WO2023195542A1 - 複数種類の標的核酸断片を検出する方法及びキット - Google Patents

複数種類の標的核酸断片を検出する方法及びキット Download PDF

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WO2023195542A1
WO2023195542A1 PCT/JP2023/014520 JP2023014520W WO2023195542A1 WO 2023195542 A1 WO2023195542 A1 WO 2023195542A1 JP 2023014520 W JP2023014520 W JP 2023014520W WO 2023195542 A1 WO2023195542 A1 WO 2023195542A1
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nucleic acid
target nucleic
acid fragment
protein
crispr
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French (fr)
Japanese (ja)
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力也 渡邉
肇 篠田
麻美 牧野
龍也 飯田
麻実 吉村
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RIKEN
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a method and kit for detecting multiple types of target nucleic acid fragments.
  • cfDNA cell-free DNA
  • ctDNA circulating tumor DNA
  • Exosomes contain various proteins, lipids, microRNAs, DNA, etc. derived from the cells that secreted them.
  • Non-Patent Documents 1 to 3 report methods for detecting target nucleic acid fragments with high sensitivity by utilizing such activities of Cas12 and Cas13.
  • Non-Patent Document 4 The inventors previously developed the CRISPR-based amplification-free digital RNA detection (STORI) method, which is a very sensitive and simple nucleic acid detection technology (see Non-Patent Document 4).
  • STORI CRISPR-based amplification-free digital RNA detection
  • SATORI method when a nucleic acid fragment for gRNA (target nucleic acid fragment) is mixed with a mixture containing CRISPR/Cas protein, guide RNA (gRNA), and nucleic acid fluorescent reporter (substrate nucleic acid fragment), Cas protein, gRNA, and target nucleic acid fragment are mixed.
  • This is a detection method that utilizes the phenomenon that Cas protein forms a ternary complex and then Cas protein expresses nuclease activity.
  • the Cas protein that has expressed nuclease activity cleaves the nucleic acid fluorescent reporter, resulting in fluorescence.
  • This reaction in an array of microwells, binarizing the presence or absence of a fluorescent signal in each well, and performing digital detection, it is possible to detect a nucleic acid fragment to be detected at a concentration of 5 fM or less.
  • An object of the present invention is to provide a technique for detecting multiple types of target nucleic acid fragments in a sample.
  • a method for detecting a plurality of types of target nucleic acid fragments in a sample comprising: detecting a plurality of types of gRNAs, CRISPR/Cas family proteins, and substrate nucleic acid fragments for each of the plurality of types of target nucleic acid fragments; in the step (a) of contacting with the CRISPR/Cas family protein, the CRISPR/Cas family protein expresses nuclease activity after forming a ternary complex with the gRNA and the target nucleic acid fragment; /Cas family protein, and the two-part complex is immobilized on the surface of the particle, and there are multiple types of particles that can be identified from each other by a combination of optical properties, and The binary complex containing a specific type of gRNA is immobilized on the particles of the type, the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and the ternary complex is
  • the fluorescent substance When the fluorescent substance is separated from the quencher by being cleaved by nuclease activity, it emits fluorescence upon irradiation with excitation light.
  • the particles are individually placed in a reaction space having a volume of 10aL to 100pL, is carried out in the reaction space, and as a result, when the target nucleic acid fragment is present in the reaction space, the three-way complex is formed, the substrate nucleic acid fragment is cleaved, and the fluorescent substance is separated from the quencher. step (a) of separating the particles, step (b) of identifying the type of the particles placed in the reaction space, and step (c) of detecting the fluorescence of the fluorescent material separated from the quenching material in the reaction space.
  • a kit for detecting multiple types of target nucleic acid fragments in a sample which uses a combination of multiple types of gRNA for each of the multiple types of target nucleic acid fragments, a CRISPR/Cas family protein, and optical properties.
  • the CRISPR/Cas family protein includes multiple types of mutually distinguishable particles and a substrate nucleic acid fragment, and the CRISPR/Cas family protein expresses nuclease activity after forming a ternary complex with the gRNA and the target nucleic acid fragment.
  • the gRNA forms a binary complex with the CRISPR/Cas family protein
  • the binary complex is immobilized on the surface of the particle
  • a specific type of particle has a specific
  • the binary complex containing the gRNA of various types is immobilized, and the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and is cleaved by the nuclease activity of the ternary complex to release the fluorescent substance.
  • the kit according to [5] further comprising a well array having a volume of 10 aL to 100 pL per well.
  • the kit according to [5] or [6], wherein the CRISPR/Cas family protein is a Cas12 protein or a Cas13 protein.
  • a technique for detecting multiple types of target nucleic acid fragments in a sample can be provided.
  • FIG. 1A is a schematic diagram illustrating the SATORI method, which is a method for detecting target nucleic acid fragments.
  • FIG. 1B is a schematic diagram illustrating the SATORI method, which is a method for detecting target nucleic acid fragments.
  • FIG. 2A is a schematic diagram showing an example of a binary complex of a CRISPR/Cas family protein and gRNA immobilized on a particle.
  • FIG. 2B is a schematic diagram showing an example of a binary complex of a CRISPR/Cas family protein and gRNA immobilized on a particle.
  • FIG. 3A is a top view of an example fluidic device.
  • FIG. 3B is a sectional view taken along line bb' in FIG. 3A.
  • FIG. 4A is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 4B is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 4C is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 4D is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 4E is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 4F is a schematic cross-sectional view illustrating each step of forming a well array.
  • FIG. 5 is a graph showing the results of Experimental Example I-3.
  • FIG. 6A is a graph showing the results of Experimental Example I-11.
  • FIG. 6B is a graph showing the results of Experimental Example I-11.
  • FIG. 7 is a graph showing the results of Experimental Example II-5.
  • FIGS. 1A and 1B are schematic diagrams illustrating the SATORI method.
  • the case where the CRISPR/Cas family protein is Cas12a protein will be explained as an example.
  • gRNA 120 partially has a base sequence complementary to target nucleic acid fragment 140.
  • the Cas12a protein 110 does not express nuclease activity, so the substrate nucleic acid fragment 150 is not cleaved.
  • substrate nucleic acid fragment 150 is a single-stranded DNA fragment labeled with fluorescent substance F and quencher Q. Even when the substrate nucleic acid fragment 150 is irradiated with excitation light, no fluorescence is generated.
  • FIG. 1A 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 substrate nucleic acid fragment 150 present around the ternary complex 100' is cleaved.
  • the fluorescent substance F of the substrate nucleic acid fragment 150 separates from the quencher Q.
  • the fluorescent substance F separated from the quencher Q emits fluorescence when irradiated with excitation light.
  • the fluorescent substance F is irradiated with excitation light and fluorescence is detected. If fluorescence is detected, it can be determined that the target nucleic acid fragment 140 was present in the sample.
  • the present invention provides a method for detecting multiple types of target nucleic acid fragments in a sample, which comprises detecting multiple types of gRNAs, CRISPR/Cas family proteins, and CRISPR/Cas family proteins for each of the multiple types of target nucleic acid fragments.
  • the gRNA forms a binary complex with the CRISPR/Cas family protein, the binary complex is immobilized on the surface of a particle, and the particles are distinguishable from each other by a combination of optical properties.
  • the contact is performed in the reaction space, and as a result, when the target nucleic acid fragment is present in the reaction space, the ternary complex is formed, the substrate nucleic acid fragment is cleaved, and the fluorescence a step (a) in which a substance leaves the quenching substance; a step (b) of identifying the type of particle placed in the reaction space; and fluorescence of the fluorescent substance separating from the quenching substance in the reaction space. (c), wherein the detection of the fluorescence indicates that the target nucleic acid fragment is present in the reaction space, and the type of the particle is different from the type of the target nucleic acid fragment. provide a corresponding method.
  • the method of this embodiment allows multiple types of target nucleic acid fragments in a sample to be detected in parallel.
  • the plurality of types refers to two or more types, for example, it may be five or more types, it may be ten or more types, it may be twenty or more types, or it may be thirty or more types. good.
  • the method of this embodiment can be realized by using particles that can be identified by a combination of optical properties.
  • Optical properties include fluorescence properties, light absorption properties, scattered light properties, and the like.
  • particles can be fluorescently labeled by binding a fluorescent substance to the particles.
  • particles can be fluorescently labeled by mixing a fluorescent substance into the particle material itself.
  • the fluorescence intensity of the fluorescently labeled particles can be changed.
  • particles are labeled with one of three gradations of red fluorescence intensity, one of three gradations of green fluorescence intensity, and one of three gradations of blue fluorescence intensity.
  • each particle is individually identified as one of the 27 types mentioned above. be able to.
  • the fluorescence intensity is labeled in four gradations instead of three, it is possible to further increase the types of particles that can be identified. Furthermore, for example, if four types of fluorescent substances are used instead of three, the types of particles that can be identified can be further increased. Furthermore, if light absorption is used in addition to fluorescence, the types of particles that can be identified can be further increased.
  • the particles are not particularly limited, and include resin beads, glass beads, and the like.
  • the resin include polyethylene, polypropylene, polystyrene, polycarbonate, cyclic polyolefin, and acrylic.
  • the particles may be magnetic beads. When the particles are magnetic beads, the particles can be collected using a magnetic holder or the like.
  • the size of the particles can be selected as appropriate; for example, particles with a diameter of about 0.1 to 100 ⁇ m can be used.
  • Examples of methods for binding fluorescent substances to particles include a method in which streptavidin-coated particles are brought into contact with biotin-bound fluorescent substances.
  • a method in which particles whose surfaces are biotinylated, streptavidin, and a fluorescent substance bound to biotin are brought into contact.
  • a method of covalently bonding a functional group present on the surface of a particle and a functional group present on a fluorescent substance using a chemical linker may also be mentioned.
  • Functional groups when using chemical linkers include hydroxyl groups, amino groups, thiol groups, and the like.
  • a click reaction using an azide group and an alkyne group may be used.
  • a commercially available fluorescent substance bound to biotin may be used, or a fluorescent substance bound to biotin may be used.
  • a method for binding biotin to a fluorescent substance includes a method using a chemical linker.
  • particles are labeled using a light-absorbing substance in the same way as fluorescent substances, particles that can be identified by light absorption can be obtained.
  • the fluorescent substance and the light-absorbing substance are not particularly limited, and those used for labeling biomolecules and the like can be used as appropriate.
  • Examples of bonding methods for particles and CRISPR/Cas family proteins include physical adsorption and covalent bonding of functional groups present on the surface of particles and functional groups present on the surface of CRISPR/Cas family proteins using chemical linkers. methods, methods that utilize hybridization of single-stranded nucleic acid fragments, methods that utilize avidin-biotin binding, and the like. These methods may be used in combination.
  • CRISPR/Cas family proteins may be immobilized on the surface of particles using, for example, a click reaction using an azide group and an alkyne group.
  • avidin-biotin binding the particles and the CRISPR/Cas family protein are bonded by contacting the particle whose surface is biotinylated, streptavidin, and the CRISPR/Cas family protein that has been biotinylated using a chemical linker. can be combined.
  • the particles and the CRISPR/Cas family protein can be bound by contacting the particle with streptavidin bound to the surface and the CRISPR/Cas family protein biotinylated using a chemical linker.
  • FIGS. 2A and 2B are schematic diagrams showing examples of binary complexes fixed to particles.
  • streptavidin 220 is bound to the surface of particle 210.
  • biotin 230 is bound to a binary complex 130 of CRISPR/Cas family protein 110 and gRNA 120 via a chemical linker. Then, by binding biotin 230 and streptavidin 220, a binary complex 130 is bound to the surface of the particle 210.
  • particles 210' which are particles that can be distinguished from each other by a combination of optical properties, are used.
  • a binary complex 130' of a CRISPR/Cas family protein 110 and gRNA 120' is immobilized on the surface of the particle 210'. That is, a binary complex 130 containing gRNA 120', which is gRNA corresponding to the type of particle 210', is immobilized on the particle 210'.
  • FIGS. 2A and 2B Multiple types of particles with immobilized binary complexes as shown in FIGS. 2A and 2B are individually placed in a reaction space having a volume of 10 aL to 100 pL, and samples containing multiple types of target nucleic acid fragments and Upon contact with the substrate nucleic acid fragment, if a target nucleic acid fragment is present in the reaction space, a ternary complex is formed, the substrate nucleic acid fragment is cleaved, and the fluorescent substance is separated from the quencher.
  • the detection of fluorescence derived from the substrate nucleic acid fragment indicates the presence of the target nucleic acid fragment within the reaction space.
  • the type of particles placed in the reaction space corresponds to the type of gRNA, that is, the type of target nucleic acid fragment present in the reaction space.
  • target nucleic acid fragments in a sample can be detected by detecting the presence or absence of fluorescence derived from the substrate nucleic acid fragment in each reaction space and the type of particles placed in the reaction space.
  • the number of target nucleic acid fragment types that can be detected simultaneously depends on the number of particle types that can be used simultaneously.
  • the sample is not particularly limited and can be appropriately selected depending on the purpose, such as saliva, blood, urine, amniotic fluid, malignant ascites, throat swab, nasal cavity swab, etc.
  • examples include samples and supernatants of cultured cells.
  • Target nucleic acid fragment examples include viral genome, cfDNA, ctDNA, microRNA, exosome-derived DNA, and the like.
  • target nucleic acid fragment examples include viral genome, cfDNA, ctDNA, microRNA, exosome-derived DNA, and the like.
  • a nucleic acid fragment containing a hotspot region of a cancer gene as a target nucleic acid fragment, it is also possible to detect a mutation in a cancer gene contained in a sample.
  • the CRISPR/Cas family protein is Cas12 protein
  • double-stranded DNA fragments can be detected as target nucleic acid fragments.
  • the CRISPR/Cas family protein is Cas13 protein
  • a single-stranded RNA fragment or a single-stranded DNA fragment can be detected as the target nucleic acid fragment.
  • the guide RNA is not particularly limited as long as it can be used for the CRISPR/Cas family protein to be used, and includes CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). It may be a complex with tracrRNA, a single gRNA (sgRNA) that is a combination of tracrRNA and crRNA, or only crRNA.
  • crRNA can have the following base sequence, for example.
  • a base sequence obtained by removing a protospacer adjacent motif (PAM) sequence from a target base sequence is defined as a spacer base sequence.
  • a base sequence is designed in which a scaffold sequence is linked to the 3' end of the spacer base sequence, and its complementary strand is used as the base sequence of crRNA.
  • the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-GCCAAGCGCACCTAATTTCC-3'" (SEQ ID NO: 1)
  • the base sequence of crRNA for Cas12a protein is "5'-AAUUUCUACUAAGUGUAGAUGGAAAUUAGGUGCGCUUGGC-3'" (SEQ ID NO: 2).
  • crRNA can have the following base sequence, for example.
  • a base sequence is designed in which a scaffold sequence is linked to the 3' end of a base sequence complementary to the target base sequence, and the complementary strand is used as the base sequence of crRNA.
  • the target base sequence is "5'-AUGGAUUACUUGGUAGAACAGCAAUCUA-3'" (SEQ ID NO: 3)
  • the base sequence of crRNA for Cas13a protein should be "5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAGUAAUCCAU-3'" (SEQ ID NO: 4). I can do it.
  • any CRISPR/Cas family protein can be used as long as it expresses nuclease activity after forming a ternary complex with gRNA and a target nucleic acid fragment.
  • a ternary complex is formed and the CRISPR/Cas family protein cleaves the target nucleic acid fragment, and then expresses nuclease activity.
  • CRISPR/Cas family proteins examples include Cas12 protein and Cas13 protein.
  • Cas12 protein and Cas13 protein may be Cas12 protein, Cas13 protein, orthologs of these proteins, variants of these proteins, and the like.
  • CRISPR/Cas family proteins that can be used in the method of this embodiment include, for example, Cas12a protein (LbCas12a, UniProtKB accession number: A0A182DWE3) derived from Lachnospiraceae bacterium ND2006, Acidamino coccus sp.
  • Cas12a protein (AsCas12a, UniProtKB accession number: U2UMQ6) derived from Francisella tularensis subsp.
  • Cas12a protein derived from A.
  • Cas13b protein derived from ZOR0009 (AspCas13b, NCBI accession number: WP_047447901), Prevotella sp.
  • Cas13b protein derived from MA2016 (PsmCas13b, NCBI accession number: WP_036929175), Cas13b protein derived from Riemerella anatipestifer (RanCas13b, NCBI accession number: WP_004919755), Prevo Cas13b protein from Tella aurantiaca (PauCas13b, NCBI accession number: WP_025000926), Cas13b protein derived from Prevotella saccharolytica (PsaCas13b, NCBI accession number: WP_051522484), Cas13b protein derived from Prevotella intermedia (Pin2Cas13b, NCBI accession number: WP_061868553), Cas13b protein from Capnocytophaga canimor
  • Cas13b protein derived from P5-125 (PspCas13b, NCBI accession number: WP_044065294), Cas13b protein derived from Porphyromonas gingivalis (PigCas13b, NCBI accession number: WP_053444417), Prevo Cas13b protein derived from tella intermedia (Pin3Cas13b, NCBI accession number: WP_050955369 ), Csm6 protein derived from Enterococcus italicus (EiCsm6, NCBI accession number: WP_007208953.1), Csm6 protein derived from Lactobacillus salivarius (LsCsm6, NCBI accession number: WP_08150) 9150.1), Csm6 protein from Thermus thermophilus (TtCsm6, NCBI Accession number: WP_011229148.1).
  • the CRISPR/Cas family protein may be a variant of the above-mentioned Cas family protein.
  • the mutant for example, a mutant with increased nuclease activity after forming a ternary complex can be used.
  • the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and when it is cleaved by the nuclease activity of the ternary complex and the fluorescent substance separates from the quenching substance, it emits fluorescence upon irradiation with excitation light. .
  • the substrate nucleic acid fragment may be appropriately selected depending on the substrate specificity of the CRISPR/Cas family protein used.
  • Cas12 protein cleaves single-stranded DNA as a substrate. Therefore, when using Cas12 protein, it is preferable to use single-stranded DNA as the substrate nucleic acid fragment.
  • Cas13 protein cleaves single-stranded RNA as a substrate. Therefore, when using Cas13 protein, single-stranded RNA is preferably used as the substrate nucleic acid fragment.
  • a combination of a fluorescent substance and a quenching substance is used that can quench the fluorescence of the fluorescent substance when placed close to each other.
  • a fluorescent substance for example, when FAM, HEX, etc. are used as the fluorescent material, Iowa Black FQ (IDT), TAMRA, etc. can be used as the quenching material.
  • reaction space As a method for accurately detecting target substances such as target nucleic acid fragments, a technique of performing an enzymatic reaction in a large number of minute reaction spaces is being considered. These methods are called digital measurements. In digital measurement, signals are detected by dividing a sample into an extremely large number of tiny reaction spaces.
  • the signals from each reaction space are binarized, only 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 quantitative performance compared to conventional ELISA, real-time PCR, and the like.
  • the method of this embodiment is performed by digital measurement. More specifically, contact between the sample, CRISPR/Cas family protein, gRNA, and substrate nucleic acid fragment is carried out by dividing the reaction space into minute reaction spaces.
  • the volume per reaction space is 10aL to 100pL, for example may be 10aL to 10pL, for example may be 10aL to 1pL, for example may be 10aL to 100fL, for example 10aL to 10fL. It's okay.
  • the reaction space is within the above range, the presence of the target nucleic acid fragment can be detected with high sensitivity even without amplifying the target nucleic acid fragment.
  • Digital measurement can be performed by carrying out the method of this embodiment under conditions in which zero or one target nucleic acid fragment is introduced into each reaction space.
  • the number of reaction spaces in which a signal is detected can be made to correspond to the number of target nucleic acid fragment molecules in the sample.
  • the reaction space may be, for example, a droplet.
  • a fluidic device can be used to form tiny droplets of aqueous phase in an oil phase.
  • one droplet can contain one particle. With such a method, particles can be individually placed in a reaction space having a volume of 10 aL to 100 pL.
  • the reaction space may be a well of a well array formed on the substrate.
  • placing the particles individually in a reaction space with a volume of 10aL to 100pL means that the size of the well is adjusted to accommodate only one particle, and each well of the well array is This can be achieved by placing one particle and then sealing the opening of the well with a sealant while the well is filled with the assay solution.
  • the sealant is immiscible with water. "Immiscible with water” means that when water and a sealant are sufficiently mixed and left to stand, they will separate into an aqueous phase and an organic phase. Moreover, it is preferable that the sealant has low water absorption. Low water absorption means that the volume change of the organic layer when mixed with an equal volume of water at 20° C. and allowed to stand and separated into an aqueous phase and an organic phase is 1% or less.
  • sealant a substance that has a boiling point of about 100° C. or higher and is liquid at room temperature can be used.
  • Specific sealants include fluorinated sealants such as FC-40, FC-43, FC-770, FC-72, FC-3283 (all manufactured by 3M), and Fomblin (registered trademark) oil (Solvay). Examples include liquids, mineral oil (Sigma-Aldrich), linear or branched saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms. These may be used alone or in combination of two or more.
  • linear or branched saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms examples include heptane (C 7 H 16 ), octane (C 8 H 18 ), nonane (C 9 H 20 ), and decane ( C 10 H 22 ), undecane (C 11 H 24 ), dodecane (C 12 H 26 ), tridecane (C 13 H 28 ), tetradecane (C 14 H 30 ), pentadecane (C 15 H 32 ), hexadecane (C 16 H 34 ), heptadecane (C 17 H 36 ), heptene (C 7 H 14 ), octene (C 8 H 16 ), nonene (C 9 H 18 ), decene (C 10 H 20 ), undecene (C 11 H 22 ), dodecene (C 12 H 24 ), tridecene (C 13 H 26 ), tetradecene (C 14 H 28 ), pentadec
  • octane isomers include 1-octane, 2-methylheptane, 3-methylheptane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,3,3-trimethylpentane, and the like.
  • octene isomers include 1-octene, 2-methyl-1-heptene, 2,3-dimethyl-1-hexene, 2-ethyl-1-hexene, 2,3,3-trimethyl-1 -Butene, etc.
  • An organic solvent containing lipid may be used as the sealant.
  • the lipid natural lipids derived from soybeans, Escherichia coli, etc., and artificial lipids such as dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG) can be used.
  • DOPE dioleoylphosphatidylethanolamine
  • DOPG dioleoylphosphatidylglycerol
  • hexadecane, chloroform, etc. can be used as an organic solvent.
  • FIG. 3A is a top view showing an example of a fluidic device including a substrate on which wells are formed, each having a volume of 10 aL to 100 pL.
  • FIG. 3B is a sectional view taken along line bb' in FIG. 3A.
  • the fluidic device 300 includes a substrate 310 on the surface of which a well 311 with a volume of 10 aL to 100 pL is formed, a spacer 320, and a lid member 330 in which a liquid introduction port 331 is formed. have.
  • a plurality of wells 311 exist and form a well array 312.
  • the space between the substrate 310 and the lid member 330 functions as a channel through which the sample, particles bound with the binary complex, substrate nucleic acid fragments, and the like flow.
  • the shape of the well is within the above-mentioned range; for example, it may be cylindrical or a polyhedron composed of multiple surfaces (for example, a rectangular parallelepiped, hexagonal prism, octagonal prism, etc.). It's okay.
  • a plurality of wells 311 of the same shape and size form a well array 312.
  • the same shape and same size may be defined as having the same shape and the same capacity to the extent required for digital measurement, and variations to the extent of manufacturing errors are acceptable.
  • FIG. 4B a film 400 is laminated on the surface of the substrate 310.
  • Examples of the material of the substrate 310 include glass, resin, and the like.
  • Examples of the resin include polyethylene, polypropylene, polystyrene, polycarbonate, cyclic polyolefin, and acrylic.
  • polycarbonate is also used as a material for CDs and DVDs that can be mass-produced at low cost, and is also suitable from the viewpoint of manufacturing well arrays at low cost.
  • the inventors have revealed that using polycarbonate as the material of the substrate 310 is preferable when detecting fluorescence with a microscope because the refractive index of light is close to that of glass.
  • Examples of the material for the membrane 400 include fluororesin, cyclic polyolefin, silicone resin, and the like.
  • a resist film 410 is laminated on the surface of the film 400.
  • the resist film 410 is exposed to active energy rays using an exposure machine.
  • development is performed with a developer to remove the resist film 410 in the portion where the well will be formed, as shown in FIG. 4D.
  • the film 400 masked by the resist film 410 is etched to form a well 311 in the film 400.
  • the resist film 410 is removed by cleaning the substrate, and an array of wells 311 is obtained. Through the above steps, a well array is obtained.
  • the invention provides a kit for detecting multiple types of target nucleic acid fragments in a sample.
  • the kit of this embodiment includes multiple types of gRNA for each of multiple types of target nucleic acid fragments, CRISPR/Cas family proteins, multiple types of particles that can be distinguished from each other by a combination of optical properties, and a substrate nucleic acid fragment.
  • the CRISPR/Cas family protein expresses nuclease activity after forming a ternary complex with the gRNA and the target nucleic acid fragment, and the gRNA forms a 2-part complex with the CRISPR/Cas family protein.
  • the two-party complex is immobilized on the surface of the particle, and the two-party complex containing a specific type of gRNA is immobilized on a specific type of the particle.
  • the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and when it is cleaved by the nuclease activity of the ternary complex and the fluorescent substance separates from the quenching substance, it emits fluorescence upon irradiation with excitation light. It is.
  • the above-described method for detecting multiple types of target nucleic acid fragments in a sample can be suitably carried out.
  • the sample, target nucleic acid fragment, gRNA, CRISPR/Cas family protein, particle, and substrate nucleic acid fragment are the same as those described above.
  • the CRISPR/Cas family protein can be Cas12 protein, Cas13 protein, etc.
  • kit of the present embodiment may further include a well array with a volume of 10 aL to 100 pL per well, a sealant, and the like.
  • the well array and sealant are the same as those described above.
  • Example I-1 (Preparation of fluidic device) A well array was produced by the same procedure as in FIGS. 4A to 4F described above. First, as shown in FIG. 4A, a glass substrate 310 was immersed in an 8M potassium hydroxide solution for about 24 hours to form hydroxyl groups on the surface.
  • a film 400 was formed on the surface of the glass substrate 310 by spin coating a fluororesin (CYTOP, manufactured by AGC Corporation).
  • the spin coating conditions were 1,000 rpm (revolutions per minute) for 30 seconds. Under this condition, the thickness of the film 400 is approximately 1.8 ⁇ m.
  • the film 400 was baked on a hot plate at 180° C. for 1 hour to dehydrate and condense the silanol groups of the film 400 (CYTOP) and the hydroxyl groups on the glass surface, thereby making the film 400 adhere to the surface of the glass substrate 310.
  • CYTOP silanol groups of the film 400
  • a resist product name "AZ-P4903", manufactured by AZ Electronic Materials
  • AZ-P4903 manufactured by AZ Electronic Materials
  • the glass substrate 310 was baked on a hot plate at 110° C. for 1 hour to evaporate the organic solvent in the resist film 410, so that the resist film 410 was brought into close contact with the surface of the film 400.
  • the resist film 410 was exposed to ultraviolet rays at 250 W for 14 seconds using a well array pattern mask using an exposure machine (manufactured by Union Optical). Subsequently, the film was developed by immersing it in a developer (AZ developer, manufactured by AZ Electronic Materials) for 1.5 minutes. As a result, the portion of the resist film 410 where the well will be formed was removed.
  • AZ developer manufactured by AZ Electronic Materials
  • the film 400 masked with the resist film 410 was dry-etched for 30 minutes under the conditions of O 2 200 sccm, pressure 5 Pa, and output 50 W using a reaction ion etching device (manufactured by YAC). By doing so, a well 311 was formed in the membrane 400.
  • the resist film 410 was removed by immersing the glass substrate 310 in acetone, washing it with isopropanol, and then washing it with pure water to obtain an array of wells 311 (well array).
  • the well array had a shape in which 1,500,000 cylindrical wells 311 with a diameter of 3.5 ⁇ m and a depth of 1.8 ⁇ m were lined up in 1 cm 2 .
  • Well 311 The volume per well was 17 fL.
  • Buffer E+500Tx means 20mM HEPES-KOH (pH 7.5), 100mM KCl, 10mM MgCl2 , 500 ⁇ M Triton X-100.
  • Example I-3 (Confirmation of fluorescently labeled magnetic beads)
  • a solution containing any of the 17 types of beads prepared in Experimental Example I-2 or a mixed solution obtained by mixing equal volumes of 17 types of beads 110 ⁇ L of the bead solution was taken, and 105 ⁇ L was dropped into the center of the well of the fluidic device prepared in Experimental Example I-1. This was followed by pipetting for 10 seconds at the center of the well. Next, a neodymium magnet was placed directly under the well. As a result, one bead was accommodated per well of the fluidic device.
  • FIG. 5 shows the fluorescence intensity distribution observed when 17 types of beads were introduced into the wells of the fluidic device. As a result, it was confirmed that each of the 17 types of beads could be identified based on the fluorescence patterns of DY405, ATTO565, and ATTO647N.
  • Table 3 below shows the results of calculating the number and probability of beads when a mixed solution of 17 types of beads was introduced into a fluidic device, imaged, and classified according to three-color fluorescence patterns.
  • “number” corresponds to the number in Table 1 above.
  • the number of each fluorescent dye indicates the ratio of the fluorescence intensity of each fluorescent dye.
  • Example I-4 (Preparation of substrate nucleic acid fragment)
  • the substrate nucleic acid fragment (single-stranded RNA fragment) was chemically synthesized outsourced (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 quencher.
  • the base sequence of the chemically synthesized substrate nucleic acid fragment (single-stranded RNA fragment) was "5'-(FAM)UUUUU(IABkFQ)-3'" (here, "IABkFQ" indicates Iowa Black FQ). .
  • RNA fragments As target nucleic acid fragments, two types of RNA fragments, CoV-N1-120nt (SEQ ID NO: 7) and CoV-N2-120nt (SEQ ID NO: 8), derived from the N gene of SARS-CoV2 were synthesized by in vitro transcription (IVT). and used it.
  • Ltr-N1-32 SEQ ID NO: 5
  • Ltr-N2-32 SEQ ID NO: 6
  • CoV-N2-120nt SEQ ID NO: 8
  • the Amicon Ultra 0.5 mL 50K column was washed and equilibrated with 500 ⁇ L of Cas stock buffer (20 mM HEPES-KOH (pH 7.5), 1.5 M NaCl). Subsequently, the mixture was centrifuged at 14,000 ⁇ g and 25° C. for 10 minutes, and the liquid remaining on the membrane and the liquid in the tube were discarded.
  • the entire solution was mixed by pipetting and briefly spun down in a tabletop centrifuge. Subsequently, the mixture was incubated for 10 minutes on a heat block at 37°C to obtain a biotinylated Cas13a protein-crRNA binary complex. Subsequently, 100 ⁇ L of Buffer F+50Tx was added to dilute the mixture 5 times. As a result, the concentration of LtrCas13a was 600 nM. Subsequently, it was aliquoted and stored at -80°C until use.
  • Example I-10 (Binding of binary complex to fluorescently labeled magnetic beads) 54 ⁇ L of buffer F+50Tx was added to 6 ⁇ L of the binary complex solution prepared in Experimental Example I-9 for dilution. As a result, the concentration of LtrCas13a was 60 nM. Subsequently, centrifugation was performed at 15,000 ⁇ g for 10 minutes, and 50 ⁇ L of supernatant was collected.
  • the fluorescently labeled magnetic beads were beads in which 1 ⁇ 10 molecules of fluorescent dye ATTO565 (Atotech) were bound to each magnetic bead (Dynabeads M280, Veritas) by the same method as in Experimental Example I-2; Beads in which 1 ⁇ 10 6 molecules of fluorescent dye ATTO647N (Atotech) were bound to each magnetic bead (Dynabeads M280, Veritas) were prepared and used.
  • streptavidin diluted to 60 ⁇ M with buffer E (20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl 2 ) was added and pipetted, and the mixture was incubated at room temperature for 10 minutes.
  • the magnetic beads were collected using a magnetic holder, and the solution other than the magnetic beads was discarded. Subsequently, washing was performed twice with 1 mL of buffer E+500Tx. Subsequently, the magnetic beads were suspended in 100 ⁇ L of the substrate nucleic acid fragment (dissolved in buffer E, 4 ⁇ M) prepared in Experimental Example I-4. As a result, the concentration of LtrCas13a was 30 nM.
  • Table 6 shows the combinations of crRNA and fluorescent substances in the two types of beads prepared.
  • Example I-11 (Detection of two types of target nucleic acid fragments with two types of beads)
  • the target nucleic acid fragments CoV-N1-120nt (SEQ ID NO: 7) and CoV-N2-120nt (SEQ ID NO: 8) prepared in Experimental Example I-6 were added to the concentrations shown in Table 7 below in 100 ⁇ L of Buffer E+50Tx. Mixed.
  • neodymium magnet directly below the well was removed, and a 20x objective lens was set. This was followed by incubation for 60 seconds. Subsequently, three types of fluorescence, ATTO565 and ATTO647N, which are fluorescent substances labeled on beads, and FAM, which is detected when the substrate nucleic acid fragment is cleaved, were detected and imaged.
  • FIGS. 6A and 6B are graphs showing the number of wells in which FAM fluorescence was detected.
  • FIG. 6A shows the concentration of the target nucleic acid fragment CoV-N1-120nt (SEQ ID NO: 7) used in the assay and the number of wells containing beads 1 and in which FAM fluorescence was detected.
  • tgRNA1 indicates the target nucleic acid fragment CoV-N1-120nt (SEQ ID NO: 7).
  • FIG. 6B shows the concentration of the target nucleic acid fragment CoV-N2-120nt (SEQ ID NO: 8) used in the assay and the number of wells in which beads 2 were accommodated and FAM fluorescence was detected.
  • tgRNA2 indicates the target nucleic acid fragment CoV-N2-120nt (SEQ ID NO: 8).
  • each target nucleic acid fragment could be analyzed in parallel with a detection limit of about 700 aM.
  • miRNA was detected by the SATORI method. In this experimental example, magnetic beads were not used.
  • Example II-1 (Preparation of gRNA and target nucleic acid fragment) Twenty types of miRNA shown in Table 8 below were detected by the SATORI method. First, a DNA fragment encoding gRNA (crRNA) was prepared by PCR amplification using an overlapping primer containing a T7 promoter sequence, a 20-base target sequence, and a scaffold sequence as a template. Subsequently, the obtained DNA fragments were subjected to an in vitro transcription reaction to prepare 20 types of gRNA (crRNA) shown in Table 8 below. Furthermore, 20 types of miRNA shown in Table 8 below were prepared by chemical synthesis.
  • crRNA DNA fragment encoding gRNA
  • Example II-4 Preparation of mixed solution of binary complex, substrate nucleic acid fragment, and Alexa647
  • Table 11 below the binary complex was prepared in Experimental Example II-2.
  • the mixed solution of the substrate nucleic acid fragment and Alexa647 was prepared in Experimental Example II-3.
  • Example II-5 (Detection of miRNA by SATORI method) The 20 types of miRNA prepared in Experimental Example II-1 were each diluted to 300 fM in 100 ⁇ L of Buffer E+50Tx.
  • FIG. 7 is a graph showing the results of detecting 20 types of miRNA.
  • the vertical axis indicates the number of wells in which FAM fluorescence was detected. The results revealed that most miRNAs can be detected by the SATORI method.
  • a technique for detecting multiple types of target nucleic acid fragments in a sample can be provided.
  • 100,100'...tripartite complex 110...CRISPR/Cas family protein, 120,120'...gRNA, 130,130'...bipartite complex, 140...target nucleic acid fragment, 150...substrate nucleic acid fragment, 210,210 '... Particle, 220... Streptavidin, 230... Biotin, 300... Fluid device, 310... Substrate, 311... Well, 312... Well array, 320... Spacer, 330... Lid member, 331... Liquid inlet, 400, 410... Film, F... fluorescent substance, Q... quencher.

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