of the present invention.

[0098] In another preferred example, the kit further includes an instruction manual, and the instruction manual guides the use of the kit for detecting a target nucleic acid.

[0099] In the sixth aspect of the present invention, there is provided a method for detecting a target nucleic acid, including the following steps:

[0100] (M1) Provide the CRISPR detection system described in the third aspect of the present invention;

[0101] (M2) Detect the fluorescence signal of the reporter molecule in the CRISPR detection system, and give the detection result of the target nucleic acid according to the fluorescence signal.

[0102] In another preferred example, detecting the target nucleic acid means detecting whether there are base mutations and / or base modifications in the target nucleic acid.

[0103] In another preferred example, in step (M2), the following sub-steps are further included:

[0104] (M2-1) Premix the target nucleic acid and the bubble-type single-stranded blocking nucleic acid BB in the CRISPR detection system to form a substrate complex;

[0105] (M2-2) React the CRISPR protein, crRNA, and reporter molecule in the CRISPR detection system with the substrate complex, detect the fluorescence signal of the reporter molecule, and give the detection result of the target nucleic acid according to the fluorescence signal.

[0106] In a seventh aspect of the present invention, a method for detecting a target nucleic acid is provided, comprising the following steps:

[0107] (N1) Provide the bubble-type single-strand blocking nucleic acid BB as described in the first aspect of the present invention according to the wild-type target nucleic acid;

[0108] (N2) In the presence of the target nucleic acid to be detected, provide a substrate complex Y1 of the target nucleic acid to be detected and the bubble-type single-strand blocking nucleic acid BB;

[0109] (N3) Detect the single-stranded and double-stranded properties of the target nucleic acid in the substrate complex; if the target nucleic acid to be detected in Y1 is single-stranded, it indicates that the target nucleic acid to be detected has a base mutation and / or modification.

[0110] In another preferred example, the detection method comprises the following steps:

[0111] (N1') Provide the bubble-type single-strand blocking nucleic acid BB' as described in the first aspect of the present invention according to the mutant and / or modified target nucleic acid;

[0112] (N2') In the presence of the target nucleic acid to be detected, provide a substrate complex Y2 of the target nucleic acid to be detected and the bubble-type single-strand blocking nucleic acid BB';

[0113] (N3') Detect the single-stranded and double-stranded properties of the target nucleic acid in the substrate complex; if the target nucleic acid to be detected in Y2 is double-stranded, it indicates that the target nucleic acid to be detected has a base mutation and / or modification.

[0114] In another preferred example, in step (N2) or step (N2'), the following sub-steps are further included:

[0115] (N2-1) Amplify the dsDNA of the target gene in the sample to be detected;

[0116] (N2-2) Unwind the dsDNA to provide the target nucleic acid to be detected;

[0117] (N2-3) Co-incubate the target nucleic acid to be detected with the bubble-type single-strand blocking nucleic acid as described in the first aspect of the present invention to provide a substrate complex of the target nucleic acid and the bubble-type single-strand nucleic acid BB.

[0118] In another preferred example, in step (N3) or step (N3'), the detection includes detection based on the CRISPR system.

[0119] In another preferred embodiment, the CRISPR system includes: a protein having trans-cleavage activity; the protein includes but is not limited to: CRISPR / Cas12 family, CRISPR / Cas13 family, CRISPR / Cas9, TnpB family, IscB family, IsrB family.

[0120] In another preferred embodiment, the CRISPR system includes: crRNA or sgRNA, a Cas nuclease, a single-stranded nucleic acid fluorescent probe, and a buffer system.

[0121] In another preferred embodiment, the single-stranded or double-stranded nature of the single-stranded target nucleic acid in the detection substrate complex refers to: whether the single-stranded target nucleic acid in the detection substrate complex is cleaved by the CRISPR system. If it is cleaved by the CRISPR system, it indicates that the single-stranded target nucleic acid is single-stranded; if it is not cleaved by the CRISPR system, it indicates that the single-stranded target nucleic acid is double-stranded.

[0122] In another preferred embodiment, after the single-stranded target nucleic acid is cleaved by the CRISPR system, a signal is released; the double-stranded target nucleic acid is not cleaved by the CRISPR system and no signal is released.

[0123] In another preferred embodiment, the signal is a fluorescent signal.

[0124] In another preferred embodiment, the detection is qualitative detection and / or quantitative detection.

[0125] In another preferred embodiment, the CRISPR system includes: a nucleic acid modification-specific binding protein.

[0126] In another preferred embodiment, the nucleic acid modification-specific binding protein binds to the site of nucleic acid modification, such as Figure 4 shown.

[0127] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0128] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] In order to more clearly illustrate the technical solutions in the preferred embodiments of the present invention, the following briefly introduces the drawings required for the description of the embodiments. It should be understood that the application of the present invention is not limited to the specific implementation schemes shown in the drawings of the present invention.

[0130] Figure 1Shows a schematic diagram of base numbering of the CRISPR / Cas class.

[0131] Figure 2 Shows a schematic diagram of the BB design of the CRISPR / Cas class.

[0132] Figure 3 Shows the schematic principle of BB for detecting single-base mutations on nucleic acids.

[0133] Figure 4 Shows the schematic principle of BB for detecting modified bases on nucleic acids.

[0134] Figure 5 Shows a schematic diagram of designing ssDNA to simulate the activation / off state of CRISPR Substrate.

[0135] Figure 6 Shows a cutting efficiency graph for studying the effect of the stable region of BB.

[0136] Figure 7 Shows a cutting efficiency graph for studying the effects of the bubble region and switch region of BB.

[0137] Figure 8 Shows a comparative cutting efficiency graph for verifying the ability of BB to distinguish multiple single-base mutations in the EGFR sequence.

[0138] Figure 9 Shows a comparative cutting efficiency graph for verifying the ability of BB to distinguish single-base mutations in sequences such as MYO7A.

[0139] Figure 10 Shows a schematic diagram of adenosine deaminase TadA 8.20 processing RNA.

[0140] Figure 11 Shows the study of BB's ability to detect RNA m 6 A modification at multiple sites such as MALAT1 2515, with a comparative cutting efficiency graph for verification.

[0141] Figure 12 Shows the study of BB's ability to detect RNA m 6 A modification at the MALAT1 2515 site, with a standard curve graph for quantitative detection.

[0142] Figure 13 Shows the study of BB's ability to quantitatively detect RNA m 6 A modification at the MALAT1 2515 site in RNA samples from different sources, with a fluorescence curve graph for quantitative detection.

[0143] Figure 14 Shows a comparative cutting efficiency graph for verifying BB's ability to detect DNA 5mC modification at multiple sites such as OCT2. Detailed implementation manners

[0144] After extensive and in-depth research, the present inventors unexpectedly discovered for the first time a bubble-type single-strand blocking nucleic acid BB for the CRISPR system. The bubble-type single-strand blocking nucleic acid BB has: a long region matching the target single-stranded nucleic acid (i.e., the stable region) to ensure specific binding of BB to the substrate single-stranded nucleic acid; a short region matching the target single-stranded nucleic acid (the switching region) to respond to mutations or modifications of the substrate single-stranded nucleic acid; and a bubble region separating the stable region from the switching region to ensure that a single-base mutation or modification can cause a structural transformation between single-stranded and double-stranded. Therefore, a general method for detecting gene mutations or nucleic acid modifications is provided based on the BB. The present invention has been completed on this basis.

[0145] Specifically, the present invention provides a bubble-type single-strand blocking nucleic acid BB for the CRISPR system, provides a general method for detecting gene mutations or nucleic acid modifications based on the BB, and provides the application of this method in the fields of gene editing, gene detection, or nucleic acid modification detection, so as to solve or at least partially solve the technical problems of low SNP resolution and / or poor generality of the CRISPR-based detection system in the prior art and the problem of inability to directly detect nucleic acid modifications.

[0146] The present invention utilizes the selectivity of the CRISPR system for single-stranded and double-stranded substrates when there is no PAM site in the detection substrate, provides a novel bubble-type single-strand blocking nucleic acid BB, and enables the complex formed after its binding to the target single-stranded nucleic acid to be detected to achieve a switch between single-stranded and double-stranded properties in the switching region. By setting the nucleotides at the complementary positions of the switching region and the mutation site, it is achieved that no mismatch is formed when binding to the wild-type nucleic acid target, the switching region is closed, and the substrate nucleic acid presents a double-stranded property. When binding to the mutant nucleic acid target, a mismatch is formed, the switching region is opened, and the substrate nucleic acid presents a single-stranded property. In this way, in the case where there is no PAM sequence in front of the CRISPR spacer of the target nucleic acid, BB cannot present a fluorescence signal when binding to the wild-type target nucleic acid, and BB can output a fluorescence signal when binding to the mutant target nucleic acid. Whether there is a mutant target nucleic acid in the detection system can be judged by the difference in fluorescence signals. The detection method based on BB provided by the present invention has great advantages in gene mutation detection and has broad application prospects. Similarly, in the case of adding a nucleic acid modification-specific binding protein to the reaction system, the protein can bind to the modified nucleotides in the substrate nucleic acid, thereby making the switching region in an open state, and further enabling the direct detection of nucleic acid modifications.

[0147] Terms

[0148] To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0149] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0150] As used herein, the terms "comprises," "comprising," "includes" may be used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0151] All numbers expressing amounts, percentages, and other numerical values used in this application should be understood to be modified in all instances by the word "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0152] Where a numerical range is provided, unless the context clearly dictates otherwise, it should be understood that each intermediate integer value, each tenth of each intermediate integer value, and any other intermediate value between the upper and lower limits of that range and within the stated range are included within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also covered by the invention, subject to any explicit exclusions set forth in the stated range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0153] As used herein, the terms "single-stranded state" and "single-stranded property" may be used interchangeably and both refer to the property or state of the structure that is similar to that of a single-stranded nucleotide during detection or response. In a specific embodiment, the single-stranded state or single-stranded property includes the form of a single nucleotide chain. In a specific embodiment, the structure being in a single-stranded state or having a single-stranded property does not mean that the structure is in the form of a single nucleotide chain.

[0154] As used herein, the terms "PAM region sequence" and "PAM sequence" may be used interchangeably and both refer to a sequence that has binding affinity for a Cas protein, enabling the Cas protein to rapidly and effectively bind to a DNA substrate containing the PAM sequence.

[0155] As used herein, the “PAM region” refers to the region where PAM sequences are frequently distributed. In a specific embodiment, the PAM region contains PAM sequences. In a specific embodiment, the PAM region does not contain PAM sequences.

[0156] CRISPR

[0157] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the accompanying Cas proteins constitute the adaptive CRISPR-Cas immune system in bacteria and archaea. This DNA-encoded, RNA-mediated defense system provides sequence-specific recognition, targeting, and degradation of foreign nucleic acids.

[0158] Cas nuclease is an RNA-mediated endonuclease and an important component of the CRISPR system found in certain bacteria and archaea. Cas nuclease binds to a class of guiding RNAs (called crRNA or CRISPR RNA, or sgRNA), which contain a constant region (CRISPR repeats) recognizable by Cas nuclease and a spacer region (CRISPR spacer) complementary to a specific DNA sequence. The spacer region is the CRISPR recognition sequence and targets it to a specific and programmable nucleic acid sequence. The Cas-crRNA complex cleaves the target DNA (or target RNA) by recognizing the protospacer adjacent motif (PAM), i.e., cis-cleavage, generating sticky ends or blunt-ended DNA double-strand breaks on the target DNA.

[0159] Some Cas nucleases, such as Cas12a (Cpf1), Cas13, Cas9, etc., while recognizing and cleaving the target DNA (or target RNA), the activated Cas-crRNA complex has trans-cleavage activity and non-specifically cleaves single-stranded DNA (or single-stranded RNA) free in the environment. Due to the signal amplification ability, simplicity, sensitivity, and specificity demonstrated by these Cas nucleases in trans-cleavage after activation, they have been widely used in the fields of biosensing and molecular detection and play an important role in pathogen diagnosis, nucleic acid detection, and other aspects.

[0160] The trans-cleavage property of the CRISPR system allows Cas12a and others to non-specifically cleave surrounding ssDNA after binding to the target DNA, thus becoming a sensitive molecular detection tool. However, for dsDNA targets, Cas12a needs to recognize a specific PAM sequence (usually TTTV, where V represents A, C, or G) to initiate editing. This PAM-site dependence limits the selection of its target sequences and restricts its versatility.

[0161] There are very few studies showing that for ssDNA targets, the binding and activation process of Cas12a can be independent of the PAM site. However, such ssDNA activation faces many challenges: for example, due to the diversity of ssDNA sequences, ssDNA activation may lead to higher background noise and Cas12a may be mis-activated.

[0162] In clinical detection, identifying specific SNPs can be used to correct pathogenic mutations for disease prevention and treatment purposes, which poses high requirements for the discrimination ability of detection technologies for SNPs. In the recognition and activation of ssDNA by Cas12a, the recognition ability for two consecutive base mutations in the 6 bases after the PAM sequence of the ssDNA target is 1 / 1000 of that in the recognition and activation process of dsDNA targets. For single-base mutations (Single Nucleotide Polymorphisms, SNPs), the discrimination factor (DF) shown by Cas12a in the recognition and activation of ssDNA is only 2 - 5 times.

[0163] In current research, the discrimination ability of the CRISPR system for SNPs on ssDNA targets far from meets the requirements of clinical detection, and the detection of dsDNA targets requires PAM sites, which forces researchers to abandon the wide coverage and convenience of the system.

[0164] Therefore, a detection technology that can ensure the convenience and wide coverage of the CRISPR system for ssDNA detection and at the same time improve its discrimination ability for SNPs is urgently needed to be developed.

[0165] The bubble-type single-strand blocking nucleic acid of the present invention

[0166] Aiming at the problems existing in the prior art, the present invention uses the method of forming a double-strand between a single-strand blocking nucleic acid and a target single-stranded nucleic acid, and uses the change of the double-strand caused by the presence of mutations or modifications in the target single-stranded nucleic acid sequence for differential detection. However, if the formed double-strand is relatively long (for example, more than 15 bp), it often will not unwind into single-stranded nucleic acids due to the change of one or several bases; if the formed double-strand is too short, it does not have sufficient specificity, is prone to mismatch, and has insufficient stability.

[0167] Therefore, the present invention creatively introduces mismatched bubble regions into single-stranded blocking nucleic acids, creating bubble-type single-stranded blocking nucleic acid BB. A stable region formed by a long matching region ensures the specific binding of BB to the substrate single-stranded nucleic acid, and a short matching region forms a switch region to respond to mutations or modifications of the substrate single-stranded nucleic acid. The bubble region separates the stable region from the switch region, ensuring that each region functions and remains as a whole.

[0168] Specifically, the present invention provides a bubble-type single-stranded blocking nucleic acid BB for the CRISPR system. The BB binds complementarily to the target nucleic acid and consists of three parts, including a gate keeper region (abbreviated as GK), a bubble region, and a stabilizer region. These three parts are arranged in sequence from the PAM proximal end to the PAM distal end. The gate keeper region can respond to the disruption or formation of one or more base pairs, resulting in a change in the single-stranded or double-stranded state of the gate keeper region, thereby causing the opening or closing of the switch.

[0169] The bubble-type single-stranded blocking nucleic acid can form a double-stranded nucleic acid substrate with the single-stranded target nucleic acid to be detected and contains a switch region and a stabilizer region separated by a bubble region. The switch region is designed to be able to form two states, namely, an open state of a free single strand that does not match the target nucleic acid and a closed state of forming a double strand that matches the target nucleic acid. A change in one or more base pairs in the switch region can cause the opening or closing of the switch. The change in base pairs can be caused by a change in the base sequence, protein binding, or other means. The opening or closing of the switch region will cause the CRISPR recognition region of the double-stranded nucleic acid substrate to exhibit single / double-stranded properties, so that the substrate can / cannot activate the CRISPR protein, and further cause the system to generate / not generate a fluorescence signal.

[0170] In another preferred example, the region of the target nucleic acid that binds complementarily to the CRISPR spacer contains 13 - 28 nt of nucleotides.

[0171] In another preferred example, the base numbering of Cas12, Cas13, or Cas9 is shown as Figure 1 shown.

[0172] In another preferred example, the PAM proximal end within the spacer, the PAM distal end within the spacer, the region near the PAM outside the spacer, or the region far from the PAM outside the spacer is shown as Figure 1 shown.

[0173] In another preferred example, the stabilizer region is located at 5 - 15 nt proximal to the PAM within the CRISPR spacer to 1 - 50 nt of nucleotides on the side far from the PAM outside the CRISPR spacer.

[0174] In another preferred example, the stable region is located at 9 nt proximal to the PAM within the CRISPR spacer to 13 nt nucleotides on the side away from the PAM outside the CRISPR spacer

[0175] In another preferred example, the length of the switch region is 2 - 12 nt nucleotides.

[0176] In another preferred example, the length of the bubble region is 2 - 22 nt nucleotides.

[0177] In another preferred example, the total length of the switch region and the bubble region is 4 - 24 nt nucleotides.

[0178] In another preferred example, the switch region is complementary to the nucleotides at 1 - 7 nt proximal to the PAM within the CRISPR spacer to 1 - 7 nt close to the PAM side outside the CRISPR spacer.

[0179] In another preferred example, the bubble region is located between the switch region and the stable region, with a length of 1 - 10 nt nucleotides.

[0180] In another preferred example, the switch region contains a sequence complementary to the nucleotides at 5 - 7 nt proximal to the PAM within the CRISPR spacer.

[0181] In another preferred example, the switch region contains a sequence complementary to the last 1 nt nucleotide of the CRISPR PAM.

[0182] In another preferred example, the bubble region contains 2 - 3 nt nucleotides that do not match the CRISPR spacer between the switch region and the stable region.

[0183] In another preferred example, the bubble-type single-strand blocking nucleic acid BB contains 1 - 40 nt nucleotides that do not match the single-stranded target nucleic acid to be detected outside the switch region, the stable region, and the bubble region.

[0184] In another preferred example, the disruption or formation of base pairs in the switch region is caused by a change in the sequence of bases.

[0185] In another preferred example, the disruption or formation of base pairs in the switch region is caused by base modification, or can also be caused by the binding of base modification to its specific recognition protein or compound.

[0186] In another preferred embodiment, the chemical nature of the bubble-type single-stranded blocking nucleic acid BB includes, but is not limited to: DNA, RNA, 2'-fluorinated modified nucleic acid (2'-FNA), 2'-O-methyl modified nucleic acid (2'-OMe), 2'-ethylene glycol modified nucleic acid (2'-O-MOE, 2-O-(2-methoxyethyl)), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino nucleic acid (PMO), or a combination thereof.

[0187] In another preferred embodiment, the switch region contains a site corresponding to the mutation site to be detected or the nucleic acid modification site to be detected on the single-stranded target nucleic acid to be detected.

[0188] In another preferred embodiment, the mutation site / nucleic acid modification site to be detected is located within the CRISPR spacer.

[0189] In another preferred embodiment, the mutation site / nucleic acid modification site to be detected is located at 1-4 nt nucleotides proximal to the PAM within the CRISPR spacer.

[0190] In another preferred embodiment, the mutation site / nucleic acid modification site to be detected is located at 1-4 nt nucleotides on the side close to the PAM outside the CRISPR spacer.

[0191] In another preferred embodiment, when all the base sequences of the switch region match the single-stranded target nucleic acid to be detected, the switch region is in the closed state; when more than one base sequence of the switch region matches the single-stranded target nucleic acid to be detected, the switch region is in the open state.

[0192] In another preferred embodiment, when the nucleic acid corresponding to the switch region of the single-stranded target nucleic acid to be detected and BB contains no modification, the switch region is in the closed state; when the nucleic acid corresponding to the switch region of the single-stranded target nucleic acid to be detected and BB has more than one nucleic acid modification, and the system contains a specific binding protein for this modification, due to the competitive effect of this binding protein, the base pairs will be disrupted, and the switch region is in the open state.

[0193] The target nucleic acid detection method of the present invention

[0194] The target nucleic acid detection method of the present invention is a general method for detecting gene mutations and / or gene modifications. Specifically, the PAM region, spacer region, and random sequence region are divided according to the mutation and / or modification site of the target nucleic acid, so that the mutation and / or modification site of the target nucleic acid is located between 1 nt and 6 nt of the spacer region; according to the target nucleic acid with the PAM region, spacer region, and random sequence region divided, the CRISPR detection system of the present invention is provided, and the detection system includes a bubble-type single-strand blocking nucleic acid BB, a crRNA sequence complementary to the spacer region of the target nucleic acid, a CRISPR protein, and a reporter molecule; the fluorescence signal of the reporter molecule in the CRISPR detection system is detected, and the detection result of the target nucleic acid is given according to the fluorescence signal.

[0195] Specifically, it includes the following steps:

[0196] (S1) Prepare the reaction substrate of CRISPR: Anneal the target nucleic acid to be detected (including wild type WT and / or mutant MUT) with the bubble-type single-strand blocking nucleic acid BB to form a partially double-stranded complex. The stable region of this complex is double-stranded, the bubble region is mismatched single-stranded, and the switch region is double-stranded if all bases match and single-stranded if there are mismatched bases;

[0197] (S2) Add the reaction substrate described in S1 to the CRISPR detection system, and the detection system includes crRNA, Cas nuclease, and a single-stranded nucleic acid fluorescent probe. A reaction system to be detected is formed, and then it reacts at an appropriate temperature and the fluorescence intensity is recorded.

[0198] In another preferred example, in the step S1, the preparation method of the substrate DNA for the CRISPR-Cas12a system includes the following steps:

[0199] (S1-1) Amplify the dsDNA of the target gene in the sample to be detected;

[0200] (S1-2) Add the bubble-type single-strand blocking nucleic acid BB to the amplified solution, denature at high temperature to unwind the double helix of the dsDNA to form target ssDNA, and the target ssDNA binds complementarily to the BB, and then anneal at low temperature to obtain the substrate DNA;

[0201] Among them, the molar concentration of the bubble-type single-strand blocking nucleic acid BB is greater than the molar concentration of the target gene dsDNA.

[0202] In another preferred example, in step S1-2, the molar concentration ratio of the BB to the target gene dsDNA is 20:1.

[0203] In another preferred example, in step S1, the substrate nucleic acid formed by BB and the mutant nucleic acid target exhibits single-stranded properties in the CRISPR detection system, while the substrate nucleic acid formed by BB and the wild-type nucleic acid target exhibits double-stranded properties.

[0204] The nucleic acid modification detection method of the present invention

[0205] The present invention provides a general nucleic acid modification detection method, comprising the following steps:

[0206] (S21) Extract the target double-stranded or single-stranded nucleic acid in the sample to be detected;

[0207] (S22) Add the bubble-type single-stranded blocking nucleic acid BB to the target gene solution, and denature at high temperature to unwind the double helix of the double-stranded or single-stranded nucleic acid to form a target single-stranded nucleic acid, which complementarily binds to the bubble-type single-stranded blocking nucleic acid BB, and then anneal at low temperature to obtain a substrate nucleic acid;

[0208] Wherein, the molar concentration of the bubble-type single-stranded blocking nucleic acid BB is greater than the molar concentration of the target nucleic acid;

[0209] (S23) Add the substrate nucleic acid in S22 to the CRISPR detection system, which includes crRNA, Cas nuclease, single-stranded nucleic acid fluorescent probe, and modified nucleic acid specific binding protein. Form a reaction system to be detected, and then carry out the reaction and record the fluorescence intensity.

[0210] In another preferred example, the nucleic acid modification includes DNA modification or RNA modification, etc.

[0211] In another preferred example, the DNA modification includes but is not limited to 5-methylcytidine (5mC), 5-hydroxymethylcytidine (5hmC), 5-formylcytidine (5fC), 5-carboxylcytidine (5caC), N6-methyladenosine (6mA), and other methylation, hydroxymethylation, glycosyl hydroxymethylation, thiolation, etc.

[0212] In another preferred example, the RNA modification includes but is not limited to: N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), N7-methylguanosine (m7G), N4-acetylcytidine (ac4C), pseudouridine (Ψ), hypoxanthine, and other methylation, hydroxymethylation, glycosyl hydroxymethylation, thiolation.

[0213] In another preferred example, the modified nucleic acid-specific binding protein includes, but is not limited to: the 5-methylcytidine (5mC)-modified SRA protein domain that specifically binds to DNA, the phosphorothioate-modified SBD protein domain that specifically binds to DNA, the N6-methyladenosine (m6A)-modified YTH domain that specifically binds to RNA, the 5-methylcytidine (m5C)-modified ALYREF domain that specifically binds to RNA, and the N7-methylguanosine (m7G)-modified CBC domain that specifically binds to RNA.

[0214] Use

[0215] The present invention provides the use of the bubble-type single-strand blocking nucleic acid BB for detecting nucleic acid modification and / or single-base gene mutation.

[0216] In another preferred example, the use is for non-therapeutic and non-diagnostic purposes.

[0217] In another preferred example, the use is for scientific research purposes.

[0218] The present invention provides the use of the bubble-type single-strand blocking nucleic acid BB for preparing a reagent or kit for detecting nucleic acid modification and / or single-base gene mutation.

[0219] The main advantages of the present invention include:

[0220] (a) The present invention utilizes the selectivity of the CRISPR system for single-stranded and double-stranded detection substrates in the absence of a PAM sequence in the detection substrate, that is, by setting the nucleotides at the complementary position of the switch region and the mutation site, no mismatch is formed when binding to the wild-type nucleic acid target, the switch region is closed, and the substrate nucleic acid exhibits double-stranded properties. When binding to the mutant nucleic acid target, a mismatch is formed, the switch region is opened, and the substrate nucleic acid exhibits single-stranded properties. Thus, when there is no PAM sequence in front of the CRISPR spacer of the target nucleic acid, BB cannot exhibit a fluorescence signal with the wild-type target nucleic acid, and BB can output a fluorescence signal with the mutant target nucleic acid. The presence of mutant target nucleic acid in the detection system can be judged by the difference in fluorescence signals. It has great advantages in gene mutation detection and broad application prospects.

[0221] (b) The substrate complex formed by the bubble-type single-strand blocking nucleic acid BB provided by the present invention and the single-stranded target nucleic acid can respond to single-base mutations and / or modifications, that is, realize the change of single-stranded / double-stranded properties, and is used for the detection of single-base mutations and / or modifications and the construction of single-stranded / double-stranded conformation-driven self-assembled nucleic acid materials.

[0222] (c) The CRISPR system containing the bubble-type single-stranded blocking nucleic acid BB provided by the present invention does not rely on the PAM sequence for the cleavage of single-stranded target nucleic acids, breaking through the site limitation, increasing the applicability of species, having good generality, reducing the screening of PAM sequences, simplifying the experimental operation, and improving the CRISPR efficiency and accuracy.

[0223] (d) The method for single-base mutation and / or modification provided by the present invention has high sensitivity, simple operation, wide application range, strong generality, and can achieve highly accurate detection of single-base mutations and / or modifications occurring in the switch region.

[0224] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The equipment and reagents used in each of the following examples and test examples can be obtained from commercial sources unless otherwise specified. The experimental methods without specific conditions are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight percentage and weight parts.

[0225] An embodiment of the present invention provides a bubble-type single-stranded blocking nucleic acid BB for a CRISPR system. The BB includes a switch region, a bubble region, and a stable region, and the three regions are arranged in sequence from the PAM proximal end to the PAM distal end.

[0226] In the context of the present invention, the CRISPR system includes a Cas nuclease and a crRNA responsible for targeting a nucleic acid substrate (CRISPR Substrate). After the crRNA binds to the Cas nuclease, it targets the nucleic acid substrate and binds to it through base complementary pairing, thereby anchoring the Cas nuclease on the substrate and activating the activity of the Cas nuclease.

[0227] The present invention provides a substrate applicable to the CRISPR system. When a target nucleic acid substrate and a bubble-type single-strand blocking nucleic acid BB adapted thereto are base-complementary bound to each other, according to the difference in the binding force formed by a base mismatch / match in the switching region, the single / double-stranded nature is changed, that is, when a base in the switching region is mismatched, the single-stranded nature is maintained, and when the switching region is completely matched, it is changed to the double-stranded nature, thereby obtaining the nucleic acid substrate applicable to the CRISPR system of the present invention. When there is no PAM site in the target nucleic acid substrate, during the process of crRNA guiding the Cas nuclease to anchor to the target nucleic acid, the nucleic acid substrate with a single-stranded nature allows the Cas nuclease to anchor to the nucleic acid substrate due to the action of the bubble region for activation; the nucleic acid substrate with a double-stranded nature maintains the double-helix structure of the nucleic acid substrate due to the strong binding force of the switching region, and the Cas nuclease cannot anchor to the nucleic acid substrate, and the Cas nuclease cannot be activated. The target nucleic acid includes dsDNA, ssDNA and RNA applicable to the CRISPR system, and the nucleic acid substrate is a substrate applicable to the CRISPR system formed after the target nucleic acid is complementary bound to the bubble-type single-strand blocking nucleic acid BB.

[0228] Through the foregoing settings, the present invention provides a bubble-type single-strand blocking nucleic acid BB, which can be base-complementary bound to the target ssDNA to form a nucleic acid substrate applicable to the CRISPR system, and presents a single / double-stranded nature according to the type of the target nucleic acid substrate. In the case where there is no PAM sequence in the PAM region, the Cas nuclease in the CRISPR system is selectively activated by the nucleic acid substrate with a single / double-stranded nature. The nucleic acid substrate formed by the bubble-type single-strand blocking nucleic acid BB of the present invention exhibits a unique PAM-independent characteristic in the CRISPR system, expanding the target substrate of the CRISPR system from isolated ssDNA to dsDNA with some special structures, expanding its versatility. Moreover, during the activation process, the CRISPR system exhibits a high-resolution ability for single-base mutations on the ssDNA target that it does not possess. When there is no base mismatch between the switching region of the bubble-type single-strand blocking nucleic acid BB and the target ssDNA, the anchoring process of the Cas nuclease to the formed substrate DNA cannot be realized, thereby hindering the activation of the Cas nuclease. Thus, compared with the single-stranded nature still presented by the substrate DNA formed when there is a base mismatch between the switching region of the bubble-type single-strand blocking nucleic acid BB and the target ssDNA, a large fluorescence signal difference is generated, greatly improving the resolution ability and sensitivity of the CRISPR system for single-base mutations on ssDNA, which helps to broaden the application scenarios of the CRISPR system.

[0229] After in-depth study of the activation degree of Cas nuclease by the substrate DNA with different single / double-stranded properties formed by the length of the switch region of the bubble-type single-stranded blocking nucleic acid BB, it was found that extending the switch region upstream by 1 nt of nucleotides to form base complementary pairing with 1 nt of nucleotides at the 3' end of the target ssDNA PAM could further improve the sensitivity of the CRISPR system to single-base mutations on partial sequence ssDNA.

[0230] Another embodiment of the present invention provides an application of the bubble-type single-stranded blocking nucleic acid BB for the CRISPR-Cas12a system in the field of gene editing or gene detection.

[0231] Exemplarily, when applied in the field of gene detection, such as the detection of a certain single-base mutation-prone site, the target gene and the bubble-type single-stranded blocking nucleic acid BB are prepared into the substrate DNA form as above. Then, the CRISPR-Cas12a system targeting the target gene and a non-specific single-stranded DNA fluorescent probe are added. The non-specific single-stranded DNA fluorescent probe adopts a dual-label design, with a fluorescent group (such as FAM) labeled at its 5' end and a quenching group (such as BHQ1) labeled at its 3' end. When the probe remains intact, the fluorescent group at the 5' end is inhibited by the quenching group at the 3' end and cannot emit fluorescence. Once the probe is cleaved, the fluorescent group at the 5' end is released from the influence of the quenching group and will emit a fluorescent signal. Therefore, once the target mutation is detected, the trans-cleavage activity of Cas12a nuclease in the CRISPR-Cas12a system is activated, thereby non-specifically cleaving the fluorescent probe and releasing a fluorescent signal. By observing the presence, absence, and intensity of the fluorescent signal, it can be determined whether the target gene is contained in the sample to be tested, thus achieving rapid, highly sensitive, and highly specific detection.

[0232] The application of the bubble-type single-stranded blocking nucleic acid BB for the CRISPR-Cas12a system in the field of gene editing or gene detection has the same advantages as the bubble-type single-stranded blocking nucleic acid BB for the CRISPR-Cas12a system compared with the prior art, which will not be repeated here.

[0233] More importantly, based on the selectivity of the CRISPR-Cas12a system for the single / double-stranded state of the above substrate DNA, that is, the difference between the bubble-type single-stranded blocking nucleic acid BB switch region and the target ssDNA with a single-base mismatch or complete match is converted into the difference in single / double-stranded properties, and further amplified into the difference in whether the CRISPR-Cas12a system can be activated, and the single-base mutation is amplified into an obvious difference in fluorescent signal, the present invention has developed its application in the field of gene mutation detection.

[0234] Another embodiment of the present invention provides a general gene mutation detection method based on the CRISPR-Cas12a system, comprising the following steps:

[0235] (S1) Prepare the reaction substrate as described above. The reaction substrate is a complex of bubble-type single-strand blocking nucleic acid BB and target ssDNA. The target ssDNA includes a wild-type target strand and / or a mutant target strand. In the substrate DNA, the bubble-type single-strand blocking nucleic acid BB forms a single-base mismatch with the mutant target ssDNA in the switch region and is completely matched with the wild-type target ssDNA;

[0236] (S2) Add the substrate DNA into the CRISPR-Cas12a detection system, which contains crRNA, Cas12a nuclease and single-stranded DNA fluorescent probe. Form the reaction system to be detected, incubate on ice, and then record the fluorescence intensity at 37°C.

[0237] In this embodiment, by setting crRNA targeting the target gene and bubble-type single-strand blocking nucleic acid BB, and ensuring that there is a single-base mismatch between the switch region of the bubble-type single-strand blocking nucleic acid BB and the nucleotide sequence of the known mutation and it is completely matched with the wild-type target gene. Thus, when there is a mutant target strand in the sample to be detected, the bubble-type single-strand blocking nucleic acid BB specifically binds to the mutant target strand to form a substrate DNA with single-strand properties. The cleavage activity of Cas12a nuclease is activated, and it non-specifically cleaves the single-stranded DNA fluorescent probe in the CRISPR-Cas12a detection system described in S2, releasing the fluorescent group in the fluorescent probe and generating a distinguishable fluorescent signal. When there is no mutant target strand, the bubble-type single-strand blocking nucleic acid BB and the wild-type target strand bind to present a double-strand property, and the cleavage activity of Cas12a nuclease cannot be activated or is only slightly activated, without generating a fluorescent signal or generating a weak fluorescent signal. Furthermore, by comparing the differences in fluorescent signals, it can be detected whether there is a mutant target strand, that is, differential statistical analysis is performed according to the rate of generating fluorescent signals in the presence and absence of the target gene. When the P value < 0.05, it is significant, indicating the existence of a single-base mismatch in the switch region, that is, there is a point mutation in the sample to be detected.

[0238] In step S1, the preparation method of the substrate DNA for the CRISPR-Cas12a system includes the following steps:

[0239] (S1-1) Amplify the dsDNA of the target gene in the sample to be detected;

[0240] (S1-2) Add the bubble-type single-strand blocking nucleic acid BB to the target gene solution, and perform high-temperature denaturation to unwind the double helix of the dsDNA to form target ssDNA. The target ssDNA binds complementarily to the bubble-type single-strand blocking nucleic acid BB, and then perform low-temperature annealing to obtain the substrate DNA;

[0241] Among them, the molar concentration of the bubble-type single-strand blocking nucleic acid BB is greater than the molar concentration of the target gene dsDNA.

[0242] In step S2, both high-temperature denaturation and low-temperature annealing are conventional means in the art, and the present invention does not limit this. Exemplarily, the conditions for high-temperature denaturation are 95°C and 10 min, and the conditions for low-temperature annealing are 0°C and 5 min.

[0243] In step S1-1, the methods for amplifying the target gene in the sample to be detected include but are not limited to conventional polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), rolling circle amplification technology (RCA), as long as the target gene can be obtained, and the present invention does not limit this.

[0244] In step S1-2, the molar concentration ratio of the bubble-type single-strand blocking nucleic acid BB to the target gene dsDNA is 20:1. Due to its high molar concentration, the bubble-type single-strand blocking nucleic acid BB can preferentially hybridize with the target ssDNA strand in the target gene dsDNA.

[0245] To further improve the specificity, preferably, the position of the base mismatch between the switch region of the bubble-type single-strand blocking nucleic acid BB and the mutant target strand is located at the 1st to 6th nucleotides proximal to the PAM of the CRISPR recognition sequence, preferably, at the 4th nucleotide proximal to the PAM of the CRISPR recognition sequence.

[0246] The absorption wavelength of the quenching group in the single-stranded DNA fluorescent probe should preferably cover or approach the emission wavelength of the fluorescent group to ensure maximum quenching efficiency, and the length is usually 15-30 bases to ensure the normal completion of fluorescence resonance energy transfer (FRET). Optionally, FAM and BHQ1, Cy5 and BHQ2 are common combinations, which can effectively reduce the background signal and increase the signal-to-noise ratio.

[0247] Example 1: Preparation and application of the BB and the substrate DNA based on BB of the present invention.

[0248] Taking the single-stranded target nucleic acid to be detected as a reference, the specific base coding schematic diagram is as Figure 1 shown. The design schematic diagram of the bubble-type single-strand blocking nucleic acid BB is as Figure 2 shown.

[0249] Specifically, the stable region contains nucleotides that match the 9 nt proximal to the PAM in the CRISPR spacer to the 13 nt on the side away from the PAM outside the CRISPR spacer, for positioning and binding to the target ssDNA. The switch region contains nucleotides that match the 6 nt proximal to the PAM in the CRISPR spacer. The bubble region contains 2 nt nucleotides that do not match the CRISPR spacer between the switch region and the stable region, for changing the single / double-stranded nature of the substrate DNA after the binding of the bubble-type single-stranded blocking nucleic acid BB and the target ssDNA. The BB contains a suspended 17 nt nucleotide tail chain outside the switch region.

[0250] A method for preparing the substrate DNA (BB-ssDNA) for the CRISPR-Cas12a system, comprising the following steps:

[0251] (1) Amplify the target gene containing the target strand in the sample to be detected by PCR technology. The specific operation includes: adding the template target strand, upstream and downstream amplification primers with a final concentration of 0.5 μM (initial concentration of 10 μM) to 2×Rapid Taq Master and supplementing with RNase-Free water, shaking and mixing well, briefly centrifuging at 1000 rpm, denaturing at 95 °C, annealing at 53 °C, and extending at 72 °C in a PCR instrument to obtain the target gene containing the target ssDNA, and adjusting the initial concentration to 0.33 μM.

[0252] (2) Take 9 μL of the target gene solution containing the target ssDNA strand in step (1), add 2 μL buffer, 9 μL of the bubble-type single-stranded blocking nucleic acid BB with the concentration adjusted to 6.6 μM, denature at 95 °C for 10 min to make the target ssDNA and the bubble-type single-stranded blocking nucleic acid BB complementarily bind, and then anneal on ice (0 °C) to obtain the substrate DNA for the CRISPR-Cas12a system.

[0253] Among them, the target ssDNA that complementarily binds to the bubble-type single-stranded blocking nucleic acid BB can be artificially synthesized according to the experimental purpose and configured into a liquid with a concentration of 0.33 μM using a RNase-Free water diluent.

[0254] To deeply study the design principles and properties of the bubble-type single-stranded blocking nucleic acid BB, the following detection system was constructed:

[0255] 1) Single-stranded DNA fluorescent probe solution: Dilute the single-stranded DNA fluorescent probe powder with a RNase-Free water diluent to configure a liquid, then measure the concentration with a Nanodrop instrument and adjust the concentration to 5 μM for standby.

[0256] 2) Preparation of CRISPR-Cas12a system: Adjust the concentration of Cas12a (Cpf1) nuclease to 1 μM for later use.

[0257] 3) Prepare an 18-μL Cas12a / crRNA mixed reaction solution: 2 μL of r2.1 buffer, 2 μL of the single-stranded DNA fluorescent probe stock solution, 8 U of recombinant RNase inhibitor (0.02 μL), 0.25 μL of Cas12a nuclease, 0.5 μL of crRNA (1 μM), 13.23 μL of RNase-Free water. The amounts of Cas12a nuclease and crRNA need to be adjusted and optimized according to the sequence. Only the reference amounts are listed here and are not the only standard.

[0258] The detection method includes the following steps

[0259] (S-a) Prepare a substrate DNA solution, where the sequences of the bubble-type single-strand blocking nucleic acid BB and crRNA need to be adjusted according to the sequence of the target ssDNA.

[0260] (S-b) Mix 2 μL of the prepared substrate DNA solution with 18 μL of the Cas12a / crRNA mixed reaction solution, incubate on ice for 10 minutes, and then record the fluorescence intensity at 37°C.

[0261] As Figure 3 shown, after the bubble-type single-strand blocking nucleic acid BB and different types of target ssDNA complementarily bind, two different forms and properties of substrate DNA are formed. Design the bubble-type single-strand blocking nucleic acid BB according to different target ssDNA sequences, and set one nucleotide within the range of 1-6 nucleotides in the switch region to be complementary to the wild-type target ssDNA and not match the mutant target ssDNA. When this complex is formed, the two different target ssDNAs can present different single-stranded and double-stranded states in the CRISPR-Cas12a system due to the difference in the single-base binding force. The mutant target ssDNA has one nucleotide that does not match the bubble-type single-strand blocking nucleic acid BB in the switch region, and under the action of the bubble region and free nucleotides, it presents a single-stranded state with the head end separated and the tail end bound; activate the Cas12a nuclease in the CRISPR-Cas12a system to non-specifically cleave the single-stranded DNA fluorescent probe in the solution, generating a strong fluorescence signal; the wild-type target ssDNA completely matches the bubble-type single-strand blocking nucleic acid BB in the switch region, with a strong binding force, which can offset the action of the bubble region and free nucleotides, presenting a double-stranded state with the head and tail ends bound and a few nucleotides in the middle bubble region not matching, and cannot activate the Cas12a nuclease in the CRISPR-Cas12a system, and cannot produce a fluorescence signal or produce a very small amount of fluorescence signal.

[0262] AsFigure 4 As shown, the bubble-type single-stranded blocking nucleic acid BB is applicable to the recognition of modified bases that can be bound by its specific recognition protein or compound. The modified base-binding protein is added to the CRISPR detection system, and crRNA targeting this base and the bubble-type single-stranded blocking nucleic acid BB are set. Thus, when there is a base modification in the sample to be detected, the modified base-binding protein binds to the modified base, making a pair of bases in the switch region between the nucleic acid template and the bubble-type single-stranded blocking nucleic acid BB unable to form complementary base pairs. The bubble-type single-stranded blocking nucleic acid BB specifically binds to the detection sample to form an activating substrate. The cleavage activity of the Cas nuclease is activated, and the single-stranded DNA fluorescent probe is non-specifically cleaved in the CRISPR detection system, releasing the fluorescent group in the fluorescent probe and generating a distinguishable fluorescent signal. The detection sample includes DNA and RNA. When there is no modified base, the modified base-binding protein has no effect on the CRISPR detection system. The bubble-type single-stranded blocking nucleic acid BB and the detection sample bind to present a non-activating property, and the cleavage activity of the Cas nuclease cannot be activated or is only slightly activated, resulting in no fluorescent signal or a weak fluorescent signal. Therefore, by comparing the differences in fluorescent signals, it can be detected whether there is a modified base, that is, differential statistical analysis is performed based on the rates of generating fluorescent signals in the presence and absence of modified bases. A P value < 0.05 is significant, indicating that a pair of bases in the switch region cannot match, that is, the base at the detection site in the sample to be detected has a modification.

[0263] Example 2: Design optimization of the BB of the present invention.

[0264] To further study the influence of the stable region on the properties and efficiency of the bubble-type single-stranded blocking nucleic acid BB, with the EGFR T790M site as the first position of the CRISPR spacer, in this example, ssDNA Primers with different lengths (corresponding to stable regions with different sequence lengths) are designed to be able to complementarily pair with different lengths of nucleotides in the CRISPR spacer (hereinafter referred to as "Block") to simulate two states after the ssDNA binds to the target ssDNA, namely the open state presenting a single-stranded property (Open state) and the closed state presenting a double-stranded property (Close state), as specifically shown in Figure 5 shown.

[0265] Under the Open state, the detection rate presented by the substrate DNA should be close to the detection of the ssDNA target; under the Close state, the detection rate presented by the substrate DNA should be close to the detection of the dsDNA target without a PAM sequence, that is, no fluorescent signal is output. And the detection rates presented by the Open state and the Close state should have a large difference to clearly distinguish the substrate types.

[0266] Plot the slope of the fluorescence value generated in the five minutes before detection, and the detection results are shown in Figure 6 , where the ordinate represents the slope value of the fluorescence value generated in the first five minutes, and each colored column represents the efficiency of activating CRISPR-Cas12a by different lengths of the stabilizing region sequence. Both the Open state and the Close state in the design can be achieved. Considering comprehensively, the stabilizing region should be a nucleotide that can match the nucleotides from 5-15 nt proximal to the PAM in the CRISPR spacer to 1-50 nt away from the PAM side outside the CRISPR spacer. Preferably, the stabilizing region should be a nucleotide that can match the nucleotides from 8 nt proximal to the PAM in the CRISPR spacer to 13 nt away from the PAM side outside the CRISPR spacer.

[0267] To further study the effects of the bubble region and the switch region on the properties and efficiency of the bubble-type single-strand blocking nucleic acid BB, based on the study of the properties of the stabilizing region, the mutant ssDNA (T_EGFR_2369T) at the EGFR T790M site was used as the target ssDNA, and the nucleotides from 8 nt proximal to the PAM in the CRISPR spacer to 13 nt away from the PAM side outside the CRISPR spacer were used as the stabilizing region. The sequences of the bubble region and the switch region complementary to the remaining 8 nt nucleotides of the CRISPR recognition sequence were optimized, with six combinations from 2+6 to 7+1. The detection results are as shown in Figure 7 shown.

[0268] Among them, the ordinate represents the slope value of the fluorescence value generated in the first five minutes, the broken line DF represents the ratio of the fluorescence value slope of the latter experimental group to that of the former experimental group, and each colored column represents the efficiency of activating CRISPR-Cas12a by the ssDNA with different bubble region and switch region sequences. When the switch region is fully matched, that is, in the Close state, the detection rate should be close to 0. When there is one nucleotide in the switch region that cannot be complementary paired, the slope should achieve a "jump" to amplify the difference between the mutant target signal and the wild-type target signal in the actual detection. The switch region is a nucleotide that can match the nucleotides from 1-7 nt proximal to the PAM in the CRISPR spacer to 1-7 nt close to the PAM side outside the CRISPR spacer, and the bubble region is a nucleotide with a length of 1-10 nt that cannot match the CRISPR spacer and is located between the switch region and the stabilizing region. Preferably, the switch region contains a nucleotide that can match the nucleotide at 6 nt proximal to the PAM in the CRISPR spacer, and the bubble region contains 2 nt nucleotides that cannot match the CRISPR spacer between the switch region and the stabilizing region.

[0269] The sequences used in the optimization process of this embodiment are shown in Table 1.

[0270] Table 1 Primer sequences for the design and optimization of bubble-type single-strand blocking nucleic acid BB

[0271]

[0272]

[0273] Note: Sequences 4-10 in the table are used to optimize and determine the stable region, and sequences 11-16 are used to optimize and determine the bubble region and the switch region; in the name, cr represents crRNA, B represents bubble-type single-strand blocking nucleic acid BB, and T represents target ssDNA; in the target ssDNA sequence, the underline represents the CRISPR spacer, and in the crRNA sequence, the underline represents the sequence matching the CRISPR spacer. In the mutant target ssDNA, the red font represents the mutation site; in the BB bubble region + switch region sequence, the underline represents the stable region complementary to the target ssDNA, the bold part represents the switch region, the red font represents the bubble region, and the italic part represents the other regions in BB that cannot match the target ssDNA.

[0274] Example 2: Application and optimization of bubble-type single-strand blocking nucleic acid BB for the CRISPR-Cas12a system in detecting single-base mutations.

[0275] To study the improvement effect of the above-mentioned bubble-type single-strand blocking nucleic acid BB on the single-base mutation resolution ability in the CRISPR-Cas12a system, mutations were simulated at the EGFR T790M site 2369T and the 1-3 nucleotides after it, which were 2370C, 2371G, and 2372G respectively (the numbers are the positions where the mutations occur on the gene, and the letters are the types of bases after the single-base mutations). According to different sequences, bubble-type single-strand blocking nucleic acids BB with various combinations of bubble regions and switch regions were designed, and the above-mentioned crRNA and wild-type target ssDNA were used for detection.

[0276] Among them, in the detections of 2369T and 2372G, the present invention introduced a PAM with the sequence TTTV in front of its dsDNA and synthesized this dsDNA for detection to obtain the difference in the single-base mutation resolution ability between the bubble-type single-strand blocking nucleic acid BB of the present invention and the CRISPR-Cas12a system in the double-strand detection state.

[0277] The detection results are shown in Figure 8As shown, where the left vertical axis represents the slope value of the fluorescence value generated in the first five minutes, the right vertical axis represents the discrimination factor (DF), and the horizontal axis represents the type of detection target or the type of bubble-type single-stranded blocking nucleic acid BB used, where B represents the number of nucleotides in the bubble region, G represents the number of nucleotides in the switch region, the green column represents the efficiency of the wild-type ssDNA target activating CRISPR-Cas12a, the orange column represents the efficiency of the mutant ssDNA target activating CRISPR-Cas12a, and the gray broken line represents the ratio of the latter to the former (DF). Among them, the sequences used are shown in Table 2.

[0278] According to the detection results, the above four bubble-type single-stranded blocking nucleic acids BB can all effectively improve the ability of the CRISPR-Cas12a system to distinguish single-base mutations in the single-stranded detection state, and there is still a tens-fold improvement compared to the ability of the CRISPR-Cas12a system to distinguish single-base mutations in the double-stranded detection state. This detection result shows that the various bubble-type single-stranded blocking nucleic acids BB developed in the present invention have a significant promoting effect on the ability of the CRISPR-Cas12a system to distinguish single-base mutations in the single-stranded detection state, and the bubble-type single-stranded blocking nucleic acid BB and the CRISPR-Cas12a system to which it is applied have high generality and can be applied to the detection of most single-base mutation sites. Preferably, the components of the highly efficient bubble-type single-stranded blocking nucleic acid BB should be: the stable region contains nucleotides that can match the nucleotides from 8 nt proximal to the PAM in the CRISPR spacer to 13 nt away from the PAM side outside the CRISPR spacer, the switch region contains nucleotides that can match the nucleotides containing 6 nt proximal to the PAM in the CRISPR spacer, the bubble region contains 2 nt nucleotides that cannot match the CRISPR spacer between the switch region and the stable region and 17 nt nucleotides that do not match the single-stranded target nucleic acid to be detected outside the switch region and the stable region.

[0279] Table 2 Primer sequences for efficiency verification of bubble-type single-stranded blocking nucleic acid BB of EGFR sequence

[0280]

[0281]

[0282]

[0283] Note: cr represents crRNA, B represents the bubble-type single-strand blocking nucleic acid BB, and T represents the target DNA (including ssDNA and dsDNA); the underlined part in the target DNA sequence represents the CRISPR spacer, and the underlined part in the crRNA sequence represents the sequence matching the CRISPR spacer. The red font in the mutant target DNA (including mutant target ssDNA and mutant target dsDNA) represents the mutation site; the underlined part in the BB sequence represents the stable region complementary to the target ssDNA, the bold part represents the switch region, the red font represents the bubble region, and the italic part represents the other regions in BB that cannot match the target ssDNA.

[0284] To further verify the improvement effect of the selected bubble-type single-strand blocking nucleic acid BB on the single-base mutation discrimination ability of other sequences in the CRISPR-Cas12a system, that is, its generality, this example selected multiple common single-base mutation sites in the human body, including MYO7A L618R, HBB E7V, G6P1C R83C, and designed the selected bubble-type single-strand blocking nucleic acid BB and crRNA according to their sequences for detection.

[0285] The detection results are shown in Figure 9 , where the meanings represented by the vertical and horizontal coordinates are the same as those in Figure 8 . The green column represents the activation efficiency of the wild-type ssDNA target for CRISPR-Cas12a, the brown column represents the activation efficiency of the mutant ssDNA target for CRISPR-Cas12a, and the gray broken line represents the ratio of the latter to the former (DF). Among them, the sequences used are shown in Table 3.

[0286] The results show that the bubble-type single-strand blocking nucleic acid BB that meets the above design conditions shows a good improvement effect on the single-base mutation discrimination ability of CRISPR-Cas12a in multiple sequences. In addition, the results verify that extending the switch region upstream by 1 nt to form a base complementary pair with the last 1 nt of the PAM region of the target ssDNA can further improve the sensitivity of the CRISPR-Cas12a system to single-base mutations on the ssDNA of some sequences.

[0287] Table 3 Primer sequences for verifying the efficiency of the bubble-type single-strand blocking nucleic acid BB for single-base mutations in other sequences

[0288]

[0289]

[0290] Note: cr represents crRNA, B represents the bubble-type single-strand blocking nucleic acid BB, and T represents the target ssDNA; the underlined part in the target ssDNA sequence represents the CRISPR spacer, and the underlined part in the crRNA sequence represents the sequence matching the CRISPR spacer. The numbers in the target ssDNA name are the positions of the mutations on the chromosome where the gene is located, and the letters represent the types of bases after mutation. The red font in the mutant target ssDNA represents the mutation site; the underlined part in the BB bubble region + switch region sequence represents the stable region complementary to the target ssDNA, the bold part represents the switch region, the red font represents the bubble region, and the italic part represents the other regions in BB that cannot match the target ssDNA.

[0291] Based on the above data, it can be concluded that the present application has discovered a novel bubble-type single-strand blocking nucleic acid BB applicable to the CRISPR-Cas12a system, which binds to the target ssDNA to form the substrate DNA, and in the CRISPR-Cas12a system, changes its single-stranded and double-stranded properties according to the difference in base-binding ability after single-base mutation, and amplifies this difference with the activated state of the Cas12a nuclease, thereby enhancing the single-base mutation discrimination ability of the CRISPR-Cas12a system in detecting single-stranded DNA. In addition, compared with the CRISPR-Cas12a double-stranded detection, the application of this novel bubble-type single-strand blocking nucleic acid BB in the CRISPR-Cas12a single-stranded detection not only has PAM independence, but also the discrimination ability for single-base mutations is dozens of times better than that of the CRISPR-Cas12a double-stranded detection. The novel bubble-type single-strand blocking nucleic acid BB of the present invention has good generality and high sensitivity, and can provide a research basis and technical means for further understanding and applying the CRISPR-Cas12a system.

[0292] Example 3: Detection of m by the bubble-type single-strand blocking nucleic acid BB for the CRISPR-Cas12a system 6 A modification.

[0293] As Figure 10 shown, by treating RNA with adenosine deaminase, the adenosine without m 6 A modification on the RNA is converted into guanosine I, while the adenosine with m 6 A modification remains unchanged. Subsequently, in reverse transcription amplification, the m 6 A modification on the RNA is converted into the difference in SNPs in the DNA. By designing crRNA and the bubble-type single-strand blocking nucleic acid BB targeting this SNP, and ensuring that there is a single-base mismatch between the switch region of the bubble-type single-strand blocking nucleic acid BB and the DNA obtained by reverse transcription amplification of the RNA with m 6 A modification, and a single-base mismatch with the DNA without m 6The DNA reverse transcribed and amplified from A-modified RNA does not form base mismatches. Thus, when there is m 6 A modification in the sample to be detected, the bubble-type single-strand blocking nucleic acid BB specifically binds to its DNA to form a substrate DNA with single-strand properties, and the cleavage activity of the Cas12a nuclease is activated, non-specifically cleaving the single-strand DNA fluorescent probe in the CRISPR-Cas12a detection system, releasing the fluorescent group in the fluorescent probe and generating a distinguishable fluorescent signal; while when there is no m 6 A modification, the bubble-type single-strand blocking nucleic acid BB and its DNA bind to present a double-strand property, and the cleavage activity of the Cas12a nuclease cannot be activated or is only slightly activated, without generating a fluorescent signal or generating a weak fluorescent signal.

[0294] By comparing the differences in fluorescent signals, it can be detected whether there is m 6 A modification, that is, according to the presence or absence of m 6 A modification, a differential statistical analysis is performed on the rate of generating fluorescent signals. When the P value < 0.05, it is significant, indicating the presence of a single-base mismatch in the switch region, that is, there is m 6 A modification at the detection site of the sample to be detected. The specific steps are as follows: Prepare the substrate DNA of the CRISPR-Cas12a system for detecting RNA m 6 A modification; add the substrate DNA to the CRISPR-Cas12a detection system, which includes crRNA, Cas12a nuclease, and single-strand DNA fluorescent probe. Form the reaction system to be detected, incubate on ice, and then record the fluorescence intensity at 37°C.

[0295] The preparation of the substrate DNA of the CRISPR-Cas12a system for detecting RNA m 6 A modification includes the following steps: Treat RNA with adenosine deaminase, reverse transcribe and amplify to obtain a dsDNA template; reverse transcribe RNA and amplify the cDNA generated by reverse transcription; add the bubble-type single-strand blocking nucleic acid BB to the dsDNA, and denature at high temperature to unwind the double helix of the dsDNA to form a target ssDNA, and the target ssDNA binds complementarily to the bubble-type single-strand blocking nucleic acid BB, and then anneal at low temperature to obtain the substrate DNA; wherein, the molar concentration of the bubble-type single-strand blocking nucleic acid BB is greater than the molar concentration of the target gene dsDNA, and the sequences of the bubble-type single-strand blocking nucleic acid BB and crRNA need to be adjusted according to the target sequence.

[0296] Among them, the molar concentration ratio of BB to the dsDNA of the target gene is 20:1. The bubble-type single-strand blocking nucleic acid BB can preferentially hybridize with the target ssDNA strand in the dsDNA of the target gene due to its high molar concentration. In this 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.

[0297] Specifically, for detecting RNA m 6 A method for preparing a substrate DNA of the CRISPR-Cas12a system modified with A includes the following steps: Before performing the in vitro deamination reaction, heat the RNA to 95 °C and keep it for 3 minutes, then immediately cool it on ice. The reaction is carried out in a deamination buffer (50 mM Tris-HCl, 2.5 mM MgCl2, 25 mM KCl, 2 mM DTT, and 10% (v:v) glycerol, pH 7.2), and 40 U of recombinant RNase inhibitor should be additionally added to the system. React 1 ng of RNA with 10 μM TadA8.20 in 20 μL of deamination buffer and incubate overnight at 37 °C; after the treatment, keep it at 95 °C for 10 minutes to terminate the reaction. Amplify the treated RNA by RT-PCR technology to obtain dsDNA. The specific operation includes: Follow the TM PrimeScript reverse transcriptase instruction manual for treatment, reverse transcribe to obtain cDNA, and use the synthesized cDNA to prepare a template for Cas12a detection through PCR reaction. Since the template GC% is relatively high, it is recommended to use TaqDNA polymerase. Use PCR&DNA Cleanup Kit to purify the PCR product and adjust the initial DNA concentration to 0.33 μM. Take 9 μL of the solution containing the target ssDNA strand, add 2 μL buffer, 9 μL of the bubble-type single-strand blocking nucleic acid BB with the concentration adjusted to 6.6 μM, denature at 95 °C for 10 min to make the target ssDNA complementarily bind to the bubble-type single-strand blocking nucleic acid BB, and then anneal on ice (0 °C) to obtain the substrate DNA for the CRISPR-Cas12a system.

[0298] As Figure 10 shown, the adenosine deaminase TadA 8.20 can convert all adenosines without m 6 A modification on the RNA into guanosine, while the adenosines with m 6 A modification remain unchanged. Subsequently, in the reverse transcription amplification, the m 6 A modification on the RNA is converted into the difference of SNP in the DNA. In this example, a detection method for C / T in the single-stranded ssDNA after reverse transcription amplification is mainly provided.

[0299] To deeply study the properties of the bubble-type single-strand blocking nucleic acid BB in the single-strand detection of the CRISPR-Cas12a system for the detection of RNA m 6 A modification detection, the following detection system was constructed:

[0300] (a1) Single-stranded DNA fluorescent probe solution: Dilute the single-stranded DNA fluorescent probe powder with RNase-Free water diluent to prepare a liquid, then measure the concentration with a Nanodrop instrument and adjust the concentration to 5 μM for use.

[0301] (a2) CRISPR-Cas12a system: Adjust the concentration of Cas12a (Cpf1) nuclease to 1 μM for use.

[0302] (a3) 18 μL of Cas12a / crRNA mixed reaction solution: 2 μL of r2.1 buffer, 2 μL of the prepared single-stranded DNA fluorescent probe solution for use, 8 U of recombinant RNase inhibitor (0.02 μL), 0.25 μL of Cas12a nuclease, 0.5 μL of crRNA (1 μM), 13.23 μL of RNase-Free water. The amounts of Cas12a nuclease and crRNA need to be adjusted and optimized according to the sequence. Only the reference amounts are listed here and do not serve as the only standard.

[0303] The detection method includes the following steps: Prepare a substrate DNA solution, where the sequences of the bubble-type single-strand blocking nucleic acid BB and crRNA are shown in Table 5; Mix 2 μL of the prepared substrate DNA solution with 18 μL of the Cas12a / crRNA mixed reaction solution, incubate on ice for 10 minutes, and then record the fluorescence intensity at 37°C.

[0304] Design bubble-type single-strand blocking nucleic acid BB and crRNA for detection at sites such as MALAT 1 2515 and ACTB (Chr.7,5527533). The detection results are as Figure 11 shown, where the vertical and horizontal coordinates represent the same meaning as Figure 9 the same. The green columns represent the efficiency of activating CRISPR-Cas12a by the ssDNA target with T at the detection site (representing m 6 A modification at the site to be detected), the brown columns represent the efficiency of activating CRISPR-Cas12a by the ssDNA target with C at the detection site (representing no m 6 A modification at the site to be detected), and the gray broken line represents the ratio of the former to the latter (DF).

[0305] The bubble-shaped single-stranded blocking nucleic acid BB improves the resolution of T / C single-base mutations in the CRISPR-Cas12a system by dozens of times, thereby distinguishing the T / C bases in DNA, because the T / C bases in DNA are determined by the presence / absence of mRNA in RNA. 6 A-modified adenosine is converted, and it can be concluded that the bubble-shaped single-stranded blocking nucleic acid BB of the present invention can be used in the CRISPR-Cas12a system to detect m in RNA. 6 A modification detection, and the detection method has good sequence versatility and high sensitivity.

[0306] In order to further study the bubble-shaped single-stranded blocking nucleic acid BB in the single-stranded detection of CRISPR-Cas12a system for RNA m 6 A modification quantitative properties, before the above detection system, the following steps are performed: Make the detection site RNA m 6 A modified abundance standard curve; extracted total cell RNA samples.

[0307] The method for preparing the detection site RNA m 6 The A modification abundance standard curve includes the following steps: adjusting the concentration of two different ssDNAs (artificially synthesized, the sites to be detected are T and C, respectively) to 0.33uM and then mixing them according to a predetermined ratio, with a total volume of 9μL (for example, 30T%: 2.7μL T ssDNA (with m6A modification) + 5.3μL C ssDNA (without m6A modification)), and preparing substrate DNA with a bubble-shaped single-stranded blocking nucleic acid BB with a concentration of 6.6uM according to the above method; recording its fluorescence intensity according to the above detection steps, and expressing the fluorescence value at the end point of the curve as F, then the ordinate R of the standard curve can be calculated as follows:

[0308]

[0309] The standard curve can be drawn with T% as the horizontal axis.

[0310] The method for extracting total RNA samples from cells comprises the following steps: culturing HeLa and HEK293T cells, subculturing, and collecting cells by centrifugation on the second day after subculturing; using RNA-easy Isolation Extract total cellular RNA, determine its concentration and set aside.

[0311] Taking the MALAT1 2515 site as an example, the sequence used is shown in Table 5. The synthetic DNA templates were mixed in proportion to prepare a standard curve, such as Figure 12 As shown, the ordinate is R calculated by the above formula, and the abscissa is the percentage of T ssDNA in the sample.

[0312] Total RNA was extracted from HEK293T cells and HeLa cells. After processing according to the steps, it was detected simultaneously with negative and positive controls of 0% T and 100% T, and a detection fluorescence curve graph was obtained, as shown in Figure 13 , where the ordinate represents the fluorescence intensity and the abscissa represents the detection time (seconds). The dark blue curve is the 100% T positive control, the orange curve is the 0% T negative control, and the yellow curve is the sample detection curve. According to the above formula, the m 6 A modification abundance at the 2515 site of MALAT1 in HEK293T cell RNA was 41.89%, and the m 6 A modification abundance in HeLa cell RNA was 63.54%. This result is similar to the reported results in the literature.

[0313] The sequences corresponding to the ACTB site are shown in Table 5. The actual sample quantification results are listed in Table 4.

[0314] Table 4 Quantitative results of other RNA m 6 A modification sites

[0315] Locus Sample source Quantitative result Literature-reported result ACTB HEK293T 63.82％ 68.54％ ACTB HeLa 69.64％ 71.11％

[0316] Table 5 RNA m 6 A modification detection primer sequences

[0317]

[0318]

[0319] Note: cr represents crRNA, B represents the bubble-type single-stranded blocking nucleic acid BB, T represents the target ssDNA; the underline in the target ssDNA (including T ssDNA and C ssDNA) sequence represents the CRISPR spacer, and the red font represents the detection site; the underline in the crRNA sequence represents the sequence matching the CRISPR spacer, the number in the target ssDNA name is the position of the mutation on the chromosome where the gene is located, and the letter represents the type of the mutated base; the underline in the BB sequence represents the stable region complementary to the target ssDNA, the bold part represents the switch region, the red font represents the bubble region, and the italic part represents the other regions in BB that cannot match the target ssDNA.

[0320] Based on the above data, it can be seen that the bubble-type single-stranded blocking nucleic acid BB provided by the present invention has good implementation effects and application prospects in the field of RNA m 6 A modification detection. After converting the presence or absence of RNA m 6 A modification into the SNP difference on DNA, the bubble-type single-stranded blocking nucleic acid BB provided by the present invention can accurately and quickly complete the detection of RNA m at specific sites.6 The qualitative and quantitative modification of A is accurate in results and has good versatility, and the application of the bubble-type single-stranded blocking nucleic acid BB in the field of gene detection has been developed.

[0321] Example 4: Detection of DNA 5mC modification using the bubble-type single-stranded blocking nucleic acid BB for the CRISPR-Cas12a system.

[0322] The DNA 5mC modification-binding protein SRC can bind to and disrupt the complementary pairing of 5mC-modified bases on dsDNA. The crRNA targeting cytosine and the bubble-type single-stranded blocking nucleic acid BB were designed as shown in Table 6.

[0323] When the sample to be detected has 5mC modification, the SRC protein binds to the 5mC-modified base, preventing the DNA strand with 5mC modification from forming complementary pairing with the bubble-type single-stranded blocking nucleic acid BB at the corresponding position in the switch region. The bubble-type single-stranded blocking nucleic acid BB specifically binds to the test sample to form a substrate with single-stranded properties. The cleavage activity of the Cas12a nuclease is activated, and it non-specifically cleaves the single-stranded DNA fluorescent probe in the CRISPR-Cas12a detection system, releasing the fluorescent group in the fluorescent probe and generating a clearly distinguishable fluorescent signal. In contrast, when there is no 5mC-modified base, the substrate with double-stranded properties cannot activate the cleavage activity of the Cas12a nuclease, enabling the determination of the presence of DNA 5mC modification at the detection site.

[0324] The specific steps include: preparing the substrate DNA for detecting DNA 5mC modification for the CRISPR-Cas12a system; adding the substrate DNA to the CRISPR-Cas12a detection system, which contains crRNA, Cas12a nuclease, and a single-stranded DNA fluorescent probe, to form a reaction system to be detected; incubating the reaction system to be detected on ice and then recording the fluorescence intensity at 37°C.

[0325] The method for preparing the substrate DNA of the CRISPR-Cas12a system for detecting DNA 5mC modification includes the following steps: synthesizing a DNA template with 5mC modification, or using a methylase to perform methylation modification at specific positions of the DNA to obtain a dsDNA or ssDNA template with 5mC modification; adding the bubble-type single-stranded blocking nucleic acid BB to the dsDNA or ssDNA template, denaturing at high temperature to unwind the double helix of the dsDNA to form a target ssDNA, and the target ssDNA hybridizes complementarily with the bubble-type single-stranded blocking nucleic acid BB, and then annealing at low temperature to obtain the substrate DNA; the operation methods of the ssDNA template and the dsDNA template are the same; mixing and incubating the obtained substrate DNA with the 5mC modification binding protein SRC; wherein the molar concentration of the 5mC modification binding protein SRC is greater than the molar concentration of the substrate DNA.

[0326] Wherein, the molar concentration ratio of the bubble-type single-stranded blocking nucleic acid BB to the dsDNA or ssDNA template with 5mC modification is 20:1. The bubble-type single-stranded blocking nucleic acid BB can preferentially hybridize with the ssDNA strand with 5mC modification in the dsDNA due to its high molar concentration. Among them, 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.

[0327] Wherein, the molar concentration ratio of the 5mC modification binding protein SRC to the substrate DNA is 10:1; the incubation process lasts for 30 minutes at 4 °C and is carried out in a buffer solution containing 10 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol.

[0328] Specifically, in order to deeply study the properties of the bubble-type single-stranded blocking nucleic acid BB in the single-strand detection of the CRISPR-Cas12a system for the detection of DNA 5mC modification, the following detection system is constructed:

[0329] (b1) Single-stranded DNA fluorescent probe solution: Dilute the single-stranded DNA fluorescent probe powder with RNase-Free water diluent to form a liquid, then measure the concentration with a Nanodrop instrument, and adjust the concentration to 5 μM for use.

[0330] (b2) Preparation of the CRISPR-Cas12a system: Adjust the concentration of the Cas12a (Cpf1) nuclease to 1 μM for use.

[0331] (b3) Prepare an 18 μL Cas12a / crRNA mixed reaction solution: 2 μL of r2.1 buffer, 2 μL of single-stranded DNA fluorescent probe stock solution, 8 U of recombinant RNase inhibitor (0.02 μL), 0.25 μL of Cas12a nuclease, 0.5 μL of crRNA (1 μM), and 13.23 μL of RNase-Free water. The amounts of Cas12a nuclease and crRNA need to be adjusted and optimized according to the sequence. Only the reference amounts are listed here and are not the only standard.

[0332] The detection method includes the following steps: Prepare a substrate DNA solution, where the sequences of the bubble-type single-stranded blocking nucleic acid BB and crRNA are shown in Table 6; Mix 2 μL of the prepared substrate DNA solution with 18 μL of the Cas12a / crRNA mixed reaction solution, incubate on ice for 10 minutes, and then record the fluorescence intensity at 37°C.

[0333] Design bubble-type single-stranded blocking nucleic acid BB and crRNA for detection at sites such as OCT2 (Chr.6,161509167) and SEPTIN9 (Chr.17,77373514). The detection results are shown in Figure 14 , where the vertical and horizontal coordinates represent the same meanings as Figure 9 The same. The green columns represent the efficiency of ssDNA targets with 5mC modification at the detection sites to activate CRISPR-Cas12a, the brown columns represent the efficiency of ssDNA targets without 5mC modification at the detection sites to activate CRISPR-Cas12a, and the gray broken line represents the ratio (DF) of the former to the latter.

[0334] The results show that the bubble-type single-stranded blocking nucleic acid BB enables the single-stranded detection in the CRISPR-Cas12a system to recognize and distinguish the 5mC modification in DNA. Furthermore, it can be concluded that the bubble-type single-stranded blocking nucleic acid BB provided by the present invention can be used for the detection of 5mC modification in DNA by the CRISPR-Cas12a system, and this detection method has good sequence generality and high sensitivity.

[0335] Table 6 DNA 5mC modification detection primer sequences

[0336]

[0337]

[0338] Note: cr represents crRNA, B represents the bubble-type single-strand blocking nucleic acid BB, and T represents the target ssDNA; the underscore in the target ssDNA sequence indicates the CRISPR spacer, the underscore in the crRNA sequence indicates the sequence matching the CRISPR spacer, and the red font in the target ssDNA indicates the modification detection site; the underscore in the BB sequence indicates the stable region complementary to the target ssDNA, the bold part indicates the switch region, the red font indicates the bubble region, and the italic part indicates the other regions in BB that cannot match the target ssDNA.

[0339] All documents mentioned in this invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A bubble-type single-strand blocking nucleic acid BB for detecting a target nucleic acid, characterized in that, The bubble-type single-strand blocking nucleic acid BB comprises a switch region, a stabilizing region, and a bubble region; Among them, the switch region comprises a nucleic acid sequence complementary to the target nucleic acid to be detected, and base mutations and / or modifications of the target nucleic acid to be detected cause a transition between the single-stranded state and the double-stranded state of the switch region; The stabilizing region forms a stable double-strand with the target nucleic acid; The bubble region is located between the switch region and the stabilizing region, and the bubble region comprises a suspended single-strand that is not complementary to the target nucleic acid.

2. The bubble-type single-strand blocking nucleic acid BB according to claim 1, wherein The bubble-type single-strand blocking nucleic acid BB comprises a structure shown in Formula I or Formula II: 5'-Z1-Z2-Z3-3' (I) 5'-Z3-Z2-Z1-3' (II) Among them, Z1 is the switch region, and the switch region responds to the target nucleic acid to be detected; Z2 is the bubble region, and the bubble region comprises a suspended single-strand; Z3 is the stabilizing region, and the stabilizing region exists in the form of a double-strand.

3. The bubble-type single-strand blocking nucleic acid BB according to claim 1 or 2, characterized in that, When the switch region of BB is completely complementary to the target nucleic acid to be detected, the BB blocks the target nucleic acid, and the target nucleic acid cannot be used as a substrate for the CRISPR protein, cannot activate the CRISPR protein, and cannot generate a signal; When the switch region of BB is not completely complementary to the target nucleic acid to be detected, the BB releases the target nucleic acid, and the target nucleic acid serves as a substrate for the CRISPR protein, activates the CRISPR protein, and thus generates a signal.

4. A reagent combination, characterized in that, The reagent combination comprises: the bubble-type single-strand blocking nucleic acid BB described in claim 1, and a crRNA complementary to the spacer region sequence of the target nucleic acid.

5. A CRISPR detection system for detecting a target nucleic acid, characterized in that, The CRISPR detection system comprises: The bubble-type single-strand blocking nucleic acid BB described in claim 1; CRISPR protein; crRNA; and Optionally, the target nucleic acid.

6. The CRISPR detection system according to claim 5, wherein The CRISPR detection system detects the target nucleic acid independently of the PAM sequence of the target nucleic acid.

7. Use of the bubble-type blocking nucleic acid BB according to claim 1, the reagent combination according to claim 4, or the CRISPR detection system according to claim 5, characterized in that, For preparing a kit for detecting a target nucleic acid.

8. A kit for detecting a target nucleic acid, characterized in that, The kit comprises the bubble-type blocking nucleic acid BB described in claim 1, the reagent combination described in claim 4, or the CRISPR detection system described in claim 5.

9. A method for detecting a target nucleic acid, characterized in that, Comprising the following steps: (M1) Provide the CRISPR detection system described in claim 5; (M2) Detect the fluorescence signal of the reporter molecule in the CRISPR detection system, and give the detection result of the target nucleic acid according to the fluorescence signal.

10. The detection method according to claim 9, characterized in that, The detection of the target nucleic acid refers to detecting whether there are base mutations and / or base modifications in the target nucleic acid.

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