Sensor array for detecting multiplex nucleic acid

Abstract

This sensor array for detecting a multiplex nucleic acid comprises a plurality of spots fixed on a solid support, wherein: the spots each contain (i) a complex containing 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; and the plurality of spots respectively contain guide RNAs including different guide sequences.

Description

Sensor arrays for multiplexed nucleic acid detection

[0001] The present invention relates to a sensor array for multiplexed nucleic acid detection, and more particularly 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.

[0002] In the diagnosis of infectious diseases, rapid and accurate identification of the causative microorganism is extremely important, and it is desirable to be able to respond quickly to genetic mutations that lead to drug resistance or aggravation. Furthermore, in personalized medicine, technologies that can rapidly, inexpensively, and comprehensively detect genetic mutations such as single nucleotide polymorphisms (SNPs) and insertion / deletions (Indels) are 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. Furthermore, some Cas proteins possess collateral cleavage activity, which randomly cleaves non-target single-stranded nucleic acids upon recognizing a target nucleic acid. Therefore, when used in combination with a single-stranded reporter nucleic acid, the detection signal of the target nucleic acid can be amplified. However, collateral cleavage lacks 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 pursued. 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 specialized equipment for droplet formation, and the numerous steps required for droplet formation make rapid analysis difficult. Furthermore, for example, a method called SHERLOCKv2 performs multiplexed detection by using Cas13 subtypes with different dinucleotide specificities in collateral cleavage (Non-Patent Document 2). However, due to its fundamental limitations, this method cannot perform multiplexed detection of more than five items.

[0004] There is a need for a multiplex 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 that uses Cas with collateral cleavage activity.

[0005] Ackerman, C.M. et al. , Nature, 2020, 582: 277-282 Gootenberg, JS. et al. Science, 2018, 360:439-444

[0006] The present invention has been made with the object of providing a nucleic acid detection method that enables comprehensive multiplex analysis without requiring any special additional device.

[0007] As a result of intensive research, the present 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, and thus completed 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 immobilized on a solid support, wherein the spots comprise (i) a complex comprising a Cas protein and a guide RNA and (ii) a reporter nucleic acid, wherein the Cas protein has collateral cleavage activity, the reporter nucleic acid is a single-stranded nucleic acid, and each of the plurality of spots comprises 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] Preferably, the reporter nucleic acid 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 2It 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-described sensor array; (2) contacting a sample containing nucleic acids with the array; and (3) detecting cleavage of the reporter nucleic acid.

[0014] The sensor array of the present invention is based on the CRISPR-Cas effector system, and is therefore highly accurate and sensitive, allowing comprehensive analysis of many types of target nucleic acids in one pot simply by detecting the detection signals from the spots. Furthermore, since no special equipment or multi-step operations are required for the nucleic acid detection reaction, highly efficient and low-cost analysis is possible.

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

[0016] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this 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, wherein the spots comprise (i) a complex comprising a Cas protein and a guide RNA and (ii) a reporter nucleic acid, wherein the Cas protein has collateral cleavage activity, the reporter nucleic acid is a single-stranded nucleic acid, and each of the plurality of spots comprises a guide RNA comprising a different guide sequence.

[0018] In the sensor array of this embodiment, both (i) a complex containing a Cas protein and a guide RNA and (ii) a reporter nucleic acid are immobilized as a single spot on a solid support. Hereinafter, the complex containing a Cas protein and a guide RNA is also referred to as a "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 of any subtype derived from any bacterium, as long as it has collateral cleavage activity (nonspecific cleavage activity against single-stranded nucleic acids surrounding the nucleic acid sequence targeted by the Cas / gRNA complex; also referred to as "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 Csm 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 the Cas protein can be obtained from a predetermined database. For example, for Cas12a derived from Lachnospiraceae bacterium, Addgene plasmid #1134131 (https: / / www.addgene.org / 113431 / ) is available, and for Cas13a derived from Leptotrichia wadei, Addgene plasmid #91909 (https: / / www.addgene.org / 91909 / ) is available.

[0022] The Cas proteins of this embodiment may include variants or homologs thereof having equivalent collateral cleavage activity. In other words, the Cas proteins of this embodiment may include proteins consisting of an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably about 95% or more identity to the amino acid sequence of the Cas protein registered in the database, as long as the collateral cleavage activity is maintained. The amino acid sequence identity can be calculated using sequence analysis software or programs commonly used in the art (e.g., FASTA, BLAST). Furthermore, the Cas proteins of this embodiment may include proteins consisting of an amino acid sequence registered in the database in which one to several amino acids have been substituted, deleted, inserted, and / or added, as long as the collateral cleavage activity is maintained. "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 these may be used in this embodiment. For example, Alt-R TM L. b. Cas12a and Alt-R TM A.s. Cas12a (Integrated DNA Technologies, Inc.), EnGen Lba Cas12a (Cpf1) (New England Biolabs), LwCas13a, Active (CS13A-E321H) (SignalChem Biotech), CRISPR Cas14a1 protein (Guangzhou Magigen Biotechnology), TtCsm6 (CS06-E311H) (SignalChem Biotech) and the like are preferred commercially available products.

[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 the crRNA and tracrRNA are linked by a linker. Methods for designing guide RNAs have already been established, and they can be designed using, for example, design tools known in the art (e.g., CRISPRdirect (http: / / crispr.dbcls.jp)).

[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 an 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, and it is sufficient that at least a portion of the reporter nucleic acid has a single-stranded structure, and therefore the reporter nucleic acid may be a linear single-stranded nucleic acid over its entire length, or a single-stranded nucleic acid containing a hairpin structure or a guanine triplex or quadruplex structure.

[0028] The reporter nucleic acid in this embodiment can be DNA or RNA depending on the collateral cleavage activity of the Cas protein used in combination, and a portion thereof (e.g., an end) may contain a modified nucleic acid. 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 preferably be linear single-stranded DNA or single-stranded RNA.

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

[0030] The reporter nucleic acid in this embodiment may contain a detectable label. The label in this embodiment may be any label used in nucleic acid biosensors, and an appropriate label may be selected depending on the detection method. Examples of the label in this embodiment include HEX TM , F.A.M. TM Fluorescent dyes such as BHQ TM -1, BHQ TMQuencher molecules such as α-2; redox probes such as methylene blue and ferrocene; chemiluminescent probes such as copper sulfide nanoparticles; Raman probes such as gold nanostar@4-mercaptobenzoic acid@goldnanoshell 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, and invertase; liposomes, etc. The label in this embodiment may preferably be a fluorescent dye.

[0031] Methods for synthesizing and modifying nucleic acids have already been well established, and the reporter nucleic acid of this embodiment can be easily prepared according to any method known in the art. Furthermore, in the reporter nucleic acid of this embodiment, a 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 the label, the fluorescent dye may be added to either the 5' or 3' end and a quencher molecule may be added to the other end, or the fluorescent dye may be added to only either the 5' or 3' end.

[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 nanotubes, or graphene. The shape of the solid support may be, for example, a plate, a membrane, a wafer, or the like, but is not limited to these.

[0033] The Cas / gRNA complex and reporter nucleic acid may be immobilized on a solid support by spotting their solutions using a micropipette, spotter, inkjet printer, or the like. The volume of the spotted solution may be, for example, 0.001 to 100 μL, preferably 0.1 to 10 μL. The concentration of the Cas / gRNA complex and reporter nucleic acid in the spotted solution may be, for example, 1 to 10,000 nM, preferably 10 to 1,000 nM. The diameter of the spot may be, for example, 0.01 to 100 mm, preferably 0.1 to 10 mm. Therefore, the density of the Cas / gRNA complex and 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 determined appropriately 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 has a density of 0.7 to 2.5 fmol / mm 2 Preferably, the spotting density is 1.25 to 2.5 fmol / mm 2 It is more preferable that the spots are spotted at a density of 1000 to 10 ...

[0035] The Cas / gRNA complex and reporter nucleic acid can be spotted sequentially using separate solutions or simultaneously using a solution containing both, but sequential spotting is preferred. For example, the reporter nucleic acid solution can be spotted first, and after the reporter nucleic acid has been immobilized, the Cas / gRNA complex solution can be spotted on top of it. The Cas / gRNA complex and reporter nucleic acid can be immobilized on the solid support by any conventionally known modification method. Examples of modification methods include, but are not limited to, covalent bonds such as amino group-amino reactive functional groups or thiol-maleimide, His tag-Ni complex interactions, avidin-biotin interactions, 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, but 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 that contains a different guide sequence.For example, when the sensor array of this embodiment contains the first, second and third spots, the first gRNA in the first spot contains the first guide sequence; the second gRNA in the second spot contains the second guide sequence; the third gRNA in the third spot contains the 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 different target nucleic acid sequences and emit detection signals, and by simply providing an analysis sample to a single array, it is possible to perform one-pot detection of multiple types (in principle, more than 1,000 types) of target nucleic acids.

[0038] According to a second embodiment, the present invention provides a method for multiplexed nucleic acid detection, comprising the steps of: (1) preparing the above-described sensor array; (2) contacting a sample containing nucleic acids 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. To bring the sample containing nucleic acid into contact with the sensor array, the sample containing nucleic acid is 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, preferably 0.001 to 0.1 μM. If the nucleic acid concentration in the sample is unknown, the sample may be serially diluted as appropriate 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 from, for example, 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, 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 exists in the sample, the corresponding Cas / gRNA complex is activated.As a result, only in the spot where the activated Cas / gRNA complex exists, the collateral cleavage activity of the Cas / gRNA complex causes random cleavage of the single-stranded reporter nucleic acid.Therefore, by detecting the cleavage of the reporter nucleic acid, the presence of the target nucleic acid in the sample can be detected.The cleavage of the reporter nucleic acid can be detected by any method known in the art, including, but not limited to, for example, fluorescence detection, chemiluminescence detection, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), biolayer interferometry (BLI), voltammetry, amperometry, potentiometry, impedance measurement, electrochemiluminescence detection, etc.

[0042] The method of this 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 equipment or complex multi-step procedures, and is therefore suitable for clinical diagnosis.

[0043] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0044] <Materials and Reagents> Alt-R TM L. b. Cas12a (hereinafter 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. Hy agarose was purchased from HydraGene. Ambion TM Nuclease-free water was purchased from Thermo Fisher Scientific. TM HS DNA polymerase, 20 bp DNA ladder, and RNase inhibitor were purchased from Takara Bio. TM PCR Purification Kit was purchased from Qiagen. 10x 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 in the target dsDNA amplicon that is replaced with TTT (a sequence corresponding to a portion of the PAM sequence). In Table 2, brackets indicate the TTT corresponding to the replaced region. In Table 1, underlines indicate the sequence targeted by the sgRNA. In Table 3, underlines indicate the target recognition sequence of the sgRNA. In Tables 1 and 3, large bold letters indicate mismatches between pEGFP-N1 and pVenus-N1.

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

[0047] Table 2. Primers

[0048] Table 3. sgRNA

[0049] Table 4. ssDNA reporters

[0050] <Apparatus> A Takara PCR Thermal Cycler Dice Touch (Takara Bio) was used for amplification of target dsDNA and denaturation of sgRNA. A NanoDrop was used for measuring the concentration of target dsDNA. TM A microspectrophotometer (Thermo Fisher Scientific) was used. A Mupid microscope was used for agarose gel electrophoresis and gel imaging of ssDNA reporters. TM -exU horizontal electrophoresis device (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 macrozoom microscope (Olympus), a U-HGLGPS light source (Olympus), and an ImagEM EM-CCD (Hamamatsu Photonics) were used to image the fluorescence 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) incorporating the PAM (protospacer adjacent motif) sequence 5'-TTTN-3'. 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 product attached to the HS DNA Polymerase was used.

[0052] Table 5. PCR mixture components and concentrations

[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) holding 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 on 96-well ELISA plate> 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. The fluorescence intensity on the plate surface increased as the concentration of the ssDNA reporter 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 ssDNA Reporter Length The ssDNA reporter HEX-PolyT-NH2-20, 40, 60, 80, or 100 nt was immobilized on the plate surface using 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. The plate was then rinsed five times with 100 μL of water, and the fluorescence intensity on the plate surface was measured. As a negative control, a collateral cleavage solution containing no 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 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. The sgRNA was denatured at 95°C for 5 minutes before mixing, and then cooled to 4°C at a rate of 0.1°C / sec.

[0058] Table 6. Collateral Cutting Solution Ingredients and Concentrations

[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 greatest when a 100-nucleotide ssDNA reporter was used. From these results, it was determined that a 100-nucleotide ssDNA reporter is optimal in length.

[0060] <4. Immobilization of Cas12a / sgRNA Complexes on 96-Well ELISA Plates> The plate surface was treated under conditions 1 to 7 listed in Table 7. Carboxyl group activation was performed using the same procedure as in 2 above. 0-25 mM NTA (in 100 μL of 100 mM phosphate buffer (pH 7.5)) was added and incubated for 12 hours to immobilize the NTA, followed by rinsing with 100 μL of water. 1 M ethanolamine hydrochloride (in 100 μL of 100 mM phosphate buffer (pH 7.5)) was added and blocked by incubating for 1 hour, followed by rinsing with 100 μL of water. 100 μL of 100 mM nickel(II) chloride was added and incubated for 1 hour to form the Ni-NTA complex, followed by rinsing with 100 μL of water.

[0061] Table 7. Plate surface treatment conditions

[0062] 500 nM Cas12a and 1 μM sgRNA (in 60 μL of 1x NEBuffer 3) were added to each well and incubated for 2 hours to prepare and immobilize the Cas12a / sgRNA complex on the plate surface. The plate was then rinsed five times with 100 μL of water. 100 nM target dsDNA and 500 nM ssDNA reporter (HEX-PolyT-NH2-80nt) were dissolved in 1x NEBuffer 3 supplemented with 0.5 U / μL RNase Inhibitor to obtain a collateral cleavage solution. 60 μL of the collateral cleavage solution was added to each well and incubated for 2 hours. The cleavage reaction solution (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. These results confirmed that the Cas12a / sgRNA complex immobilized on the plate surface exhibited collateral cleavage activity. Cleavage activity was highest under conditions 4, 5, 6, and 7, followed by conditions 3 and 2. This trend suggests that increasing the density of NHS esters may disrupt 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. Simultaneous immobilization of Cas12a / sgRNA complex and ssDNA reporter on a 96-well ELISA plate> Carboxyl groups were activated using 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. Blocking and Ni-NTA complex formation were then performed using the same procedure as in 4 above. Simultaneously, sgRNA was denatured using the same procedure as in 3 above. Cas12a / sgRNA complexes were then prepared and immobilized on the plate surface using the same procedure as in 4 above. 60 μL of 0 nM (negative control) or 100 nM target dsDNA solution (in 1×NEBuffer 3 supplemented with 0.5 U / μL RNase Inhibitor) was added to each well, and the plate was incubated at 37°C for 2 hours, followed by incubation at 70°C for 10 minutes to stop the collateral cleavage reaction. The plate was then 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 any NTA concentration, the addition of target dsDNA reduced the fluorescence intensity on the plate surface, demonstrating that target dsDNA can be detected on a plate co-immobilized with Cas12a / sgRNA complex and ssDNA reporter. The rate of decrease in fluorescence intensity increased as the NTA concentration decreased.

[0066] <6. Optimization of ssDNA reporter modification density> A 96-well ELISA plate (hereinafter referred to as "modified plate") modified with Cas12a / sgRNA complex and ssDNA reporter was prepared using the same procedure as in 5 above. 0 nM (negative control) or 100 nM target dsDNA solution was added, and the collateral cleavage reaction was carried out for 2 hours using the same procedure as in 5 above. After that, the plate was rinsed five times with 300 μL of water and vacuum dried. Then, 9 μL of nuclease-free water was added dropwise, 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 into an unmodified 96-well ELISA plate (9 μL each per well), and the plate was sealed with a 6.3 mm diameter PET film to prepare a thin layer of each solution. The ratio of 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 2It 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 1000 μg / ml.

[0069] 7. Preparation of Duplex Detection Arrays A duplex detection array containing two spots for detecting different target dsDNAs was prepared in each well of a 96-well ELISA plate. As shown in Figure 6, sealing tape was cut into a circle (6 mm diameter) with two holes (2 mm diameter). The seal was peeled off the backing film and attached to the bottom of a 96-well ELISA plate. The ssDNA reporter and Cas12a / sgRNA complex were spotted using the same procedure as in 5 above. The sgRNA for the pVenus-N1 amplicon was used in one spot, and the sgRNA for the pEGFP-N1 amplicon was used in the other spot. The volumes of each solution and wash water were 2.5 μL and 4 μL per spot.

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

[0071] Various concentrations of pVenus-N1 amplicon solution were added dropwise, and the collateral cleavage reaction was 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. According to Student's t-test, the fluorescence intensity was significantly higher at 10 -9 It was confirmed that a significant difference in fluorescence intensity occurred at 10 M compared to 0 M. From these results, it was determined that 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 dsDNAs using a duplex detection array (1) a sample containing no dsDNA (negative control), (2) a sample containing 100 nM of pEGFP-N1 amplicon, (3) a sample containing 100 nM of pVenus-N1 amplicon, or (4) a sample containing 100 nM of pEGFP-N1 amplicon and 100 nM of pVenus-N1 amplicon was dropped onto the detection spot, and a 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 Figure 9. In the figure, n.s. 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 to which 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 to which the sample containing the dsDNA different from the target was dropped showed no significant difference from the negative control. These results demonstrate that the duplex detection array of this example can distinguish dsDNAs that differ by two bases in one pot without cross-reaction between spots.

Claims

1. A sensor array for multiplexed nucleic acid detection, comprising a plurality of spots immobilized on a solid support, wherein the spots comprise (i) a complex comprising a Cas protein and a guide RNA and (ii) a reporter nucleic acid, wherein the Cas protein has collateral cleavage activity, the reporter nucleic acid is a single-stranded nucleic acid, and each of the plurality of spots comprises a guide RNA comprising a different guide sequence.

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

Images