Methods and kits for detecting target nucleic acids using crispr associated proteases

Abstract

Provided herein are methods for determining whether a target nucleic acid is present in a sample. In some cases, the methods comprise producing a reaction mixture by combining a sample with: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and ii) a substrate for the Craspase; and assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample. Also provided are analyzers and kits comprising devices and analyzers that are designed for performing the methods disclosed herein. Further provided are lateral flow assay devices designed for performing the methods disclosed herein. Also provided are protocols for performing the methods disclosed herein using the devices, analyzers, and lateral flow assay devices of the disclosure.

Description

[0001] Atty. Docket No.: GRIP-013WO

[0002] METHODS AND KITS FOR DETECTING TARGET NUCLEIC ACIDS USING CRISPR ASSOCIATED PROTEASES

[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 / 743,548, filed January 9, 2025, the disclosure of which application is herein incorporated by reference.

[0005] INTRODUCTION

[0006] Clustered regularly interspaced short palindromic repeats (CRISPR) associated proteases (Craspase) exhibit dual function of endonucleases and proteases. Activation of Craspase requires interaction between a Craspase and its target nucleic acid sequence via CRISPR RNA (crRNA) that specifically binds to the target nucleic acid. Typically, Craspase activation in presence of a target nucleic acid is a part of a bacterial defense mechanism.

[0007] Practical applications of such Craspase activity are desired.

[0008] SUMMARY

[0009] The inventors have realized that the sequence specific interaction of crRNA of a Craspase and its target nucleic acid can be used to determine whether the target nucleic acid is present in a sample. As described herein, certain 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.

[0010] The methods disclosed herein utilize a Craspase comprising a crRNA that specifically hybridizes with the target nucleic acid. If the target nucleic acid is present in a sample, the Craspase exhibits protease activity towards its substrate. Thus, the presence of the Craspase protease activity towards its substrate indicates the presence in the sample of the target nucleic acid. This disclosure provides methods in which the presence or absence of the target nucleic acid is determined based on detecting the Craspase activity towards its substrate.Atty. Docket No.: GRIP-013WO

[0011] Certain embodiments of the disclosure provide a method for determining whether a target nucleic acid is present in a sample, the method comprising:

[0012] (a) producing a reaction mixture by combining the sample with:

[0013] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0014] ii) a substrate for the Craspase;

[0015] and

[0016] (b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample.

[0017] In some cases, the substrate is labeled and assaying the reaction mixture for cleavage of the substrate comprises detecting a labeled substrate fragment. For example, the labeled substrate 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.

[0018] Alternatively, in some cases, the substrate is bound to a solid support and, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0019] In certain such cases, the solid support is a sensor and assaying the reaction mixture for cleavage of the substrate 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 property 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.

[0020] In some cases, the substrate is bound to a solid support such that Craspase mediated cleavage of the bound substrate produces a free substrate fragment and a solid support bound substrate fragment. In some cases, the solid support is a bead, such as a magnetic bead.

[0021] In certain such cases, assaying the reaction mixture for cleavage of the substrate comprises detecting the free substrate fragment in the reaction mixture after separating the solid support from the reaction mixture. In some cases, detecting the free substrate fragment comprises contacting the reaction mixture after separating the solid support to a sensor comprising an attached probe that specifically binds the free substrate fragment. Such binding produces a change in a property of the sensor, which can be detected to detect the binding of the free substrate fragment to the sensor. In some cases, the sensor is a graphene sensor.Atty. Docket No.: GRIP-013WO

[0022] 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.

[0023] In certain embodiments, the disclosure provides methods of utilizing an analyzer to determine whether a target nucleic acid is present in a sample. Certain such methods comprise:

[0024] (a) introducing the sample into a sample chamber of an analyzer to produce a reaction mixture,

[0025] wherein the sample chamber comprises: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and ii) a substrate for the Craspase, the substrate bound to a solid support;

[0026] (b) incubating the reaction mixture, and

[0027] (c) assaying for cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0028] The solid support can be a sensor and assaying the cleavage of the substrate 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 methods utilizing an analyzer.

[0029] In some cases, the substrate is bound to a solid support such that Craspase mediated cleavage of the bound substrate produces a free substrate fragment and a solid support bound substrate fragment. In some cases, the solid support is a bead, such as a magnetic bead. Assaying the cleavage of the substrate comprises detecting the free substrate fragment in the reaction mixture after separating the beads from the reaction mixture. Certain details of such methods are disclosed elsewhere in this disclosure and such embodiments are applicable to detecting a free substrate fragment in methods utilizing an analyzer.

[0030] In certain embodiments, the methods of detecting a target nucleic acid are performed using lateral flow assay devices. Thus, in certain embodiments, determining whether a target nucleic acid is present in a sample comprises:

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

[0032] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0033] ii) a substrate for the Craspase, the substrate comprising a label;Atty. Docket No.: GRIP-013WO

[0034] and

[0035] (b) assaying the reaction mixture in a lateral flow assay device for cleavage of substrate to determine whether the target nucleic acid is present in the sample.

[0036] 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 sample into the reaction region of the lateral flow assay device. The reaction region of the lateral flow assay device can comprise 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.

[0037] 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.

[0038] Further embodiments of the disclosure also provide kits for determining whether a target nucleic acid is present in a sample. In some cases, a target nucleic acid detection kit comprises:

[0039] a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid;

[0040] a substrate bound to a solid support, and

[0041] a sensor configured to detect cleavage of the substrate bound to the solid support. In some embodiments, the solid support is a sensor, for example, a graphene sensor. Alternatively, the solid support can comprise beads and the cleavage of the substrate can be assayed by detecting the free substrate fragment using a sensor comprising a probe that specifically binds to the free substrate fragment. Such sensor can also be a graphene sensor.

[0042] Even further embodiments of the disclosure provide devices comprising a sensor in contact with a reaction mixture. In certain such cases, a reaction is produced by:

[0043] incubating a sample suspected of containing a target nucleic acid with: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid, and ii) a substrate bound to a solid support,

[0044] wherein the sensor detects the cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.Atty. Docket No.: GRIP-013WO

[0045] Furthermore, 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:

[0046] (a) a reaction region comprising:

[0047] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Graspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0048] ii) a substrate for the Graspase, the substrate immobilized in the reaction region; (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 Graspase.

[0049] BRIEF DESCRIPTION OF THE FIGURES FIGS. 1 A-1 B depict proteolytic activity of Graspase system on Csx30. A. Graspase protease activity on Csx30. B. SDS-PAGE image showing cleavage of Csx30 into 50 kD and 18 kD fragments.

[0050] FIGS. 2A-2C depict characterization of Graspase proteolytic activity. A. Time course of Csx30 cleavage upon addition of target RNA at 45°C. B. Dose-dependent cleavage of Csx30 by Graspase. C. Csx30 cleavage across temperatures.

[0051] FIGS. 3A-3B depict exemplary Craspase-based lateral flow immunoassays.

[0052] FIG. 4 depicts schematic of an exemplary Craspase-based electrochemical sensor. FIG. 5 depicts schematic of an exemplary Craspase-based assay that utilizes substrate bound to beads and comprises detecting free substrate fragment using a graphene sensor.

[0053] FIG. 6 depicts schematic of an exemplary Craspase-based assay that utilizes a sensor comprising a Graspase substrate comprising a reporter that releases the reporter upon Craspase mediated cleavage of the substrate.

[0054] FIG. 7 depicts schematic of an exemplary Craspase-based assay that utilizes substrate bound to beads and comprises detecting free substrate fragment using a graphene sensor using Dirac point shift or an electrochemical sensor using square wave voltammetry.

[0055] FIG. 8 depicts schematic of an exemplary Craspase-based assay that utilizes a graphene sensor comprising a substrate comprising a reporter that releases the reporter upon Craspase activity thereby causing a Dirac point shift of the graphene sensor.Atty. Docket No.: GRIP-013WO

[0056] FIG. 9 depicts schematic of an exemplary Craspase-based assay that utilizes an HRP conjugated Csx30 bound to beads and optically detecting the cleavage of the substrate using a detection solution comprising hydrogen peroxide and TMB.

[0057] FIG. 10 depicts the efficiency of Craspase mediated Csx30 cleavage.

[0058] FIG. 11 depicts Craspase mediated Csx30 cleavage dependent on the target RNA concentration.

[0059] FIG. 12 depicts that Craspase is highly active even at room temperature.

[0060] FIG. 13 depicts that Craspase is highly active within cell lysis workflow.

[0061] FIG. 14 depicts that Craspase mediated Csx30 cleavage can be used to detect Neisseria gonorrhoeae.

[0062] FIG. 15 depicts a lateral flow assay that utilizes Craspase mediated Csx30 cleavage. FIG. 16 depicts electrochemical data for the detection of N. gonorrhoeae utilizing Craspase mediated Csx30 cleavage.

[0063] FIG. 17 depicts a calcium-responsive RTX17 System.

[0064] FIG. 18 depicts the affinity depletion scheme.

[0065] DETAILED DESCRIPTION

[0066] As summarized above, methods of detecting whether a target nucleic acid is present or absent in a sample are provided.

[0067] In certain embodiments, a method for determining whether a target nucleic acid is present in a sample comprises:

[0068] (a) producing a reaction mixture by combining the sample with:

[0069] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0070] ii) a substrate for the Craspase;

[0071] and

[0072] (b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample.

[0073] 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:

[0074] (a) introducing the sample into a sample chamber of an analyzer to produce a reaction mixture,Atty. Docket No.: GRIP-013WO

[0075] wherein the sample chamber comprises: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and ii) a substrate for the Craspase, the substrate bound to a solid support;

[0076] (b) incubating the reaction mixture, and

[0077] (c) assaying for cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0078] 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:

[0079] (a) producing a reaction mixture by combining the sample with:

[0080] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

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

[0082] and

[0083] (b) assaying the reaction mixture in a lateral flow assay device for cleavage of substrate to determine whether the target nucleic acid is present in the sample.

[0084] 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:

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

[0086] a substrate bound to a solid support, and

[0087] a sensor configured to detect cleavage of the substrate bound to the solid support. Certain embodiments of the disclosure provide devices in which the methods of the disclosure are performed. Thus, certain embodiments of the disclosure provide:

[0088] a device comprising a sensor in contact with a reaction mixture,

[0089] wherein the reaction mixture is produced by:

[0090] incubating a sample suspected of containing a target nucleic acid with: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid, and ii) a substrate bound to a solid support,Atty. Docket No.: GRIP-013WO

[0091] wherein the sensor detects the cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0092] Even further embodiments of the disclosure provide lateral flow assay devices suitable for performing the methods disclosed herein. In some cases, a lateral flow assay device for determining whether a target nucleic acid is present in a sample comprises:

[0093] (a) a reaction region comprising:

[0094] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0095] ii) a substrate for the Craspase, the substrate immobilized in the reaction region; (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.

[0096] 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.

[0097] 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.

[0098] 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.Atty. Docket No.: GRIP-013WO

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 nucleic 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 canAtty. Docket No.: GRIP-013WO

[0104] 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.

[0105] 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.

[0106] 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 or 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 saltAtty. Docket No.: GRIP-013WO

[0107] concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0108] 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).

[0109] 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 / , 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.

[0110] "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, theAtty. Docket No.: GRIP-013WO

[0111] 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 10“1° M, 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.

[0112] 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.

[0113] 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.

[0114] In further describing various aspects of the disclosure, the methods, the devices, analyzers, kits, and lateral flow assay devices are described first in greater detail, followed by the description of exemplary clauses and embodiments.

[0115] METHODS OF DETECTING A TARGET NUCLEIC ACID

[0116] Certain aspects of the present disclosure provide methods for determining whether a target nucleic acid is present in a sample. These methods utilize CRISPR associated proteases (Craspases) and their activation in presence of a target nucleic acid, which specifically hybridizes with the Craspase crRNA.

[0117] 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),Atty. Docket No.: GRIP-013WO

[0118] 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.

[0119] On the other hand, Beljouw et al. ((2024), ACS Chem. Biol., 19, 1051-1055 characterized the Craspase system from Candidates “Jettenia caeni’ (Jc-Craspase)

[0120] and Candidates “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.

[0121] The first component of the 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.

[0122] 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. This is shown in FIGS. 1 A-1 B. FIG. 1 A shows schematic of RNA detection with Craspase programmed to detect a specific target RNA. FIG. 1 B shows an SDS-PAGE gel image after Csx29-crRNA cleavage of Csx30 showing larger 50 kD fragment and 18 kD fragment.

[0123] FIGS. 2A-2B show certain enzyme kinetics of Craspase. For example, Csx29 cleaves a 100-fold molar excess of the Csx30 within 1-2 minutes at 45°C (FIG. 2A), demonstrating fast multiple substrate turnover. Notably, Craspase displays proteolytic activity even at 1 :1 ,000 molar concentrations in under an hour (FIG. 2B). Craspase activity is optimal at 45°C, but Craspase still maintains comparable activity at 37°C (FIG. 2C).

[0124] Thus, 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.

[0125] Also, Craspase can be activated in the presence of a target nucleic acid and its resultant protease activity can be used in detecting the target nucleic acid, particularly, in an amplification-free assay.Atty. Docket No.: GRIP-013WO

[0126] Accordingly, in certain embodiments of the disclosure, a Craspase, such as Csx29 is used as a surveillance complex with a target-specific crRNA, as shown in FIG. 1. 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.

[0127] To that end, a “substrate” such as “Csx30” is provided in a reaction mixture comprising a sample and a Craspase and cleavage of the substrate is assayed. In some cases, substrate in an immobilized form is provided in the reaction mixture. For example, substrate can be bound to beads, such as magnetic beads, and added to the reaction mixture. Alternatively, a substrate can be immobilized on an inner wall of the chamber in which the reaction is conducted. A substrate can also be immobilized on a sensor and the cleavage of the substrate is assayed using the sensor.

[0128] When a substrate is bound to a solid support, cleavage of the substrate produces a free substrate fragment, i.e., a fragment of the substrate that is not bound to the solid support, such as the beads or an inner wall of the reaction chamber. After Craspase mediated target nucleic acid detection and substrate cleavage, the free substrate fragment, if produced, is separated from the reaction mixture. For example, the reaction mixture can be passed through a filter or centrifuged to separate the beads. Alternatively, the reaction mixture in a reaction chamber having a substrate bound on an inner wall can be transferred from the chamber. Such free substrate fragment can be detected for assaying the cleavage of the substrate.

[0129] 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.

[0130] In further embodiments, a substrate is immobilized in a reaction region of a lateral flow assay device. The reaction region also comprises 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 label.

[0131] Depending on the point of attachment of the label to the substrate, Craspase mediated cleavage of a labeled substrate bound to a solid support produces either 1) a labeled free substrate fragment and an unlabeled bound substrate fragment or 2) a labeled bound substrateAtty. Docket No.: GRIP-013WO

[0132] 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.

[0133] With such general description of the disclosure, certain specific embodiments of the methods, devices, and kits of the disclosure will be described below in further detail.

[0134] Certain embodiments of the disclosure provide a method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0135] (a) producing a reaction mixture by combining the sample with:

[0136] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0137] ii) a substrate for the Craspase;

[0138] and

[0139] (b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample.

[0140] As discussed above, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate.

[0141] In some cases, the Craspase is Csx29. 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.

[0142] 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 the target nucleic acid is present in the subject, the Craspase cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment.

[0143] Accordingly, in some cases, assaying the reaction mixture for cleavage of the substrate comprises detecting the labeled substrate fragment. Alternatively, in some cases, assaying the reaction mixture for cleavage of the substrate comprises detecting the unlabeled substrate fragment.

[0144] 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 reaction mixture and detecting a signal produced from the free labeled substrate fragment.Atty. Docket No.: GRIP-013WO

[0145] Such signal can be a fluorescent signal, a chromogenic signal, a chemiluminescence signal, or the like.

[0146] Alternatively, such assaying can also comprise contacting the 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.

[0147] In some cases, assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample comprises separating the labeled substrate fragment from the reaction mixture even when the labeled substrate is not bound to a support. In certain such embodiments, the substrate is conjugated to a tag that, in the presence of an appropriate coagulator (e.g., a metal chelator that coagulates the tag), coagulates the unlabeled substrate fragment thereby allowing separating the labeled substrate fragment, if produced, from the unlabeled substrate fragment. Certain non-limiting examples of peptide that can be used as tags to coagulate in the presence of appropriate coagulators are described by Dooley et al. (2012), Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation, Biomacromolecules, 13, 1758-1764, which is herein incorporated by reference in its entirety.

[0148] Repeats-in-toxin (RTX) peptide as a tag that coagulates the conjugated substrate

[0149] In some cases, a tag that coagulates the conjugated substrate or its fragment, i.e., either the labeled substrate or the unlabeled substrate fragment, is a repeats-in-toxin (RTX) peptide. RTX peptides are derived from the RTX domains of certain secreted pathogenic proteins. RTX peptides are intrinsically disordered in the absence of calcium but in high calcium conditions, RTX peptides bind Ca++ions and coagulate. Therefore, the RTX peptides have been used for separating a target tagged with the RTX peptide.

[0150] Certain details of RTX peptides are described by Scott Banta, "Developing the calciumdependent conformational behavior of the RTX peptide domain for novel protein capture and recovery applications" in "Biochemical and Molecular Engineering XXI", Christina Chan,Atty. Docket No.: GRIP-013WO

[0151] Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). This reference is incorporated herein in its entirety.

[0152] In certain embodiments, a labeled substrate is further covalently conjugated to an RTX peptide. An example of such RTX peptide is RTX17. Certain details of the RTX17 peptide and its implementation in separating a labeled substrate fragment are described by Shur et al. (2013), A Designed, Phase Changing RTX-Based Peptide for Efficient Bioseparations, BioTechniques, 54:4, 197-206; which is herein incorporated by reference in its entirety. One such embodiment is schematically depicted in FIG. 17. The RTX17 peptide contains 17 repeats of a 9 amino acid sequence. Calcium, such as Ca2+ions or sources of Ca2+ions, e.g., CaCh, induces coagulation of the RTX17 peptide thereby causing coagulation of any molecules bound to the RTX17 peptide, e.g., a peptide covalently bound to the RTX17. Additional examples of RTX peptides are known to a person of ordinary skill in the art and use of such peptides in the methods and devices of the disclosure is within the purview of the disclosure.

[0153] Accordingly, in one embodiment, the Craspase reaction is carried out with a labeled substrate that is further covalently conjugated to an RTX peptide (e.g., the RTX17 peptide). After the reaction is completed, for example, after incubation of the reaction mixture for 10 minutes, a source of Ca2+ions, e.g., CaCk, is added to the reaction mixture. Ca2+ions induce coagulation of the RTX peptide thereby also coagulating any portion of the substrate that is conjugated to the RTX peptide. Thus, if a target nucleic acid is present in the sample, the Craspase cleaves labeled substrate conjugated to the RTX peptide and produces a labeled substrate fragment and an unlabeled substrate fragment that is conjugated to the RTX peptide. The label can be HRP as shown in FIG. 17. Any other suitable label can also be used.

[0154] The coagulated unlabeled substrate fragment conjugated to the RTX peptide can be separated from the reaction mixture, for example, by centrifugation or filtration. The labeled substrate fragment can be assayed in the remaining portion (the non-coagulated portion) of the reaction mixture, e.g., supernatant or filtrate. Thus, the presence of the labeled substrate fragment in the non-coagulated portion of the reaction mixture indicates the presence of the target nucleic acid in the sample.

[0155] On the other hand, if a target nucleic acid is not present in the sample, Craspase does not cleave the labeled substrate conjugated to the RTX peptide. Therefore, coagulation of RTX peptide also causes coagulation of the labeled substrate. The coagulated labeled substrate conjugated to the RTX peptide can be separated from the reaction mixture and the remaining portion (the non-coagulated portion) of the reaction mixture does not contain any labeledAtty. Docket No.: GRIP-013WO

[0156] substrate fragment. Thus, the absence of the labeled substrate fragment in the non-coagulated portion of the reaction mixture indicates that the sample does not contain the target nucleic acid.

[0157] In further cases, assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample comprises separating the labeled substrate fragment from the reaction mixture even when the labeled substrate is not bound to a support. In certain such embodiments, the substrate is conjugated to a tag that, in the presence of an appropriate binding partner (e.g., an antibody against the tag), immobilizes the unlabeled substrate fragment thereby allowing separating the labeled substrate fragment, if produced, from the unlabeled substrate fragment. For example, an antibody that specifically binds to a peptide tag, e.g., an epitope tag, can be used to immobilize the tagged-fragment of the substrate.

[0158] Epitope-tag as a tag that immobilizes the conjugated substrate

[0159] In some cases, an affinity tag that immobilizes the conjugated substrate or its fragment, i.e. , either the labeled substrate or the unlabeled substrate fragment is an epitope tag. Certain non-limiting examples of epitope tags include hemagglutinin (HA-tag), V5-tag, 6xHis-tag, FLAG-tag, Myc-tag, glutathione S-transferase (GST -tag), and maltose binding protein (MBP-tag). Additional examples of epitope tags are known in the art and use of such tags in the methods and devices disclosed herein is within the purview of the disclosure.

[0160] In a specific embodiment, an epitope tag is the a-tag. The a-tag binds strongly and specifically to an anti-a-tag antibody, which can be present in solution or immobilized to a surface, such as a bead. Certain details of the a-tag are described in Gotzke et al. (2019), Nature Communications, Volume 10, Article number: 4403 (2019), which is herein incorporated by reference in its entirety. One such embodiment is schematically depicted in FIG. 18. An antibody against the a-tag captures the a-tag thereby immobilizing any molecules conjugated to it, e.g., a peptide covalently bound to the a-tag.

[0161] Accordingly, in certain embodiments, a labeled substrate is further covalently conjugated to the a-tag. The Craspase reaction is carried out with the labeled substrate that is covalently conjugated to the a-tag. After the reaction is completed, for example, after incubation of the reaction mixture for 10 minutes, an antibody against the a-tag is added to the reaction mixture. The antibody against a-tag captures the a-tag, thereby also captures any portion of the substrate that is conjugated to the a-tag.Atty. Docket No.: GRIP-013WO

[0162] Thus, if a target nucleic acid is present in the sample, the Craspase cleaves labeled substrate conjugated to the a-tag and produces a labeled substrate fragment and an unlabeled substrate fragment that is conjugated to the a-tag. The label can be HRP as shown in FIG. 18. Any other suitable label can also be used.

[0163] The unlabeled substrate fragment conjugated to the a-tag can be separated from the reaction mixture by immobilization to the antibody. The labeled substrate fragment can be assayed in the supernatant portion (the non-immobilized portion) of the reaction mixture, e.g., supernatant or filtrate. Thus, the presence of the labeled substrate fragment in the nonimmobilized portion of the reaction mixture indicates the presence of the target nucleic acid in the sample.

[0164] On the other hand, if a target nucleic acid is not present in the sample, Craspase does not cleave the labeled substrate conjugated to the a-tag. Therefore, immobilization of a-tag also causes immobilization of the labeled substrate. The immobilized labeled substrate conjugated to the a-tag can be separated from the reaction mixture and the remaining portion (the nonimmobilized portion) of the reaction mixture does not contain any labeled substrate fragment. Thus, the absence of the labeled substrate fragment in the non-immobilized portion of the reaction mixture indicates that the sample does not contain the target nucleic acid.

[0165] In further embodiments, a combination of coagulation-based and immobilization-based approaches is used to separate the unlabeled substrate fragment from the labeled substrate fragment. For example, an RTX peptide as well as an antigen is conjugated to a labeled substrate. In one such embodiment, an RTX peptide (e.g., the RTX17 peptide) and an antigen (e.g., the a-tag) are conjugated to the labeled substrate.

[0166] For example, a-tag-RTX17-Csx30-HRP conjugate is produced. If the target RNA is present in a sample, a Craspase cleaves the substrate to produce an a-tag-RTX17 fragment and Csx30-HRP fragment. Because Csx30-HRP is cleaved from a-tag-RTX17, it will neither coagulate with RTX nor immobilize when the a-tag binds to an anti-a-tag antibody. The free labeled substrate fragment can be separated and detected. For example, the free Csx30-HRP fragment can be contacted with a sensor surface for detection.

[0167] Regardless of the tag used to coagulate or immobilize the unlabeled substrate fragment, the free labeled substrate fragment separated from the substrate fragment conjugated to the tag (e.g., an RTX peptide or the a-tag) can be detected by any suitable methods, for example, those described elsewhere in this disclosure.Atty. Docket No.: GRIP-013WO

[0168] Detecting labeled substrate fragment in a lateral flow assav device

[0169] 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. In some cases, a sample 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.

[0170] 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.

[0171] 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 reaction mixture migrates via laminar flow from the reaction region to the detection region.

[0172] 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.

[0173] 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.Atty. Docket No.: GRIP-013WO

[0174] 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.

[0175] 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.

[0176] Further details of such devices are described elsewhere in this disclosure. Exemplary embodiments of the methods disclosed herein using a lateral flow assay device are provided in FIGS. 3A-3B and described below in Example 1 .

[0177] 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 substrate fragment can precipitate to provide a visible signal.

[0178] Alternatively, an anti-horse-radish peroxidase (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 (3, 3', 5, 5'-tetramethylbenzidine) present in the detection region produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.

[0179] 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.

[0180] Substrate bound to a solid

[0181]

[0182] In certain embodiments of the disclosure, the substrate in a reaction mixture is bound to a solid support and, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.Atty. Docket No.: GRIP-013WO

[0183] The substrate can be bound to the 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.

[0184] In some cases, the substrate is bound to the 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 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.

[0185] Solid Support is a sensor

[0186] As noted above, in some cases, the substrate in a reaction mixture is bound to a solid support. In certain such cases, the 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 et al. (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.

[0187] 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.Atty. Docket No.: GRIP-013WO

[0188] 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 (Bi2Ses), 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.

[0189] 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. Such assay is schematically represented in FIG. 4 and is described in further details in Example 2 below.

[0190] In certain other cases, the label is an enzyme that produces a change in a redox active species. For example, the enzyme can be HRP and it can cause degradation of hydrogen peroxide near the sensor surface.

[0191] 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.

[0192] Other solid supports

[0193] In certain embodiments of the methods disclosed herein where the substrate is bound to a solid support, the solid support is a bead, such as a magnetic bead or a binding partner coated beads. Thus, when a target nucleic acid is present, Craspase cleaves the bead bound substrate into a free substrate fragment and a solid support bound substrate fragment.

[0194] When the bead is a magnetic bead, a free substrate fragment can be separated from the solid support by capturing the magnetic beads using a magnet. The portion of the reaction mixture other than magnetic beads can thus be separated.

[0195] When the bead is a binding partner coated bead, a free substrate fragment can be separated from the solid support by capturing the binding partner coated beads using an immobilized binding agent that specifically binds to the binding partner on the beads. For example, streptavidin coated beads could be captured using immobilized biotin and vice versa.Atty. Docket No.: GRIP-013WO

[0196] Similarly, antigen coated 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.

[0197] In certain embodiments, 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 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.

[0198] In further embodiments, 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 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.

[0199] The reaction mixture after separating the beads can be assayed for free substrate fragments. Thus, in some cases, the method comprises detecting the free substrate fragments in the reaction mixture after separating the solid support from the reaction mixture.

[0200] In certain such embodiments, detecting the free substrate fragment comprises contacting the reaction mixture after separating the solid support from the reaction mixture to 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 lack of any change in a property of the sensor indicates that the sample does not contain the target nucleic acid.

[0201] Additional sensor surfaces described above, such as rGO, 2D-TMDs, M0S2, W-Sulfide, Selenide, Selenide compounds, such as copper indium selenide (CIS) and Bi2Se3, Mxenes, silicon IS-FET, CNTs (carbon nanotubes), and Si nanowires can also be used in such embodiments of the disclosure.

[0202] In specific embodiments, the sensor comprising the probe is a graphene sensor. The graphene sensor can be in a graphene field effect transistor (GFET) device. In certain such methods, the GFET device comprises:

[0203] a source electrode electrically connected to a graphene sensor;Atty. Docket No.: GRIP-013WO

[0204] a drain electrode electrically connected to the graphene sensor and separated from the source electrode; and

[0205] a gate electrode separated from the graphene sensor and the source and drain electrodes.

[0206] In further embodiments, the GFET device further comprises:

[0207] a platform supporting the GFET device and comprising an electrically insulating layer; and

[0208] a channel region located between the source and drain electrodes, wherein the graphene sensor is present within the channel region.

[0209] 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.

[0210] 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 graphene sensor where such interaction affects) a property, such as an electrical property of the sensor, for example, a graphene sensor of a GFET device.

[0211] 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.

[0212] 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

[0213] concentration.Atty. Docket No.: GRIP-013WO

[0214] Certain details of reporters that could be conjugated to sensors or free substrate fragments are provided in United States Provisional Patent Application No. 63 / 718,476, which is incorporated by reference in its entirety. Also, in some cases, the graphene sensor and / or the GFET devices used in the methods disclosed herein are as described in United States Provisional Patent Application No. 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.

[0215] 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.

[0216] 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.

[0217] A reaction mixture comprising a Craspase and a sample that is tested for the presence of a target nucleic acid can comprise additional reagents beyond the Craspase 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.

[0218] Reagents and conditions appropriate for Craspase reaction are well-known to a person of ordinary skill in the art and selection and maintenance of appropriate reagents and conditions for a reaction mixture are within the purview of this disclosure.

[0219] DEVICES HAVING SENSORS

[0220] Certain embodiments of the disclosure provide devices comprising sensors in contact with reaction mixtures produced in the methods disclosed herein.Atty. Docket No.: GRIP-013WO

[0221] Accordingly, in some cases, the disclosure provides a device comprising a sensor in contact with a reaction mixture, wherein the reaction mixture is produced by:

[0222] incubating a sample suspected of containing a target nucleic acid with: i) a Craspase comprising a crRNA that specifically hybridizes with the target nucleic acid, and ii) a substrate bound to a solid support,

[0223] wherein the sensor detects the cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0224] In some cases, the sensors in the devices provide the solid support to which the substrate is attached. In such cases, Craspase mediated cleavage of the substrate causes a change in a property of the sensor, which is detected by the device to determine whether the sample contains a target nucleic acid. The sensors in such devices can be electrochemical sensors or graphene sensors. Graphene sensors can be a part of GFET devices.

[0225] In some cases, the solid support to which a substrate is bound comprises beads, such as magnetic beads. In such cases, free substrate fragments, if produced in a reaction mixture, are separated from the reaction mixture by separating the beads from the reaction mixture. After separating the beads, the remaining reaction mixture is detected for the presence of free substrate fragments. Such detection can be conducted in devices comprises a sensor comprising a probe that specifically binds the free substrate fragment. Binding of the free substrate fragment to the probe causes a change in a property of the sensor, which is detected in the device to determine whether the sample contains a target nucleic acid.

[0226] Certain details of the sensors and devices comprising such sensors, such as electrochemical sensors, graphene sensors as well as GFET devices comprising graphene sensors are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” above. These details are also applicable to the devices as 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 devices as described herein and these details include the Craspase, crRNA, target nucleic acids to be detected, substrates, solid support and their binding to substrates, types of probes used, etc. Certain exemplary details are summarized below under “Clauses” and such embodiments are within the purview of the disclosure.

[0227] LATERAL FLOW ASSAY DEVICES

[0228] As discussed above, in certain embodiments of the methods disclosed herein, assaying the reaction mixture for cleavage of substrate is performed in a lateral flow assay devices.Atty. Docket No.: GRIP-013WO

[0229] Accordingly, certain embodiments of the disclosure provide lateral flow assay devices that are designed for performing certain of the methods disclosed herein.

[0230] In certain such embodiments, the disclosure provides a lateral flow assay device for determining whether a target nucleic acid is present in a sample, the lateral flow assay device comprising:

[0231] (a) a reaction region comprising:

[0232] i) a Craspase comprising a crRNA that specifically hybridizes with the target nucleic acid; and

[0233] ii) a substrate for the Craspase, the substrate immobilized in the reaction region; (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. In some cases, the lateral flow assay device further comprises a sample loading port for loading the sample. The sample loading port can be fluidically connected to the reaction region.

[0234] Alternatively, in some embodiments of the lateral flow devices disclosed herein, samples can be loaded by a user into the reaction region.

[0235] As noted above, a Craspase can be Csx29 and the substrate can be Csx30. Thus, when the target nucleic acid is present in a sample introduced into the reaction region, the Craspase, Csx29, cleaves the substrate, Csx30, into an 18 kD fragment and a 50 kD fragment.

[0236] Because the substrate for the Craspase is immobilized in the reaction region, when the substrate is cleaved by a Craspase, it produces a free substrate fragment and a reaction region bound substrate fragment. The substrate immobilized in the reaction region can also comprise a label. Typically, the label is attached to the substrate such that labeled substrate fragment is not attached, i.e., is free from the reaction region. Thus, when the substrate comprises a label and, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce an unlabeled substrate fragment that remains immobilized to the reaction region and a free labelled substrate fragment that migrates to the detection region.

[0237] In the lateral flow assay devices disclosed herein, the reaction region is fluidically connected to the detection region. As discussed elsewhere in this disclosure, the such fluidic connection typically comprises adsorbent material that allows flow of liquid from the reaction region to the detection region.

[0238] However, 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 surfacesAtty. Docket No.: GRIP-013WO

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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 theAtty. Docket No.: GRIP-013WO

[0246] 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.

[0247] ANALYZERS

[0248] Certain embodiments of the disclosure provide analyzers that are specifically designed to carry out the methods disclosed herein.

[0249] Certain details of methods, sensors, such as electrochemical sensors and graphene sensors, and devices comprising such sensors, for example GFET devices, are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid” and “Devices” above and these details are also applicable to the analyzers as described herein. The features of the devices include sensors described elsewhere in this disclosure include types of sensors, such as electrochemical sensors or graphene sensors as well as GFET devices having such sensors. These details are applicable to the analyzers disclosed herein.

[0250] 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” and “Devices” above are also applicable to the analyzers as described herein and these details include the Craspases, crRNA, target nucleic acids to be detected, labeled or unlabeled substrates, solid support (either sensor or beads), and binding of substrates to solid support, types of probes used, etc. Certain exemplary details are summarized below under “Clauses” and such embodiments are within the purview of the disclosure.

[0251] In some cases, analyzers disclosed herein comprise a sample chamber and a device comprising a sensor. The sensor can be an electrochemical sensor. The sensor can be a graphene sensor, which can be a part of a GFET device.

[0252] The sample chamber can be fluidically connected to the device comprising sensor such that a reaction mixture from the sample chamber can be transported to the sensor, for example, the electrochemical sensor or a graphene sensor of the GFET device.

[0253] In some cases, analyzers disclosed herein comprise a sample chamber and a device comprising a sensor that are not fluidically connected. In certain such cases, a user may transport a reaction mixture from the sample chamber to the device comprising a sensor.Atty. Docket No.: GRIP-013WO

[0254] A sample chamber of the analyzer is configured to carry out the Craspase reaction. Accordingly, the sample chamber can comprise one or more reagents, such as 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.

[0255] In some cases, an analyzer comprises a sample chamber providing the interior surface or a portion thereof as a sensor which provides a solid support to which the substrate is bound. Thus, in some cases, the interior surface of a sample chamber is a sensor and substrate is bound to the interior surface. If a target nucleic acid is present in a sample, the Craspase would cleave the substrate to produce free substrate fragment, which would be released into the sample chamber. The reaction mixture containing such free substrate fragment can be transported out of the sample chamber and analyzed for the presence of the free substrate fragment. Such transport can be performed using a transporting means, such as a pump, to move the reaction mixture from the sample chamber into the device comprising the sensor. Alternatively, such transport can be performed manually by a user.

[0256] In certain embodiments where an interior surface or a portion thereof of a sample chamber is used as a sensor to which substrate is bound, the interior surface or the portion thereof is a graphene sensor of the GFET device. Thus, in such embodiments, the interior surface of the sample chamber is a part of the GFET device. The substrate is bound to the graphene sensor of the GFET device, when cleaved by a Craspase, changes a property of the graphene sensor. In some cases, the substrate can further comprise a reporter, such as an electrochemical reporter. A change in a property can be a change in an electrical property, such as conductivity of Dirac voltage.

[0257] In certain embodiments where an interior surface or a portion thereof of a sample chamber is used as a sensor to which substrate is bound, the interior surface or the portion thereof is an electrochemical sensor of the device. Thus, in such embodiments, the interior surface of the sample chamber is a part of the device. The substrate is bound to the electrochemical sensor of the device, when cleaved by a Craspase, changes a property of the electrochemical sensor. In some cases, the substrate can further comprise a reporter, such as an electrochemical reporter. A change in a property can be a change in an electrochemical property, such as a change in the redox signal.

[0258] In some cases, an analyzer comprises a filter between a sample chamber and fluidic connection to the device comprising the sensor. Thus, when a reaction mixture is transported from the sample chamber into the device, beads comprising the substrate, if used in the reaction mixture, are separated from the rest of the reaction mixture. The filtered reactionAtty. Docket No.: GRIP-013WO

[0259] mixture can then be introduced to a sensor of the device for determining the presence or absence of free substrate fragments.

[0260] In some cases, a user can manually filter a reaction mixture from the reaction chamber and introduce filtered reaction mixture to a sensor of a device for determining the presence or absence of free substrate fragments.

[0261] METHODS OF DETECTING A TARGET NUCLEIC ACID IN SPECIFIC ANALYZERS / DEVICES

[0262] In certain embodiments, the disclosure provides methods of determining whether a target nucleic acid is present in a sample, wherein the method is performed using a suitable analyzer. Accordingly, certain embodiments of the disclosure provide a method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0263] (a) introducing the sample into a sample chamber of an analyzer to produce a reaction mixture, wherein the sample chamber comprises: i) a Craspase comprising a crRNA that specifically hybridizes with the target nucleic acid; and ii) a substrate for the Craspase, the substrate bound to a solid support;

[0264] (b) incubating the reaction mixture, and

[0265] (c) assaying for cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0266] As discussed above, the substrate bound to the solid support is cleaved from the solid support by the Craspase when the target nucleic acid is present in the sample.

[0267] Certain details of analyzers are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid,” “Devices,” and “Analyzers” above and these details are also applicable to the methods of detecting target nucleic acids in specific analyzers as described herein. The features of the sensors, such as electrochemical sensors or graphene sensors, described elsewhere in this disclosure are applicable to the methods of detecting target nucleic acids using specific analyzers described herein.

[0268] 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,” “Devices,” and “Analyzers” above and these details are also applicable to the methods of detecting target nucleic acids in specific devices as described herein. Certain such details include the Craspases, crRNA, target nucleic acids to be detected, substrate and its labels, solid support and their binding to substrates, types of probes used, etc. Certain exemplary details are summarized below under “Clauses” and such embodiments are within the purview of the disclosure.Atty. Docket No.: GRIP-013WO

[0269] KITS FOR DETECTING A TARGET NUCLEIC ACID

[0270] In some cases, the disclosure provides kits designed for detecting target nucleic acids. In some cases, a target nucleic acid detection kit comprises:

[0271] a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid;

[0272] a substrate bound to a solid support, and

[0273] a sensor configured to detect cleavage of the substrate bound to the solid support. In some cases, the devices that detects the cleavage of the substrate bound to the solid support by detecting the presence of any free substrate fragment produced by the cleavage of the substrate bound to the 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 substrate fragment. Binding of the free substrate 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 substrate fragment.

[0274] Alternatively, the solid support is sensor, such as an electrochemical sensor or a graphene sensor, and the device detects the cleavage of the substrate by detecting the change caused by the cleavage of the substrate in a property of the sensor. Certain details of the devices and methods are provided elsewhere in this disclosure, including under “Methods of detecting a target nucleic acid,” “Devices,” and “Analyzers” above and these details are also applicable to the target nucleic acid detection kits as described herein. The features of the devices described elsewhere in this disclosure and also applicable to the target nucleic acid detection kits as disclosed herein. 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,” “Devices,” and “Analyzers” above and these details are also applicable to the target nucleic acid detection kits described herein and include the Craspases, crRNA, target nucleic acids to be detected, substrates and their labels, solid support and their binding to the substrates, types of probes used, etc. Certain exemplary details are summarized below under “Clauses” and such embodiments are within the purview of the disclosure.

[0275] 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, forAtty. Docket No.: GRIP-013WO

[0276] 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.

[0277] 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, for example, a battery or an electrical connector for connecting the power supply to an external power source such as a plug-in power source.

[0278] 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.

[0279] 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.

[0280] 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 orAtty. Docket No.: GRIP-013WO

[0281] aspects of surface functionalization (e.g., probe and electrochemical reporter) of the graphene sensor, as described herein.

[0282] 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 to 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.

[0283] 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.

[0284] APPLICATIONS

[0285] 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.Atty. Docket No.: GRIP-013WO

[0286] 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 0043, 229E and HK, human metapneumovirus, rhinoviruses, enteroviruses, hepatitis A, B, G 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, 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.

[0287] 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. coli such as 0157:H7 E. coli or 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.Atty. Docket No.: GRIP-013WO

[0288] 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.

[0289] 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, BCR / ABL1, CMYC, NMYC, EGER, EML4AK, KRAS, and RAS 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.

[0290] 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.

[0291] Certain genetic diseases are caused by specific mutation in certain genes. For example, a mutation in the HBA1 or HBA2 gene 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.

[0292] 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.

[0293] 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.Atty. Docket No.: GRIP-013WO

[0294] Contamination of agricultural land with undesirable microorganisms can be detected by analyzing a soil sample for target nucleic acids specific for such microorganisms.

[0295] 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.

[0296] 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.

[0297] 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) toAtty. Docket No.: GRIP-013WO

[0298] 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).

[0299] 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).

[0300] 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.

[0301] 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.

[0302] 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 sampleAtty. Docket No.: GRIP-013WO

[0303] (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.

[0304] 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 (PCR)), reverse transcription to obtain complementary DNA (cDNA), reverse transcription PCR, nucleic acid denaturation and isothermal amplification.

[0305] 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.

[0306] 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.

[0307] The following example(s) is / are offered by way of illustration and not by way of limitation.

[0308] EXAMPLES

[0309] Example 1 - Detecting nucleic acids and diagnosing an infection using a Craspase and an immobilized substrate and detecting free substrate fragment in a lateral flow assay device Certain exemplary embodiments of the disclosure are described in FIGS. 3A and 3B. For the embodiment shown in FIG. 3A, 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 reaction mixture by combining with Csx29 having a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reactionAtty. Docket No.: GRIP-013WO

[0310] mixture can then be added to a reaction region of a lateral flow assay device. The reaction region of the lateral flow assay device comprises immobilized therein Csx30. Csx30 will also comprise protein A label and gold nanoparticles.

[0311] If the sample contains the target nucleic acid, Csx29 would cleave the protein A labeled and tagged Csx30 to produce a protein A and gold nanoparticle containing free Csx30 fragment. The free Csx30 fragment would then migrate from the reaction region to a flu idically connected detection region of the lateral flow assay device. The free protein A labeled substrate would then encounter a IgG immobilized in the detection region and bind to the immobilized IgG via protein A label. Accumulation of protein A labeled free substrate fragment would produce a detectable signal because of the accumulation of gold nanoparticles tag. 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 bacterial or viral infection in the subject.

[0312] For the embodiment shown in FIG. 3B, 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 reaction mixture by combining with Csx29 having a crRNA that specifically hybridizes with a target RNA from the bacterium or virus and Csx30 conjugated to beads and labeled with HRP enzyme (Steps A and B). During incubation with Csx30 and if the target RNA from the bacterium or virus is present, Craspase Csx29 cleaves the substrate to produce HRP labeled free substrate fragment and bead bound substrate fragment. The reaction mixture can then be filtered to remove beads and bead bound substrate fragments. The reaction mixture can then be added to a reaction region of a lateral flow assay device (Step C).

[0313] The HRP labeled free 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 HRP enzyme. Accumulation of HRP labeled free substrate fragment would produce a detectable signal because of the action of HRP on hydrogen peroxide (provided in the detection region) and TMB, 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 bacterial or viral infection in the subject.

[0314] In addition to the “test zone” having immobilized anti-HRP antibodies (shown in FIG. 3B,Atty. Docket No.: GRIP-013WO

[0315] steps C and D), 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 with the reaction mixture.

[0316] 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.

[0317] Example 2 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to an electrochemical sensor and detecting cleavage of the substrate This exemplary embodiment of the disclosure is described in FIG. 4. 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 mixed with Csx29 comprising a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reaction will be carried out in the presence of an electrochemical sensor having immobilized on the sensor Csx30 tagged with an electrochemically active species. The electrochemically active species can be a redox agent, such as methylene blue or ferrocene. Alternatively, the sensor can comprise an enzyme, such as HRP, alkaline phosphatase, and tyrosinase, which produces redox changes on the electrochemical sensor in presence of appropriate chemicals. Release of electrochemically active species results in reduction of charge on the electrochemical sensor An electrochemical property, such as redox signal, of the electrochemical sensor can be monitored throughout the reaction. If the sample contains the target nucleic acid, the Csx29 would cleave Csx30 bound to the sensor and induce a change in an electrochemical property of the electrochemical sensor. Such change in a electrochemical property will be detected using square wave voltammetry. Any change in the electrochemical property of the sensor will be enhanced by the release of methylene blue from the vicinity of the electrochemical sensor as induced by the cleavage of the substrate and release of methylene blue containing free substrate fragment.

[0318] If the sample does not contain the target nucleic acid, Csx29 would not cleave Csx30 from the electrochemical sensor and would not induce any change in an electrical property of the electrochemical sensor.Atty. Docket No.: GRIP-013WO

[0319] Thus, monitoring the electrical property of the electrochemical sensor would indicate the presence or absence of the target nucleic acid in the sample.

[0320] Example 3 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to a bead and detecting cleavage of the substrate by a graphene sensor This exemplary embodiment of the disclosure is described in FIG. 5. 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 using a complex comprising Csx29 and a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reaction mixture will also comprise Csx30 for the Craspase immobilized on magnetic beads. If the sample contains the target nucleic acid, Csx30 would cleave Csx29 from the magnetic beads to produce free substrate fragments. Steps A and B will be carried out as shown in FIG. 5. The reaction mixture at the end of Step B will be filtered to separate free substrate fragments, if present. Alternatively, the reaction mixture at the end of Step B will be separated using a magnet, which would capture magnetic beads.

[0321] Regardless, the filtrate / supernatant will then be contacted with a GFET device comprising a graphene sensor comprising a probe that specifically binds to the free substrate fragment.

[0322] If the sample contains the target nucleic acid, the filtrate will contain the free substrate fragments and the substrate fragments would bind to the probe on the graphene sensor. Such binding would change the electrical properties of the graphene sensor, which will be monitored to determine whether the target RNA is present in the patient sample.

[0323] Example 4 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to a sensor and detecting cleavage of the substrate by detecting release of a reporter from the sensor

[0324] This exemplary method (600) of the disclosure is described in FIG. 6. 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 using a complex comprising Csx29 (605) and a crRNA (606) that specifically hybridizes with a target RNA (607) from the bacterium or virus. The reaction mixture will also comprise Csx30 (comprising fragments 602, 603, and reporter 604) for the Craspase immobilized on the sensor (601 ). If the sample contains the target nucleic acid, Csx30 would cleave Csx29 from the sensor to produce free substrate fragments comprising the reporter. Such release of the reporter would change theAtty. Docket No.: GRIP-013WO

[0325] properties of the graphene sensor, which will be monitored to determine whether the target RNA is present in the patient sample.

[0326] Example 5 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to a bead and detecting cleavage of the substrate by detecting changes in electrochemical properties of a sensor

[0327] This exemplary embodiment of the disclosure is described in FIG. 7. 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 using a complex comprising Csx29 and a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reaction mixture will also comprise Csx30 for the Craspase conjugated to HRP and immobilized on magnetic beads. If the sample contains the target nucleic acid, Gsx30 would cleave Csx29 from the magnetic beads to produce free substrate fragments comprising HRP. The reaction mixture will be filtered to separate free substrate fragments, if present.

[0328] Alternatively, the reaction mixture will be separated using a magnet, which would capture magnetic beads.

[0329] Regardless, in one embodiment, the filtrate / supernatant will then be contacted with a GFET device comprising a graphene sensor in the presence of TMB and hydrogen peroxide. HRP would then cause degradation of hydrogen peroxide and in the process, change Dirac point of the graphene sensor.

[0330] Alternatively, in one embodiment, the filtrate / supernatant will then be contacted with an electrochemical sensor in the presence of TMB and hydrogen peroxide. HRP would then cause degradation of hydrogen peroxide and in the process, change an electrochemical readout of the electrochemical sensor, which can be observed by square wave voltammetry.

[0331] Example 6 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to a graphene sensor

[0332] This exemplary embodiment of the disclosure is described in FIG. 8. 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 using a complex comprising Csx29 and a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reaction mixture will be contacted with a graphene sensor comprising a charged Csx30. If the sample contains the target nucleic acid, Csx29 would cleave charged Csx30 from the sensor to produce free substrate fragments. Cleavage of the charged Csx30 would change theAtty. Docket No.: GRIP-013WO

[0333] Dirac point readout of the graphene sensor, which can be observed by measuring Dirac point shift.

[0334] Example 7 - Detecting nucleic acids and diagnosing an infection using a Craspase and a substrate bound to beads and optically detecting the cleavage of the substrate

[0335] This exemplary embodiment of the disclosure is described in FIG. 9. 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 using a complex comprising Csx29 and a crRNA that specifically hybridizes with a target RNA from the bacterium or virus. The reaction mixture will be contacted with a beads comprising Csx30 conjugated to HRP. If the sample contains the target nucleic acid, Csx29 would cleave Csx30 from the bead to produce free Csx30 fragments comprising HRP. Cleavage of the charged Csx30 would change the Dirac point readout of the graphene sensor, which can be observed by measuring Dirac point shift.

[0336] The reaction mixture will be filtered to separate free substrate fragments, if present. Alternatively, the reaction mixture will be separated using a magnet, which would capture magnetic beads.

[0337] Regardless, the supernatant / filtrate is added into a detection solution comprising TMB and hydrogen peroxide. If the supernatant / filtrate contains free Csx30 fragment comprising HRP, the HRP would degrade hydrogen peroxide and cause TMB to turn blue, thereby turning the solution blue. The blue solution can be analyzed in a spectrophotometer to qualitatively or quantitatively determine the presence or the quantity of the target nucleic acid in the sample.

[0338] As an alternative to the colorimetric changes in the solution, other optical signals can be detected. Certain such alternatives include fluorescence (small molecule or label like GPP, or fluorophore quencher pair), SPR (surface plasmon resonance), Surface enhanced Raman spec (SERS), or luminescence. A person of ordinary skill in the art can implement appropriate methods according to the detected signal.

[0339] Example 8 - Efficiency of Craspase in cleaving Csx30

[0340] This example provides characterization of a Craspase that can be used in the methods and devices of the disclosure.

[0341] Cas7-11 -crRNA-Csx29 complex was used in this assay. Cas7-11-crRNA-Csx29 complex is a Type lll-E CRISPR-Cas system component. Cas7-11 is a large, single effector protein and Csx29 is an associated Craspase enzyme of this complex. When activated, Csx29Atty. Docket No.: GRIP-013WO

[0342] cleaves the substrate (Csx30).

[0343] A reaction mixture was produced containing, among other components, the Cas7-11-crRNA-Csx29 complex (150 nM), target RNA (250 nM), and Csx30 (3.75 pM). The reaction was incubated at 37°C. Reaction mixtures were analyzed after 10 minutes, 30 minutes, and 60 minutes of incubation. The reaction mixtures were processed and analyzed by SDS-PAGE electrophoresis. The gels were stained to identify different proteins in the reaction mixtures.

[0344] As shown in FIG. 10, after 10 minutes of incubation, the tested Graspase cleaved almost all of Csx30 (molecular weight around 70 kDa) present in the reaction mixture to produce a fragment of around 55 kDa and a fragment of around 15 kDa.

[0345] In a similar experimental set up, reaction mixtures were prepared with varying concentrations of the target RNA, from 100 nM to 0.1 nM. These data are represented in FIG.

[0346] 11.

[0347] The tested Graspase cleaved almost all of Csx30 present in the reaction mixture at the target RNA concentrations of as low as 2.5 nM. This is evidenced in FIG. 11 by the bands of 55 kDa and 15 kDa fragments with almost no bands at the 70 kDa positions.

[0348] At the target RNA concentrations of 1 nM and 0.25 nM, the tested Graspase cleaved sufficient amount of Csx30 to produce clearly visible bands on the SDS-PAGE for 50 kDa and 15 kDa fragments. At the target RNA concentration of 0.1 nM, very faint 50 kDa fragment band was observed but band was not clearly visible for the 15 kDa fragment. However, any fragments produced with the target RNA concentration of 0.1 nM and lower may be detectable via methods that are more sensitive than staining the SDS-PAGE gels.

[0349] In a similar experimental set up, reaction mixtures were incubated at varying temperatures, e.g., at 20°C and 28°C. These data are represented in FIG. 12. FIG. 12 shows that, at both 20°C and 28°C, Graspase cleaved almost all of Csx30 present in the reaction mixture at the target RNA concentrations of as low as 2.5 nM. Also, at the target RNA concentrations of 1 nM and 0.25 nM and at both 20°C and 28°C, the tested Graspase cleaved sufficient amount of Csx30 to produce clearly visible bands on the SDS-PAGE for 50 kDa and 15 kDa fragments.

[0350] These data show that the methods and devices disclosed herein can operate at room temperature thereby indicating that an end-user based lateral flow assays and devices disclosed herein would be operable to detect target nucleic acids.

[0351] In a similar experimental set up, reaction mixtures were incubated at 25°C with the target RNA in different solutions, such as lysis buffer, urine, or a mixture of lysis buffer and urine. These data are represented in FIG. 13.Atty. Docket No.: GRIP-013WO

[0352] FIG. 13 shows that the tested Craspase detected the target nucleic acid in different solutions, including a urine sample. These data show that the methods and devices disclosed herein can be used for detecting nucleic acids in biological samples, such as urine samples.

[0353] Example 9 - Detection of N. gonorrhoeae NCTC 8375

[0354] This example describes implementing the methods disclosed herein to detect N. gonorrhoeae NCTC 8375. A sample containing N. gonorrhoeae NCTC 8375 was incubated in a reaction mixture with a Craspase having a crRNA that specifically binds a section of the 16S ribosomal RNA from N. gonorrhoeae NCTC 8375. Aliquots of the reaction mixture taken after 1 , 5, and 10 minutes were run on an SDS-PAGE gel and stained. The stained gel is shown in FIG.

[0355] 14.

[0356] As shown in FIG. 14, 55 kDa and 15 kDa fragments of Csx30 were produced after 2 minutes of incubation and a significant amount of these fragments were produced after 10 minutes of incubation. These data show that the methods and devices disclosed herein can be used for detecting nucleic acids from N. gonorrhoeae NCTC 8375. These methods can be modified to detect any other desired pathogens or organisms of other interest.

[0357] Example 10- Detection of N. qonorrhoeae NCTC 8375 in lateral flow assay devices

[0358] This example describes an exemplary lateral flow assay device for detecting a target nucleic acid. This is described in FIG. 15.

[0359] The top panel of FIG. 15 depicts pictures of lateral flow assay devices showing test lines and control lines. A sample containing 0 (left panel, negative control) or 8 pM (right panel) of the labeled substrate fragment (Csx30-HRP) is introduced to the lateral flow assay device. This figure shows detection of Csx30-HRP fragment at the picomolar concentration range, while no signal is detected in the absence of the labeled substrate (negative control). The bottom panel of FIG. 15 is a graph showing quantified intensities of the lines developed in the lateral flow assay devices depicted in the top panel of FIG. 15.

[0360] Example 11 -Detection of N. gonorrhoeae NCTC 8375 based on changes in electrochemical properties of a sensor

[0361] This example describes an exemplary sensor based detection of a target nucleic acid from N. gonorrhoeae NCTC 8375. This is described in FIG. 16.

[0362] For the data presented in FIG. 16, 104cells of N. gonorrhoeae NCTC 8375 were lysedAtty. Docket No.: GRIP-013WO

[0363] using a proprietary lysis buffer containing a denaturant, surfactant, and buffering agent. The resultant RNA from this lysis was used as target RNA to activate a cognate Craspase complex, resulting in the cleavage of Csx30-HRP immobilized to a magnetic bead surface after the reaction is performed for 10 minutes at ambient temperature. After separation of the beads using a magnet, the labeled substrate fragment was introduced to the sensor surface along with the TMB substrate. Electrochemical current generated from the reduction of TMB was measured, showing a marked difference between positive (solid line) and negative samples (dotted lines).

[0364] Notwithstanding the appended claims, the invention may be defined by the following clauses:

[0365] 1. A method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0366] (a) producing a reaction mixture by combining the sample with:

[0367] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0368] ii) a substrate for the Craspase;

[0369] and

[0370] (b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample.

[0371] 2. The method of Clause 1 , wherein, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate.

[0372] 3. The method of Clause 1 or 2, wherein the substrate for the Craspase is Csx30.

[0373] 4. The method of any one of Clauses 1 to 3, wherein, when the target nucleic acid is present in the sample, 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.

[0374] 5. The method of any one of preceding Clauses, wherein the substrate comprises a label and, when the target nucleic acid is present in the subject, the Craspase cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment.

[0375] 6. The method of Clause 5, wherein assaying the reaction mixture for cleavage of the substrate comprises detecting the labeled substrate fragment.Atty. Docket No.: GRIP-013WO

[0376] 7. The method of Clause 6, wherein detecting the labeled substrate fragment comprises contacting the reaction mixture with a binding partner for the label, wherein the binding partner for the label is immobilized on a matrix.

[0377] 8. The method of Clause 7, wherein the matrix is an adsorbent material in a detection region of a lateral flow assay device.

[0378] 9. The method of Clause 8, wherein the method comprises loading the sample into a reaction region of the lateral flow assay device, the reaction region comprising the Craspase comprising the 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.

[0379] 10. The method of any one of Clauses 7 to 9, 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.

[0380] 11. The method of any one of Clauses 7 to 10, wherein binding of the binding partner and the label produces a detectable signal.

[0381] 12. The method of Clause 11 , wherein the detectable signal is produced from a tag bound to the label.

[0382] 13. The method of Clause 12, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0383] 14. The method of any one of Clauses 1 to 5, wherein the substrate is bound to a solid support and, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment.

[0384] 15. The method of Clause 14, wherein the substrate is bound to the solid support via a covalent bond.

[0385] 16. The method of Clause 14, wherein the substrate is bound to the solid support via a non-covalent bond.

[0386] 17. The method of any one of Clauses 14 to 16, wherein the solid support is a sensor and cleavage of the substrate produces the free substrate fragment thereby causing a change in a property of the sensor.

[0387] 18. The method of Clause 17, wherein the change in the property of the sensor is a change in an electrochemical property.

[0388] 19. The method of Clause 18, wherein the change in the electrochemical property is a change in the redox signal.Atty. Docket No.: GRIP-013WO

[0389] 20. The method of Clause 19, 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.

[0390] 21. The method of Clause 17, wherein the change in the property of the sensor is a change in an electrical property.

[0391] 22. The method of Clause 21 , wherein the electrical property is electrical resistance or Dirac voltage of the sensor.

[0392] 23. The method of any one of Clauses 17 to 22, wherein the sensor is a graphene sensor.

[0393] 24. The method of any one of Clauses 14 to 23, wherein the free substrate fragment is the labeled substrate fragment and the solid support bound fragment is the unlabeled substrate fragment.

[0394] 25. The method of any one of Clauses 14 to 16, wherein the solid support is a bead.

[0395] 26. The method of Clause 25, comprising separating the solid support from the reaction mixture.

[0396] 27. The method of Clause 26, wherein separating the solid support from the reaction mixture comprises centrifuging the reaction mixture.

[0397] 28. The method of Clause 26, wherein the beads are magnetic beads and separating the magnetic beads from the reaction mixture comprises capturing the magnetic beads with a magnet.

[0398] 29. The method of any one of Clauses 25 to 28, further comprising detecting the free substrate fragment in the reaction mixture after separating the solid support from the reaction mixture.

[0399] 30. The method of Clause 29, wherein detecting the free fragment comprises contacting the reaction mixture after separating the solid support from the reaction mixture to a graphene sensor comprising a probe that specifically binds the free substrate fragment and detecting a change in a property of the graphene sensor caused by binding of the free substrate fragment to the probe.

[0400] 31. The method of Clause 30, wherein the graphene sensor is in a graphene field effect transistor (GFET) device comprising:

[0401] a source electrode electrically connected to the graphene sensor;

[0402] a drain electrode electrically connected to the graphene sensor and separated from the source electrode; and

[0403] a gate electrode separated from the graphene sensor and the source and drain electrodes.Atty. Docket No.: GRIP-013WO

[0404] 32. The method of Clause 31 , wherein the GFET device further comprises:

[0405] a platform supporting the GFET device and comprising an electrically insulating layer; and

[0406] a channel region located between the source and drain electrodes, wherein the graphene sensor is present within the channel region.

[0407] 33. The method of any one of Clauses 30 to 32, wherein the probe that specifically binds to the free substrate fragment is a protein.

[0408] 34. The method of any one of Clauses 30 to 32, wherein the probe that specifically binds to the free substrate fragment is an aptamer.

[0409] 35. The method of any one of Clauses 30 to 34, wherein the probe comprises an electrochemical reporter.

[0410] 36. The method of any one of Clauses 30 to 35, wherein the property of the graphene sensor is an electrical property of the graphene sensor.

[0411] 37. The method of Clause 36, wherein the electrical property of the graphene sensor is conductivity of the graphene sensor.

[0412] 38. The method of Clause 36, wherein the electrical property of the graphene sensor is Dirac voltage of the graphene sensor.

[0413] 39. The method of any one of the preceding Clauses, wherein the Craspase is Csx29. 40. The method of any one of the preceding Clauses, wherein the target nucleic acid is a nucleic acid from a microorganism.

[0414] 41. The method of Clause 40, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0415] 42. A method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0416] (a) introducing the sample into a sample chamber of an analyzer to produce a reaction mixture,

[0417] wherein the sample chamber comprises: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and ii) a substrate for the Craspase, the substrate bound to a solid support;

[0418] (b) incubating the reaction mixture, and

[0419] (c) assaying for cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0420] 43. The method of Clause 42, wherein the substrate for the Craspase is Csx30.Atty. Docket No.: GRIP-013WO

[0421] 44. The method of Clause 43, wherein, when the target nucleic acid is present in the sample, the Craspase cleaves Csx30 into an 18 kD fragment and a 50 kD fragment.

[0422] 45. The method of any one of Clauses 42 to 44, wherein, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate bound to the solid support to produce a free substrate fragment and a solid support bound substrate fragment.

[0423] 46. The method of Clause 45, wherein the assaying the reaction mixture for cleavage of the substrate comprises detecting with a graphene sensor the presence of any free substrate fragment produced by the cleavage of the substrate bound to the solid support, and wherein the graphene sensor comprises a probe that specifically binds to the free substrate fragment.

[0424] 47. The method of Clause 46, wherein the graphene sensor is a part of a GFET device, the GFET device further comprising:

[0425] a source electrode electrically connected to the graphene sensor;

[0426] a drain electrode electrically connected to the graphene sensor and separated from the source electrode; and

[0427] a gate electrode separated from the graphene sensor and the source and drain electrodes.

[0428] 48. The method of Clause 47, wherein the GFET device further comprises:

[0429] a platform supporting the GFET device and comprising an electrically insulating layer; and

[0430] a channel region located between the source and drain electrodes, wherein the graphene sensor is present within the channel region.

[0431] 49. The method of any one of Clauses 46 to 48, comprising separating the solid support from the reaction mixture before said contacting the reaction mixture with the graphene sensor.

[0432] 50. The method of any one of Clauses 42 to 49, wherein the solid support comprises beads.

[0433] 51. The method of Clause 50, wherein the beads are magnetic beads.

[0434] 52. The method of any one of Clauses 42 to 45, wherein the solid support comprises a sensor.

[0435] 53. The method of Clause 52, wherein the sensor is an electrochemical sensor.

[0436] 54. The method of Clause 52 or 53, wherein the substrate comprises a label.

[0437] 55. The method of Clause 54, wherein the Craspase mediated cleavage of the substrate releases a labeled substrate fragment from the electrochemical sensor thereby changing a property of the electrochemical sensor.

[0438] 56. The method of Clause 54 or 55, wherein the label is a redox active agent or an enzyme.

[0439] 57. The method of Clause 56, wherein the redox active agent is methylene blue.Atty. Docket No.: GRIP-013WO

[0440] 58. The method of Clause 56, wherein the enzyme is horseradish peroxidase.

[0441] 59. The method of any one of Clauses 42 to 58, wherein the sample chamber comprises the Craspase in a dried form.

[0442] 60. The method of any one of Clauses 42 to 59, wherein the substrate is bound to the solid support via a covalent bond.

[0443] 61. The method of any one of Clauses 42 to 59, wherein the substrate is bound to the solid support via a non-covalent bond.

[0444] 62. The method of any one of Clauses 42 to 61 , wherein the target nucleic acid is a nucleic acid from a microorganism.

[0445] 63. The method of Clause 62, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0446] 64. A method of determining whether a target nucleic acid is present in a sample, the method comprising:

[0447] (a) producing a reaction mixture by combining the sample with:

[0448] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0449] ii) a substrate for the Craspase, the substrate comprising a label;

[0450] and

[0451] (b) assaying the reaction mixture in a lateral flow assay device for cleavage of substrate to determine whether the target nucleic acid is present in the sample.

[0452] 65. The method of Clause 64, wherein the substrate for the Craspase is Csx30.

[0453] 66. The method of Clause 64 or 65, wherein, when the target nucleic acid is present in the sample, the Craspase cleaves Csx30 into a fragment of between 16 kD and 20 kD and a fragment of between 45 kD and 50 kD.

[0454] 67. The method of any one of Clauses 64 to 66, wherein, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment.

[0455] 68. The method of any one of Clauses 64 to 67, wherein the lateral flow assay device comprises a reaction region comprising the Craspase and the labeled substrate, and wherein the method comprises introducing the sample into the reaction region of the lateral flow assay device.Atty. Docket No.: GRIP-013WO

[0456] 69. The method of Clause 68, 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, a free labeled substrate fragment is produced.

[0457] 70. The method of any one of Clauses 64 to 69, comprising introducing the reaction mixture in a sample loading region of the lateral flow assay device.

[0458] 71. The method of any one of Clauses 64 to 70, 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.

[0459] 72. The method of Clause 71 , 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.

[0460] 73. The method of Clause 71 or 72, wherein binding of the binding partner and the label produces a detectable signal.

[0461] 74. The method of Clause 73, wherein the detectable signal is produced from a tag bound to the label.

[0462] 75. The method of Clause 74, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0463] 76. A target nucleic acid detection kit, comprising:

[0464] a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid;

[0465] a substrate bound to a solid support, and

[0466] a sensor configured to detect cleavage of the substrate bound to the solid support.

[0467] 77. The kit of Clause 76, wherein the substrate for the Craspase is Csx30.

[0468] 78. The kit of Clause 77, wherein, when the crRNA hybridizes with the target 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.

[0469] 79. The kit of any one of Clauses 76 to 78, wherein, when the crRNA hybridizes with the target nucleic acid, the Craspase cleaves the substrate bound to the solid support to produce a free substrate fragment.

[0470] 80. The kit of any one of Clauses 76 to 79, wherein the substrate is bound to the solid support via a covalent bond.Atty. Docket No.: GRIP-013WO

[0471] 81. The kit of any one of Clauses 76 to 79, wherein the substrate is bound to the solid support via a non-covalent bond.

[0472] 82. The kit of any one of Clauses 76 to 81 , wherein the solid support is a bead.

[0473] 83. The kit of Clause 80, wherein the bead is a magnetic bead.

[0474] 84. The kit of any one of Clauses 76 to 83, 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.

[0475] 85. The kit of Clause 84, wherein the sensor is a graphene sensor.

[0476] 86. The kit of Clause 85, wherein the graphene sensor is a part of a GFET device, the GFET device further comprising:

[0477] a source electrode electrically connected to the graphene sensor;

[0478] a drain electrode electrically connected to the graphene sensor and separated from the source electrode; and

[0479] a gate electrode separated from the graphene sensor and the source and drain electrodes.

[0480] 87. The kit of Clause 86, wherein the GFET device further comprises:

[0481] a platform supporting the GFET device and comprising an electrically insulating layer; and

[0482] a channel region located between the source and drain electrodes, wherein the graphene sensor is present within the channel region.

[0483] 88. The kit of any one of Clauses 84 to 87, wherein the probe that specifically binds to the free substrate fragment is a protein.

[0484] 89. The kit of any one of Clauses 84 to 87, wherein the probe that specifically binds to the free substrate fragment is an aptamer.

[0485] 90. The kit of any one of Clauses 84 to 89, wherein the probe comprises an electrochemical reporter.

[0486] 91. The kit of any one of Clauses 86 to 90, wherein the GFET device detects the cleavage of the substrate by detecting a change in property of the graphene sensor caused by the binding of the free substrate fragment to the probe on the graphene sensor of the GFET device.

[0487] 92. The kit of Clause 91 , wherein the property of the graphene sensor is an electrical property of the graphene sensor.

[0488] 93. The kit of Clause 92, wherein the electrical property of the graphene sensor is conductivity of the graphene sensor or Dirac voltage of the graphene sensor.Atty. Docket No.: GRIP-013WO

[0489] 94. The kit of any one of Clauses 76 to 81 , wherein the solid support to which the substrate is bound is the sensor configured to detect cleavage of the substrate.

[0490] 95. The kit of Clause 94, wherein the sensor is an electrochemical sensor.

[0491] 96. The kit of Clause 94 or 95, wherein the substrate comprises a label.

[0492] 97. The kit of Clause 96, wherein the Craspase mediated cleavage of the substrate causes release of the label from the sensor thereby changing a property of the electrochemical sensor.

[0493] 98. The kit of Clause 96 or 97, wherein the label is a redox active agent or an enzyme.

[0494] 99. The kit of Clause 98, wherein the redox active agent is methylene blue.

[0495] 100. The kit of Clause 98, wherein the enzyme is horseradish peroxidase.

[0496] 101. The kit of any one of Clauses 76 to 100, wherein the Craspase is Csx29 and the substrate for the Craspase is Csx30.

[0497] 102. The kit of any one of Clauses 76 to 101 , wherein the target nucleic acid is a nucleic acid from a microorganism.

[0498] 103. The kit of Clause 102, wherein the microorganism is a bacterium, virus, algae, archaeon, fungus, or protozoan.

[0499] 104. A device comprising a sensor in contact with a reaction mixture,

[0500] wherein the reaction mixture is produced by:

[0501] incubating a sample suspected of containing a target nucleic acid with: i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid, and ii) a substrate bound to a solid support,

[0502] wherein the sensor detects the cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

[0503] 105. The device of Clause 104, wherein the substrate for the Craspase is Csx30.

[0504] 106. The device of Clause 104 or 105, wherein, when the target nucleic acid is present in the sample, 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.

[0505] 107. The device of any one of Clauses 104 to 106, wherein the sensor is a graphene sensor and the device is the GFET device.

[0506] 108. The GFET device of Clause 107, wherein the substrate bound to the solid support is cleaved from the solid support by the Craspase when the target nucleic acid is present in the sample.

[0507] 109. The device of any one of Clauses 104 to 108, wherein the reaction mixture is further produced by separating the reaction mixture from the solid support after said incubating, andAtty. Docket No.: GRIP-013WO

[0508] wherein the GFET device detects the presence of a fragment of the substrate cleaved from the solid support.

[0509] 110. The device of any one of Clauses 104 to 106, wherein the 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.

[0510] 111. The device of Clause 110, wherein the sensor is an electrochemical sensor.

[0511] 112. The device of Clause 110 or 111 , wherein the substrate comprises a label.

[0512] 113. The device of Clause 112, wherein the Craspase mediated cleavage of the substrate releases the label from the sensor thereby changing the property of the electrochemical sensor.

[0513] 114. The device of Clause 112 or 113, wherein the label is a redox active agent or an enzyme.

[0514] 115. The device of Clause 114, wherein the redox active agent is methylene blue.

[0515] 116. The device of Clause 114, wherein the enzyme is horseradish peroxidase.

[0516] 117. The device of any one of Clauses 104 to 116, wherein the Craspase is Csx29 and the substrate for the Craspase is Csx30.

[0517] 118. The device of any one of Clauses 104 to 117, wherein the target nucleic acid is a nucleic acid from a microorganism.

[0518] 119. The device of Clause 118, wherein the microorganism is a bacterium, virus, fungus, or protozoan.

[0519] 120. A lateral flow assay device for determining whether a target nucleic acid is present in a sample, the lateral flow assay device comprising:

[0520] (a) a reaction region comprising:

[0521] i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; and

[0522] ii) a substrate for the Craspase, the substrate immobilized in the reaction region; (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.

[0523] 121. The lateral flow assay device of Clause 120, wherein the substrate for the Craspase is Csx30.

[0524] 122. The lateral flow assay device of Clause 121, wherein, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate into a fragment between 15 and 20 kD and a fragment between 45 kD and 55 kD.Atty. Docket No.: GRIP-013WO

[0525] 123. The lateral flow assay device of any one of Clauses 120 to 122, wherein, when the target nucleic acid is present in the sample introduced into the reaction region, the Craspase cleaves the substrate.

[0526] 124. The lateral flow assay device of any one of Clauses 120 to 123, further comprising a sample loading port for loading the sample, the sample loading port fluidically connected to the reaction region.

[0527] 125. The lateral flow assay device of any one of Clauses 120 to 124, wherein the substrate comprises a label and, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce an unlabeled substrate fragment that remains immobilized to the reaction region and a labelled substrate fragment that migrates to the detection region. 126. The lateral flow assay device of any one of Clauses 120 to 125, wherein binding of the binding partner and the substrate or the labeled substrate fragment produces a detectable signal.

[0528] 127. The lateral flow assay device of Clause 126, wherein the binding partner for the substrate specifically binds the label of the labeled substrate fragment.

[0529] 128. The lateral flow assay device of Clause 127, wherein the binding partner for the labeled substrate fragment 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.

[0530] 129. The lateral flow assay device of Clause 128, wherein the detectable signal is produced from a tag bound to the label.

[0531] 130. The lateral flow assay device of Clause 129, wherein the tag comprises gold-nanoparticles, a fluorophore, or a chromophore.

[0532] 131. A kit for determining whether a target nucleic acid is present in a sample, the kit comprising a lateral flow assay device of any one of Clauses 120 to 130 and one or more reagents for processing the sample.

[0533] 132. The kit of Clause 131 , wherein the one or more reagents for processing the sample comprise one or more reagents for purifying nucleic acids from the sample.

[0534] 133. The kit of Clause 132, wherein the nucleic acids purified from the sample are RNA.

[0535] 134. The kit of Clause 132, wherein the nucleic acids purified from the sample are DNA. 135. The kit of any one of Clauses 120 to 134, wherein the Craspase is Csx29.Atty. Docket No.: GRIP-013WO

[0536] 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.

[0537] 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 and 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.

[0538] 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

1. Atty. Docket No.: GRIP-013WOClaimsWe claim:

1. A method of determining whether a target nucleic acid is present in a sample, the method comprising:(a) producing a reaction mixture by combining the sample with:i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; andii) a substrate for the Craspase;wherein, when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a labeled substrate fragment and an unlabeled substrate fragment, and (b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample,wherein:the assaying the reaction mixture for cleavage of the substrate comprises detecting whether the labeled substrate fragment is produced by separating the reaction mixture in a portion suspected of containing the unlabeled substrate fragment and a portion suspected of containing the labeled substrate fragment and detecting whether the labeled substrate fragment is present in the portion suspected of containing the labeled substrate fragment.

2. The method of claim 1 , wherein detecting whether the labeled substrate fragment is produced comprises contacting the portion suspected of containing the labeled substrate fragment with a binding partner for the label, wherein the binding partner for the label is immobilized on a matrix,wherein the matrix is an adsorbent material in a detection region of a lateral flow assay device, and the method comprises:loading the sample into a reaction region of the lateral flow assay device, wherein the reaction region comprises the Craspase comprising the 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.Atty. Docket No.: GRIP-013WO3. The method of claim 2, 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, andwherein binding of the binding partner and the label produces a detectable signal.

4. The method of claim 1 , wherein separating the reaction mixture in a portion suspected of containing the unlabeled substrate fragment comprises contacting the reaction mixture with a coagulant that coagulates the unlabeled substrate fragment or an immobilization agent that immobilizes the unlabeled substrate fragment, wherein the coagulation or immobilization of the unlabeled substrate fragment is mediated by a tag covalently conjugated to the substrate.

5. The method of claim 4, wherein:i) the method comprises contacting the reaction mixture with the coagulant that coagulates the unlabeled substrate fragment, wherein the tag is an RTX peptide and the coagulant is a source of Ca2+ions; orii) the method comprises contacting the reaction mixture with an immobilization agent that immobilizes the unlabeled substrate fragment, wherein the tag is an epitope-tag and the immobilization agent is an antibody that specifically binds to the epitope-tag.

6. A method of determining whether a target nucleic acid is present in a sample, the method comprising:(a) producing a reaction mixture by combining the sample with:i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; andii) a substrate for the Craspase;(b) assaying the reaction mixture for cleavage of the substrate to determine whether the target nucleic acid is present in the sample,wherein:the substrate is bound to a solid support and when the target nucleic acid is present in the sample, the Craspase cleaves the substrate to produce a free substrate fragment and a solid support bound substrate fragment, andAtty. Docket No.: GRIP-013WOwherein assaying the reaction mixture for cleavage of the substrate comprises detecting cleavage of the substrate bound to the solid support.

7. The method of claim 6, wherein the solid support is a sensor and cleavage of the substrate produces the free substrate fragment thereby causing a change in a property of the sensor.

8. The method of claim 7, wherein the change in the property of the sensor is a change in an electrochemical property, a redox signal, or an electrical property.

9. The method of claim 6, wherein the solid support is a bead, and the method comprises separating the solid support from the reaction mixture and detecting the free substrate fragment in the reaction mixture after separating the solid support from the reaction mixture.

10. The method of claim 9, wherein detecting the free substrate fragment comprises contacting the reaction mixture after separating the solid support from the reaction mixture to a sensor comprising a probe that specifically binds the free substrate fragment and detecting a change in a property of the sensor caused by binding of the free substrate fragment to the probe.

11. The method of claim 10, wherein the probe that specifically binds to the free substrate fragment is a protein or an aptamer, and wherein the probe comprises an electrochemical reporter.

12. The method of claim 10 or 11 , wherein the change in the property of the sensor caused by binding of the free substrate fragment to the probe is a change in an electrochemical property or an electrical property.

13. The method of any one of claims 6 to 12, the method comprising:(a) introducing the sample into a sample chamber of an analyzer to produce a reaction mixture,wherein the sample chamber comprises: i) the Craspase comprising the crRNA that specifically hybridizes with the target nucleic acid; and ii) the substrate for the Craspase;(b) incubating the reaction mixture, andAtty. Docket No.: GRIP-013WO(c) assaying for cleavage of the substrate bound to the solid support to determine whether the target nucleic acid is present in the sample.

14. A target nucleic acid detection kit, comprising:a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid;a substrate bound to a solid support, anda sensor configured to detect cleavage of the substrate bound to the solid support.

15. A lateral flow assay device for determining whether a target nucleic acid is present in a sample, the lateral flow assay device comprising:(a) a reaction region comprising:i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated protease (Craspase) comprising a CRISPR RNA (crRNA) that specifically hybridizes with the target nucleic acid; andii) a substrate for the Craspase, the substrate immobilized in the reaction region; and(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, andwherein, when the target nucleic acid is present in the sample introduced into the reaction region, the Craspase cleaves the substrate to produce an unlabeled substrate fragment that remains immobilized to the reaction region and a labelled substrate fragment that migrates to the detection region, wherein binding of the binding partner and the labeled substrate fragment produces a detectable signal in the detection region.

Images