Crispr cascade methods for detecting target nucleic acids and kits and devices for practicing the same

Abstract

Provided herein are methods for determining whether a target nucleic acid is present in a sample. In some cases, the methods comprise combining: i) the sample, ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid, iii) a first bait nucleic acid, iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and v) a reactant for the second CRISPR associated enzyme; and assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample. Also provided are analyzers and kits that are designed for performing the methods disclosed herein.

Description

[0001] Attorney Docket No.: GRIP-014WO

[0002] CRISPR CASCADE METHODS FOR DETECTING TARGET NUCLEIC ACIDS AND KITS AND DEVICES FOR PRACTICING THE SAME

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 63 / 755,631, filed February 7, 2025, the disclosure of which application is herein incorporated by reference.

[0005] INTRODUCTION

[0006] Clustered regularly interspaced short palindromic repeats (CRISPR) associated enzymes, such as CRISPR associated nucleases are increasingly used for target specific genetic modification. Interaction between a CRISPR associated nuclease and its target involves sequence specific interaction of CRISPR RNA (crRNA) and its target, which also activates the CRISPR associated nucleases.

[0007] CRISPR associated proteases (Craspase) exhibit dual function of endonucleases and proteases. Activation of Craspase also requires interaction between a Craspase and its target nucleic acid sequence via crRNA that specifically binds to the target nucleic acid.

[0008] Sequence specific interaction of crRNA of a CRISPR associated enzyme and its target nucleic acid can also be used to determine whether the target nucleic acid is present in a sample. For example, certain methods of detecting a target nucleic acid using CRISPR associated nucleases are described in PCT Publication W02023201203.

[0009] Conventional methods of target nucleic acid detection using CRISPR associated nucleases either use (i) nucleic acid amplification, which is time-consuming and often requires specialized equipment, or (ii) a fluorescent or colorimetric indicator that requires for detection expensive / specialized equipment such as a fluorometer or colorimeter, e.g. Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). Therefore, conventional technologies preclude use of similar detection methods in an at-home settings.

[0010] SUMMARY

[0011] Improvements are desired in CRISPR associated enzyme based methods for determining whether a target nucleic acid is present in a sample.

[0012] The inventors of the instant disclosure realized that the sequence specific interaction of crRNA of a CRISPR associated enzyme and its target nucleic acid can be used to determine whether the target nucleic acid is present in a sample. As described herein, certainAttorney Docket No.: GRIP-014WO

[0013] embodiments of the disclosure describe methods of detecting whether a target nucleic acid is present or absent in a sample. The target nucleic acid can be any nucleic acid of interest, for example, a nucleic acid from microorganisms, such as bacteria, archaea, alga (e.g., marine alga), viruses, fungi (e.g., yeast and mold), and protozoa.

[0014] The methods disclosed herein utilize a CRISPR cascade in which a first CRISPR associated nuclease comprising a crRNA that specifically hybridizes with the target nucleic acid is initially contacted with the sample. If the target nucleic acid is present in a sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity (also referenced as trans-cleavage activity) towards a first bait nucleic acid, for example, a single-stranded DNA (ssDNA), single-stranded RNA (ssRNA). Certain aspects of such collateral-cleavage activity are described in East-Seletsky et al., “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection,” Nature, volume 538, pages 270-273, 2016, which is incorporated by reference in its entirety.

[0015] In certain aspects of the disclosure, the collateral-cleavage activity of the first CRISPR associated nuclease is assayed in two CRISPR enzyme mediated reactions, namely, a first reaction and a second reaction, where the first and second CRISPR enzyme mediated reactions may be referred to as a CRISPR cascade. The first reaction and the second reaction can be conducted in the same reaction mixture or in separate reaction mixtures.

[0016] When the first reaction and the second reaction are conducted in the same reaction mixture, the following reaction components are combined in one reaction mixture: i) a sample suspected of containing a target nucleic acid, ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid, iii) a first bait nucleic acid, iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and v) a reactant for the second CRISPR associated enzyme.

[0017] If the target nucleic acid is present in the sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards the first bait nucleic acid, which is cleaved to produce a cleaved first bait nucleic acid. If the cleaved first bait nucleic acid is produced, the second CRISPR associated enzyme having a crRNA that specifically hybridizes with the cleaved first bait nucleic acid is activated. Activated second CRISPR associated enzyme then acts on a reactant for the second CRISPR associated enzyme. The reaction mixture is assayed for a product resulting from the activity of the second CRISPR associated enzyme on the reactant.Attorney Docket No.: GRIP-014WO

[0018] The presence in the reaction mixture of a product resulting from the activity of the second CRISPR associated enzyme on the reactant indicates that the cleaved first bait nucleic acid is present in the reaction mixture. This in turn indicates that the target nucleic acid is present in the reaction mixture. Therefore, the presence in the reaction mixture of a product resulting from the activity of the second CRISPR associated enzyme on the reactant indicates that the target nucleic acid is present in the sample.

[0019] When the first reaction and the second reaction are conducted in separate reaction mixtures, a first CRISPR associated nuclease having a crRNA that specifically hybridizes with the target nucleic acid is combined with a sample in a first reaction mixture. If the target nucleic acid is present in the sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards a first bait nucleic acid conjugated to a solid support, which is cleaved to produce a free first bait nucleic acid. The free first bait nucleic acid, if produced, is separated from the first solid support to prepare a first product mixture. The first product mixture is then combined in a second reaction mixture with a second CRISPR associated enzyme having a crRNA that specifically hybridizes with the free first bait nucleic acid. If the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated enzyme is activated and acts on a reactant for the second CRISPR associated enzyme. The second reaction mixture is then assayed for a product resulting from the activity of the second CRISPR associated enzyme on the reactant.

[0020] The presence in the second reaction mixture of a product resulting from the activity of the second CRISPR associated enzyme on the reactant indicates that the free first bait nucleic acid is present in the second reaction mixture. This in turn indicates that the target nucleic acid is present in the first reaction mixture. Therefore, the presence in the second reaction mixture of a product resulting from the activity of the second CRISPR associated enzyme on the reactant indicates that the target nucleic acid is present in the sample.

[0021] Thus, the disclosure provides a cascade of reactions involving a first CRISPR associated nuclease followed by a reaction involving a second CRISPR associated enzyme, i.e. , a CRISPR cascade, to determine whether a target nucleic acid is present in a sample.

[0022] Accordingly, certain embodiments of the disclosure provide a method for determining whether a target nucleic acid is present in a sample,

[0023] (a) combining in one or more reaction mixtures:

[0024] i) the sample,Attorney Docket No.: GRIP-014WO

[0025] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0026] iii) a first bait nucleic acid,

[0027] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0028] v) a reactant for the second CRISPR associated enzyme; and

[0029] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0030] When the first reaction and the second reaction occur in separate reaction mixtures, the method comprises:

[0031] (a) preparing a first reaction mixture by combining the sample with:

[0032] (i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0033] ii) a first bait nucleic acid bound to a first solid support;

[0034] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0035] (c) preparing a second reaction mixture by combining the first product mixture with:

[0036] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid; and

[0037] ii) a reactant for the second CRISPR associated enzyme, and

[0038] (d) assaying the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.

[0039] In certain embodiments, the second CRISPR associated enzyme cleaves the reactant. For example, the second CRISPR associated enzyme can be a CRISPR associated protease (Craspase), which cleaves a substrate for the Craspase. Alternatively, the second CRISPR associated enzyme can also be a second CRISPR associated nuclease, which cleaves a nucleic acid, such as a second bait nucleic acid.

[0040] In some cases, the reactant, for example, a substrate for the Craspase or the second bait nucleic acid, is labeled and assaying the second reaction mixture for cleavage of the reactant comprises detecting a labeled reactant fragment.Attorney Docket No.: GRIP-014WO

[0041] The labeled reactant fragment can be detected by specific binding to a binding partner for the label. In some cases, such detection of the label using a binding partner for the label is performed in a lateral flow assay device.

[0042] Alternatively, in some cases, the reactant is bound to a second solid support and, when the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated enzyme cleaves the reactant to produce a free reactant fragment and a second solid support bound reactant fragment. In certain such cases, the second solid support is a sensor and assaying the reaction mixture for cleavage of the reactant comprises detecting a change in a property of the sensor. The change in the property can be a change in an electrochemical property, such as a redox signal of the sensor, or an electrical property, such as electrical resistance or Dirac voltage of the sensor. In specific embodiments, the sensor is a graphene sensor.

[0043] In some cases, the reactant is bound to a second solid support such that the CRISPR associated enzyme mediated cleavage of the second solid support bound reactant produces a free reactant fragment and a solid support bound reactant fragment. In some cases, the second solid support is a bead, such as a magnetic bead.

[0044] In certain such cases, assaying the second reaction mixture for cleavage of the reactant comprises detecting the free reactant fragment in the second reaction mixture after separating the second solid support from the second reaction mixture. In some cases, detecting the free reactant fragment comprises contacting the second reaction mixture after separating the second solid support to a sensor comprising an attached probe that specifically binds the free reactant fragment. Such binding produces a change in a property of the sensor, which can be assayed to detect the binding of the free reactant fragment to the sensor. In some cases, the sensor is a graphene sensor.

[0045] Any desired target nucleic acid can be detected according to the methods disclosed herein. For example, such nucleic acid can be from a microorganism, such as bacteria, archaea, alga (e.g., marine alga), viruses, fungi (e.g., yeast and mold), and protozoa.

[0046] In certain embodiments, the disclosure provides methods of utilizing an analyzer to determine in a first reaction and a second reaction whether a target nucleic acid is present in a sample. In some cases, the method comprises:

[0047] (a) combining in an analyzer one or more reaction mixtures comprising:

[0048] i) the sample,Attorney Docket No.: GRIP-014WO

[0049] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0050] iii) a first bait nucleic acid,

[0051] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0052] v) a reactant for the second CRISPR associated enzyme; and

[0053] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample

[0054] When the first reaction and the second reaction occur in separate reaction mixtures, the method comprises:

[0055] (a) preparing a first reaction mixture in a first reaction chamber of an analyzer, wherein the first reaction mixture comprises:

[0056] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0057] ii) a first bait nucleic acid bound to a first solid support;

[0058] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0059] (c) preparing a second reaction mixture in a second reaction chamber of the analyzer, wherein the second reaction mixture comprises:

[0060] i) the first product mixture,

[0061] ii) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0062] iii) a reactant for the second CRISPR associated enzyme; and

[0063] (d) assaying the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.

[0064] In some cases, the reactant for the second CRISPR associated enzyme is bound to a second solid support. In certain such cases, the second solid support is a sensor and assaying the cleavage of the reactant can comprise detecting a change in a property of the sensor. Certain details of such methods are disclosed elsewhere in this disclosure and such embodiments are applicable to detecting a change in a property of the sensor in the methods utilizing an analyzer.Attorney Docket No.: GRIP-014WO

[0065] In some cases, the reactant is bound to a second solid support such that a second CRISPR associated enzyme mediated cleavage of the second solid support bound reactant produces a free reactant fragment and a second solid support bound reactant fragment. In some cases, the second solid support is a bead, such as a magnetic bead. Assaying the cleavage of the reactant comprises detecting the free reactant fragment in the second reaction mixture after separating the beads from the second reaction mixture. Certain details of such methods are disclosed elsewhere in this disclosure and such embodiments are applicable to detecting a free reactant fragment in methods utilizing an analyzer.

[0066] In certain embodiments, the methods of detecting a target nucleic acid are performed using lateral flow assay devices. Thus, in certain embodiments, determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample comprises:

[0067] (a) combining in one or more reaction mixtures:

[0068] i) the sample,

[0069] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0070] iii) a first bait nucleic acid,

[0071] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0072] v) a reactant for the second CRISPR associated enzyme; and

[0073] (b) assaying in a lateral flow assay device whether a product resulting from activity of the second CRISPR associated enzyme on the substrate is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0074] In some cases, the first reaction and the second reaction occur in separate reaction mixtures. In certain such cases, the disclosure provides a method comprising:

[0075] (a) in a first reaction mixture, combining the sample with:

[0076] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0077] ii) a first bait nucleic acid bound to a first solid support;

[0078] (b) in a second reaction mixture, combining:

[0079] i) a first product mixture produced by separating the first reaction mixture from the first solid support,

[0080] ii) a Craspase comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, andAttorney Docket No.: GRIP-014WO

[0081] iii) a substrate for the Craspase, the substrate comprising a label; and

[0082] (c) assaying the second reaction mixture in a lateral flow assay device for the cleavage of the substrate for the Craspase to determine whether the target nucleic acid is present in the sample.

[0083] Accordingly, certain embodiments of the disclosure provide lateral flow assay devices for determining whether a target nucleic acid is present in a sample. In certain such cases, the lateral flow assay device comprises:

[0084] (a) a reaction region comprising:

[0085] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the first bait nucleic acid; and

[0086] ii) a substrate for the Craspase, the substrate immobilized in the reaction region;

[0087] (b) a detection region fluidically connected to the reaction region, the detection region comprising immobilized therein a binding partner that specifically binds to a substrate fragment produced from the substrate by the protease action of the Craspase.

[0088] In certain such methods, the lateral flow assay device comprises a reaction region comprising the Craspase and a labeled substrate, and wherein the method comprises introducing the first product mixture into the reaction region of the lateral flow assay device. The reaction region of the lateral flow assay device can comprises an absorbent material to which the labeled substrate is immobilized such that when the labeled substrate is cleaved by the Craspase, a free labeled substrate fragment is produced. Such free labeled substrate fragment can migrate to a detection region, which comprises immobilized therein a binding partner for the label. Binding of the binding partner for the label and the free labeled substrate fragment can produce a detectable signal. Development of such detectable signal can be further facilitated by a tag bound to the free labeled substrate fragment.

[0089] Further embodiments of the disclosure also provide kits for determining whether a target nucleic acid is present in a sample.

[0090] In some cases, a target nucleic acid detection kit comprises:

[0091] a first bait nucleic acid;

[0092] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with a cleaved first bait nucleic acid; and

[0093] a reactant for the second CRISPR associated enzyme bound to a solid support.

[0094] In some cases, a target nucleic acid detection kit comprises:Attorney Docket No.: GRIP-014WO

[0095] a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0096] a first bait nucleic acid bound to a first solid support;

[0097] a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid;

[0098] a reactant for the second CRISPR associated enzyme, the reactant bound to a second solid support, and

[0099] a sensor configured to detect cleavage of the reactant bound to the second solid support.

[0100] In some embodiments, the second solid support is the sensor configured to detect cleavage of the reactant. In some cases, the sensor is a graphene sensor. Alternatively, the second solid support can comprise beads and the cleavage of the reactant can be assayed by detecting the free reactant fragment using a sensor comprising a probe that specifically binds to the free reactant fragment. Such sensor can also be a graphene sensor.

[0101] Even further embodiments of the disclosure provide devices comprising a sensor in contact with a second reaction mixture. In certain such cases, a second reaction is produced by:

[0102] (a) preparing a first reaction mixture by combining a sample suspected of containing a target nucleic acid with:

[0103] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0104] ii) a first bait nucleic acid bound to a first solid support;

[0105] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0106] (c) preparing a second reaction mixture by combining the first product mixture with:

[0107] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0108] ii) a reactant for the second CRISPR associated enzyme, wherein the reactant is bound to a second solid support;

[0109] wherein the sensor detects the cleavage of the reactant bound to the second solid support to determine whether a free first bait nucleic acid is present in the first product mixture.

[0110] The second CRISPR associated enzyme can be a second CRISPR associated nuclease or a Craspase. Accordingly, a substrate for the second CRISPR associated enzyme can be aAttorney Docket No.: GRIP-014WO

[0111] substrate for a Craspase or a second bait nucleic acid for a second CRISPR associated nuclease.

[0112] In some cases, the reactant for the second CRISPR associated enzyme is bound to a second solid support. In certain such cases, the second solid support is a sensor and assaying the cleavage of the reactant can comprise detecting a change in a property of the sensor.

[0113] Certain details of such sensors are disclosed elsewhere in this disclosure and such embodiments are applicable to the sensors in contact with second reaction mixtures as disclosed herein.

[0114] In some cases, the reactant is bound to a second solid support such that a second CRISPR associated enzyme mediated cleavage of the second solid support bound reactant produces a free reactant fragment and a second solid support bound reactant fragment. In some cases, the second solid support is a bead, such as a magnetic bead. The free reactant fragment can be detected using a sensor. Certain details of such sensors are disclosed elsewhere in this disclosure and such embodiments are applicable to the sensors in contact with second reaction mixtures as disclosed herein.

[0115] In further embodiments, the disclosure provides a composition comprising:

[0116] i) a sample suspected of containing a target nucleic acid,

[0117] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0118] iii) a first bait nucleic acid,

[0119] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0120] v) a reactant for the second CRISPR associated enzyme.

[0121] BRIEF DESCRIPTION OF THE FIGURES FIGS. 1 A-1 E depict an exemplary method of determining whether a target nucleic acid is present in a sample. A shows cleavages of a first bait nucleic acid by a first CRISPR associated nuclease when a target nucleic acid is present in a sample. B shows a second reaction mixture comprising filtrate from the first reaction mixture and a Craspase Csx29. C shows cleavage of a substrate for Craspase if free first bait nucleic acid is present in a second reaction mixture. D shows colorimetric method of detecting horseradish peroxidase (HRP) labeled substrate fragment using hydrogen peroxide and 3, 3', 5, 5'-tetramethylbenzidine (TMB). E showsAttorney Docket No.: GRIP-014WO

[0122] electrochemical sensor and voltammetry based detection of HRP labeled Csx30 using TMB and hydrogen peroxide.

[0123] FIGS. 2A-2B depict an exemplary method of determining whether a target nucleic acid is present in a sample. A shows that a filtrate from the first reaction mixture as shown in FIG. 1 A is contacted with an electrochemical sensor comprising an HRB labeled substrate for the Craspase. B shows the effect of the release of HRP labeled substrate fragment on the redox signal of the sensor.

[0124] FIGS. 3A-3C depict an exemplary method of determining whether a target nucleic acid is present in a sample. A exemplifies a first reaction comprising combining a first CRISPR associated nuclease with a sample suspected of containing a target nucleic acid. B exemplifies contacting a first product mixture produced in A with a sensor configured to detect the presence in the second product mixture of a free first bait nucleic acid. G shows results obtained by the sensor depicted in B depending on whether a sample contains a target nucleic acid.

[0125] FIGS. 4A-4B depict an exemplary method of determining in a single reaction chamber whether a target nucleic acid is present in a sample. A shows conducting a first CRISPR associated nuclease reaction with a first bait nucleic acid that is cleaved by a first CRISPR associated nuclease if a target nucleic acid is present in a sample, as shown in the middle panel of A. Cleavage of the first bait nucleic acid produces free first bait nucleic acid, which acts as a second bait nucleic acid for a second CRISPR associated enzyme, exemplified as a Craspase shown in the right panel of A. B shows contacting filtered Csx30-HRP onto an electrochemical sensor and readout of a change in redox signal of the electrochemical sensor using a square wave voltammetry as shown in FIG. 4B.

[0126] FIG. 5 depicts an exemplary lateral flow assay device of the disclosure.

[0127] DETAILED DESCRIPTION

[0128] As summarized above, methods of detecting whether a target nucleic acid is present or absent in a sample are provided.

[0129] In certain embodiments, a method for determining whether a target nucleic acid is present in a sample comprises:

[0130] (a) combining in one or more reaction mixtures:

[0131] i) the sample,Attorney Docket No.: GRIP-014WO

[0132] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0133] iii) a first bait nucleic acid,

[0134] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0135] v) a reactant for the second CRISPR associated enzyme; and

[0136] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0137] In some cases, the methods disclosed herein utilize a specific analyzer. In certain such cases, a method of determining whether a target nucleic acid is present in a sample comprises:

[0138] (a) combining in an analyzer one or more reaction mixtures comprising:

[0139] i) the sample,

[0140] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0141] iii) a first bait nucleic acid,

[0142] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0143] v) a reactant for the second CRISPR associated enzyme; and

[0144] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0145] In some cases, the methods disclosed herein are performed in a lateral flow assay devices. In certain such embodiments, a method of determining whether a target nucleic acid is present in a sample comprises:

[0146] (a) combining in one or more reaction mixtures:

[0147] i) the sample,

[0148] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0149] iii) a first bait nucleic acid,Attorney Docket No.: GRIP-014WO

[0150] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0151] v) a reactant for the second CRISPR associated enzyme; and

[0152] (b) assaying in a lateral flow assay device whether a product resulting from activity of the second CRISPR associated enzyme on the substrate is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0153] Further embodiments of the disclosure provide a kit that is designed to perform the methods disclosed herein. Accordingly, certain embodiments of the disclosure provide a target nucleic acid detection kit, comprising:

[0154] a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid;

[0155] a first bait nucleic acid;

[0156] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with a cleaved first bait nucleic acid; and

[0157] a reactant for the second CRISPR associated enzyme bound to a solid support.

[0158] Certain embodiments of the disclosure also provide a target nucleic acid detection kit, comprising:

[0159] a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0160] a first bait nucleic acid bound to a first solid support;

[0161] a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid;

[0162] a reactant for the second CRISPR associated enzyme, the reactant bound to a second solid support, and

[0163] a sensor configured to detect cleavage of the reactant bound to the second solid support.

[0164] Certain embodiments of the disclosure provide devices in which the methods of the disclosure are performed. Thus, certain embodiments of the disclosure provide:

[0165] a device comprising a sensor in contact with a second reaction mixture,

[0166] wherein the second reaction mixture is produced by:

[0167] (a) preparing a first reaction mixture by combining a sample suspected of containing a target nucleic acid with:Attorney Docket No.: GRIP-014WO

[0168] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0169] ii) a first bait nucleic acid bound to a first solid support;

[0170] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0171] (c) preparing a second reaction mixture by combining the first product mixture with:

[0172] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0173] ii) a reactant for the second CRISPR associated enzyme, wherein the reactant is bound to a second solid support;

[0174] wherein the sensor detects the cleavage of the reactant bound to the second solid support to determine whether a free first bait nucleic acid is present in the first product mixture.

[0175] Further embodiments of the disclosure provide a composition comprising:

[0176] i) a sample suspected of containing a target nucleic acid,

[0177] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0178] iii) a first bait nucleic acid,

[0179] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0180] v) a reactant for the second CRISPR associated enzyme.

[0181] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, and various aspects of the disclosure as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0182] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.Attorney Docket No.: GRIP-014WO

[0183] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0184] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0185] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0186] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0187] The term “nucleic acid” used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, term “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multistranded DNA; single-stranded RNA; doublestranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0188] By “hybridizable” or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleicAttorney Docket No.: GRIP-014WO

[0189] acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine / adenosine) (A) pairing with thymine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine) (G) pairing with cytosine / cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (dsRNA duplex) of a crRNA molecule; of a target nucleic acid base pairing with a guide RNA) is considered complementary to both a U and to C. For example, when a G / U base -pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0190] Hybridization conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W , Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. These conditions can be adjusted to determine the permitted variance in the detected target nucleic acid. For example, if minimal sequence variation from the target nucleic acid is desired, high stringency conditions can be used in the reaction mixture, whereas if moderate or higher sequence variation from the target nucleic acid is desired, moderate to low stringency conditions can be used in the reaction mixture. A person of ordinary skill in the art can determine appropriate conditions according to the other reagents used in the reaction mixture and desired stringency.

[0191] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or fewer, 30 or fewer, 25 orAttorney Docket No.: GRIP-014WO

[0192] fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides) the position of mismatches can become important (see Sambrook et aL, supra, 11 .7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). The temperature and solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0193] The sequence of a nucleic acid need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a nucleic acid may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). A nucleic acid can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Exemplary methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et aL, J. Mol. Biol. , 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0194] A nucleic acid has a certain percent "sequence identity" to another nucleic acid, meaning that, when aligned, that percentage of bases are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including: world-wide-website: ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / ,Attorney Docket No.: GRIP-014WO

[0195] mafft.cbrc.jp / alignment / software / , http: / / www.sbg.bio.ic.ac.uk / ~phyre2 / . See, e.g., Altschul etal. (1990), J. Mol. Biol. 215:403-10.

[0196] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a protein, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10“6M, less than 10“7M, less than 10“8M, less than 10-9M, less than 1O-10M, less than 10“11M, less than 10“12M, less than 10“13M, less than 10“14M, or less than 10-15M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0197] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0198] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0199] In further describing various aspects of the disclosure, the methods, the devices, analyzers, and kits are described first in greater detail, followed by the description of examples.Attorney Docket No.: GRIP-014WO

[0200] METHODS OF DETECTING A TARGET NUCLEIC ACID

[0201] Certain aspects of the present disclosure provide methods for determining whether a target nucleic acid is present in a sample. In some embodiments, the disclosure provides a method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:

[0202] (a) combining in one or more reaction mixtures:

[0203] i) the sample,

[0204] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0205] iii) a first bait nucleic acid,

[0206] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0207] v) a reactant for the second CRISPR associated enzyme; and

[0208] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0209] In some cases, the first reaction and the second reaction occur in separate reaction mixtures. In certain such embodiments, the disclosure provides a method for determining whether a target nucleic acid is present in a sample, the method comprising:

[0210] (a) preparing a first reaction mixture by combining the sample with:

[0211] (i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0212] ii) a first bait nucleic acid bound to a first solid support;

[0213] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0214] (c) preparing a second reaction mixture by combining the first product mixture with:

[0215] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid; and

[0216] ii) a reactant for the second CRISPR associated enzyme, and

[0217] (d) assaying the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.Attorney Docket No.: GRIP-014WO

[0218] A first step of the methods disclosed herein is carried out in a first reaction mixture. A first reaction mixture comprises, among other ingredients, a sample to be tested for the presence of a target nucleic acid and a first CRISPR associated nuclease comprising a crRNA that specifically hybridizes with the target nucleic acid. If the target nucleic acid is present in the sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards a first bait nucleic acid bound to a solid support, which is cleaved to produce a free first bait nucleic acid. A first bait nucleic acid can be a single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), a partially single stranded and double stranded DNA, or a partially single stranded DNA / RNA hybrid.

[0219] Certain aspects of such collateral-cleavage activity are described in East-Seletsky etal., “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection,” Nature, volume 538, pages 270-273, 2016, which is incorporated by reference in its entirety. Thus, the presence of the collateral-cleavage activity of a first CRISPR associated nuclease indicates the presence in the sample of the target nucleic acid. The presence of the collateral-cleavage activity of the first CRISPR associated nuclease is detected in a second step using a second CRISPR associated enzyme. The free first bait nucleic acid, if produced, is separated from the first solid support to prepare a first product mixture. The first product mixture is then tested in a second step in a second reaction mixture.

[0220] A second reaction mixture comprises, among other ingredients, a second CRISPR associated enzyme and the first product mixture. The second CRISPR associated enzyme comprises a crRNA that specifically hybridizes with the free first bait nucleic acid. If the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated enzyme acts on a reactant for the second CRISPR associated enzyme. The second reaction mixture is then assayed for a product resulting from the activity of the second CRISPR associated enzyme on the reactant.

[0221] In certain cases, the second CRISPR associated enzyme is a second CRISPR associated nuclease. In certain such cases, a reactant upon which the second CRISPR associated nuclease exerts its enzyme activity is a second bait nucleic acid. If the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards the second bait nucleic acid.

[0222] Accordingly, the second reaction mixture is assayed for the cleavage of the second bait nucleic acid. If the second reaction mixture contains a free second bait nucleic acid, it indicates the presence in the second reaction mixture of the free first bait nucleic acid, which in turn indicates the presence in the sample of the target nucleic acid. On the other hand, if the second reactionAttorney Docket No.: GRIP-014WO

[0223] mixture does not contain the free second bait nucleic acid, it indicates the absence in the second reaction mixture of the free first bait nucleic acid, which in turn indicates the absence in the sample of the target nucleic acid.

[0224] In some cases, the second CRISPR associated enzyme is a Craspase. The Craspase system was discovered from the bacterium Desulfonema ishimotonii, and contains two components. For example, Strecker et al. ((2022), Science 378, 874-881 (2022), characterized the system from Desulfonema ishimotonii. In this system, Csx30 produces 16 kD and 48 kD fragments (not considering any protein tags, labels, etc.). The disclosure of Strecker et al. is incorporated herein by reference in its entirety.

[0225] On the other hand, Beljouw et al. ((2024), ACS Chem. Biol., 19, 1051-1055 characterized the Craspase system from Candidatus “Jettenia caeni’ (Jc-Craspase) and Candidatus “Scalindua brodae” (Sb-Craspase). Jc-Csx30 produces a 16 kD and ~50 kD fragment, whereas Sb-Csx30 produces a 18 kD and ~50 kD fragment. The disclosure of Beljouw et al. is also incorporated herein by reference in its entirety.

[0226] The first component of the Craspase system is a Craspase comprising a crRNA that specifically hybridizes with the target nucleic acid. An example of such Craspase comprising a crRNA is Cas7-11-crRNA-Csx29 complex. This complex can specifically bind to a target nucleic acid via hybridization between the target nucleic acid and the crRNA. The second component is a protein substrate for the Craspase. An example of the protein substrate is Csx30.

[0227] In the Craspase system, the crRNA determines the system’s target specificity. Upon binding of the crRNA to a target nucleic acid, the Craspase, e.g., Csx29 is activated. The activated Craspase then cleaves the substrate, e.g., Csx30. In one such example, Craspase Csx29 cleaves the target Csx30 to produce an 18 kD fragment and a 50 kD fragment. Certain details of the Craspase system are described in the United States Provisional Patent Application No. 63 / 743,548 and which is incorporated by reference in its entirety.

[0228] In embodiments, a Craspase system comprises a Craspase enzyme that cleavages a substrate into at least two fragments, one ranging from 15 kD to 20 kD and the other ranging from 45 kD to 55 kD, excluding any labels or modifications of the substrate.

[0229] Accordingly, in certain embodiments of the disclosure, a Craspase, such as Csx29 is used as a surveillance complex. A reactant upon which the Craspase exerts its enzyme activity is a substrate for the Craspase. For example, upon target detection, Craspase, such as Csx29 exhibits protease activity against its substrate, Csx30. This protease activity is detected in the methods disclosed herein as indicative of the presence in the sample of the target nucleic acid.Attorney Docket No.: GRIP-014WO

[0230] Thus, in certain cases, the second CRISPR associated enzyme is a Craspase comprising a second crRNA that specifically hybridizes with the free first bait nucleic acid. A substrate for the Craspase, such as Csx30 is provided in a second reaction mixture comprising a first product mixture and a Craspase. The second reaction mixture is assayed for the cleavage of the substrate.

[0231] The First Reaction

[0232] As noted above, in a method disclosed herein, a first reaction mixture is prepared by combining a sample with: (i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and ii) a first bait nucleic acid bound to a first solid support.

[0233] As noted above, a CRISPR associated nuclease suitable for use as a first CRISPR associated nuclease in the methods disclosed herein has a collateral-cleavage activity in the presence of a target nucleic acid. Certain details of such collateral-cleavage activity and CRISPR associated nucleases that exhibit such activity are described in Varble et al. (2019), Trends Genet;35(6):446-456, which is incorporated by reference in its entirety.

[0234] A “first bait nucleic acid” as used herein refers to a nucleic acid having a sequence that does not match the sequence of the target nucleic acid. Depending upon the type of CRISPR associated nuclease used, for example, Type III, V, or VI, different types of first bait nucleic acids could be used. For example, if Type III CRISPR associated nuclease is used as a first CRISPR associated nuclease, a first bait nucleic acid could be RNA or single stranded DNA. If Type V CRISPR associated nuclease is used as a first CRISPR associated nuclease, a first bait nucleic acid could be single stranded DNA. Moreover, if Type VI CRISPR associated nuclease is used as a first CRISPR associated nuclease, a first bait nucleic acid could be RNA.

[0235] Further, in some cases, a first bait nucleic acid is a partially single stranded and double stranded DNA or a partially single stranded DNA / RNA hybrid. When a partially single stranded and double stranded DNA is used as a first bait nucleic acid, cleavage of the first bait nucleic may occur in the single stranded portion thereby producing a shorter partially single stranded double stranded DNA or completely double stranded DNA. Similarly, when a partially single stranded DNA / RNA hybrid is used as a first bait nucleic acid, cleavage of the first bait nucleic acid may occur in the single stranded portion thereby producing a partially single stranded DNA / RNA hybrid or completely double stranded DNA / RNA hybrid.

[0236] Depending on a first CRISPR associated nuclease used in a first step as well as the CRISPR associated enzyme used in a second step of the methods disclosed herein, a personAttorney Docket No.: GRIP-014WO

[0237] of ordinary skill in the art can determine appropriate first bait nucleic acid to be used under appropriate conditions and such embodiments are within the purview of the disclosure.

[0238] As shown in Figure 1 of Varble et al., Type VI CRISPR associated nuclease, for example, Cas13a, shows collateral-cleavage activity against RNA. Therefore, when a Type VI CRISPR associated nuclease, such as Cas13a can be used in the methods disclosed herein, RNA is used as a first bait nucleic acid. Certain aspects and examples of Type VI CRISPR associated nucleases are provided in O’Connell (2019), J Mol Bio, 4:431 (1 ):66-87, which is herein incorporated by reference in its entirety. Use as first CRISPR associated nucleases of Type VI CRISPR associated nucleases described in O’Connell and additional examples otherwise known in the art is within the purview of the disclosure.

[0239] Type V CRISPR associated nuclease, for example, Cas12, shows collateral-cleavage activity against single stranded DNA. Therefore, when a Type V CRISPR associated nuclease, such as Cas12 or Cas14 is used in the methods disclosed herein, single stranded DNA can be used as a first bait nucleic acid. Certain aspects and examples of Type V CRISPR associated nucleases are provided in Urbaitis et al. (2022), EMBO Rep, 23(12):e55481 , which is herein incorporated by reference in its entirety. Use as a first CRISPR associated nuclease of Type V CRISPR associated nucleases described in Urbaitis etal. and additional examples otherwise known in the art is within the purview of the disclosure.

[0240] Type III CRISPR associated nucleases, for example, Casio, shows collateral-cleavage activity against RNA or single stranded DNA. Therefore, when Type III CRISPR associated nuclease, such as Casio is used in the methods disclosed herein, single stranded DNA or RNA can be used as a first bait nucleic acid. Certain aspects and examples of Type III CRISPR associated nucleases are provided in McMahon et al. (2020), Nat Comm, 11 , Article No. 500, which is herein incorporated by reference in its entirety. Use as a first CRISPR associated nuclease of Type III CRISPR associated nucleases described in McMahon etal. and additional examples otherwise known in the art is within the purview of the disclosure.

[0241] A first crRNA used in the methods disclosed herein is a small RNA molecule that guides a CRISPR associated nuclease to a specific target sequence. A complex of a crRNA and a trans-activating CRISPR RNA (tracrRNA), guide the CRISPR associated nuclease complex to a target nucleic. The first crRNA binds to a target nucleic acid and the first CRISPR associated nuclease then cleaves the first bait nucleic acid.

[0242] Therefore, for use in the methods disclosed herein, a first crRNA is designed to specifically hybridize with the target nucleic acid. Thus, depending upon the desired target nucleic acid, a first crRNA has a sequence that is complementary to the target nucleic acid orAttorney Docket No.: GRIP-014WO

[0243] matches with the target nucleic acid. For example, if the target nucleic acid is double stranded, a first crRNA is complementary to one of the two strands of the double stranded target nucleic acid and has a sequence that matches with the other strand of the double stranded target nucleic acid. Alternatively, if the target nucleic acid is single stranded, such as single stranded DNA or RNA, the first crRNA has a sequence that is complementary to the target nucleic acid.

[0244] In some cases, a first crRNA and a first tracrRNA are in one single stranded nucleic acid, which is called a single guide RNA (sgRNA). A first sgRNA contains a target nucleic acid specific first crRNA sequence fused to a first tracrRNA sequence by a linker loop.

[0245] Any suitable nucleic acid could be used as a target nucleic acid. Typically, a nucleic acid sequence that is unique to the target being detected is used as a target nucleic acid. For example, to test the presence of a bacterium in a sample, the 16S ribosomal RNA of the bacterium could be used as a target nucleic acid. Alternatively, if a target being detected has a unique genetic sequence, such as a unique gene, such gene or a unique sequence within the gene could be used as a target nucleic acid. Other specific nucleic acids that could be used as target nucleic acids are well known in the art and a person of ordinary skill in the art can readily determine an appropriate target nucleic acid to be used in a given situation. Such embodiments are within the purview of the disclosure.

[0246] In some cases, the target nucleic acid has a sequence of from 17 to 30, such as 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or base pairs. Accordingly, a crRNA can have a length of from 17 to 30, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0247] A reaction mixture comprising a first CRISPR associated nuclease and a sample that is tested for the presence of a target nucleic acid can comprise additional reagents beyond the first CRISPR associated nuclease and the sample. Such additional reagents can contain one or more of: salts like magnesium chloride and sodium chloride; chelating agents, such as ethylene diamine tetra-acetic acid; reducing agent, such as dithiothreitol; stabilizing agent, such as glycerol; and a buffer such as Tris or HEPES to maintain pH.

[0248] Further reagents may include one or more of a denaturant (e.g. urea or guanidine-HCI), surfactant / detergent (e.g. sodium dodecyl sulfate (SDS) or Triton X-100), RNase inhibitor (e.g., RNasin or polyvinylsulfonic acid (PVSA)), and enhancer that increases collateral cleavage catalytic activity (e.g. dithiothreitol (DTT) or polyvinyl alcohol (PVA)). Certain details of such additional reagents are provided in Deng etal., Sensors & Actuators: B. Chemical 373 (2022) 132767, which is herein incorporated by reference in its entirety. In some cases, an enhancers of collateral cleavage catalytic activity can be a protein, such as Csm6, auxiliary CRISPR-Attorney Docket No.: GRIP-014WO

[0249] associated enzyme. Certain such options are described by Gootenberg, et al. Science. 2018, 360(6387): 439-444, which is herein incorporated by reference in its entirety.

[0250] Reagents and conditions appropriate for CRISPR associated nuclease reactions are well-known to a person of ordinary skill in the art and selection and maintenance of appropriate reagents and conditions for different reactions are within the purview of this disclosure.

[0251] As noted above, a “first bait nucleic acid” as used herein refers to a nucleic acid having a sequence that does not match to the sequence of the target nucleic acid. A first bait nucleic acid is cleaved by a first CRISPR associated nuclease when a target nucleic acid is present in a sample. A first bait nucleic acid can have a length from 10 to 100 nucleotides, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some cases, a first bait nucleic acid has a unique and known sequence that can be detected in the methods disclosed herein.

[0252] In some cases, a first bait nucleic acid has a suitable secondary structure, e.g. a stemloop structure. When appropriate, such secondary structure can prevent non-specific RNase activity.

[0253] In some cases, a first bait nucleic acid is bound to a first solid support and cleavage of a first bait nucleic acid by a first CRISPR associated nuclease causes the first bait nucleic to be converted into “a free first bait nucleic acid.” A first bait nucleic acid can be bound to the first solid support via a covalent bond. A covalent bond can be through an appropriate reactive group, such as an amine, sulfhydryl, aldehyde, or carboxylic group. Examples of covalently binding a nucleic acid to a solid support are described in United States Patent Application Publication No. US20020137045, which is incorporated by reference in its entirety. Additional examples of covalently binding a nucleic acid to a solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0254] In some cases, a first bait nucleic acid is bound to a first solid support via a non-covalent bond, such as an ionic bond, hydrogen bridge, hydrophobic bond, or van der Waals interaction. For example, a first bait nucleic acid can be conjugated to a binding partner and such first bait nucleic acid can be bound to a first solid support comprising an agent that specifically binds to the binding partner. Such pairs of binding partner and specific binding agents include antigenantibody, other protein binding partners, and nucleic acid that specifically hybridizes to a complementary nucleic acid. Additional examples of non-covalently binding a first bait nucleic acid to a first solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0255] In certain such cases, a first solid support is a first bead, such as a first magnetic bead or a first binding partner coated beads. In some cases, the beads are agarose beads.Attorney Docket No.: GRIP-014WO

[0256] When the first bead is a magnetic bead, a free first bait nucleic acid can be separated from the first solid support by capturing the first magnetic beads using a magnet. Thus, the first reaction mixture other than the first magnetic beads can be separated to produce a first product mixture.

[0257] When the first bead is a first binding partner coated bead, a free first bait nucleic acid can be separated from the first solid support by capturing the first binding partner coated beads using an immobilized binding agent that specifically binds to the first binding partner on the beads. For example, streptavidin coated first beads could be captured using immobilized biotin and vice versa. Similarly, an antigen coated first beads could be captured using immobilized antibodies specific to the antigen and vice versa. Additional examples of binding partners and specific binding agent combinations are well-known in the art and their use is within the purview of the disclosure.

[0258] In certain embodiments, the first beads having bound first bait nucleic acid are separated from the first reaction mixture by centrifuging the first reaction mixture. The pellet produced after centrifugation would contain the first beads having bound first bait nucleic acids and the free first bait nucleic acids, if present, would remain in the supernatant, which can be separated and tested for the presence of the free first bait nucleic acids.

[0259] In further embodiments, first beads having bound first bait nucleic acid are separated from the reaction mixture by filtering the first reaction mixture. The residue produced after filtration would contain the first beads having bound first bait nucleic acids and the free first bait nucleic acids, if present, would be in the filtrate, which can be tested for the presence of free first bait nucleic acids.

[0260] In additional embodiments, a first solid support is a functionalized surface to which a first bait nucleic acid is bound. When a first bait nucleic acid is cleaved by a first CRISPR associated nuclease, it can be separated by physically separating the first reaction mixture from the functionalized surface to produce a first product mixture.

[0261] The second reaction using a Craspase

[0262] As noted above, in certain embodiments, the disclosure provides a method for determining whether a target nucleic acid is present in a sample, the method comprising a first reaction and a second reaction. In some cases, the method comprises:

[0263] preparing a second reaction mixture by combining a first product mixture with:

[0264] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid; andAttorney Docket No.: GRIP-014WO

[0265] ii) a reactant for the second CRISPR associated enzyme, and

[0266] assaying the second reaction mixture for a product resulting from activity.

[0267] In certain embodiments, a second CRISPR associated enzyme is a Craspase having a second crRNA that specifically hybridizes with the first bait nucleic acid. If a free first bait nucleic acid is present in the second reaction mixture, the Craspase exhibits protease activity towards its substrate. Thus, the presence of the Craspase protease activity towards its substrate indicates the presence in the second reaction mixture of a free first bait nucleic acid, which in turn indicates the presence in a sample of the target nucleic acid.

[0268] Certain exemplary Craspases are described elsewhere in this disclosure, for example, as described by Strecker et al. and Beljouw et al. Typically a Craspase system comprises a Craspase enzyme that cleavages a substrate into at least two fragments, one ranging from 15 kD to 20 kD and the other ranging from 45 kD to 55 kD, excluding any labels or modifications of the fragments.

[0269] Accordingly, in certain embodiments of the disclosure, a Craspase, such as Csx29 with a free first bait nucleic acid specific second crRNA is used as a second CRISPR associated enzyme. Upon binding of the second crRNA to free first bait nucleic acid, Craspase, such as Csx29 exhibits protease activity against its substrate, such as Csx30. This protease activity is detected in the methods disclosed herein as indicative of the presence in the sample of the target nucleic acid.

[0270] A substrate for a Craspase, such as “Csx30” is provided in a second reaction mixture comprising a first product mixture and a Craspase and cleavage of the substrate is assayed.

[0271] In some cases, a Craspase substrate bound to a second solid support is provided in the second reaction mixture. For example, a substrate can be bound to second beads, such as second magnetic beads, and added to the second reaction mixture. Alternatively, a substrate can be bound to an inner wall of a chamber in which the second reaction is conducted. A substrate can also be bound to a sensor and the cleavage of the substrate can be assayed using the sensor.

[0272] When a substrate is bound to a second solid support, cleavage of the substrate produces a free substrate fragment, i.e., a fragment of the substrate that is not bound to the second solid support, such as the beads or an inner wall of the reaction chamber.

[0273] After Craspase mediated free first bait nucleic acid detection and substrate cleavage, the free substrate fragment, if produced, is separated from the second solid support to produce a second product mixture. For example, the second reaction mixture can be passed through a filter or centrifuged to separate the second beads. Alternatively, second magnetic beads fromAttorney Docket No.: GRIP-014WO

[0274] the second reaction mixture can be captured on a magnet. Even further, the second reaction mixture in a reaction chamber having a substrate bound on an inner wall can be transferred from the chamber. Such second product mixture can be assayed for the free substrate fragment.

[0275] Alternatively, a substrate can be bound to a sensor, where cleavage of the substrate changes a property of the sensor. Thus, changes in a property of the sensor indicates cleavage of the substrate and, hence, such changes in the property can be used to assay the cleavage of the substrate.

[0276] In further embodiments, a substrate is immobilized in a reaction region of a lateral flow assay device. The reaction region also comprises a Craspase comprising a crRNA and the reaction region is fluidically connected to a detection region such that a free substrate fragment produced in the reaction region migrates to the detection region. In the detection region, a binding agent that specifically binds to the substrate or to a label bound to the substrate and produces a detectable signal. In some cases, the free labeled substrate fragment also contains a label that produces a detectable signal.

[0277] Depending on the point of attachment of the label to the substrate, Craspase mediated cleavage of a labeled substrate bound to a second solid support produces either 1) a labeled free substrate fragment and an unlabeled bound substrate fragment or 2) a labeled bound substrate fragment and an unlabeled free substrate fragment. Detection of the free or bound labeled substrate fragment can be done by detecting a signal from the detectable label.

[0278] With such general description of the second reaction using a Craspase, certain specific embodiments will be described below in further detail.

[0279] In some cases, the Craspase is Csx29, particularly, a complex of Csx29, Cas7-11 , and the crRNA. In some cases, the substrate for the Craspase is Csx30. However, any suitable substrate, for example, a peptide fragment of Csx30 towards which a Craspase, particularly, Csx29 exhibits protease activity can be used. A person of ordinary skill in the art can determine such peptides and use of such peptides is within the purview of the disclosure. When Csx30 is used as a substrate and the target nucleic acid is present in the sample, the Craspase Csx29 cleaves Csx30 into a fragment between 15 kD and 20 kD, particularly, 18 kD and a fragment between 45 kD and 55 kD, particularly, 50 kD.

[0280] In some cases, the substrate comprises a label, for example, a label that produces a detectable signal. Such label can be a fluorophore, chromophore, an enzyme, and the like. When a free first bait nucleic acid is present in the second reaction mixture, the CraspaseAttorney Docket No.: GRIP-014WO

[0281] cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment.

[0282] Accordingly, in some cases, assaying the second reaction mixture for cleavage of the substrate comprises detecting the labeled substrate fragment. Alternatively, in some cases, assaying the second reaction mixture for cleavage of the substrate comprises detecting the unlabeled substrate fragment.

[0283] When the method comprises assaying the free labeled substrate fragment, in some cases, such assaying can comprise separating the labeled substrate fragment from the rest of the second reaction mixture and detecting a signal produced from the free labeled substrate fragment. Such signal can be a fluorescent signal, a chromogenic signal, a chemiluminescence signal, or the like.

[0284] Alternatively, such assaying can also comprise contacting the second reaction mixture with a binding partner for the label, wherein the binding partner for the label is immobilized on a matrix. For example, if the label is a protein, the binding partner for the protein can be an antibody or an antibody body fragment of an antibody that specifically binds to the protein. For example, the label can be protein A and the binding partner for protein A can be immunoglobin G (IgG) or a binding fragment of IgG. A binding partner can also be an aptamer or a nucleic acid that specifically binds the label. Thus, binding of the label with the binding partner immobilized on a matrix precipitates or accumulates the label in the area of the matrix where the binding partner is immobilized. Such area can be visualized using a signal produced by the label or a tag bound to the label.

[0285] Detecting labeled substrate fragment in a lateral flow assay device

[0286] In certain cases, binding of a label on a free substrate fragment produced from a bound substrate is used to detect the free substrate fragment. In certain such cases, the matrix to which a binding partner of the label is immobilized is an adsorbent material in a detection region of a lateral flow assay device. Thus, certain embodiments of the disclosure provide a method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0287] (a) in a first reaction mixture, combining the sample with:

[0288] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0289] ii) a first bait nucleic acid bound to a first solid support;

[0290] (b) in a second reaction mixture, combining:

[0291] i) the first reaction mixture separated from the first solid support,Attorney Docket No.: GRIP-014WO

[0292] ii) a Craspase comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0293] iii) a substrate for the Craspase, the substrate comprising a label; and

[0294] (c) assaying the second reaction mixture in a lateral flow assay device for the cleavage of the substrate for the Craspase to determine whether the target nucleic acid is present in the sample.

[0295] Accordingly, certain embodiments of the disclosure provide lateral flow assay devices for determining whether a target nucleic acid is present in a sample. In certain such cases, the lateral flow assay device comprises:

[0296] (a) a reaction region comprising:

[0297] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the first bait nucleic acid; and

[0298] ii) a substrate for the Craspase, the substrate immobilized in the reaction region;

[0299] (b) a detection region fluidically connected to the reaction region, the detection region comprising immobilized therein a binding partner that specifically binds to a substrate fragment produced from the substrate by the protease action of the Craspase.

[0300] In the lateral flow assay devices disclosed herein, the reaction region is fluidically connected to the detection region. As discussed elsewhere in this disclosure, such fluidic connection typically comprises adsorbent material that allows flow of liquid from the reaction region to the detection region.

[0301] However, a fluidic connection can also comprise connection that causes a fluid to migrate from the reaction region to the detection region in a laminar flow or capillary flow. For example, a laminar flow involves flow of fluid / liquid between two typically flat or substantially flat surfaces that are close to each other, for example, at a distance of between 100 microns and 2 mm. In certain such cases, the reaction region and the detection region are connected with two or more surfaces that are substantially parallel to each other and close to each other such that a reaction mixture migrates via laminar flow from the reaction region to the detection region.

[0302] A capillary flow involves flow of fluid / liquid through tubes of small cross-section area, for example, between 50 square microns and 10,000 square microns. In certain such cases, the reaction region and the detection region are connected with two or more channels such that a reaction mixture migrates via capillary flow from the reaction region to the detection region. In some cases, the channels are microfluidic channels.Attorney Docket No.: GRIP-014WO

[0303] A detection region in the devices disclosed herein comprises immobilized therein a binding partner that specifically binds to the free substrate fragment produced from the substrate by the protease action of the Craspase. Production of the free substrate fragment is indicated by binding of the binding partner and the substrate or the labeled substrate fragment, which produces a detectable signal. The binding partner for the substrate can specifically bind the label of the labeled substrate fragment. Alternatively, the binding partner for the substrate can specifically bind the substrate portion of the labeled substrate fragment.

[0304] In some cases, the binding partner is a protein, such as a protein that specifically binds to a substrate fragment or its label. A protein can also be an antibody or a binding fragment of an antibody that specifically binds to a substrate fragment or its label. A binding partner can also be a nucleic acid, such as an aptamer.

[0305] The binding partner for the labeled substrate fragment can be an antibody or a binding fragment thereof that specifically binds the substrate portion of the labeled substrate fragment or the label. For example, the label can be protein A and the antibody can be IgG and vice versa. Alternatively, the label can be streptavidin and the binding partner can be biotin and vice versa. Additional combinations of label and its binding partners are known in the art and use of such combinations in the lateral flow assay devices is within the purview of the disclosure.

[0306] In some cases, accumulation of the label of the labeled substrate fragment in the detection region because of the binding of the free labeled substrate fragment to its binding partner produces a detectable signal. Alternatively, a detectable signal is produced by accumulation of a tag conjugated to the labeled substrate fragment. Such tag can comprise gold-nanoparticles, a fluorophore, or a chromophore.

[0307] Certain details of the lateral flow assay devices comprising sample ports (optional), reaction regions, and detection region are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above. These details are also applicable to the latera flow assay devices described herein. Moreover, certain details of the methods of detecting target nucleic acids provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” are also applicable to the lateral flow assay devices described herein. Certain such details include the Craspase, crRNA, target nucleic acids to be detected, substrates, binding partners of the substrates, types of labels, tags that produce detectable signal, etc. Certain exemplary details are summarized below under “Clauses” and such embodiments are within the purview of the disclosure. An exemplary lateral flow assay device is depicted in FIG. 5.

[0308] For the embodiment shown in FIG. 5, a sample suspected of containing a target nucleicAttorney Docket No.: GRIP-014WO

[0309] acid is analyzed in a first reaction mixture by combining the sample with a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid and a first bait nucleic acid bound to a first solid support. The first reaction mixture separated from the solid support is introduced into the reaction region of the lateral flow assay device as shown in FIG 5, left panel. The reaction region of the lateral flow assay device comprises: i)_a Craspase (e.g., Csx29) comprising a crRNA that specifically hybridizes with the first bait nucleic acid and ii) a substrate for the Craspase (e.g., Csx30), the substrate immobilized in the reaction region. The substrate, e.g. Csx30, can also comprise an HRP label.

[0310] If the sample contains the target nucleic acid, the first CRISPR associated nuclease comprising a first crRNA would specifically hybridize with the target nucleic acid. The first CRISPR associated nuclease would then cleave the first bait nucleic acid bound to the first solid support and the cleaved first bait nucleic acid would be presented in the first reaction mixture separated from the first solid support.

[0311] Accordingly, if the target nucleic acid is present in the sample, a cleaved first bait nucleic acid would be present in the first reaction mixture separated from the first solid support. In the presence of such cleaved first bait nucleic acid, the Craspase (e.g., Csx29) would cleave the labeled substrate for the Craspase (e.g., Csx30 labeled with HRP). This would produce free labeled Csx30 fragment (e.g., free HRP labeled Csx30). The free HRP labeled Csx30 fragment would then migrate from the reaction region to a fluidically connected detection region of the lateral flow assay device. The free HRP labeled substrate would then encounter an anti-HRP antibody immobilized in the detection region and bind to the immobilized antibody via the HRP label. Accumulation of labeled free HRP substrate fragment would produce a detectable signal because of the action of the HRP enzyme on its substrate, e.g., a combination of hydrogen peroxide and TMB (provided in the detection region), which produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP. Thus, the presence of a detectable signal in the detection region of the lateral flow assay device would indicate the presence of a free substrate fragment and, consequently, the presence of a target nucleic acid in the analyzed sample.

[0312] In addition to the “test zone” having immobilized anti-HRP antibodies (shown in FIG. 5), the detection region of the lateral flow assay device can also contain a “control zone” that is designed to indicate proper functioning of the lateral flow assay. For example, the control zone can contain anti-Protein A antibody and Protein A conjugated to gold nanoparticles can be provided within the fluidic connection between the reaction region and the detection region. Protein A conjugated with gold nanoparticles can then migrate to the detection region along withAttorney Docket No.: GRIP-014WO

[0313] the reaction mixture.

[0314] In addition to the test and control zones, in some cases, the detection region can also contain a third invisible zone, which can be designed to capture substrate cleavage product that is not detected in the test zone. This may reduce interference within the control zone with nonspecific binding and improve clarity of the results. In some cases, a first product mixture can be loaded into a reaction region of the lateral flow assay device, the reaction region comprising the Craspase comprising a crRNA and the labeled substrate immobilized in the reaction region. The reaction region is fluidically connected to the detection region such that the free labeled substrate fragment produced in the reaction region migrates to the detection region.

[0315] In some cases, the fluidic connection between the reaction region and the detection region can be an adsorbent material, such as a porous material that allows fluid migration.

[0316] In further embodiments, a fluidic connection can also comprise connection that cause a fluid to migrate from the reaction region to the detection region in a laminar flow or capillary flow. For example, a laminar flow involves flow of fluid / liquid between two typically flat or substantially flat surfaces that are close to each other, for example, at a distance of between 100 microns and 2 mm. In certain such cases, the reaction region and the detection region are connected with two or more surfaces that are substantially parallel to each other and close to each other such that a second reaction mixture migrates via laminar flow from the reaction region to the detection region.

[0317] A capillary flow involves flow of fluid / liquid through tubes of small cross-section area, for example, between 50 square microns and 10,000 square microns. In certain such cases, the reaction region and the detection region are connected with two or more channels such that a second reaction mixture migrates via capillary flow from the reaction region to the detection region. In some cases, the channels are microfluidic channels.

[0318] Once a free labeled substrate fragment produced in the reaction region migrates to the detection region (either through lateral flow, laminar flow, or capillary flow), it encounters the binding partner for the label and specifically binds to the binding partner. In some cases, the binding partner for the label is an antibody or a binding fragment thereof that specifically binds the label. Alternatively, the binding partner for the label can be a protein binding partner that specifically binds the label. The binding partner for the label can also be an aptamer or a nucleic acid that specifically binds the label. Additional examples of specific binding partners that could be used in the methods disclosed herein are well known to a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.Attorney Docket No.: GRIP-014WO

[0319] When the binding partner interacts / specif ically binds to the label, such binding / interaction produces a detectable signal. In certain such cases, the detectable signal is produced from a tag bound to the label or another suitable attachment point to the free substrate fragment. For example, the tag can be gold-nanoparticles, wherein the detectable signal is produced from gold nanoparticles accumulated in the detection region. The tag can also be a fluorophore, wherein the detectable signal is produced from the fluorophore accumulated in the detection region. Similarly, the tag can be a chromophore, wherein the detectable signal is a color produced from the chromophore accumulated in the detection region.

[0320] In some cases, the detection of the free labeled substrate fragment using a binding partner immobilized on an adsorbent material is performed in a lateral flow assay device.

[0321] Further details of such devices are described elsewhere in this disclosure.

[0322] In certain such cases, an IgG is immobilized in the detection region of a lateral flow assay device and a protein A labeled free substrate fragment migrates from a reaction region to the detection region. Protein A specifically binds to IgG thereby immobilizing protein A and the substrate fragment. Gold nanoparticles tagged on the free substrate fragment can precipitate to provide a visible signal.

[0323] Alternatively, an anti-HRP antibody is immobilized in the detection region of a lateral flow assay device and an HRP labeled free substrate fragment migrates from a reaction region to the detection region. TMB present in the detection region produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.

[0324] Additional examples of enzymes and their corresponding reactions that produce detectable signals are well-known in the art and use of such enzymes are within the purview of the disclosure.

[0325] Certain details of the lateral flow assay devices comprising reaction regions and detection region are provided elsewhere in this disclosure. Additional details of the lateral flow assay devices used in the methods disclosed herein are also provided in United States Provisional Application No. 63 / 743,548, which is incorporated by reference in its entirety. These details are also applicable to the methods of using lateral flow assay devices described herein. Certain such details include the Craspase, crRNA, target nucleic acids to be detected, substrates, binding partners of the substrates, types of labels, tags that produce detectable signal, etc. Implementation of such details is within the purview of the disclosure.

[0326] Substrate bound to a second solidAttorney Docket No.: GRIP-014WO

[0327] In certain embodiments of the disclosure, the substrate in a second reaction mixture is bound to a second solid support and, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0328] The substrate can be bound to the second solid support via a covalent bond. A covalent bond can be through an appropriate reactive group, such as an amine, sulfhydryl, aldehyde, or carboxylic group. Examples of covalently binding a protein or peptide to a solid support are described in United States Patent Application Publication No. 20060014232, which is incorporated by reference in its entirety. Additional examples of covalently binding a protein or peptide to a solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0329] In some cases, the substrate is bound to the second solid support via a non-covalent bond, such as an ionic bond, hydrogen bridge, hydrophobic bond, or van der Waals interaction. For example, a substrate can be conjugated to a binding partner and such substrate can be bound to a second solid support comprising an agent that specifically binds to the binding partner. Such pairs of binding partner and specific binding agents include antigen-antibody, other protein binding partners, and aptamers that specifically bind to a protein. Additional examples of non-covalently binding a substrate to a solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0330] Second solid Support is a sensor

[0331] As noted above, in some cases, the substrate in a second reaction mixture is bound to a second solid support. In certain such cases, the second solid support is a sensor and cleavage of the substrate produces a free substrate fragment thereby causing a change in a property of the sensor. Such change can be in an electrochemical property of a sensor, for example, a change in a redox signal. In some cases, determining the change in the redox signal of the sensor is performed by voltammetry, such as square wave voltammetry. Certain details of voltammetry, particularly, square wave voltammetry are provided in Gulaboski et al. (2023), Journal of Solid State Electrochemistry, Volume 28, pages 1121-1130; Chen eta!. (2013), Analytical Methods, Issue 9, pages 2137-2428; and US Patent Application Publication No. 20120187000, all of which are incorporated herein by reference in their entirety. Additional details of performing square wave voltammetry are well-known to a person of ordinary skill in the art and application of such details to the methods disclosed herein is within the purview of the disclosure.Attorney Docket No.: GRIP-014WO

[0332] In certain embodiments, the change in the property of the sensor is a change in an electrical property. In some cases, the change in an electrical property of a sensor can be a change in redox signal. The change in the electrical property can also be a change in an electrical resistance or Dirac voltage of the sensor. In certain embodiments, the sensor is a graphene sensor. An exemplary embodiment of detecting a target nucleic acids using a first CRISPR associated nuclease, a Craspase as a second GRISPR associated enzyme, and an electrochemical sensor comprising an HRP labeled substrate is described in FIGS. 2A-2C and in Example 2 below.

[0333] A person of ordinary skill in the art can recognize that any suitable conductive surfaces other than graphene could also be used to measure a change in an electrical property, such as voltage or resistance of a sensor. Certain such surfaces include rGO (reduced graphene oxide, “graphene flakes”), 2D-TMDs (Two-dimensional transition metal dichalcogenides), M0S2 (Molybdenum Disulfide), W-Sulfide (Tungsten Sulfide), Selenide (Selenide compounds, such as copper indium selenide (CIS) and bismuth selenide (Bi2Se3), Mxenes (a class of 2D transition metal carbides, nitrides, or carbonitrides), silicon IS-FET (Silicon-based ion-sensitive field-effect transistors), CNTs (carbon nanotubes), and Si nanowires. Use of any such surfaces is within the purview of the disclosure.

[0334] In certain embodiments, where the substrate is bound to a sensor, the substrate can also comprise a label. The label can be attached to the substrate such that the Craspase mediated cleavage of the substrate produces free substrate fragment that comprises the label and the sensor bound substrate fragment that is unlabeled. Thus, the loss of label on the substrate can further facilitate a change in a property of the sensor. In certain such cases, the label is an electrochemically active species, such as methylene blue.

[0335] In certain other cases, the label is an enzyme that produces a change in a redox active species. In certain such cases, an enzyme is an oxidoreductase, such as horseradish peroxidase (HRP). For example, the enzyme can be HRP and it can cause degradation of hydrogen peroxide near the sensor surface. An example of such assay is schematically represented in FIGS. 1 A-1 D and is described in further details in Example 1 below.

[0336] In certain embodiments, a solid support is a sensor which is a part of the inner wall of a container or a reaction chamber in which the Craspase reaction is conducted.

[0337] Other solid

[0338] In certain embodiments of the methods disclosed herein where the substrate is bound to a second solid support, the second solid support is a bead, such as a second magnetic bead orAttorney Docket No.: GRIP-014WO

[0339] a binding partner coated second beads. In some cases, the beads are agarose beads. Thus, when a free first bait nucleic acid is present in a second reaction mixture, Craspase cleaves the second bead bound substrate into a free substrate fragment and a second solid support bound substrate fragment.

[0340] When the second bead is a magnetic bead, a free substrate fragment can be separated from the second solid support by capturing the second magnetic beads using a magnet. The portion of the reaction mixture other than second magnetic beads can thus be separated.

[0341] When the second bead is a binding partner coated bead, a free substrate fragment can be separated from the second solid support by capturing the binding partner coated second beads using an immobilized binding agent that specifically binds to the binding partner on the beads. For example, streptavidin coated second beads could be captured using immobilized biotin and vice versa. Similarly, antigen coated second beads could be captured using immobilized antibodies specific to the antigen and vice versa. Additional examples of binding partners and specific binding agent combinations are well-known in the art and their use in the methods disclosed herein is within the purview of the disclosure.

[0342] In certain embodiments, second beads having solid support bound substrate fragment are separated from the reaction mixture by centrifuging the reaction mixture. The pellet produced after centrifugation would contain the second beads having bound substrate fragments and the free substrate fragments, if present, would remain in the supernatant, which can be separated and tested for the presence of the free substrate fragments.

[0343] In further embodiments, second beads having solid support bound substrate fragments are separated from the reaction mixture by filtering the reaction mixture. The residue produced after filtration would contain the second beads having solid support bound substrate fragments, and the free substrate fragments, if present, would be in the filtrate, which can be assayed for the presence of free substrate fragments.

[0344] The second reaction mixture after separating the second beads, i.e. , the second product mixture can be assayed for free substrate fragments. Thus, in some cases, the method comprises detecting the free substrate fragments in the second product mixture.

[0345] In certain such embodiments, detecting the free substrate fragment in a second product mixture comprises contacting the second product mixture with a sensor, such as a graphene sensor or an electrochemical sensor, comprising a probe that specifically binds the free substrate fragment. Binding of a free substrate fragment to the probe on the sensor produces a change in a property of the sensor. Thus, the method comprises detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe. The lackAttorney Docket No.: GRIP-014WO

[0346] of any change in a property of the sensor indicates that the second product mixture does not contain the free first bait nucleic acid and, hence, the sample does not contain the target nucleic acid.

[0347] Additional sensor surfaces described above, such as rGO, 2D-TMDs, M0S2, W-Sulfide, Selenide, Selenide compounds, such as copper indium selenide (CIS) and BisSes, Mxenes, silicon IS-FET, CNTs (carbon nanotubes), and Si nanowires can also be used in such embodiments of the disclosure.

[0348] A probe that specifically binds to the free substrate fragment can be a protein, such as a binding partner of the free substrate fragment or a label conjugated to a free substrate fragment. A probe can also be an antibody or an antigen binding fragment of an antibody that specifically binds to the free substrate fragment or a label conjugated to it. Alternatively, a probe can also be an aptamer that specifically binds to the free substrate fragment or a label conjugated to it. Additional examples of molecules that can specifically bind to the free substrate fragment or its label and that can be used as probes in the methods disclosed herein are well known to a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0349] To further enhance the effects of binding of a free substrate fragment to a probe bound to a sensor, the probe can further comprise a reporter, such as an electrochemical reporter. In certain such embodiments, when a free substrate fragment binds to the probe, such binding causes the electrochemical reporter to affect (or facilitate an interaction between the electrochemical reporter and the sensor where such interaction affects) a property, such as an electrical property of the sensor, for example, a graphene sensor.

[0350] In some cases, binding of a free substrate fragment to a probe (with or without a reporter) on a sensor, such as a graphene sensor, changes the property of the sensor, such as an electrical property of the sensor. The electrical property of the sensor can be conductivity of the sensor or Dirac voltage of the sensor.

[0351] Probes and electrochemical reporters may be employed in any convenient orientation or location or density or concentration, and such may be associated with the sensors, such as graphene sensors utilizing any convenient technique. Certain embodiments of the present invention may comprise a plurality of probes comprising electrochemical reporters and such may be present on the device in any convenient orientation or location or density or concentration.

[0352] Certain details of reporters that could be conjugated to sensors or free substrate fragments are provided in United States Provisional Patent Application Nos. 63 / 718,476 andAttorney Docket No.: GRIP-014WO

[0353] 63 / 743,548, each of which is incorporated by reference in its entirety. Also, in some cases, the graphene sensor and / or the “Graphene Field Effect Transistor (GFET)” devices used in the methods disclosed herein are as described in United States Provisional Patent Application No.

[0354] 63 / 708,186, which is incorporated by reference in its entirety, particularly, pages 9 to 53 under “Graphene Field Effect Transistor (GFET) devices and systems” and FIGS. 1 to 6.

[0355] The second reaction using a second CRISPR associated nuclease

[0356] As noted above, in certain embodiments, the disclosure provides a method for determining whether a target nucleic acid is present in a sample, the method comprising a first reaction and a second reaction. In some cases, the method comprises:

[0357] preparing a second reaction mixture by combining the first product mixture with:

[0358] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid; and

[0359] ii) a reactant for the second CRISPR associated enzyme, and

[0360] assaying the second reaction mixture for a product resulting from activity.

[0361] In certain embodiments, a second CRISPR associated enzyme is a second CRISPR associated nuclease having a second crRNA that specifically hybridizes with the first bait nucleic acid. If a free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease exhibits nuclease activity towards its reactant, e.g., a second bait nucleic acid. Thus, the presence of the second CRISPR associated nuclease activity towards a second bait nucleic acid indicates the presence in the second reaction mixture of a free first bait nucleic acid, which in turn indicates the presence in the sample of the target nucleic acid.

[0362] A CRISPR associated nuclease suitable for use as a second CRISPR associated nuclease in the methods disclosed herein has a collateral-cleavage activity in the presence of a target nucleic acid. As noted above, certain details of such collateral-cleavage activity and CRISPR associated nucleases that exhibit such activity are described in Varble et al. (2019), Trends Genet;35(6):446-456, which is incorporated by reference in its entirety.

[0363] A “second bait nucleic acid” as used herein refers to a nucleic acid having a sequence that does not match the sequence of the target nucleic acid. Depending upon the type of CRISPR associated nuclease used as a second CRISPR associated nuclease, for example, Type III, V, or VI, different types of second bait nucleic acids could be used. For example, if Type III CRISPR associated nuclease is used as a second CRISPR associated nuclease, a second bait nucleic acid could be RNA or single stranded DNA. If Type V CRISPR associated nuclease is used as a second CRISPR associated nuclease, a second bait nucleic acid couldAttorney Docket No.: GRIP-014WO

[0364] be single stranded DNA. Moreover, if Type VI CRISPR associated nuclease is used as a second CRISPR associated nuclease, a second bait nucleic acid could be RNA.

[0365] Further, in some cases, a second bait nucleic acid is a partially single stranded and double stranded DNA or a partially single stranded DNA / RNA hybrid. When a partially single stranded and double stranded DNA is used as a second bait nucleic acid, cleavage of the second bait nucleic may occur in the single stranded portion thereby producing a shorter partially single stranded double stranded DNA or completely double stranded DNA. Similarly, when a partially single stranded DNA / RNA hybrid is used as a second bait nucleic acid, cleavage of the second bait nucleic acid may occur in the single stranded portion thereby producing a partially single stranded DNA / RNA hybrid or completely double stranded DNA / RNA hybrid.

[0366] Depending on a first CRISPR associated nuclease used in the first reaction as well as the second CRISPR associated nuclease used in the second reaction of the methods disclosed herein, a person of ordinary skill in the art can determine appropriate second bait nucleic acid to be used under appropriate conditions and such embodiments are within the purview of the disclosure.

[0367] As shown in Figure 1 of Varble et al., Type VI CRISPR associated nuclease, for example, Cas13a, shows collateral-cleavage activity against RNA. Therefore, when a Type VI CRISPR associated nuclease, such as Cas13a is used as a second CRISPR associated nuclease in the methods disclosed herein, RNA is used as a second bait nucleic acid. Certain aspects and examples of Type VI CRISPR associated nucleases are provided in O’Connell (2019), J Mol Bio, 4:431 (1 ):66-87, which is herein incorporated by reference in its entirety. Use as second CRISPR associated nucleases of Type VI CRISPR associated nucleases described in O’Connell and additional examples otherwise known in the art is within the purview of the disclosure.

[0368] Type V CRISPR associated nuclease, for example, Cas12, shows collateral-cleavage activity against single stranded DNA. Therefore, when a Type V CRISPR associated nuclease, such as Cas12 or Cas14 is used in the methods disclosed herein, single stranded DNA is used as a second bait nucleic acid. Certain aspects and examples of Type V CRISPR associated nucleases are provided in Urbaitis et al. (2022), EMBO Rep, 23(12):e55481 , which is herein incorporated by reference in its entirety. Use as a second CRISPR associated nuclease of Type V CRISPR associated nucleases described in Urbaitis et al. and additional examples otherwise known in the art is within the purview of the disclosure.Attorney Docket No.: GRIP-014WO

[0369] Type III CRISPR associated nucleases, for example, Casio, shows collateral-cleavage activity against RNA or single stranded DNA. Therefore, when Type III CRISPR associated nuclease, such as Casio is used as a second CRISPR associated nuclease in the methods disclosed herein, single stranded DNA or RNA is used as a second bait nucleic acid. Certain aspects and examples of Type III CRISPR associated nucleases are provided in McMahon et al. (2020), Nat Comm, 11 , Article No. 500, which is herein incorporated by reference in its entirety. Use as a second CRISPR associated nuclease of Type III CRISPR associated nucleases described in McMahon et al. and additional examples otherwise known in the art is within the purview of the disclosure.

[0370] A second crRNA used in the methods disclosed herein is a small RNA molecule that guides a second CRISPR associated nuclease to a second bait nucleic acid. A complex of a second crRNA and a second trans-activating CRISPR RNA (tracrRNA), guide a CRISPR associated nuclease complex to a second bait nucleic acid. When a second crRNA binds to a first bait nucleic acid, the CRISPR associated nuclease cleaves a second bait nucleic acid.

[0371] Therefore, for use in the methods disclosed herein, a second crRNA is designed to specifically hybridize with the free first bait nucleic acid. Thus, depending upon the second bait nucleic acid used, a second crRNA has a sequence that is complementary to the free first bait nucleic acid or matches with the free first bait nucleic acid. For example, if the free first bait nucleic acid is double stranded, a second crRNA is complementary to one of the two strands of the double stranded free first bait nucleic acid and has a sequence that matches with the other strand of the double stranded free first bait nucleic acid. Alternatively, if the free first bait nucleic acid is single stranded, such as single stranded DNA or RNA, a second crRNA has a sequence complementary to the free first bait nucleic acid.

[0372] In some cases, a second crRNA and a second tracrRNA are in one single stranded nucleic acid, which is called a single guide RNA (sgRNA). A second sgRNA contains a second bait nucleic acid specific second crRNA sequence fused to a second tracrRNA sequence by a linker loop.

[0373] A second reaction mixture comprising a second CRISPR associated nuclease and a first product mixture that is tested for the presence of a free first bait nucleic acid can comprise additional reagents beyond the second CRISPR associated nuclease and the first product mixture. Such additional reagents can contain one or more of: salts like magnesium chloride and sodium chloride; chelating agents, such as ethylene diamine tetra-acetic acid; reducing agent, such as dithiothreitol; stabilizing agent, such as glycerol; and a buffer such as Tris or HEPES to maintain pH.Attorney Docket No.: GRIP-014WO

[0374] Further reagents in a second reaction mixture may include one or more of a denaturant (e.g. urea or guanidine-HCI), surfactant / detergent (e.g. sodium dodecyl sulfate (SDS) or Triton X-100), RNase inhibitor (e.g., RNasin or polyvinylsulfonic acid (PVSA)), and enhancer that increases collateral cleavage catalytic activity (e.g. dithiothreitol (DTT) or polyvinyl alcohol (PVA)). Certain details of such additional reagents are provided in Deng et al., Sensors & Actuators: B. Chemical 373 (2022) 132767, which is herein incorporated by reference in its entirety. In some cases, an enhancers of collateral cleavage catalytic activity can be a protein, such as Csm6, auxiliary CRISPR-associated enzyme. Certain such options are described by Gootenberg, et al. Science. 2018, 360(6387): 439-444, which is herein incorporated by reference in its entirety.

[0375] Reagents and conditions appropriate for CRISPR associated nuclease reaction are well-known to a person of ordinary skill in the art and selection and maintenance of appropriate reagents and conditions for a second reaction mixture in the methods disclosed herein are within the purview of this disclosure.

[0376] As noted above, a “second bait nucleic acid” as used herein refers to a nucleic acid having a sequence that does not match to the sequence of the target nucleic acid. A second bait nucleic acid is cleaved by a second CRISPR associated nuclease when a free first bait nucleic acid is present in a first product mixture and, hence, in a second reaction mixture. A second bait nucleic acid can have a length from 10 to 100 nucleotides, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some cases, a second bait nucleic acid has a unique and known sequence that can be detected in the methods disclosed herein.

[0377] In some cases, a second bait nucleic acid has a suitable secondary structure, e.g. a stem-loop structure. When appropriate, such secondary structure can prevent non-specific RNase activity.

[0378] Second bait nucleic acid bound to a second solid support

[0379] In certain embodiments of the disclosure, a second bait nucleic acid in a second reaction mixture is bound to a second solid support and, when the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid to produce a free second bait nucleic acid.

[0380] In some cases, a second bait nucleic acid is bound to a second solid support and cleavage of a second bait nucleic acid by a second CRISPR associated nuclease causes the second bait nucleic to be converted into “a free second bait nucleic acid.” A second bait nucleic acid can be bound to the second solid support via a covalent bond. A covalent bond can beAttorney Docket No.: GRIP-014WO

[0381] through an appropriate reactive group, such as an amine, sulfhydryl, aldehyde, or carboxylic group. Examples of covalently binding a nucleic acid to a solid support are described in United States Patent Application Publication No. US20020137045, which is incorporated by reference in its entirety. Additional examples of covalently binding a nucleic acid to a solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0382] In some cases, the second bait nucleic acid is bound to a second solid support via a non-covalent bond, such as an ionic bond, hydrogen bridge, hydrophobic bond, or van der Waals interaction. For example, a second bait nucleic acid can be conjugated to a binding partner and such second bait nucleic acid can be bound to a second solid support comprising an agent that specifically binds to the binding partner. Such pairs of binding partner and specific binding agents include antigen-antibody, other protein binding partners, and nucleic acid that specifically hybridizes to a complementary nucleic acid. Additional examples of non-covalently binding a second bait nucleic acid to a second solid support are well-known to a person of ordinary skill in the art and their use is within the purview of the disclosure.

[0383] Second solid Support is a sensor

[0384] As noted above, in some cases, the second bait nucleic acid in a second reaction mixture is bound to a second solid support. In certain such cases, the second solid support is a sensor and cleavage of the second bait nucleic acid produces a free second bait nucleic acid thereby causing a change in a property of the sensor. Such change can be in an electrochemical property of a sensor, for example, a change in a redox signal. In some cases, determining the change in the redox signal of the sensor is performed by voltammetry, such as square wave voltammetry. Certain details of voltammetry, particularly, square wave voltammetry are provided in Gulaboski etal. (2023), Journal of Solid State Electrochemistry, Volume 28, pages 1121-1130; Chen etal. (2013), Analytical Methods, Issue 9, pages 2137-2428; and US Patent Application Publication No. 20120187000, all of which are incorporated herein by reference in their entirety. Additional details of performing square wave voltammetry are well-known to a person of ordinary skill in the art and application of such details to the methods disclosed herein is within the purview of the disclosure.

[0385] In certain embodiments, the change in the property of the sensor is a change in an electrical property. In some cases, the change in an electrical property of a sensor can be a change in redox signal. The change in the electrical property can also be a change in anAttorney Docket No.: GRIP-014WO

[0386] electrical resistance or Dirac voltage of the sensor. In certain embodiments, the sensor is a graphene sensor.

[0387] A person of ordinary skill in the art can recognize that any suitable conductive surfaces other than graphene could also be used to measure a change in an electrical property, such as voltage or resistance of a sensor. Certain such surfaces include rGO (reduced graphene oxide, “graphene flakes”), 2D-TMDs (Two-dimensional transition metal dichalcogenides), M0S2 (Molybdenum Disulfide), W-Sulfide (Tungsten Sulfide), Selenide (Selenide compounds, such as copper indium selenide (CIS) and bismuth selenide (Bi2Se3), Mxenes (a class of 2D transition metal carbides, nitrides, or carbonitrides), silicon IS-FET (Silicon-based ion-sensitive field-effect transistors), CNTs (carbon nanotubes), and Si nanowires. Use of any such surfaces is within the purview of the disclosure.

[0388] In certain embodiments, where the second bait nucleic acid is bound to a sensor, the second bait nucleic acid can also comprise a label. The label can be attached to the second bait nucleic acid such that the second CRISPR associated nuclease mediated cleavage of the second bait nucleic acid produces free second bait nucleic acid that comprises the label and the sensor bound fragment of the second bait nucleic acid that is unlabeled. Thus, the loss of label on the second bait nucleic acid can further facilitate a change in a property of the sensor. In certain such cases, the label is an electrochemically active species, such as methylene blue. An example of such assay is schematically represented in FIGS. 3A-3B and is described in further details in Example 3 below.

[0389] In certain other cases, the label is an enzyme that produces a change in a redox active species. In certain such cases, an enzyme is an oxidoreductase, such as horseradish peroxidase (HRP). For example, the enzyme can be HRP and it can cause degradation of hydrogen peroxide near the sensor surface.

[0390] In certain embodiments, a solid support is a sensor which is a part of the inner wall of a container or a reaction chamber in which the second reaction is conducted.

[0391] Other solid supports

[0392] In certain embodiments of the methods disclosed herein where the second bait nucleic acid is bound to a second solid support, the second solid support is a bead, such as a second magnetic bead or a binding partner coated second beads. In some cases, the beads are agarose beads. Thus, when a free first bait nucleic acid is present in a second reaction mixture, a second CRISPR associated nuclease cleaves the second bait nucleic acid bound to a second bead into a free second bait nucleic acid.Attorney Docket No.: GRIP-014WO

[0393] In certain such cases, the second solid support is a second bead, such as a second magnetic bead or a second binding partner coated beads. In some cases, the beads are agarose beads.

[0394] When the second bead is a magnetic bead, a free second bait nucleic acid can be separated from the second solid support by capturing the second magnetic beads using a magnet. Thus, the second reaction mixture other than the second magnetic beads can be separated to produce a third product mixture.

[0395] When the second bead is a second binding partner coated bead, a free second bait nucleic acid can be separated from the second solid support by capturing the second binding partner coated beads using an immobilized binding agent that specifically binds to the second binding partner on the beads. For example, streptavidin coated second beads could be captured using immobilized biotin and vice versa. Similarly, an antigen coated second beads could be captured using immobilized antibodies specific to the antigen and vice versa.

[0396] Additional examples of binding partners and specific binding agent combinations are well-known in the art and their use is within the purview of the disclosure.

[0397] In certain embodiments, second beads having bound second bait nucleic acid are separated from the second reaction mixture by centrifuging the second reaction mixture. The pellet produced after centrifugation would contain the second beads having bound second bait nucleic acids and the free second bait nucleic acids, if present, would remain in the supernatant, which can be separated and tested for the presence of the free second bait nucleic acids.

[0398] In further embodiments, second beads having bound second bait nucleic acid are separated from the second reaction mixture by filtering the second reaction mixture. The residue produced after filtration would contain the second beads having bound second bait nucleic acids and the free second bait nucleic acids, if present, would be in the filtrate, which can be tested for the presence of free second bait nucleic acids.

[0399] In additional embodiments, a second support is a functionalized surface to which a second bait nucleic acid is bound. When a second bait nucleic acid is free by a second CRISPR associated nuclease, it can be separated by physically separating the second reaction mixture from the functionalized surface to produce a third product mixture. For example, in some cases, a second bait nucleic acid is immobilized on one or more inner walls of a second reaction chamber in which the second CRISPR associated nuclease reaction is conducted. Cleavage of the second bait nucleic acid produces free second bait nucleic acid, i.e. , a second bait nucleic acid that is not immobilized to the one or more inner walls of the second reaction chamber. Such free second bait nucleic acid can be readily removed by simply moving theAttorney Docket No.: GRIP-014WO

[0400] second reaction mixture from the second reaction chamber. Such removal can be performed manually, for example, by a user using a pipette, or in a device having a mechanism for moving a second reaction mixture from the reaction chamber.

[0401] The second reaction mixture after separating the second beads having the second bait nucleic acids bound to it, i.e., the third product mixture can be assayed for free second bait nucleic acid. Thus, in some cases, the method comprises detecting the free second bait nucleic acid in the second product mixture.

[0402] In certain such embodiments, detecting the free second bait nucleic acid comprises contacting the third product mixture with a sensor, such as a graphene sensor or an electrochemical sensor, comprising a probe that specifically binds the free second bait nucleic acid. Binding of a free second bait nucleic acid to the probe on the sensor produces a change in a property of the sensor. Thus, the method comprises detecting a change in a property of the sensor caused by binding of the free second bait nucleic acid to the probe. The lack of any change in a property of the sensor indicates that the third product mixture does not contain the free second bait nucleic acid and, hence, the sample does not contain the target nucleic acid.

[0403] Additional sensor surfaces described above, such as rGO, 2D-TMDs, M0S2, W-Sulfide, Selenide, Selenide compounds, such as copper indium selenide (CIS) and BisSes, Mxenes, silicon IS-FET, CNTs (carbon nanotubes), and Si nanowires can also be used in such embodiments of the disclosure.

[0404] A probe that specifically binds to the free second bait nucleic acid can be a protein that specifically binds to the free second bait nucleic acid. For example, if the free second bait nucleic acid is DNA, the protein that specifically binds to the free second bait DNA is a DNA binding protein, such as a transcription factor. Alternatively, if the free second bait nucleic acid is RNA, the protein that specifically binds to the free second bait RNA is an RNA binding protein, such as pentatricopeptide repeat (PPR) proteins. Certain examples of RNA binding protein are described in Jolma et al. (2020), Genome Res. 30(7):962-973, which is incorporated herein by reference in its entirety. Use of the RNA binding proteins described in Jolma et al. as well as additional such proteins known-in the art is within the purview of this disclosure.

[0405] In some cases, the probe that specifically binds to the free second bait nucleic acid is a nucleic acid that specifically binds to the free second bait nucleic acid, for example, through complementarity between the probe nucleic acid and the free second bait nucleic acid. The probe nucleic acid can be DNA or RNA, which may contain naturally occurring or chemically modified nucleotides or a combination thereof.Attorney Docket No.: GRIP-014WO

[0406] In certain embodiments, the probe that specifically binds to the free second bait nucleic acid is an aptamer that specifically binds to the free bait nucleic acid.

[0407] To further enhance the effects of binding of a free second bait nucleic acid to a probe bound to a sensor, the probe can further comprise a reporter, such as an electrochemical reporter. In certain such embodiments, when a free second bait nucleic acid binds to the probe, such binding causes the electrochemical reporter to affect (or facilitate an interaction between the electrochemical reporter and the sensor where such interaction affects) a property, such as an electrical property of the sensor, for example, a graphene sensor.

[0408] In some cases, binding of a free second bait nucleic acid to a probe (with or without a reporter) on a sensor, such as a graphene sensor, changes the property of the sensor, such as an electrical property of the sensor. The electrical property of the sensor can be conductivity of the sensor or Dirac voltage of the sensor.

[0409] Probes and electrochemical reporters may be employed in any convenient orientation or location or density or concentration, and such may be associated with the sensors, such as graphene sensors utilizing any convenient technique. Certain embodiments of the present invention may comprise a plurality of probes comprising electrochemical reporters and such may be present on the device in any convenient orientation or location or density or concentration.

[0410] Certain details of reporters that could be conjugated to sensors or free second bait nucleic acid are provided in United States Provisional Patent Application Nos. 63 / 718,476 and 63 / 743,548, each of which is incorporated by reference in its entirety. Also, in some cases, the graphene sensor and / or the “Graphene Field Effect Transistor (GFET)” devices used in the methods disclosed herein are as described in United States Provisional Patent Application No.

[0411] 63 / 708,186, which is incorporated by reference in its entirety, particularly, pages 9 to 53 under “Graphene Field Effect Transistor (GFET) devices and systems” and FIGS. 1 to 6.

[0412] A first reaction and a second reaction in the same reaction mixture

[0413] As noted above, certain embodiments of the disclosure provide a method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample. In some cases, the first reaction and the second reaction occur in the same reaction mixture. In certain such cases, the methods utilize a first bait nucleic acid that is designed such that a crRNA of a second CRISPR associated enzyme specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.Attorney Docket No.: GRIP-014WO

[0414] This design of a first bait nucleic acid avoids the necessity that a first bait nucleic acid be bound to a solid support. This also avoids the necessity to separate a solid support from a first reaction mixture, as described above in methods where two reaction mixtures are used.

[0415] Also, this design of a first bait nucleic acid ensures that a second CRISPR associated enzyme is only activated when a first CRISPR associated nuclease cleaves a first bait nucleic acid, i.e. , only when a target nucleic acid is present in a sample. In certain such cases, the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with an uncleaved first bait nucleic acid. Such secondary structure can be a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure. These secondary structures of the first bait nucleic acid are designed so that, only in the presence of a target nucleic acid, the first CRISPR associated nuclease cleaves the first bait nucleic acid thereby producing a cleaved first bait nucleic acid. The cleaved first bait nucleic acid has sequences that are accessible to the crRNA of a second CRISPR associated nuclease.

[0416] For example, certain non-limiting examples of toehold switches are described in the U.S. Patent No. 11 ,788,156, which is incorporated herein in its entirety. Certain examples of inverted repeats, hairpin, triplex, slipped, and cruciform structures in the context of secondary structures of nucleic acids is described in Bowater et al. (2022), Int. J. Mol. Sci., 23(11 ):6171 , which is also incorporated herein by reference in its entirety.

[0417] Thus, when the same reaction mixture is used for a first reaction as well as a second reaction in the methods disclosed herein, the reaction mixture is assayed to determine whether a product resulting from activity of the second CRISPR associated enzyme on its reactant is produced in the reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0418] Various alternatives for assaying whether a product resulting from the activity of the second CRISPR associated enzyme on the reactant is produced in a reaction mixture are essentially the same as the methods utilized when separate reaction mixtures are utilized and the second reaction mixture is assayed for the product resulting from activity of the second CRISPR associated enzyme on the reactant. Certain such details are provided throughout this disclosure and include the use of different solid supports, sensors, labels, substrates, second bait nucleic acids, etc. Implementation of such alternatives is envisioned in the embodiments of the disclosure where a first reaction and the second reaction are performed in the same reaction mixture and such embodiments are within the purview of the disclosure. Certain such details are provided in Example 4 below.Attorney Docket No.: GRIP-014WO

[0419] ANALYZERS

[0420] Certain embodiments of the disclosure provide analyzers that are specifically designed to carry out the methods disclosed herein.

[0421] In some cases, analyzers disclosed herein comprise a first reaction chamber, a second reaction chamber, and a device for assaying the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme on the reactant. The first reaction chamber can be fluidically connected to the second reaction chamber, which in turn can be fluidically connected to the device for assaying the second reaction mixture. Thus, a first reaction mixture or a part thereof from the first reaction chamber can be transported to the second reaction chamber and the second reaction mixture or a part thereof can be transported to the device for assaying the second reaction mixture or a part thereof.

[0422] In some cases, analyzers disclosed herein comprise a first reaction chamber, a second reaction chamber, and a device for assaying the second reaction mixture, but these components are not fluidically connected. In certain such cases, a user may transport a first reaction mixture or a part thereof from the first reaction chamber to the second reaction chamber. Similarly, a user may transport a second reaction mixture or a part thereof from the second reaction chamber to the device for assaying the second reaction mixture or a part thereof.

[0423] A first reaction chamber of the analyzer is configured to carry out the first CRISPR associated nuclease reaction. Accordingly, the first reaction chamber can comprise one or more reagents, such as a first CRISPR associated nuclease and / or additional compounds used in the reaction. In some cases, such reagents are provided in a dried form, for example, a lyophilized form.

[0424] In some cases, an analyzer comprises a first reaction chamber providing the interior surface or a portion thereof as a solid support to which the first bait nucleic acids are bound. Thus, in some cases, the interior surface of a first reaction chamber is functionalized and the first bait nucleic acids are bound to the interior surface. If a target nucleic acid is present in a sample, the first CRISPR associated nuclease would cleave the first bait nucleic acid to produce free first bait nucleic acid, which would be released into the first reaction chamber. The first reaction mixture containing such free first bait nucleic acid can be transported out of the first reaction chamber and analyzed for the presence of the free first bait nucleic acid. Such transport can be performed using a transporting means, such as a pump, to move the first reaction mixture or a part thereof from the first reaction chamber into a second reaction chamber. Alternatively, such transport can be performed manually by a user.Attorney Docket No.: GRIP-014WO

[0425] A second reaction mixture of the analyzer is configured to carry out the second CRISPR associated enzyme reaction. Accordingly, the second reaction chamber can comprise one or more reagents, such as a first CRISPR associated enzyme (nuclease or Craspase) and / or additional compounds used in the reaction. In some cases, such reagents are provided in a dried form, for example, a lyophilized form.

[0426] In some cases, an analyzer comprises a second reaction chamber providing the interior surface or a portion thereof as a solid support to which a reactant (e.g., second bait nucleic acids for a second CRISPR associated nuclease or substrate for Craspase) is bound. Thus, in some cases, the interior surface of a second reaction chamber is functionalized and a reactant (e.g., second bait nucleic acids or substrate) is bound to the interior surface. If a free first bait nucleic acid is present in a sample, the second CRISPR associated enzyme (e.g., nuclease or Craspase) would cleave the reactant (e.g., second bait nucleic acid or substrate) to produce free reactant fragment (e.g., free second bait nucleic acid or free substrate fragment), which would be released into the second reaction chamber. The second reaction mixture containing such free reactant (e.g., free second bait nucleic acid or free substrate fragment) can be transported out of the second reaction chamber and analyzed for the presence of the free reactant fragment (e.g., free second bait nucleic acid or free substrate fragment). Such transport can be performed using a transporting means, such as a pump, to move the second reaction mixture or a part thereof from the second reaction chamber into a device for assaying the free reactant fragment or a part thereof. Alternatively, such transport can be performed manually by a user.

[0427] In certain embodiments where an interior surface or a portion thereof of a second reaction chamber is used as a second solid support to which the reactants for the second CRISPR associated enzyme are bound, the interior surface or the portion thereof is a sensor, for example, a graphene sensor. Thus, in such embodiments, the interior surface of the second reaction chamber is a part of device that assays the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme.

[0428] For example, a second bait nucleic acids bound to a graphene sensor of the device, when cleaved by a CRISPR associated nuclease, changes a property of the graphene sensor. Similarly, a substrate for a Craspase bound to a graphene sensor of the device, when cleaved by a Craspase, changes a property of the graphene sensor.

[0429] In some cases, the second bait nucleic acid or a substrate can further comprise a reporter, such as an electrochemical reporter. A change in a property of a sensor can be a change in an electrical property, such as conductivity of Dirac voltage.Attorney Docket No.: GRIP-014WO

[0430] In some cases, an analyzer comprises a filter between a first sample chamber and a second sample chamber. An analyzer can also comprise a filter between the second sample chamber and the device that assays the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme. Thus, when a first reaction mixture is transported from the first sample chamber into the second sample chamber, beads comprising first bait nucleic acid, if used in the reaction mixture, are separated from the rest of the reaction mixture. The filtered first reaction mixture, i.e., a first product mixture can then be introduced to a second reaction mixture.

[0431] Similarly, when a second reaction mixture is transported from the second sample chamber into a device that assays the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme, beads comprising reactants, if used in the second reaction mixture, are separated from the rest of the second reaction mixture. The filtered second reaction mixture, i.e., a second or third product mixture can then be introduced to the device that assays the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme.

[0432] Certain details of methods provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above are also applicable to the analyzers as described herein. Certain such details include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids to be detected, bait nucleic acids, solid support and their binding to reactants of the second CRISPR associated enzymes, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0433] In some cases, analyzers described herein comprise a single reaction chamber where a first reaction and the second reaction occurs. In such embodiments, a first bait nucleic acid that is not bound to a solid support are used. Such first bait nucleic acid is also designed such that a crRNA of a second CRISPR associated enzyme specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid. Certain details of such first bait nucleic acids are provided elsewhere in this disclosure and use of such details in the analyzers described herein is within the purview of the disclosure.

[0434] Moreover, certain details of analyzers are elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above are also applicable to the analyzers that utilize a single reaction mixture as described herein. Certain such details include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids toAttorney Docket No.: GRIP-014WO

[0435] be detected, bait nucleic acids, solid support and their binding to reactants of the second CRISPR associated enzymes, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0436] METHODS OF DETECTING A TARGET NUCLEIC ACID IN A SPECIFIC DEVICE

[0437] In some cases, the methods of detecting target nucleic acids are performed using devices and / or analyzers disclosed herein. Certain such embodiments provide a method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:

[0438] (a) combining in an analyzer one or more reaction mixtures comprising:

[0439] i) the sample,

[0440] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0441] iii) a first bait nucleic acid,

[0442] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0443] v) a reactant for the second CRISPR associated enzyme; and

[0444] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0445] In certain such cases, the first reaction occurs in a first reaction mixture and the second reaction occurs in a second reaction mixture. Accordingly, certain embodiments of the disclosure provide a method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0446] (a) preparing a first reaction mixture in a first reaction chamber of an analyzer, wherein the first reaction mixture comprises:

[0447] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0448] ii) a first bait nucleic acid bound to a first solid support;

[0449] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;Attorney Docket No.: GRIP-014WO

[0450] (c) preparing a second reaction mixture in a second reaction chamber of the analyzer, wherein the second reaction mixture comprises:

[0451] i) the first product mixture,

[0452] ii) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0453] iii) a reactant for the second CRISPR associated enzyme; and

[0454] (d) assaying the second reaction mixture for a product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.

[0455] As discussed above, if the target nucleic acid is present in the sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards a first bait nucleic acid, which is cleaved to produce a free first bait nucleic acid. The free first bait nucleic acid, if produced, is separated from the first solid support to prepare a first product mixture. The first product mixture is then combined in a second reaction mixture with a second CRISPR associated enzyme having a second crRNA that specifically hybridizes with the free first bait nucleic acid. If the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated enzyme is activated and acts on a reactant for the second CRISPR associated enzyme. The second reaction mixture is then assayed for a product resulting from the activity of the second CRISPR associated enzyme on the reactant.

[0456] Certain details of analyzers suitable for use in the methods disclosed herein are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” and “Analyzers” above and these details are also applicable to the methods of detecting target nucleic acids in specific devices as described herein. The features described elsewhere in this disclosure and applicable to the methods of detecting target nucleic acids using specific devices as disclosed herein include the first CRISPR associated nucleases, second CRISPR associated enzymes (including nucleases and Craspases), crRNAs, tracrRNAs, sgRNAs, target nucleic acids to be detected, first and second bait nucleic acids, substrates for Craspase, first and second solid support and their binding to reactants, such as bait nucleic acids or substrate, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0457] In some cases, methods described herein utilize analyzers that are designed to have a single reaction chamber where a first reaction and a second reaction occurs. In such embodiments, a first bait nucleic acid is designed such that a crRNA of a second CRISPR associated enzyme specifically hybridizes with the cleaved first bait nucleic acid but does notAttorney Docket No.: GRIP-014WO

[0458] hybridize with an uncleaved first bait nucleic acid. Certain details of such first bait nucleic acids are provided elsewhere in this disclosure and use of such details in the analyzers described herein is within the purview of the disclosure.

[0459] Moreover, certain details provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above are also applicable to the analyzers that utilize a single reaction mixture as described herein. Certain such details include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids to be detected, bait nucleic acids, solid support and their binding to reactants of the second CRISPR associated enzymes, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0460] DEVICES HAVING SENSORS

[0461] Certain embodiments of the disclosure provide devices comprising sensors in contact with second reaction mixtures produced in the methods disclosed herein.

[0462] Accordingly, in some cases, the disclosure provides a device comprising a sensor in contact with a second reaction mixture,

[0463] wherein the second reaction mixture is produced by:

[0464] (a) preparing a first reaction mixture by combining a sample suspected of containing a target nucleic acid with:

[0465] i) a first CRISPR associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[0466] ii) a first bait nucleic acid bound to a first solid support;

[0467] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0468] (c) preparing a second reaction mixture by combining the first product mixture with:

[0469] i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0470] ii) a reactant for the second CRISPR associated enzyme, wherein the reactant is bound to a second solid support;

[0471] wherein the sensor detects the cleavage of the reactant bound to the second solid support to determine whether a free first bait nucleic acid is present in the first product mixture.Attorney Docket No.: GRIP-014WO

[0472] As discussed above, if the target nucleic acid is present in the sample, the first CRISPR associated nuclease exhibits nonspecific collateral-cleavage activity towards a first bait nucleic acid, which is cleaved to produce a free first bait nucleic acid. The free first bait nucleic acid, if produced, is separated from the first solid support to prepare a first product mixture. The first product mixture is then combined in a second reaction mixture with a second CRISPR associated enzyme having a second crRNA that specifically hybridizes with the free first bait nucleic acid. If the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated enzyme is activated and acts on a reactant for the second CRISPR associated enzyme. The second reaction mixture is then assayed for a product resulting from the activity of the second CRISPR associated enzyme on the reactant.

[0473] Certain details of sensors suitable for use in the methods disclosed herein are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” and “Analyzers” above and these details are also applicable to the devices having sensors in contact with a second reaction mixture as described herein. The features described elsewhere in this disclosure and applicable to the devices having sensors in contact with a second reaction mixture include the first CRISPR associated nucleases, second CRISPR associated enzymes (including nucleases and Craspases), crRNAs, tracrRNAs, sgRNAs, target nucleic acids to be detected, first and second bait nucleic acids, substrates for Craspase, first and second solid support and their binding to reactants, such as bait nucleic acids or substrate, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0474] KITS FOR DETECTING A TARGET NUCLEIC ACID

[0475] In some cases, the disclosure provides kits designed for detecting target nucleic acids. In some cases, a target nucleic acid detection kit comprises:

[0476] a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and a first bait nucleic acid bound to a first solid support;

[0477] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with the first bait nucleic acid;

[0478] a reactant for the second CRISPR associated enzyme, the reactant bound to a second solid support, and

[0479] a sensor configured to detect cleavage of the reactant bound to the second solid support.Attorney Docket No.: GRIP-014WO

[0480] In some cases, a devices detects the cleavage of the reactant bound to the second solid support by detecting the presence of any free reactant fragment produced by the cleavage of the reactant bound to the second solid support. In certain such embodiments, the device comprises a sensor, such as an electrochemical sensor or graphene sensor comprising a probe that specifically binds to the free reactant fragment. Binding of the free reactant fragment to the probe on the sensor causes a change in a property of the sensor, which is detected in the devices described herein thereby detecting the presence of the free reactant fragment.

[0481] Alternatively, the solid support is sensor, such as an electrochemical sensor or a graphene sensor, and the device detects the cleavage of the reactant by detecting the change caused by the cleavage of the reactant in a property of the sensor.

[0482] Certain details of the devices and methods are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid,” and “Analyzers” above and these details are also applicable to the target nucleic acid detection kits as described herein.

[0483] Moreover, certain details of the methods of detecting target nucleic acids are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid,” and “Analyzers” above and these details are also applicable to the target nucleic acid detection kits described herein and include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids to be detected, bait nucleic acids, solid support and their binding to reactants of the second CRISPR associated enzymes, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0484] Also provided are kits that include a device, e.g., as described above, as well as packaging for the device, which packaging may be sterile, as desired. Components of the kit may be disposable or reusable, as desired. In some cases, kits may comprise a plurality of devices including multiple versions of the same device with different characteristics, such as, for example, different electrical characteristics or different surface functionalization (e.g., different probes and / or different electrochemical reporters and / or different combinations thereof) tailored to different target analyte.

[0485] Kits according to the present invention may also include a power supply for the device. Any convenient power supply capable of causing the device to operate in the intended manner may be applied. Such a power supply may include a voltage source configured to provide voltage potentials to the drain electrode and / or the source electrode and / or the gate electrode of the device, e.g., VD, Vsor VG, as described above. Power supplies of interest may include, forAttorney Docket No.: GRIP-014WO

[0486] example, a battery or an electrical connector for connecting the power supply to an external power source such as a plug-in power source.

[0487] In embodiments of kits according to the present invention, the packaging for the device comprises a cartridge configured to house the device. Any convenient cartridge may be applied, such as, for example, a cartridge that facilitates the device being held and manipulated by hand or a cartridge that facilitates, e.g., provides a platform for, sample collection or connecting a power supply or connecting a transmitter.

[0488] Kits according to the present invention may also include a sample collection device. Any convenient sample collection device capable of collecting a sample of interest may be applied. For example, sample collection devices may be configured to collect nasal swabs, throat swabs, check swabs, saliva, urine or the like. Sample collection devices of interest may be configured to interface with a device according to the present invention or packaging therefor such that any sample collected by the sample collection device can be delivered to the device, e.g., a sensor or a lateral flow assay device.

[0489] In some embodiments, the sample collection device comprises sample transport media. Any convenient sample transport media may be applied, such as, for example, sample transport media configured to maximize sample stability and / or preserve aspects of the sample prior to and / or while the sample is exposed to the device. For example, sample transport media may be configured to preserve target analyte present in the sample. In some cases, transport media may comprise an inert buffer or dilutant. In other cases, transport media contain one or more constituents that preserve certain sample characteristics (e.g., prevent the breakdown of a cell wall or a cell membrane by cell lysis). In addition, some of these constituents may serve the dual purpose of preservation and decontamination of the sample. Embodiments may comprise a transport media that does not affect the electrical characteristics of the device, e.g., via the graphene sensor, as well as probe and electrochemical reporter thereof, of the device or aspects of surface functionalization (e.g., probe and electrochemical reporter) of the graphene sensor, as described herein.

[0490] In some embodiments, the kits comprise one or more controls. The control may be any experimental control of interest, such as a control configured to confirm that a sample was exposed to the device and / or that the device is functioning correctly to evaluate the presence of a target analyte in a sample. In some cases, the control comprises a positive control. Such a positive control may be configured to confirm that a sample has been exposed to the device and / or that the device is capable of evaluating the presence of an analyte correctly. In other cases, the control comprises a negative control. Such a negative control may be configured toAttorney Docket No.: GRIP-014WO

[0491] confirm that the device is capable of evaluating the presence of an analyte correctly. In still other cases, the control comprises both a positive and a negative control.

[0492] Also present in the kit may be instructions for using the kit components. The instructions may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD- or CD-ROM, etc. The instructions may take any form, including complete instructions for how to use the device or system or as a website address with which instructions posted on the world wide web may be accessed.

[0493] In some cases, a target nucleic acid detection kit is designed to allow conducting a first reaction and a second reaction in the same reaction mixture. In certain such cases, a kit comprises:

[0494] a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid;

[0495] a first bait nucleic acid;

[0496] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with a cleaved first bait nucleic acid; and

[0497] a reactant for the second CRISPR associated enzyme bound to a solid support.

[0498] As noted above, when a first reaction and a second reaction is performed in the same reaction mixture, the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid. Accordingly, in certain embodiments, a kit comprises a first bait nucleic acid having a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid. A secondary structure can be a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0499] Additional details of the kits described above for performing a first reaction and a second reaction in separate reaction mixtures are also applicable to the kits that allow performing a first reaction and a second reaction in the same reaction mixture. Certain such details include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids to be detected, bait nucleic acids, solid support and their binding toAttorney Docket No.: GRIP-014WO

[0500] reactants of the second CRISPR associated enzymes, types of probes used, etc. and implementation of such details is within the purview of the disclosure.

[0501] COMPOSITIONS

[0502] In some embodiments, the disclosure provides a composition comprising:

[0503] i) a sample suspected of containing a target nucleic acid,

[0504] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0505] iii) a first bait nucleic acid,

[0506] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0507] v) a reactant for the second CRISPR associated enzyme; and

[0508] The composition described herein allow performing methods described herein where a first reaction and a second reaction are performed in the same reaction mixture. Accordingly, in some cases, the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid. For example, the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid. Such secondary structure can be selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0509] Certain details of methods provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above are also applicable to the compositions as described herein. Certain such details include the first CRISPR associated nucleases, the second CRISPR associated enzymes (including Craspases and second CRISPR associated nucleases), crRNA, tracrRNA, sgRNA, target nucleic acids to be detected, bait nucleic acids, solid support and their binding to reactants of the second CRISPR associated enzymes, types of probes used, etc. Implementation of such details is within the purview of the disclosure.

[0510] MULTIPLEX APPLICATIONS

[0511] In some embodiments, the methods disclosed herein are used to detect one or more target nucleic acids in one or more samples. In certain such cases, multiple first reactions are performed. In such multiple reactions, multiple first CRISPR associated nucleases are used in multiple first reactions. The multiple first CRISPR associated nucleases can comprise crRNAsAttorney Docket No.: GRIP-014WO

[0512] that bind to the same or different target nucleic acids. The same first bait nucleic acid can be used in the multiple first reactions. Multiple first product mixtures can then be produced by separating the multiple first reactions from the multiple first solid supports. Because the same free first nucleic acid would be present in the multiple first product mixtures, the same reagents in multiple second reactions could be used to detect the free first bait nucleic acid in the multiple first product mixtures.

[0513] Based on the presence or absence of free first bait nucleic acids in different first product mixtures, one can determine the presence or absence of multiple target nucleic acids in multiple samples tested in multiple first reactions.

[0514] Various details of the first and the second reactions disclosed in this disclosure can be applied to methods of multiplexing as described herein and such embodiments are within the purview of the disclosure.

[0515] APPLICATIONS

[0516] Methods, devices, analyzers, and kits disclosed herein find use in a variety of applications. In some instances, devices, analyzers, and kits find use in detecting the presence of a target nucleic acid with medical implications. For example, methods, devices, analyzers, and kits may be configured to detect the presence of an infection, for example, a bacterial, viral, algal, fungal, or protozoan infection. Similarly, methods, devices, analyzers, and kits may be configured to detect a disease such as cancer, genetic disease, and the like. Further, methods, devices, analyzers, and kits may be configured to detect a target nucleic acid in a sample obtained from an environmental site. Such analysis can be used to detect contamination of a site. Certain such sites can be tested by analyzing sources such as soil, water, air and the like. In further cases, methods, devices, analyzers, and kits may be configured to detect contamination of goods, such as clothing, appliances, working surfaces, and the like and samples can be obtained and tested accordingly.

[0517] With respect to viral detection applications, examples of viruses (e.g., potentially infectious viruses) that can be detected using the methods, devices, and kits provided herein include, without limitation, human immunodeficiency virus (e.g., HIV1 and HIV2), Zika virus, influenza virus A and B, adenovirus 4, RSV, parainfluenza types 1 , 2 and 3, human coronaviruses OG43, 229E and HK, human metapneumovirus, rhinoviruses, enteroviruses, hepatitis A, B, C and E viruses, rotavirus, human papillomavirus, measles viruses, caliciviruses, astrovirus, West Nile virus, Ebola virus, Dengue fever virus, African swine fever, herpes simplex virus (e.g., HSV-2), Norwalk and Norwalk-like viruses, enteric adenoviruses, yellow fever virus,Attorney Docket No.: GRIP-014WO

[0518] chikungunya virus, Epstein-Barr virus, parvovirus, varicella zoster virus and Ross River virus, as well as seasonal influenza viruses or coronaviruses, e.g., SARS-CoV-2 viruses, such as SARS-CoV-2 variants, e.g., SARS-CoV-2 Alpha, SARS-CoV-2 Beta, SARS-CoV-2 Delta or SARS-CoV-2 Delta Plus, SARS-CoV-2 Gamma or SARS-CoV-2 Omicron.

[0519] In some cases, the methods, devices, analyzers, and kits provided herein can be used to identify the presence of a microorganism (bacteria, archaea, alga (e.g., marine alga), viruses, fungi (e.g., yeast and mold), and protozoa) based, at least in part, on the presence, absence or amount of a target nucleic acid in a sample. In some cases, methods, devices, analyzers, and kits provided herein can be used to identify the presence of an antimicrobial resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA)). Examples of microorganisms (e.g., potentially infecting microorganisms) that can be detected using the methods, devices, analyzers, and kits provided herein include, without limitation, bacterial microorganisms such as Staphylococcus aureus (e.g., MRSA and MSSA), Streptococcus pyogenes, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Bordelella pertussis, Mycobacterium tuberculosis, Escherichia. coli(e.g., enterohaemorrhagic E. co / / such as 0157:H7 E. colior enteropathogenic E. coli), Salmonella species (e.g., Salmonella enterica), Listeria monocytogenes, Acinetobacter baumanni, Klebsiella oxytoca, Giardia intestinalis, Sarcoptes scabiei, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Campylobacter species (e.g., thermophilic strains of Campylobacter jejuni, C. lari or C. coli), Bacillus cereus, Vibrio species, Yersinia enterocolitica, Shigella species, Enterococcus species (e.g., Enterococcus faecalis or E. faecium), Helicobacter pylori and Clostridium species (e.g., Clostridium botulinum or Clostridium perfringens), fungal microorganisms such as Aspergillus species (e.g., A. flavus, A. fumigatus and A. niger), yeast (e.g., Candida norvegensis and C. albicans), Penicillium species, Rhizopus species and Alternaria species and protozoan microorganisms such as Cryptosporidium parvum, Giardia lamblia and Toxoplasma gondii.

[0520] For detection of cancer, nucleic acids that are specifically produced in cancer cells or nucleic acids that are increased in cancer cells compared to healthy cells can be detected as target nucleic acids according to the methods disclosed herein. Moreover, certain mutations in specific genes may indicate increased likelihood that a cancer would develop in a subject and such mutated genes can also be detected as target nucleic acids according to the methods disclosed herein.

[0521] For example, oncogenes that are specifically produced in cancer cells can be detected using the methods disclosed herein. Non-limiting examples of such oncogenes include HER2,Attorney Docket No.: GRIP-014WO

[0522] BCR / ABL1, CMYC, NMYC, EGER, EML4AK, KRAS, and HAS genes. Similarly, certain mutations in some of these genes are known to increase likelihood of cancer development and such mutations can be detected using the methods disclosed herein to determine if a subject has high likelihood of cancer development.

[0523] Further, cancer cells often release fragmented DNA into the bloodstream of a patient. Such DNA is called cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). In some cases, the methods disclosed herein can be used to detect as target nucleic acids cfDNA or ctDNA. Detection of cfDNA or ctDNA can be used for cancer diagnosis as well as to monitor progress of cancer therapy by monitoring the presence / absence and / or the amount of cfDNA or ctDNA.

[0524] Certain genetic diseases are caused by specific mutation in certain genes. For example, a mutation in the HBA1 or HBA2gene causes thalassemia. Accordingly, mutations in HBA1 or HBA2 gene can be detected to identify the presence / absence of determine a specific type of thalassemia in a subject.

[0525] Fetal genetic material is known to be present in pregnant mother’s blood circulation. In some cases, fetal genetic material can be detected in pregnant mother’s blood to screen for a potential genetic abnormalities in the growing fetus. Certain such genetic abnormalities include: disorders caused by single gene mutations, such as cystic fibrosis, sickle cell anemia, Tay-Sachs disease, hemophilia, and Marfan syndrome; chromosomal abnormalities, such as Down syndrome; multifactorial or complex disorders, such as heart defects, cleft lip or cleft palate, and spina bifida.

[0526] In some cases, environmental sites can be analyzed for the presence or absence of a target nucleic acid. Samples obtained from environmental sites, such as soil, water, air, or other materials can be analyzed for the presence of target nucleic acids that indicate contamination of the environmental sites. For example, contamination of an area with sewage or fecal matter can be detected by analyzing a sample for target nucleic acids specific for E. coli.

[0527] Contamination of agricultural land with undesirable microorganisms can be detected by analyzing a soil sample for target nucleic acids specific for such microorganisms.

[0528] In further embodiments, the methods disclosed herein can be used to detect target nucleic acids in foods to determine food safety. Food borne illnesses often include infections caused by E. coli, Salmonella sp., Clostridium botulinum, and hepatitis A. Target nucleic acids that are specific for such food borne pathogens can be detected using the methods disclosed herein.

[0529]

[0530] Attorney Docket No.: GRIP-014WO

[0531] Any appropriate sample can be evaluated, e.g., for the presence, absence or amount of target nucleic acids using the methods, devices, analyzers, and kits provided herein. In some cases, a sample can be a biological sample. In some cases, a sample can be an environmental sample. A sample can contain whole cells, cellular fragments, DNA, RNA, viruses, virus fragments and / or proteins. Examples of samples that can be used in the methods, devices, analyzers, and kits described herein include, without limitation, biological samples (e.g., blood (e.g., whole blood, a blood spot, serum or plasma) samples, urine samples, saliva samples, mucus samples, sputum samples, bronchial lavage samples, fecal samples, buccal samples, nasal samples, amniotic fluid samples, cerebrospinal fluid samples, synovial fluid samples, pleural fluid samples, pericardial fluid samples, peritoneal fluid samples, urethral samples, cervical samples, genital sore samples, hair samples and skin samples), environmental samples (e.g., water samples, soil samples and air samples), food samples (e.g., meat samples, produce samples or drink samples), plant samples (e.g., leaf samples, root samples, flower samples, stem samples, pollen samples and seed samples), industrial samples (e.g., air filter samples, samples collected from work stations, samples collected from storage facilities and / or products (e.g., grain silos), and samples collected from transportation machinery (e.g., railroad cars, trucks or pipelines)). In some cases, the methods, devices, and kits provided herein can retain the sample for safe and clean disposal.

[0532] A sample to be evaluated (e.g., for the presence, absence or amount of a target nucleic acid) using the methods, devices, analyzers, and kits provided herein can be obtained using any appropriate technique. For example, biological samples can be obtained using non-invasive (e.g., swab) techniques or invasive techniques (e.g., venipuncture, finger stick or biopsy). For example, an environmental sample and / or an industrial sample can be obtained using a surface swab technique. In some cases, a sample can be a liquid sample. A liquid sample can be any appropriate volume. For example, a liquid sample can include from about 10 microliters (pL) to about 10 mL (e.g., from about 10 pL to about 8 mL, from about 10 pL to about 5 mL, from about 10 pL to about 3 mL, from about 10 pL to about 2 mL, from about 10 pL to about 1 mL, from about 10 pL to about 500 pL, from about 10 pL to about 250 pL, from about 10 pL to about 100 pL, from about 10 pL to about 50 pL, from about 25 pL to about 8 mL, from about 50 pL to about 7 mL, from about 100 pL to about 5 mL, from about 250 pL to about 2 mL, from about 500 pL to about 1 mL, from about 25 pL to about 20 mL, from about 50 pL to about 20 mL, from about 250 pL to about 20 mL, from about 500 pL to about 20 mL, from about 1 mL to about 20 mL, from about 5 mL to about 20 mL, from about 10 mL to about 20 mL, from about 15 mL to about 20 mL).Attorney Docket No.: GRIP-014WO

[0533] A sample to be evaluated using the methods, devices, analyzers, and kits provided herein can be obtained from any appropriate species. In some cases, a sample to be assessed as described herein can be obtained from an animal. In some cases, a sample to be assessed as described herein can be obtained from a mammal (e.g., a human). Examples of mammals that samples can be obtained from include, without limitation, primates (e.g., humans and monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits and rodents (e.g., mice and rats). Other examples of animals that samples can be obtained from include, without limitation, fish, avian species (e.g., chickens, turkeys, ostrich, emus, cranes, and falcons) and non-mammalian animals (e.g., mollusks, frogs, lizards, snakes and insects).

[0534] A sample to be evaluated using the methods, devices, analyzers, and kits provided herein can be obtained from any appropriate plant. In some cases, a sample to be assessed as described herein can be obtained from a crop plant (e.g., corn). Examples of plants include, without limitation, corn, soybeans, wheat, rice, trees, flowers, shrubs, grains, grasses, legumes and fruits.

[0535] In some cases, a sample to be evaluated using the methods, devices, analyzers, and kits provided herein can be obtained from a source (e.g., a mammal or surface) and processed prior to being introduced to a device or system provided herein (e.g., can be pre-processed). Samples that are pre-processed can be pre-processed using one or more appropriate reagents (e.g., enzymes, acids, bases, buffers, detergents, anticoagulants, and / or aptamers) and / or techniques (e.g., purification techniques, centrifugation techniques, amplification techniques, culturing techniques and / or denaturing techniques). For example, a blood sample can be obtained from a mammal (e.g., a human) and treated with one or more anticoagulants.

[0536] Examples of anticoagulants that can be used to pre-process a sample (e.g., a blood sample) include, without limitation, EDTA, citrate (trisodium citrate), heparinates (e.g., sodium, lithium, or ammonium salt of heparin or calcium-titrated heparin), and hirudin. In some cases, a sample (e.g., a sample suspected to contain a microorganism) to be to be introduced to a device or system provided herein can be obtained from a source (e.g., a food preparation surface) and pre-processed by culturing the sample with appropriate culture media for a period of time (e.g., four hours to 24 hours) prior to being introduced to a device or system described herein.

[0537] Examples of other pre-processing techniques that can be performed prior to introducing the sample to a device or system provided herein include, without limitation, centrifugation to obtain cell containing material, centrifugation to obtain cell-free material, filtration to remove cell containing material, cell lysis, nucleic acid purification, protein purification, nucleic acid amplification (e.g., polymerase chain reaction (PGR)), reverse transcription to obtainAttorney Docket No.: GRIP-014WO

[0538] complementary DNA (cDNA), reverse transcription PCR, nucleic acid denaturation and isothermal amplification.

[0539] In some cases, a sample does not require any processing prior to or after being introduced into a device provided herein. For example, a sample (e.g., a sample without any pre-processing or a sample that was pre-processed) can be introduced into a device provided herein and directly evaluated via such device without any sample processing being performed within such device.

[0540] In some cases, the methods, devices, analyzers, and kits provided herein can be designed to process a sample (e.g., a sample without any pre-processing or a sample that was pre-processed) after the sample is introduced into a device provided herein. For example, a sample can be introduced into a device provided herein, subjected to one or more processing steps within such device or system (e.g., one or more processing steps designed to lyse cells and / or one or more processing steps designed to denature nucleic acid) and evaluated by such device.

[0541] The following is offered by way of illustration and not by way of limitation.

[0542] Examples

[0543] The following example(s) is / are offered by way of illustration and not by way of limitation.

[0544] Example 1 - Detecting nucleic acids and diagnosing an infection using a first CRISPR associated nuclease, a Craspase as a second CRISPR associated enzyme, and HRP labeled substrate

[0545] Certain exemplary embodiments of the disclosure are described in FIGS. 1 A to 1 D. A sample will be obtained from a patient suspected of having an infection, for example, a bacterial or a viral infection. RNA will be isolated from the sample and analyzed in a first reaction by combining it with a Cas13a as a first CRISPR associated nuclease having a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The first reaction mixture will also contain a first bait nucleic acid bound to first beads. If the target RNA from the bacterium or virus is present in the sample, Cas13a would cleave the first bait nucleic acids to produce free first bait nucleic acids, as shown in the right panel of FIG. 1 A.

[0546] The first reaction mixture from the right panel of FIG. 1 A will then be filtered to separate the first bait nucleic acids bound to the first beads from the free first bait nucleic acids. The filtrate would constitute a first product mixture. Whether the first product mixture contains theAttorney Docket No.: GRIP-014WO

[0547] free first bait nucleic acid will be analyzed in the second reaction.

[0548] In the second reaction, the first product mixture will be combined with Csx29 having a crRNA that specifically hybridizes with the free first bait nucleic acid and Csx30 fused to HRP bound to a second bead. If the free first bait nucleic acid is present in the second reaction mixture, Csx29 would cleave Csx30 to produce the free HRP labeled Csx30 fragment and the second bead bound Csx30 fragment. This reaction is shown in FIG. 10. The free HRP labeled Csx30 fragment, if produced, will be separated from the second reaction mixture by filtration, centrifugation, or magnet depending on the type of second beads. The second reaction mixture after separating the beads would constitute the second product mixture.

[0549] The presence of free HRP labeled Csx30 fragment in the second product mixture will be detected using HRP based colorimetric reaction. This is shown in FIG. 1D. The second product mixture will be combined with TMB (3, 3', 5, 5'-tetramethylbenzidine) and hydrogen peroxide. If free HRP labeled Csx30 fragment is present in the second product mixture, HRP would oxidize TMB to produce blue solution thereby indicating the presence in the second reaction mixture of the free first bait nucleic acids. This in turn would indicate the presence in the sample of the target RNA from the virus or bacterium.

[0550] Similarly, HRP labeled Csx30 fragment can be detected using a mixture of hydroquinone hydrogen peroxide. HRP catalyzes the oxidation of hydroquinone into benzoquinone using hydrogen peroxide. Hydroquinone is light-colored or colorless, whereas benzoquinone is bright yellow. Thus, the development of yellow color indicates that free HRP labeled Csx30 fragment is present in the second product mixture.

[0551] Alternatively, the presence of free HRP labeled Csx30 fragment in the second product mixture will be detected using a changed in a redox signal of an electrochemical sensor. This is shown in FIG. 1 E. The second product mixture will be filtered onto a surface of an electrochemical sensor, where hydrogen peroxide and TMB are provided in the vicinity of the sensor. When cleaved HRP labeled Csx30 fragment is present in the second product mixture, HRP would oxidize TMB to produce a change in the redox signal of the sensor. This change will be measured by square wave voltammetry as shown in FIG. 1 E. This in turn would indicate the presence in the sample of the target RNA from the virus or bacterium.

[0552] Example 2 - Detecting nucleic acids and diagnosing an infection using a first CRISPR associated nuclease, a Craspase as a second CRISPR associated enzyme, and an electrochemical sensor comprising an HRP labeled substrate

[0553] This exemplary embodiment of the disclosure is described in FIGS. 2A-2B. A firstAttorney Docket No.: GRIP-014WO

[0554] product mixture would be produced as described in Example 1 and FIGS. 1A and 1B. Whether the first product mixture contains the free first bait nucleic acid will be analyzed in the second reaction.

[0555] In the second reaction, the first product mixture will be contacted with Csx29 having a crRNA that specifically hybridizes with the free first bait nucleic acid and a sensor comprising HRP labeled Csx30 in presence of TMP and hydrogen peroxide. If the first product mixture contains free first bait nucleic acid, Csx29 would cleave HRP labeled Csx30 fragment, taking it away from the sensor. Removing HRP from the sensor would attenuate TMB related redox signal. This change will be measured by square wave voltammetry as shown in FIG. 2B.

[0556] Change in the redox signal of the sensor will indicate the presence in the second reaction mixture of the free first bait nucleic acids. This in turn would indicate the presence in the sample of the target RNA from the virus or bacterium.

[0557] Example 3 - Detecting nucleic acids and diagnosing an infection using a first CRISPR associated nuclease, a second CRISPR associated nuclease, and an electrochemical sensor comprising an methylene blue labeled second bait nucleic acid

[0558] This exemplary embodiment of the disclosure is described in FIGS. 3A to 3C. A sample will be obtained from a patient suspected of having an infection, for example, a bacterial or a viral infection. RNA will be isolated from the sample and analyzed in a first reaction mixture by combining with a Cas13a as a first CRISPR associated nuclease having a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The first reaction mixture will also contain a first bait nucleic acid bound to first beads. If the target RNA from the bacterium or virus is present in the sample, Cas13a would cleave the first bait nucleic acids to produce free first bait nucleic acids, as shown in the middle panel of FIG. 3A. The first reaction mixture from the middle panel of FIG. 3A will then be filtered to separate the first bait nucleic acids bound to the beads from the free first bait nucleic acids. The filtrate would constitute a first product mixture, which is shown in the right panel of FIG. 3A. Whether the first product mixture contains the free first bait nucleic acid will be analyzed in the second reaction.

[0559] In the second reaction, the first product mixture is contacted with Cas12a having a crRNA that specifically hybridizes with the free first bait nucleic acid and a sensor comprising methylene blue labeled DNA (labeled second bait nucleic acid) conjugated to an electrochemical sensor. If the first product mixture contains free first bait nucleic acid, Cas12a cleaves methylene-blue labeled DNA. This removes methylene blue away from the sensor. This is shown in FIG. 3B.Attorney Docket No.: GRIP-014WO

[0560] Removing methylene blue from the sensor would attenuate methylene blue related redox signal. This change will be measured by square wave voltammetry as shown in FIG. 3C.

[0561] Change in the redox signal of the sensor would indicate the presence in the second reaction mixture of the free first bait nucleic acids. This in turn would indicate the presence in the sample of the target RNA from the virus or bacterium.

[0562] Example 4 -In a single reaction chamber, detecting nucleic acids and diagnosing an infection using a first CRISPR associated nuclease, a second CRISPR associated enzyme, and an electrochemical sensor

[0563] This exemplary embodiment of the disclosure is described in FIGS. 4A to 4B. In this exemplary embodiment, both the first CRISPR associated nuclease reaction and the second CRISPR associated enzyme (Craspase or nuclease) reactions are performed in the same reaction chamber.

[0564] A sample will be obtained from a patient suspected of having an infection, for example, a bacterial or a viral infection. RNA will be isolated from the sample and analyzed in a first reaction mixture by combining with a Cas13a as a first CRISPR associated nuclease having a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The first reaction mixture will also contain a first bait nucleic acid having a secondary structure that prevents the second CRISPR associated enzyme from binding to an uncleaved first bait nucleic acid. Certain such secondary structures include toehold switch, inverted repeat, hairpins, triplex, slipped structures, or cruciform structure.

[0565] If the target RNA from the bacterium or virus is present in the sample, Cas13a would cleave the first bait nucleic acids to produce free first bait nucleic acids, as shown in the middle panel of FIG. 4A. The first reaction mixture also further contains a Craspase having a crRNA that specifically hybridizes with the free first bait nucleic acid and, conjugated to a bead, an HRP-labeled Csx30 substrate for the Craspase.

[0566] If the free first bait nucleic acid is produced in the first reaction mixture, the Craspase detects the free first bait nucleic acid and cleaves the HRP-labeled Csx30 substrate into an HRP-labeled fragment and an unlabeled fragment. This is shown in the right panel of FIG. 4A.

[0567] The presence of free HRP labeled Csx30 fragment in the first reaction mixture will be detected using a changed in a redox signal of an electrochemical sensor. This is shown in FIG.

[0568] 4B. The first reaction mixture will be filtered onto a surface of an electrochemical sensor, where hydrogen peroxide and TMB are provided in the vicinity of the sensor. When cleaved HRP labeled Csx30 fragment is present in the filtered first reaction mixture, HRP would oxidize TMBAttorney Docket No.: GRIP-014WO

[0569] to produce a change in the redox signal of the sensor. This change will be measured by square wave voltammetry as shown in FIG. 4B. This in turn would indicate the presence in the sample of the target RNA from the virus or bacterium.

[0570] Notwithstanding the appended claims, the invention may be defined by the following clauses:

[0571] 1. A method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:

[0572] (a) combining in one or more reaction mixtures:

[0573] i) the sample,

[0574] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0575] iii) a first bait nucleic acid,

[0576] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0577] v) a reactant for the second CRISPR associated enzyme; and

[0578] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0579] 2. The method of Clause 1 , wherein the first reaction occurs in a first reaction mixture and the second reaction occurs in a second reaction mixture.

[0580] 3. The method of Clause 2, comprising:

[0581] (a) preparing the first rection mixture by combining the sample with:

[0582] (i) the first CRISPR associated nuclease comprising the first crRNA; and ii) the first bait nucleic acid bound to a first solid support;

[0583] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0584] (c) preparing the second reaction mixture by combining the first product mixture with:

[0585] i) the second CRISPR associated enzyme comprising the second crRNA that specifically hybridizes with the cleaved first bait nucleic acid; and

[0586] ii) the reactant for the second CRISPR associated enzyme, andAttorney Docket No.: GRIP-014WO

[0587] (d) assaying the second reaction mixture for the product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.

[0588] 4. The method Clause 3, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

[0589] 5. The method of Clause 4, wherein, when the target nucleic acid is present in the sample in the first reaction mixture, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support to produce a free first bait nucleic acid.

[0590] 6. The method of Clause 4 or 5, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate.

[0591] 7. The method of Clause 6, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0592] 8. The method of any one of Clauses 4 to 7, wherein the substrate is bound to a second solid support and, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a free substrate fragment and a second solid support bound substrate fragment.

[0593] 9. The method of any one of Clauses 4 to 8, wherein the substrate comprises a label and, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled second solid support bound substrate fragment.

[0594] 10. The method of Clause 9, wherein assaying the second reaction mixture for cleavage of the substrate comprises detecting the free labeled substrate fragment.

[0595] 11. The method of Clause 10, wherein detecting the free labeled substrate fragment comprises contacting the second reaction mixture with a binding partner for the label, wherein the binding partner for the label is immobilized on a matrix.

[0596] 12. The method of Clause 11, wherein the matrix is an adsorbent material in a detection region of a lateral flow assay device.

[0597] 13. The method of Clause 12, wherein the method comprises loading the first product mixture into a reaction region of the lateral flow assay device, the reaction region comprising the Craspase comprising the second crRNA and the labeled substrate immobilized in the reaction region, and wherein the reaction region is fluidically connected to the detection region such that the labeled substrate fragment produced in the reaction region migrates to the detection region.Attorney Docket No.: GRIP-014WO

[0598] 14. The method of any one of Clauses 11 to 13, wherein the binding partner for the label is: i) an antibody or a binding fragment thereof that specifically binds the label, ii) an aptamer that specifically binds the label, iii) a protein that specifically binds the label, or iv) a nucleic acid that specifically binds the label.

[0599] 15. The method of any one of Clauses 11 to 14, wherein binding of the binding partner and the label produces a detectable signal.

[0600] 16. The method of Clause 15, wherein the detectable signal is produced from a tag bound to the label.

[0601] 17. The method of Clause 16, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0602] 18. The method of Clause 9 or 10, wherein the label is an electrochemically active species.

[0603] 19. The method of Clause 18, wherein the electrochemically active species is methylene blue.

[0604] 20. The method of Clause 9 or 10, wherein the label is an enzyme.

[0605] 21. The method of Clause 20, wherein the enzyme is an oxidoreductase.

[0606] 22. The method of Clause 21 , wherein the oxidoreductase is horseradish peroxidase.

[0607] 23. The method of any one of Clauses 9 to 10 and 18 to 22, wherein the second solid support is a sensor and cleavage of the substrate produces the labeled free substrate fragment thereby causing a change in a property of the sensor.

[0608] 24. The method of Clause 23, wherein the change in the property of the sensor is a change in an electrochemical property.

[0609] 25. The method of Clause 24, wherein the electrochemical property is a redox signal.

[0610] 26. The method of Clause 25, wherein assaying the second reaction mixture for cleavage of the substrate comprises determining the change in the redox signal of the sensor by square wave voltammetry.

[0611] 27. The method of Clause 23, wherein the change in the property of the sensor is a change in an electrical property.

[0612] 28. The method of Clause 27, wherein the electrical property is electrical resistance or Dirac voltage.

[0613] 29. The method of any one of Clauses 23 to 28, wherein the sensor is a graphene sensor.

[0614] 30. The method of any one of Clauses 9 to 10 and 18 to 22, wherein the second solid support is a second bead.Attorney Docket No.: GRIP-014WO

[0615] 31. The method of Clause 30, comprising separating the second bead from the second reaction mixture to produce a second product mixture.

[0616] 32. The method of Clause 31 , wherein separating the second bead from the second reaction mixture comprises centrifuging or filtering the Asecond reaction mixture.

[0617] 33. The method of Clause 32, wherein the second bead is a second magnetic bead and separating the second magnetic bead from the second reaction mixture comprises capturing the second magnetic bead with a magnet.

[0618] 34. The method of any one of Clauses 31 to 33, further comprising detecting the free substrate fragment in the second product mixture.

[0619] 35. The method of Clause 34, wherein the free substrate fragment is an oxidoreductase labeled substrate fragment.

[0620] 36. The method of Clause 35, wherein the oxidoreductase is horseradish peroxidase.

[0621] 37. The method of Clause 36, wherein the method comprises contacting the second product mixture with: 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide.

[0622] 38. The method of Clause 37, wherein, if the second product mixture comprises the free substrate fragment comprising horseradish peroxidase, the horseradish peroxidase degrades hydrogen peroxide and oxidizes TMB to produce blue color.

[0623] 39. The method of any one of Clauses 8 to 10 and 18 to 22, wherein assaying the second reaction mixture for cleavage of the substrate comprises detecting with a sensor the presence of any free substrate fragment produced by the cleavage of the substrate bound to the second solid support, and wherein the sensor comprises a probe that specifically binds to the free substrate fragment, and wherein the method comprises detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe.

[0624] 40. The method of Clause 38, wherein the probe that specifically binds to the free substrate fragment is a protein or an aptamer.

[0625] 41. The method of any one of Clauses 39 to 40, wherein the probe comprises an electrochemical reporter.

[0626] 42. The method of any one of Clauses 39 to 41 , wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0627] 43. The method of Clause 42, wherein the electrical property of the sensor is conductivity of the sensor or Dirac voltage of the sensor.

[0628] 44. The method of any one of Clauses 39 to 41 , wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.Attorney Docket No.: GRIP-014WO

[0629] 45. The method of Clause 44, wherein the electrochemical property of the sensor is a redox signal.

[0630] 46. The method of any one of Clauses 39 to 45, wherein the sensor is a graphene sensor.

[0631] 47. The method of any one of Clauses 4 to 46, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate is Csx30.

[0632] 48. The method of Clause 3, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid, wherein the second bait nucleic acid is bound to a second solid support.

[0633] 49. The method of Clause 48, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support to produce a free first bait nucleic acid.

[0634] 50. The method of Clause 49, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the second solid support.

[0635] 51. The method of Clause 49 or 50, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[0636] 52. The method of any one of Clauses 48 to 51 , wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[0637] 53. The method of Clause 51 , wherein the second complex comprises a second single guide RNA (sgRNA).

[0638] 54. The method of any one of Clauses 48 to 53, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[0639] 55. The method of Clause 54, wherein the CRISPR-Cas Type V is CRISPR-Cas12.

[0640] 56. The method of any one of Clauses 48 to 55, wherein the second solid support is a second bead.

[0641] 57. The method of Clause 56, wherein the second bead is a second magnetic bead.

[0642] 58. The method of Clause 56 or 57, further comprising separating the second reaction mixture from the second solid support to produce a third product mixture.

[0643] 59. The method of Clause 58, wherein the second solid support is the second magnetic bead and separating the second reaction mixture from the second solid support comprises capturing the second magnetic bead with a magnet.

[0644] 60. The method of Clause 58, wherein separating the second reaction mixture from the second solid support comprises centrifuging or filtering the second reaction mixture.Attorney Docket No.: GRIP-014WO

[0645] 61. The method of any one of Clauses 58 to 60, wherein the assaying the second reaction mixture for the cleavage of the second bait nucleic acid comprises assaying the third product mixture with a sensor.

[0646] 62. The method of Clause 61 , wherein assaying the third product mixture with the sensor comprises detecting with the sensor the presence in the third product mixture of any free second bait nucleic acid produced by the cleavage of the second bait nucleic acid bound to the second solid support.

[0647] 63. The method of Clause 61 or 62, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[0648] 64. The method of Clause 63, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[0649] 65. The method of Clause 64, wherein the protein is an RNA binding protein.

[0650] 66. The method of Clause 64, wherein the protein is a DNA binding protein.

[0651] 67. The method of Clause 63, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[0652] 68. The method of Clause 67, wherein the nucleic acid is an RNA.

[0653] 69. The method of Clause 67, wherein the nucleic acid is a DNA.

[0654] 70. The method of Clause 63, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[0655] 71. The method of any one of Clauses 63 to 70, wherein the probe comprises an electrochemical reporter.

[0656] 72. The method of any one of Clauses 48 to 55, wherein the second solid support is a sensor, and wherein the assaying the second reaction mixture with the sensor for cleavage of the second bait nucleic acid comprises detecting a change in a property of the sensor caused by the cleavage of the second bait nucleic acid.

[0657] 73. The method of Clause 72, wherein the property of the sensor is an electrical property of the sensor.

[0658] 74. The method of Clause 73, wherein the electrical property is conductivity or Dirac voltage.

[0659] 75. The method of Clause 72, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0660] 76. The method of Clause 75, wherein the electrochemical property is a redox signal.Attorney Docket No.: GRIP-014WO

[0661] 77. The method of any one of Clauses 72 to 76, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a labeled free second bait nucleic acid.

[0662] 78. The method of Clause 77, wherein the label is an electrochemically active species.

[0663] 79. The method of Clause 78, wherein the electrochemically active species is methylene blue.

[0664] 80. The method of Clause 78, wherein the label is an enzyme.

[0665] 81. The method of Clause 80, wherein the enzyme is an oxidoreductase.

[0666] 82. The method of Clause 81 , wherein the oxidoreductase is horseradish peroxidase.

[0667] 83. The method of any one of Clauses 61 to 82, wherein the sensor is a graphene sensor.

[0668] 84. The method of any one of Clauses 3 to 83, wherein the first solid support is a first bead.

[0669] 85. The method of Clause 84, wherein separating the first reaction mixture from the first solid support comprises centrifuging or filtering the first reaction mixture.

[0670] 86. The method of Clause 84, wherein the first bead is a first magnetic bead.

[0671] 87. The method of Clause 86, wherein separating the first reaction mixture from the first solid support comprises capturing the first magnetic bead with a magnet.

[0672] 88. The method of Clause 1 , wherein the first reaction and the second reaction occur in the same reaction mixture.

[0673] 89. The method of Clause 88, wherein when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid to produce a cleaved first bait nucleic acid.

[0674] 90. The method of Clause 89, wherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

[0675] 91. The method of Clause 90, wherein the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid.

[0676] 92. The method of Clause 91 , wherein the secondary structure is selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0677] 93. The method any one of Clauses 88 to 92, wherein the second CRISPR associated enzyme is a Craspase and the reactant is a substrate for the Craspase.Attorney Docket No.: GRIP-014WO

[0678] 94. The method of Clause 93, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate.

[0679] 95. The method of Clause 94, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0680] 96. The method of any one of Clauses 93 to 95, wherein the substrate is bound to a solid support and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0681] 97. The method of Clause 96, wherein the substrate comprises a label and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled solid support bound substrate fragment.

[0682] 98. The method of any one of Clauses 96 or 97, wherein the solid support is a sensor and cleavage of the substrate produces the free labeled substrate fragment thereby causing a change in a property of the sensor.

[0683] 99. The method of Clause 98, wherein the change in the property of the sensor is a change in an electrochemical property.

[0684] 100. The method of Clause 99, wherein the electrochemical property is a redox signal.

[0685] 101. The method of Clause 100, wherein assaying the reaction mixture for cleavage of the substrate comprises determining the change in the redox signal of the sensor by square wave voltammetry.

[0686] 102. The method of Clause 98, wherein the change in the property of the sensor is a change in an electrical property.

[0687] 103. The method of Clause 102, wherein the electrical property is electrical resistance or Dirac voltage.

[0688] 104. The method of any one of Clauses 98 to 103, wherein the sensor is a graphene sensor.

[0689] 105. The method of Clause 96 or 97, wherein the solid support is a bead.

[0690] 106. The method of Clause 105, comprising separating the bead from the reaction mixture to produce a product mixture.

[0691] 107. The method of Clause 106, wherein separating the bead from the reaction mixture comprises centrifuging or filtering the reaction mixture.Attorney Docket No.: GRIP-014WO

[0692] 108. The method of Clause 105, wherein the bead is a magnetic bead and separating the magnetic bead from the reaction mixture comprises capturing the magnetic bead with a magnet.

[0693] 109. The method of any one of Clauses 106 to 108, further comprising detecting the free substrate fragment in the product mixture.

[0694] 110. The method of Clause 109, wherein the free substrate fragment is an oxidoreductase labeled free substrate fragment.

[0695] 111. The method of Clause 110, wherein the oxidoreductase is horseradish peroxidase.

[0696] 112. The method of Clause 111 , wherein the method comprises contacting the product mixture with: 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide.

[0697] 113. The method of Clause 112, wherein, if the product mixture comprises the free substrate fragment comprising horseradish peroxidase, the horseradish peroxidase degrades hydrogen peroxide and oxidizes TMB to produce blue color.

[0698] 114. The method of any one of Clauses 105 to 113, wherein assaying the reaction mixture for cleavage of the substrate comprises detecting with a sensor the presence of any free substrate fragment produced by the cleavage of the substrate bound to the solid support, and wherein the sensor comprises a probe that specifically binds to the free substrate fragment, and wherein the method comprises detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe.

[0699] 115. The method of Clause 114, wherein the probe that specifically binds to the free substrate fragment is a protein or an aptamer.

[0700] 116. The method of Clause 114 or 115, wherein the probe comprises an electrochemical reporter.

[0701] 117. The method of any one of Clauses 114 to 116, wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0702] 118. The method of Clause 117, wherein the electrical property of the sensor is conductivity of the sensor or Dirac voltage of the sensor.

[0703] 119. The method of any one of Clauses 114 to 116, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0704] 120. The method of Clause 119, wherein the electrochemical property of is a redox signal.

[0705] 121. The method of any one of Clauses 114 to 120, wherein the sensor is a graphene sensor.

[0706] inAttorney Docket No.: GRIP-014WO

[0707] 122. The method of any one of Clauses 93 to 121, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate is Csx30.

[0708] 123. The method any one of Clauses 88 to 92, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid, wherein the second bait nucleic acid is bound to a solid support.

[0709] 124. The method of Clause 123, wherein, when the cleaved first bait nucleic acid is present in the reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the solid support.

[0710] 125. The method of any one of Clauses 123 to 124, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[0711] 126. The method of any one of Clauses 123 to 125, wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[0712] 127. The method of Clause 126, wherein the second complex comprises a second single guide RNA (sgRNA).

[0713] 128. The method of any one of Clauses 123 to 127, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[0714] 129. The method of Clause 128, wherein the CRISPR-Cas Type V is CRISPR-Cas12.

[0715] 130. The method of any one of Clauses 123 to 129, wherein the solid support is a bead.

[0716] 131. The method of Clause 130, wherein the bead is a magnetic bead.

[0717] 132. The method of Clause 130 or 131 , further comprising separating the reaction mixture from the solid support to produce a product mixture.

[0718] 133. The method of Clause 132, wherein the solid support is the magnetic bead and separating the reaction mixture from the solid support comprises capturing the magnetic bead with a magnet.

[0719] 134. The method of Clause 132, wherein separating the reaction mixture from the solid support comprises centrifuging or filtering the reaction mixture.

[0720] 135. The method of any one of Clauses 132 to 134, wherein the assaying the reaction mixture for the cleavage of the second bait nucleic acid comprises assaying the product mixture with a sensor.

[0721] 136. The method of Clause 135, wherein assaying the product mixture with the sensor comprises detecting with the sensor the presence in the product mixture of any free second bait nucleic acid produced by the cleavage of the second bait nucleic acid bound to the solid support.Attorney Docket No.: GRIP-014WO

[0722] 137. The method of Clause 135 or 136, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[0723] 138. The method of Clause 137, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[0724] 139. The method of Clause 138, wherein the protein is an RNA binding protein.

[0725] 140. The method of Clause 138, wherein the protein is a DNA binding protein.

[0726] 141. The method of Clause 138, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[0727] 142. The method of Clause 141 , wherein the nucleic acid is an RNA.

[0728] 143. The method of Clause 141 , wherein the nucleic acid is a DNA.

[0729] 144. The method of Clause 138, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[0730] 145. The method of any one of Clauses 137 to 144, wherein the probe comprises an electrochemical reporter.

[0731] 146. The method of any one of Clauses 123 to 129, wherein the solid support is a sensor, and wherein the assaying the reaction mixture with the sensor for cleavage of the second bait nucleic acid comprises detecting a change in a property of the sensor caused by the cleavage of the second bait nucleic acid.

[0732] 147. The method of Clause 146, wherein the property of the sensor is an electrical property of the sensor.

[0733] 148. The method of Clause 147, wherein the electrical property is conductivity or Dirac voltage.

[0734] 149. The method of Clause 146, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0735] 150. The method of Clause 149, wherein the electrochemical property is a redox signal.

[0736] 151. The method of any one of Clauses 146 to 150, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a labeled free second bait nucleic acid.

[0737] 152. The method of Clause 151 , wherein the label is an electrochemically active species.

[0738] 153. The method of Clause 152, wherein the electrochemically active species is methylene blue.

[0739] 154. The method of Clause 152, wherein the label is an enzyme.Attorney Docket No.: GRIP-014WO

[0740] 155. The method of Clause 154, wherein the enzyme is an oxidoreductase.

[0741] 156. The method of Clause 155, wherein the oxidoreductase is horseradish peroxidase.

[0742] 157. The method of any one of Clauses 135 to 156, wherein the sensor is a graphene sensor.

[0743] 158. The method of any one of the preceding Clauses, wherein the target sequence has a length from 17 to 30 nucleotides.

[0744] 159. The method of any one of the preceding Clauses, wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[0745] 160. The method of any one of the preceding Clauses, wherein the first crRNA is in a first complex comprising a first trans-activating crRNA (tracrRNA).

[0746] 161. The method of Clause 160, wherein the first complex comprises a first single guide RNA (sgRNA).

[0747] 162. The method of any one of the preceding Clauses, wherein the first CRISPR associated nuclease is a CRISPR-Cas Type VI.

[0748] 163. The method of Clause 162, wherein the CRISPR-Cas Type VI is CRISPR-Cas13.

[0749] 164. The method of any one of the preceding Clauses, wherein the target nucleic acid is a nucleic acid from a microorganism.

[0750] 165. The method of Clause 164, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0751] 166. A method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:

[0752] (a) combining in an analyzer one or more reaction mixtures comprising:

[0753] i) the sample,

[0754] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0755] iii) a first bait nucleic acid,

[0756] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0757] v) a reactant for the second CRISPR associated enzyme; and

[0758] (b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.Attorney Docket No.: GRIP-014WO

[0759] 167. The method of Clause 166, wherein the first reaction occurs in a first reaction mixture and the second reaction occurs in a second reaction mixture.

[0760] 168. The method of Clause 167, comprising:

[0761] (a) preparing the first reaction mixture in a first reaction chamber of an analyzer, wherein the first reaction mixture comprises:

[0762] i) the first CRISPR associated nuclease comprising the first crRNA; and ii) the first bait nucleic acid bound to a first solid support;

[0763] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0764] (c) preparing the second reaction mixture in a second reaction chamber of the analyzer, wherein the second reaction mixture comprises:

[0765] i) the first product mixture,

[0766] ii) the second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[0767] iii) the reactant for the second CRISPR associated enzyme; and (d) assaying the second reaction mixture for the product resulting from activity of the second CRISPR associated enzyme on the reactant to determine whether the target nucleic acid is present in the sample.

[0768] 169. The method of Clause 168, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant for the second CRISPR associated enzyme is a substrate for the Craspase, wherein the substrate is bound to a second solid support.

[0769] 170. The method of Clause 169, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.

[0770] 171. The method of Clause 169 or 170, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate bound to the second solid support.

[0771] 172. The method of any one of Clauses 169 to 171 , wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0772] 173. The method of any one of Clauses 169 to 172, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrateAttorney Docket No.: GRIP-014WO

[0773] bound to the second solid support to produce a free substrate fragment and a second solid support bound substrate fragment.

[0774] 174. The method of any one of Clauses 169 to 173, wherein the substrate comprises a label and, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled second solid support bound substrate fragment.

[0775] 175. The method of Clause 174, wherein the label is an electrochemically active species.

[0776] 176. The method of Clause 175, wherein the electrochemically active species is methylene blue.

[0777] 177. The method of Clause 174, wherein the label is an enzyme.

[0778] 178. The method of Clause 177, wherein the enzyme is an oxidoreductase.

[0779] 179. The method of Clause 178, wherein the oxidoreductase is horseradish peroxidase.

[0780] 180. The method of any one of Clauses 175 to 178, wherein the second solid support is a sensor and cleavage of the substrate produces the labeled free substrate fragment thereby causing a change in a property of the sensor.

[0781] 181. The method of Clause 180, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0782] 182. The method of Clause 181 , wherein the electrochemical property is a redox signal.

[0783] 183. The method of Clause 182, wherein assaying the second reaction mixture for cleavage of the substrate comprises determining the change in the redox signal of the sensor by square wave voltammetry.

[0784] 184. The method of Clause 180, wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0785] 185. The method of Clause 184, wherein the electrical property is electrical resistance or Dirac voltage.

[0786] 186. The method of any one of Clauses 180 to 185, wherein the sensor is a graphene sensor.

[0787] 187. The method of any one of Clauses 173 to 179, wherein the second solid support is a second bead.Attorney Docket No.: GRIP-014WO

[0788] 188. The method of Clause 187, further comprising detecting the free substrate fragment in a second product mixture produced by separating the second bead from the second reaction mixture.

[0789] 189. The method of Clause 188, wherein the free substrate fragment is an oxidoreductase labeled substrate fragment.

[0790] 190. The method of Clause 189, wherein the oxidoreductase is horseradish peroxidase.

[0791] 191. The method of Clause 190, wherein the method comprises contacting the second product mixture with: 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide.

[0792] 192. The method of Clause 191 , wherein, if the second product mixture comprises the free substrate fragment comprising horseradish peroxidase, the horseradish peroxidase degrades hydrogen peroxide and oxidizes TMB to produce blue color.

[0793] 193. The method of any one of Clauses 173 to 179, wherein the assaying the second reaction mixture for cleavage of the substrate comprises detecting with a sensor the presence of any free substrate fragment produced by the cleavage of the substrate bound to the second solid support, and wherein the sensor comprises a probe that specifically binds to the free substrate fragment, and wherein the method comprises detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe.

[0794] 194. The method of Clause 193, wherein the probe that specifically binds to the free substrate fragment is a protein or an aptamer.

[0795] 195. The method of Clause 193 or 194, wherein the probe comprises an electrochemical reporter.

[0796] 196. The method of any one of Clauses 193 to 195, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0797] 197. The method of Clause 196, wherein the electrochemical property is a redox signal.

[0798] 198. The method of any one of Clauses 193 to 195, wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0799] 199. The method of Clause 198, wherein the electrical property of the sensor is conductivity of the sensor or Dirac voltage.

[0800] 200. The method of any one of Clauses 193 to 199, wherein the sensor is a graphene sensor.

[0801] 201. The method of any one of Clauses 173 to 200, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate is Csx30.Attorney Docket No.: GRIP-014WO

[0802] 202. The method of Clause 168, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid, wherein the second bait nucleic acid is bound to a second solid support.

[0803] 203. The method of Clause 202, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support to produce a free first bait nucleic acid.

[0804] 204. The method of Clause 203, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the second solid support.

[0805] 205. The method of any one of Clauses 202 to 204, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[0806] 206. The method of any one of Clauses 202 to 205, wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[0807] 207. The method of Clause 206, wherein the second complex comprises a second single guide RNA (sgRNA).

[0808] 208. The method of any one of Clauses 202 to 207, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[0809] 209. The method of Clause 208, wherein the CRISPR-Cas Type V is CRISPR-Cas12.

[0810] 210. The method of any one of Clauses 202 to 209, wherein the second solid support is a second bead.

[0811] 211. The method of Clause 210, wherein the second bead is a second magnetic bead.

[0812] 212. The method of Clause 210 or 211 , further comprising separating the second reaction mixture from the second solid support to produce a third product mixture.

[0813] 213. The method of Clause 212, wherein the second solid support is the second magnetic bead and separating the second reaction mixture from the second solid support comprises capturing the second magnetic bead with a magnet.

[0814] 214. The method of Clause 211 or 212, wherein separating the second reaction mixture from the second solid support comprises centrifuging or filtering the second reaction mixture.

[0815] 215. The method of any one of Clauses 211 to 214, wherein assaying the second reaction mixture for the cleavage of the second bait nucleic acid comprises assaying the third product mixture with a sensor.

[0816] 216. The method of Clause 215, wherein assaying the third product mixture with the sensor comprises detecting with the sensor the presence in the third product mixture of any freeAttorney Docket No.: GRIP-014WO

[0817] second bait nucleic acid produced by the cleavage of the second bait nucleic acid bound to the second solid support.

[0818] 217. The method of Clause 215 or 216, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[0819] 218. The method of Clause 217, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[0820] 219. The method of Clause 218, wherein the protein is an RNA binding protein.

[0821] 220. The method of Clause 218, wherein the protein is a DNA binding protein.

[0822] 221. The method of Clause 217, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[0823] 222. The method of Clause 221 , wherein the nucleic acid is an RNA.

[0824] 223. The method of Clause 221 , wherein the nucleic acid is a DNA.

[0825] 224. The method of Clause 217, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[0826] 225. The method of any one of Clauses 217 to 224, wherein the probe comprises an electrochemical reporter.

[0827] 226. The method of any one of Clauses 202 to 209, wherein the second solid support is the sensor, and wherein the assaying the second reaction mixture with the sensor for cleavage of the second bait nucleic acid comprises detecting a change in a property of the sensor caused by the cleavage of the second bait nucleic acid.

[0828] 227. The method of Clause 226, wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0829] 228. The method of Clause 227, wherein the electrical property is conductivity or Dirac voltage.

[0830] 229. The method of Clause 228, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0831] 230. The method of Clause 229, wherein the electrochemical property is a redox signal.

[0832] 231. The method of any one of Clauses 226 to 230, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a labeled free second bait nucleic acid.

[0833] 232. The method of Clause 231 , wherein the label is an electrochemically active species.Attorney Docket No.: GRIP-014WO

[0834] 233. The method of Clause 232, wherein the electrochemically active species is methylene blue.

[0835] 234. The method of Clause 231 , wherein the label is an enzyme.

[0836] 235. The method of Clause 234, wherein the enzyme is an oxidoreductase.

[0837] 236. The method of Clause 235, wherein the oxidoreductase is horseradish peroxidase.

[0838] 237. The method of any one of Clauses 215 to 236, wherein the sensor is a graphene sensor.

[0839] 238. The method of any one of the Clauses 168 to 237, wherein the first solid support is a first bead.

[0840] 239. The method of Clause 238, wherein separating the first reaction mixture from the first solid support comprises centrifuging or filtering the first reaction mixture.

[0841] 240. The method of Clause 238, wherein the first bead is a first magnetic bead.

[0842] 241. The method of Clause 240, wherein separating the first reaction mixture from the first solid support comprises capturing the first magnetic bead with a magnet.

[0843] 242. The method of Clause 166, wherein the first reaction and the second reaction are conducted in the same reaction chamber of the analyzer.

[0844] 243. The method of Clause 242, wherein when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid to produce a cleaved first bait nucleic acid.

[0845] 244. The method of Clause 243, wherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

[0846] 245. The method of Clause 244, wherein the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid.

[0847] 246. The method of Clause 245, wherein the secondary structure is selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0848] 247. The method any one of Clauses 242 to 246, wherein the second CRISPR associated enzyme is a Craspase and the reactant is a substrate for the Craspase.

[0849] 248. The method of Clause 247, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate.Attorney Docket No.: GRIP-014WO

[0850] 249. The method of Clause 248, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0851] 250. The method of any one of Clauses 247 to 249, wherein the substrate is bound to a solid support and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0852] 251. The method of Clause 250, wherein the substrate comprises a label and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled solid support bound substrate fragment.

[0853] 252. The method of any one of Clause 250 or 251 , wherein the solid support is a sensor and cleavage of the substrate produces the free labeled substrate fragment thereby causing a change in a property of the sensor.

[0854] 253. The method of Clause 252, wherein the change in the property of the sensor is a change in an electrochemical property.

[0855] 254. The method of Clause 253, wherein the electrochemical property is a redox signal.

[0856] 255. The method of Clause 254, wherein assaying the reaction mixture for cleavage of the substrate comprises determining the change in the redox signal of the sensor by square wave voltammetry.

[0857] 256. The method of Clause 252, wherein the change in the property of the sensor is a change in an electrical property.

[0858] 257. The method of Clause 256, wherein the electrical property is electrical resistance or Dirac voltage.

[0859] 258. The method of any one of Clauses 252 to 257, wherein the sensor is a graphene sensor.

[0860] 259. The method of Clause 250 to 251 , wherein the solid support is a bead.

[0861] 260. The method of Clause 259, comprising separating the bead from the reaction mixture to produce a product mixture.

[0862] 261. The method of Clause 260, wherein separating the bead from the reaction mixture comprises centrifuging or filtering the reaction mixture.Attorney Docket No.: GRIP-014WO

[0863] 262. The method of Clause 261 , wherein the bead is a magnetic bead and separating the magnetic bead from the reaction mixture comprises capturing the magnetic bead with a magnet.

[0864] 263. The method of any one of Clauses 260 to 262, further comprising detecting the free substrate fragment in the product mixture.

[0865] 264. The method of Clause 263, wherein the free substrate fragment is an oxidoreductase labeled free substrate fragment.

[0866] 265. The method of Clause 264, wherein the oxidoreductase is horseradish peroxidase.

[0867] 266. The method of Clause 265, wherein the method comprises contacting the product mixture with: 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide.

[0868] 267. The method of Clause 266, wherein, if the product mixture comprises the free substrate fragment comprising horseradish peroxidase, the horseradish peroxidase degrades hydrogen peroxide and oxidizes TMB to produce blue color.

[0869] 268. The method of any one of Clauses 259 to 267, wherein assaying the reaction mixture for cleavage of the substrate comprises detecting with a sensor the presence of any free substrate fragment produced by the cleavage of the substrate bound to the solid support, and wherein the sensor comprises a probe that specifically binds to the free substrate fragment, and wherein the method comprises detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe.

[0870] 269. The method of Clause 268, wherein the probe that specifically binds to the free substrate fragment is a protein or an aptamer.

[0871] 270. The method of Clause 168 or 269, wherein the probe comprises an electrochemical reporter.

[0872] 271. The method of any one of Clauses 268 to 270, wherein the change in the property of the sensor is a change in an electrical property of the sensor.

[0873] 272. The method of Clause 271 , wherein the electrical property of the sensor is conductivity of the sensor or Dirac voltage of the sensor.

[0874] 273. The method of any one of Clauses 268 to 270, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0875] 274. The method of Clause 273, wherein the electrochemical property of is a redox signal.

[0876] 275. The method of any one of Clauses 268 to 274, wherein the sensor is a graphene sensor.Attorney Docket No.: GRIP-014WO

[0877] 276. The method of any one of Clauses 247 to 275, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate is Csx30.

[0878] 277. The method any one of Clauses 242 to 246, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid, wherein the second bait nucleic acid is bound to a solid support.

[0879] 278. The method of Clause 277, wherein, when the cleaved first bait nucleic acid is present in the reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the solid support.

[0880] 279. The method of any one of Clauses 277 to 278, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[0881] 280. The method of any one of Clauses 277 to 279, wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[0882] 281. The method of Clause 280, wherein the second complex comprises a second single guide RNA (sgRNA).

[0883] 282. The method of any one of Clauses 277 to 281 , wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[0884] 283. The method of Clause 282, wherein the CRISPR-Cas Type V is CRISPR-Cas12.

[0885] 284. The method of any one of Clauses 277 to 283, wherein the solid support is a bead.

[0886] 285. The method of Clause 284, wherein the bead is a magnetic bead.

[0887] 286. The method of Clause 284 or 285, further comprising separating the reaction mixture from the solid support to produce a product mixture.

[0888] 287. The method of Clause 286, wherein the solid support is the magnetic bead and separating the reaction mixture from the solid support comprises capturing the magnetic bead with a magnet.

[0889] 288. The method of Clause 286, wherein separating the reaction mixture from the solid support comprises centrifuging or filtering the reaction mixture.

[0890] 289. The method of any one of Clauses 284 to 288, wherein the assaying the reaction mixture for the cleavage of the second bait nucleic acid comprises assaying the product mixture with a sensor.

[0891] 290. The method of Clause 289, wherein assaying the product mixture with the sensor comprises detecting with the sensor the presence in the product mixture of any free second bait nucleic acid produced by the cleavage of the second bait nucleic acid bound to the solid support.Attorney Docket No.: GRIP-014WO

[0892] 291. The method of Clause 289 or 290, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[0893] 292. The method of Clause 291 , wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[0894] 293. The method of Clause 292, wherein the protein is an RNA binding protein.

[0895] 294. The method of Clause 292, wherein the protein is a DNA binding protein.

[0896] 295. The method of Clause 291 , wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[0897] 296. The method of Clause 295, wherein the nucleic acid is an RNA.

[0898] 297. The method of Clause 295, wherein the nucleic acid is a DNA.

[0899] 298. The method of Clause 291 , wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[0900] 299. The method of any one of Clauses 291 to 298, wherein the probe comprises an electrochemical reporter.

[0901] 300. The method of any one of Clauses 277 to 283, wherein the solid support is a sensor, and wherein the assaying the reaction mixture with the sensor for cleavage of the second bait nucleic acid comprises detecting a change in a property of the sensor caused by the cleavage of the second bait nucleic acid.

[0902] 301. The method of Clause 300, wherein the property of the sensor is an electrical property of the sensor.

[0903] 302. The method of Clause 301 , wherein the electrical property is conductivity or Dirac voltage.

[0904] 303. The method of Clause 300, wherein the change in the property of the sensor is a change in an electrochemical property of the sensor.

[0905] 304. The method of Clause 303, wherein the electrochemical property is a redox signal.

[0906] 305. The method of any one of Clauses 300 to 304, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a labeled free second bait nucleic acid.

[0907] 306. The method of Clause 305, wherein the label is an electrochemically active species.

[0908] 307. The method of Clause 306, wherein the electrochemically active species is methylene blue.Attorney Docket No.: GRIP-014WO

[0909] 308. The method of Clause 305, wherein the label is an enzyme.

[0910] 309. The method of Clause 308, wherein the enzyme is an oxidoreductase.

[0911] 310. The method of Clause 309, wherein the oxidoreductase is horseradish peroxidase.

[0912] 311. The method of any one of Clauses 289 to 310, wherein the sensor is a graphene sensor.

[0913] 312. The method of any one of Clauses 166 to 311 , wherein the target sequence has a length from 17 to 30 nucleotides.

[0914] 313. The method of any one of Clauses 166 to 312, wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[0915] 314. The method of any one of Clauses 166 to 313, wherein the first crRNA is in a complex comprising a trans-activating crRNA (tracrRNA).

[0916] 315. The method of Clause 314, wherein the complex comprises a single guide RNA (sgRNA).

[0917] 316. The method of any one of Clauses 166 to 315, wherein the CRISPR associated nuclease is a CRISPR-Cas Type VI.

[0918] 317. The method of Clause 316, wherein the CRISPR-Cas Type VI is CRISPR-Cas13.

[0919] 318. The method of any one of Clauses 166 to 317, wherein the target nucleic acid is a nucleic acid from a microorganism.

[0920] 319. The method of Clause 318, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0921] 320. A method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:

[0922] (a) combining in one or more reaction mixtures:

[0923] i) the sample,

[0924] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[0925] iii) a first bait nucleic acid,

[0926] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[0927] v) a reactant for the second CRISPR associated enzyme; andAttorney Docket No.: GRIP-014WO

[0928] (b) assaying in a lateral flow assay device whether a product resulting from activity of the second CRISPR associated enzyme on the substrate is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

[0929] 321. The method of Clause 320, wherein the first reaction occurs in a first reaction mixture and the second reaction occurs in a second reaction mixture.

[0930] 322. The method of Clause 321 , comprising:

[0931] (a) in the first reaction mixture, combining the sample with:

[0932] i) the CRISPR associated nuclease comprising the first crRNA; and ii) the first bait nucleic acid bound to a first solid support;

[0933] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[0934] (c) in the second reaction mixture, combining:

[0935] i) the first product mixture,

[0936] ii) the Craspase comprising the second crRNA, and

[0937] iii) the substrate for the Craspase; and

[0938] (d) assaying the second reaction mixture in a lateral flow assay device for the cleavage of the substrate for the Craspase to determine whether the target nucleic acid is present in the sample.

[0939] 323. The method of Clause 322, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.

[0940] 324. The method of Clause 322 or 323, wherein the first solid support is a first bead.

[0941] 325. The method of Clause 324, wherein the first bead is a first magnetic bead.

[0942] 326. The method of Clause 324 or 325, comprising separating the first bead from the first reaction mixture.

[0943] 327. The method of any one of Clauses 324 to 326, wherein separating the first solid support from the first reaction mixture comprises centrifuging or filtering the first reaction mixture.

[0944] 328. The method of Clause 327, wherein separating the first solid support from the first reaction mixture comprises capturing the first magnetic bead with a magnet.

[0945] 329. The method of Clause 322 or 328, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate.Attorney Docket No.: GRIP-014WO

[0946] 330. The method of any one of Clauses 322 to 329, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0947] 331. The method of any one of Clauses 322 to 330, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment.

[0948] 332. The method of any one of Clauses 322 to 331 , wherein the lateral flow assay device comprises a reaction region comprising the Craspase and the labeled substrate, and wherein the method comprises introducing the first product mixture into the reaction region of the lateral flow assay device.

[0949] 333. The method of Clause 332, wherein the reaction region of the lateral flow assay device comprises an absorbent material to which the labeled substrate is immobilized such that when the labeled substrate is cleaved by the Craspase, the free labeled substrate fragment is produced.

[0950] 334. The method of any one of Clauses 322 to 333, comprising introducing the first product mixture in a sample loading region of the lateral flow assay device.

[0951] 335. The method of any one of Clauses 322 to 334, wherein a detection region of the lateral flow assay device comprises a binding partner for the label, wherein the binding partner for the label is immobilized on a matrix in the detection region.

[0952] 336. The method of Clause 335, wherein the binding partner for the label is: i) an antibody or a binding fragment thereof that specifically binds the label, ii) an aptamer that specifically binds the label, iii) a protein that specifically binds the label, or iv) a nucleic acid that specifically binds the label.

[0953] 337. The method of Clause 330 or 336, wherein binding of the binding partner and the label produces a detectable signal.

[0954] 338. The method of Clause 337, wherein the detectable signal is produced from a tag bound to the label.

[0955] 339. The method of Clause 338, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0956] 340. The method of Clause 320, wherein the first reaction and the second reaction occur in the same reaction mixture.

[0957] 341. The method of Clause 340, wherein when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid to produce a cleaved first bait nucleic acid.Attorney Docket No.: GRIP-014WO

[0958] 342. The method of Clause 341 , wherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

[0959] 343. The method of Clause 342, wherein the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid.

[0960] 344. The method of Clause 343, wherein the secondary structure is selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0961] 345. The method of any one of Clauses 341 to 344, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate.

[0962] 346. The method of Clause 345, wherein, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0963] 347. The method of Clause 345 or 346, wherein the substrate is bound to a solid support and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0964] 348. The method of Clause 347, wherein the substrate comprises a label and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled solid support bound substrate fragment.

[0965] 349. The method of Clause 348, comprising separating the solid support from the rest of the reaction mixture to produce a product mixture and introducing the product mixture into a sample loading region of the lateral flow assay device.

[0966] 350. The method of Clause 349, wherein the solid support is a bead.

[0967] 351. The method of Clause 350, wherein the bead is a magnetic bead and separating the reaction mixture from the solid support comprises capturing the magnetic bead with a magnet.

[0968] 352. The method of Clause 350, wherein separating the reaction mixture from the solid support comprises centrifuging or filtering the reaction mixture.

[0969] 353. The method of any one of Clauses 349 to 352, wherein the sample loading region of the lateral flow assay device is fluidically connected to a detection region.

[0970] 354. The method of Clause 353, wherein the detection region of the lateral flow assay device comprises a binding partner for the label immobilized on a matrix.Attorney Docket No.: GRIP-014WO

[0971] 355. The method of Clause 354, wherein the binding partner for the label is: i) an antibody or a binding fragment thereof that specifically binds the label, ii) an aptamer that specifically binds the label, iii) a protein that specifically binds the label, or iv) a nucleic acid that specifically binds the label.

[0972] 356. The method of Clause 354 or 355, wherein binding of the binding partner and the label produces a detectable signal.

[0973] 357. The method of Clause 356, wherein the detectable signal is produced from a tag bound to the label.

[0974] 358. The method of Clause 357, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0975] 359. The method of any one of Clauses 320 to 358, wherein the target nucleic acid is a nucleic acid from a microorganism.

[0976] 360. The method of Clause 359, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0977] 361. The method of any one of Clauses 320 to 360, wherein the target sequence has a length from 17 to 30 nucleotides.

[0978] 362. The method of any one of Clauses 320 to 361 , wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[0979] 363. The method of any one of Clauses 320 to 362, wherein the first crRNA is in a complex comprising a trans-activating crRNA (tracrRNA).

[0980] 364. The method of Clause 363, wherein the complex comprises a single guide RNA (sgRNA).

[0981] 365. The method of any one of Clauses 320 to 364, wherein the CRISPR associated nuclease is a CRISPR-Cas Type VI.

[0982] 366. The method of Clause 365, wherein the CRISPR-Cas Type VI is CRISPR-Cas13.

[0983] 367. A target nucleic acid detection kit, comprising:

[0984] a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid;

[0985] a first bait nucleic acid;

[0986] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with a cleaved first bait nucleic acid; and

[0987] a reactant for the second CRISPR associated enzyme bound to a solid support.Attorney Docket No.: GRIP-014WO

[0988] 368. The kit of Clause 367, wherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

[0989] 369. The kit of Clause 368, wherein the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid.

[0990] 370. The kit of Clause 369, wherein the secondary structure is selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.

[0991] 371. The kit of any one of Clauses 367 to 370, further comprising a sensor configured to detect cleavage of the reactant bound to the second solid support.

[0992] 372. The kit of any one of Clauses 367 to 371 , wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

[0993] 373. The kit of Clause 372, wherein, when the first crRNA hybridizes with the target nucleic acid, the first CRISPR associated nuclease cleaves the first bait nucleic acid.

[0994] 374. The kit of Clause 372 or 373, wherein, when the second crRNA hybridizes with the cleaved first bait nucleic acid, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0995] 375. The kit of Clause 374, wherein, when the second crRNA hybridizes with the cleaved first bait nucleic acid, the Craspase cleaves the substrate bound to the solid support to produce a free substrate fragment and a second solid support bound substrate fragment.

[0996] 376. The kit of any one of Clauses 367 to 375, wherein the solid support is a bead.

[0997] 377. The kit of Clause 376, wherein the bead is a magnetic bead.

[0998] 378. The kit of any one of Clauses 371 to 377, wherein the sensor comprises a probe that specifically binds to the free substrate fragment produced by cleavage of the substrate bound to the solid support.

[0999] 379. The kit of Clause 378, wherein the sensor is a graphene sensor.

[1000] 380. The kit of Clause 378 or 379, wherein the probe that specifically binds to the free substrate fragment is a protein.

[1001] 381. The kit of any one of Clauses 378 to 380, wherein the probe that specifically binds to the free substrate fragment is an aptamer.

[1002] 382. The kit of any one of Clauses 378 to 381 , wherein the probe comprises an electrochemical reporter.Attorney Docket No.: GRIP-014WO

[1003] 383. The kit of any one of Clauses 378 to 382, wherein the sensor detects the cleavage of the substrate by detecting a change in property of the sensor caused by the binding of the free substrate fragment to the probe on the sensor.

[1004] 384. The kit of Clause 383, wherein the property of the sensor is an electrical property of the sensor.

[1005] 385. The kit of Clause 384, wherein the electrical property is conductivity of the sensor or Dirac voltage.

[1006] 386. The kit of Clause 383, wherein the property of the sensor is an electrochemical property of the sensor.

[1007] 387. The kit of Clause 386, wherein the electrochemical property sensor a redox signal.

[1008] 388. The kit of any one of Clauses 378 to 387, wherein the solid support to which the substrate is bound is the sensor configured to detect cleavage of the substrate.

[1009] 389. The kit of Clause 388, wherein the sensor is an electrochemical sensor.

[1010] 390. The kit of Clause 388 or 389, wherein the substrate comprises a label.

[1011] 391. The kit of Clause 390, wherein the Craspase mediated cleavage of the substrate causes release of the label from the sensor thereby changing a property of the electrochemical sensor.

[1012] 392. The kit of Clause 390 or 391 , wherein the label is an electrochemically active species.

[1013] 393. The kit of Clause 392, wherein the electrochemically active species is methylene blue.

[1014] 394. The kit of Clause 390 or 391 , wherein the label is an enzyme.

[1015] 395. The kit of Clause 394, wherein the enzyme is an oxidoreductase.

[1016] 396. The kit of Clause 395, wherein the oxidoreductase is horseradish peroxidase. 397. The kit of any one of Clauses 367 to 396, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate for the Craspase is Csx30.

[1017] 398. The kit of any one of Clauses 367 to 371 , wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid.

[1018] 399. The kit of Clause 398, wherein, when the first crRNA hybridizes with the target nucleic acid, the first CRISPR associated nuclease cleaves the first bait nucleic acid.Attorney Docket No.: GRIP-014WO

[1019] 400. The kit of Clause 398 or 399, wherein, when the second crRNA hybridizes with the cleaved first bait nucleic acid, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the solid support.

[1020] 401. The kit of any one of Clauses 398 to 400, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[1021] 402. The kit of any one of Clauses 398 to 401 , wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[1022] 403. The kit of Clause 402, wherein the second complex comprises a second single guide RNA (sgRNA).

[1023] 404. The kit of any one of Clauses 398 to 403, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[1024] 405. The kit of Clause 404, wherein the CRISPR-Cas Type V is CRISPR-Cas12. 406. The kit of any one of Clauses 398 to 405, wherein the solid support is a bead. 407. The kit of Clause 406, wherein the second bead is a second magnetic bead. 408. The kit of any one of Clauses 398 to 407, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[1025] 409. The kit of Clause 408, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[1026] 410. The kit of Clause 409, wherein the protein is an RNA binding protein.

[1027] 411. The kit of Clause 409, wherein the protein is a DNA binding protein.

[1028] 412. The kit of Clause 408, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[1029] 413. The kit of Clause 412, wherein the nucleic acid is an RNA.

[1030] 414. The kit of Clause 412, wherein the nucleic acid is a DNA.

[1031] 415. The kit of Clause 408, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[1032] 416. The kit of any one of Clauses 408 to 415, wherein the probe comprises an electrochemical reporter.

[1033] 417. The kit of Clause 416, wherein the electrochemical reporter is methylene blue. 418. The kit of any one of Clauses 398 to 405, wherein the solid support is the sensor.

[1034] 419. The kit of Clause 418, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a change in a property of the sensor.

[1035] 420. The kit of Clause 419, wherein the change in the property of the sensor is a change in an electrochemical property.Attorney Docket No.: GRIP-014WO

[1036] 421. The kit of Clause 420, wherein the change in the electrochemical property is a change in the redox signal.

[1037] 422. The kit of Clause 419, wherein the change in the property of the sensor is a change in an electrical property.

[1038] 423. The kit of Clause 422, wherein the change in the electrical property of the sensor is a change in electrical resistance or Dirac voltage of the sensor.

[1039] 424. The kit of any one of Clauses 419 to 423, wherein the label is an electrochemically active species.

[1040] 425. The kit of Clause 424, wherein the electrochemically active species is methylene blue.

[1041] 426. The kit of any one of Clauses 419 to 423, wherein the label is an enzyme.

[1042] 427. The kit of Clause 426, wherein the enzyme is an oxidoreductase.

[1043] 428. The kit of Clause 427, wherein the oxidoreductase is horseradish peroxidase. 429. The kit of any one of Clauses 371 to 428, wherein the sensor is a graphene sensor.

[1044] 430. A target nucleic acid detection kit, comprising:

[1045] a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[1046] a first bait nucleic acid bound to a first solid support;

[1047] a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with the first bait nucleic acid;

[1048] a reactant for the second CRISPR associated enzyme, the reactant bound to a second solid support, and

[1049] a sensor configured to detect cleavage of the reactant bound to the second solid support.

[1050] 431. The kit of Clause 430, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

[1051] 432. The kit of Clause 431 , wherein, when the first crRNA hybridizes with the target nucleic acid, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.Attorney Docket No.: GRIP-014WO

[1052] 433. The kit of Clause 431 or 432, wherein, when the second crRNA hybridizes with the free first bait nucleic acid, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[1053] 434. The kit of Clause 433, wherein, when the second crRNA hybridizes with the free first bait nucleic acid, the Craspase cleaves the substrate bound to the second solid support to produce a free substrate fragment and a second solid support bound substrate fragment.

[1054] 435. The kit of any one of Clauses 431 to 434, wherein the second solid support is a second bead.

[1055] 436. The kit of Clause 435, wherein the second bead is a magnetic bead.

[1056] 437. The kit of any one of Clauses 431 to 436, wherein the sensor comprises a probe that specifically binds to the free substrate fragment produced by cleavage of the substrate bound to the second solid support.

[1057] 438. The kit of Clause 437, wherein the sensor is a graphene sensor.

[1058] 439. The kit of Clause 437 or 438, wherein the probe that specifically binds to the free substrate fragment is a protein.

[1059] 440. The kit of any one of Clauses 437 to 438, wherein the probe that specifically binds to the free substrate fragment is an aptamer.

[1060] 441. The kit of any one of Clauses 437 to 440, wherein the probe comprises an electrochemical reporter.

[1061] 442. The kit of any one of Clauses 437 to 441 , wherein the sensor detects the cleavage of the substrate by detecting a change in property of the sensor caused by the binding of the free substrate fragment to the probe on the sensor.

[1062] 443. The kit of Clause 442, wherein the property of the sensor is an electrical property of the sensor.

[1063] 444. The kit of Clause 443, wherein the electrical property is conductivity of the sensor or Dirac voltage.

[1064] 445. The kit of Clause 442, wherein the property of the sensor is an electrochemical property of the sensor.

[1065] 446. The kit of Clause 445, wherein the electrochemical property sensor a redox signal.

[1066] 447. The kit of any one of Clauses 431 to 434, wherein the second solid support to which the substrate is bound is the sensor configured to detect cleavage of the substrate.

[1067] 448. The kit of Clause 447, wherein the sensor is an electrochemical sensor.

[1068] 449. The kit of Clause 447 or 448, wherein the substrate comprises a label.Attorney Docket No.: GRIP-014WO

[1069] 450. The kit of Clause 449, wherein the Craspase mediated cleavage of the substrate causes release of the label from the sensor thereby changing a property of the electrochemical sensor.

[1070] 451. The kit of Clause 449 or 450, wherein the label is an electrochemically active species.

[1071] 452. The kit of Clause 451 , wherein the electrochemically active species is methylene blue.

[1072] 453. The kit of Clause 449 or 450, wherein the label is an enzyme.

[1073] 454. The kit of Clause 453, wherein the enzyme is an oxidoreductase.

[1074] 455. The kit of Clause 454, wherein the oxidoreductase is horseradish peroxidase. 456. The kit of any one of Clauses 431 to 455, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate for the Craspase is Csx30.

[1075] 457. The kit of Clause 430, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid.

[1076] 458. The kit of Clause 457, wherein, when the first crRNA hybridizes with the target nucleic acid, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.

[1077] 459. The kit of Clause 457 or 458, wherein, when the second crRNA hybridizes with the free first bait nucleic acid, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the second solid support.

[1078] 460. The kit of any one of Clauses 457 to 459, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[1079] 461. The kit of any one of Clauses 457 to 460, wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[1080] 462. The kit of Clause 461 , wherein the second complex comprises a second single guide RNA (sgRNA).

[1081] 463. The kit of any one of Clauses 457 to 462, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[1082] 464. The kit of Clause 463, wherein the CRISPR-Cas Type V is CRISPR-Cas12. 465. The kit of any one of Clauses 457 to 464, wherein the second solid support is a second bead.

[1083] 466. The kit of Clause 465, wherein the second bead is a second magnetic bead. 467. The kit of any one of Clauses 457 to 466, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.Attorney Docket No.: GRIP-014WO

[1084] 468. The kit of Clause 467, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[1085] 469. The kit of Clause 468, wherein the protein is an RNA binding protein.

[1086] 470. The kit of Clause 468, wherein the protein is a DNA binding protein.

[1087] 471. The kit of Clause 467, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.

[1088] 472. The kit of Clause 471 , wherein the nucleic acid is an RNA.

[1089] 473. The kit of Clause 471 , wherein the nucleic acid is a DNA.

[1090] 474. The kit of Clause 467, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[1091] 475. The kit of any one of Clauses 467 to 474, wherein the probe comprises an electrochemical reporter.

[1092] 476. The kit of Clause 475, wherein the electrochemical reporter is methylene blue. 477. The kit of any one of Clauses 457 to 464, wherein the solid support is the sensor.

[1093] 478. The kit of Clause 477, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a change in a property of the sensor.

[1094] 479. The kit of Clause 478, wherein the change in the property of the sensor is a change in an electrochemical property.

[1095] 480. The kit of Clause 479, wherein the change in the electrochemical property is a change in the redox signal.

[1096] 481. The kit of Clause 478, wherein the change in the property of the sensor is a change in an electrical property.

[1097] 482. The kit of Clause 481 , wherein the change in the electrical property of the sensor is a change in electrical resistance or Dirac voltage of the sensor.

[1098] 483. The kit of any one of Clauses 478 to 482, wherein the label is an electrochemically active species.

[1099] 484. The kit of Clause 483, wherein the electrochemically active species is methylene blue.

[1100] 485. The kit of any one of Clauses 478 to 482, wherein the label is an enzyme.

[1101] 486. The kit of Clause 485, wherein the enzyme is an oxidoreductase.

[1102] 487. The kit of Clause 486, wherein the oxidoreductase is horseradish peroxidase. 488. The kit of any one of Clauses 457 to 487, wherein the sensor is a graphene sensor.Attorney Docket No.: GRIP-014WO

[1103] 489. The kit of any one of Clauses 430 to 488, wherein the target sequence has a length from 17 to 30 nucleotides.

[1104] 490. The kit of any one of Clauses 430 to 489, wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[1105] 491. The kit of any one of Clauses 430 to 490, wherein the first crRNA is in a first complex comprising a first trans-activating crRNA (tracrRNA).

[1106] 492. The kit of Clause 491 , wherein the first complex comprises a first single guide RNA (sgRNA).

[1107] 493. The kit of any one of Clauses 430 to 492, wherein the first CRISPR associated nuclease is a CRISPR-Cas Type VI.

[1108] 494. The kit of Clause 493, wherein the CRISPR-Cas Type VI is CRISPR-Cas13. 495. The kit of any one of Clauses 430 to 494, wherein the first solid support is a first bead.

[1109] 496. The kit of Clause 495, wherein the first bead is a first magnetic bead.

[1110] 497. The kit of any one of Clauses 430 to 496, wherein the target nucleic acid is a nucleic acid from a microorganism.

[1111] 498. The kit of Clause 497, wherein the microorganism is a bacterium, virus, fungus, or a protozoa.

[1112] 499. A device comprising a sensor in contact with a second reaction mixture, wherein the second reaction mixture is produced by:

[1113] (a) preparing a first reaction mixture by combining a sample suspected of containing a target nucleic acid with:

[1114] i) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid; and

[1115] ii) a first bait nucleic acid bound to a first solid support;

[1116] (b) separating the first solid support from the first reaction mixture to produce a first product mixture;

[1117] (c) preparing a second reaction mixture by combining the first product mixture with: i) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with the first bait nucleic acid, and

[1118] ii) a reactant for the second CRISPR associated enzyme, wherein the reactant is bound to a second solid support;Attorney Docket No.: GRIP-014WO

[1119] wherein the sensor detects the cleavage of the reactant bound to the second solid support to determine whether a free first bait nucleic acid is present in the first product mixture.

[1120] 500. The device of Clause 499, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

[1121] 501. The device of Clause 500, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.

[1122] 502. The device of Clause 500 or 501 , wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate.

[1123] 503. The device of any one of Clauses 500 to 502, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[1124] 504. The device of any one of Clauses 500 to 503, wherein the sensor is a graphene sensor.

[1125] 505. The device of any one of Clauses 500 to 504, wherein the second reaction mixture is further processed by separating the second reaction mixture from the second solid support after said combining the first product, and wherein the device detects the presence of a fragment of the substrate cleaved from the second solid support.

[1126] 506. The device of any one of Clauses 500 to 503, wherein the second solid support is the sensor, wherein the device detects the cleavage of the substrate by detecting the change in a property of the sensor caused by the cleavage of the substrate.

[1127] 507. The device of Clause 506, wherein the sensor is an electrochemical sensor. 508. The device of Clause 506, wherein the sensor is a graphene sensor.

[1128] 509. The device of any one of Clauses 506 to 508, wherein the substrate comprises a label.

[1129] 510. The device of Clause 509, wherein the Craspase mediated cleavage of the substrate releases the label from the sensor thereby changing the property of the sensor.

[1130] 511. The device of Clause 509 or 510, wherein the label is an electrochemically active species.

[1131] 512. The device of Clause 511 , wherein the electrochemically active species is methylene blue.

[1132] 513. The device of Clause 509 or 510, wherein the label is an enzyme.Attorney Docket No.: GRIP-014WO

[1133] 514. The device of Clause 513, wherein the enzyme is an oxidoreductase.

[1134] 515. The device of Clause 514, wherein the oxidoreductase is horseradish peroxidase.

[1135] 516. The device of any one of Clauses 500 to 515, wherein the Craspase is a complex of Csx29, Cas7-11 , and the crRNA and the substrate for the Craspase is Csx30.

[1136] 517. The device of Clause 499, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid.

[1137] 518. The device of Clause 517, wherein, when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid bound to the first solid support.

[1138] 519. The device of Clause 518, wherein, when the free first bait nucleic acid is present in the second reaction mixture, the second CRISPR associated nuclease cleaves the second bait nucleic acid bound to the second solid support.

[1139] 520. The device of any one of Clauses 517 to 519, wherein the second bait nucleic acid has a length from 10 to 100 nucleotides.

[1140] 521. The device of any one of Clauses 517 to 520, wherein the second crRNA is in a second complex comprising a second trans-activating crRNA (tracrRNA).

[1141] 522. The device of Clause 521 , wherein the second complex comprises a second single guide RNA (sgRNA).

[1142] 523. The device of any one of Clauses 517 to 522, wherein the second CRISPR associated nuclease is a CRISPR-Cas Type V.

[1143] 524. The device of Clause 523, wherein the CRISPR-Cas Type V is CRISPR-Cas12.

[1144] 525. The device of any one of Clauses 517 to 524, wherein the second solid support is a second bead.

[1145] 526. The device of Clause 525, wherein the second bead is a second magnetic bead.

[1146] 527. The device of any one of Clauses 517 to 526, wherein the sensor comprises a probe that specifically binds to the free second bait nucleic acid.

[1147] 528. The device of Clause 527, wherein the probe that specifically binds to the free second bait nucleic acid is a protein.

[1148] 529. The device of Clause 528, wherein the protein is an RNA binding protein.

[1149] 530. The device of Clause 529, wherein the protein is a DNA binding protein.

[1150] 531. The device of Clause 527, wherein the probe that specifically binds to the free second bait nucleic acid is a nucleic acid.Attorney Docket No.: GRIP-014WO

[1151] 532. The device of Clause 531 , wherein the nucleic acid is an RNA.

[1152] 533. The device of Clause 531 , wherein the nucleic acid is a DNA.

[1153] 534. The device of Clause 527, wherein the probe that specifically binds to the free second bait nucleic acid is an aptamer.

[1154] 535. The device of any one of Clauses 527 to 534, wherein the probe comprises an electrochemical reporter.

[1155] 536. The device of Clause 535, wherein the electrochemical reporter is methylene blue.

[1156] 537. The device of any one of Clauses 526 to 524, wherein the second solid support is the sensor.

[1157] 538. The device of Clause 537, wherein the second bait nucleic acid comprises a label and cleavage of the second bait nucleic acid produces a change in a property of the sensor.

[1158] 539. The device of Clause 538, wherein the change in the property of the sensor is a change in an electrochemical property.

[1159] 540. The device of Clause 539, wherein the electrochemical property is a change in the redox signal.

[1160] 541. The device of Clause 538, wherein the change in the property of the sensor is a change in an electrical property.

[1161] 542. The device of Clause 541 , wherein the change in the electrical property is a change in electrical resistance or Dirac voltage.

[1162] 543. The device of any one of Clauses 538 to 542, wherein the label is an electrochemically active species.

[1163] 544. The device of Clause 543, wherein the electrochemically active species is methylene blue.

[1164] 545. The device of any one of Clauses 538 to 542, wherein the label is an enzyme.

[1165] 546. The device of Clause 545, wherein the enzyme is an oxidoreductase.

[1166] 547. The device of Clause 546, wherein the oxidoreductase is horseradish peroxidase.

[1167] 548. The device of any one of Clauses 517 to 547, wherein the sensor is a graphene sensor.

[1168] 549. The device of any one of Clauses 499 to 548, wherein the target sequence has a length from 17 to 30 nucleotides.Attorney Docket No.: GRIP-014WO

[1169] 550. The device of any one of Clauses 499 to 549, wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[1170] 551. The device of any one of Clauses 499 to 550, wherein the first crRNA is in a first complex comprising a first trans-activating crRNA (tracrRNA).

[1171] 552. The device of Clause 551 , wherein the first complex comprises a first single guide RNA (sgRNA).

[1172] 553. The device of any one of Clauses 499 to 552, wherein the first CRISPR associated nuclease is a CRISPR-Cas Type VI.

[1173] 554. The device of Clause 553, wherein the CRISPR-Cas Type VI is CRISPR-Cas13.

[1174] 555. The device of any one of Clauses 499 to 554, wherein the first solid support is a first bead.

[1175] 556. The device of Clause 555, wherein the first bead is a first magnetic bead.

[1176] 557. The device of any one of Clauses 499 to 556, wherein the target nucleic acid is a nucleic acid from a microorganism.

[1177] 558. The device of Clause 557, wherein the microorganism is a bacterium, virus, fungus, or a protozoa.

[1178] 559. A composition comprising:

[1179] i) a sample suspected of containing a target nucleic acid,

[1180] ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,

[1181] iii) a first bait nucleic acid,

[1182] iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, and

[1183] v) a reactant for the second CRISPR associated enzyme.

[1184] 560. The composition of Clause 559, wherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

[1185] 561. The composition of Clause 560, wherein the first bait nucleic acid has a secondary structure that prevents the second crRNA from hybridizing with the uncleaved first bait nucleic acid.

[1186] 562. The composition of Clause 561 , wherein the secondary structure is selected from a toehold switch, inverted repeat, hairpin, triplex, slipped structure, or cruciform structure.Attorney Docket No.: GRIP-014WO

[1187] 563. The composition of any one of Clauses 559 to 562, wherein the reactant for the second CRISPR associated enzyme is bound to a solid support.

[1188] 564. The composition of Clause 563, wherein the solid support is a sensor.

[1189] 565. The composition of Clause 564, wherein the sensor is a graphene sensor.

[1190] 566. The composition of Clause 563, wherein the solid support is a bead.

[1191] 567. The composition of Clause 566, wherein the bead is a magnetic bead.

[1192] 568. The composition of any one of Clauses 559 to 567, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

[1193] 569. The composition of any one of Clauses 559 to 567, wherein the second CRISPR associated enzyme is a second CRISPR associated nuclease and the reactant is a second bait nucleic acid.

[1194] 570. The composition of any one of Clauses 559 to 569, wherein the target sequence has a length from 17 to 30 nucleotides.

[1195] 571. The composition of any one of Clauses 559 to 570, wherein the first bait nucleic acid has a length from 10 to 100 nucleotides.

[1196] 572. The composition of any one of Clauses 559 to 571 , wherein the first crRNA is in a first complex comprising a first trans-activating crRNA (tracrRNA).

[1197] 573. The composition of Clause 572, wherein the first complex comprises a first single guide RNA (sgRNA).

[1198] 574. The composition of any one of Clauses 559 to 573, wherein the first CRISPR associated nuclease is a CRISPR-Cas Type VI.

[1199] 575. The composition of Clause 574, wherein the CRISPR-Cas Type VI is CRISPR-Cas13.

[1200] 576. A sensor in contact with the composition of any one of Clauses 569 to 563 and 556 to 575.

[1201] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[1202] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention andAttorney Docket No.: GRIP-014WO

[1203] are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[1204] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase “means for” or the exact phrase “step for” is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

Attorney Docket No.: GRIP-014WOCLAIMS WE CLAIM:

1. A method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:(a) combining in one or more reaction mixtures:i) the sample,ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,iii) a first bait nucleic acid,iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, andv) a reactant for the second CRISPR associated enzyme; and(b) assaying whether a product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

2. The method claim 1 , wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) and the reactant is a substrate for the Craspase.

3. The method of claim 2, wherein the first reaction occurs in a first reaction mixture and the second reaction occurs in a second reaction mixture.

4. The method of claim 3, comprising:(a) preparing the first rection mixture by combining the sample with:(i) the first CRISPR associated nuclease comprising the first crRNA; and ii) the first bait nucleic acid bound to a first solid support;(b) separating the first solid support from the first reaction mixture to produce a first product mixture;(c) preparing the second reaction mixture by combining the first product mixture with:i) the Craspase comprising the second crRNA that specifically hybridizes with the cleaved first bait nucleic acid; andAttorney Docket No.: GRIP-014WOii) the substrate for the Craspase, and(d) assaying the second reaction mixture for the product resulting from activity of the Craspase on the substrate to determine whether the target nucleic acid is present in the sample.

5. The method of claim 4, wherein the substrate is bound to a second solid support and comprises a label,wherein, when the free first bait nucleic acid is present in the second reaction mixture, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled second solid support bound substrate fragment, andwherein assaying the second reaction mixture for cleavage of the substrate comprises detecting the free labeled substrate fragment.

6. The method of claim 5, wherein assaying the second reaction mixture for cleavage of the substrate comprises:i) loading the first product mixture into a reaction region of a lateral flow assay device, the reaction region comprising the Craspase comprising the second crRNA and the labeled substrate immobilized on the second solid support in the reaction region, and wherein the reaction region is fluidically connected to the detection region such that the labeled substrate fragment produced in the reaction region migrates to the detection region, and wherein binding of a binding partner in the detection region and the label produces a detectable signal in the detection region; orii) detecting a change in a property of the second solid support, wherein the second solid support is a sensor, and the cleavage of the substrate to produce the free labeled substrate fragment causes a change in a property of the sensor.

7. The method of claim 5, wherein assaying the second reaction mixture for cleavage of the substrate comprises separating the second solid support, which is a second bead, from the second reaction mixture to produce a second product mixture, and detecting the substrate fragment in the second product mixture by:i) contacting the second product mixture to a sensor comprising a probe that specifically binds to the substrate fragment, wherein binding of the substrate fragment to the probe causes a change in a property of the sensor; orAttorney Docket No.: GRIP-014WOii) contacting the second product mixture with a composition comprising a signal producing agent, wherein the cleaved substrate fragment comprises a label that produces a signal from the signal producing agent.

8. The method of claim 2, wherein the first reaction and the second reaction occur in the same reaction mixture,wherein when the target nucleic acid is present in the sample, the first CRISPR associated nuclease cleaves the first bait nucleic acid to produce a cleaved first bait nucleic acid, andwherein the second crRNA specifically hybridizes with the cleaved first bait nucleic acid but does not hybridize with an uncleaved first bait nucleic acid.

9. The method of claim 8, wherein the substrate comprises a label and, when the cleaved first bait nucleic acid is produced in the first reaction, the Craspase cleaves the substrate to produce a free labeled substrate fragment and an unlabeled solid support bound substrate fragment, and wherein assaying whether the product resulting from activity of the Craspase on the substrate is produced comprises detecting the free labeled substrate fragment.

10. The method of claim 9, wherein:i) the solid support is a sensor and cleavage of the substrate produces the free labeled substrate fragment thereby causing a change in a property of the sensor; orii) the solid support is a bead and the method comprises:a) separating the bead from the reaction mixture to produce a product mixture, andb) detecting in the product mixture the free substrate fragment.

11. The method of any one of claims 1 to 10, the method comprising:(a) combining in an analyzer one or more reaction mixtures comprising:i) the sample,ii) the first CRISPR associated nuclease comprising the first crRNA that specifically hybridizes with the target nucleic acid,iii) the first bait nucleic acid,iv) the second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, andv) the reactant for the second CRISPR associated enzyme; andAttorney Docket No.: GRIP-014WO(b) assaying whether the product resulting from activity of the second CRISPR associated enzyme on the reactant is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

12. A method of determining in a first reaction and a second reaction whether a target nucleic acid is present in a sample, the method comprising:(a) combining in one or more reaction mixtures:i) the sample,ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,iii) a first bait nucleic acid,iv) a CRISPR associated protease (Craspase) comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, andv) a substrate for the Craspase; and(b) assaying in a lateral flow assay device whether a product resulting from activity of the Craspase on the substrate is produced in the one or more reaction mixtures to determine whether the target nucleic acid is present in the sample.

13. A target nucleic acid detection kit, comprising:a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first crRNA that specifically hybridizes with the target nucleic acid;a first bait nucleic acid;a second clustered regularly interspaced short palindromic repeats (CRISPR) associated enzyme comprising a second CRISPR RNA (crRNA) that specifically hybridizes with a cleaved first bait nucleic acid; anda reactant for the second CRISPR associated enzyme bound to a solid support.

14. The target nucleic acid detection kit of claim 13, wherein the second CRISPR associated enzyme is a CRISPR associated protease (Craspase) comprising the crRNA that specifically hybridizes with the first bait nucleic acid,wherein the kit comprises a lateral flow assay devices comprising:(a) a reaction region comprising: i) the Craspase; and ii) a substrate for the Craspase, wherein the substrate is immobilized in a reaction region; andAttorney Docket No.: GRIP-014WO(b) a detection region fluidically connected to the reaction region, the detection region comprising immobilized therein a binding partner that specifically binds to a substrate fragment produced from the substrate by the protease action of the Craspase.

15. A composition comprising:i) a sample suspected of containing a target nucleic acid,ii) a first clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease comprising a first CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid,iii) a first bait nucleic acid,iv) a second CRISPR associated enzyme comprising a second crRNA that specifically hybridizes with a cleaved first bait nucleic acid, andv) a reactant for the second CRISPR associated enzyme.

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