Sensor array for multiplexed nucleic acid detection

Abstract

To provide a sensor array for multiplexed analysis of nucleic acid samples.SOLUTION: Provided is a sensor array for multiplexed nucleic acid detection including a plurality of spots fixed on a solid support, the spots including (i) a complex including a Cas protein and a guide RNA and (ii) a reporter nucleic acid, the Cas protein having collateral cleavage activity, the reporter nucleic acid being a single-stranded nucleic acid, and each of the plurality of spots including a guide RNA including a different guide sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a sensor array for multiplexed nucleic acid detection. In particular, the present invention relates to a sensor array for multiplexed nucleic acid detection based on a CRISPR-Cas effector system and a method for analyzing a nucleic acid sample using the same. [Background technology]

[0002] In the diagnosis of infectious diseases, it is extremely important to quickly and accurately identify the causative microorganism, and it is desirable to be able to quickly respond to genetic mutations that lead to drug resistance or aggravation. In addition, in personalized medicine, a technology that can quickly, cheaply, and comprehensively detect genetic mutations such as single nucleotide polymorphisms (SNPs) and insertion / deletion (Indel) is important. In recent years, nucleic acid detection methods based on the CRISPR-Cas effector system have attracted attention as a technology that can meet these demands. The CRISPR-Cas effector system has high sequence specificity and a fast reaction rate. In addition, some Cas proteins have collateral cleavage activity that randomly cleaves non-target single-stranded nucleic acids when they recognize a target nucleic acid, so there is an advantage that the detection signal of the target nucleic acid can be amplified by using it in combination with a single-stranded reporter nucleic acid. However, on the other hand, there is a problem that collateral cleavage does not have sequence specificity, making it difficult to apply to multiplex detection.

[0003] Approaches to multiplexed nucleic acid detection based on the CRISPR-Cas effector system using Cas with collateral cleavage activity have also been undertaken. For example, in a method called CARMEN, multiple detection reactions are performed simultaneously by separating the detection reaction solution into droplets (Non-Patent Document 1). However, this method is expensive because it requires dedicated equipment for dropletization, and rapid analysis is difficult because it requires many procedures for dropletization. In addition, for example, in a method called SHERLOCKv2, multiplexed detection is performed by using Cas13 subtypes with different dinucleotide specificities in collateral cleavage (Non-Patent Document 2). However, this method cannot perform multiplexed detection of more than five items due to its fundamental limitations.

[0004] There is a need for a multiplexed nucleic acid detection method that is rapid, low-cost, simple, and suitable for clinical implementation, while taking advantage of the high specificity and high sensitivity of the CRISPR-Cas effector system using Cas with collateral cleavage activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ackerman,CM.et al.,Nature,2020,582:277-282 [Non-Patent Document 2] Gootenberg,JS.et al.Science,2018,360:439-444 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made with an object to provide a nucleic acid detection method that enables comprehensive multiplex analysis without requiring any special additional device. [Means for solving the problem]

[0007] As a result of intensive research, the inventors discovered that the above-mentioned problems can be solved by immobilizing both the Cas / gRNA complex and the reporter nucleic acid on a solid support, thereby completing the present invention.

[0008] That is, according to one embodiment, the present invention provides a sensor array for multiplexed nucleic acid detection comprising a plurality of spots fixed on a solid support, the spots comprising (i) a complex comprising a Cas protein and a guide RNA and (ii) a reporter nucleic acid, the Cas protein having collateral cleavage activity, the reporter nucleic acid being a single-stranded nucleic acid, and each of the plurality of spots comprising a guide RNA comprising a different guide sequence.

[0009] Preferably, the Cas protein is selected from the group consisting of Cas3, Cas12, Cas13, Cas14 and Csm.

[0010] The reporter nucleic acid is preferably at least 20 nucleotides in length.

[0011] The reporter nucleic acid preferably comprises a detectable label.

[0012] The detectable label is preferably a fluorescent dye, and the density of the reporter nucleic acid in the spot is 0.7 to 2.5 fmol / mm 2 It is preferable that:

[0013] According to one embodiment, the present invention also provides a method for multiplexed nucleic acid detection, comprising the steps of: (1) preparing the above-mentioned sensor array; (2) contacting a sample containing a nucleic acid with the array; and (3) detecting cleavage of the reporter nucleic acid. Effect of the Invention

[0014] The sensor array according to the present invention is based on the CRISPR-Cas effector system, and is therefore highly accurate and sensitive, and can comprehensively analyze many types of target nucleic acids in one pot simply by detecting the detection signals from the spots. In addition, since no special equipment or multi-step operations are required for the nucleic acid detection reaction, analysis can be performed efficiently and at low cost. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a graph showing the fluorescence intensity on the plate surface on which ssDNA reporters were immobilized at various concentrations. [Diagram 2] FIG. 2 is a graph comparing the rate of decrease in fluorescence intensity due to collateral cleavage on the plate surface on which ssDNA reporters of various lengths were immobilized. [Diagram 3] Figure 3 shows agarose gel electrophoresis images confirming the collateral cleavage activity of the Cas12a / sgRNA complex immobilized on the plate surface under various conditions (conditions 1 to 7). [Figure 4] Figure 4 is a graph comparing the rate of decrease in fluorescence intensity due to collateral cleavage on a plate surface on which the Cas12a / sgRNA complex and the ssDNA reporter were co-immobilized under various conditions. [Diagram 5] Figure 5 shows the results of converting the fluorescence intensity (a) on the plate surface to which the Cas12a / sgRNA complex and the ssDNA reporter were co-immobilized and to which 0 nM or 100 nM target dsDNA solution was added, into the density of the ssDNA reporter on the plate surface (b). [Figure 6] FIG. 6 is a diagram showing an outline of the procedure for fabricating a duplex detection array. [Figure 7] FIG. 7 shows the time course of fluorescence intensity upon addition of a pVenus-N1 amplicon solution in a pVenus-N1 amplicon detection spot. [Figure 8]FIG. 8 is a graph showing the fluorescence intensity in the pVenus-N1 amplicon detection spots to which various concentrations of pVenus-N1 amplicon solutions were added. [Figure 9] FIG. 9 is a graph showing the fluorescence intensity in the pVenus-N1 amplicon detection spots and the pEGFP-N1 amplicon detection spots to which a solution containing the pVenus-N1 amplicon and / or the pEGFP-N1 amplicon was added. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in the present specification.

[0017] According to a first embodiment, the present invention provides a sensor array for multiplexed nucleic acid detection, comprising a plurality of spots immobilized on a solid support, the spots comprising (i) a complex comprising a Cas protein and a guide RNA, and (ii) a reporter nucleic acid, the Cas protein having collateral cleavage activity, the reporter nucleic acid being a single-stranded nucleic acid, and each of the plurality of spots comprising a guide RNA comprising a different guide sequence.

[0018] In the sensor array of this embodiment, (i) the complex containing Cas protein and guide RNA and (ii) the reporter nucleic acid are both fixed as a single spot on a solid support. Hereinafter, the complex containing Cas protein and guide RNA is also referred to as "Cas / gRNA complex".

[0019] "Cas protein" refers to an RNA-guided endonuclease that constitutes the CRISPR-Cas system. The Cas protein in this embodiment may be any subtype derived from any bacterium, as long as it has collateral cleavage activity (non-specific cleavage activity against single-stranded nucleic acids surrounding the nucleic acid sequence targeted by the Cas / gRNA complex; also called "trans-cleavage activity"). Examples of Cas proteins with collateral cleavage activity include, but are not limited to, Cas3; Cas12, such as Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d, Cas12e (also known as CasX), Cas12g to Cas12j and CasDx1; Cas13, such as Cas13a (also known as C2c2), Cas13b, Cas13c and Cas13d; Cas14, such as Cas14a to Cas14h; and Csms, such as Csm1 (also known as Cas10) and Csm2 to Csm5.

[0020] The Cas protein in the method of this embodiment may be selected appropriately depending on the target nucleic acid to be detected. For example, if the target nucleic acid is double-stranded DNA, Cas12a can be used, and if the target nucleic acid is single-stranded RNA, Cas13a can be used.

[0021] Information on the amino acid sequence of Cas protein can be obtained from a given database. For example, Addgene plasmid#1134131 (https: / / www.addgene.org / 113431 / ) is available for Cas12a derived from Lachnospiraceae bacterium, and Addgene plasmid#91909 (https: / / www.addgene.org / 91909 / ) is available for Cas13a derived from Leptotrichia wadei.

[0022] The Cas protein in this embodiment may include variants or homologs thereof having the same collateral cleavage activity. In other words, the Cas protein in this embodiment may include a protein consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably about 95% or more identity with the amino acid sequence of the Cas protein registered in the database, as long as the collateral cleavage activity is maintained. The identity of the amino acid sequence can be calculated using sequence analysis software or a program commonly used in the art (FASTA, BLAST, etc.). The Cas protein in this embodiment may also include a protein consisting of an amino acid sequence in which one to several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence registered in the database, as long as the collateral cleavage activity is maintained. The term "one to several" may mean, for example, 1 to 30, preferably 1 to 10, and particularly preferably 1 to 5.

[0023] The Cas protein in this embodiment can be prepared by any conventionally known genetic engineering method. Specifically, a nucleic acid encoding the Cas protein is prepared and introduced into a host cell such as E. coli to express the Cas protein. Cas proteins are also commercially available, and may be used in this embodiment. For example, the Alt-R TM LbCas12a and Alt-R TM Preferred commercially available products include AsCas12a (Integrated DNA Technologies, Inc.), EnGen Lba Cas12a (Cpf1) (New England Biolabs), LwCas13a, Active (CS13A-E321H) (SignalChem Biotech), CRISPR Cas14a1 protein (Guangzhou Magigen Biotechnology), and TtCsm6 (CS06-E311H) (SignalChem Biotech).

[0024] "Guide RNA" (gRNA) refers to an RNA that contains a guide sequence complementary to a target nucleic acid sequence and has the function of guiding a Cas / gRNA complex to a target nucleic acid and specifically binding thereto. The structure of the guide RNA is not particularly limited as long as it contains a guide sequence and has the above-mentioned function in the CRISPR-Cas system. That is, the guide RNA in this embodiment may be a dual RNA of a crRNA and a trans-activating crRNA (tracrRNA), or may be a single-stranded guide RNA (sgRNA) in which a crRNA and a tracrRNA are linked by a linker. A method for designing a guide RNA has already been established, and it can be designed using, for example, a design tool known in the art (e.g., CRISPRdirect (http: / / crispr.dbcls.jp), etc.).

[0025] The guide RNA in this embodiment can be easily prepared by any conventionally known nucleic acid synthesis method. For example, the guide RNA in this embodiment can be produced by preparing a template DNA and transcribing it with RNA polymerase.

[0026] The Cas / gRNA complex can be prepared by a method known in the art. Specifically, the Cas protein and gRNA (e.g., 1 to 100 μM each) are mixed in a buffer (e.g., 1 to 100 mM HEPES (pH 7.0 to 8.0), 1 to 100 mM Tris (pH 7.0 to 8.0), etc.) and incubated for a certain period of time.

[0027] The reporter nucleic acid in this embodiment is a single-stranded nucleic acid. The reporter nucleic acid in this embodiment may have at least a portion of a single-stranded structure, and therefore may be a linear single-stranded nucleic acid over its entire length, or may be a single-stranded nucleic acid that includes a hairpin structure or a guanine triplex or quadruplex structure.

[0028] In this embodiment, the reporter nucleic acid may be DNA or RNA depending on the collateral cleavage activity of the Cas protein used together, and may include a modified nucleic acid in a part (e.g., an end, etc.). Examples of modified nucleic acids include phosphorothioated RNA, boranophosphated RNA, 2'-O-methylated RNA, 2'-F-modified RNA, and 2',4'-BNA (also known as LNA (Locked Nucleic Acid)). The reporter nucleic acid in this embodiment may be preferably a linear single-stranded DNA or single-stranded RNA.

[0029] The reporter nucleic acid in this embodiment may have any length and nucleotide sequence. The length of the reporter nucleic acid in this embodiment may be, for example, at least 20 nucleotides, and preferably 20 to 100 nucleotides.

[0030] The reporter nucleic acid in this embodiment may contain a detectable label. The label in this embodiment may be any label used in a nucleic acid biosensor, and an appropriate label may be selected depending on the detection method. The label in this embodiment may be, for example, HEX TM , F.A.M. TM Fluorescent dyes such as BHQ TM -1, BHQ TM quenching molecules such as Zn-2; redox probes such as methylene blue and ferrocene; chemiluminescence probes such as copper sulfide nanoparticles; Raman probes such as gold nanostar@4-mercaptobenzoic acid@gold nanoshell structures (AuNS@4-MBA@Au), AgNPs@4ATP, and Prussian blue nanoparticles; cadmium sulfide quantum dots, [Ru(phen)2dppz] 2+ Examples of the label include, but are not limited to, electrochemiluminescent probes such as glucose oxidase, horseradish peroxidase, invertase, and the like; enzymes such as glucose oxidase, horseradish peroxidase, invertase, and the like; liposomes, etc. The label in this embodiment may preferably be a fluorescent dye.

[0031] The method of synthesizing nucleic acid and the method of modifying nucleic acid have been well established, and the reporter nucleic acid in this embodiment can be easily prepared according to any method known in the art.In addition, in the reporter nucleic acid in this embodiment, the label may be added to both or either of the 5' and 3' ends of the reporter nucleic acid.For example, when a fluorescent dye is used as a label, the fluorescent dye may be added to either one of the 5' and 3' ends and a quenching molecule may be added to the other end, or the fluorescent dye may be added to only one of the 5' and 3' ends.

[0032] The material of the solid support in the sensor array of this embodiment is not particularly limited, and may be, for example, a polymeric substance such as polystyrene or polyethylene terephthalate, an inorganic substance such as glass, a semiconductor such as silicon, a metal such as platinum, gold, silver, palladium, rhodium, iridium, tungsten, stainless steel, or mercury, or a carbon material such as graphite, carbon nanotube, or graphene, etc. The shape of the solid support may be, for example, a plate, a membrane, a wafer, or the like, but is not limited thereto.

[0033] The Cas / gRNA complex and the reporter nucleic acid may be fixed on the solid support by spotting the solution thereof using a micropipette, a spotter, an inkjet printer, or the like. The amount of the solution to be spotted may be, for example, 0.001 to 100 μL, and preferably 0.1 to 10 μL. The concentration of the Cas / gRNA complex and the reporter nucleic acid in the solution to be spotted may be, for example, 1 to 10,000 nM, and preferably 10 to 1,000 nM. The diameter of the spot may be, for example, 0.01 to 100 mm, and preferably 0.1 to 10 mm. Therefore, the density of the Cas / gRNA complex and the reporter nucleic acid in the spot is not particularly limited.

[0034] In the sensor array of this embodiment, the density of the reporter nucleic acid in the spot may be appropriately determined depending on the type of label introduced into the reporter nucleic acid. For example, when the reporter nucleic acid is labeled with a fluorescent dye, the reporter nucleic acid is 0.7 to 2.5 fmol / mm 2 The density of spots is preferably 1.25 to 2.5 fmol / mm 2 It is more preferable that the spots are spotted at a density of 0.1 to 100 μm.

[0035] The Cas / gRNA complex and the reporter nucleic acid can be spotted sequentially using separate solutions or simultaneously using a solution containing both, but it is preferable to spot them sequentially. For example, the reporter nucleic acid solution can be spotted first, the reporter nucleic acid can be fixed, and then the Cas / gRNA complex solution can be spotted on top of it. The Cas / gRNA complex and the reporter nucleic acid can be immobilized on the solid support by any modification method known in the art. Modification methods include, but are not limited to, covalent bonds such as amino group-amino reactive functional group or thiol-maleimide, His tag-Ni complex interaction, avidin-biotin interaction, and thiol self-assembly.

[0036] The sensor array of this embodiment includes a plurality of spots. The number of spots arranged on the solid support is not particularly limited, and may be, for example, 10 or more, 100 or more, or 1,000 or more. The spot pitch (i.e., the distance between the midpoints of the spots) may be, for example, 0.01 to 100 mm, and preferably 0.1 to 10 mm.

[0037] In the sensor array of this embodiment, each of the multiple spots contains a gRNA containing a different guide sequence. For example, when the sensor array of this embodiment contains a first, second, and third spot, the first gRNA in the first spot contains a first guide sequence; the second gRNA in the second spot contains a second guide sequence; the third gRNA in the third spot contains a third guide sequence; the first, second, and third guide sequences are different from each other. Therefore, in the sensor array of this embodiment, each of the multiple spots can recognize a different target nucleic acid sequence and emit a detection signal, and one-pot detection of multiple types of target nucleic acids (in principle, more than 1,000 types) is possible by simply providing an analysis sample to a single array.

[0038] According to a second embodiment, the present invention is a method for multiplexed nucleic acid detection, comprising the steps of (1) preparing the above-mentioned sensor array, (2) contacting a sample containing a nucleic acid with the array, and (3) detecting cleavage of the reporter nucleic acid.

[0039] In the method of this embodiment, a sample containing nucleic acid is brought into contact with a sensor array. In order to bring the sample containing nucleic acid into contact with the sensor array, the sample containing nucleic acid may be loaded onto the sensor array and incubated for a certain period of time. The nucleic acid concentration in the sample may be, for example, 0.0001 to 1 μM, and preferably 0.001 to 0.1 μM. When the nucleic acid concentration in the sample is unknown, the sample may be appropriately diluted stepwise and added. The incubation time may be, for example, 10 minutes to 2 hours.

[0040] The "sample containing nucleic acid" in this embodiment is not particularly limited, and may be prepared, for example, from biological fluids such as blood, serum, urine, saliva, etc., extracts from cells or tissues, culture supernatants of cells or tissues, etc. The biological fluids, cells, and tissues may be derived from any vertebrate or invertebrate animal, preferably from mammals such as mice, rats, rabbits, monkeys, and humans, and particularly preferably from humans.

[0041] Then, the cleavage of the reporter nucleic acid is detected. When the target nucleic acid is present in the sample, the corresponding Cas / gRNA complex is activated. As a result, only in the spot where the activated Cas / gRNA complex is present, the single-stranded reporter nucleic acid is randomly cleaved by the collateral cleavage activity of the Cas / gRNA complex. Therefore, the presence of the target nucleic acid in the sample can be detected by detecting the cleavage of the reporter nucleic acid. The cleavage of the reporter nucleic acid can be detected by any method known in the art, such as, but not limited to, fluorescence detection, chemiluminescence detection, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), biolayer interference (BLI), voltammetry, amperometry, potentiometry, impedance measurement, electrochemiluminescence detection, etc.

[0042] The method of the present embodiment can comprehensively analyze nucleic acids in a sample by simply contacting the sample containing nucleic acids with a sensor array and detecting detection signals from the spots by any known method, without requiring any special device or complicated multi-step operations, and therefore the method of the present embodiment is suitable for clinical diagnosis. EXAMPLES

[0043] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0044] <Materials and Reagents> Alt-R TMLbCas12a (hereafter simply referred to as “Cas12a”) was purchased from Integrated DNA Technologies. sgRNA was purchased from Thermo Fisher Scientific. Double-stranded DNA (dsDNA) plasmids (pEGFP-N1 and pVenus-N1) were obtained from the Life Imaging Group of the Advanced Photonics and Biosensing Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology (AIST PhotoBIO-OIL). PCR primers and single-stranded DNA (ssDNA) reporter were purchased from Eurofins Genomics. N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), Nα,Nα-bis(carboxymethyl)-L-lysine hydrate (NTA), and ethanolamine hydrochloride were purchased from Sigma-Aldrich. 2-morpholinoethanesulfonic acid (MES) was purchased from Dojindo Laboratories. Sodium hydroxide (NaOH), sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, and nickel(II) chloride were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Hy agarose was purchased from HydraGene. Ambion TM Nuclease-free water was purchased from Thermo Fisher Scientific. SpeedSTAR TM HS DNA Polymerase, 20bp DNA ladder and RNase Inhibitor were purchased from Takara Bio. TM PCR Purification Kit was purchased from Qiagen. 10×NEBuffer TM 3 was purchased from New England Biolabs. TBE buffer (10x) was purchased from SERVA Electrophoresis. C-type ELISA plates were purchased from Sumitomo Bakelite. Microplate sealing tape polyolefin (#9795) was purchased from 3M.

[0045] The nucleotide sequences of the target dsDNA, primers, sgRNA and ssDNA reporter are shown in Tables 1 to 4. In Table 1, brackets indicate the region to be replaced with TTT (a sequence corresponding to a part of the PAM sequence) in the target dsDNA amplicon. In Table 2, brackets indicate TTT corresponding to the replaced region. In Table 1, underlines indicate the sequence targeted by sgRNA. In Table 3, underlines indicate the target recognition sequence of sgRNA. In Tables 1 and 3, large bold letters indicate mismatches between pEGFP-N1 and pVenus-N1.

[0046] Table 1. Amplification region of target dsDNA (sense strand) [Table 1]

[0047] Table 2. Primers [Table 2]

[0048] Table 3.sgRNA [Table 3]

[0049] Table 4. ssDNA reporters [Table 4]

[0050] <Device> The amplification of the target dsDNA and the denaturation of the sgRNA were performed using a Takara PCR Thermal Cycler Dice Touch (Takara Bio). The concentration of the target dsDNA was measured using a NanoDrop. TM A microvolume spectrophotometer (Thermo Fisher Scientific) was used. A Mupid was used for agarose gel electrophoresis and gel imaging of ssDNA reporters. TM-exU horizontal electrophoresis system (Takara Bio) and ChemiDoc TM An imaging system (Bio-Rad Laboratories) was used. A MOV-112(U) thermostatic dryer (Sanyo Electric) was used to control the temperature of the collateral cleavage reaction. An MVX10 macro zoom microscope (Olympus), a U-HGLGPS light source device (Olympus), and an ImagEM EM-CCD (Hamamatsu Photonics) were used to perform fluorescence imaging of the ELISA plate surface. A Silhouette Cameo 4 cutter (Graphtec) was used to cut the sealing tape.

[0051] <1. Preparation of target dsDNA> Using pEGFP-N1 and pVenus-N1 as templates, PCR was performed with the above primers to prepare target dsDNA (Table 1) with the PAM (protospacer adjacent motif) sequence 5'-TTTN-3' introduced. The components and concentrations of the PCR mixture are shown in Table 5. In the table, 10x Fast Buffer I and dNTP Mixture were prepared using SpeedSTAR TM The one attached to the HS DNA Polymerase was used.

[0052] Table 5. PCR mixture components and concentrations [Table 5]

[0053] The reaction conditions were as follows: (1) 98°C for 30 seconds (1 cycle), (2) 98°C for 10 seconds, (3) 59°C for 10 seconds, (4) 72°C for 10 seconds (40 cycles of (2) to (4)), (5) 72°C for 2 minutes (1 cycle), and (6) held at 4°C. After the reaction, QIAquick TM The amplified target dsDNA was purified using a PCR Purification Kit.

[0054] 2. Immobilization of ssDNA reporter onto 96-well ELISA plates First, 200 mM EDC / NHS (in 100 μL of 50 mM MES-NaOH buffer (pH 6.0)) was added to a 96-well ELISA plate and incubated for 1 hour. This activated the carboxyl groups on the plate surface. Then, the plate was rinsed three times with 100 μL of water. 0-500 nM HEX-PolyA-NH2-20nt (in 100 μL of 100 mM phosphate buffer (pH 7.5)) was added and incubated for 12 hours. This immobilized the ssDNA reporter on the plate surface. Then, the plate was rinsed five times with 100 μL of water, and the fluorescence intensity on the plate surface was measured (λ ex =535-555nm,λ em =570-625nm).

[0055] The results of measuring the fluorescence intensity on the plate surface are shown in Figure 1. As the concentration of the ssDNA reporter increased, the fluorescence intensity on the plate surface increased. A weighted calibration curve confirmed a linear response in the range of 0 to 250 nM, so 250 nM of ssDNA reporter was used in subsequent experiments.

[0056] 3. Optimization of the length of the ssDNA reporter The ssDNA reporter HEX-PolyT-NH2-20, 40, 60, 80 or 100 nt was immobilized on the plate surface by the procedure in 2 above. The collateral cleavage solution (60 μL) described below was added to each well with the immobilized ssDNA reporter and incubated at 37°C for 1 hour. After that, the plate was rinsed five times with 100 μL of water, and the fluorescence intensity on the plate surface was measured. For the negative control, a collateral cleavage solution not containing the target dsDNA was used.

[0057] The components and concentrations of the collateral cleavage solution are shown in Table 6. The reagents were added to the tube in the order from top to bottom of the table and mixed to prepare a collateral cleavage solution containing the Cas12a / sgRNA complex and the target dsDNA. Before mixing, the sgRNA was denatured at 95°C for 5 minutes and then cooled to 4°C at a rate of 0.1°C / s.

[0058] Table 6. Components and concentrations of collateral cutting solution [Table 6]

[0059] The results are shown in Figure 2. In the figure, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005, **** indicates p<0.001, and ***** indicates p<0.0005 (analysis by Student's t-test). Comparing the rate of decrease in fluorescence intensity due to collateral cleavage, the rate of decrease (-81.8%) was the largest when a 100-nucleotide long ssDNA reporter was used. From these results, it was determined that the optimal length for the ssDNA reporter was 100 nucleotides.

[0060] <4. Immobilization of Cas12a / sgRNA complex on 96-well ELISA plate> The plate surface was treated under conditions 1 to 7 in Table 7. The activation of the carboxyl group was performed by the same procedure as in 2 above. 0 to 25 mM NTA (in 100 μL of 100 mM phosphate buffer (pH 7.5)) was added, and NTA was immobilized by incubating for 12 hours, and then rinsed with 100 μL of water. 1 M ethanolamine hydrochloride (in 100 μL of 100 mM phosphate buffer (pH 7.5)) was added, and blocking was performed by incubating for 1 hour, and then rinsed with 100 μL of water. 100 μL of 100 mM nickel (II) chloride was added, and Ni-NTA complex was formed by incubating for 1 hour, and then rinsed with 100 μL of water.

[0061] Table 7. Plate surface treatment conditions [Table 7]

[0062] Cas12a / sgRNA complexes were prepared and immobilized on the plate surface by adding 500 nM Cas12a and 1 μM sgRNA (in 60 μL of 1×NEBuffer3) to each well and incubating for 2 hours, followed by rinsing five times with 100 μL of water. 100 nM of target dsDNA and 500 nM of ssDNA reporter (HEX-PolyT-NH2-80nt) were dissolved in 1×NEBuffer3 supplemented with 0.5 U / μL of RNase Inhibitor to obtain a collateral cleavage solution. 60 μL of collateral cleavage solution was added to each well and incubated for 2 hours. The solution after the cleavage reaction (12 μL) was subjected to agarose gel electrophoresis, and the band images were analyzed.

[0063] The results are shown in Figure 3. Bands of 20 nucleotides or less were detected under all conditions 2 to 7 in which the plate surface was treated. This result confirmed that the Cas12a / sgRNA complex immobilized on the plate surface exhibited collateral cleavage activity. The cleavage activity was highest under conditions 4, 5, 6, and 7, followed by conditions 3 and 2. This trend suggested that an increase in the density of NHS ester may destroy the active site of Cas12a. On the other hand, no difference in cleavage activity was observed depending on whether or not blocking was performed.

[0064] <5. Co-immobilization of Cas12a / sgRNA complex and ssDNA reporter on 96-well ELISA plate> The carboxyl group was activated by the same procedure as in 2 above. 250 nM ssDNA reporter (HEX-PolyT-NH2-100nt) and 12.5, 25 or 250 nM NTA (in 100 μL of 100 mM phosphate buffer (pH 7.5)) were added and incubated for 12 hours to immobilize the ssDNA reporter and NTA on the plate surface. Then, blocking and Ni-NTA complex formation were performed by the same procedure as in 4 above. At the same time, sgRNA was denatured by the same procedure as in 3 above. Then, Cas12a / sgRNA complex was prepared and immobilized on the plate surface by the same procedure as in 4 above. 60 μL of 0 nM (negative control) or 100 nM target dsDNA solution (in 1×NEBuffer3 with 0.5 U / μL RNase Inhibitor added) was added to each well, incubated at 37 ° C for 2 hours, and then incubated at 70 ° C for 10 minutes to stop the collateral cleavage reaction. Thereafter, the plate was rinsed five times with 300 μL of water, and the fluorescence intensity on the plate surface was measured (λ ex =535-555nm,λ em =570-625nm).

[0065] The results are shown in Figure 4. At all NTA concentration conditions, the addition of target dsDNA reduced the fluorescence intensity on the plate surface, demonstrating that it is possible to detect target dsDNA on a plate in which the Cas12a / sgRNA complex and ssDNA reporter were co-immobilized. The rate of decrease in fluorescence intensity increased with decreasing NTA concentration.

[0066] 6. Optimization of modification density of ssDNA reporter A 96-well ELISA plate modified with Cas12a / sgRNA complex and ssDNA reporter (hereinafter referred to as "modified plate") was prepared by the same procedure as in 5 above. 0 nM (negative control) or 100 nM target dsDNA solution was added, and a collateral cleavage reaction was carried out for 2 hours by the same procedure as in 5 above, followed by rinsing five times with 300 μL of water and drying in vacuum. Then, 9 μL of nuclease-free water was dropped, the plate was sealed with a PET film with a diameter of 6.3 mm, and the fluorescence intensity was measured in the same manner as in 5 above.

[0067] Separately, 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, or 15.0 nM HEX-PolyT-NH2-100nt solutions were dropped (9 μL each / well) onto an unmodified 96-well ELISA plate, and a thin layer of each solution was prepared by sealing with a PET film of φ=6.3 mm. The ssDNA reporter concentration × ssDNA reporter solution volume (9 μL) / film area (31 mm 2 ), the density of HEX-PolyT-NH2-100nt in each well was 0.06, 0.12, 0.17, 0.23, 0.29, 0.58, 1.15, 1.73, 2.31, 2.89, or 4.33 fmol / mm 2 The fluorescence intensity was measured in the same manner as in 5 above.

[0068] The results are shown in Figure 5. Figure 5(a) shows the fluorescence intensity in the modified plate to which 0 nM or 100 nM target dsDNA solution was added, and Figure 5(b) shows the fluorescence intensity in the thin layer of ssDNA reporter solution. From these measurement results, it was found that the ssDNA reporter was added at 0.7 to 2.5 fmol / mm 2 It was shown that a decrease in fluorescence intensity due to the collateral cleavage reaction (i.e., the presence of target dsDNA) could be detected by spotting at a density of 100 nm.

[0069] 7. Preparation of duplex detection array A duplex detection array containing two spots for detecting different target dsDNA in one well of a 96-well ELISA plate was prepared. As shown in Figure 6, a sealing tape was cut into a circle (φ6 mm) with two holes (φ2 mm). The seal was peeled off from the backing film and attached onto the bottom surface of a 96-well ELISA plate. The ssDNA reporter and Cas12a / sgRNA complex were spotted by the same procedure as in 5 above. In one spot, sgRNA for the pVenus-N1 amplicon was used, and in the other spot, sgRNA for the pEGFP-N1 amplicon was used. The amount of each solution and washing water was 2.5 μL / spot and 4 μL / spot.

[0070] The measurement results of the fluorescence intensity at the detection spot where 100 nM of pVenus-N1 amplicon solution was dropped are shown in Figure 7. The decrease in fluorescence intensity due to the collateral cleavage reaction continued for 1 hour, but a significant difference was confirmed in 20 minutes according to Student's t-test. From this result, it was shown that the duplex detection array of this example can detect 100 nM of target dsDNA in 20 minutes.

[0071] Various concentrations of pVenus-N1 amplicon solutions were dropped, and collateral cleavage reactions were carried out for 2 hours using the same procedure as in 5 above. The fluorescence intensity was then measured, and the results are shown in Figure 8. As the concentration of pVenus-N1 amplicon increased, the fluorescence intensity decreased, and according to the Student's t-test, the fluorescence intensity decreased by 10 -9 It was confirmed that a significant difference in fluorescence intensity occurred at 10 M compared to 0 M. From these results, the detection limit concentration of the duplex detection array of this example was 10 -9 It was shown to be M.

[0072] <8. Simultaneous detection of two target dsDNA using duplex detection array> (1) A sample containing no dsDNA (negative control), (2) a sample containing 100 nM pEGFP-N1 amplicon, (3) a sample containing 100 nM pVenus-N1 amplicon, or (4) a sample containing 100 nM pEGFP-N1 amplicon and 100 nM pVenus-N1 amplicon were dropped onto the detection spot, and the collateral cleavage reaction was carried out for 2 hours using the same procedure as in 5 above, after which the fluorescence intensity was measured.

[0073] The results are shown in FIG. 9. In the figure, ns indicates no significant difference, * indicates p<0.05, and ** indicates p<0.01 (analysis by Student's t-test). The fluorescence intensity of the spots where the sample containing the target dsDNA was dropped decreased by 42.1 to 57.3%. On the other hand, the fluorescence intensity of the spots where the sample containing a dsDNA different from the target was dropped was not significantly different from the negative control. From these results, it was demonstrated that the duplex detection array of this embodiment can distinguish dsDNAs with two base differences in one pot without cross-reaction between spots.

Claims

1. A sensor array for multiplexed nucleic acid detection, comprising a plurality of spots fixed on a solid support, The spot comprises (i) a complex comprising a Cas protein and a guide RNA, and (ii) a reporter nucleic acid; The Cas protein has collateral cleavage activity, the reporter nucleic acid is a single-stranded nucleic acid, Each of the plurality of spots contains a guide RNA having a different guide sequence. Sensor array.

2. 2. The array of claim 1, wherein the Cas protein is selected from the group consisting of Cas3, Cas12, Cas13, Cas14 and Csm.

3. The array of claim 1 or 2, wherein the reporter nucleic acid is at least 20 nucleotides in length.

4. The array of any one of claims 1 to 3, wherein the reporter nucleic acid comprises a detectable label.

5. The detectable label is a fluorescent dye, and the density of the reporter nucleic acid in the spot is 0.7 to 2.5 fmol / mm ２ The array of claim 4,

6. (1) preparing a sensor array according to any one of claims 1 to 5; (2) contacting a sample containing nucleic acid with the array; (3) detecting cleavage of the reporter nucleic acid; 1. A method for multiplexed nucleic acid detection comprising:

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