WO2024111543A1 - 標的核酸の検出方法及びキット - Google Patents

標的核酸の検出方法及びキット Download PDF

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WO2024111543A1
WO2024111543A1 PCT/JP2023/041610 JP2023041610W WO2024111543A1 WO 2024111543 A1 WO2024111543 A1 WO 2024111543A1 JP 2023041610 W JP2023041610 W JP 2023041610W WO 2024111543 A1 WO2024111543 A1 WO 2024111543A1
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stranded dna
grna
nucleic acid
double
acid fragment
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French (fr)
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力也 渡邉
肇 篠田
麻美 牧野
麻実 吉村
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RIKEN
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

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  • the present invention relates to a method and kit for detecting a target nucleic acid. More specifically, the present invention relates to a method for detecting fully double-stranded DNA in a sample containing fully double-stranded DNA and incomplete double-stranded DNA, a method for detecting human hepatitis B virus covalently closed circular DNA (HBV cccDNA), a method for screening a therapeutic agent for chronic hepatitis B, a kit for detecting fully double-stranded DNA in a sample containing fully double-stranded DNA and incomplete double-stranded DNA, and a kit for detecting HBV cccDNA in a sample.
  • HBV cccDNA human hepatitis B virus covalently closed circular DNA
  • Non-Patent Documents 1 to 3 report a method for detecting target nucleic acid fragments with high sensitivity by utilizing such activity of Cas12 and Cas13.
  • the present invention aims to provide a technology that uses the CRISPR-Cas protein to detect fully double-stranded DNA in a sample that contains fully double-stranded DNA and incomplete double-stranded DNA.
  • a method for detecting a completely double-stranded DNA in a sample containing a completely double-stranded DNA and an incomplete double-stranded DNA comprising: a step (a) of contacting the sample with a first gRNA, a CRISPR-Cas12 protein, and a first substrate nucleic acid fragment; wherein the incomplete double-stranded DNA is a completely double-stranded DNA in which a part of one strand of the completely double-stranded DNA has been replaced with RNA, a part of the completely double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one strand of the completely double-stranded DNA; the first gRNA is a gRNA that is complementary to a region of the completely double-stranded DNA that corresponds to an incomplete portion of the incomplete double-stranded DNA; and The method includes: step (a) in which a first substrate nucleic acid fragment is labeled with a first gRNA, a CRISPR-C
  • the rcDNA forms a ternary complex, it is cleaved by the nuclease activity expressed by the CRISPR-Cas13 protein, causing the second fluorescent substance to separate from the second quenching substance and emitting a second fluorescence when irradiated with a second excitation light; in the step (b), the second fluorescent substance is further irradiated with the second excitation light and the second fluorescence
  • a method for screening a therapeutic agent for chronic hepatitis B comprising: incubating cells infected with human hepatitis B virus (HBV) in the presence of a test substance; and detecting HBV cccDNA in the cells, wherein a decrease in the amount of the detected HBV cccDNA compared to that in the absence of the test substance indicates that the test substance is a candidate for a therapeutic agent for chronic hepatitis B, and the detection of the HBV cccDNA is a step (a) of contacting the cell-derived sample with a first gRNA, a CRISPR-Cas12 protein, and a first substrate nucleic acid fragment, wherein the first gRNA is a gRNA complementary to at least a part of an RNA region of HBV rcDNA, the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance, and the first gRNA, the CRISPR-Cas12 protein, and
  • a kit for detecting a completely double-stranded DNA in a sample containing a completely double-stranded DNA and an incomplete double-stranded DNA comprising a first gRNA, a CRISPR-Cas12 protein, and a first substrate nucleic acid fragment, wherein the incomplete double-stranded DNA is a completely double-stranded DNA in which a part of one strand of the completely double-stranded DNA has been replaced with RNA, a part of the completely double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one strand of the completely double-stranded DNA, and the first gRNA is a nuclease that is a nucleic acid fragment of the completely double-stranded DNA.
  • the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance; and when the first gRNA, the CRISPR-Cas12 protein, and the completely double-stranded DNA form a ternary complex, the first substrate nucleic acid fragment is cleaved by nuclease activity expressed by the CRISPR-Cas12 protein, and the first fluorescent substance is separated from the first quenching substance, and emits a first fluorescence when irradiated with a first excitation light.
  • kits according to [8] for detecting HBV cccDNA in a sample wherein the complete double-stranded DNA is HBV cccDNA, the incomplete double-stranded DNA is HBV rcDNA, and the first gRNA is a gRNA complementary to at least a portion of the RNA region of HBV rcDNA.
  • kits according to [8] for further detecting HBV rcDNA in a sample further comprising a second gRNA, a CRISPR-Cas13 protein, and a second substrate nucleic acid fragment, wherein the second gRNA is a gRNA complementary to an RNA region of HBV rcDNA, and the second substrate nucleic acid fragment is labeled with a second fluorescent substance and a second quencher, and when the second gRNA, the CRISPR-Cas13 protein, and the HBV rcDNA form a ternary complex, the second substrate nucleic acid fragment is cleaved by nuclease activity expressed by the CRISPR-Cas13 protein, and the second fluorescent substance is separated from the second quencher, and emits a second fluorescence when irradiated with a second excitation light.
  • the present invention provides a technique for detecting fully double-stranded DNA in a sample that contains fully double-stranded DNA and incomplete double-stranded DNA.
  • FIG. 1 is a schematic diagram illustrating a method for detecting a target nucleic acid.
  • FIG. 2 is a schematic diagram showing an example of the structure of an incomplete double-stranded DNA.
  • FIG. 3 is a schematic diagram illustrating the life cycle of human hepatitis B virus (HBV).
  • Figure 4 is a diagram illustrating the structures of a nucleic acid fragment that mimics HBV cccDNA and a nucleic acid fragment that mimics HBV rcDNA.
  • Figure 5 illustrates the recognition of a nucleic acid fragment mimicking HBV cccDNA by gRNA.
  • Figure 6 is a diagram illustrating the recognition of a nucleic acid fragment mimicking HBV rcDNA by gRNA.
  • FIG. 7 is a set of representative fluorescence micrographs showing the results of Experimental Example 1.
  • FIG. 8 is a graph created based on FIG.
  • FIG. 9 is a schematic diagram illustrating Experimental Example 2.
  • FIG. 10 is a representative fluorescence micrograph showing the results of Experimental Example 2.
  • the present invention provides a method for detecting a completely double-stranded DNA in a sample containing a completely double-stranded DNA and an incomplete double-stranded DNA, comprising the step (a) of contacting the sample with a first gRNA, a CRISPR-Cas12 protein, and a first substrate nucleic acid fragment, wherein the incomplete double-stranded DNA is a completely double-stranded DNA in which a part of one strand of the completely double-stranded DNA has been replaced with RNA, a part of the completely double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one strand of the completely double-stranded DNA, and the first gRNA is a gRNA complementary to a region of the completely double-stranded DNA that corresponds to the incomplete portion of the incomplete double-strand
  • the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quencher, and when the first gRNA, the CRISPR-Cas12 protein, and the completely double-stranded DNA form a ternary complex, the first substrate nucleic acid fragment is cleaved by nuclease activity expressed by the CRISPR-Cas12 protein, causing the first fluorescent substance to separate from the first quencher and emitting a first fluorescence upon irradiation with a first excitation light; and a step (b) of irradiating the first fluorescent substance with the first excitation light and detecting the first fluorescence, wherein detection of the first fluorescence indicates the presence of the completely double-stranded DNA in the sample.
  • FIG. 1 is a schematic diagram illustrating the method of this embodiment.
  • the Cas12 protein 110 and the first gRNA 120 are brought into contact with each other, they bind to each other to form a binary complex 130.
  • the first gRNA 120 has a base sequence complementary to the target nucleic acid (completely double-stranded DNA) 140 in a part thereof.
  • the Cas12 protein 110 does not express nuclease activity, so the first substrate nucleic acid fragment 150 is not cleaved.
  • the substrate nucleic acid fragment 150 is a single-stranded DNA fragment labeled with a first fluorescent substance F and a first quenching substance Q. No fluorescence is generated even when the first substrate nucleic acid fragment 150 is irradiated with excitation light.
  • the Cas12 protein 110 cleaves the target site of the target nucleic acid 140.
  • the target site of the target nucleic acid 140 is indicated by an arrowhead.
  • the lower part of FIG. 1 is a schematic diagram showing a ternary complex 100' in which the target site of the target nucleic acid fragment 140 has been cleaved.
  • the ternary complex 100' expresses nuclease activity.
  • the first substrate nucleic acid fragment 150 present around the ternary complex 100' is cleaved.
  • the first fluorescent substance F of the first substrate nucleic acid fragment 150 is separated from the first quenching substance Q.
  • the first fluorescent substance F separated from the first quenching substance Q emits a first fluorescence when irradiated with the first excitation light.
  • the first excitation light is irradiated onto the first fluorescent substance F, and the first fluorescence is detected. If the first fluorescence is detected, it can be determined that the target nucleic acid 140 is present in the sample.
  • the sample, the first gRNA 120, the CRISPR-Cas12 protein 110, and the first substrate nucleic acid fragment 150 may be mixed and contacted in any order.
  • the first gRNA 120 and the CRISPR-Cas12 protein 110 may first be brought into contact with each other to form a binary complex 130, and then the sample may be brought into contact with the sample.
  • the target nucleic acid 140 binds to the binary complex 130, forming a ternary complex 100.
  • the first substrate nucleic acid fragment 150 may be brought into contact with the sample.
  • the target nucleic acid 140 and the first substrate nucleic acid fragment 150 may be contacted simultaneously.
  • the first gRNA 120, the CRISPR-Cas12 protein 110, and the sample may be contacted simultaneously. Even in this case, if the target nucleic acid 140 is present in the sample, a ternary complex 100 is ultimately formed. After that, the first substrate nucleic acid fragment 150 may be contacted.
  • the sample, the first gRNA 120, the CRISPR-Cas12 protein 110, and the first substrate nucleic acid fragment 150 may be contacted simultaneously. Even in this case, if the target nucleic acid 140 is present in the sample, a ternary complex 100 is eventually formed, and when the target site of the target nucleic acid 140 is cleaved in the ternary complex 100, it is converted to a ternary complex 100', which expresses nuclease activity and cleaves the first substrate nucleic acid fragment 150.
  • the fully double-stranded DNA is the target nucleic acid 140 that is cleaved as a target by the binary complex 130 of the first gRNA 120 and the CRISPR-Cas12 protein 110.
  • the first gRNA 120 is selected so that the incomplete double-stranded DNA is not targeted by the binary complex 130 of the first gRNA 120 and the CRISPR-Cas12 protein 110. This makes it possible to detect the complete double-stranded DNA in a sample that contains complete double-stranded DNA and incomplete double-stranded DNA.
  • Incomplete double-stranded DNA is DNA in which part of one of the strands of a fully double-stranded DNA has been replaced with RNA, part of the fully double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one of the strands of a fully double-stranded DNA.
  • Figure 2 is a schematic diagram showing an example of the structure of incomplete double-stranded DNA.
  • the solid line represents DNA
  • the dashed line represents RNA.
  • a part of a completely double-stranded DNA is single-stranded DNA when, for example, it has a base sequence that is not complementary to a part of the completely double-stranded DNA, making that region single-stranded DNA, or when part of one of the strands of the completely double-stranded DNA is missing.
  • the first gRNA is a gRNA that is complementary to a region of the completely double-stranded DNA that corresponds to an incomplete portion of the incomplete double-stranded DNA.
  • the incomplete portion is, as described above, an RNA region when part of one strand of the completely double-stranded DNA is replaced with RNA, a single-stranded region when part of the completely double-stranded DNA becomes single-stranded DNA, a deleted region when part of one strand of the completely double-stranded DNA is deleted, a region containing a nick when a nick is introduced into one strand of the completely double-stranded DNA, etc.
  • the first gRNA is a gRNA that is complementary to a region on the completely double-stranded DNA that corresponds to these incomplete portions.
  • the first gRNA forms a ternary complex with the Cas12 protein and fully double-stranded DNA, and is capable of cleaving the fully double-stranded DNA to express nuclease activity.
  • the first gRNA is unable to form a ternary complex with the Cas12 protein and incompletely double-stranded DNA, or is unable to express nuclease activity even if it does form a ternary complex. Therefore, according to the method of this embodiment, it becomes possible to detect fully double-stranded DNA in a sample containing fully double-stranded DNA and incompletely double-stranded DNA.
  • the Cas12 protein expresses nuclease activity after forming a ternary complex with the gRNA and the target nucleic acid (completely double-stranded DNA).
  • the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance, and when it is cleaved by the nuclease activity of the ternary complex and the first fluorescent substance is separated from the first quenching substance, it emits a first fluorescence when irradiated with a first excitation light.
  • the above-mentioned ternary complex is formed, the first substrate nucleic acid fragment is cleaved, and the first fluorescent substance separates from the first quenching substance. Then, when the first excitation light is irradiated, the first fluorescence is detected.
  • the method of this embodiment may be a method in which the complete double-stranded DNA is human hepatitis B virus covalently closed circular DNA (HBV cccDNA), the incomplete double-stranded DNA is human hepatitis B virus relaxed circular DNA (HBV rcDNA), the first gRNA is a gRNA complementary to at least a portion of the RNA region of HBV rcDNA, and in which the detection of the first fluorescence in step (b) indicates the presence of the HBV cccDNA in the sample.
  • HBV cccDNA human hepatitis B virus covalently closed circular DNA
  • HBV rcDNA human hepatitis B virus relaxed circular DNA
  • the first gRNA is a gRNA complementary to at least a portion of the RNA region of HBV rcDNA
  • FIG 3 is a schematic diagram explaining the life cycle of human hepatitis B virus (HBV).
  • HBV is known to infect humans through body fluids such as blood, and cause acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma.
  • HBV virus particles contain a genome consisting of HBV rcDNA of approximately 3,200 bases.
  • HBV rcDNA is an incomplete double-stranded DNA that is partially RNA.
  • HBV infects human hepatocytes HBV rcDNA is transported to the nucleus, where the host's gene repair pathway forms a completely double-stranded closed circular DNA (HBV cccDNA).
  • HBV cccDNA is structurally extremely stable, and it is known that once an individual is infected with HBV, it remains in the nucleus for a long period of time even after the HBs antigen in the blood has disappeared.
  • RNA required for HBV replication is transcribed from HBV cccDNA.
  • Viral transcription products include pregenomic (pg)RNA, which is the viral RNA genome, in addition to mRNA that codes for the viral surface protein (HBs) and mRNA for the HBx protein.
  • pg pregenomic
  • HBs viral surface protein
  • pgRNA has an RNA higher-order structure called the epsilon structure.
  • the viral reverse transcriptase P protein recognizes this epsilon structure, the viral RNP complex is formed.
  • the RNP complex is incorporated into an icosahedral structure composed of the core protein, forming the nucleocapsid.
  • the P protein converts pgRNA to HBV rcDNA, and when the nucleocapsid assembles with the viral surface protein (HBs), it is released from the hepatocyte as an infectious virus.
  • HBs viral surface protein
  • HBV-infected cells contain HBV rcDNA, as well as double-stranded linear DNA (dslDNA), which is produced intermediately during the production process of HBV rcDNA, and the total amount of these is 100 to 1,000 times that of HBV cccDNA. For this reason, it has previously been difficult to specifically detect only HBV cccDNA.
  • dslDNA double-stranded linear DNA
  • the method of this embodiment makes it possible to specifically detect only HBV cccDNA, even in the presence of HBV crcDNA.
  • the first gRNA is not particularly limited as long as it can detect HBV cccDNA in a sample and does not react with HBV rcDNA.
  • the first gRNA may be complementary to at least a part of the RNA region of HBV rcDNA, for example, a gRNA complementary to a region on HBV cccDNA corresponding to a region including the RNA region of HBV rcDNA and the DNA region adjacent thereto.
  • the first gRNA may be complementary to a region on HBV cccDNA corresponding to the RNA region of HBV rcDNA.
  • gRNAs shown in SEQ ID NOs: 4 and 5 can be suitably used as the first gRNA.
  • the method of this embodiment may further detect HBV rcDNA in the sample.
  • the sample is further contacted with a second gRNA, a CRISPR-Cas13 protein, and a second substrate nucleic acid fragment, where the second gRNA is a gRNA complementary to the RNA region of HBV rcDNA, and the second substrate nucleic acid fragment is labeled with a second fluorescent substance and a second quenching substance.
  • the second substrate nucleic acid fragment is cleaved by the nuclease activity expressed by the CRISPR-Cas13 protein, and the second fluorescent substance is separated from the second quenching substance and emits a second fluorescence when irradiated with a second excitation light.
  • the second fluorescent substance is further irradiated with a second excitation light, and the second fluorescence is detected, and the detection of the second fluorescence indicates the presence of the HBV rcDNA in the sample.
  • the method of this embodiment is a method for detecting HBV cccDNA and HBV rcDNA in a sample, and includes a step (a) of contacting the sample with a first gRNA, a CRISPR-Cas12 protein, a first substrate nucleic acid fragment, a second gRNA, a CRISPR-Cas13 protein, and a second substrate nucleic acid fragment, in which the first gRNA is a gRNA complementary to at least a portion of the RNA region of HBV rcDNA, the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance, and when the first gRNA, the CRISPR-Cas12 protein, and the HBV cccDNA form a ternary complex, the first substrate nucleic acid fragment is cleaved by the nuclease activity expressed by the CRISPR-Cas12 protein, the first fluorescent substance is separated from the first quenching substance, and emit
  • the Cas13 protein can detect single-stranded RNA or single-stranded DNA as the target nucleic acid.
  • the second gRNA is not particularly limited as long as it can detect HBV rcDNA in a sample and does not react with HBV cccDNA.
  • the second gRNA may be complementary to the RNA region of HBV rcDNA. This allows the second gRNA, Cas13 protein, and HBV rcDNA to form a three-component complex, and the second substrate nucleic acid fragment can be cleaved by the nuclease activity expressed by the Cas13 protein.
  • the gRNA shown in SEQ ID NO: 6 can be suitably used as the second gRNA.
  • the guide RNA is not particularly limited as long as it can be used with the CRISPR-Cas protein used, and may be a complex of CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA), a single gRNA (sgRNA) that combines tracrRNA and crRNA, or crRNA alone.
  • examples of the CRISPR-Cas12 protein include Cas12a protein derived from Lachnospiraceae bacterium ND2006 (LbCas12a, UniProtKB accession number: A0A182DWE3), Cas12a protein derived from Acidaminococcus sp. (AsCas12a, UniProtKB accession number: U2UMQ6), and Cas12a protein derived from Francisella tularensis subsp. Cas12a protein derived from B.
  • novicida FnCas12a, UniProtKB accession number: A0Q7Q2
  • Cas12b protein derived from Alicyclobacillus acidoterrestris AaCas12b, UniProtKB accession number: T0D7A2
  • orthologs of these proteins and modified forms of these proteins can be used.
  • Modified forms of the CRISPR-Cas12 protein include, for example, mutants with improved efficiency in forming a ternary complex and mutants with increased nuclease activity after forming a ternary complex.
  • CRISPR-Cas13 proteins include the Cas13a protein derived from L. trevisanii JMUB4039 (LtrCas13a, NCBI accession number: BBM56601.1), the Cas13a protein derived from Leptotrichia wadei (LwaCas13a, NCBI accession number: WP_021746774.1), the Cas13a protein derived from Lachnospiraceae bacterium NK4A179 (LbaCas13a, NCBI accession number: WP_022785443.1), and the Cas13a protein derived from Leptotrichia buccalis C-1013-b.
  • Cas13b protein from Bergeyella zoohelcum (BzoCas13b, NCBI accession number: WP_002664492), Cas13b protein from Prevotella intermedia (PinCas13b, NCBI accession number: WP_036860899), Cas13b protein from Prevotella buccae (PbuCas13b, NCBI accession number: WP_004343973), Alistipes sp.
  • Cas13b protein derived from ZOR0009 (AspCas13b, NCBI accession number: WP_047447901), Prevotella sp.
  • the Cas13b protein derived from MA2016 (PsmCas13b, NCBI accession number: WP_036929175), the Cas13b protein derived from Riemerella anatipestifer (RanCas13b, NCBI accession number: WP_004919755), the Cas13b protein derived from Prevotella aurantiaca (PauCas13b, NCBI accession number: WP_025000926), the Cas13b protein derived from Prevotella saccharolytica (PsaCas13b, NCBI accession number: WP_025000926), I accession number: WP_051522484), Cas13b protein derived from Prevotella intermedia (Pin2Cas13b, NCBI accession number: WP_061868553), Cas13b protein derived from Capnocytophaga canimorsus (CcaCas13b, NCBI accession number: WP_013997271),
  • Cas13b protein derived from P5-125 (PspCas13b, NCBI accession number: WP_044065294), Cas13b protein derived from Porphyromonas gingivalis (PigCas13b, NCBI accession number: WP_053444417), Cas13b protein derived from Prevotella intermedia (Pin3Cas13b, NCBI accession number: WP_050955369), Cas13b protein derived from Enterococcus italicus
  • the Csm6 protein derived from Lactobacillus salivarius (EiCsm6, NCBI accession number: WP_007208953.1), the Csm6 protein derived from Lactobacillus salivarius (LsCsm6, NCBI accession number: WP_081509150.1), the Csm6 protein derived from Thermus thermophilus (TtCsm6, NCBI accession number: WP_011229148.1), ortholog
  • the sample is not particularly limited and can be appropriately selected depending on the purpose, and examples include biological samples such as saliva, blood, urine, amniotic fluid, malignant ascites, pharyngeal swabs, and nasal swabs, as well as cultured cell supernatants and cultured cell homogenates.
  • biological samples such as saliva, blood, urine, amniotic fluid, malignant ascites, pharyngeal swabs, and nasal swabs, as well as cultured cell supernatants and cultured cell homogenates.
  • liver biopsy samples from patients with chronic hepatitis B, homogenates of HBV-infected cells, etc. can be suitably used.
  • the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance, and is cleaved by the nuclease activity of the ternary complex of gRNA, Cas12 protein, and target nucleic acid.
  • the first fluorescent substance separates from the first quenching substance, the first fluorescence is emitted by irradiation with the first excitation light.
  • the Cas12 protein cleaves single-stranded DNA as a substrate by the nuclease activity expressed after the formation of the ternary complex. Therefore, it is preferable to use single-stranded DNA as the first substrate nucleic acid fragment.
  • the second substrate nucleic acid fragment is labeled with a second fluorescent substance and a second quenching substance, and is cleaved by the nuclease activity of the ternary complex of gRNA, Cas13 protein, and target nucleic acid.
  • the second fluorescent substance separates from the second quenching substance, the second fluorescence is emitted by irradiation with second excitation light.
  • the Cas13 protein cleaves single-stranded RNA as a substrate by the nuclease activity expressed after the formation of the ternary complex. Therefore, it is preferable to use single-stranded RNA as the second substrate nucleic acid fragment.
  • the combination of fluorescent substance and quenching substance is one that can quench the fluorescence of the fluorescent substance when they are placed close to each other.
  • fluorescent substance for example, when FAM, HEX, JOE, etc. are used as the fluorescent substance, Iowa Black FQ (IDT) or TAMRA, etc. can be used as the quenching substance.
  • Iowa Black FQ (IDT) or TAMRA, etc. can be used as the quenching substance.
  • Alexa Fluor 647, TAMRA, ROX, etc. are used as the fluorescent substance
  • Iowa Black RQ (IDT) etc. can be used as the quenching substance.
  • step (a) is preferably carried out in a reaction space having a volume of 10 aL to 100 pL.
  • Technologies for carrying out enzyme reactions in a large number of minute reaction spaces are being considered as a method for detecting target substances with high accuracy. These methods are called digital measurement. In digital measurement, the sample is divided into an extremely large number of minute reaction spaces to detect signals.
  • the signal from each reaction space is binarized, and the presence or absence of the target substance is determined, and the number of molecules of the target substance is measured.
  • Digital measurement can significantly improve detection sensitivity and quantitation compared to conventional ELISA and real-time PCR methods.
  • the method of this embodiment is preferably performed by digital measurement. More specifically, the sample, Cas12 protein, first gRNA, first substrate nucleic acid fragment, Cas13 protein, second gRNA, and second substrate nucleic acid fragment are contacted in a minute reaction space.
  • the volume of each reaction space may be, for example, 10 aL to 10 pL, for example, 10 aL to 1 pL, for example, 10 aL to 100 fL, or for example, 10 aL to 10 fL.
  • the reaction space may be, for example, a droplet.
  • the reaction space may be a well formed on a substrate.
  • the present invention provides a method for screening a therapeutic agent for chronic hepatitis B, comprising: incubating a cell infected with human hepatitis B virus (HBV) in the presence of a test substance; and detecting HBV cccDNA in the cell, wherein a decrease in the amount of the detected HBV cccDNA compared to the absence of the test substance indicates that the test substance is a candidate for a therapeutic agent for chronic hepatitis B, and the detection of the HBV cccDNA is a step (a) of contacting the cell-derived sample with a first gRNA, a CRISPR-Cas12 protein, and a first substrate nucleic acid fragment, wherein the first gRNA is a gRNA complementary to at least a part of an RNA region of HBV rcDNA, and the first substrate nucleic acid fragment is labeled with a first fluorescent substance and
  • the first gRNA, Cas12 protein, first substrate nucleic acid fragment, first fluorescent substance, first quenching substance, first excitation light, first fluorescence, etc. are the same as those described above.
  • step (a) it is preferable to carry out step (a) in a reaction space having a volume of 10 aL to 100 pL, and to detect HBV cccDNA by digital measurement under conditions in which 0 or 1 HBV cccDNA is introduced per reaction space.
  • test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.
  • Examples of cells infected with HBV include primary human hepatocytes and cell lines expressing the sodium-taurocholate cotransporting molecule NTCP (Na + Taurocholate Co-transporting Polypeptide).
  • NTCP Na + Taurocholate Co-transporting Polypeptide
  • the second gRNA, Cas13 protein, and second substrate nucleic acid fragment described above may be further used to further detect HBV rcDNA together with HBV cccDNA.
  • the present invention provides a kit for detecting a completely double-stranded DNA in a sample containing a completely double-stranded DNA and an incomplete double-stranded DNA, the kit comprising: a first gRNA; a CRISPR-Cas12 protein; and a first substrate nucleic acid fragment; wherein the incomplete double-stranded DNA is a completely double-stranded DNA in which a part of one strand of the completely double-stranded DNA has been replaced with RNA, a part of the completely double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one strand of the completely double-stranded DNA; and the first gRNA is a completely double-stranded DNA in which a part of the completely double-stranded DNA has been replaced with RNA, a part of the completely double-stranded DNA has become single-stranded DNA, or a nick has been introduced into one strand of the completely double-stranded DNA.
  • A is a gRNA complementary to a region corresponding to the incomplete part of the incomplete double-stranded DNA
  • the first substrate nucleic acid fragment is labeled with a first fluorescent substance and a first quenching substance
  • the first substrate nucleic acid fragment is cleaved by nuclease activity expressed by the CRISPR-Cas12 protein
  • the first fluorescent substance is separated from the first quenching substance, and emits a first fluorescence when irradiated with a first excitation light.
  • the kit of this embodiment can be used to suitably carry out the above-mentioned method of detecting fully double-stranded DNA in a sample containing fully double-stranded DNA and incomplete double-stranded DNA, the method of detecting HBV cccDNA, and the method of screening for a therapeutic drug for chronic hepatitis B.
  • the first gRNA, Cas12 protein, first substrate nucleic acid fragment, first fluorescent substance, first quenching substance, first excitation light, first fluorescence, etc. are the same as those described above.
  • the kit of this embodiment may be for detecting HBV cccDNA in a sample.
  • the fully double-stranded DNA is HBV cccDNA
  • the incompletely double-stranded DNA is HBV rcDNA
  • the first gRNA is a gRNA complementary to at least a portion of the RNA region of HBV rcDNA.
  • the kit of this embodiment may be for detecting HBV rcDNA in a sample in addition to HBV cccDNA.
  • the kit of this embodiment further includes a second gRNA, a CRISPR-Cas13 protein, and a second substrate nucleic acid fragment, the second gRNA being a gRNA complementary to the RNA region of HBV rcDNA, the second substrate nucleic acid fragment being labeled with a second fluorescent substance and a second quenching substance, and when the second gRNA, the CRISPR-Cas13 protein, and the HBV rcDNA form a ternary complex, the second substrate nucleic acid fragment is cleaved by the nuclease activity expressed by the CRISPR-Cas13 protein, the second fluorescent substance is separated from the second quenching substance, and emits a second fluorescence when irradiated with a second excitation light.
  • the second gRNA, Cas13 protein, second substrate nucleic acid fragment, second fluorescent substance, second quenching substance, second excitation light, second fluorescence, etc. are the same as those described above.
  • the kit of this embodiment may further include a well array with a volume of 10 aL to 100 pL per well. This makes it possible to detect completely double-stranded DNA or incompletely double-stranded DNA in a reaction space with a volume of 10 aL to 100 pL and perform digital measurement.
  • Cas12 protein As the Cas12 protein, the D156R/G532R/K538R mutant (enLbCas12a) of Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) was used.
  • the expression vector of enLbCas12a was transfected into Escherichia coli Rosetta 2 (DE3) strain to express it.
  • the expression vector was a pET-based vector having a 6xHis tag and a FLAG tag at the N-terminus.
  • the expressed Cas12 protein was purified using Ni-NTA resin. Subsequently, cation exchange chromatography was performed using a HiTrap SP HP column (Cytiva), and further purification was performed by gel filtration chromatography using an Enrich SEC 650 column (Bio-Rad).
  • Cas13 protein As the Cas1 protein, the Cas13a protein (LtrCas13a) derived from L. trevisanii JMUB4039 was used. The expression vector of LtrCas13a was transfected into the E. coli Rosetta 2 (DE3) strain to express it. The expression vector was a pET-based vector having a 6xHis tag and a FLAG tag at the N-terminus. The expressed Cas13 protein was purified using Ni-NTA resin. Subsequently, cation exchange chromatography was performed using a HiTrap SP HP column (Cytiva), and further purification was performed by gel filtration chromatography using an Enrich SEC 650 column (Bio-Rad).
  • Target Nucleic Acid Fragment As target nucleic acid fragments, nucleic acid fragments mimicking regions in which the structures of HBV cccDNA and HBV rcDNA are different were prepared.
  • ccc1 a complete double-stranded nucleic acid fragment that mimics HBV cccDNA.
  • Figure 4 The structure of ccc1 is shown in Figure 4.
  • nucleic acid fragments Fw and Rv_rc_A shown in Table 1 below were annealed to prepare a nucleic acid fragment (hereinafter sometimes referred to as "rc1") that mimics HBV rcDNA.
  • rc1 a nucleic acid fragment that mimics HBV rcDNA.
  • the lowercase letters represent RNA and the uppercase letters represent DNA.
  • a portion of the nucleic acid fragment rc1 is RNA, and furthermore, the 5' end of the RNA portion has a region that is not complementary to the nucleic acid fragment Fw.
  • the structure of rc1 is shown in Figure 4.
  • rc2 nucleic acid fragments in which the 3'-base of rcDNA (+) extends to the RNA flap site.
  • rc2 was prepared by annealing the nucleic acid fragments Fw, Rv_rc_A, and Rv_rc_B shown in Table 1 below.
  • rc3 was prepared by annealing the nucleic acid fragments Fw, Rv_rc_A, and Rv_rc_C shown in Table 1 below.
  • the structures of rc2 and rc3 are shown in Figure 4. In Figure 4, "(" in rc2 and rc3 represents a nick.
  • Each annealed sample was subjected to agarose gel electrophoresis, and the target nucleic acid fragment was excised and then purified using NucleoSpin Gel and PCR Clean-up (U0609C, Takara).
  • gRNA for Cas13 The nucleic acid fragments shown in Table 3 below were used as gRNA (crRNA) for Cas13. In Table 3, the lowercase letters represent RNA.
  • Substrate nucleic acid fragment for Cas12 A single-stranded DNA fragment was used as the substrate nucleic acid fragment for Cas12.
  • the substrate nucleic acid fragment was chemically synthesized by outsourcing (IDT).
  • the 5' end of the substrate nucleic acid fragment was labeled with FAM, a fluorescent substance, and the 3' end was labeled with Iowa Black FQ (IDT), a quenching substance.
  • the base sequence of the chemically synthesized substrate nucleic acid fragment for Cas12 was "5'-(FAM)TTATT(IABkFQ)-3'" (where "IABkFQ” stands for Iowa Black FQ).
  • substrate nucleic acid fragment for Cas13 A single-stranded RNA fragment was used as the substrate nucleic acid fragment for Cas13.
  • the substrate nucleic acid fragment single-stranded RNA fragment was chemically synthesized by outsourcing (IDT).
  • IDTT The 5' end of the substrate nucleic acid fragment was labeled with Alexa Fluor 647, a fluorescent substance, and the 3' end was labeled with Iowa Black RQ (IDT), a quenching substance.
  • the base sequence of the chemically synthesized substrate nucleic acid fragment was "5'-(Ax647)UUUUU(IABkRQ)-3'" (where "Ax647” represents Alexa Fluor 647 and "IABkRQ” represents Iowa Black RQ).
  • HBV cccDNA or HBV rcDNA was performed using a device (hereinafter sometimes referred to as a "CD device") in which a sealing material (keshimir) was applied in a circular pattern with a diameter of 7 mm and intervals of 9 mm onto a well array containing approximately 1 million cylindrical wells, each 3.5 ⁇ m in diameter and 3.5 ⁇ m deep, arranged in an area of 1 cm2.
  • a sealing material keshimir
  • Buffers The compositions of the buffers used are as follows: Buffer A (20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 10 mM MgCl 2 ), Buffer F (20 mM HEPES-KOH (pH 6.8), 60 mM NaCl, 6 mM MgCl 2 ), Buffer N (10 mM Tris-NaCl (pH 9.0), 10 mM NaCl, 15 mM MgCl 2 ).
  • Figure 5 is a diagram explaining the recognition by gRNA of a nucleic acid fragment (ccc1 described above) that mimics HBV cccDNA.
  • the top of Figure 5 is a schematic diagram of HBV cccDNA, and the position surrounded by a dotted line in a square corresponds to the nucleic acid fragment ccc1.
  • the bottom of Figure 5 shows the case where the above-mentioned LbCas12a_crRNA_H5 is used as the gRNA.
  • the lower case letters represent RNA
  • the upper case letters represent DNA.
  • the gRNA, Cas12 protein, and nucleic acid fragment ccc1 form a ternary complex, and it is believed that the Cas12 protein expresses nuclease activity to cleave the substrate nucleic acid fragment for Cas12.
  • Figure 6 is a diagram explaining the recognition by gRNA of nucleic acid fragments (rc1, rc2, and rc3 mentioned above) that mimic HBV rcDNA.
  • the top row of Figure 6 is a schematic diagram of HBV rcDNA, and the positions enclosed by dotted lines in the squares correspond to the nucleic acid fragments rc1, rc2, and rc3.
  • the second row from the top in Figure 6 shows the recognition site of the nucleic acid fragment rc1 by gRNA LbCas12a_crRNA_H5.
  • the lowercase letters represent RNA, and the uppercase letters represent DNA.
  • the recognition site of the nucleic acid fragment rc1 by LbCas12a_crRNA_H5 is a DNA-RNA hybrid, and only a portion of the base sequence is identical. Since the Cas12 protein binds to and activates DNA fragments, it is thought that when LbCas12a_crRNA_H5 is used as the gRNA, the Cas12 protein will not be activated by the nucleic acid fragment rc1.
  • the third row from the top in Figure 6 shows the recognition site of the nucleic acid fragment rc2 by gRNA LbCas12a_crRNA_H5.
  • the lowercase letters represent RNA, and the uppercase letters represent DNA.
  • "(" represents a nick.
  • the recognition site of the nucleic acid fragment rc2 by LbCas12a_crRNA_H5 is a DNA-RNA hybrid, and also contains a nick. For this reason, when LbCas12a_crRNA_H5 is used as the gRNA, it is thought that the Cas12 protein will not be activated by the nucleic acid fragment rc2.
  • the fourth row from the top in Figure 6 shows the recognition site of the nucleic acid fragment rc3 by gRNA LbCas12a_crRNA_H5.
  • the lowercase letters represent RNA, and the uppercase letters represent DNA.
  • "(" represents a nick.
  • the recognition site of the nucleic acid fragment rc2 by LbCas12a_crRNA_H5 contains a nick. For this reason, when LbCas12a_crRNA_H5 is used as the gRNA, it is thought that the Cas12 protein is unlikely to be activated by the nucleic acid fragment rc2.
  • the reagents shown in Table 4 below were mixed and incubated at room temperature for 10 minutes to prepare a gRNA-Cas12 binary complex.
  • the gRNA used was the LbCas12a_crRNA_H5 described above.
  • the gRNA-Cas12 binary complex and the substrate nucleic acid fragment for Cas12 were mixed and diluted with Buffer A + 50 ⁇ M Triton X-100 so that the final concentration of Cas12 protein was 120 nM, the final concentration of gRNA was 30 nM, and the final concentration of the substrate nucleic acid fragment for Cas12 was 12 ⁇ M, to prepare an assay solution.
  • sealant (Fomblin Y-LVAC 25/6) was dripped from the edge of the well at 5 ⁇ L/sec. Then, 15 ⁇ L of the mixture was aspirated from the edge of the well opposite the location where the sealant was dripped and discarded. This was followed by incubation at 45°C for 15 minutes. The well array was then observed under a fluorescence microscope. An excitation wavelength of 488 nm was applied and FAM fluorescence was detected.
  • nucleic acid fragments rc1, rc2, and rc3 were used instead of nucleic acid fragment ccc1.
  • Figure 7 is a representative fluorescence microscope photograph showing the results of the assay.
  • Figure 8 is a graph created based on Figure 7. As a result, it was revealed that when nucleic acid fragment ccc1 was reacted, a fluorescent signal that was approximately 1,000 times higher than when nucleic acid fragments rc1, rc2, and rc3 were reacted was detected. This result shows that the method of this experimental example can distinguish between HBV cccDNA and HBV rcDNA, and specifically detect only HBV cccDNA.
  • the upper part of Figure 9 shows the position on the nucleic acid fragment ccc1 that is recognized by the Cas12 protein when the above-mentioned LbCas12a_crRNA_H5 is used.
  • the lined region is recognized by the Cas12 protein, and it is believed that the gRNA, Cas12 protein, and nucleic acid fragment ccc1 form a ternary complex, and the Cas12 protein expresses nuclease activity to cleave the substrate nucleic acid fragment for Cas12.
  • the lower part of Figure 9 shows the position on the nucleic acid fragment rc1 that is recognized by the Cas13 protein when the above-mentioned LtrCas13a_crRNA_rc is used.
  • the lower case letters represent RNA
  • the upper case letters represent DNA.
  • the Cas13 protein uses a single-stranded RNA fragment or a single-stranded DNA fragment as a substrate, when LtrCas13a_crRNA_rc is used as the gRNA, the lined region in the lower part of Figure 9 is recognized by the Cas13 protein, and the gRNA, Cas13 protein, and nucleic acid fragment rc1 form a ternary complex, and it is believed that the Cas13 protein expresses nuclease activity to cleave the substrate nucleic acid fragment for Cas13.
  • the reagents shown in Table 5 below were mixed and incubated at room temperature for 10 minutes to prepare a gRNA-Cas12 binary complex.
  • the gRNA used was the LbCas12a_crRNA_H5 described above.
  • the reagents shown in Table 6 below were mixed and incubated at 37°C for 10 minutes to prepare a gRNA-Cas13 binary complex.
  • the gRNA used was the LtrCas13a_crRNA_rc described above.
  • the gRNA-Cas12 binary complex, the gRNA-Cas13 binary complex, the substrate nucleic acid fragment for Cas12, and the substrate nucleic acid fragment for Cas13 were mixed and diluted with Buffer N + 50 ⁇ M Triton X-100 so that the final concentration of Cas12 protein was 120 nM, the final concentration of gRNA (LbCas12a_crRNA_H5) was 30 nM, the final concentration of Cas13 protein was 300 nM, the final concentration of gRNA (LtrCas13a_crRNA_rc) was 75 nM, the final concentration of the substrate nucleic acid fragment for Cas12 was 12 ⁇ M, and the final concentration of the substrate nucleic acid fragment for Cas13 was 12 ⁇ M, to prepare an assay solution.
  • Triton X-100 Triton X-100
  • sealant (Fomblin Y-LVAC 25/6) was dripped from the edge of the well at 5 ⁇ L/sec. 15 ⁇ L of the mixture was then aspirated and discarded from the edge of the well opposite the sealant drip position. The well was then incubated at room temperature for 5 minutes and at 45°C for 15 minutes. The well array was then observed under a fluorescence microscope. FAM fluorescence was detected by irradiating with an excitation wavelength of 488 nm, and Alexa Fluor 647 fluorescence was detected by irradiating with an excitation wavelength of 640 nm.
  • Figure 10 is a representative fluorescence microscope photograph showing the results of the assay.
  • the top row shows the results of reacting with Cas12 protein and detecting FAM fluorescence
  • the bottom row shows the results of reacting with Cas13 protein and detecting Alexa Fluor 647 fluorescence.
  • the left column shows the results of reacting with nucleic acid fragment ccc1
  • the right column shows the results of reacting with nucleic acid fragment rc1.
  • nucleic acid fragment ccc1 and the nucleic acid fragment rc1 could be detected independently in two color channels. This result shows that the method of this experimental example can simultaneously detect HBV cccDNA and HBV rcDNA.
  • the present invention provides a technique for detecting fully double-stranded DNA in a sample that contains fully double-stranded DNA and incomplete double-stranded DNA.

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