Use of Cas proteins, methods for detecting target nucleic acid molecules, and kits

Abstract

The present invention provides a method and kit for detecting a target nucleic acid molecule using a Cas protein, which comprises adding a guide RNA, Cas12a, and a nucleic acid probe to a reaction system containing the target nucleic acid molecule to be detected, and detecting the nucleic acid probe after the reaction is complete. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention is in the field of biotechnology, and in particular, to a method for detecting a target nucleic acid molecule. [Background technology]

[0002] Specific nucleic acid detection methods have important application value in pathogen detection, genetic disease detection, and the like. In one aspect of pathogen detection, because each pathogenic microorganism has its own unique, characteristic nucleic acid molecular sequence, it is possible to develop specific species of nucleic acid molecular detection, also known as nucleic acid diagnostics (NAD), which is extremely important in fields such as food safety, environmental microbial contamination detection, and human pathogen infection. Another aspect is the detection of single nucleotide polymorphisms (SNPs) in humans or other species. Understanding the relationship between genetic variation and biological function at the genome level brings new perspectives to modern molecular biology. Because SNPs are closely related to biological function, evolution, and disease, the development of SNP detection and analysis technologies is particularly important.

[0003] Currently, many NAD methods are established primarily for the detection of specific DNA molecules, with several methods also available for RNA molecules. DNA molecules are generally very stable, requiring careful handling of test samples obtained from a complex array of biological samples. RNA is highly susceptible to degradation and must be handled with care. In the 1970s, detection methods using restriction endonuclease digestion were established. Subsequently, Southern, Northern, and dot-blot hybridization techniques were developed for the specific detection of nucleic acid molecules. In 1985, PCR became a standard laboratory technique, bringing about exponential improvements in molecular biology. Currently, the detection of specific nucleic acid molecules typically requires two steps: the first step is amplification of the target nucleic acid, and the second step is detection of the target nucleic acid. PCR technology was the first established and is currently the most commonly used amplification method. Currently, fluorescently labeled probes have been introduced to PCR to enable real-time detection of target amplification, a process known as real-time PCR. Real-time PCR is not only a rapid and sensitive detection method, but also a quantitative analysis method. In addition to PCR amplification, many alternative methods have been established, including ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and recombinase polymerase amplification (RPA). The advantage of many of these alternative methods is that they are isothermal; that is, only one temperature is required to complete the reaction, without the need for a thermal cycling device like that used in PCR. Among nucleic acid detection methods, in addition to real-time PCR, which can complete amplification and detection directly, FISH (fluorescence in situ hybridization) is the most commonly used detection method, which involves in situ hybridization of labeled molecular probes with complementary target sequences. Furthermore, detection methods such as next-generation sequencing and Oxford nanopore sequencing have also been developed, but these methods typically require expensive laboratory equipment.

[0004] The detection of SNPs also requires amplification by methods such as PCR to obtain sufficient fragments of the SNP site-containing region for further detection. Commonly used methods include primer extension, hybridization, ligation, and enzymatic cleavage. After the above methods are completed, specific detection methods such as mass spectrometry, fluorescence, chemiluminescence, etc., must be used.

[0005] As mentioned above, many detection methods have been developed for nucleic acid detection. However, in certain cases, such as the rapid on-site detection of pathogens or the rapid detection of drug-susceptibility SNPs, faster, simpler, and cheaper detection methods remain an important development trend. In 2016, Collins et al. developed a rapid and inexpensive method for detecting Zika virus based on the CRISPR-Cas9's ability to specifically recognize and cleave target sequences. In 2017, Feng Zhang et al. established a rapid nucleic acid detection method utilizing the "collateral action" feature of CRISPR-Cas13a. "Collateral action" refers to the random cleavage of non-target RNAs by Cas13a after binding to a specific target RNA (in this case, the RNA molecule is designed as an RNA fluorescent reporter system). This rapid target RNA detection was achieved by combining it with the isothermal amplification technique RPA. Feng Zhang's team named this detection method SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking). Because the SHERLOCK method involves binding to an RNA template, if DNA detection is required, the DNA must first be transcribed into an RNA template for detection, and given the instability of RNA, this method undoubtedly increases the difficulty of operation.

[0006] In 2015, Feng Zhang et al. discovered a new CRISPR-associated endoproteinase, Cas12a (previously known as Cpf1), which, like the commonly used Cas9 protein, is an RNA-guided specific DNA endonuclease; however, compared to Cas9, Cas12a has unique properties, such as requiring only crRNA to induce specific cleavage of double-stranded DNA, resulting in the production of sticky ends. Summary of the Invention [Means for solving the problem]

[0007] It is an object of the present invention to provide a method for detecting a target nucleic acid molecule.

[0008] Another object of the present invention is to provide the use of Cas proteins in methods for detecting target nucleic acid molecules.

[0009] In a first aspect of the present invention, a kit is provided comprising a guide RNA, a Cas protein, a nucleic acid probe, and a buffer solution.

[0010] The method for detecting a target nucleic acid molecule includes adding a guide RNA, a Cas protein, a nucleic acid probe, and a buffer solution to a reaction system containing the target nucleic acid molecule to be detected, and then detecting the target nucleic acid (particularly by detecting fluorescence intensity).

[0011] Preferably, the Cas protein is Cas12a or a Cas protein having collateral single-strand DNA cleavage activity similar to that of Cas12a.

[0012] Preferably, the Cas protein is Cas12a.

[0013] The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a.

[0014] Preferably, the Cas12a is LbCas12a.

[0015] Preferably, guide RNA refers to an RNA that guides a Cas protein to specifically bind to a target DNA.

[0016] In another preferred embodiment, the nucleic acid probe is a single-stranded DNA, and the single-stranded DNA is preferably a fluorescently labeled single-stranded DNA, and the single-stranded DNA is preferably a fluorescent probe labeled with the fluorescent group HEX at the 5' end and the quenching group BHQ1 at the 3' end.

[0017] In another preferred embodiment, the method for detecting the nucleic acid probe is preferably a fluorescence detection method, and the fluorescence detection method is preferably a detection method using a microplate reader or a fluorescence spectrophotometer.

[0018] Preferably, the target nucleic acid molecule to be detected in the reaction system is obtained after amplification.

[0019] Preferably, the detection method of the present invention can be used to detect pathogenic microorganisms, genetic mutations, or specific target DNA.

[0020] In another preferred embodiment, the Cas protein comprises Cas12b (C2c1).

[0021] In a second aspect of the present invention there is provided the use of a Cas protein in a method for the detection of a target nucleic acid molecule or in the preparation of a formulation for the detection of a target nucleic acid molecule.

[0022] In another preferred embodiment, when the target DNA, guide RNA, and Cas protein form a ternary complex, this complex cleaves other single-stranded DNA molecules in the system.

[0023] Preferably, guide RNA refers to an RNA that guides a Cas protein to specifically bind to a target DNA.

[0024] In a third aspect of the present invention, a kit is provided comprising a guide RNA, a Cas protein, and a nucleic acid probe.

[0025] In another preferred embodiment, the kit further comprises a buffer solution.

[0026] In a fourth aspect of the present invention, there is provided a detection system for detecting a target nucleic acid molecule, the system comprising: (a) a Cas protein that is Cas12a or a Cas protein that has collateral single-strand DNA cleavage activity similar to that of Cas12a; (b) a guide RNA that guides the Cas protein to specifically bind to a target nucleic acid molecule; and (c) a nucleic acid probe that is single-stranded DNA; A detection system is provided in which the target nucleic acid molecule is target DNA.

[0027] In another preferred embodiment, the detection system further comprises (d) a buffer solution.

[0028] In another preferred embodiment, the detection system further comprises a target nucleic acid molecule to be detected.

[0029] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 100 copies / microliter or 10 15 copies / microliter, preferably 1 to 10 copies / microliter, more preferably 1 to 5 copies / microliter.

[0030] In another preferred embodiment, the detection system has a molar ratio of nucleic acid probe to target nucleic acid molecule of 10 3 :1~10 14 :1, preferably 10 4 :1~10 7 :1.

[0031] In another preferred embodiment, the detection site of the target nucleic acid molecule is located at positions 1 to 12 downstream of the PAM sequence of the guide RNA.

[0032] In another preferred embodiment, the length of the guide RNA is 15 to 30 nt, preferably 15 to 18 nt.

[0033] In another preferred embodiment, the target DNA comprises cDNA.

[0034] In another preferred embodiment, the target DNA is selected from the group consisting of single-stranded DNA, double-stranded DNA, or a combination thereof.

[0035] In another preferred embodiment, the nucleic acid probe comprises a fluorescent group and a quencher group.

[0036] In another preferred embodiment, the fluorescent group and the quencher group are each independently located at the 5' end, the 3' end, and in the middle of the nucleic acid probe.

[0037] In another preferred embodiment, the length of the nucleic acid probe is 3 to 300 nt, preferably 5 to 100 nt, more preferably 6 to 50 nt, and most preferably 8 to 20 nt.

[0038] In another preferred embodiment, the target nucleic acid molecule comprises a target nucleic acid molecule derived from the group consisting of a plant, an animal, an insect, a microorganism, a virus, or a combination thereof.

[0039] In another preferred embodiment, the target DNA is artificially synthesized or naturally occurring DNA.

[0040] In another preferred embodiment, the target DNA comprises wild-type or mutant DNA.

[0041] In another preferred embodiment, the target DNA comprises DNA obtained by reverse transcription or amplification of RNA (eg, cDNA, etc.).

[0042] In another preferred embodiment, Cas12a is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, or a combination thereof; more preferably, Cas12a is LbCas12a.

[0043] In another preferred embodiment, the Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from the group consisting of Cas12b (i.e., C2c1).

[0044] In another preferred embodiment, the Cas12b protein is selected from the group consisting of AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus), AkCas12b (Alicyclobacillus kakegawensis), AmCas12b (Alicyclobacillus macrosporangiidus), AhCas12b (Alicyclobacillus herbarius), and AcCas12b (Alicyclobacillus contaminans).

[0045] In another preferred embodiment, the nucleic acid probe comprises a single-stranded DNA having a detectable label.

[0046] In another preferred embodiment, the single-stranded DNA is a fluorescently labeled and biotin-labeled single-stranded DNA.

[0047] In another preferred embodiment, the single-stranded DNA is a fluorescently labeled single-stranded DNA.

[0048] In another preferred embodiment, the single-stranded DNA is a fluorescent probe labeled at the 5' end with the fluorescent group HEX and at the 3' end with the quenching group BHQ1.

[0049] In a fifth aspect of the present invention, there is provided a kit for detecting a target nucleic acid molecule, the kit comprising: i) a first container, and a Cas protein in the first container, wherein the Cas protein is Cas12a or a Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a; ii) an optional second container, and a guide RNA in the second container, wherein the guide RNA guides the Cas protein to specifically bind to the target nucleic acid molecule; iii) a third container and a nucleic acid probe in the third container; iv) an optional fourth container and a buffer solution within the fourth container; A kit is provided wherein the target nucleic acid molecule is target DNA.

[0050] In another preferred embodiment, two, three, or four (or all) of the first, second, third, and fourth containers may be the same or different containers.

[0051] In another preferred embodiment, the nucleic acid probe comprises a fluorescent group and a quencher group.

[0052] In a sixth aspect of the present invention, there is provided a method for detecting the presence of a target nucleic acid molecule in a sample, comprising the steps of: (a) providing a detection system for detecting a target nucleic acid molecule according to the fourth aspect of the present invention, the detection system further comprising a sample to be detected; (b) detecting whether the nucleic acid probe in the detection system is cleaved by the Cas protein, wherein the cleavage is trans-cleavage of collateral single-stranded DNA; If the nucleic acid probe is cleaved by the Cas protein, it indicates the presence of the target nucleic acid molecule in the sample; if the nucleic acid probe is not cleaved by the Cas protein, it indicates the absence of the target nucleic acid molecule in the sample. Methods for detecting are provided.

[0053] In another preferred embodiment, the sample to be detected includes an unamplified sample and an amplified (or nucleic acid amplified) sample.

[0054] In another preferred embodiment, the sample to be detected is a sample obtained by amplification.

[0055] In another preferred embodiment, the method for amplifying nucleic acids is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2.

[0056] In another preferred embodiment, the PCR includes high temperature PCR, ambient temperature PCR, and low temperature PCR.

[0057] In another preferred embodiment, the method is used to detect whether SNPs, point mutations, deletions, and / or insertions are present in the nucleic acid at the target site.

[0058] In another preferred embodiment, when the upstream and downstream of the target site (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, more preferably in the range of -10 nt to +10 nt) lack a PAM sequence, nucleic acid amplification is performed using a PAM-introducing primer.

[0059] In another preferred embodiment, the PAM-introducing primer has the structure of formula I in the 5'-3' portion: P1-P2-P3(I) having the structure During the ceremony, P1 is a 5' segment sequence located at the 5' end, which is complementary or non-complementary to the sequence of the target nucleic acid molecule; P2 is the PAM sequence, P3 is a 3' segment sequence located at the 3' end and complementary to the sequence of the target nucleic acid molecule.

[0060] In another preferred embodiment, the PAM primer specifically binds upstream or downstream of the target nucleic acid molecule.

[0061] In another preferred embodiment, the length of P1 is 0 to 20 nt.

[0062] In another preferred embodiment, the length of P3 is 5 to 20 nt.

[0063] In another preferred embodiment, the length of the PAM primer is 18 to 50 nt, preferably 20 to 35 nt.

[0064] In another preferred embodiment, complementarity includes full complementarity and partial complementarity.

[0065] In another preferred embodiment, at least one primer comprising a PAM sequence is used for nucleic acid amplification.

[0066] In another preferred embodiment, when the upstream and downstream of the target site (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, more preferably in the range of -10 nt to +10 nt) contain a PAM sequence, primers with or without a PAM sequence can be used, and the amplification product contains a PAM sequence.

[0067] In another preferred embodiment, the detection in step (b) comprises a fluorescent detection method.

[0068] In another preferred embodiment, the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer for detection.

[0069] In a seventh aspect of the present invention, there is provided the use of a Cas protein in the preparation of a detection reagent or kit for detecting a target nucleic acid molecule based on collateral single-stranded DNA cleavage, wherein the Cas protein is Cas12a or a Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a.

[0070] In another preferred embodiment, Cas12a is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, or a combination thereof; more preferably, Cas12a is LbCas12a.

[0071] In another preferred embodiment, the Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from the group consisting of Cas12b (or C2c1).

[0072] In another preferred embodiment, the Cas12b protein is selected from the group consisting of AacCas12b. It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features detailed below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here further due to length limitations. In certain embodiments, for example, the following are provided: (Item 1) A method for detecting a target nucleic acid molecule, the method comprising adding a guide RNA, a Cas protein, a nucleic acid probe, and a buffer solution to a system containing the target nucleic acid molecule to be detected, and then detecting the nucleic acid probe. (Item 2) the Cas protein is Cas12a or a Cas protein having collateral single-strand DNA cleavage activity similar to that of Cas12a; the Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a, and the Cas12a is preferably LbCas12a; Item 1. A method for detecting a target nucleic acid molecule according to item 1. (Item 3) 2. The method for detecting a target nucleic acid molecule according to Item 1, wherein the guide RNA refers to an RNA that guides the Cas protein to specifically bind to a target DNA. (Item 4) The nucleic acid probe is a single-stranded DNA, and the single-stranded DNA is preferably a fluorescently labeled single-stranded DNA. The single-stranded DNA is preferably a fluorescent probe labeled with a fluorescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end, and preferably The method for detecting the nucleic acid probe is preferably a fluorescence detection method, and the fluorescence detection method is preferably a detection method using a microplate reader or a fluorescence spectrophotometer. Item 1. A method for detecting a target nucleic acid molecule according to item 1. (Item 5) 5. The method for detecting a target nucleic acid molecule according to any one of items 1 to 4, wherein the target nucleic acid molecule to be detected in the reaction system is obtained by amplification. (Item 6) 6. The method for detecting a target nucleic acid molecule according to item 5, which can detect pathogenic microorganisms, gene mutations, or specific target DNA. (Item 7) 2. The method of claim 1, wherein the Cas protein comprises Cas12b (i.e., C2c1). (Item 8) Use of Cas proteins in methods for detecting target nucleic acid molecules. (Item 9) 9. The use according to item 8, wherein when the target DNA, the guide RNA, and the Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system, and preferably the guide RNA refers to an RNA that guides the Cas protein to specifically bind to the target DNA. (Item 10) A kit for detecting a target nucleic acid molecule, comprising a guide RNA, a Cas protein, and a nucleic acid probe. (Item 11) 1. A detection system for detecting a target nucleic acid molecule, comprising: (a) a Cas protein, which is Cas12a or a Cas protein having collateral single-strand DNA cleavage activity similar to that of Cas12a; (b) a guide RNA that guides the Cas protein to specifically bind to the target nucleic acid molecule; and (c) a nucleic acid probe that is single-stranded DNA; The above detection system, wherein the target nucleic acid molecule is a target DNA. (Item 12) Item 12. The detection system of item 11, wherein the Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from the group consisting of Cas12b (i.e., C2c1). (Item 13) 12. The detection system of claim 11, wherein the nucleic acid probe comprises a single-stranded DNA having a detectable label. (Item 14) 1. A kit for detecting a target nucleic acid molecule, comprising: i) a first container and a Cas protein in the first container, wherein the Cas protein is Cas12a or a Cas protein having collateral single-stranded DNA cleavage activity similar to that of the Cas12a; ii) an optional second container, and a guide RNA in the second container, wherein the guide RNA guides the Cas protein to specifically bind to the target nucleic acid molecule; iii) a third container and a nucleic acid probe in the third container; iv) an optional fourth container, and a buffer solution in said fourth container; The above kit, wherein the target nucleic acid molecule is a target DNA. (Item 15) 1. A method for detecting the presence of a target nucleic acid molecule in a sample, comprising the steps of: (a) providing a detection system for detecting the target nucleic acid molecule according to item 11, wherein the detection system further comprises a sample to be detected; (b) detecting whether the nucleic acid probe of the detection system is cleaved by a Cas protein, wherein the cleavage is trans-cleavage of collateral single-stranded DNA; If the nucleic acid probe is cleaved by the Cas protein, it indicates the presence of the target nucleic acid molecule in the sample; if the nucleic acid probe is not cleaved by the Cas protein, it indicates the absence of the target nucleic acid molecule in the sample. Methods for detecting prophase. (Item 16) 16. The method of claim 15, wherein the method for amplifying the nucleic acid is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2. (Item 17) Item 16. The method according to Item 15, wherein nucleic acid amplification is performed using a PAM-introducing primer when the upstream and downstream of the target site (within a range of -20 nt to +20 nt, preferably within a range of -15 nt to +15 nt, more preferably within a range of -10 nt to +10 nt) lack a PAM sequence. (Item 18) The PAM-introducing primer has a structure represented by the formula I in the 5'-3' region. P1-P2-P3(I) having the structure During the ceremony, P1 is a 5' segment sequence located at the 5' end, which is complementary or non-complementary to the sequence of the target nucleic acid molecule; P2 is the PAM sequence, P3 is a 3' segment sequence located at the 3' end and complementary to the sequence of the target nucleic acid molecule; Item 17. The method according to item 17. (Item 19) Item 16. The method according to Item 15, wherein when the PAM sequence is contained upstream and downstream of the target site (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, more preferably in the range of -10 nt to +10 nt), a primer containing or not containing the PAM sequence can be used, and the amplification product contains the PAM sequence. (Item 20) Use of a Cas protein in the preparation of a detection reagent or kit for detecting a target nucleic acid molecule based on collateral single-stranded DNA cleavage, wherein the Cas protein is Cas12a or a Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a. (Item 21) 21. The use of item 20, wherein the Cas protein having collateral single-stranded DNA cleavage activity similar to that of Cas12a is selected from the group consisting of Cas12b (or C2c1). [Brief explanation of the drawings]

[0073] [Figure 1] Figure 1 shows the cis-cleavage properties of Cas12a in cleaving target single-stranded DNA. [Figure 2] We demonstrate that Cas12a does not depend on the PAM sequence required to make double-stranded breaks when cleaving target single-stranded DNA. [Figure 3] Figure 1 shows the trans-cleavage properties of Cas12a in cleaving single-stranded DNA. [Figure 4] We present ten different sources of tested Cas12as, all of which have cis- and trans-cleavage activity against single-stranded DNA. [Figure 5]Single-site mutagenesis experiments of Cas12a identify sites likely involved in the cis- and trans-cleavage activity of Cas12a against single-stranded DNA. [Figure 6] 1 shows the structures of Cas12a and Cas12b (i.e., C2c1) monomers and their complexes with guide RNA and target DNA. [Figure 7] The fluorescence values ​​obtained by different Cas12as assays using specific double-stranded DNA substrates and single-stranded DNA (HEX-N12-BHQ1) as a fluorescent detection probe are shown. A negative control group was used without the specific substrate. [Figure 8] 1 shows a schematic flow chart of the HOLMES method for detecting target DNA based on target DNA amplification and the trans-cleavage activity of Cas12a on collateral single-stranded DNA. [Figure 9] We demonstrate sensitivity testing of target DNA by using FnCas12a or LbCas12a directly or in combination with the HOLMES method. [Figure 10] Figure 1 shows the fluorescence detection values ​​of target sequences with different single point mutations detected by the HOLMES method using crRNAs with different guide sequence lengths in combination with FnCas12a or LbCas12a. [Figure 11] By using a FAM-labeled fluorescent probe and 10 Cas12a proteins, we tested whether the FAM-labeled single-stranded DNA probe was trans-cleaved after the addition of target single-stranded DNA. [Figure 12] Using HEX-N12-BHQ1 as a probe and 10 Cas12a proteins, the fluorescence value was tested after adding target single-stranded DNA. [Figure 13](A) HOLMES detection values ​​are shown for a single-stranded DNA fluorescent probe, both ends of which are labeled with HEX and BHQ1, using a gyrB gene fragment as the target sequence. Different concentrations of pure cultured Escherichia coli MG1655 are used as the positive control template. The fluorescence response of Escherichia coli MG1655 decreases with decreasing concentration. (B) Detection values ​​for environmental water samples from different locations. [Figure 14] 1 shows a schematic flow chart of the HOLMES method for detecting SNPs and the fluorescence detection values ​​of five SNP sites. [Figure 15] This shows the fluorescence detection values ​​of the main regions of the TP53 gene (a cancer-related gene) detected by the HOLMES method. [Figure 16] The detection values ​​of five SNP sites (associated with gout) detected by the HOLMES method are shown. [Figure 17] The detection value of one SNP site (associated with gout) detected by the HOLMES method is shown, and the samples are from 21 volunteers. [Figure 18] 1 shows an example primer design scheme of the present invention that can be used for HOLMES detection of SNPs at any site. [Figure 19]A combination of LAMP and HOLMES was used to detect Escherichia coli in the system. (A) Electropherogram of the gyrB gene of Escherichia coli amplified by LAMP. A total of two sets of primers, gyrB-1 and gyrB-2, were used for amplification. gyrB is a characteristic gene of Escherichia coli. (B) The HOLMES detection system was used to detect the LAMP amplification product. Negative control: The sample was sterile water, and gyrB-1 amplification primers were used to amplify or detect the gyrB gene product; gyrB-1: The sample was Escherichia coli to be detected, and the first set of gyrB gene amplification primers were used to amplify or detect the gyrB gene product; gyrB-2: The sample was Escherichia coli to be detected, and the second set of gyrB gene amplification primers were used to amplify or detect the gyrB gene product. [Figure 20] The genotype of human HEK293T cells was detected using a combination of LAMP and HOLMES. (A) Electropherograms of the corresponding SNP detection templates of human HEK293T cells amplified by LAMP. Negative control: The sample was sterile water, and the results were obtained by amplification using rs5082 amplification primers; rs5082: The sample was the whole genome of human HEK293T cells, and the results were obtained by amplification using rs5082 amplification primers; rs1467558: The sample was the whole genome of human HEK293T cells, and the results were obtained by amplification using rs1467558 amplification primers. (B) The HOLMES detection system was used to detect the LAMP amplification products. The rs5082 site was detected using two crRNAs, crRNA-G and crRNA-T, respectively (Sequence Table 5), and the rs1467558 site was detected using two crRNAs, crRNA-C and crRNA-T, respectively (Sequence Table 5). [Figure 21]A combination of RPA and HOLMES is used to detect Escherichia coli in the system. (A) Amplification of the gyrB gene of Escherichia coli by RPA. A total of two sets of primers, gyrB-1 and gyrB-2, are used for amplification. gyrB is a characteristic gene of Escherichia coli. (B) The HOLMES detection system is used to detect the RPA amplification product. Negative control: The sample is sterile water, and gyrB-1 amplification primers are used to amplify or detect the results of the gyrB gene; gyrB-1: The sample is Escherichia coli to be detected, and the first set of gyrB amplification primers are used to amplify or detect the results of the gyrB gene; gyrB-2: The sample is Escherichia coli to be detected, and the second set of gyrB amplification primers are used to amplify or detect the results of the gyrB gene. [Figure 22] This figure shows the detection of collateral single-stranded DNA cleavage activity of Cas12b when single-stranded DNA was used as the target DNA. After the collateral cleavage reaction was completed, the reaction mixture was separated by 12% urea denaturing gel electrophoresis and detected using a fluorescent imaging system. The numbers in parentheses represent the final concentrations of the reactants in nM. The target DNA is a 66-nt single-stranded DNA at a dose of 50 nM; the single-stranded DNA probe is a single-stranded DNA with a FAM label at the 5' end at a dose of 50 nM. As shown in the figure, after adding Cas12b, guide RNA, and target DNA, the FAM-labeled single-stranded DNA is cleaved into fragments, demonstrating that Cas12b possesses collateral single-stranded DNA cleavage activity. [Figure 23]This figure shows the detection of collateral single-stranded DNA cleavage activity of Cas12b when using single-stranded and double-stranded DNA as target DNA. After the collateral cleavage reaction was completed, the reaction mixture was detected using a fluorescent microplate reader. The doses of Cas12b and guide RNA were both 500 nM; the target DNA was 66-nt single-stranded or double-stranded DNA at a dose of 50 nM; and the single-stranded DNA probe was a single-stranded DNA probe (HEX-N12-BHQ1) containing a fluorescent reporter group and a quencher group at a dose of 500 nM. As shown in the figure, regardless of whether a single-stranded or double-stranded DNA template was used, collateral single-stranded DNA cleavage activity was detectable after the addition of Cas12b and guide RNA. [Figure 24] Figure 1 shows the collateral single-stranded DNA trans-cleavage activity of Cas12b against target DNA at low concentrations after combined with LAMP amplification. DETAILED DESCRIPTION OF THE INVENTION

[0074] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are only a part, but not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0075] Through extensive and thorough research and studies on the cleavage properties of Cas enzymes (such as Cas12a and Cas12b enzymes), the present inventors have developed a technical solution for target nucleic acid detection. Experimental results show that by adopting the above technical solution, nucleic acids can be successfully and rapidly detected, for example, to identify whether a specific concentration of microorganisms such as Escherichia coli exists in water and to rapidly identify SNP genotypes. The present invention is based on this.

[0076] term

[0077] The term "guide RNA" refers to an RNA that guides a Cas protein to specifically bind to a target DNA sequence.

[0078] The term "crRNA" refers to CRISPR RNA, which is a short RNA that guides Cas12a to bind to a target DNA sequence.

[0079] The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats, which are the immune systems of many prokaryotes.

[0080] The term "Cas protein" refers to a CRISPR-associated protein, which is an associated protein of the CRISPR system.

[0081] The term "Cas12a" (previously called "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system classification.

[0082] The terms "Cas12b" and "C2c1" are used interchangeably and refer to the crRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0083] The term "LAMP" stands for loop-mediated isothermal amplification technology, which is an isothermal nucleic acid amplification technology suitable for genetic diagnosis.

[0084] The term "PAM" refers to the protospacer adjacent motif required for Cas12a cleavage. The PAM of FnCas12a is a TTN sequence, the PAM of LbCas12a is a TTTN sequence, and the PAM of AacCas12b is TTN.

[0085] The present invention discloses a method for detecting a target nucleic acid molecule, comprising adding a guide RNA, a Cas protein, a nucleic acid probe, and a buffer solution to a reaction system containing the target nucleic acid molecule to be detected, and then performing fluorescent detection of the target nucleic acid molecule.

[0086] The Cas protein is Cas12a or Cas12b.

[0087] The Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a, and the Cas12a is preferably LbCas12a.

[0088] Cas12b is preferably AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b, or AcCas12b.

[0089] Guide RNA refers to RNA that directs the Cas protein to specifically target a DNA sequence.

[0090] In the reaction system of the target nucleic acid molecule to be detected, the target nucleic acid molecule to be detected is obtained by amplification.

[0091] The detection method can detect pathogenic microorganisms, genetic mutations, or specific target DNA.

[0092] Use of Cas proteins in methods for detecting target nucleic acid molecules.

[0093] When the target DNA, guide RNA, and Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system.

[0094] Guide RNA refers to RNA that directs the Cas protein to specifically target a DNA sequence.

[0095] The present invention also provides a kit comprising a guide RNA, a Cas protein, and a nucleic acid probe. The kit of the present invention may further comprise a buffer solution.

[0096] The present invention provides a detection method for rapidly detecting target nucleic acid molecules with high specificity. When a (single- or double-stranded) target DNA, crRNA, and Cas12a protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system. By design, crRNA targets the target DNA (a segment of the DNA sequence to be detected). When crRNA and Cas12a protein are added to a detection system, Cas12a forms a ternary complex with the crRNA and target DNA in the presence of the target DNA. Meanwhile, the complex exerts its collateral cleavage activity, cleaving the single-stranded DNA labeled with a fluorescent signal (both ends of the single-stranded DNA are connected to a luminescent group and a quencher group, respectively, and the luminescent group emits light after cleavage), thereby emitting fluorescence. Therefore, the presence of a target DNA molecule in the detection system can be confirmed by fluorescence detection. The method of the present invention can rapidly detect whether a specific DNA sequence is present in a sample. The sensitivity of the detection method can be significantly improved by combining it with PCR technology. The nucleic acid probe in the present invention is preferably a fluorescent probe.

[0097] HOLMES criteria test: The present invention provides the use of Cas12 enzymes, such as Cas12a and Cas12b, in nucleic acid detection. In the following description, Cas12a is taken as an example.

[0098] Cas12a Selection: Research has shown that Cas12a has trans-cleavage activity. That is, when the target DNA, crRNA, and Cas12a protein form a ternary complex, other single-stranded DNA (collateral single-stranded DNA) in the system is cleaved. Based on this principle, a specific DNA detection method was designed. First, the collateral DNA was designed as a fluorescent probe, consisting of a 12-nt random sequence, labeled with the fluorescent group HEX at the 5' end and the quenching group BHQ1 (HEX-N12-BHQ1) at the 3' end. When the target DNA fragment was included in the system, a ternary complex of the target DNA, crRNA, and Cas12a protein was formed. At this point, the probe was cleaved, during which the HEX fluorescent group emitted fluorescence (excitation light at 535 nM and emission light at 556 nM) that was detected by a fluorescence detector. Next, 10 types of Cas12a were tested, and the target sequence was double-stranded DNA, as shown in Figure 7. It can be seen that the complex consisting of the target double-stranded DNA and each Cas12a protein can achieve trans-cleavage activity.

[0099] HOLMES response sensitivity: Next, we tested the response sensitivity of FnCas12a and LbCas12a to target DNA. That is, we investigated the lowest concentration of target DNA at which a response could occur. As shown in Figure 9, when the test target was added directly, a response to the target DNA was observed at concentrations above 0.1 nM, and a significant response was observed at concentrations above 1 nM. As shown in Figure 8, when PCR technology (HOLMES method) was combined, i.e., PCR was followed by amplification of the target fragment via Cas12a cleavage reaction, the response sensitivity could be reduced to 10 aM, as shown in Figure 9.

[0100] SNP testing: Next, we tested whether the HOLMES method can detect SNP genotypes. We considered T1 as the target sequence and mutated the PAM at this site or mutated positions 1–18 of the target sequence. We then compared the detection difference between the unmutated sequence and the mutated sequence using crRNAs of different lengths.

[0101] As shown in Figure 10, when the target complementary sequence is a 24-nt crRNA (crRNA-24nt), single-point mutations at positions 8–18 do not significantly affect the wild-type fluorescence. However, after PAM mutations and mutations at positions 1–7, the fluorescence intensity significantly decreased. When the crRNA is cleaved and the paired target sequence is 18 nt long, the fluorescence intensity at the mutated positions 8–16 nt is significantly reduced compared to the fluorescence intensity of the 24-nt target sequence. When the crRNA is further shortened to 16 or 17 nt, the fluorescence intensity of the mutated target sequence is further reduced. When further shortened to 15 nt, the fluorescence intensity of the mutated target sequence is weaker compared to that of this target sequence, but the fluorescence intensity of the mutated target sequence can still be higher than that of other target sequences and can therefore be used for detection. In summary, crRNAs of 15 nt, 16 nt, and 17 nt are optimal for SNP detection.

[0102] In this study, we demonstrate that Cas12a cleaves single-stranded DNA in a PAM sequence-independent programmed cleavage manner, called cis-cleavage, but when the ternary complex Cas12a / crRNA / target DNA is formed, it exhibits trans-cleavage activity, i.e., the activity of cleaving non-target single-stranded DNA in the system.

[0103] Based on the properties of Cas12a, we have developed a method for detecting specific nucleic acid molecules called HOLMES (one HOur Low-cost Multipurpose Efficient Simple assay). As the name of the technology suggests, it is fast (one hour), low-cost, multi-channel, highly efficient, and simple to use. This method can be used in fields such as rapid pathogen detection and SNP detection.

[0104] Nucleic acid detection based on collateral cleavage activity The present invention also provides nucleic acid detection methods based on the collateral cleavage activity of Cas12 enzymes (including Cas12a or Cas12b).

[0105] Preferably, the detection of the present invention can be carried out for SNPs, in particular, PCR amplification is first carried out and then detection is carried out.

[0106] Referring to Figure 18, a primer design scheme is provided.

[0107] Case 1: When a PAM site is present near the SNP site, crRNA synthesized based on a guide sequence designed according to the PAM site can be used for HOLMES detection. When the HOLMES method is used for detection, a relatively low background signal is observed, and the signal difference between different SNP templates for the same guide sequence is very large.

[0108] Case 2. If there is no PAM site near the SNP site or no suitable PAM site exists, a PAM site can be introduced according to the experimental scheme above.

[0109] A typical process involves designing a primer near the SNP site and incorporating a PAM site on the primer, such that the sequence located at the 3' end of the PAM site should pair with the template DNA. There are no special requirements for the primer at the other end, as long as the primer can pair with the template DNA and be used for PCR amplification. As shown in Figure 18, the PAM site can be successfully introduced after PCR amplification.

[0110] Referring to Figure 10, when designing the introduction of a PAM site in the present invention, the SNP site is typically located in the first 16 bases of the 5' end of the crRNA guide sequence, preferably at positions 1-14, more preferably at positions 1-12, even more preferably at positions 1-11 or 1-10, and most preferably at positions 1-8 or 1-7.

[0111] The present invention has the following main advantages: (1) High speed: Once the test conditions are met, it only takes about one hour from the time of sample collection to obtaining the test results. (2) Low cost: Since no special materials or enzymes are required for the experiment and only small amounts of materials and reagents are required, it can be used for microanalysis. (3) High efficiency: The present invention is highly sensitive and can detect DNA at a concentration of 10 aM. (4) Multiple applications: different nucleic acid samples can be detected, including DNA samples and RNA samples. (5) Simplicity: There are no special or complicated steps, and once the kit is prepared and the program is set, only simple operations such as adding a sample are required.

[0112] The present invention will be described in detail below with reference to specific examples. It should be understood that the following examples are intended only to illustrate the present invention, and not to limit the scope of the present invention. Experimental methods in the following examples that are not specified in specific conditions are generally carried out according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0113] Unless specifically stated otherwise, the experimental materials involved in the present invention can be obtained from commercial sources.

[0114] material 1. RNase inhibitors are purchased from TaKaRa, and high-fidelity DNA polymerase KOD FX is purchased from ToYoBo; primers (oligonucleotides) are synthesized by Sangon Biotech (Shanghai) Co., Ltd., and T7 RNA polymerase is purchased from Thermo; RNA purification and concentration kit (RNA Clean & Concentrator™_5) is purchased from Zymo Research; Wizard® SV Gel and PCR Clean-Up System is purchased from Promega; and media (e.g., tryptone, yeast extract, etc.) are all purchased from OXOID.

[0115] 2. Media formulation: Liquid LB (1% tryptone, 0.5% yeast extract, 1% NaCl), and if you are preparing solid LB, simply add 2% agar to the liquid LB. [Example]

[0116] Example 1: Cas12a protein detection capable of detecting single-stranded DNA targets (probe labeled with FAM) Single-stranded DNA (target-T1-R) was selected as the target sequence, and the response values ​​of its detection by different Cas12a proteins were tested.

[0117] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-T1-24-R, as shown in Table 5. Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C (Thermo Fisher Scientific), diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0118] 2. Cas12a reaction: The purified crRNA (0.5 μM) from step 1, Cas12a (0.25 μM), target single-stranded DNA (Target-T1-R) (0.01 μM), nucleic acid probe (N25-5' FAM) (0.01 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction system. A blank control reaction was performed in which all other components except the single-stranded DNA target sequence were added. The reaction was run at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0119] 3. Fluorescence detection: The reaction mixture was subjected to urea-acrylamide gel electrophoresis (Urea-PAGE) and then detected using a fluorescence imager. As shown in Figure 11, different Cas12as have different detection effects on the target. For example, HkCas12a cleaved the probe even without adding target single-stranded DNA. LbCas12a cleaved the probe only when target single-stranded DNA was added, making it a better candidate for Cas12a protein.

[0120] Example 2: Detection of Cas12a protein capable of detecting single-stranded DNA targets (probes labeled with two labels: HEX and BHQ1) Single-stranded DNA (target-T1-R) was selected as the target sequence, and the response values ​​of its detection by different Cas12a proteins were tested.

[0121] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0122] 2. Cas12a reaction: The purified crRNA (0.5 μM) from step 1, Cas12a (0.25 μM), target single-stranded DNA (target-T1-R) (0.01 μM), fluorescent probe (HEX-N12-BHQ1, i.e., a 12-nt single-stranded DNA labeled with HEX at the 5' end and BHQ1 at the 3' end) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction system. A control reaction was performed in which all other components except the single-stranded DNA target sequence were added. The reaction was run at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0123] 3. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 12, different Cas12as have different detection effects on targets. For example, HkCas12a cleaved the probe even without the addition of target single-stranded DNA. FnCas12a cleaved the probe only when target single-stranded DNA was added, making it a better candidate for Cas12a protein.

[0124] Example 3: Detection of Cas12a protein capable of detecting double-stranded DNA targets Double-stranded DNA (target-T1) was selected as the target sequence, and the response values ​​of its detection by different Cas12a proteins were tested.

[0125] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0126] 2. Cas12a reaction: The purified crRNA (0.5 μM), Cas12a (0.25 μM), target double-stranded DNA (target-T1, obtained by annealing primer target-T1-F to target-T1-R) (0.01 μM), fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction system. The reaction was carried out at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0127] 3. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 7, different Cas12a proteins have different detection efficiencies for the target. LbCas12a is a better candidate for Cas12a protein because probe cleavage only occurred when target double-stranded DNA was added.

[0128] Example 4 Testing Varying Concentrations of Targets with FnCas12a and LbCas12a Target-T1 was selected as the target DNA and then diluted to different concentrations in a gradient to test the response sensitivity of FnCas12a and LbCas12a to them. To increase sensitivity, a PCR amplification step was added.

[0129] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0130] 2. PCR amplification (optional): A plasmid containing the target target-T1 (pUC18-T1) was used as a template, diluted with a gradient, and then used in a PCR reaction. The total volume of each reaction was 20 μL, and 0.25 μM M13F-47 and M13R-48 were used as primers (Table 4). The high-fidelity enzyme KOD FX (ToYoBo) was used in the PCR reaction. The PCR reaction procedure was 95°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 10 seconds. After PCR was completed, the PCR amplified product was directly used in the Cas12a reaction.

[0131] 3. Cas12a reaction: The purified crRNA (0.5 μM), FnCas12a or LbCas12a (0.25 μM), 1 μL of PCR product (or target DNA diluted directly to different concentrations), fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was carried out at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0132] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 9, when the test target was added directly, all target DNA concentrations above 0.1 nM responded, and concentrations above 1 nM showed significant responses. When PCR technology was combined (i.e., amplification of the target fragment by PCR followed by Cas12a cleavage), the response sensitivity could be reduced to 10 aM.

[0133] Example 5 Testing single point mutation targets with FnCas12a and LbCas12a Target-T1 was selected as the target and subjected to single point mutations in the PAM region and positions 1–18, respectively, to test the response values ​​of wild-type and several crRNAs of different lengths from the same one after single point mutations.

[0134] 1. Preparation of crRNA: First, transcription templates were prepared by annealing T7-crRNA-F to synthetic oligonucleotides T7-T1-24-R, T7-T1-15-R, T7-T1-16-R, T7-T1-17-R, and T7-T1-18-R (Table 5). Specifically, paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by an annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. RNA was then purified using an RNA purification and concentration kit, quantified on a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at −80°C.

[0135] 2. PCR amplification: A plasmid containing the target target-T1 (pUC18-T1) was used as a template. The total volume of each reaction system was 20 μL, and 0.25 μM of primers M13R-48 and target-T1-F mutation primers were used (Table 4). The high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was 95 ° C for 2 minutes, followed by 35 cycles of 98 ° C for 10 seconds, 60 ° C for 15 seconds, and 68 ° C for 10 seconds. After PCR was completed, the product was directly used in the Cas12a reaction.

[0136] 3. Cas12a reaction: The purified crRNA (0.5 μM), FnCas12a or LbCas12a (0.25 μM), 1 μL of PCR product, fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was carried out at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0137] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 10, when the target complementary sequence was 24 nt crRNA (crRNA-24nt), single point mutations at positions 8–18 did not significantly differ from the wild-type, whereas PAM mutations and point mutations at positions 1–7 significantly reduced the fluorescence intensity. When the crRNA was cleaved and the paired target sequence was 18 nt long, the fluorescence intensity at the mutated positions 8–16 nt was significantly reduced compared to that of the 24 nt sequence. When the length was 16 nt or 17 nt, the decrease in fluorescence intensity of the mutated target sequence was more pronounced. When the length was 15 nt, the fluorescence intensity of both the target sequence and the mutant target sequence was very weak, but the fluorescence intensity of the mutant target sequence was still higher than that of the other target sequences and could therefore be used for detection. In summary, 15nt, 16nt and 17nt crRNAs are optimal for SNP detection.

[0138] Example 6 Testing of Escherichia coli and other microorganisms in environmental water The gyrB gene of Escherichia coli was selected as the detection target to indirectly test the concentration of microorganisms such as Escherichia coli in water. Escherichia coli MG1655 was used as a positive control to measure the content of microorganisms in environmental water (such as sewage and tap water).

[0139] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0140] 2. PCR amplification: Positive control sample Escherichia coli MG1655 was diluted to OD 600 When the cultured samples reached a pH of approximately 0.5, they were diluted 10-fold and then used as templates. The samples were ambient water samples (including tap water and muddy water in the environment). The total volume of each reaction was 20 μL, and 0.25 μM of primers gyrB-F and gyrB-R were used (Table 4). The high-fidelity enzyme KOD FX (ToYoBo) was used for PCR. The PCR reaction procedure was 95°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 10 seconds. After PCR was completed, the PCR product was directly used in the Cas12a reaction.

[0141] 3. Cas12a reaction: The purified crRNA (0.5 μM), LbCas12a (0.25 μM), 1 μL of PCR product, fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was carried out at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0142] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 13, the fluorescence response value of Escherichia coli MG1655 decreased with decreasing concentration. Among them, microorganisms were more clearly detected in samples 2, 4, 5, and 6.

[0143] Example 7 Testing of Human SNPs For the SNP study, five human SNP sites, namely rs5082, rs1467558, rs2952768, rs4363657, and rs601338, were selected to test the feasibility of the HOLMES method.

[0144] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to synthetic oligonucleotides (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 High-Throughput Transcription Kit and incubated overnight (approximately 16 hours) at 37°C. RNA was purified using an RNA Clean & Concentrator™-5 (Zymo Research), quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0145] 2. PCR amplification: The total volume of the reaction system was 20 μL, and 0.25 μM of each primer (Table 4) was used. 1 ng of human genome (HEK293T) or directly exfoliated oral epithelial mucosa was used as the template. The high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was 95°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 10 seconds. After PCR was completed, the product was directly used in the Cas12a reaction. (Primers 1-rs5082-FT, 2-rs1467558-FT, and 3-rs2952768-RC were used to directly introduce the mutation products corresponding to the SNPs.)

[0146] 3. Cas12a reaction: The corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of PCR product, and fluorescent probe (HEX-N12-BHQ1) (0.5 μM) were added to a 20 μL reaction mixture. The reaction was run at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0147] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 14, a higher fluorescence response value was observed only when the crRNA corresponded to the corresponding target sequence, whereas the response value was significantly reduced when a single point mutation was present. The genotype of the corresponding SNP could be determined by the fluorescence value, and these results were confirmed by sequencing.

[0148] Example 8 Testing for cancer-related genes The TP53 gene was selected as the test gene. TP53 harbors a nonsense mutation in human T24 cells, which results in gene inactivation. Cells with a normal gene at this site (HEK293T), individual genes, and mutant T24 cells were tested.

[0149] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotides T7-crRNA-34-TP53-T24-C-16nt and T7-crRNA-34-TP53-T24-G-16nt (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. RNA was purified using RNA Clean&Concentrator™-5 (Zymo Research), quantified on a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at −80°C.

[0150] 2. PCR amplification: The total volume of the reaction system was 20 μL, and 0.25 μM primers 34-TP53-T24-F and 34-TP53-T24-R were used (Table 4). 1 ng of human genome (HEK293T, T24) or directly exfoliated oral epithelial mucosa was used as template. The high-fidelity enzyme KOD FX (ToYoBo) was used in the PCR reaction. The PCR reaction procedure was 95 ° C for 2 minutes, followed by 35 cycles of 98 ° C for 10 seconds, 60 ° C for 15 seconds, and 68 ° C for 10 seconds. After PCR was completed, the product was directly used in the Cas12a reaction.

[0151] 3. Cas12a reaction: The corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of PCR product, and fluorescent probe (HEX-N12-BHQ1) (0.5 μM) were added to a 20 μL reaction mixture. The reaction was run at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0152] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nM, emission 556 nM). As shown in Figure 15, when the normal TP53 gene at this site was used as the template, the detected value of crRNA-C was significantly higher than that of crRNA-G, but the crRNA-G level in the mutant cell T24 was significantly increased.

[0153] Example 9 Testing of human SNPs (gout-associated genes) For SNP testing, five human SNP sites, namely rs1014290, rs6449213, rs737267, rs1260326, and rs642803, were selected to test the HOLMES method.

[0154] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to synthetic oligonucleotides (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 High-Throughput Transcription Kit and incubated overnight (approximately 16 hours) at 37°C. RNA was purified using an RNA Clean & Concentrator™-5 (Zymo Research), quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C.

[0155] 2. PCR amplification: The total volume of the reaction system was 20 μL, and 0.25 μM of each primer (Table 4) was used. 1 ng of human genome (HEK293T) or directly exfoliated oral epithelial mucosa was used as the template. The high-fidelity enzyme KOD FX (ToYoBo) was used for the PCR reaction. The PCR reaction procedure was 95°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 10 seconds. After PCR was completed, the product was directly used in the Cas12a reaction. (Primers 1-rs5082-FT, 2-rs1467558-FT, and 3-rs2952768-RC were used to directly introduce the mutation products corresponding to the SNPs.)

[0156] 3. Cas12a reaction: The corresponding crRNA (1 μM), LbCas12a (0.5 μM), 1 μL of PCR product, and fluorescent probe (HEX-N12-BHQ1) (0.5 μM) were added to a 20 μL reaction mixture. The reaction was run at 37°C for 15 minutes and then stopped at 98°C for 2 minutes.

[0157] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). As shown in Figure 16, a higher fluorescence response was observed only when the crRNA corresponded to the corresponding target sequence, whereas a single point mutation significantly reduced the response. The genotype of the corresponding SNP could be determined by the fluorescence intensity, and these results were confirmed by sequencing.

[0158] Example 10 SNP testing of clinical samples (gout-related genes) from volunteers using the kit The kit was prepared by adding the premixed solution to a 96-well plate, and the genomic DNA of 21 volunteers was added to the kit to test for the rs1014290 site associated with gout risk.

[0159] 1. Kit Preparation: First, a transcription template was prepared by annealing T7-crRNA-F to synthetic oligonucleotides (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). The crRNA was synthesized using the T7 High-Throughput Transcription Kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was purified using an RNA Clean & Concentrator™-5 (Zymo Research), quantified using a NanoDrop 2000C, and diluted to a concentration of 10 μM.

[0160] 2. Premixing in a 96-well plate for PCR: 19 μL of the reagents required for PCR reaction was added to the system, and the primers were 41-rs1014290-F and 41-rs1014290-R.

[0161] 3. Premixing in a 96-well plate for fluorescence detection: crRNA (1 μM), LbCas12a (0.5 μM), and fluorescent probe (HEX-N12-BHQ1) (0.5 μM) were added to the 19 μL system and added to a 96-well plate.

[0162] 4. PCR amplification: The volunteers' genomic DNA was added to a premixed 96-well plate for PCR, and then subjected to PCR reaction. The PCR reaction procedure was 95°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 10 seconds.

[0163] 5. Cas12a reaction: 1 μL of PCR reaction solution was taken and added to a premixed 96-well plate for fluorescence detection, reacted at 37°C for 15 minutes, and then stopped at 98°C for 2 minutes.

[0164] 6. Fluorescence detection: Detection was performed using a microplate reader (excitation light 535 nm, emission light 556 nm). As shown in Figure 17, the A:A genotype group had a high risk of gout, and people other than volunteers 5, 7, and 9 had the A:G or G:G genotype, so more attention should be paid to the risk of gout.

[0165] Example 11 Detection of microorganisms such as Escherichia coli in environmental water by LAMP combined with Cas protein The gyrB gene of Escherichia coli was selected as the detection target to indirectly test whether microorganisms such as Escherichia coli exist in water.

[0166] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X Taq DNA polymerase reaction buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, and finally diluted to a concentration of 10 μM. It was then stored in a refrigerator at -80°C for later use.

[0167] 2. LAMP amplification: Sterile water and a contaminated solution containing Escherichia coli were used as negative controls and detection samples, respectively. The total volume of each reaction system was 25 μL, and 1.6 μM each of LAMP-FIP and LAMP-BIP, 0.2 μM each of LAMP-F3 and LAMP-B3, and 0.4 μM each of LAMP-LoopF ​​and LAMP-LoopB primers were used. The kit used for the LAMP reaction was the WarmStart® LAMP Kit (NEB). The LAMP reaction procedure was at 65°C for 30 minutes. After completion of LAMP, the product was annealed at 80°C for 10 minutes and directly used in the Cas12a reaction.

[0168] 3. Cas12a reaction: The purified crRNA (0.5 μM), Cas12a (0.25 μM), 1 μL of LAMP product, fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was carried out at 37°C for 15 minutes.

[0169] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation light 535 nm, emission light 556 nm). The results are shown in Figure 19.

[0170] Example 12 Detection of SNPs by using LAMP amplification in combination with Cas proteins

[0171] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotides T7-crRNA-rs5082-T / T7-crRNA-rs5082-G / T7-crRNA-rs1467558-T / T7-crRNA-rs1467558-C (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X Taq DNA polymerase reaction buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and allowed to react overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified on a NanoDrop 2000C, and finally diluted to a concentration of 10 μM and stored in a refrigerator at −80°C for later use.

[0172] 2. LAMP amplification: Human genome HEK293T was used as the sample. The total volume of each reaction system was 25 μL, and 1.6 μM each of LAMP-FIP and LAMP-BIP, 0.2 μM each of LAMP-F3 and LAMP-B3, and 0.4 μM each of LAMP-LoopF ​​and LAMP-LoopB primers were used. The LAMP reaction was performed using the WarmStart® LAMP Kit (NEB). The LAMP reaction procedure was at 65°C for 30 minutes. After LAMP was completed, annealing was performed at 80°C for 10 minutes, and the product was directly used in the Cas12a reaction.

[0173] 3. Cas12a reaction: The purified crRNA (0.5 μM), Cas12a (0.25 μM), 1 μL of LAMP product, fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was carried out at 37°C for 15 minutes.

[0174] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation light 535 nm, emission light 556 nm). The results are shown in Figure 20.

[0175] Example 13: Detection of Escherichia coli and other microorganisms in environmental water by RPA amplification combined with Cas proteins The gyrB gene of Escherichia coli was selected as the detection target to indirectly test whether microorganisms such as Escherichia coli exist in water.

[0176] 1. Preparation of crRNA: First, a transcription template was prepared by annealing T7-crRNA-F to the synthetic oligonucleotide T7-crRNA-gyrB (Table 5). Specifically, the paired oligonucleotides (4 μM) were annealed in 1X PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). crRNA was synthesized using a T7 high-throughput transcription kit and incubated overnight (approximately 16 hours) at 37°C. The RNA was then purified using an RNA purification and concentration kit, quantified using a NanoDrop2000C, and finally diluted to a concentration of 10 μM. It was then stored in a refrigerator at -80°C for later use.

[0177] 2. RPA amplification: Sterile water and a contaminated solution containing Escherichia coli were used as a negative control and a detection sample, respectively. The total volume of each reaction system was 25 μL, and 0.5 μM primers RPA-gyrB-F (or RPA-gyrB-F2) and RPA-gyrB-R2 were used, respectively. The TwistAmp® Basic Kit (TwistDX) was used for the RPA reaction. The RPA reaction procedure was at 37°C for 30 minutes. After RPA was completed, annealing was performed at 80°C for 10 minutes, and the product was directly used in the Cas12a reaction.

[0178] 3. Cas12a reaction: The purified crRNA (0.5 μM), Cas12a (0.25 μM), 1 μL of RPA product, fluorescent probe (HEX-N12-BHQ1) (0.5 μM), NEB buffer 3.1, and 0.5 μL of RNase inhibitor were added to a 20 μL reaction mixture. The reaction was allowed to proceed at 37°C for 15 minutes.

[0179] 4. Fluorescence detection: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation light 535 nm, emission light 556 nm). The results are shown in Figure 21.

[0180] Example 14: Cas12b with collateral cleavage activity

[0181] 1. Preparation of guide RNA (sgRNA) First, the plasmid pUC18-guide RNA-T1 was constructed using pUC18 as the plasmid backbone. In this plasmid, a T7 promoter and a template DNA sequence for guide RNA transcription were inserted into pUC18 (Note: The guide RNA transcribed from this template targeted a sequence referred to as T1 in this study). In this method, a series of PCRs was performed using the pUC18 plasmid as a template and pUC18-1-F and pUC18-1-R as primers. The PCR products were ligated with T4 DNA ligase, transformed into DH10b, and sequenced to obtain the correct clone, referred to as pUC18-guide RNA-T1-pre. A second PCR was then performed using pUC18-guide RNA-T1-pre as a template and pUC18-2-F and pUC18-2-R as primers, ligating and transforming the PCR products in the same manner. Finally, the correct plasmid, pUC18-guide RNA-T1, was obtained, as sequenced.

[0182] Next, guide RNA was synthesized using the plasmid pUC18-guideRNA-T1 as a template using a T7 high-throughput transcription kit (Thermo) and allowed to react overnight (12–16 h) at 37°C.

[0183] Finally, DNase I was added to the transcription system (2 μL of DNase I was added per 50 μL of transcription system), and the system was placed in a water bath at 37°C for 30 minutes to remove the plasmid DNA. The RNA was purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, diluted to a concentration of 10 μM, and stored in a refrigerator at -80°C for later use.

[0184] 2. Target DNA Preparation

[0185] (1) When the target DNA was single-stranded, a 66-bp-long oligonucleotide was directly synthesized as the target DNA (target-T1-R) containing a 20-bp target sequence (T1) recognized by the guide RNA.

[0186] (2) When the target DNA was double-stranded, two 66-bp-long complementary oligonucleotides (Target-T1-F; Target-T1-R) containing a 20-bp target sequence (T1) recognized by the guide RNA were directly synthesized. The two oligonucleotides were annealed to obtain a short target DNA. Specifically, paired oligonucleotides (1 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 20 μL, and then subjected to the annealing procedure (initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler).

[0187] 3.Cas12b reaction

[0188] (1) Guide RNA annealing: The guide RNA was diluted to an appropriate concentration (10 μM) and annealed in a PCR machine. Annealing procedure: After denaturing at 75°C for 5 minutes, the temperature was cooled from 75°C to 20°C at a rate of 1°C / min.

[0189] (2) Incubation of guide RNA and C2c1: The annealed guide RNA was mixed with C2c1 at an equimolar concentration and left at 30°C for 20–30 min.

[0190] (3) Cas12b reaction: A mixture of the guide RNA and C2c1 incubated in step (2) (both at final concentrations of 250 μM or 500 μM), target DNA (final concentration 50 nM), FAM-labeled oligonucleotide (target-DNMT1-3-R-FAM-5') or fluorescent quenching probe (HEX-N12-BHQ1, final concentration 500 nM), 2 μL of 10X NEB buffer 3.1, and 0.5 μL of RNase inhibitor (40 U / μL) was added to a 20 μL reaction mixture. After uniform mixing, the reaction was carried out at 48°C for 30 minutes. The mixture was then inactivated by heating at 98°C for 5 minutes in a PCR machine.

[0191] 4. Detection of Cas12b trans-cleavage activity by urea denaturing gel electrophoresis: 20 μL of the inactivated reaction solution was separated by urea denaturing gel electrophoresis and then imaged using a fluorescent imaging system, ImageQuant LAS 4000 mini (GE Healthcare). The results are shown in Figure 22.

[0192] 5. Detection of Cas12b trans-cleavage activity using a fluorescent microplate reader: 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation light 535 nm, emission light 556 nm). The results are shown in Figure 23.

[0193] Example 15: Sensitivity test of Cas12b reaction (trans-cleavage) By detecting the excitation fluorescence intensity of the fluorescent probe (HEX-N12-BHQ1), we determined the target DNA concentration required for Cas12b to exert its trans-cleavage activity, i.e., the sensitivity of the Cas12b trans-cleavage reaction.

[0194] 1. Guide RNA Preparation First, using pUC18-guide RNA-T1 as a template and guide RNA-DNMT1-3-F and guide RNA-DNMT1-3-R as primers, 20 bases of the guide RNA targeting the target DNA of T1 were replaced with guide RNA targeting DNMT1-3 by PCR, resulting in another plasmid, pUC18-guide RNA-DNMT1-3.

[0195] Next, guide RNA was synthesized using the plasmid pUC18-guide RNA-DNMT1-3 as a template using a T7 high-throughput transcription kit (Thermo) and reacted overnight (12–16 h) at 37 °C.

[0196] Finally, DNase I was added to the transcription system (2 μL of DNase I was added per 50 μL of transcription system), and the system was placed in a water bath at 37°C for 30 minutes to remove the plasmid DNA. The RNA was purified using an RNA purification and concentration kit, quantified using a NanoDrop 2000C, and stored in a refrigerator at -80°C for later use.

[0197] 2. Target DNA Preparation Regarding the target DNA, the first method was to add the target DNA directly to the Cas12b reaction system without amplification. The method was as follows: (1) When the target DNA was single-stranded, a 50-bp-long oligonucleotide was directly synthesized as the target DNA (DNMT1-3(TTC PAM)-R), containing a 20-bp target sequence (DNMT1-3) recognized by the guide RNA. (2) When the target DNA was double-stranded, two 50-bp-long complementary oligonucleotides (DNMT1-3(TTC PAM)-F; DNMT1-3(TTC PAM)-R) containing the 20-bp target sequence (DNMT1-3) recognized by the guide RNA were directly synthesized. The two oligonucleotides were annealed to obtain a short target DNA. Specifically, the paired oligonucleotides (2 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 20 μL, and then subjected to the annealing procedure (denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C at a rate of 1°C / min using a thermal cycler). (3) Single-stranded or double-stranded target DNA was diluted in a gradient of 2 μM, 0.2 μM, 0.02 μM, 0.002 μM, and 0.0002 μM for later use.

[0198] The second was to insert a fragment containing the target sequence (DNMT1-3) into a plasmid vector for amplification by the LAMP reaction.

[0199] (1) Fragments containing the target sequences (DNMT1-3) were inserted into the pEasy-Blunt Zero cloning vector using Transgen's pEasy-Blunt Zero cloning kit, and correct clones were obtained after verification by sequencing.

[0200] (2) LAMP expansion reaction LAMP amplification reactions were carried out using the above plasmids as templates. The templates were added at 0 nM, 1 nM, and 0.1 nM, respectively, and diluted 10 times in a 10-fold gradient. -11The primers were diluted to 100 nM. The total volume of each reaction was 25 μL, and the primers used were 1.6 μM each of LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 μM each of LAMP-DNM-F3 and LAMP-DNM-B3, and 0.4 μM each of LAMP-DNM-LoopF ​​and LAMP-DNM-LoopB. The kit used for the LAMP reaction was the WarmStart® LAMP Kit (NEB). The LAMP reaction procedure was at 65°C for 30 minutes. After completion of the LAMP, the product was inactivated at 80°C for 10 minutes. The product was directly used in the Cas12b reaction.

[0201] 3.Cas12b reaction

[0202] (1) Annealing of guide RNA: The guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR machine. Annealing procedure: After denaturing at 75°C for 5 minutes, the temperature was cooled from 75°C to 20°C at a rate of 1°C / min.

[0203] (2) Incubation of guide RNA and Cas12b: The annealed guide RNA was mixed with Cas12b at an equimolar concentration and left at 30°C for 20–30 min.

[0204] (3) Cas12b reaction: The mixture of guide RNA and Cas12b incubated in step (2) (final concentrations of guide RNA and Cas12b were both 250 μM), 1 μL of target DNA or 1 μL of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), 2 μL of 10X NEB buffer 3.1, and 0.5 μL of RNase inhibitor (40 U / μL) were added to a 20 μL reaction system. After uniform mixing, the reaction was carried out at 48°C for 30 minutes. The mixture was then inactivated by heating at 98°C for 5 minutes in a PCR machine.

[0205] 4. Detection of Cas12b trans-cleavage activity by fluorescence microplate reader method:

[0206] 20 μL of the inactivation reaction solution was added to a 96-well plate and detected using a microplate reader (excitation 535 nm, emission 556 nm). Combined with LAMP amplification, Cas12b can induce significant collateral single-strand DNA trans-cleavage activity at target DNA concentrations as low as 10 aM. The results are shown in Figure 24.

[0207] Cis-cleavage properties of Cas12a in cleaving target single-stranded DNA:

[0208] First, to test the single-stranded DNA cleavage properties of Cas12a, several crRNAs targeting short single-stranded DNAs (DNMT1-3) (Table 1) were designed and labeled with 5(6)-carboxyfluorescein (FAM) at the 3' end. After cleavage by FnCas12a, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (urea-PAGE). It was found that single-stranded DNA cleavage by Cas12a was programmed. That is, the cleavage site was located near the 22nd base of the target sequence (between the 21st and 23rd bases) counting from the 3'-end base of the first target sequence paired with the crRNA guide sequence to the 5'-end, as shown in Figure 1A and Figure 1C. While Cas12a requires a PAM sequence for double-stranded DNA cleavage, Cas12a does not require a PAM sequence for single-stranded DNA cleavage (Figure 1A, Figure 1B, and Figure 2), which is similar to single-stranded DNA cleavage mediated by Cas9. However, Cas12a-mediated single-stranded DNA cleavage activity depends on the stem-loop structure of the crRNA, as shown in Figure 1A. Cas9 still exhibits weak cleavage activity against single-stranded DNA with only a 20-nt complementary RNA sequence. The stem-loop structure of the crRNA is important for stabilizing the structure of Cas12a, which is why the circular structure of the crRNA is necessary for Cas12a-mediated cleavage of single-stranded DNA. We further tested whether short lead sequence crRNAs can pass through the Cas12a single-stranded DNA cleavage site in such a way that the cleavage occurs outside the recognition site. When the guide sequence length was 16 nt, 18 nt, and 20 nt, all of these crRNAs induced Cpf1 cleavage near base 22, as shown in Figures 1B and 1D, meaning that the cleavage site was 4 nt, 2 nt, or 0 nt outside the recognition site. Next, the cleavage efficiency of Cas12a on different substrates was tested using double-stranded and single-stranded DNA substrates, respectively, as shown in Figure 1F. Similar to the situation with Cas9 cleavage, cleavage of single-stranded DNA is slower than cleavage of double-stranded DNA, as shown in Figures 1E-1G.These results suggest that the mechanism by which Cas12a recognizes and cleaves single-stranded DNA may differ from that for double-stranded DNA, that this is a PAM-independent, low-efficiency recognition and cleavage method, and that the PAM sequence accelerates the recognition and / or cleavage of target double-stranded DNA by Cas12a.

[0209] Trans-cleavage properties of Cas12a in single-strand DNA cleavage:

[0210] When the target single-stranded DNA is labeled at the 3' end, Cas12a cleaves it near base 22, as shown in Figure 1. However, when the target single-stranded DNA is labeled at the 5' end, the predicted size cleavage product band is not observed, but a short (less than 6 nt) FAM-labeled product is generated, as shown in Figure 3B. Through detailed experiments, we found that the formation of the ternary complex Cas12a / crRNA / target single-stranded DNA cleaves the 5'-end-labeled target single-stranded DNA (DNMT1-3) (Table 1), generating a short FAM-labeled product, as shown in Figure 3C. In addition, the ternary complex also cleaves single-stranded DNA that does not have a sequence complementary to the crRNA in other reaction systems (i.e., collateral single-stranded DNA), as shown in Figures 3C and 3D. This cleavage phenomenon is called trans-cleavage and is distinct from programmable cis-cleavage. When the target single-stranded DNA is labeled at the 3' end, trans-cleavage is also observed, but many cis-cleavage products remain, as shown in Figure 3B. This may be due to the complex formed by Cas12a / crRNA / target single-stranded DNA, where the target single-stranded DNA is protected so that its labeled 3' end is protected from exposure to the nuclease active site of the ternary complex. These cleavage processes may be as shown in Figure 3A.

[0211] In addition to the FnCas12a tested above, nine Cas12as from other species sources were also tested (Table 2 and Figure 4A). Except for Lb4Cas12a, all Cas12as possess good endonuclease activity on plasmid DNA (as shown in Figure 4B), and all Cas12a ternary complexes exhibit cis- and trans-cleavage activity on single-stranded DNA (as shown in Figures 4C and 4D). This indicates that the cis- and trans-activities of Cas12a on single-stranded DNA are a general phenomenon.

[0212] Cis and trans key sites and mechanism for single-stranded DNA cleavage by Cas12a. To determine the critical amino acid residues involved in the cis- and trans-cleavage activity of Cas12a against single-stranded DNA, we mutated several candidate residues in Cas12a and performed activity studies. First, we purified and tested three single amino acid mutants of FnCas12a (H843A, K852A, and K869A) to determine the residues involved in RNase activity. The results of the trans-cleavage activity against single-stranded DNA, as shown in Figures 5A and 5C, indicate that there is no significant difference in the cis- and trans-cleavage activity against single-stranded DNA between wild-type FnCas12a and the three mutants.

[0213] Next, when the endonuclease active sites of FnCas12a, i.e., the RuvC domain (D917A, E1006A, or D1255A) and the Nuc domain (R1218A) sites, were mutated, both the cis- and trans-cleavage activities of these mutant Cas12a against single-stranded DNA were affected, as shown in Figures 5B and 5D. These results indicate that the critical sites of Cas12a for cleavage of target double-stranded DNA are closely related to the cis- and trans-cleavage activities against single-stranded DNA.

[0214] Recent structural studies of Cas12b (i.e., C2c1) (including complexes with extended target DNA or extended non-target DNA) indicate that both strands reside in the RuvC pocket, as shown in Figures 6A and 6B. Comparing the endonuclease catalytic residues of Cas12b (i.e., C2c1) and Cas12a, these sites most likely play similar roles in the cleavage and function of Cas12b (i.e., C2c1) and Cas12a. Results from in vitro single-amino acid mutation experiments are consistent with the above hypothesis. Thus, Cas12a may cleave both strands using only one RuvC catalytic pocket.

[0215] Trans-cleavage activity of the Cas12a complex: In the structure of the complex of Cas12b (i.e., C2c1) with an additional single-stranded DNA, as shown in Figure 6C, the sequence-independent single-stranded DNA is also located on the surface of the catalytic pocket, which is similar to that of the collateral single-stranded DNA substrate of Cas12a. Combined with single-amino acid mutation experiments, as shown in Figures 6D, 6E, and 6F, it is proposed that target DNA, non-target DNA, and collateral single-stranded DNA are all cleaved in a single RuvC pocket of Cas12a. The reason why the ternary Cas12a complex possesses collateral single-stranded DNA trans-cleavage activity, while the monomeric or binary complexes do not, can be explained by comparing the structures of the monomeric, binary, and ternary complexes. The structure of monomeric Cas12a is disordered, and the binary complex Cas12a / crRNA has a triangular structure, as shown in Figure 6G, while the ternary complex Cas12a / crRNA / target DNA converts to a two-leaf structure, exposing the catalytic pocket to achieve trans-cleavage of collateral single-stranded DNA (as shown in Figure 6H).

[0216] Establishment of nucleic acid detection methods Based on the properties of Cas12a, a specific nucleic acid molecule detection method has been developed, called HOLMES (1-hour Low-Cost Multipurpose Efficient Simple Assay). As the name of the technology suggests, it is a simple test method that can be performed in 1 hour, at low cost, for multiple uses, and with high efficiency.

[0217] The entire reaction system of this method can be divided into two major steps: amplification of the template nucleic acid and specific nucleic acid detection using the Cas12a protein. Here, PCR is used for nucleic acid amplification, but in fact, any amplification method, such as the isothermal amplification method RPA, can be combined with the second step of nucleic acid detection. The initial nucleic acid is not limited to double-stranded DNA; it can also be single-stranded DNA; or RNA can remain detectable after reverse transcription, making this method suitable for various types of nucleic acid molecules. In the nucleic acid detection phase, three components are key to the experiment: Cas12a, crRNA, and a nucleic acid probe. In addition to the 10 Cas12a proteins mentioned in the examples (these 10 proteins are randomly selected), other Cas12a proteins are also suitable for this method. In addition, other types of Cas proteins (e.g., C2c1 proteins) are also suitable for the claims of the present invention: experimental results show that Alicyclobacillus acidoterrestris Cas12b (i.e., C2c1) also has collateral single-stranded DNA trans-cleavage activity similar to that of Cas12a, and its complex with crRNA / target DNA is also capable of cleaving collateral single-stranded DNA.

[0218] The crRNA that functions as a guide is more stable in the system after manipulation, such as manual modification. Regarding the selection of the nucleic acid probe, the present invention selected a short single-stranded DNA labeled with HEX and BHQ1, but other detectable labeling methods could theoretically be applied as long as a detectable difference occurs upon cleavage of the nucleic acid probe. Alternatively, the nucleic acid probe could be designed to emit fluorescence after binding to a compound, allowing detection of whether the probe has been cleaved.

[0219] Furthermore, after reading the above teachings of the present invention, one skilled in the art will recognize that various changes or modifications can be made to the present invention, and that equivalents thereof are also encompassed within the scope of the present invention as defined by the claims appended hereto. Table 1. Characteristic experimentally relevant cleavage substrate sequences of Cas12a [Table 1-1] [Table 1-2] Table 2. Names and GI numbers of Cas12a and Cas12b (i.e., C2c1) proteins. [Table 2] Table 3. Plasmid information [Table 3-1] [Table 3-2] Table 4 Primers used in Holmes method tests [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] Table 5. Template sequences for transcription of crRNA [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] Primers used for detection by LAMP-mediated DNA amplification in combination with Cas12a: Table 6. Primers used for amplification of gyrB-1 [Table 6] Table 7. Primers used for amplification of gyrB-2 [Table 7] Table 8. Primers used for amplification of the rs1467558 site [Table 8] Table 9. Primers used for amplification of the rs5082 site [Table 9] Table 10. Primers used for detection by RPA amplification in combination with Cas12 [Table 10] Table 11 Primers used to determine Cas12b with trans-cleavage activity: [Table 11] Table 12. Primers used in sensitivity testing of Cas12b transfection [Table 12] Table 13. Other sequences involved in the present invention [Table 13]

[0220] All documents referred to in this application are incorporated by reference herein as if each document were individually incorporated by reference. Furthermore, after reading the above teachings of the invention, those skilled in the art will recognize that they can make various changes or modifications to the invention, and that equivalents thereof are also encompassed within the scope of the invention as defined by the claims appended hereto.

Claims

1. A method for detecting a target nucleic acid molecule, comprising the steps of adding a guide RNA, a Cas protein, a nucleic acid probe, and a buffer solution to a system containing the target nucleic acid molecule, and then detecting cleavage of the nucleic acid probe, wherein the Cas protein is a Cas12 protein, the Cas12 protein forms a ternary complex with the guide RNA and the target nucleic acid molecule, and has collateral single-stranded DNA cleavage activity, and the nucleic acid probe is single-stranded DNA.

2. 2. The method of claim 1, wherein the guide RNA guides the Cas12 protein to specifically bind to the target nucleic acid molecule.

3. The method according to any one of claims 1 to 2, wherein the nucleic acid probe is a single-stranded DNA having a detectable label.

4. The method of any one of claims 1 to 3, wherein the target nucleic acid molecule is amplified.

5. 2. The method of claim 1, wherein the Cas12 protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a and Cas12b (i.e., C2c1).

6. The method of any one of claims 1 to 5, wherein detecting the nucleic acid probe comprises a fluorescent detection method.

7. The method of any one of claims 1 to 6, wherein the nucleic acid probe comprises a fluorescent label.

8. The method according to any one of claims 1 to 6, wherein the nucleic acid probe comprises a fluorescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

9. The method of any one of claims 1 to 8, further comprising the step of amplifying the target nucleic acid molecule.

10. 10. The method of claim 9, wherein the step of amplifying the target nucleic acid molecule comprises PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, or SMAP2.

11. 11. The method of claim 9 or 10, wherein the target nucleic acid molecule is amplified using primers, and at least one primer comprises a PAM sequence.

12. The method of any one of claims 1 to 11, wherein the target nucleic acid molecule comprises a sequence found only in a pathogen.

13. The method of any one of claims 1 to 12, wherein the target nucleic acid molecule comprises a single nucleotide polymorphism (SNP) site in human or other species.

14. The method of any one of claims 1 to 13, wherein the target nucleic acid molecule comprises a genetic mutation.

15. 15. The method of any one of claims 1 to 14, wherein the guide RNA comprises a crRNA of 15 nt, 16 nt or 17 nt in length.

16. A detection system comprising: (a) a Cas12 protein; and (b) a target nucleic acid molecule; and (c) a guide RNA that guides the Cas12 protein to specifically bind to the target nucleic acid molecule; and (d) a nucleic acid probe that is single-stranded DNA; wherein the Cas12 protein forms a ternary complex with the guide RNA and the target nucleic acid molecule and has collateral single-strand DNA cleavage activity.

17. 17. The detection system of claim 16, wherein the nucleic acid probe comprises a detectable label.

18. The detection system of any one of claims 16 to 17, wherein the Cas12 protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a and Cas12b (i.e., C2c1).

19. The detection system according to any one of claims 16 to 17, wherein the Cas12 protein is LbCas12a.

20. The detection system according to any one of claims 16 to 19, wherein the nucleic acid probe comprises a fluorescent label.

21. The detection system according to any one of claims 16 to 19, wherein the nucleic acid probe comprises a fluorescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

22. The detection system according to any one of claims 16 to 21, wherein the target nucleic acid molecule is a target DNA.

23. 23. The detection system of claim 22, wherein the target nucleic acid molecule is amplified.

24. 24. The detection system of claim 23, wherein the target nucleic acid molecule is amplified by a method selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2.

25. 25. The detection system of claim 23 or 24, wherein the target nucleic acid molecule is amplified using primers, and at least one primer comprises a PAM sequence.

26. The detection system according to any one of claims 16 to 25, wherein the target nucleic acid molecule comprises a PAM sequence upstream or downstream of the binding site of the guide RNA.

27. 27. The detection system of claim 26, wherein the PAM sequence is −20 nt to +20 nt upstream or downstream of the binding site of the guide RNA.

28. The detection system of any one of claims 16 to 27, wherein the target nucleic acid molecule comprises a sequence found only in a pathogen.

29. The detection system according to any one of claims 16 to 27, wherein the target nucleic acid molecule comprises a single nucleotide polymorphism (SNP) site in humans or other species.

30. The detection system according to any one of claims 16 to 27, wherein the target nucleic acid molecule comprises a genetic mutation.

31. 31. The detection system of any one of claims 16 to 30, wherein the guide RNA comprises a crRNA of 15 nt, 16 nt, or 17 nt in length.

32. The detection system according to any one of claims 16 to 31, further comprising a buffer solution.

33. A kit comprising: i) a first container containing a Cas protein, wherein the Cas protein is a Cas12 protein; ii) an optional second container containing a guide RNA, wherein the guide RNA guides the Cas protein to specifically bind to a target nucleic acid molecule; iii) a third container containing a nucleic acid probe; iv) an optional fourth container containing a buffer; and wherein the Cas12 protein forms a ternary complex with the guide RNA and the target nucleic acid molecule and has collateral single-stranded DNA cleavage activity, and the nucleic acid probe is single-stranded DNA.

34. The kit of claim 33, wherein the Cas12 protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a and Cas12b (i.e., C2c1).

35. 34. The kit of claim 33, wherein the Cas12 protein is LbCas12a.

36. The kit of any one of claims 33 to 35, wherein the nucleic acid probe comprises a fluorescent label.

37. The kit according to any one of claims 33 to 35, wherein the nucleic acid probe comprises a fluorescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

38. The kit according to any one of claims 33 to 37, wherein the target nucleic acid molecule is a target DNA.

39. 39. The kit of any one of claims 33 to 38, wherein the guide RNA comprises a crRNA of 15 nt, 16 nt, or 17 nt in length.

40. Use of a Cas protein in the preparation of the kit described in claim 33, wherein the Cas protein is a Cas12 protein, and the Cas12 protein forms a ternary complex with the guide RNA and the target nucleic acid molecule and has collateral single-stranded DNA cleavage activity.

41. The use of claim 40, wherein the Cas12 protein is a Cas12a protein.

42. The use of claim 41, wherein the Cas12a protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a and Cas12b (i.e., C2c1).

43. The use of claim 42, wherein the Cas12a protein is LbCas12a.

44. The use according to any one of claims 40 to 43, wherein the target nucleic acid molecule comprises a sequence found only in a pathogen.

45. The use according to any one of claims 40 to 43, wherein the target nucleic acid molecule comprises a single nucleotide polymorphism (SNP) site in humans or other species.

46. The use according to any one of claims 40 to 43, wherein the target nucleic acid molecule comprises a genetic mutation.

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