Nucleic acid detection method

Abstract

The present invention relates to a method for detecting the presence of a nucleic acid sequence in a sample using CRISPR technology. The method of the present invention enables the detection of a low nucleic acid amount of 100 fM with a high signal-to-noise ratio and without the need to amplify the nucleic acid of interest. The present invention also relates to a kit for carrying out a method for detecting the presence of a nucleic acid sequence in a sample, and the use of the method or kit of the present invention for the diagnosis or prognosis of a disease or condition.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to a method for detecting the presence of a nucleic acid sequence in a sample using CRISPR technology. The method of the present invention enables the detection of a low amount of nucleic acid of 100 fM with a high signal-to-noise ratio without the need for amplifying the nucleic acid of interest. The present invention also relates to a kit for carrying out a method for detecting the presence of a nucleic acid sequence in a sample.

[0002] [Background of the Invention] The ability of CRISPR-Cas systems programmed to recognize specific nucleic acid sequences has been driving their biotechnological applications. One of these is the detection of pathogen genetic material or gene markers in diagnostics. Systems for detecting specific nucleic acid sequences based on CRISPR-Cas technology, namely SHERLOCK, DETECTR, and their derivatives, have been recently developed and are expected to revolutionize point-of-care diagnostics in the near future. The basis of these systems relies, on the one hand, on the specificity and versatility brought about by the mechanism of target recognition and cleavage. This mechanism is directed by base-pair hybridization with a guide RNA (gRNA) sequence and forms a Cas ribonucleoprotein (RNP) together with a Cas effector protein. And, on the other hand, it is the potent collateral nuclease activity that some of these Cas proteins acquire when cleaving their target sequences. This collateral activity lies in the endonucleolytic degradation of single-stranded nucleic acids of the same type as the sequence-specific target, by a sequence-independent and unexpected endonuclease. The detection of this collateral activity by means of either a nuclease reporter substrate, fluorescence, or a lateral flow device adapted thereto can thus be used, provided the appropriate Cas RNP is designed, as a readout indicating the presence of the nucleic acid of interest (e.g., pathogen genetic material) in a given sample. The gRNA represents either a single guide RNA molecule or a combination of a tracrRNA and a crRNA molecule, and examples of widely used Cas effector proteins with collateral activity are proteins of the Cas13 and Cas12 (including Cas14 / Cas12f) families, which are activated by RNA and DNA, respectively, and act on RNA and DNA, although some Cas12a proteins can also degrade dsDNA and ssRNA in addition to ssDNA.

[0003] These CRISPR-Cas diagnostic systems have already been proven to be useful for the detection of a wide range of pathogens and genetic markers. However, despite the high specificity and versatility they offer, these systems are currently limited by sensitivity levels that are not within the concentration ranges required for diagnostic purposes and currently rely on pre-amplification of the target sequence. This introduces complexity into the reaction and limits their current use in direct point-of-care applications.

[0004] Therefore, improving the sensitivity of these assays will have a major impact on the widespread application of CRISPR-Cas technology as a reliable diagnostic tool.

[0005] Current solutions are moving in the direction of simplifying the pre-amplification step, for example by isothermal nucleic acid amplification, and enabling the reaction to be coupled within a minimal number of steps and in a possible time frame. However, this does not prevent the need for at least some minimal equipment to enable incubation at a constant (usually high) temperature.

[0006] A conceptual alternative to overcoming this problem is to amplify Cas activation once triggered by the target nucleic acid, rather than amplifying the target sequence. One way to achieve this is to include molecules that induce further activation of Cas RNPs when processed by collateral Cas activity. This is the type of approach described by Zeng et al., using a system in which a target-activated Cas13a RNP (T-RNP) recognizes, binds to, and cleaves target RNA. As a result, the collateral activity of activated Cas13a can act on ssRNA loops located in the hairpin RNA structure that Zeng et al. call "mediators," the dsRNA region of which contains the target sequence of a second mediator-activated Cas13a RNP (M-RNP). This system relies on the fact that unprocessed, hairpin-shaped mediators are difficult or not recognized at all by M-RNPs. On the other hand, a portion of the cleaved molecule leaves a short ssRNA sequence, which can serve as a "scaffold" to facilitate annealing with M-RNPs, and theoretically an exponential chain reaction occurs, such that further mediator molecules are processed by its activation and subsequent recognition by M-RNPs is enhanced. However, the fact that the ability of hairpin loops to stimulate recognition by M-RNPs is limited is also evident from the fact that true exponential reaction rates are not induced by low concentrations of the target. A similar approach has also been used by Sha et al., where signals are substantially but limitedly amplified by successive activation of Cas13a and Cas14a, rather than a chain reaction.

[0007] The present invention overcomes the problems of previously known detection methods by an improved Cas signal amplification strategy that enables the detection and even quantification of nucleic acids present in trace amounts in a sample, simply, rapidly, and on-site.

[0008] [Summary of the Invention]

[0009] A first aspect of the present invention relates to a method for detecting the presence of a nucleic acid sequence in a sample, comprising the following steps: (a) adding to said sample at least one Cas ribonucleoprotein (Cas RNP) having collateral activity, at least one amplifier, at least one reporter substrate, and at least one of exonuclease, or optionally polymerase combined with dNTP and / or NTP; (b) incubating the mixture of step (a) for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours, at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 20°C to 37°C, and (c) reading the signal of the reporter substrate; wherein at least one Cas RNP (target-activatable RNP; T-RNP) is designed to recognize a specific sequence (primary target) in the nucleic acid of interest; the presence of the nucleic acid of interest activates the collateral nuclease activity of the T-RNP; the amplifier is: i) a non-target strand (NTS) containing a sequence that is reverse complementary to the target of a second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) a target strand (TS) containing a sequence that is at least partially reverse complementary to the NTS, and is a nucleic acid; the 5', 3' or both ends of at least one strand of the amplifier are modified by chemical modification or by cyclization; the collateral activities of the activated T-RNP and A-RNP on the amplifier result in the generation of a TS that is recognizable by and accessible to the A-RNP, together with the activity of at least one exonuclease or polymerase combined with dNTP and / or NTP; and the reporter substrate contains the type of nucleic acid that is the target for the collateral activity of the A-RNP, and optionally its ends are protected from degradation or polymerization; and Preferably, amplification of the nucleic acid sequence of interest is not performed.

[0010] A second aspect of the present invention relates to a kit for detecting the presence or absence or quantifying a nucleic acid of interest in a sample, the kit comprising at least one Cas RNP having collateral activity, at least one amplifying agent, at least one reporter substrate, and at least one of an exonuclease or a polymerase optionally combined with dNTPs and / or NTPs, wherein at least one Cas RNP (target-activatable RNP; T-RNP) is designed to recognize a specific sequence (primary target) in the nucleic acid of interest; the amplifying agent is: i) a non-target strand (NTS) comprising a sequence that is reverse complementary to the target of a second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) a target strand (TS) comprising a sequence that is at least partially reverse complementary to the NTS, wherein the 5', 3' or both ends of at least one strand of the amplifying agent are modified by chemical modification or by circularization; and the reporter substrate comprises the type of nucleic acid that is the target for the collateral activity of the A-RNP, optionally with its ends protected from degradation or polymerization; and preferably, the kit does not include means for amplifying the nucleic acid sequence of interest in the sample.

[0011] A third aspect of the present invention relates to the use of the method of the first aspect or the kit of the second aspect for detecting the presence or absence or quantifying a nucleic acid of interest in a sample, preferably, the nucleic acid is present in the sample at a concentration of 100 fM to 10 nM.

[0012] A fourth aspect of the present invention relates to the use of the method of the first aspect or the kit of the second aspect for diagnosing or prognosticating a disease or condition, wherein the presence or absence or amount of the nucleic acid is related to the disease or condition.

[0013] Furthermore, a fifth aspect of the present invention relates to a method for detecting the presence of a nucleic acid sequence in a sample, the method comprising the following steps: (a) adding to the sample at least one Cas ribonucleoprotein (Cas RNP) having collateral activity, at least one amplifier, at least one reporter substrate, and optionally at least one of an exonuclease or a polymerase optionally combined with dNTP and / or NTP; (b) incubating the mixture of step (a) for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours, at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 20°C to 37°C, and (c) reading the signal of the reporter substrate, wherein at least one Cas RNP (target-activatable RNP; T-RNP) is designed to recognize a specific sequence in the nucleic acid of interest; the presence of the nucleic acid of interest activates the collateral nucleolytic activity of the T-RNP; the amplifier is: i) a non-target strand (NTS) containing a sequence that is reverse complementary to the target of a second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) a target strand (TS) containing a sequence that is at least partially reverse complementary to the NTS, and is a nucleic acid; at least one of the 5', 3' or both ends of at least one strand of the amplifier is modified by chemical modification or by cyclization; the collateral activities of the activated T-RNP and A-RNP on the amplifier result in the generation of a TS that is recognizable by and accessible to the A-RNP; the reporter substrate contains the type of nucleic acid that is the target for the collateral activity of the A-RNP, and optionally its ends are protected from degradation or polymerization; and The amplification agent is adhered to the solid substrate via one or more collateral regions containing at least one extension in TS and / or NTS, and the gRNA of the A-RNP also contains at least one collateral region and is adhered to a different solid substrate via the collateral region; and the two solid substrates are spatially separated.

[0014] The present invention constitutes the most advantageous method alternative to the methods in the art aimed at generating a signal amplification cascade based on the collateral activity of Cas proteins. The method of the present invention works more efficiently than previous methods and results in a true exponential chain reaction, as demonstrated by the sigmoidal nature of the graph of the present invention as opposed to the linear nature of the graph corresponding to conventional methods; provides a reduction in background signal when compared to previous methods; and provides a tool for detecting both RNA and DNA, which improves the functionality of preliminary methods capable of only enabling the detection of RNA.

[0015] All these effects are achieved by a specific design of the present invention that enables no basal signal to be generated in the absence of the target nucleic acid to be detected. In particular, this is achieved by: · Providing an amplification agent that contains the complete target region in TS but does not contain the PAM motif, and the collateral region is located either 3' or 5' of the target region that coincides with the polarity of the exonuclease used in the system on NTS, terminally protecting a given nucleic acid end from exonuclease activity. The activated T-RNP and the subsequent activated A-RNP cleave the collateral region, enabling access for the exonuclease to degrade NTS and enabling recognition of TS in single-stranded form by the A-RNP. · An amplifying agent is provided for Cas12a A-RNP acting as a template, comprising an NTS for Cas12a A-RNP acting as a template and a TS nucleic acid that is partially hybridized with a region complementary to the 3'-end and a non-complementary collateral region, and protecting against exonucleolytic degradation and polymerization from the 3'-end. The activated T-RNP and the subsequent activated A-RNP cleave the collateral region that generates a primer to which DNA polymerase can extend, and synthesize a complete dsDNA A-RNP target. The target region needs to contain a PAM motif for A-RNP recognition in double-stranded form. · Establish a spatial separation between the amplifying agent and the A-RNP. Both are adhered to the solid substrate via the collateral region. The interaction between the amplifying agent and the A-RNP cannot occur until the activated T-RNP and the subsequent activated A-RNP cleave the collateral region and release both the target region of the amplifying agent and the A-RNP.

[0016] Thus, the present invention increases the amount of target available for activation of A-RNP by any of (i) a combination of an amplifying agent and an exonuclease (or a polymerase proficient in exonuclease), (ii) a combination of an amplifying agent, a polymerase, and dNTP and / or NTP, or (iii) cleavage of a collateral region where the amplifying agent and the A-RNP are adhered to a solid substrate and spatially separated.

[0017] Furthermore, unlike the methods in the art, when RNA and / or DNA sequences are included in the collateral region and Cas 13 and / or Cas 12-based RNPs are used, the same amplifying agent may be useful for both RNA and DNA detection.

[0018] [Description of the Invention]

[0019] The basis of the present invention is the reaction of Cas effector protein activity, by which its in trans collateral nucleic acid degrading activity against a specific nucleic acid molecule, the amplifier, leads to the release or emergence of more target molecules. More precisely, when activated by the nucleic acid sequence of interest, the action of the first target-induced Cas T-RNP on the amplifier molecule leads to the activation of the second amplifier-induced Cas A-RNP as the second target, which in turn acts on the amplifier molecule (Figure 1). Some conceptual bases for this essential approach seem to be shared by Zeng Hongwei et al.

[0020] The present invention provides a more efficient and reliable method of Cas signal amplification, in particular, beyond the disclosure of the knowledge in the art by Zeng Hongwei et al., through the design of novel amplifier molecules and coupling to additional enzymatic activities, which altogether result in a highly efficient activation of the amplifier as a secondary target and a truly exponential chain amplification of Cas activity. Thus, the system of the present invention can unleash the self-propagating chain reaction of Cas and additional enzymatic activities, and ultimately reach the complete activation of the reporter substrate even when the initial concentration of the target of interest is very low. In this regard, the amplifier contains a target region that is initially inactive towards A-RNP recognition, but due to the collateral activity of Cas RNP and subsequent additional one or more enzymatic steps, when a specific region (collateral region) in its structure is degraded, a target recognizable to A-RNP is released and / or reconfigured. This is achieved either by having a hidden target (Figure 2) exposed by the action of exonuclease or by the absence of a target synthesized by polymerase (Figure 3). It should be noted that the amplifier molecule is also protected from the action of these exonuclease and / or polymerase activities until the Cas RNP collateral activity acts on the collateral region. Furthermore, the present invention improves the systems in the art by providing a spatial separation between the target region of the amplifier and A-RNP (Figure 4). In this case, the amplifier contains sequences that are fully recognizable by A-RNP, but this is prevented by being physically separated until these two molecules are released by the degradation of their collateral regions. This spatial separation can also be combined with exonuclease-based approaches and polymerase-based approaches. That is, the spatial separation approach can be implemented in combination with enzymatic steps including either exonuclease or polymerase approaches.

[0021] Regardless of the specific configuration, and similar to PCR, endpoint measurements can be used for the qualitative detection of the nucleic acid of interest, or real-time reactions can be used for quantitative analysis.

[0022] In the context of the present invention, CRISPR-Cas endonuclease chain reaction, 3CR, CCR and ECR are synonymous terms and can be used interchangeably.

[0023] The direct detection of specific nucleic acid sequences using CRISPR-Cas systems at concentrations commonly found in biological or clinical samples is currently limited and typically requires methods such as PCR to amplify the target of interest. This requires more complex reactions and sophisticated equipment, limiting the capabilities of point-of-care testing. As described above, the present invention circumvents this need by dramatically improving the sensitivity of direct CRISPR-Cas nucleic acid detection. In contrast to current amplification-based methods, the present invention does not amplify the nucleic acid sequence of interest, but rather amplifies the activation of the Cas protein and simply enhances the signal without the need for an additional pre-amplification step. Thus, it is faster and easier, occurring in a single reaction, or at least a continuous reaction, without the need for further liquid handling, and occurs over a wide range of temperatures compatible with point-of-care testing.

[0024] As used herein, the term "T-RNP" or "Target-activatable RNP" refers to a Cas RNP that is activated by the presence of a target sequence, i.e., the nucleic acid of interest detected by the methods of the present invention. The T-RNP contains a gRNA that hybridizes to the nucleic acid detected by the methods of the present invention. The term "A-RNP" or "Amplifier-activatable RNP", as used herein, refers to a Cas RNP that is activated by the presence of a target sequence within the TS of an amplifier molecule. Thus, the A-RNP contains a gRNA that hybridizes to the target sequence in the TS of the amplifier. As used herein, the term "activated" refers to a Cas RNP that is activated when its gRNA hybridizes to a target sequence and the target sequence is cleaved. The activated Cas RNP used in the present invention exhibits collateral activity. Either a single guide RNA molecule, or a combination of a tracrRNA and a crRNA molecule, means a gRNA.

[0025] In step (a) of the method of the first aspect, the Cas RNP can be added as already pre-combined components, or can also be added as individual components, i.e., the Cas protein and the guide RNA (gRNA); the remaining components can be added together and incubated, or some can be pre-incubated in different sub-steps before adding the remaining components. In step (a), a buffer suitable for the Cas RNP enzymatic reaction is used, similar to the exonuclease and / or polymerase reaction. The buffer can be, for example, Buffer 2.1 NEB (New England Biolabs), which contains 50 mM NaCl, 10 mM TrisHCl, 10 mM MgCl2, and 100 μg / ml BSA at 1× concentration.

[0026] In a preferred embodiment of the first aspect, the nucleic acid to be detected is a single-stranded or double-stranded nucleic acid. In a preferred embodiment of the first aspect, the nucleic acid to be detected is RNA or DNA. In another preferred embodiment of the method of the first aspect, the nucleic acid of interest is RNA. In another preferred embodiment of the method of the first aspect, the nucleic acid of interest is DNA. In a preferred embodiment, the nucleic acid of interest is derived from SARS-CoV2. This preferred embodiment applies to the method of the first aspect, the kit of the second aspect, and the use of the third aspect.

[0027] The method of the first aspect, the kit of the second aspect, and the use of the third aspect all, as preferred embodiments, have those in which the nucleic acid of interest is derived from a virus, bacterium, mammal, human, or is a disease marker such as a genetic trait, mutation, cancer marker, for example, a single nucleotide polymorphism.

[0028] The term "exonuclease" refers to the activity of an enzyme that removes consecutive nucleotides from the 3'-end (3'-exonuclease) or 5'-end (5'-exonuclease) of a polynucleotide molecule. The term "polymerase" refers to the enzyme activity that generates and / or elongates a polynucleotide chain by continuously adding nucleotides to its 3'-end. Generally, it is understood that a single enzyme can have both polymerase activity and exonuclease activity. Thus, if necessary, polymerization can be achieved using a polymerase having exonuclease activity, in which case the addition of dNTP and / or NTP is required, or degradation by exonuclease can be achieved, in which case dNTP and / or NTP are not required.

[0029] The term "exonuclease" in the present invention includes any enzyme that may exhibit exonuclease activity. This includes, for example, polymerases having exonuclease activity. When such polymerases having exonuclease activity are included to exhibit exonuclease activity, a combination with dNTP / NTP is not necessary.

[0030] In a preferred embodiment of the first aspect, the exonuclease, the polymerase having exonuclease activity and / or the polymerase is selected from the group consisting of exonuclease I, exonuclease III, exonuclease T, Rec JF, T7 exonuclease, lambda exonuclease, T4 DNA polymerase, T7 DNA polymerase, DNA polymerase I, Klenow fragment, Phi29 DNA polymerase.

[0031] In a preferred embodiment of the first aspect, a phosphatase and / or a Cas RNP collateral activity kinase is added in step (a), preferably a polynucleotide kinase-phosphatase, more preferably a T4 polynucleotide kinase-phosphatase, preferably when Cas13 RNP is used.

[0032] In a preferred embodiment of the method of the first aspect, the TS completely contains, partially contains, or does not contain the target region of the A-RNP.

[0033] In a preferred embodiment of the method of the first aspect, the amplifier includes one or more nucleic acid molecules, preferably less than three molecules in total, more preferably including two molecules: one single molecule for each NTS and TS.

[0034] In a preferred embodiment of the method of the first aspect, at least one of the 3', 5' or both ends of the molecule of the amplifying agent is chemically modified to prevent degradation by exonuclease and / or priming of nucleic acid synthesis by polymerase. In another preferred embodiment, at least one of the nucleic acid molecules is ligated to another DNA strand or circularized by ligation between its 5' and 3' ends (Figure 5).

[0035] In a preferred embodiment of the method of the first aspect, the amplifying agent includes a collateral region constituted by at least one extension in the TS and / or NTS, preferably, the collateral region is single-stranded DNA (ssDNA) and / or RNA (ssRNA); and optionally, this collateral region includes at least one strand (TS or NTS) protected from the collateral activity of the Cas RNP.

[0036] Regardless of the structure (Figures 2-6), this collateral region is a preferred target for the collateral activity of the Cas RNP. The presence of this so-called collateral region enhances the effectiveness of the method of the present invention because the collateral activity is directed towards a region that is productive for the release and / or synthesis of the TS by including, for example, the nucleic acid type that is the preferred target of the collateral activity of the selected Cas RNP (e.g., RNA vs. DNA, ssDNA vs. dsDNA, etc.) (Figures 2-4 and 6f). The collateral activity can be further prevented from acting on specific strands and / or regions that are non-productive by including, for example, chemical modifications such as phosphorothioate linkages, or specific nucleotide sequences that are resistant (such as poly-G of Cas12a) (Figures 6d-g).

[0037] In another preferred embodiment of the method of the first aspect, at least one exonuclease is added in step (a), the amplifying agent includes the complete target region in the TS but does not include the PAM motif, and the collateral region is located either 3' (Figure 2) or 5' (Figure 7) of the target region that coincides with the polarity of the exonuclease used on the NTS.

[0038] As used herein, the term "PAM motif" refers to a protospacer adjacent motif (PAM), which is a 2- to 6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas RNP. The presence of this PAM motif near the target sequence is required for the dsDNA Cas RNP to bind and cleave the molecule in the target sequence. The PAM motif is not associated with ssRNA-directed Cas proteins such as Cas13.

[0039] In another preferred embodiment of the method of the first aspect, in step (a), at least one polymerase and dNTP (deoxynucleotide triphosphate) or NTP (nucleotide triphosphate) are added, wherein the TS does not completely contain the target region, or contains at least one mutated or deleted nucleotide, or is at least partially contained by a nucleic acid type not recognized by the A-RNP; the collateral region is located 5' of these modifications on the TS (FIGS. 3 and 8).

[0040] In a preferred embodiment of the method of the first aspect (FIG. 4), the amplifier contains a PAM motif and the complete target region in the TS, is adhered to the solid substrate via the collateral region, where the gRNA of the A-RNP also contains at least one collateral region adhered to a different solid substrate; and the two solid substrates are spatially separated;

[0041] In a preferred embodiment of the method of the first aspect, a single Cas RNP is used, and the target region of the NTS of the amplifier contains a sequence that is reverse complementary to the primary target sequence in the nucleic acid of interest, and thus the T-RNP also acts as the A-RNP.

[0042] In another preferred embodiment of the method of the first aspect, the first and second Cas RNPs contain different gRNAs but the same Cas protein, or contain different Cas proteins.

[0043] In a preferred embodiment, at least one Cas RNP comprises a Cas12 protein, preferably Cas12a, more preferably Lachnospiraceae bacterium Lba Cas12a. In another preferred embodiment, at least one Cas RNP comprises a Cas13 protein, preferably Cas13a, more preferably Leptotrichia wadei Lwa Cas13a.

[0044] In a preferred embodiment, both the T-RNP and the A-RNP comprise a Cas12 protein, preferably Cas12a, more preferably Lba Cas12a.

[0045] In a preferred embodiment, both the T-RNP and the A-RNP comprise a Cas13 protein, preferably Cas13a, more preferably Lwa Cas13a.

[0046] In a preferred embodiment, the T-RNP comprises a Cas13 protein, preferably Cas13a, more preferably Lwa Cas13a, and the A-RNP comprises a Cas12 protein, preferably Cas12a, more preferably Lba Cas12a.

[0047] In another preferred embodiment of the method of the first aspect, the amplifier is prepared by mixing the TS and the NTS in a ratio of 1:1 to 1:5, preferably 1:1 to 1:3, more preferably 1:1 to 1:2.

[0048] In another preferred embodiment of the method of the first aspect, the unannealed TS and / or NTS are removed from the reaction or quenched with the reverse complementary strand; in another preferred embodiment, the correctly assembled amplifier is purified by PAGE or HPLC before the addition of the amplifier in step (a).

[0049] In another preferred embodiment of the method of the first aspect, the amplifier is dsDNA lacking a PAM motif and containing the target sequence of Cas12 A-RNP, and the NTS has a 3' extension of ssDNA and / or RNA that is protected from degradation by exonuclease; and a 3' exonuclease is added in step (a), and optionally, a phosphatase is also added in step (a).

[0050] In a preferred embodiment, the reporter substrate is a nucleic acid of 2 to 100 nucleotides. In a preferred embodiment, the reporter substrate is 3 to 10, more preferably 4 to 8, even more preferably 4 to 6 or 5 nucleotides in length. In a preferred embodiment, the reporter substrate contains a marker, and the marker can be, for example, a fluorophore / quencher pair bound to each end of the nucleic acid, or a molecule such as biotin or fluorescein that can be further detected by streptavidin or a specific antibody.

[0051] For fluorescence detection, the reporter comprises a fluorophore / quencher pair attached to each end of the nucleic acid. The nucleic acid is of a length (typically 5 nucleotides) that allows quenching of fluorescence unless it is cleaved, but is long enough to enable the action of the activated Cas RNP. The structure of the reporter is known to those skilled in the art and can be designed using general knowledge in the field. Fluorescence is usually measured using a fluorometer. For quantitative analysis, a standard curve prepared with nucleic acids of known concentrations is used. In a method similar to quantitative PCR, in the quantitative assay, fluorescence is measured and the time at which half of the maximum fluorescence signal is detected is used for quantification. For example, in lateral flow detection, the reporter comprises two motifs that can be specifically recognized, one at each end of the nucleic acid. Since one is used for capture / immobilization and the other for detection, cleaved and uncleaved molecules can be distinguished. Preferably, biotin is used for immobilization and fluorescein is used for detection with a specific antibody. Since the length of the molecule is less relevant in this case, the amplification agent itself can be used as the reporter by adding biotin and fluorescein moieties to the strand that undergoes cleavage. Thus, in a preferred embodiment, the amplification agent is also a reporter substrate.

[0052] As used herein, the term "56-FAM" or "FAM" refers to the marker fluorescein. The term "3IABkFQ" as used herein means the quencher Iowa Black® FQ, which has a broad absorbance spectrum from 420 to 620 nm and a peak absorbance at 531 nm. This quencher is ideal for use with fluorescein.

[0053] In another preferred embodiment of the method of the first aspect, the reporter is a nucleic acid molecule having a fluorescent marker at one end and a quencher at the other end.

[0054] In another preferred embodiment of the method of the first aspect, steps (a) and (b) are carried out in sub-steps: (a1) Adding at least one T-RNP having collateral activity and at least one amplifying agent to the sample; (b1) Mixing and incubating the mixture of step (a1) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C for 1 minute to 2 hours, preferably 10 minutes to 1 hour, more preferably 20 minutes to 40 minutes; (a2) Optionally, adding at least one of a further amplifying agent and / or its components (TS and NTS), preferably adding additional NTS of the amplifying agent; (b2) Optionally, mixing and incubating the mixture of step (a2) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C for 1 minute to 1 hour, preferably 10 minutes to 1 hour, more preferably 15 minutes to 30 minutes; (a3) Adding at least one A-RNP, at least one reporter substrate, and at least one of an exonuclease or a polymerase combined with dNTP and / or NTP; and (b3) Incubating the mixture of step (a3) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours.

[0055] In another preferred embodiment, sub-steps (a2) and (b2) are omitted.

[0056] In a preferred embodiment of the first aspect, the T-RNP contains a Cas13 protein, the amplifying agent contains a target region for Cas12 A-RNP, a DNA TS having at least one phosphorothioate bond, and a DNA NTS that does not contain a PAM motif, contains at least one phosphorothioate bond, and has a single-stranded collateral region having at least three ribonucleotides.

[0057] In another preferred embodiment of the method of the first aspect, the T-RNP comprises the Lwa Cas13a protein and the gRNA of SEQ ID NO: 6, the A-RNP comprises the Lba Cas12a protein and the gRNA of SEQ ID NO: 3, and the amplifiers comprise SEQ ID NO: 4 and SEQ ID NO: 5 as TS and NTS, respectively.

[0058] In a preferred embodiment of the first aspect (Figure 9), the A-RNP comprises a Cas12 protein, and the amplifier comprises a circularized NTS lacking a PAM motif, and a TS that hybridizes completely or partially to the NTS but has at least one mutation in the target region, and a non-annealing DNA and / or RNA flap having at least one phosphorothioate bond and an InvdT motif at its 3' end, and a DNA polymerase having strand displacement ability and 3'-exonuclease activity, preferably phi29pol, is added in step (a).

[0059] In a preferred embodiment of the first aspect (Figure 10), both the T-RNP and the A-RNP comprise a Cas13 protein; the amplifier comprises an NTS having at least one phosphorothioate bond and an InvdT motif at its 3' end, and a TS that hybridizes partially to the 3' end of the NTS containing a promoter sequence for the non-transcribed strand and RNA polymerase, preferably T7 RNA polymerase, for the target of the Cas13 A-RNP, and a flap containing at least three ribonucleotides, at least one phosphorothioate bond and an InvdT motif at its 3' end. A DNA and RNA polymerase, preferably T7 RNA polymerase, is added in step (a).

[0060] The method of the present invention preferably occurs in the absence of amplification of the nucleic acid sequence of interest, but may also be compatible with one or more additional amplification steps. In other words, whether or not amplification of the nucleic acid sequence of interest is performed in addition to each method, kit or use of the present invention is not relevant to the method, kit or use of the present invention.

[0061] In another preferred embodiment, the method of the first aspect of the present invention is used in a diagnostic method (in vitro method): the diagnostic method includes a method for detecting the presence of a nucleic acid sequence in a sample of the first aspect, and also includes associating the presence, absence or amount of the nucleic acid sequence in the sample with a disease or condition. The method can be carried out in a qualitative or quantitative manner, for example, using a lateral-flow device. The method can be carried out for qualitative detection, while quantitative detection is possible by measuring the fluorescence of the reporter in real time using a fluorometer. As described above, the time when the fluorescence level reaches half of the maximum fluorescence is used for quantification. A specific embodiment of interest is, as shown in the examples, a SARS-CoV2 quantitative detection method or a SARS-CoV2 qualitative detection method using a lateral-flow device.

[0062] In relation to the second aspect of the present invention, the kit can be a kit-of-parts or can also be a device such as a lateral-flow device. Thus, as used herein, the term "kit" means both a kit-of-parts and a device.

[0063] In relation to the kit of the second aspect of the present invention, the means for amplifying the nucleic acid sequence of interest in the sample is, for example, a heat-resistant DNA polymerase for PCR, a reverse polymerase that copies cDNA from RNA, or a specific primer pair for amplifying the sequence by PCR or other amplification means, such as Loop-mediated isothermal amplification (LAMP) or Recombinase polymerase amplification (RPA).

[0064] In a preferred embodiment, the kit comprises a Cas13a protein, preferably the Lwa Cas13a protein, and a T-RNP comprising the gRNA of SEQ ID NO: 6, a Cas12a protein, preferably Lba Cas12a, and an A-RNP comprising the gRNA of SEQ ID NO: 3, and an amplifying agent comprising SEQ ID NO: 4 and SEQ ID NO: 5 as TS and NTS, respectively. This kit is useful for detecting the presence of and quantifying the amount of nucleic acid derived from SARS-CoV2.

[0065] In a preferred embodiment of the second aspect, the kit further comprises the components necessary for lateral-flow detection or is a lateral-flow device as described in Gootenberg et al. 2018 (Science, 360(6387):439-444) and Broughton et al. 2020 (Nature Biotechnology, 38(7):870-874), and the kit optionally comprises means for collecting a sample and / or means for extracting nucleic acid from the sample.

[0066] In a lateral flow kit, the sample is applied onto one end (pad) of a solid support and moves through it by capillary action. The lateral flow reporter substrate is a nucleic acid molecule having two different marker molecules at both ends, preferably biotin and fluorescein. The solid support includes two regions having immobilized molecules that bind to each of these markers, through which the sample sequentially moves, preferably first through a stripe coated with streptavidin proximal to the pad and then preferably through a stripe coated with an anti-fluorescein antibody distal to the pad. One of the markers, preferably fluorescein, is used for detection of the reporter by an anti-fluorescein antibody conjugated to gold nanoparticles preferably embedded in the pad, and the other marker, preferably biotin, is used for retention at the first stripe, preferably by interaction with streptavidin. When the reporter reaches the first stripe, all intact molecules are retained by interaction with the retention marker, while the cleaved reporter fragments containing the detection marker continue to move through the solid support until they reach the second stripe. If a signal is visualized only in the first stripe (control region), it indicates that the target nucleic acid is not present in the sample, while if a signal is visualized in the second stripe (test region), it indicates that the target nucleic acid is present in the sample. This type of lateral flow test is shown in FIG. 21.

[0067] As used herein, the expression “at least one” means one, two, three or more, preferably one or two, more preferably one.

[0068] In preferred embodiments of the first and second aspects, the amplifier is modified to act directly as a lateral flow reporter by including, at either end of the collateral region, on the same strand or one of the markers on each strand, the retention marker and the visualization marker (as described above).

[0069] The methods, kits, and uses of the present invention can be adapted for the detection of a protein or metabolite of interest by binding nucleic acids to specific antibodies or binding molecules and then detecting these nucleic acids. Thus, this is a preferred embodiment relating to four aspects of the present invention.

[0070] All of the above embodiments of the first aspect also apply to the second, third, and fourth aspects. All embodiments of the second aspect apply to the first, third, and fourth aspects. All embodiments of the third aspect apply to the first, second, and fourth aspects. All embodiments of the fourth aspect apply to the first, second, and third aspects. The present disclosure includes all combinations of all aspects disclosed above. The terms "comprising," "comprise," or "comprises" include the terms "consisting of," "consist of," or "consists of."

[0071] [Brief Description of the Drawings]

[0072] Figure 1: General scheme of the CRISPR Cas chain reaction (3CR). The target activates the first Cas RNP (target-activatable RNP or T-RNP). Each activated T-RNP acts on an amplifier molecule that releases or generates a target for a second amplifier-activatable Cas RNP (A-RNP), and the A-RNP is activated. The activated A-RNP acts on the amplifier, causing a chain reaction of target release and RNP activation. The amplified Cas activity is detected by a reporter substrate (fluorescence, lateral flow device, etc.).

[0073] Figure 2: Scheme of an example of a target-exposure 3CR approach by 3'-exonuclease activity. The amplifier contains the target (target region) of the Cas12a A-RNP in dsDNA form, but recognition is prevented by the absence of a PAM motif. The non-target strand (NTS) has a 3'-extension (collateral region) of ssDNA or RNA and is protected from degradation by exonuclease (asterisk). The activated T-RNP degrades the overhang, generates a substrate for the 3'-exonuclease that degrades the NTS, and releases the target strand (TS) in the form of ssDNA. The accessible TS here activates the A-RNP, and the A-RNP can further remove overhangs from more amplifiers and unleash a chain reaction.

[0074] Figure 3: Scheme of a target-synthesis 3CR approach. The amplifier contains the non-target strand (NTS) of the Cas12a A-RNP that acts as a template, and a partially hybridized target strand (TS) nucleic acid with a region complementary and a non-complementary flap at the 3'-end (collateral region), and is protected from degradation by 3'-exonuclease (asterisk). The activated T-RNP degrades the flap, generates a primer that can be extended by DNA polymerase, and synthesizes a complete dsDNA A-RNP target. Note that the target region needs to contain a PAM motif for the A-RNP to recognize it in dsDNA form. The reconstituted target activates the A-RNP, and the A-RNP further removes flaps from more amplifiers and unleashes a chain reaction.

[0075] Figure 4: Scheme of a spatial separation approach. The amplifier with a fully functional target region is spatially separated from the A-RNP that is attached to a solid substrate via the collateral region and to a different solid substrate via the collateral region of the gRNA. The activated T-RNP cleaves the collateral region, releasing both the target region of the amplifier and the A-RNP, so that the two can interact, the latter is activated, and its collateral activity releases more molecules, unleashing 3CR amplification.

[0076] Figure 5: Examples of amplifier configurations in which DNA ends are protected by circularization. The NTS and TS are connected in a hairpin loop (a) or dumbbell (b) structure. Alternatively, the NTS alone (c) or both the NTS and TS (d) are circularized.

[0077] Figure 6: Examples of the configurations of various collateral regions (left) and strategies for inducing Cas activity (right). Loops (a), bubbles (b), and array-like mismatches (c) are used as collateral regions. If necessary, the RNP collateral activity is directed to the NTS by protecting the NTS with chemical modifications (d, e) or by using different nucleic acid types (f) or sequences (g).

[0078] Figure 7: Scheme of an example of a 3CR target exposure approach by 5' exonuclease activity. It is the same as in Figure 1, but has a 5' extension on the NTS and 5' exonuclease activity.

[0079] Figure 8: Examples of various strategies for protecting the NTS template from degradation by activated RNP. The NTS is protected by chemical modification (a) and / or by hybridization with RNA (b) or DNA, in which case deletions (c) or mutant (d) nucleotides are included to prevent direct activation of A-RNP.

[0080] Figure 9: Scheme of the rolling circle amplification (RCA) 3CR approach. The amplifier anneals to a target strand (TS) that contains a circular non-target strand (NTS) for Cas12a A-RNP, lacks the PAM motif, and has mutations that further prevent direct recognition by A-RNP. The TS is not circularized and contains an ssDNA / RNA non-complementary flap (asterisk) on the 3'-end (collateral region) that is protected from degradation by 3'-exonuclease. The activated T-RNP degrades the flap that generates a substrate for a DNA polymerase with strand displacement ability that initiates the RCA reaction, synthesizing and releasing in-tandem repeats of the non-mutated TS in ssDNA form. Thus, each event activates multiple A-RNPs, further removing flaps from more amplifiers, and unleashing a more powerful chain reaction.

[0081] Figure 10: Scheme of the in vitro transcription (IVT) 3CR approach. This approach is target-synthesis based as in Figure 13, but with the following modifications: The TS and NTS correspond to the transcribed and non-transcribed strands, respectively, and when reconstituted in dsDNA form by DNA polymerase, the target region is transcribed by RNA polymerase to generate an RNA molecule that is the target for Cas13a A-RNP. For this purpose, the NTS contains a promoter sequence. Thus, each event can activate multiple A-RNPs, further removing flaps from more amplifiers, and unleashing a more powerful chain reaction.

[0082] Figure 11: Design and validation of a target-exposed 3'-exonuclease amplifier. (a) Scheme of an amplifier molecule with a TS represented by SEQ ID NO: 4 and an NTS represented by SEQ ID NO: 5. Ribonucleotides are indicated by (r). The target region is highlighted in bold. Protected phosphorothioate linkages are indicated by asterisks. This amplifier was incubated with (b) LwaCas13a and (c, d) LbaCas12a in NEB Buffer 2.1 at 37 °C for 1 h in the presence (+) or absence (-) of the indicated enzyme, run on a 3% agarose gel, and stained with ethidium bromide.

[0083] Figure 12: Background activation of A-RNP by an amplifying agent molecule. The amplifying agent described in Figure 3 was prepared by annealing the indicated ratio of TS:NTS and incubated at 37 °C for the indicated times in the presence of the corresponding LbuCas12a A-RNP and a fluorescent DNAse reporter substrate. Fluorescence was measured every 5 minutes for 2 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0084] Figure 13: Example 1: Comparison of one-step target-exposed 3CR DNA detection and direct detection at room temperature. The 3CR (a) and direct detection (b) reactions were performed at 25 °C in the presence of the indicated concentrations of target DNA as described in Example 1. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 2 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0085] Figure 14: Example 2: Two-step target-exposed 3CR DNA detection by ExoIII. The 3CR reaction was performed as described in Example 2 in the presence of the indicated concentrations of target DNA. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 2 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0086] Figure 15: Example 3: Two-step target-exposed 3CR DNA detection by T7 pol. The 3CR reaction was performed as described in Example 3 in the presence of the indicated concentrations of target DNA. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 2 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0087] Figure 16: Example 4: Three-step target-exposed 3CR DNA detection by T4 pol. The 3CR reaction was performed as described in Example 4 in the presence of the indicated concentrations of target DNA. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 3 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0088] Figure 17: Example 5: Two-step target exposure 3CR RNA detection by ExoIII and comparison with direct detection. 3CR (a) and direct detection (b) reactions were performed as described in Example 5 in the presence of the indicated concentrations of target RNA. Fluorescence emission of the DNAse (a) or RNAse (b) reporter substrate was measured every 5 minutes for 2 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0089] Figure 18: Design and validation of a target synthetic amplifier with partial TS coverage. (a) Scheme of the amplifier containing SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The PAM motif is underlined and the target region is highlighted in bold. Asterisks indicate phosphorothioate bonds and the 3’ end modified with an inverted dT nucleotide ( / InvdT / ). To validate the strategy, it was shown that only fully dsDNA-reconstituted amplifiers induce a Cas12a response (b), and that Klenow (Kle), which reconstitutes the complete dsDNA sequence of the amplifier, requires Cas12a activity.

[0090] Figure 19: Example 6: Target synthesis-3CR DNA detection using Klenow and partial TS coverage. The 3CR reaction was performed as described in Example 6 in the presence of the indicated concentrations of target RNA. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 1.5 hours. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0091] Figure 20: Example 7: Target synthesis-3CR detection DNA detection using Klenow and complete TS coverage. (a) Scheme of the amplifier containing SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 11. The PAM motif is underlined and the nucleotides in the target region recognized by A-RNP are highlighted in bold. Asterisks indicate phosphorothioate bonds and the 3’ end modified with an inverted dT nucleotide ( / InvdT / ). (b) The 3CR reaction was carried out as described in Example 7 in the presence of the indicated concentrations of target RNA. Fluorescence emission of the DNAse reporter substrate was measured every 5 minutes for 1 hour. The horizontal axis indicates time in minutes and the vertical axis indicates fluorescence in arbitrary units.

[0092] Figure 21: Scheme of the double-hit 3CR approach. The combination of the approaches for target synthesis (Figure 13) and 5′ target exposure (Figure 7) imposes the need for two events to achieve productive 3CR amplification and limits potential background signals due to spontaneous hydrolysis of the collateral region. After activation of the T-RNP on the collateral region of the target strand (TS) and subsequent polymerization from the generated primer, the dsDNA target region is reconstituted, but without a PAM motif, the activation of Cas12a A-RNP, which requires degradation of the collateral region of the NTS by the T-RNP, is prevented unless the NTS is degraded by a dsDNA-specific 5′ exonuclease. Once initiated, the reaction proceeds by the action of the activated A-RNP, as with other 3CR approaches. The NTS is protected from the action of the activated RNP, as explained in Figure 14.

[0093] Figure 22: Example of a dual amplifier-reporter molecule for lateral flow detection. As shown in (a), by adding biotin and FAM molecules to the 3′ ends of the NTS and TS, the amplifier also functioned as a reporter for lateral flow detection. (b) Biotin was retained in the control region by a streptavidin-coated area, restricting the mobility of anti-FAM gold nanoparticles if the amplifier was intact. However, when the amplifier was cleaved and / or degraded, the nanoparticles continued to the test region. (c) Treatment of these dual amplifier-reporter molecules with activated LbuCas12a was detected as a complete shift to the test position.

[0094] Examples The following examples illustrate the present invention and are not to be construed as limiting the present invention. In the examples, nucleic acid sequences related to SARS-CoV-2 have been detected, but the claimed technology described herein can be readily adapted by those skilled in the art to any nucleic acid of interest, including other viruses, bacteria, genetic traits, cancer markers, etc., and includes the identification of single nucleotide polymorphisms.

[0095] In the simplest embodiment, the method of the present invention is based on the use of a Cas protein, such as the Cas12 family, which recognizes only its target sequence in double-stranded form (dsDNA for Cas12 protein) when containing an adjacent PAM motif, while in single-stranded form it does not require detection of its target strand. The same applies to Cas proteins, such as the Cas13 family, which recognize only single-stranded nucleic acids (RNA in this case) and do not have a PAM motif. With this in mind, as seen in Figure 2, an amplifier molecule containing a dsDNA region (target region) composed of both a target strand and a non-target strand (TS and NTS respectively) was envisioned for Cas12a RNP (amplifier-activated Cas12a RNP; A-RNP). The non-target strand contains a non-annealing ssDNA region at its 3' end, which is the preferred substrate for collateral cleavage by activated Cas12a (collateral region). Both TS and NTS are protected from degradation by 3' exonucleases present during the reaction by chemical modification and / or binding to bulky adducts. Although not essential, the 5' end can also be protected in the same way to prevent degradation by other exonucleases that may be present in the sample. Thus, only in the presence of a predetermined target sequence of interest does the endonucleolytic collateral activity of the target-activated Cas12a RNP (T-RNP) act on the collateral region of the NTS and release a free 3' nucleic acid end that can be used by the 3' exonucleases to release the TS in ssDNA form recognizable by the A-RNP, and this free 3' nucleic acid end unleashes exponential 3CR signal amplification, such as by acting on the collateral region of additional amplifiers.

[0096] This target-exposure approach is also useful for detecting RNA, for example by using a Cas13a-based T-RNP, where the collateral region of the amplifier contains ribonucleotides that enable collateral cleavage by the activated Cas13a. With this in mind, based on the above-described DNA targets of LbaCas12a containing mutated PAMs, an 8-uracil ribonucleotide 3’ extension was added onto the NTS, and an amplifier molecule (Figure 11a) was generated in which the last 5 contain phosphorothioate linkages (see the oligonucleotide list of Example 1), and the actions of LwaCas13a, LbaCas12a, and the 3’ exonuclease Exo III of E. coli on this substrate were confirmed. As seen in Figure 11b, the amplifier was completely protected from the action of Exo III. However, when incubated with active LwaCas13a or RNAse If (as a positive control), the overhang was cleaved, and when Exo III was included in the reaction, partial degradation of the molecule with release of the target strand occurred. Furthermore, as expected, the addition of T4 polynucleotide kinase-phosphatase (PNK) strongly stimulated the reaction. This is because the phosphatase activity of the enzyme removed the 2’-3’ cyclophosphate product of the collateral activity of Cas13a that restricts subsequent 3’ exonuclease activity. Interestingly, incubation with active LbaCas12a also resulted in cleavage of the overhang due to its ability for collateral activity that acts on any of ssDNA, dsDNA, or ssRNA (Figure 11c). Furthermore, incubation with Exo III, as expected, resulted in complete degradation of the amplifier, which is because the collateral activity of Cas12a also degrades the released ssDNA TS. These results demonstrate the validity of RNA overhang-based amplifiers to amplify both Cas13a and Cas12a activities and thus specifically detect both RNA and DNA sequences. Amplifiers having both ssDNA and ssRNA, or combinations of ssDNA-based and ssRNA-based amplifiers, can also be used.Similar results were obtained for Cas12a activation using two other enzymes such as T4 and T7 DNA polymerases (Fig. 11d), which have strong 3' exonuclease activity against both ssDNA and dsDNA in the absence of dNTPs.

[0097] Since any of the free TSs present may be sufficient to trigger the reaction, all TS molecules of the amplifiers should anneal to the corresponding NTSs. To confirm this, we tested the ability of amplifier molecules annealed at different TS:NTS ratios to activate the corresponding Cas12a A-RNP. As shown in Fig. 12, TS alone (1:0 ratio) induced robust activation of the A-RNP as measured by the time-dependent luminescence of fluorescence by the single-stranded DNAse reporter substrate, while, as expected, NTS alone (0:1 ratio) did not result in measurable A-RNP activation. Furthermore, the presence of an equimolar ratio of 1:1 NTS significantly decreased TS-dependent A-RNP activation but was not completely removed unless used in excess (ratio of 1:2 or higher). We concluded from this that free, unannealed TS is a background signal source that can be removed by preparing the amplifier at a TS:NTS ratio of 1:2.

[0098] The overhang is the simplest configuration of the collateral region, but additional structures such as loops, bubbles, continuous mismatches, etc. can also be used (Figs. 5a-c). In this case, various approaches can be adopted to direct the collateral activity towards the productive collateral region strand for the purpose of TS release if necessary. For example, in a bubble structure, TS can be protected by chemical modifications (e.g., phosphorothioate) within the collateral region or by the nucleic acid type or sequence that is an unfavorable substrate for collateral activity (e.g., Cas12a RNA or poly-G region) (Figs. 5d-g). Regarding the structure of the amplifier, it is worth noting that in addition to chemical modifications, circularization of a given strand, which can be achieved in different configurations shown in Fig. 4, is another possibility for protecting the DNA ends. Furthermore, for DNA detection, by basing the amplifier on the target sequence, a single Cas12a RNP can be used, and it is worth noting that the same Cas12a RNP can recognize both the main target and the TS released from the amplifier. Finally, when a 5' exonuclease is used instead of a 3' exonuclease; in this case, the collateral region of the amplifier is located at the 5' end of the NTS (Fig. 6).

[0099] Examples 1-6 show some representative results corresponding to the exonuclease version of the system of the present invention. Examples 7 and 8 correspond to the polymerase version, with the former protecting the NTS from Cas-mediated degradation by partial coverage of the NTS and the latter by complete coverage with the addition of mismatches.

[0100] Example 1: One-step detection of an ssDNA sequence related to SARS-CoV2 using LbaCas12a and E. coli exonuclease III (Exo III).

[0101] The above-mentioned amplifier (Figure 11) and the LbaCas12a T-RNP directed against the already verified ssDNA region derived from the SARS-CoV2 gene S were used. In the 3CR reaction, synthetic ssDNA of different concentrations of the target region was mixed with the newly prepared amplifier at a ratio of 1:2 of TS:NTS, the LbaCas12a T-RNP, the LbaCas12a A-RNP, Exo III, PNK, and a fluorescent DNAse reporter protected from degradation by exonucleases (see oligonucleotide list for details), and then incubated at 25 °C for 2 hours in a Victor Nivo instrument (Perkin Elmer), and the fluorescence of the reporter was measured every 5 minutes. The results in Figure 13a show that a target at a concentration of 100 pM can be clearly detected in less than 90 minutes. In contrast, for the detection of a 100 pM target in the absence of the amplifier under room temperature conditions, only weak reporter fluorescence is obtained (Figure 13b). In addition to the improved sensitivity, the self-activating property of 3CR allows for complete reporter fluorescence regardless of the target concentration, and it is worth noting that, similar to real-time PCR, the time required to achieve exponential amplification can be easily determined. This property not only enables quantitative measurements using real-time detection, as demonstrated in this example, but is also important for adapting this method for qualitative analysis in low-sensitivity detection devices such as lateral flow systems. This indicates the potential of 3CR as a method without amplification for the room temperature detection of nucleic acids.

[0102] Oligonucleotide list:

Table 1

[0103] Reaction conditions:

Table 2

[0104] Example 2: Two-step detection of SARS-CoV2-related ssDNA sequences using LbaCas12a and Escherichia coli exonuclease III (Exo III).

[0105] The main problem with the sensitivity of the 3CR determined in Example 1 is the background signal that begins to appear around 100 minutes in the absence of the target DNA (Figure 13a). This is presumably the result of the intrinsic ability of the amplifier to activate the A-RNP even in dsDNA form and / or the residual action of Exo III acting on the amplifier to release very small amounts of TS for A-RNP activation. This problem was minimized to some extent by pre-incubating the target with the T-RNP and the amplifier and then adding the remaining components that caused this background signal. As can be seen from Figure 14, this pre-incubation delayed the appearance of the non-specific signal and enabled complete activation of the reporter with as little as 10 pM of the target in 60 minutes.

[0106] Oligonucleotide list: Same as Example 1. Reaction conditions: [Table 3] Temperature: 37 °C Time: 45 minutes

[0107] Addition: [Table 4] Temperature: 25 °C. Time: 120 minutes, measurement every 5 minutes.

[0108] Example 3: Two-step detection of SARS-CoV2-related ssDNA sequences using the 3’exonuclease activity of LbaCas12a and T7 DNA polymerase (T7 pol).

[0109] The 3'-exonuclease activity of other exonucleases besides ExoIII was tested, and attention was paid to T7 pol in accordance with the results of Fig. 11d. Basically, the 3CR reaction was set up as described in Example 2, but T7 pol was used instead of ExoIII. As seen in Fig. 15, complete detection of 1 pM of the target was achieved in the incubation around 80 minutes, and the background at this time was very low.

[0110] Oligonucleotide list: Same as Example 1. Reaction conditions: [Table 5] Time: 45 minutes Temperature: 37 °C

[0111] Addition: [Table 6]

[0112] Temperature: 37 °C. Time: 120 minutes, measured every 5 minutes.

[0113] Example 4: Three-step detection of an ssDNA sequence related to SARS-CoV2 using the 3'-exonuclease activities of LbaCas12a and T4 DNA polymerase (T4 pol).

[0114] T4 pol was also tested. A pre-incubation step was included using the amplifiers generated at a ratio of 1:1 of TS:NTS. Then, excess NTS was added to make this a 1:2 ratio, and finally, it was incubated with the remaining components. As seen in Fig. 16, this three-step reaction further enhanced the sensitivity of the assay and reached the detection of 100 fM at 160 minutes before the background signal began to appear.

[0115] Oligonucleotide list: Same as Example 1. Reaction conditions:

Table 7

[0116] Addition:

Table 8

[0117] Time: 30 minutes. Temperature: 37 °C.

[0118] Addition:

Table 9

[0119] Temperature: 37 °C. Time: 180 minutes, measured every 5 minutes.

[0120] Example 5: Two-step detection of an ssRNA sequence related to SARS-CoV2 using LwaCas13a, LbaCas12a, and E. coli exonuclease III (Exo III).

[0121] Using the amplifiers and two-step incubation approach described in Example 2, the ability of 3CR was extended to RNA detection. For this purpose, LwaCas13a, which is specifically directed against the target sequence of the SARS-CoV2 gene S, was used as the T-RNP. In addition, ExoIII, which has been demonstrated to have activity in extending ribonucleic acid cleavage, was used (Figure 11b). The results in Figure 17a show that, in contrast to the detection sensitivity being lower than 1 pM in the absence of the amplifier, the in vitro transcribed RNA target could be detected at 100 fM in 120 minutes (Figure 17b). This indicates to what extent 3CR can enhance RNA detection.

[0122] Oligonucleotide list:

Table 10

[0123] Reaction conditions:

Table 11

[0124] Time: 45 minutes. Temperature: 37 °C.

[0125] Addition:

Table 12

[0126] Temperature: 25 °C. Time: 120 minutes, measured every 5 minutes.

[0127] Therefore, 3CR has been demonstrated to be a useful method for detecting specific sequences of both DNA and RNA at room temperature and with a sensitivity up to 100 fM without target amplification.

[0128] As described above, excess NTS is advantageous for restricting direct activation by the amplifier. Another advantageous possibility is to prepare the amplifier with a 1:2 TS:NTS ratio as described in the examples and then quench the excess NTS with inactivated TS (e.g., containing mutations in the target sequence). Other advantageous embodiments include steps for purifying the correctly assembled amplifier by PAGE or HPLC.

[0129] The absence of hybridization of the gRNA with the four last PAM-distal nucleotides of the target strongly affects Cas12a cleavage and activation by dsDNA, even in the presence of the PAM motif, but has little effect in the ssDNA form. Another advantageous embodiment is achieved by using this target or other reduced targets by changing either the sequence in the amplifying agent or the gRNA used in the A-RNP. Systematic and / or large-scale screening methods are available to those skilled in the art to identify optimal target sequences for this purpose. In another embodiment, other natural or artificial Cas proteins, which are more preferred with respect to ssDNA without PAM than Cas12a, are used.

[0130] In another embodiment, the target sequence is not initially present in the amplifying agent but is instead synthesized during the course of the reaction. In the simplest configuration (Figure 3), the TS does not cover the target region but only the adjacent upstream region, like a primer annealed to the 3’ end of the NTS. This TS primer contains a flap that is not annealed to its 3’ end and is complementary to the collateral region of the amplifying agent, thus preventing elongation from the primer unless it is pre-degraded. All 3’ ends in the amplifying agent are blocked from exonuclease activity as described above and include additional modifications (inverted dT, spacers, biotin-streptavidin, circularization, etc.) to block priming. The collateral activity of Cas results in complete degradation of the flap along with the proofreading 3’ exonuclease activity of DNA polymerase, such that the annealed 3’ terminus can be used for complete synthesis of the TS using the NTS as a template and for reconstitution of the dsDNA target molecule for the A-RNP, thus initiating the 3CR reaction. Note that this amplifying agent sequence contains the PAM motif, being dsDNA and in contrast to the target-exposure approach. Further, the ssDNA region in the target strand is protected from degradation by the collateral activity of the activated RNP (Figure 7). This is achieved by including a protecting modification such as phosphorothioate or by hybridizing to DNA or RNA, in which case the major nucleotides in the TS are deleted or mutated to prevent direct activation of the A-RNP. The different adaptations and structures described above for target-exposure also apply to these target-synthesis approaches, including the detection of both DNA and RNA, the possibility of different end-protections, and the composition of the collateral region, in which case a particular strand of the collateral region is selectively protected (not as in Figure 5d-g but protecting the NTS).

[0131] Example 6: Target-synthesis 3CR DNA detection by partial coverage of the NTS using LbaCas12a and the Klenow fragment of E. coli DNA polymerase I (Klenow).

[0132] In the target-synthesis approach, the same target sequence as in Example 1 was used in the amplifier, and a structure formed by the NTS and a primer containing a 25-nucleotide ssDNA flap annealed to its 3'-end was designed (Figure 18a). The protective DNA oligonucleotide also annealed to the 5'-target region of the NTS, leaving a 10-nucleotide ssDNA gap, which was sufficient to prevent the activation of A-RNP (Figure 18b). Except for the protective oligonucleotide, all 3'-ends in the amplifier were protected by phosphorothioate bonds and contained inverted dT residues to prevent their use as primers. Note that an excess of NTS is not required for the preparation of this type of amplifier. As confirmed by agarose gel electrophoresis, the amplifier molecules were not affected by incubation with Klenow alone, but complete conversion to the dsDNA form was observed when activated LbaCas12a was included in the reaction.

[0133] Using this amplifier molecule, a 3CR reaction was performed basically as described in Example 1, but Klenow was used instead of T4 pol / PNK, and dNTPs were added for DNA synthesis. Pre-incubation was not included, so the reaction was carried out directly at 37 °C for 2 hours, and the activation of the reporter was continuously analyzed by fluorescence emission. As can be seen in Figure 19, 1 pM of the target could be detected under these conditions with a 50-minute incubation.

[0134] Oligonucleotide list: [Table 13] InvdT means inverted deoxythymidine 3'-monophosphate.

[0135] Reaction conditions:

Table 14

[0136] Example 7: Target-synthesis 3CR DNA detection using the Klenow fragment of LbaCas12a and E. coli DNA polymerase I (Klenow).

[0137] Another strategy for protecting the NTS was also tested, which is basically the same as in Example 6, but is based on the complete coverage of the portion of the NTS not covered by the TS primer. To avoid direct activation of the A-RNP by the amplifier, eight mismatches that interfere with the hybridization of the A-RNP with the gRNA were included as generally described in Fig. 8d, resulting in the amplifier depicted in Fig. 20a. These 3CR reactions resulted in the detection of 100 pM in 35 minutes (Fig. 20b).

[0138] Oligonucleotide list:

Table 15

[0139] Reaction conditions:

Table 16

[0140] Figure 8 shows an approach based on the spatial separation of the amplifier and A-RNP. The gRNA of A-RNP contains a terminal biotin moiety, or a collateral region with any modification that enables it to bind to a solid substrate. The amplifier is also bound to another solid substrate by a similar means. This reaction is carried out with both solid substrates, i.e., the amplifier and A-RNP, spatially separated. The collateral activity of the RNP liberates both the target region of the amplifier and A-RNP, enabling the activation of the latter and triggering the 3CR reaction. Advantageous possibilities from the perspective of specificity are to use molecules with a collateral-region-dependent binding to two or more solid substrates, and / or to modify the amplifier and include enzymatic activity in the reaction as described above, thereby combining this spatial separation approach with target exposure or synthesis.

[0141] Figure 9 shows an approach using a circularized NTS, an annealed TS that covers the entire circle, and a flap that constitutes a collateral region at the 3'-end. To strongly prevent direct recognition by A-RNP, the target region does not contain a PAM motif and the TS has mutations. The advantage of this embodiment is that once polymerization is triggered by flap cleavage, if a DNA polymerase with strand displacement ability (such as phi29pol) is used, DNA synthesis proceeds by rolling-circle amplification (RCA), and multiple in-tandem copies of the ssDNA-form TS without mutations are released, and as a result, they become fully recognized by A-RNP. In this system, as a result of the activation of each amplifier, multiple targets are generated (one after each "lap" of the RCA synthesis), and signal amplification is truly enhanced.

[0142] Figure 10 shows an approach involving the reconstitution of a transcription unit for an RNA polymerase (e.g., T7 RNA polymerase, etc.), the transcription of which generates a target RNA molecule that activates a Cas13-based A-RNP. This has two advantages. First, as in the above RCA-based example, each activated amplifier results in the generation of multiple targets of the amplifier. Second, when applied to RNA detection, there is no DNAse present during the reaction, and thus there is no need to protect the NTS from degradation.

[0143] Figure 21 shows an approach for background reduction, in which case the amplifier requires two independent nuclease events that combine a subsequent target synthesis and exposure system.

[0144] 3CR can be adapted for qualitative diagnosis using a lateral flow device. To do so, by simply adding biotin and FAM moieties to the ends of the NTS, or biotin and FAM moieties to one end of the TS and the other end of the NTS (Figure 22a), the amplifier itself is used as a reporter, and thus only non-cleaved molecules are retained on the control strip (Figure 22b). Such an amplifier enables the detection of Lba Cas12a DNAse activity by a lateral flow strip (Figure 22c), eliminating the need to use specific reporter molecules in this setting.

[0145] References Zeng Hongwei et al. Rapid RNA detection though intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification. Analytica Chimica Acta, Elsevier, Amsterdam, NL, vol. 1217, 31 May 2022. DOI: 10.1016 / J.ACA.2022.340009. Sha Yong et al. Cascade CRISPR / cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity. Chem. Commun., 2021, vol. 57, 247-250. DOI: 10.1039 / D0CC06412B.

Brief Description of the Drawings

[0146]

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Claims

1. A method for detecting the presence of a nucleic acid sequence in a sample, comprising the following steps: (a) Adding to the sample at least one Cas ribonucleoprotein (Cas RNP) having collateral activity, at least one amplification agent, at least one reporter substrate, and at least one exonuclease or polymerase of dNTPs and / or NTPs, (b) Incubate the mixture from step (a) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 20°C to 37°C, for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours, and (c) Reading the signal of the reporter substrate, Here, at least one Cas RNP (Target-Activatable RNP; T-RNP) is designed to recognize a specific sequence in the nucleic acid of interest; The presence of the aforementioned target nucleic acid activates the collateral nucleolytic activity of T-RNP; The aforementioned amplifying agent is: i) a non-target chain (NTS) containing a sequence that is inversely complementary to the target of the second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) A nucleic acid comprising a target strand (TS) which includes a sequence that is at least partially inversely complementary to the NTS; At least one chain of the amplification agent has its 5', 3', or both ends modified by chemical modification or by cyclization; The collateral activity of activated T-RNP and A-RNP on the amplification agent results in the generation of TS that are recognizable by and accessible to the A-RNP; and The reporter substrate comprises a type of nucleic acid that is a target for the collateral activity of the A-RNP, and optionally its ends are protected from degradation or polymerization; and Preferably, amplification of the target nucleic acid sequence is not performed.

2. The method according to claim 1, wherein the amplification agent comprises a collateral region in the TS and / or NTS having at least one extension portion, preferably the collateral region comprising ssDNA and / or ssRNA; and optionally the collateral region comprising at least one strand in the TS or NTS protected from the collateral activity of the T-RNP or the A-RNP.

3. The method according to claim 2, wherein at least one exonuclease, for example, a polymerase having exonuclease activity, is added in step (a), and the amplification agent includes a complete target region in the TS but does not include a PAM motif, and the collateral region is located on the NTS at either 3' or 5' of the target region, which is consistent with the polarity of the exonuclease used.

4. The method according to claim 2, wherein at least one polymerase and dNTP or NTP are added in step (a), and the TS does not completely contain the target region or contains at least one mutated or deleted nucleotide; and the collateral region is located 5' of these modifications on the TS.

5. The method according to claim 1, wherein the T-RNP and the A-RNP have the same guide RNA and / or the same Cas protein.

6. The method according to any one of claims 1 to 5, wherein the target nucleic acid is RNA.

7. The method according to claim 1, wherein the amplifying agent is prepared by mixing the TS and the NTS in a ratio of 1:1 to 1:5, preferably 1:1 to 1:3, more preferably 1:1 to 1:

2.

8. The method according to any one of claims 1 or 7, wherein the amplification agent is a dsDNA lacking a PAM motif containing a target sequence of Cas12 A-RNP, and the NTS has a 3' extension of ssDNA and / or RNA that is terminally protected from degradation by exonuclease; and an exonuclease is added in step (a), and optionally a phosphatase is also added in step (a).

9. The method according to claim 1, wherein the reporter substrate is a nucleic acid molecule having a fluorescent marker at one end and a quencher at the other end.

10. The method according to claim 1, wherein step (a) is carried out in the following sub-steps: (a1) Adding at least one T-RNP having collateral activity and at least one amplification agent to the sample, (b1) Mix and incubate the mixture from step (a1) for 1 minute to 2 hours, preferably 10 minutes to 1 hour, more preferably 20 minutes to 40 minutes, at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C. (a2) Optionally, further adding at least one of the amplifying agents and / or components thereof (TS and NTS), preferably adding an additional NTS of the amplifying agent. (b2) Optionally, mix and incubate the mixture from step (a2) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C, for 1 minute to 1 hour, preferably 10 minutes to 1 hour, more preferably 15 minutes to 30 minutes. (a3) Adding at least one A-RNP, at least one reporter substrate, and at least one exonuclease or polymerase combined with dNTP and / or NTP, (b3) Incubate the mixture from step (a3) ​​at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 25°C to 37°C, for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours.

11. The method according to any one of claims 1, 7, 9, or 10, wherein the T-RNP comprises Lwa Cas13a protein and a guide RNA represented by SEQ ID NO: 6, the A-RNP comprises Lba Cas12a protein and a guide RNA represented by SEQ ID NO: 3, and the amplification agent comprises TS and NTS represented by SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

12. A kit for detecting the presence or absence of a target nucleic acid in a sample, or for quantifying a target nucleic acid in a sample, comprising at least one collaterally active Cas RNP, at least one amplification agent, at least one reporter substrate, and at least one exonuclease or dNTP and / or NTP combined polymerase, Here, at least one Cas RNP (target-activatable RNP; T-RNP) is designed to recognize a specific sequence (primary target) in the nucleic acid of interest; The aforementioned amplifying agent is: i) a non-target chain (NTS) containing a sequence that is inversely complementary to the target of the second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) A nucleic acid comprising a target strand (TS) which includes a sequence that is at least partially inversely complementary to the NTS; The 5', 3', or both ends of at least one chain of the amplification agent are modified by chemical modification or by cyclization; and The reporter substrate comprises a type of nucleic acid that is a target for the collateral activity of the A-RNP, and optionally its ends are protected from degradation or polymerization; and Preferably, the kit does not include means for amplifying the target nucleic acid sequence in the sample.

13. The kit according to claim 12, wherein the kit comprises a lateral-flow device including a solid support, and the reporter substrate is a nucleic acid molecule having two different marker molecules, preferably biotin and fluorescein, at each end, and the kit optionally includes means for collecting a sample and / or means for extracting nucleic acids from a sample.

14. Use of the kit according to claim 12 or 13, For use in vitro for the in vitro detection of the presence or absence of a target nucleic acid in a sample, or for the in vitro quantification of a target nucleic acid in a sample.

15. A method for detecting the presence of nucleic acid sequences in a sample, comprising the following steps: (a) Adding to the sample at least one Cas ribonucleoprotein (Cas RNP) having collateral activity, at least one amplification agent, at least one reporter substrate, and optionally at least one exonuclease or polymerase of dNTPs and / or NTPs, (b) Incubate the mixture from step (a) at a temperature of 15°C to 45°C, preferably 20°C to 40°C, more preferably 20°C to 37°C, for 1 minute to 8 hours, preferably 15 minutes to 2 hours, more preferably 30 minutes to 1.5 hours, and (c) Reading the signal of the reporter substrate, Here, at least one Cas RNP (Target-Activatable RNP; T-RNP) is designed to recognize a specific sequence in the nucleic acid of interest; The presence of the aforementioned target nucleic acid activates the collateral nucleolytic activity of T-RNP; The aforementioned amplifying agent is: i) a non-target chain (NTS) containing a sequence that is inversely complementary to the target of the second Cas RNP (amplifier-activatable RNP; A-RNP); and ii) A nucleic acid comprising a target strand (TS) which includes a sequence that is at least partially inversely complementary to the NTS; At least one chain of the amplification agent has its 5', 3', or both ends modified by chemical modification or by cyclization; The collateral activity of activated T-RNP and A-RNP on the amplification agent results in the generation of TS that is recognizable by and accessible to the A-RNP; The reporter substrate comprises a type of nucleic acid that is a target for the collateral activity of the A-RNP, and optionally its ends are protected from degradation or polymerization; and The amplification agent is attached to a solid substrate via one or more collateral regions containing at least one extension portion in the TS and / or NTS, the gRNA of the A-RNP also contains at least one collateral region and is attached to a different solid substrate via the collateral region; and the two solid substrates are spatially separated.

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