Toehold-mediated strand displacement-based composition and method for RPA-crispr nucleic acid detection

Abstract

The present invention relates to: a Cas protein function-regulating switch oligonucleotide, which is an oligonucleotide that hybridizes complementarily to a spacer of a guide RNA and consists of 5 to 20 nucleotides starting from the 5' end; and a target nucleic acid amplification composition comprising same. In addition, the present invention provides the Cas protein function-regulating switch oligonucleotide, which is an oligonucleotide that hybridizes complementarily to a spacer of a guide RNA and consists of 5 to 20 nucleotides starting from the 5' end, thereby regulating the rate at which a Cas protein binds to a target nucleic acid so as to cause an RPA-CRISPR reaction to occur within a single tube without physical separation or additional steps, and thus can be used in a point-of-care diagnostic device.

Description

Composition and detection method for RPA-CRISPR nucleic acid detection based on Tohold-mediated strand substitution

[0001] The present invention relates to a switch oligonucleotide that regulates the function of a Cas protein, which is a nucleotide that hybridizes complementarily to a spacer of guide RNA and consists of 5 to 20 nucleotides starting from the 5' portion of said nucleotide, and more specifically, said oligonucleotide forms a tohold region and then detaches from the guide RNA.

[0002]

[0003] Nucleic acid amplification is the foundation of molecular diagnostic technology and is used in various fields, such as diagnosing infectious diseases or detecting gene mutations by amplifying specific genetic material. The most widely used traditional nucleic acid amplification method is qPCR (Real-Time Polymerase Chain Reaction); while it offers high sensitivity and accuracy, it is not suitable for point-of-care diagnostics due to the need for thermal cycling and long execution times. In contrast, isothermal amplification, another nucleic acid amplification method, is suitable for point-of-care diagnostics because it performs nucleic acid amplification at a single temperature, eliminating the need for thermal cycling and offering a short reaction time.

[0004] However, there are problems such as the generation of non-specific amplification products and lower sensitivity compared to PCR, so a new diagnostic technology combining the CRISPR-Cas system and isothermal amplification technology is being developed to overcome these issues. This technology implements a diagnostic method with high sensitivity and accuracy by artificially designing crRNA to cleave desired DNA and activating the CRISPR-Cas system. In particular, it provides sensitivity at the level of qPCR regardless of non-specific amplification products and enables simple analysis suitable for point-of-care diagnosis. However, the above technology has the problem that false positives may occur when the amplification product is transferred to the CRISPR-Cas system after amplification, and that sensitivity is low when amplification and the CRISPR-Cas reaction are performed simultaneously in a single tube, as a weak signal is generated due to the cleavage of specific amplification products and primers at the beginning of the reaction.

[0005] To solve these problems, the researchers of the present invention implemented a 'one pot RPA CRIPSR' without additional steps in a single tube by applying toehold-mediated strand displacement (TMDS) to crRNA.

[0006]

[0007] One objective of the present invention is to provide a switch oligonucleotide that regulates the function of a Cas protein, which is a nucleotide that hybridizes complementarily to a spacer of guide RNA and consists of 5 to 20 nucleotides starting from the 5' portion of said nucleotide, and a composition for nucleic acid amplification containing said oligonucleotide.

[0008] Another objective of the present invention is to provide a method for amplifying and detecting a target nucleic acid, comprising a switch oligonucleotide that regulates the function of a Cas protein, which is a nucleotide that hybridizes complementarily to a spacer of guide RNA and consists of 5 to 20 nucleotides starting from the 5' portion of said nucleotide.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010]

[0011] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0012] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0013]

[0014] In this specification, the term "oligonucleotide" refers to a molecule consisting of a short sequence of linked nucleotides, generally meaning single-stranded DNA or RNA composed of 2 to 25 nucleotides.

[0015] In this specification, the term "nucleotide" refers to a basic unit that constitutes DNA and RNA and serves to store and transmit genetic information of living organisms. A nucleotide consists of a base, a sugar, and a phosphate group.

[0016] In this specification, the term "guide RNA (gRNA)" refers to a short RNA sequence that serves as a guide for other Cas proteins that can be used for gene editing by cutting double-stranded DNA. In bacteria and archaea, guide RNA is part of the CRISPR-Cas system, which serves as an adaptive immune defense protecting the organism from viruses; short guide RNA acts as a detector of foreign DNA and directs Cas proteins to degrade foreign nucleic acids. Guide RNA targets complementary sequences, while single-stranded guide RNA (sgRNA) directs Cas enzymes to target specific regions of the genome for target DNA cutting. Additionally, guide RNA consists of crRNA and tracrRNA, and some Cas proteins, such as Cas12 and Cas13, require only crRNA as guide RNA.

[0017] In this specification, the term "crRNA" refers to CRISPR RNA, and the components of crRNA serve to bind to target-specific DNA regions. Transcription of a CRISPR locus generates crRNA containing a spacer region surrounded by a repeat sequence, which is typically 20 to 24 base pairs (bp) in length. crRNA forms an effector complex with a single nuclease or multiple Cas proteins, and modification of the crRNA sequence within a single-stranded guide RNA changes the binding site to accurately target various DNA regions, thereby becoming a programmable system for effective genome editing.

[0018] In this specification, the term "tracrRNA (trans-activating CRISPR RNA)" refers to trans-encoded RNA that binds complementarily to crRNA to form an activation complex and plays a role in helping Cas proteins recognize and cleave target DNA.

[0019] In this specification, the term "spacer" refers to a sequence complementary to the target DNA included in crRNA, which enables the Cas protein to cleave the target sequence by recognizing the target sequence of foreign DNA or RNA. Furthermore, spacers are a key component of the immune response that remembers foreign genes and defends against them upon infection.

[0020] In this specification, the term "hybridization" refers to the process in which two nucleic acid strands (DNA or RNA) combine through complementary binding between gene sequences. This is a fundamental property of nucleotide sequences and is utilized in various molecular biology techniques.

[0021] In this specification, the term "PAM (Protospacer Adjacent Motif)" refers to a DNA sequence of 2 to 6 base pairs located immediately following the DNA sequence targeted by the Cas nuclease in the CRISPR bacterial adaptive immune system. It is a short DNA sequence essential to the CRISPR-Cas system, serving to help the spacer accurately recognize the target gene and enable the Cas protein to cleave the corresponding gene. The PAM is not included in the crRNA itself but is a sequence located immediately before the target gene.

[0022] PAM is a 4-base pair DNA sequence immediately following the DNA sequence targeted by Cas12 nuclease, and may be any one of TTTA, TTTC, and TTTG.

[0023] In this specification, the term "CRISPR (Clustered regularly interspaced short palindromic repeats)" refers to a genetic element used by bacteria as a form of acquired immunity to protect against viruses. When a phage or plasmid enters a bacterium, it inserts a fragment of foreign DNA between the repetitive sequences within the bacterial genome, and CRISPR refers to these repetitive sequences.

[0024] In this specification, the term "Cas protein" refers to a nuclease capable of cleaving target DNA or RNA and working in conjunction with a CRISPR sequence.

[0025] Although not limited to these, the Cas protein may be any one of Cas12, Cas13, and Cas14.

[0026] In this specification, the term "Cas12" refers to an enzyme that binds to crRNA to recognize and cleave a target DNA sequence. Cas12 cleaves one strand of a DNA double strand first and then the remaining strand, and also has the ability to cleave non-specific single-stranded DNA.

[0027] In this specification, the term "Cas13" refers to an enzyme that binds to crRNA to recognize and cleave a target RNA sequence, and there are four subtypes: Cas13a, Cas13b, Cas13c, and Cas13d. Cas13 possesses unique cleavage activity capable of recognizing and binding to a specific RNA sequence to cleave the target RNA and surrounding non-specific RNA. Cas13 consists of a recognition lobe (REC) that serves to recognize and bind to the target RNA and a nuclease lobe (NUC) that performs RNA cleavage.

[0028] In this specification, the term "Cas14" refers to an enzyme that recognizes and cleaves a target DNA sequence without relying on PAM, and has particularly excellent sensitivity to ssDNA. Cas14 binds to a guide RNA, recognizes the target ssDNA, and forms an R-loop structure. Subsequently, Cas14 induces cleavage activity that non-specifically cleaves the ssDNA.

[0029] In this specification, the term "respiratory infectious RNA virus" refers to a virus that has RNA as its genome and causes infection in the human body primarily through the respiratory tract, and includes viruses that cause infection in the upper or lower respiratory tract through droplets or airborne transmission.

[0030] Specific examples include, but are not limited to, influenza A virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), respiratory syncytial virus (RSV), parainfluenza virus, human metapneumovirus, rhinovirus, coronavirus OC43 or 229E.

[0031] In this specification, the term "Influenza A" refers to a type of influenza virus, specifically an RNA virus that causes influenza in various animals. Influenza viruses are broadly classified into types A, B, C, and D; among these, type A causes infection not only in humans but also in animals such as birds and pigs. Influenza A is a negative single-stranded RNA virus that undergoes frequent genetic mutations, making it prone to the emergence of new variants. In this specification, "Influenza A" may be used to include its derived RNA sequence, nucleic acid sequences targeting the virus, derived genetic material, etc.

[0032] In this specification, the term "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)" refers to a positive (+) single-stranded RNA virus that was first identified in late 2019 and infects humans to cause Coronavirus Disease 19 (COVID-19). The virus belongs to the Family Coronaviridae and is transmitted primarily through the respiratory system and can cause various respiratory symptoms such as high fever, cough, and pneumonia. In this specification, SARS-CoV-2 may be used to include its derived RNA sequence, or nucleic acid sequences targeting the virus, derived genetic material, etc.

[0033] In this specification, the term "Toehold-Mediated Strand Displacement (TMDS)" refers to a mechanism in which a strand based on a specific sequence replaces an existing strand in a DNA or RNA strand substitution reaction. This process is initiated by a short complementary sequence called a toehold and is widely utilized primarily in DNA nanotechnology, molecular diagnostics, and synthetic biology.

[0034] In this specification, the term "toehold" refers to a short sequence that plays an important role in DNA or RNA strand substitution reactions, meaning a complementary single-stranded sequence that provides a starting point for a new strand to bind to an existing double strand. It is generally about 4 to 8 base pairs long and is usually located at one end or an exposed portion of double-stranded DNA or RNA.

[0035] In this specification, the term "switch" refers to a nucleotide that hybridizes complementarily to a spacer of guide RNA, and is a switch oligonucleotide that controls the function of a Cas protein, consisting of 5 to 20 nucleotides starting from the 5' portion of said nucleotide, and plays a role in controlling the binding rate of the Cas protein and the target double strand.

[0036] In this specification, the term "PCR (Polymerase Chain Reaction)" refers to a method used to amplify a specific DNA sequence by repeating the three steps of denaturation, binding, and extension of template DNA to amplify it millions of times. It is a representative gene amplification technique that requires a thermal cycler and DNA polymerase.

[0037] In this specification, the term "RT-PCR (Reverse Transcription Polymerase Chain Reaction)" refers to a method of converting RNA into DNA and then amplifying the resulting DNA. RNA is converted into cDNA using reverse transcriptase, and then the DNA is amplified through a standard PCR process. It is primarily used for the detection of RNA viruses and the analysis of gene expression.

[0038] In this specification, the term "RPA (recombinant enzyme polymer amplification)" refers to an isothermal amplification technique capable of amplifying DNA at room temperature. Template DNA is amplified at 37-42°C using a recombinant enzyme and a DNA polymerase, providing fast and efficient amplification without thermal cycling.

[0039] In this specification, the term "LAMP (Loop-mediated Isothermal Amplification)" refers to an isothermal amplification method that amplifies DNA at a constant temperature. It uses 4 to 6 specialized primers to form a loop-shaped DNA structure and amplifies at a very high speed, so it is mainly used in point-of-care diagnostics.

[0040] In this specification, the term "NASBA (nucleic acid sequence-based amplification)" refers to a technique for amplifying RNA at an isothermal temperature. Reverse transcriptase, T7 RNA polymerase, RNaseH, etc., are used, and it is primarily utilized for the amplification of RNA viruses or RNA targets. It is used for real-time detection and diagnostic purposes.

[0041] In this specification, the term "reporter" refers to a single-stranded nucleic acid probe comprising a fluorescent dye and a quencher, and is a single-stranded DNA or RNA molecule used to detect a specific gene sequence using a fluorescent signal. The fluorescent dye and the quencher are attached to the respective ends, so that light emitted from the fluorescent dye is blocked by the quencher, and when the reporter binds to or is cleaved from a target sequence, the quencher and the fluorescent dye separate, thereby generating a fluorescent signal.

[0042] In this specification, the term "fluorescent dye" refers to a compound or molecule that absorbs light of a specific wavelength and then emits light of a unique different wavelength, utilizing a fluorescence phenomenon process that generates a fluorescent signal by using the light emitted after transitioning to an excited state. In single-stranded nucleic acid probes, a fluorescent signal is generated when a target sequence is present or when specific conditions are met.

[0043] Fluorescent dyes may include, but are not limited to, FAM, CY3, Alexa Fluor, etc.

[0044] In this specification, the term "quencher" refers to a molecule that absorbs and blocks the fluorescent signal emitted from a fluorescent dye. When the quencher is near the fluorescent dye, it absorbs or transmits energy to prevent the fluorescent dye from emitting light, thereby suppressing the fluorescent signal.

[0045] Although not limited to these, matting agents may include BHQ1, DABCYL, etc.

[0046] In this specification, the term "primer" refers to a short single-stranded nucleotide sequence that binds to a template strand during DNA or RNA synthesis to help initiate the synthesis of a new strand. It is typically composed of about 18 to 30 nucleotides and provides a starting point where DNA or RNA polymerase can bind in nucleic acid amplification techniques such as PCR. Since the primer has a sequence complementary to the template strand, it can recognize that specific sequence, and DNA or RNA synthesis begins after the DNA or RNA polymerase binds.

[0047] In this specification, the term "polymerase" refers to an enzyme that synthesizes a new DNA or RNA strand by linking nucleotides, and includes DNA polymerases that use DNA as a template strand and RNA polymerases that use RNA as a template strand. Polymerases are enzymes that form phosphate bonds of nucleotides and are involved in gene replication, expression, and amplification.

[0048] In this specification, the term "reverse transcriptase" refers to an enzyme that synthesizes complementary DNA (cDNA) using RNA as a template strand. This process is called reverse transcription and is generally the process in which the conversion from RNA to DNA occurs. Reverse transcriptase is primarily found in retroviruses and enables the virus to replicate its RNA genes into the DNA of a host cell.

[0049] In this specification, the term "kit" refers to a set of materials or tools assembled for a specific purpose, and includes reagents, tools, manuals, etc., necessary to perform a specific experiment or test.

[0050]

[0051] In one embodiment of the invention, a switch oligonucleotide for regulating the function of a Cas protein is provided, which consists of 5 to 20 nucleotides starting from the 5' portion of the nucleotide, as nucleotides that hybridize complementarily to a spacer of guide RNA.

[0052] In the above embodiment, a switch oligonucleotide for regulating the function of a Cas protein is provided, consisting of 10 to 16 nucleotides starting from the 5' portion of the nucleotide.

[0053] A switch oligonucleotide that regulates the function of a Cas protein is provided, consisting of 12 to 14 nucleotides starting from the 5' portion of the above nucleotide.

[0054] In the above specific example, the target of the Cas protein is influenza A, and

[0055] The above oligonucleotide provides a switch oligonucleotide that creates a tohold region and then detaches from the guide RNA.

[0056] In one embodiment of the invention, a composition for target nucleic acid amplification is provided, comprising a switch oligonucleotide that regulates the function of a Cas protein, which is a nucleotide that hybridizes complementarily to a spacer of guide RNA and consists of 5 to 20 nucleotides starting from the 5' portion of said nucleotide.

[0057] In the above embodiment, the composition further comprises a primer that hybridizes complementarily to a target nucleotide for a nucleic acid amplification method of RPA (recombinant enzyme polymer amplification), LAMP (loop-mediated isothermal amplification), or NASBA (nucleic acid sequence-based amplification), the composition further comprises a Cas protein and a guide RNA, the composition further comprises MgCl₂, dNTPs, and a DNA or RNA polymerase, and the Cas protein is any one of Cas12, Cas13, and Cas14, thereby providing a composition for target nucleic acid amplification.

[0058] In one embodiment of the invention, a method for amplifying a target nucleic acid is provided by adding the target nucleic acid amplification composition to a sample containing the target nucleic acid and amplifying the target nucleic acid isothermally.

[0059] In the above embodiment, the step of amplifying the target nucleic acid provides a method for amplifying the target nucleic acid, wherein the step of amplifying the target nucleic acid is performed at an isothermal temperature between 35 and 45°C.

[0060] In another embodiment of the invention, a method for detecting a target nucleic acid is provided, comprising the steps of: preparing a composition for amplification and detection by further adding a single-stranded nucleic acid probe comprising a fluorescent dye and a quencher to the composition; and adding the composition to a sample containing the target nucleic acid and amplifying the target nucleic acid isothermally. In the above embodiment

[0061] The step of amplifying the target nucleic acid is performed at an isotherm between 35 and 45°C.

[0062] A method for detecting target nucleic acids is provided.

[0063] In one embodiment of the invention, a kit for amplifying a target nucleic acid is provided, comprising the switch oligonucleotide, a primer that binds complementarily to the target nucleic acid, a Cas protein, and a guide RNA.

[0064] In one embodiment of the invention, a kit for target nucleic acid amplification and detection is provided, comprising a single-stranded nucleic acid probe comprising the switch oligonucleotide, a primer that binds complementarily to the target nucleic acid, a Cas protein, a guide RNA, and a fluorescent dye and a quencher.

[0065] In one embodiment of the invention, a switch oligonucleotide consisting of 5 to 20 nucleotides starting from the 5' portion of said nucleotide is provided as a nucleotide that hybridizes complementarily to a spacer of guide RNA for regulating the function of a Cas protein in target nucleic acid amplification.

[0066] In the above embodiment, the target of the Cas protein is influenza A, the switch oligonucleotide is used, and the oligonucleotide is used to detach from the guide RNA after forming a tohold region.

[0067]

[0068] By providing a switch oligonucleotide that regulates the function of a Cas protein, consisting of 5 to 20 nucleotides starting from the 5' portion of the guide RNA as a hybridized nucleotide, and a composition for target nucleic acid amplification containing the same, the RPA-CRIPSR reaction can occur within a single tube.

[0069] The present invention provides a switch oligonucleotide that binds to the crRNA of a Cas protein, thereby adjusting the binding rate between the Cas protein and the target double strand, enabling the RPA-CRISPR reaction to occur within a single tube without physical separation or additional steps.

[0070] The present invention provides a switch oligonucleotide that regulates the function of a Cas protein, which is a nucleotide that hybridizes complementarily to a switch of guide RNA and consists of 5 to 20 nucleotides starting from the 5' portion of said nucleotide, thereby confirming that the fluorescent signal increases in the target nucleic acid amplification and detection reaction.

[0071] Furthermore, the effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0072]

[0073] Figure 1 illustrates the process of applying Tohold-mediated strand substitution technology to crRNA.

[0074] Figure 2 illustrates the sequential process of applying Tohold-mediated strand substitution technology to crRNA. The switch controls whether the amplification product is cleaved or accumulated initially, and as the amplification product accumulates, the switch bound to Cas12a is released. Cas12a generates a fluorescent signal by cleaving the reporter after cleaving the amplification product.

[0075] Figure 3 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when LbCas12a is used targeting influenza A virus RNA.

[0076] Figure 4 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when AsCas12a is used targeting influenza A virus RNA.

[0077] Figure 5 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when enAsCas12a is used targeting influenza A virus RNA.

[0078] Figure 6 shows the degree of RPA reaction product generation by reaction time in the case without a switch and in the case with a switch.

[0079] Figure 7 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when LbCas12a is used targeting SARS-CoV-2 RNA.

[0080] Figure 8 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when AsCas12a is used targeting SARS-CoV-2 RNA.

[0081] Figure 9 shows the degree of fluorescence signal detection according to the length of the switch oligonucleotide when enAsCas12a is used targeting SARS-CoV-2 RNA.

[0082]

[0083] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0084]

[0085] Example 1. One-pot RT-RPA CRISPR reaction

[0086] Influenza A virus (#VR-1938, ATCC) was extracted using the QIAamp Virus RNA Mini Kit (#52906, Qiagen). The RNA switch was denatured at 95°C for 5 minutes using a thermal cycler (#C1000, Bio-Rad) and heat-treated from 95°C to 12°C at 0.1°C / s.

[0087] 10 μl of CRISRP-Cas complex was assembled by incubating the final 1xr2.1 buffer, 120 nM cas protein, and 120 nM crRNA-switch complex in a 25°C thermal cycler (#C1000, Bio-Rad, USA) for 10 minutes. Subsequently, a 60 nM CRISPR-Cas complex, 1 μM ssFQ (FAM-TTATT-BHQ1) reporter, 140 nM of each primer, a final 4 U / μl reverse transcriptase (EP0441, Thermo Scientific), 5.9 μl of one lyophilized RPA (#TABAS03KIT, Twistdx) rehydrated with 29.5 μl of rehydration buffer (#TABAS03KIT, Twistdx), and 14 mM MgOAc (#TABAS03KIT, Twistdx) were injected into a 10 μl tube, and a one-pot RT-RPA CRISPR reaction was performed for 60 minutes using a thermal cycler (#CFX96, Bio-Rad) to detect fluorescence signals in real time.

[0088]

[0089] The switch oligonucleotide sequence used in the embodiments of the present invention is as follows.

[0090] Influenza A switch oligonucleotide sequence

[0091] Sequence No. 1: UCCCAG

[0092] Sequence No. 2: UUUCCCAG

[0093] Sequence No. 3: UUUUUCCCAG

[0094] Sequence No. 4: UGUUUUUCCCAG

[0095] Sequence No. 5: UGUGUUUUUCCCAG

[0096] Sequence No. 6: UCUGUGUUUUUCCCAG

[0097]

[0098] SARS-CoV-2 switch oligonucleotide sequence

[0099] Sequence No. 7: GAUGAC

[0100] Sequence No. 8: CAGAUGAC

[0101] Sequence No. 9: UCCAGAUGAC

[0102] Sequence No. 10: AGUCCAGAUGAC

[0103] Sequence No. 11: GCAGUCCAGAUGAC

[0104]

[0105] Example 2. Fluorescent signal detection

[0106] The above process was performed on the Cas proteins LbCas12a (#M0653S, New England Biolabs), AsCas12a (#Z03502, GenScript), and enAsCas12a (#114072, AdGene). Subsequently, fluorescence signals were verified using a thermal cycler (#CFX96, Bio-Rad). The fluorescence signal results for each Cas protein according to the switch length are shown in Table 1 below.

[0107] Switch Length LbCas12aAsCas12aenAsCas12aTargetNTCRNANTCRNANTCRNA(-)212.2612278.2827164.5126368.2509117.5291109.02276nt233.7691475.7272190.1642477.9604126.2845149.81828nt244.9263297.2088213.6083313.851124.3391201.05821 0nt246.6248303.6119251.84747972.494144.75581295.47612nt232.5272274.178377.86629850.461140.81012970.249 14nt269.2974823.5227223.28744443.684144.13711169.2216nt237.5116426.5237198.88822027.679134.6964801.5646

[0108]

[0109] Through the results of Table 1 and Figures 3 to 5 above, it was confirmed that the fluorescence signal was highest when the switch length was between 10nt and 14nt, and that the fluorescence signal was highest when the length was 12nt in AsCas12a and enAsCas12a.

[0110] In addition, as shown in the results in Figure 6, it was confirmed that more RPA reaction products were generated when the switch was used compared to when it was not.

[0111]

[0112] Additionally, to confirm that the switch-based CRISPR-Cas reaction described above is applicable to other RNA viruses, fluorescence signals were analyzed under the same reaction conditions targeting SARS-CoV-2 (#VR-1986HK, ATCC) RNA. The results of fluorescence signal measurements performed using LbCas12a, AsCas12a, and enAsCas12a, respectively, are shown in Figures 7 to 9.

[0113] From FIGS. 7 to 9, it was confirmed that the switch oligonucleotide significantly regulates Cas protein activity against SARS-CoV-2 and induces fluorescence signal amplification.

[0114] The fluorescence signal results for each Cas protein according to the length of the switch are as shown in Table 2 below.

[0115] Switch Length LbCas12aAsCas12aenAsCas12aTargetNTCRNANTCRNANTCRNA(-)125.7345127.597870.7548375.71295125.6331189.75486nt132.8894132.529987.441681.72798133.0254182.79628nt162.3522126.501184.964 2589.91399115.18112234.88710nt194.7171779.6672654.12612233.375403.56513803.7112nt257.8692 3530.92972.9077239.409432.38751978.85514nt186.61061657.864512.35862439.513269.8341702.2305

[0116]

[0117] Through the results of Table 2 and Figures 7 to 9 above, it was confirmed that the fluorescence signal was highest when the switch length was between 10nt and 14nt, and that the fluorescence signal was highest when the switch length was 12nt in LbCas12a and AsCas12a.

[0118]

[0119] The sequences according to the length of the switch oligonucleotides used in the above experiment are summarized in Table 3 below.

[0120] Virus Type / Switch Length 6nt 8nt 10nt 12nt 14nt 16nt Influenza AUCCCAG(Sequence No. 1)UUUCCCAG(Sequence No. 2)UUUUUCCCAG(Sequence No. 3)UGUUUUUCCCAG(Sequence No. 4)UGUGUUUUUCCCAG(Sequence No. 5)UCUGUGUUUUUCCCAG(Sequence No. 6)SARS-CoV-2GAUGAC(Sequence No. 7)CAGAUGAC(Sequence No. 8)UCCAGAUGAC(Sequence No. 9)AGUCCAGAUGAC(Sequence No. 10)GCAGUCCAGAUGAC(Sequence No. 11)-

[0121]

[0122] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0123]

[0124] Sequence No. 1: UCCCAG

[0125] Sequence No. 2: UUUCCCAG

[0126] Sequence No. 3: UUUUUCCCAG

[0127] Sequence No. 4: UGUUUUUCCCAG

[0128] Sequence No. 5: UGUGUUUUUCCCAG

[0129] Sequence No. 6: UCUGUGUUUUUCCCAG

[0130] Sequence No. 7: GAUGAC

[0131] Sequence No. 8: CAGAUGAC

[0132] Sequence No. 9: UCCAGAUGAC

[0133] Sequence No. 10: AGUCCAGAUGAC

[0134] Sequence No. 11: GCAGUCCAGAUGAC

Claims

1. As a nucleotide that hybridizes complementarily to the spacer of guide RNA, A composition for target nucleic acid amplification comprising a switch oligonucleotide that regulates the function of a Cas protein, consisting of 5 to 20 nucleotides starting from the 5' portion of the above nucleotide.

2. In Paragraph 1, A composition for target nucleic acid amplification comprising a switch oligonucleotide that regulates the function of a Cas protein, consisting of 10 to 16 nucleotides starting from the 5' portion of the above nucleotide.

3. In Paragraph 1, A composition for target nucleic acid amplification comprising a switch oligonucleotide that regulates the function of a Cas protein, consisting of 12 to 14 nucleotides starting from the 5' portion of the above nucleotide.

4. In Paragraph 1, The target of the above Cas protein is a respiratory infectious RNA virus, and the composition for amplifying the target nucleic acid is a target nucleic acid composition.

5. In Paragraph 1, The above switch oligonucleotide is a composition for target nucleic acid amplification that detaches from the guide RNA after creating a tohold region.

6. In Paragraph 1, The above composition is a composition for target nucleic acid amplification, further comprising a primer that hybridizes complementarily to a target nucleotide for nucleic acid amplification methods of RPA (recombinant enzyme polymer amplification), LAMP (loop-mediated isothermal amplification), or NASBA (nucleic acid sequence-based amplification).

7. In Paragraph 1, The above composition is a composition for target nucleic acid amplification that further comprises Cas protein and guide RNA.

8. In Paragraph 1, The above composition is a composition for target nucleic acid amplification that further comprises MgCl₂, dNTPs, and DNA or RNA polymerase.

9. In Paragraph 7, The above Cas protein is a composition for target nucleic acid amplification that is any one of Cas12, Cas13, and Cas14.

10. A method for amplifying a target nucleic acid isothermally by adding the composition for amplifying a target nucleic acid of claim 1 to a sample containing the target nucleic acid.

11. In Paragraph 10, The step of amplifying the target nucleic acid is a method for amplifying the target nucleic acid performed at an isotherm between 35 and 45°C.

12. A step of preparing a composition for amplification and detection by further adding a single-stranded nucleic acid probe comprising a fluorescent dye and a quencher to the composition for amplifying a target nucleic acid of claim 1; and A method for detecting a target nucleic acid comprising the step of adding the composition to a sample containing the target nucleic acid and amplifying the target nucleic acid isothermally.

13. In Paragraph 12, A method for detecting target nucleic acid, wherein the step of amplifying the target nucleic acid is performed at an isotherm between 35 and 45°C.

14. A kit for amplifying a target nucleic acid comprising a composition for amplifying a target nucleic acid according to claim 1, a primer that binds complementarily to the target nucleic acid, a Cas protein, and a guide RNA.

15. A kit for target nucleic acid amplification and detection comprising a composition for target nucleic acid amplification of claim 1, a primer that binds complementarily to the target nucleic acid, a Cas protein, a guide RNA, and a single-stranded nucleic acid probe comprising a fluorescent dye and a quencher.

16. Use of a switch oligonucleotide consisting of 5 to 20 nucleotides starting from the 5' portion of said nucleotide, which hybridizes complementarily to a spacer of guide RNA for regulating the function of a Cas protein in target nucleic acid amplification.

17. In Paragraph 16, The target of the above Cas protein is a switch oligonucleotide, which is a respiratory infectious RNA virus.

18. In Paragraph 16, The above oligonucleotide is used as a switch oligonucleotide that detaches from the guide RNA after creating a tohold region.

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