Method and assay kit for detecting single base substitution in short-chain RNA group having sequence diversity on 3' - side

Abstract

To provide a technique for identifying one base substitution on a micro RNA having sequence diversity on its 3' side in a short time at a low cost.SOLUTION: The method according to the embodiment is a method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity on the 3 ' side. The method comprises contacting a solution containing the short-chain RNA group as a test nucleic acid, a guide RNA containing a complementary sequence of a target sequence comprising a single base substitution site, a genome editing enzyme corresponding to the guide RNA, and a detection nucleic acid comprising a label that can be detected by activation of the genome editing enzyme, measuring a signal derived from the label, and determining the presence or absence of the single base substitution in the test nucleic acid from the intensity of the signal. The guide RNA comprises a single-stranded structure and an intramolecular hairpin structure on the 5 ' side of the single-stranded structure. The single-stranded structure contains a complementary sequence of the target sequence, and the length from the 5 ' end of the complementary sequence to the 1-base substitution site is 0 to 10 bases.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] An embodiment of the present invention relates to a method for detecting a single base substitution in a group of short RNAs having sequence diversity on the 3' side. [Background technology]

[0002] Genome editing has made it possible to perform various genetic modifications in microorganisms, animals, and plants. In recent years, in addition to editing by DNA cleavage, techniques for sequence-specific modification and labeling have also been developed by introducing functional domains such as DNA-modifying proteins. A typical genome editing technique involves using a DNA cleavage enzyme to induce specific double-stranded DNA breaks in target genes, and then precisely modifying the genes by utilizing the repair process.

[0003] On the other hand, it has been reported that microRNAs may be used to detect and identify various cancers. For example, it has been proposed to use the sequence ratio of single-nucleotide substitutions (i.e., single-nucleotide polymorphisms) in microRNAs as an indicator. However, general sequence analysis requires a lot of time and cost, making it difficult to apply to multi-sample diagnosis, such as primary screening. Summary of the Invention [Problem to be solved by the invention]

[0004] The problem to be solved by the present invention is to provide a technique for detecting single-base substitutions in a group of short RNAs having sequence diversity on the 3' side in a short time and at low cost. [Means for solving the problem]

[0005] A method according to an embodiment is a method for detecting a single-base substitution in a group of short-stranded RNAs having sequence diversity on the 3' side. The method includes the following steps: contacting a solution containing the group of short-stranded RNAs as test nucleic acids with a guide RNA containing a sequence complementary to a target sequence having a single-base substitution site, a genome editing enzyme corresponding to the guide RNA, and a label that becomes detectable upon activation of the genome editing enzyme; measuring a signal derived from the label; and determining the presence or absence of a single-base substitution in the test nucleic acid based on the intensity of the signal. The guide RNA comprises a single-stranded structure and an intramolecular hairpin structure on the 5' side of this single-stranded structure. The single-stranded structure comprises a sequence complementary to the target sequence. The length from the 5' end of the complementary sequence to the single-base substitution site is 0 to 10 bases. [Brief explanation of the drawings]

[0006] [Figure 1] 1 is a schematic diagram illustrating the flow of a method according to a first embodiment; [Figure 2] FIG. 1 is a schematic diagram illustrating the concept of a first embodiment. [Figure 3] FIG. 1 is a schematic diagram illustrating the concept of a first embodiment. [Figure 4] Graph showing experimental results. DETAILED DESCRIPTION OF THE INVENTION

[0007] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially identical components are denoted by the same reference numerals, and some of their descriptions may be omitted. The drawings are schematic, and the relationship between the thickness of each component and the planar dimensions, the thickness ratio of each component, etc. may differ from the actual ones.

[0008] (First embodiment) The first embodiment is a method for detecting single-base substitutions in a group of short RNAs with sequence diversity at the 3' end. As shown in Figure 1, the method includes the following three steps: contacting a solution containing a group of short-stranded RNAs with sequence diversity at the 3' end as test nucleic acids with a guide RNA containing a sequence complementary to a target sequence containing a single-base substitution site, a genome editing enzyme corresponding to the guide RNA, and a detection nucleic acid containing a label that can be detected upon activation of the genome editing enzyme (S11); measuring a signal derived from the label (S12); and The presence or absence of a single base substitution in the short-chain RNA group in the test nucleic acid is determined based on the signal intensity (S13).

[0009] The test nucleic acid may be a group of short-stranded RNAs having a single-base substitution site to be detected, or a group of nucleic acids containing such a group of short-stranded RNAs. For example, a solution containing such a test nucleic acid may be an aqueous solution containing nucleic acids derived from humans, other animals, or plants. The short-stranded RNA may be relatively short, for example, having a length of more than 10 bases, such as 100 to 50 bases, 15 to 30 bases, or 15 to 25 bases. Such short-stranded RNAs may be, for example, microRNAs, piRNAs, and siRNAs. A solution containing a group of short-stranded RNAs as test nucleic acids may also be referred to as a test solution.

[0010] The target nucleic acid 11 and the guide RNA 21 will be described with reference to FIG. 2. The target nucleic acid 11 includes a sequence diversity portion 32 on the 3' side and a target sequence 22a on the 5' side thereof. The target sequence 22a has a single-base substitution site 12a within its sequence. In other words, the portion of the target nucleic acid 11 on the 3' side of the target sequence 22a may be any sequence. Furthermore, the target nucleic acid 11 may include any sequence on the 5' side of the target sequence 22a. The target sequence 22a can be selected from, for example, sequences on a microRNA database or a portion of a naturally occurring miRNA sequence obtained from the results of miRNA sequence analysis. The target sequence 22a may be selected in advance as desired.

[0011] The guide RNA 21 includes a single-stranded structure 22b and an intramolecular hairpin structure 23. The single-stranded structure 22b includes a complementary sequence 22c of a target sequence 22a of the target nucleic acid 11. Therefore, the single-stranded structure 22b can also be said to include a target sequence recognition site 22c. The target sequence recognition site 22c has a single-base substitution site 12b at a position corresponding to the single-base substitution site 12a of the target nucleic acid 11. The length from the 5' end of the target sequence recognition site 22c to the single-base substitution site can be 0 to 10 bases. Here, the target sequence recognition site 22c can be, for example, a complementary sequence of the target sequence 22a. However, the single-stranded structure 22b may include one or two bases that are non-complementary to the target sequence 22a at a site other than the single-base substitution site 12b. While FIG. 2 shows an example in which the length of the target sequence recognition site and the length of the single-stranded structure are equal, this is not limiting, and the single-stranded structure may be longer. The intramolecular hairpin structure 23 is a genome editing enzyme recognition site (i.e., a direct repeat) that recognizes and binds to a corresponding genome editing enzyme. For example, the genome editing enzyme may be an enzyme having collateral activity, such as a Cas protein, for example, CRISPR-Cas13.

[0012] The sequence of the target sequence recognition site 22c of the guide RNA 21 is selected according to the sequence of the target nucleic acid to be detected. This allows the target nucleic acid to recognize and specifically bind to a specific group of short-stranded RNAs from a solution containing a group of short-stranded RNAs with sequence diversity on the 3' side. This activates the genome editing enzyme. Here, the single-base substitution site 12b of the target sequence recognition site 22c is selected to have the type of base to be detected. For example, a base corresponding to either the wild type or the mutant type may be selected. That is, even for single-nucleotide polymorphism sites, the genome editing enzyme is activated when the guide RNA 21 and the target nucleic acid 11 have complementary bases. This configuration allows the presence or absence of single-base substitutions in the target nucleic acid group and the state of the polymorphism to be detected while maintaining polymorphism without being selected based on the diversity on the 3' side. In the case of microRNAs, information on the presence of polymorphisms or mutations in sequences that share a target nucleic acid sequence, regardless of the miRNA name, can be useful for detecting or identifying diseases such as cancer.

[0013] FIG. 3 illustrates a specific example of distinguishing between a target mutant type A and a non-target wild-type C. For example, when determining the base sequence of a single-base substitution site for miR_1 contained in a test nucleic acid group, the sequence diversity portion includes the miR_1-standard (FIG. 3(a)) containing the standard sequence 32a and the miR_1-3'-modified (FIG. 3(b)) containing the modified sequence 32b, which are simultaneously target sequences. For each of these, for example, the single-base substitution site 12a is assumed to be the targeted mutant type A and the non-target wild-type C. In other words, a microRNA having a standard sequence containing a single-base substitution is miR_1-standard, specifically, the target nucleic acid 11a containing the mutant type A and the non-target nucleic acid 31a containing the wild-type C. A microRNA having a 3'-side modified sequence containing a single-base substitution is miR_1-3'-modified, specifically, the target nucleic acid 11b containing the mutant type A and the non-target nucleic acid 31b containing the wild-type C.

[0014] The guide RNA 21 has a complementary base U of the target mutant A at the single-base substitution site. Alternatively, wild-type C may be targeted, in which case the base at the single-base substitution site is the complementary base G of wild-type C. In this method, when the guide RNA 21 encounters the corresponding genome editing enzyme 35, they bind to each other to form a complex 37 (also referred to as a "guide RNA-genome editing enzyme complex"). When the target sequence recognition site 22c of the complex 37 encounters the target sequence 11a (i.e., the target nucleic acid 11) containing its complementary strand, a double strand is formed. Furthermore, the genome editing enzyme 35 is activated, and genome editing enzyme activity (i.e., nucleic acid cleavage activity) 38 is expressed, causing cleavage at the cleavage recognition site (not shown) of the target nucleic acid 11a.

[0015] For example, by forming a base sequence-specific duplex for such a target sequence, if the guide RNA 21 is a crRNA, it may bind to the target sequence as an identification molecule that can distinguish between single base differences, activating a genome editing enzyme (e.g., a Cas protein) and cleaving the target sequence.

[0016] Meanwhile, this reaction system contains a label that can be detected upon activation of the genome editing enzyme. An example of such a label is the detection nucleic acid 41a shown below. The detection nucleic acid 41a comprises a signal substance 42, a masking substance 45, and an oligonucleotide portion. The oligonucleotide portion comprises a cleavage recognition site for the genome editing enzyme. The length of the oligonucleotide portion can be, for example, 50 bases or less. The detection nucleic acid 41a is cleaved at the cleavage recognition site of the genome editing enzyme by the nucleic acid cleavage activity 38 of the genome editing enzyme 35 activated by double-strand formation. As a result, the signal from the signal substance 42, which was inhibited by the masking substance 45, becomes detectable. For example, the signal substance 42 may be a fluorescent substance, a chemiluminescent substance, a chromogenic substance, or the like. The signal possessed by such a substance becomes detectable when the detection nucleic acid 41a is cleaved by the cleavage activity 38 due to activation of the genome editing enzyme 35. Due to this configuration and function, the signal substance 42 can be said to be a substance that specifically transmits specific information.

[0017] The signal 43 from the signal substance 42 is detected by an appropriate detection mechanism or detection device depending on the type of the signal substance. For example, the method may be performed in a real-time PCR system or a microplate reader equipped with such a detection mechanism. The contacting (S11) can be performed by reacting for 1 to 120 minutes under conditions of 25°C to 55°C. The reaction temperature is preferably 37°C. Depending on the type of signal substance, the signal 43 may be detected over time in parallel with the reaction caused by the contacting, or may be measured once, 1 to 2 times, or more than 2 times, for example, 1 to 120 times, at different time points after the reaction has progressed for a certain period of time.

[0018] In the method, determining the presence or absence of a single-base substitution in the test nucleic acid may further include determining the presence or absence of a single-base substitution in the test nucleic acid based on the presence or absence of a difference between the signal of a standard nucleic acid and the signal of a nucleic acid derived from the test substance. The signal of the standard nucleic acid may be measured in advance, or may be measured before, after, or in parallel with the measurement of the signal from the test substance, i.e., the test nucleic acid. The presence or absence of a single-base substitution may be determined based on the presence or absence of a detection signal, for example, the presence or absence of fluorescence intensity, or the magnitude of the detection signal, for example, differences in fluorescence intensity. For example, even when a standard nucleic acid is not used, determination can be made based on the presence or absence of a detection signal or the relative magnitude of the detection signal. The standard nucleic acid can be designed based on a target sequence, for example, a microRNA sequence in a database containing the target sequence or a sequence obtained by sequence analysis such as next-generation sequencing. If desired, multiple types of guide RNAs 21 with different bases assigned to the single-base substitution site may be used in combination. In this case, determination may be made for each type of guide RNA 21 and then comprehensive determination may be made, or labels may be designed so that the signals to be detected can be distinguished for each type of RNA 21.

[0019] In conventional sequence analysis, detecting a single base substitution requires a lot of time and cost, making it difficult to apply to multi-sample diagnosis. However, according to the method of the embodiment, detection can be performed in a short time and at low cost. Furthermore, the test nucleic acid group to be detected is not differentiated based on the diversity on the 3' side, and it is possible to detect the presence or absence of a single base substitution and the state of polymorphism in the target nucleic acid group while maintaining diversity. Considering the sequence diversity on the 3' side of the single base substitution site in the target nucleic acid, for example, by using microRNA or the like as the test substance, can be used to detect or identify diseases such as cancer while also incorporating information on the diversity on the 3' side of the target nucleic acid.

[0020] (Second embodiment) The second embodiment is an assay kit for detecting single-base substitutions in a group of short RNAs with sequence diversity at the 3' end. The kit includes a guide RNA having a sequence complementary to a target nucleic acid containing a single-base substitution site, a genome editing enzyme corresponding to the guide RNA, and a label that can be detected upon activation of the genome editing enzyme.

[0021] 2 and 3, guide RNA 21 includes a single-stranded structure 22b and an intramolecular hairpin structure 23 on its 5' side. The single-stranded structure includes a complementary sequence 22c of the target sequence on its 5' side, and the length from the 5' end of complementary sequence 22c to the one-base substitution site is 0 to 10 bases.

[0022] The genome editing enzyme may be an enzyme having collateral activity, such as a Cas protein, for example, CRISPR-Cas13, etc. The label that becomes detectable upon activation of such a genome editing enzyme may be, for example, the detection nucleic acid 41a described below.

[0023] As shown in FIG. 3 , the detection nucleic acid 41a comprises a signal substance 42, a masking substance 45, and an oligonucleotide portion. The oligonucleotide portion comprises a cleavage recognition site for the genome editing enzyme. The length of the oligonucleotide portion can be, for example, 50 bases or less. The detection nucleic acid 41a is cleaved at the cleavage recognition site of the genome editing enzyme by the nucleic acid cleavage activity 38 of the genome editing enzyme 35 activated by double-strand formation. As a result, the signal from the signal substance 42 that was inhibited by the masking substance 45 becomes detectable. For example, the signal substance 42 can be a fluorescent substance, a chemiluminescent substance, a chromogenic substance, or the like. For example, examples of a fluorescent substance and a quencher as the masking substance 45 are as follows: Fluorescent materials: FAM, HEX, VIC, ROX, Cy5, Texas Red, JOE Masking materials: Iowa Black, BHQ, TAMRA, ZEN For example, at least one fluorescent substance and one masking substance selected from the above group may be used in combination, such as, but not limited to, FAM and Iowa Black® FQ, FAM and BHQ-1, ROX and Iowa Black® RQ, ROX and BHQ-2, Cyanine 5 and Iowa Black® RQ, and Cyanine 5 and BHQ-2.

[0024] The assay kit may include each component in a container in a dried state, such as by freeze-drying. Alternatively, each component may be maintained in an appropriate solution and stored in an appropriate container, which may then be provided as an assay kit. Providing an assay kit enables detection of single-base substitutions in a short time and at low cost. Furthermore, the test nucleic acids to be detected are not differentiated based on 3'-side diversity, and the presence or absence of single-base substitutions and polymorphisms in target nucleic acids can be detected while maintaining diversity. The importance of considering sequence diversity on the 3' side of the single-base substitution site in target nucleic acids is emphasized by using, for example, microRNA as a test substance, which can be used to detect or identify diseases and cancers while also incorporating information on the 3'-side diversity of the target nucleic acid.

[0025] [example] An example of carrying out the method according to the embodiment will be described below.

[0026] Experiment 1. Detection of single base substitutions in two microRNA groups with sequence diversity at the 3' end Four types of microRNAs were prepared, as shown in Figure 3. The microRNA used was miR_1. The sequence diversity portion targeted miR_1-standard (Figure 3(a)) containing the standard sequence 32aUUUGUG (SEQ ID NO: a) and miR_1-3'-modified (Figure 3(b)) containing the modified sequence 32bUGUUGU (SEQ ID NO: b). Furthermore, the single-base substitution site 12a was designated as the targeted mutant type A and the non-target wild-type C. That is, the microRNA with the standard sequence containing the single-base substitution was miR_1-standard, specifically, the target nucleic acid 11a (SEQ ID NO: 1) containing the mutant type A and the non-target nucleic acid 31a (SEQ ID NO: 2) containing the wild-type C. The microRNA with the 3'-side modified sequence containing the single-base substitution was miR_1-3'-modified, specifically, the target nucleic acid 11b (SEQ ID NO: 3) containing the mutant type A and the non-target nucleic acid 31b (SEQ ID NO: 4) containing the wild-type C. These nucleic acids were synthesized and the resulting nucleic acids were maintained in their respective buffer solutions.

[0027] Similarly, guide RNA21 (SEQ ID NO: 5) was synthesized. Guide RNA21 contains the target mutant A base at the single-base substitution site. Separately, a fluorescent substance FAM was bound to the 5' end of the oligonucleotide shown in SEQ ID NO: c, and Iowa Black (registered trademark) FQ was bound to the 3' end as a quencher to obtain a detection nucleic acid.

[0028] The full-length base sequences of these are shown in Table 1. Furthermore, Table 2 lists these in association with the guide RNA (abbreviated as "gRNA") for mutation detection, target sequence, and non-target sequence. Table 3 summarizes the design features of the miR-1 standard. Specifically, for each of the miR-1 standard and miR-1-3' modified gRNAs, the number of complementary bases in the sequence downstream of the mutation is 4, the number of diversity-tolerant sequences on the 3' side is 6, the insertion position of the mutation recognition site is the miRNA sequence site, i.e., the fifth position from the 5' end of the complementary sequence of the target sequence, and the insertion position of the mismatch contained in the complementary sequence of the target sequence is the ninth position from the 5' end of the complementary sequence of the target sequence (Table 3). Furthermore, a poly-U reporter nucleic acid was used as the reporter nucleic acid, and the fluorescent substance FAM and quencher Iowa Black® FQ were used; these are summarized in Table 4. The complementary base pair is a combination of AU and GC.

[0029] [Table 1]

[0030] [Table 2]

[0031] [Table 3]

[0032] [Table 4]

[0033] The genome editing enzyme used was the Cas protein CRISPR-Cas13 (Genscript, product number Z03472). The recognition sequence, cleavage sequence, and guide RNA structure corresponding to the genome editing enzyme can be selected as needed.

[0034] The target nucleic acid 11a (SEQ ID NO: 1, "target (mutant A)" in FIG. 3(a)) containing mutant A and the non-target nucleic acid 31a (SEQ ID NO: 2, "non-target (wild-type C)" in FIG. 3) containing wild-type C are used. The microRNA having a 3'-side modified sequence containing a single base substitution is miR_1-3' modified. Specifically, the target nucleic acid 11b (SEQ ID NO: 3, "target (mutant A)" in FIG. 3(b)) containing mutant A and the non-target nucleic acid 31b (SEQ ID NO: 4, "non-target (wild-type C)" in FIG. 3(b)) were each diluted to 1 × 10 10 The mixture was suspended in buffer at a concentration of 100 copies / μL. 2 μL of this mixture was dispensed into a microplate. 18 μL of a reaction solution containing guide RNA21 (SEQ ID NO: 5) at a final concentration of 100 nM in the final reaction solution and CRISPR-Cas13 at a final concentration of 100 nM in the final reaction solution was added and incubated at 37°C for 5 minutes. Fluorescence intensity was measured using a plate reader at an excitation wavelength of 470 nm and an emission wavelength of 520 nm, and relative fluorescence intensity was calculated using a real-time PCR system. The results are shown in Figure 4 and Table 5.

[0035] [Table 5]

[0036] The results of FIG. 4 and Table 5 demonstrate that the presence or absence of a single base substitution can be detected regardless of differences in the 3'-terminal sequence, that is, regardless of the sequence diversity present on the 3'-terminal side.

[0037] Further exemplary embodiments are described below. [1] A method for detecting a single base substitution in a group of short RNAs having sequence diversity on the 3' side, comprising: contacting a solution containing a group of short-stranded RNAs with sequence diversity on the 3' side as test nucleic acids with a guide RNA containing a complementary sequence of a target sequence having a single-base substitution site, a genome editing enzyme corresponding to the guide RNA, and a label that can be detected upon activation of the genome editing enzyme; measuring a signal derived from said label; and determining the presence or absence of a single-base substitution in the group of short-chain RNAs in the test nucleic acid based on the intensity of the signal, The guide RNA comprises a single-stranded structure and an intramolecular hairpin structure on the 5' side of the single-stranded structure, the single-stranded structure comprises a complementary sequence of the target sequence on its 5' side, and the length from the 5' end of the complementary sequence to the single-base substitution site is 0 to 10 bases. [2] The method described in [1], wherein the intramolecular hairpin structure is a genome editing enzyme recognition site. [3] The method according to [1] or [2], wherein the short-chain RNA is a microRNA. [4] The method according to any one of [1] to [3], wherein the genome editing enzyme has collateral activity. [5] The method according to any one of [1] to [4], wherein the label is a detection nucleic acid comprising a signal substance that emits a detection signal, a masking substance that inhibits the detection signal, and an oligonucleotide to which the signal substance and the masking substance are attached. [6] The method according to [5], wherein the length of the oligonucleotide is 50 bases or less. [7] The method according to [5], wherein the ligand is a fluorescent substance and the masking substance is a quencher. [8] The method according to any one of [1] to [7], wherein the signal from the detection nucleic acid is measured by a means selected from the group consisting of a real-time PCR system, a microplate reader, a digital PCR system, a fluorescence microscope, or a fluorescence detection device. [9] The method according to any one of [1] to [8], wherein the determination of the presence or absence of the single-base substitution is carried out by determining the presence or absence of the single-base substitution in the test nucleic acid based on the presence or absence of a difference between a detection signal obtained in a similar manner for a standard nucleic acid and a signal obtained from the test nucleic acid.

[10] An assay kit for use in the method according to any one of [1] to [9], comprising: a guide RNA comprising a sequence complementary to a target sequence comprising a single base substitution site; a genome editing enzyme corresponding to the guide RNA; and a detection nucleic acid comprising a signal substance that can be detected by activation of the genome editing enzyme and a base sequence recognized by the genome editing enzyme; An assay kit in which the guide RNA comprises a single-stranded structure and an intramolecular hairpin structure on its 5' side, the single-stranded structure comprises a complementary sequence to the target sequence on its 5' side, and the length from the 5' end of the complementary sequence to the single-base substitution site is 0 to 10 bases.

[0038] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0039] 11...target nucleic acid, 11a, 11b...target nucleic acid (mutant), 12, 12a, 12b...single base substitution site, 21...guide RNA, 22a...target sequence, 22b...single-stranded structure, 22c...target sequence recognition site, 23...intramolecular hairpin structure (genome editing enzyme recognition site (direct repeat)), 31a, 31b...non-target nucleic acid (wild type), 32, 32a, 32b...sequence diversity portion, 35...genome editing enzyme, 37...[guide RNA-genome editing enzyme] complex, 38...genome editing enzyme activity (nucleic acid cleavage activity), 41a, 41b...label (detection nucleic acid), 42...signal substance, 43...detection signal, 45...masking substance

Claims

1. A method for detecting a single base substitution in a group of short RNAs having sequence diversity on the 3' side, comprising: - Contacting a solution containing the group of short-chain RNAs as test nucleic acids with a guide RNA containing a complementary sequence of a target sequence having a single-base substitution site, a genome editing enzyme corresponding to the guide RNA, and a label that can be detected by activation of the genome editing enzyme; - measuring the signal derived from said label; and determining the presence or absence of a single-base substitution in the group of short-stranded RNAs in the test nucleic acid based on the intensity of the signal, The guide RNA comprises a single-stranded structure and an intramolecular hairpin structure on the 5' side of the single-stranded structure, the single-stranded structure comprises a complementary sequence of the target sequence, and the length from the 5' end of the complementary sequence to the single-base substitution site is 0 to 10 bases.

2. The method of claim 1, wherein the intramolecular hairpin structure is a genome editing enzyme recognition site.

3. The method of claim 1, wherein the short RNA is a microRNA.

4. The method of claim 1, wherein the genome editing enzyme has collateral activity.

5. The method according to claim 1, wherein the label is a detection nucleic acid comprising a signal substance that emits a detection signal, a masking substance that inhibits the detection signal, and an oligonucleotide to which the signal substance and the masking substance are attached.

6. The method of claim 5, wherein the oligonucleotide is 50 bases or less in length.

7. 6. The method of claim 5, wherein the signal substance is a fluorescent substance and the masking substance is a quencher.

8. 2. The method of claim 1, wherein the signal is measured by a means selected from the group consisting of a real-time PCR system, a microplate reader, a digital PCR system, a fluorescence microscope, and a fluorescence detection device.

9. The method according to claim 1, wherein the presence or absence of the single base substitution is determined by determining the presence or absence of a single base substitution in the test nucleic acid based on the presence or absence of a difference between a detection signal obtained in a similar manner for a standard nucleic acid and a signal obtained from the test nucleic acid.

10. An assay kit for use in the method of claim 1, comprising: a guide RNA comprising a sequence complementary to a target sequence comprising a single-base substitution site; a genome editing enzyme corresponding to the guide RNA; and a label that can be detected upon activation of the genome editing enzyme; The assay kit includes a guide RNA having a single-stranded structure and an intramolecular hairpin structure on the 5' side of the single-stranded structure, the single-stranded structure having a complementary sequence to the target sequence, and the length from the 5' end of the complementary sequence to the single-base substitution site is 0 to 10 bases.

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