Thermonuclear cascade with t7 RNA polymerase

Abstract

The present disclosure relates to multiplexed assay methods used to detect and identify several to many target nucleic acids of interest in a sample without amplification of the target nucleic acids of interest using blocked RNP2 activator molecules that reduce or eliminate erroneous activation of ribonucleoprotein complexes in the absence of a target nucleic acid of interest.

Description

TITLE: THERMONUCLEAR CASCADE WITH T7 RNA POLYMERASERELATED CASES

[0001] This International PCT application claims priority to U.S. Ser. Nos. 63 / 665,028, filed 27 June 2024; 63 / 686,919, filed 26 August 2024; and 63 / 768,155, filed 06 March 2025, all of which are incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to multiplexed assay methods used to detect and identify several to many target nucleic acids of interest in a sample without amplification of the target nucleic acids of interest.BACKGROUND OF THE INVENTION

[0003] In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[0004] Rapid and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of diseases such as cancer, and / or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment including identification of biothreats. Classic PCR and nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of nucleic acids are therefore needed for timely diagnosis andtreatment of disease, to identify toxins in consumables and the environment, as well as in other applications.SUMMARY OF THE INVENTION

[0005] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[0006] The present disclosure provides cascade assay compositions and methods to detect target nucleic acids of interest in a sample without amplification of the target nucleic acids of interest. The “nucleic acid-guided nuclease cascade assays” or “signal boost cascade assays” or “signal boost assays” or “cascade assays” or, specifically, “thermonuclear cascade with T7 RNA polymerase” (“TNT”) assays or “thermonuclear assays” described herein comprise two different ribonucleoprotein (RNP) complexes, blocked RNP2 activator molecules, reporter (“detector”) moieties, a DNA / RNA repair enzyme or 3' phosphatase, DNA and RNA polymerases, and dNTPs and rNTPs, all of which allow for massive multiplexing and rapid time to detection.

[0007] Thus, there is provided a method for identifying a target nucleic acid of interest in a sample using a thermonuclear cascade with RNA polymerase assay comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP Is) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits gRNA-target hybridization dependent trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) each comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest and comprises a crRNA of X nucleotides in length, where X = 12 to 35 nucleotides, wherein the crRNA is capable of hybridizing to an RNP2 activator transcript product, wherein the second nucleic acidguided nuclease is an RNA-cleaving nucleic acid-guided nuclease that exhibits gRNA- target hybridization-dependent trans-cleavage activity; a plurality of blocked RNP2 split activator molecules each comprising a blocked partial transcription template and a nontemplate strand; wherein the blocked partial transcription template comprises from 5' to 3': a sequence of at least four nucleotides complementary to a 5' portion of the second gRNA, a first linker region having a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least two adjacent ribonucleotides, and a terminal region comprising a first 3' blocking moiety; and wherein the non-template stand comprises from 5' to 3': a sequence at least X minus 4 nucleotides in length having substantially a same nucleotide sequence as a 3' portion of the second gRNA, a sequence of nucleotides coding for one strand of an RNA polymerase promoter sequence, a second linker region having a length equivalent to at least 15 nucleotides with complementarity to the first linker region, and a terminal region comprising a second 3' blocking moiety; a 3' phosphatase; a DNA polymerase optionally comprising both polymerase and 3' — > 5' exonuclease activity; a RNA polymerase; dNTPs and rNTPs; and reporter moieties that produce a signal upon trans-cleavage by the first or second nucleic acid-guided nuclease; and contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample, if present, to bind to RNP1; wherein upon binding of the target nucleic acid of interest, RNP1 becomes active initiating trans-cleavage of a bond between the at least two adjacent nucleotides of the blocked partial transcription template thereby unblocking the at least one blocked RNP2 activator molecule by removing the first 3' terminal blocking moiety; allowing the DNA polymerase to remove single-strand nucleotides from the 3' cleaved single-strand region of the blocked partial transcription template, if present, and to extend the blocked partial transcription template by copying the sequence complementary to the 3' portion of the second gRNA of non-template stand and the RNA polymerase promoter sequence creating a polymerization product; allowing the RNA polymerase to use the polymerization product to transcribe RNP2 activator transcript products, wherein the RNP2 activator transcript products are capable of binding to the second gRNAs and activating trans-cleavage of the RNP2s thereby unblocking at least one additional blocked RNP2 activator molecule; and

[0008] detecting a presence or absence of the signal from the reporter moieties.

[0009] In some aspects of this embodiment, the crRNA of the second gRNA is 20-30 nucleotides in length.

[0010] In some aspects of this embodiment, the blocked partial transcription template comprises a sequence of at least 6 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises a sequence at least X minus 6 nucleotides in length. In other aspects of this embodiment, the blocked partial transcription template comprises a sequence of at least 7 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises at least X minus 7 nucleotides in length, or at least X minus 10 nucleotides in length, or at least X minus 12 nucleotides in length.

[0011] In some embodiments, the first linker region has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least four adjacent ribonucleotides, or

[0012] has a length equivalent to at least 20 nucleotides and wherein the first linker comprises at least four adjacent ribonucleotides, or has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least six adjacent ribonucleotides, or has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least eight adjacent ribonucleotides or has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least ten adjacent ribonucleotides.

[0013] In some aspects, linker comprises only deoxyribonucleotides and ribonucleotides, and in some embodiments, the dNTPs and rNTPs may be modified dNTPs and rNTPs.

[0014] In some embodiments, the reporter moieties are separate molecules from RNP1, RNP2 and the plurality of blocked RNP2 activator molecules; however, in some embodiments, the blocked RNP2 activator molecules comprise the reporter moieties.

[0015] In some embodiments, w the RNA polymerase is selected from T7 RNA polymerase, Enterobacteria phage T3 RNA polymerase), Salmonella phage SP6 RNA polymerase, Synechococcus phage Syn5 RNA polymerase, Pseudomonas phage VSW-3 RNA polymerase, Pseudomonas phage phi6 RNA polymerase, E. coll RNA polymerase containing subunit beta, human RNA polymerase I containing POLR1A, human RNA polymerase II containing POLR2A, Human RNA polymerase III containing POLR3A, human mitochondrial RNA polymerase or chimeras thereof, and in some embodiments, the DNA polymerase exhibits 3' — > 5' exonuclease activity; yet in other embodiments, the DNA polymerase does not exhibit 3' — > 5' exonuclease activity.

[0016] These aspects and other features and advantages of the invention are described below in more detail.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of exemplary embodiments taken in conjunction with the accompanying drawings in which:

[0018] FIG. 1 is an overview of the general principles underlying the signal boost cascade assay described in detail herein, where target nucleic acids of interest from a sample do not need to be amplified before detection.

[0019] FIG. 2 is a diagram showing the sequence of steps in an exemplary prior art signal boost cascade assay utilizing blocked nucleic acid molecules.

[0020] FIG. 3 is a simplified graphic showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

[0021] FIG. 4 is a simplified diagram of the “correct” pathway for unblocking the blocked nucleic acid molecule, as well as a “failure” pathway for unblocking the blocked nucleic acid molecule.

[0022] FIG. 5A is a simplified illustration of an exemplary thermonuclear cascade with an RNA polymerase (“TNT”) detection and signal amplification cascade system.

[0023] FIG. 5B is a simplified illustration one exemplary TNT detection and signal amplification cascade system (“TNT1”) and one point of failure that reduces the effectiveness of this embodiment of the TNT cascade system.

[0024] FIG. 6A is a simplified illustration of the exemplary TNT detection and signal amplification cascade system shown in FIG. 5A (“TNT1”).

[0025] FIG. 6B is a simplified illustration of an exemplary blocked RNP2 activator molecule suitable for use with the exemplary TNT1 detection and signal amplification cascade system in FIG. 6A and an example sequence therefor.

[0026] FIG. 7 A is a simplified illustration of an alternative exemplary TNT detection and signal amplification cascade system (“TNT2”).

[0027] FIG. 7B is a simplified illustration of an exemplary blocked RNP2 activator molecule suitable for use with the alternative exemplary TNT2 detection and signal amplification cascade system in FIG. 7 A and an example sequence therefor.

[0028] FIG. 8 is a simplified illustration of yet another alternative exemplary TNT detection and signal amplification cascade system (“TNT3”).

[0029] FIG. 9A is a simplified illustration of a twist on the TNT3 embodiment employing three rNTPs and one drNTP, which lacks a 3'-OH group (“RASTA”).

[0030] FIG. 9B shows an exemplary sequence for a RASTA blocked RNP2 activator molecule that was determined to be difficult to unblock.

[0031] FIG. 9C is a simplified illustration of an exemplary blocked RNP2 activator molecule suitable for use with the RASTA detection and signal amplification cascade system in FIG. 9A and an example sequence therefor.

[0032] FIG. 9D is a simplified illustration of an alternative to the exemplary blocked RNP2 activator molecule shown in FIG. 9C. The exemplary blocked RNP2 activator molecule shown in FIG. 9D is a “split activator” configuration, which splits the target sequence for the gRNA2 into two regions separated by a linker. This split activator embodiment is also suitable for use with the RASTA detection and signal amplification cascade system in FIG. 9A and example sequences are provided.

[0033] It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.DEFINITIONS

[0034] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

[0035] All of the functionalities described in connection with one embodiment of the compositions and / or methods described herein are intended to be applicable to theadditional embodiments of the compositions and / or methods except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

[0036] Note 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. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as "left," "right," "top," "bottom," "front," "rear," "side," "height," "length," "width," "upper," "lower," "interior," "exterior," "inner," "outer" that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as "first," "second," "third," etc., merely identify one of a number of portions, components, steps, operations, functions, and / or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0037] 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. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5' to 3' direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).

[0038] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening valuein that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in 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.

[0039] The term “and / or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art given the present description that the present methods may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

[0041] As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0042] As used herein, the terms “binding affinity” or “dissociation constant” or “Kd” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high Kd (which in the context of the present disclosure refers to blocked RNP2 activator molecules binding to the second ribonucleoprotein complex (RNP2)) indicates the presence of more unbound molecules, and a low Kd (which in the context of the present disclosure refers to an RNP2 activator transcription product binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the bindingof blocked RNA2 activator molecules or RNP2 activator transcript products to RNP2, low Kd values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kd values are in the range of 100 nM - 100 pM (10 mM) and thus are about 105- to 1010- fold or higher as compared to low Kd values.

[0043] As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and / or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule may serve as a binding domain for a different nucleic acid molecule. Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.

[0044] As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second ribonucleoprotein complex (RNP) (i.e., RNP1 or RNP2) of the cascade assay to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules; that is, an “unblocked nucleic acid molecule” is the target nucleic acid for RNP2. In the context of the present disclosure, the term “blocked RNP2 activator molecule” refers to nucleic acid molecules that cannot bind to the first or second ribonucleoprotein complex (RNP) (i.e., RNP1 or RNP2) of the cascade assay to activate cis- or trans-cleavage. “Unblocked RNP2 activator molecule” refers to a formerly blocked RNP2 activator molecule that can serve as a template for an RNA polymerase to synthesize RNP2 activator transcript products to bind to the second RNP complex (RNP2), which then activates trans-cleavage of additional blocked RNP2 activator molecules.

[0045] The terms “Cas RNA-guided endonuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided endonuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.

[0046] As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”, “cismediated endonuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence- specific cleavage of a target nucleic acid ofinterest, including an RNP2 activator transcript product, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

[0047] The term "complementary" as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'; and the nucleotide sequence 3'-ATCGAT-5' is 100% complementary to a region of the nucleotide sequence 5'-GCTAGCTAG-3'.

[0048] As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in samples or in vivo by administering an agent to a subject.

[0049] A “control” is a reference standard of a known value or range of values.

[0050] The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1 ) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein complex (RNP) containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid.

[0051] “Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In oneembodiment, a nucleic acid molecule (for example, a blocked RNP2 activator molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and / or internucleoside linkages. In another embodiment, a modified protein (e.g., a nucleic acid- guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

[0052] The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSLBLAST, or MEGALIGN™ software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5): 1792- 1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)).

[0053] As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest or in the context of the present disclosure, an RNP2 activator transcript product, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a nucleic acid target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, casl2a or casl4a for a DNA target nucleic acid, or casl3a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an RNP2 activator transcript product, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.

[0054] As used herein, the terms "protein" and "polypeptide" are used interchangeably. Proteins may or may not be made up entirely of amino acids.

[0055] RNA polymerases are polypeptides capable of templated-directed synthesis of polyribonucleotides. RNA polymerases include but are not limited to naturally occurring or variants of: Escherichia phage T7 RNA polymerase (Gene ID: 1261050), Enterobacteria phage T3 RNA polymerase (Gene ID: 54983143), Salmonella phage SP6 RNA polymerase (Gene ID: 1481778), Synechococcus phage Syn5 RNA polymerase (Gene ID: 5220157), Pseudomonas phage VSW-3 RNA polymerase (Gene ID: 40071755), Pseudomonas phage phi6 RNA polymerase (Gene ID: 95643) , E. coli RNA polymerase (containing subunit beta: Gene ID: 948488), human RNA polymerase I (containing POLR1A: Gene ID: 25885), human RNA polymerase II (containing POLR2A: Gene ID: 5430), Human RNA polymerase III (containing POLR3A Gene ID: 11128), human mitochondrial RNA polymerase (Gene ID:5442), and an artificial polypeptide such as a chimeric protein.

[0056] As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimen or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water, air, or sewer sample; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0057] The terms "target DNA sequence", “target RNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or invivo. The “target strand” of a target nucleic acid of interest is the strand of a doublestranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as pre- mRNA, mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.

[0058] As used herein, the terms “trans-cleavage”, “trans-endonuclease activity”, “transmediated endonuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non-sequence-specific cleavage of a nucleic acid molecule by an endonuclease (such as by a Casl2, Casl3, and Casl4) which is triggered by gRNA-target nucleic acid interactions with or without cis- (sequencespecific) cleavage. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation.

[0059] Type V CRISPR / Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR / Cas effector nucleases such as, but not limited to, engineered Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasY (Casl2d) nucleases or naturally-occurring proteins, such as a Casl2a isolated from, for example, Francisellatidarensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and Eubacterium eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

[0060] Type VI CRISPR / Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR / Cas effector nucleases such as, but not limited to, engineered Casl3a, Casl3d, Casl3c, Casl3bl, Casl3b2 nucleases or naturally-occurring proteins, isolated from, for example, Leptotrichia shahii, Ruminococcus cicirculans, Fusobacterium perfoetens, Prevotella buccae, Bergeyella zoohelcum. Casl3a enzymes include but are not limited to naturally occurring or engineered variants including from Leptotrichia buccalis, Leptotrichia wadei, Thermoclostridium caenicola, Herbinix hemicelhdosilytica, etc., and an artificial polypeptide, such as a chimeric protein.

[0061] The term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications-including chemical modifications-to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

[0062] A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and / or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.DETAILED DESCRIPTION

[0063] The present disclosure provides compositions of matter and cascade assay methods for detecting target nucleic acids of interest in a sample without the need for amplifying the target nucleic acids of interest. The compositions and methods provide for multiplexing, high fidelity, low background, high signal-to-noise ratios, low cost, minimum workflow, with rapid results at ambient temperatures.

[0064] The cascade assays described herein comprise first and second ribonucleoprotein complexes, blocked RNP2 activator molecules, a DNA / RNA repair enzyme or 3' phosphatase, a DNA polymerase that optionally exhibits 3' exonuclease activity, and an RNA polymerase. The blocked RNP2 activator molecules by and large keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. By “locked” it is meant that the blocked RNP2 activator molecules are designed in such a way that they are largely blocked from serving as a template to synthesize targets that activate the ribonucleoprotein complexes; therefore, the ribonucleoprotein complexes remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. However, an improvement to the cascade assay described herein involves both an unblocking step and a synthesis step, the combination of which allows for use of a robust blocked RNP2 activator molecule configuration to minimize false positive signals yet still provides signal amplification. The methods comprise the steps of providing TNT cascade assay components, contacting the TNT cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample.

[0065] Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of such diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. However, currently available state-of-the-art nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) relies on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for qPCR assays is due to the fact that there is a significant lag phase early in the amplificationprocess where fluorescence above background cannot be detected. That is, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified.

[0066] The present disclosure describes a signal boost cascade assay and improvements thereto that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and / or cDNA) without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the cascade assays utilize a signal amplification or signal boost mechanism comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked RNP2 activator molecules, reporter moieties, a kinase, a DNA polymerase that optionally exhibits 3' exonuclease activity, dNTPs, RNA polymerases, and rNTPs. A particularly advantageous feature of the cascade assay is that with the exception of the gRNA (gRNAl) in RNP1 the cascade assay components can be essentially identical, if desired, no matter what target nucleic acid(s) of interest are being detected, and gRNAl is easily programmable.

[0067] The improvement to the signal amplification or signal boost cascade assay described herein is drawn to being able to employ a tightly “locked” blocked RNP2 activator molecule that can be unblocked and then serve as a template to synthesize an RNP2 via a combination of trans-cleavage activity by a nucleic acid-guided nuclease in the ribonucleoprotein complexes in the reaction mixture, a DNA polymerase that exhibits 3' — > 5' exonuclease activity and an RNA polymerase. This combination of enzymatic activity unblocks the blocked RNP2 activator molecules by removing a blocking moiety from the 3' end of the blocked RNP2 activator molecule followed by synthesis of RNP2 activator transcript products complementary to gRNA2 in RNP2; that is, the combination of enzymatic activity “frees up” the portion of the blocked RNP2 activator molecule that can prime synthesis of the target strand for RNP2 (i.e., the RNP2 activator transcript product), making it available for binding to and activating RNP2 as described in detail below.

[0068] As noted above, the downside to currently available nucleic acid-guided nuclease detection assays is that they rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is, at least in-part, due to cis-cleavage of a target nucleic acid of interest being a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample; thus, PCR amplification affects the rapidity of currently available nucleic acid-guided nuclease detection systems. Once the ribonucleoprotein complex is activated , transcleavage activity of the reporter moieties that are initially quenched is generated. However, the turnover (Kcat) of, e.g., activated Casl3a complex is ~20 / sec for RNA targets. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 30-60 minutes.

[0069] FIG. 1 provides a simplified diagram demonstrating a method (100) of a cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mixture — as opposed to separate nucleic acid-guided nucleases and gRNAs — to facilitate rapid detection of the target nucleic acid(s) of interest, if desired.

[0070] “Activation” of RNP 1 (106) in the context of the cascade assay refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by first initiating cis-cleavage where the target nucleic acid of interest is cleaved by the nucleic acid-guided nuclease, or at least upon specific binding of N nucleotide bases of a target nucleic acid of interest to the ribonucleoprotein complex. This cis-cleavage activity then initiates trans- cleavage activity (i.e., multi-turnover activity) by the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, leading to non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP 1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) via blockednucleic acid molecules — or in the context of the present disclosure, blocked RNP2 activator molecules — which are described in detail below. Each newly activated RNP2 (110) activates more RNP2s (108 — > 110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5' end and a quencher on the 3' end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties may also be incorporated into blocked RNP2 activator molecules.

[0071] As more RNP2s are activated (108 — > 110), more trans -cleavage activity is activated and more reporter moieties (118) are unquenched; thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially, hence, the terms signal amplification or signal boost. The cascade assay thus comprises a single turnover event that triggers a multiturnover event that then triggers another multi-turnover event. The reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non- covalently linked to the blocked RNP2 activator molecules.Target Nucleic Acids of Interest

[0072] The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT- PCR). The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep (and other livestock), and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrialsites and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

[0073] In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and / or Gardnerella vaginalis.

[0074] As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumo virus, human rhinovirus, enterovirus, influenza A, influenza A / Hl, influenza A / H3, influenza A / Hl-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and / or human parvovirus B19 (B19V).

[0075] Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and / or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis, Bonamia exitiosa, Bonamia ostreae,Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Marteilia refringens, Perkinsus marinus, Perkinsus olseni, Theileria annulata, Theileria equi, Theileria parva, Tritrichomonas foetus, Trypanosoma brucei, Trypanosoma congolense, Trypanosoma equiperdum, Trypanosoma evansi and, Trypanosoma vivax.

[0076] Additionally, the target nucleic acid of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses, mycoplasma and other bovine mastitis pathogens, Clostridium perfringens, Campylobacter sp., Salmonella sp., Pospirivoidae, Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasma sp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, or Botryotinis fuckleina.

[0077] In some embodiments, other target nucleic acids of interest may be for non- infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g, trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia. Other target nucleic acids of interest and samples are described herein. Target nucleic acids of interest may include engineered biologies, including cells such as chimeric antigen receptor T (CAR-T) cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing.

[0078] The cascade assays described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Further testing may be used to identify the specific member of the pool, if warranted.

[0079] While the methods described herein do not require the target nucleic acid of interest to be DNA — and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA — it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting thebiological sample with the composition. Alternatively, RNA target nucleic acids of interest can be detected directly via RNA-specific nucleic acid nucleases such as Cas 13a or Cas 12g.Nucleic Acid-Guided Nucleases

[0080] The cascade assays comprise nucleic acid-guided nucleases in the reaction mixture, either provided as a protein, a coding sequence for the protein, or, in most embodiments, in a pre-assembled ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mixture may be, for example, a Cas endonuclease. Any nucleic acid-guided nuclease having both cis- and trans-endonuclease activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNP complexes or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. Note that trans-cleavage activity is not triggered unless and sequence-specific target nucleic acid binding occurs. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain, or a HEPN or HEPN- like domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly- discovered systems include BioGRID ORCS (orcs: thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).

[0081] The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA- cleaving nucleic acid-guided nuclease (e.g., a Cas 12a, Cas 14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA-cleaving nucleic acid- guided nuclease (e.g., Casl3a or Casl2g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA endonucleases, such as Cas3, Cas 12a (e.g., AsCasl2a, LbCasl2a), Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2h, Casl2i, Casl2j,and other Casl2 subvariants; Cas RNA-guided RNA endonucleases, such as Casl3a (LbaCasl3, LbuCasl3, LwaCasl3), Casl3b (e.g., CccaCasl3b, PsmCasl3b), and Casl2g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as a Casl2aor Cas 14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI (such as Cas 13a) nucleic acid-guided nucleases may also be employed.Guide RNA (gRNA)

[0082] The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two different guide RNAs (gRNAs) each incorporated into an RNP complex (i.e., RNP1 or RNP2). Suitable gRNAs each include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP 1 (gRNAl) is specific to a target nucleic acid of interest and the gRNA of RNP2 (gRNA2) is specific to an RNP2 activator transcript product (described in detail below). Like the nucleic acid-guided nuclease, the gRNA in most embodiments is provided in the cascade assay reaction mixture in a preassembled RNP but may also be provided as an RNA molecule or as a DNA sequence to be transcribed, in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNP complex (i.e., RNP1 or RNP2) is preferred if rapid assay kinetics are preferred. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site is encoded between neighboring gRNA sequences and more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementarity to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

[0083] The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP 1 which triggers non-sequence-specific trans-cleavage of other molecules in the reaction mixture. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest in the case of gRNAl or RNP2 activator transcript products in the case of gRNA2. Target nucleic acids of interest may include a protospacer- adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest.

[0084] In any of the foregoing embodiments, the gRNA may be a modified or non- naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and / or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and / or internucleoside linkage, such as a 2'-O-methyl (2'-0-Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.Ribonucleoprotein (RNP) Complex

[0085] As described above, although the assay “reaction mixture” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assay reaction mixtures preferably comprise preassembled ribonucleoprotein complexes (RNPs), allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes, RNP1 and RNP2, each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid- guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 pM of a given RNP1, or about 1 pM to about 1 pM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6 x 104to about 6 x 1012complexes per microliter (pl) of a given RNP1, or about 6 x 106to about 6 x 1010complexes per microliter (pl) of a given RNP1. In some embodiments, the reaction mixture containsabout 1 fM to about 500 M of a given RNP2, or about 1 pM to about 250 pM of a given RNP2, or about 10 pM to about 100 pM of a given RNP2. In some embodiments the reaction mixture contains about 6 x 104to about 6 x 1012complexes per microliter (pl) of a given RNP2 or about 6 x 106to about 6 x 1012complexes per microliter (pl) of a given RNP2.

[0086] In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP Is (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP Is), where different RNP Is comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique RNP 1 -gRNA (or RNP 1 -crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. That is, more than one RNP1 may be present for the purpose of targeting one target nucleic acid of interest from many sources or more than one RNP1 may be present for targeting more than one target nucleic acid of interest from a single organism or condition.

[0087] In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNPl(s) present in the reaction mixture. A homologous mixture of RNP 1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and / or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twentyheterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty- five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables multiplex testing.

[0088] As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNPls having different gRNAs targeting parainfluenza virus 1; a number of RNPls having different gRNAs targeting human metapneumovirus; a number of RNPls having different gRNAs targeting human rhino virus; a number of RNPls having different gRNAs targeting human enterovirus; a number of RNP1 having different gRNAs targeting respiratory syncytial virus; and a number of RNP 1 s having different gRNAs targeting coronavirus HKU 1. As a second nonlimiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNPls containing a gRNA each targeting a SARS-Co-V-2 variant, e.g., B.1.1.7, B.1.351, P.l, B.l.617.2, BA.l, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.

[0089] As another non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain RNPls targeting two or more target nucleic acids of interest from, e.g., organisms that infect vineyards, such as Guignardia bidwellii, Uncinida necator, Botrytis cincerea, Plasmopara viticola, and Botryotinia fuckleina.Reporter Moieties

[0090] The cascade assay in many embodiments detects a target nucleic acid of interest via detection of a signal generated in the reaction mixture by a reporter moiety. Depending on the type of reporter moiety used, trans- and / or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone (e.g., not bound to any blocked RNP2 activator molecules) reporter moieties may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown at bottom in FIG. 2). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal; thus, when the reporter moiety is a separate molecule, the reporter moieties are activated quickly by the trans-cleavage activity. The reporter moiety can comprise DNA, RNA, a chimera of DNA and RNA, oran oligonucleotide with modified nucleic acids. The reporter moiety also can comprise both single- and double-stranded portions.

[0091] In alternative embodiments, the reporter moiety may be bound to the blocked RNP2 activator molecule, where trans-cleavage of the blocked RNP2 activator molecule and conversion to an unblocked RNP2 activator molecule may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time. In this embodiment, the reaction kinetics of signal generation match that of the cascade assay reaction rate. The signal is generated as each blocked RNP2 activator molecule is unblocked.

[0092] The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TAQMAN® probe with a dye label (e.g., FAM or FITC) on the 5 ' end and a quencher on the 3 ' end. The reporter and quencher may be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety.

[0093] Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as1251,35S,14C, or3H. Reporters can also include a change in pH or charge of the cascade assay reaction mixture.

[0094] The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. The classic example of an LFA is the home pregnancy test.

[0095] For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mixture with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher / phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor / acceptor) suitable for use include, but are not limited to, EDANS / fluorescein, lAEDANS / fluorescein, fluorescein / tetramethylrhodamine, fluorescein / Cy 5, IEDANS / DABCYL, fluorescein / QSY™ (succinimidyl ester) -7, fluorescein / LC Red 640, fluorescein / Cy 5.5, Texas Red / DABCYL, BODIPY™ (4,4- difluoro-4-bora-3A,4A-diaza-s-indacene) / DABCYL, Lucifer yellow / DABCYL, coumarin / DABCYL, and fluorescein / LC Red 705. In addition, a fluorophore / quantum dot donor / acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-l- sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl]amino)naphthalene-l- sulfonic acid); DABCYL is 4-(4- dimethylaminophenyl) diazenylbenzoic acid. Usefulquenchers include, but are not limited to, DABCYL, QSY™ (succinimidyl ester) 7 and QSY™ (succinimidyl ester) 33.

[0096] In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2'-O-methyl (2'- O-Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

[0097] The TNT cascade embodiments may employ alternatives to using the reporter moieties described above and shown, e.g., in FIGs. 1, and 2, which utilize quencher pairs. In some aspects, aptamer-based RNA imaging may be employed. Light-up aptamers (also called fluorescent light-up aptamers or FLAPs) are genetically encoded RNA reporter moieties designed to bind specific fluorogenic dyes that fluoresce only in a bound state, such that fluorescence is “switched on” upon RNA expression. Light-up aptamers include spinach, squash, mango, corn, and broccoli. The TNT cascade embodiments thus could utilize a blocked RNP2 activator molecule and instead of only transcribing the RNA target for RNA2, a FLAP such as spinach would be transcribed as well and the fluorogen difluoro 4-hydroxybenzylidene imidazolinone (DFHBI) (or other appropriate fluorogen) would be added to the reaction mix. (See, e.g., Paige, et al., Science, 333(6042):642-46 (2011) and Wang, et al., Sensors & Actuators, B. Chemical, 362:131765 (2022).)Nucleic Acid Modifications

[0098] For any of the nucleic acid molecules described herein (e.g., blocked RNP2 activator molecules, gRNAs, and / or reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form and for the blocked RNP2 activator molecules, it is specifically contemplated that at least the 3' terminal nucleotide(s) will be modified, and likely at least one or more of the nucleotides in a single-strand template or non-template or hairpin loop region (if present) may be modified as well. Typically, modifications to the blocked RNP2 activator molecules, gRNAs, and / or reporter moieties described herein are introduced to optimize the molecule’s biophysical properties, for example, to increase endonuclease resistance and / or increase thermal stability. Modifications typically areachieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and / or alternative internucleoside linkages.

[0099] For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5 '-nitroindole, 5 -methylcytosine (5-me- C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CHs) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3 -deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (i.e., blocked RNP2 activator molecules, gRNAs, and / or reporter moieties) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in USPN 3,687,808; Kroschwitz, ed., The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

[0100] In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2' sugar modifications, including 2'-O-methyl (2’-0-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'- MOE), 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'- DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2'-modifications that can modify the nucleic acid molecules described herein (i.e., blocked RNP2 activator molecules, gRNAs, and / or reporter moieties) may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl(mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0101] Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodi thioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates, 5 '-alkylene phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalky Iphosphoramidates, thionophosphoramidates , thionoalkylphosphonates , thionoalky Iphospho triesters , selenophosphates, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.The Signal Boosting Cascade Assay Employing Blocked Nucleic Acids

[0102] Before getting to the details relating to aiding the unblocking of the blockingRNP2 activator molecules via the TNT cascade assay, understanding the basic cascade assay itself is key. FIG. 1 , described above, depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acids is depicted in FIG. 2 and described in detail below, as well in USPNs 11,693,520; 11,702,686; 11,821,025; 11,970,730; 11,987,839; 11,884,921; 11,820,983; 11,859,182; 11,884,922; and 11,946,052, which are incorporated by reference in their entirety. In this embodiment known in the art, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method (200) in FIG. 2 begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleicacid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, Cas 12a or Cas 14 for a DNA target synthesized from an unblocked nucleic acid molecule or a Cas 13a for an RNA target synthesized from an unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and target synthesized from the unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.

[0103] In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mixture. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP 1 cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, which then activates non-specific trans-cleavage of other nucleic acids present in the reaction mixture, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. “Blocking moiety” typically refers to one or more nucleoside modifications, topographical configurations such as secondary structures, and / or structural modifications.

[0104] Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then interact with and activate an RNP2 (208). Because the nucleic acid-guided nucleases in the RNP Is (205) and RNP2s (208) have both cis- and trans-cleavage activity, more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of more RNP2s (208) and more trans-cleavage activity in a cascade. FIG. 2 at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties(212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activation of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed.

[0105] FIG. 3 is a diagram showing an exemplary prior art blocked nucleic acid molecule (320) and an exemplary technique for unblocking the blocked nucleic acid molecules also as described in, e.g., USPNs 11,693,520; 11,702,686; 11,821,025; 11,970,730; 11,987,839; 11,884,921; 11,820,983; 11,859,182; 11,884,922; and 11,946,052. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (320) comprising a target strand (322) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (324) containing unhybridized and cleavable secondary loop structures (326) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNPls or RNP2s, generates short strand nucleotide sequences (328) which, because of the short length and low melting temperature Tm, can dehybridize at room temperature (e.g., 15°-25°C), thereby unblocking the blocked nucleic acid molecule (320) to create an unblocked nucleic acid molecule (330), enabling the internalization of the unblocked nucleic acid molecule (330) (target strand) into an RNP2, leading to RNP2 activation.

[0106] A blocked nucleic acid molecule may be single-stranded or doublestranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified in FIG. 3. Such blocked nucleic acid molecules typically have a low binding affinity, or high dissociation constant (Kd) in relation to binding to RNP2 and may be referred to herein as a high Kd nucleic acid molecule. In the context of the present disclosure, the binding of blocked RNP2 activator molecules or RNP2 activator transcript products to RNP2, low Kd values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kd values are in the range of 100 nM to about 10-100 10 mM and thus are about 105-, 106-, 107-, 108-, 109-to 1010-fold or higher as compared to low Kd values. Of course, the ideal blocked nucleic acid molecule would have an “infinite Kd.”

[0107] The blocked nucleic acid molecules (high Kd molecules) described herein can be converted into unblocked nucleic acid molecules (low Kd molecules - also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNPls and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked nucleic acid molecule. Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop or cascade.

[0108] In embodiments where blocked nucleic acid molecules are linear and / or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double-strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-cleavage activity of RNP2.Improving the Signal Boosting Cascade Assay Employing Blocked RNP2 Activator Molecules

[0109] The present disclosure provides reaction mix compositions and methods for a signal boost cascade assay that reduces false positive signals from erroneous, non-specific unblocking of blocked nucleic acid molecules by configuring the blocked nucleic acid molecules as blocked RNP2 activator molecules. Further, the blocked RNP2 activator molecules are configured such that many RNP2 activator transcript products are synthesized from a single unblocked RNP2 activator molecule, thereby increasing the rateof specific exponential signal amplification. Specificity in the present assays is important, as non-specific unblocking of blocked RNP2 activator molecules leads to non-specific activation of RNP2 in the absence of a target nucleic acid of interest. This concept is shown generally in FIG. 4. FIG. 4 at top is a simplified diagram of the “correct” pathway for unblocking the blocked nucleic acid molecule, where the unblocking is due to transcleavage of single-strand regions of a blocked nucleic acid molecule leading to activation of RNP2 (see, e.g., FIG. 3). FIG. 4 at bottom illustrates a “failure” pathway, where the unblocking of the blocked nucleic acid molecule is due not to trans-cleavage of singlestrand regions of the blocked nucleic acid molecule and dissociation of the non-target strand oligonucleotide segments from the target strand, but instead is due to erroneous enzyme-mediated unwinding of the blocked nucleic acid molecule. In the failure pathway, the unblocking is due not to trans-cleavage of the blocked nucleic acid molecule from target nucleic acids of interest binding to RNP1, but instead is due to enzyme-mediated unwinding of the blocked nucleic acid molecule by the nucleic acid-guided nuclease in RNP2.

[0110] The unwinding of the blocked nucleic acid molecule may be triggered by the presence of a PAM in the blocked nucleic acid molecule (if present) but even in blocked nucleic acid molecules lacking a PAM sequence, unwinding can take place simply by the sequence complementarity between the blocked nucleic acid molecule (i.e., target molecule for RNP2) and gRNA2 which leads to DNA unpairing of the blocked nucleic acid molecule, R-loop formation, and subsequent activation of RNP2. This erroneous activation of RNP2 triggers trans-cleavage activity of RNP2 and subsequent signal generation. In this failure pathway, a target nucleic acid of interest is not present and RNP1 trans-cleavage activity has not been activated, yet RNP2 becomes activated leading to a false positive signal and kicking off the signal cascade.

[0111] A solution, as described herein, involves not having an RNP2 target sequence in the blocked RNP2 activator molecules and designing them to maintain a tight “lock” on RNP2 in the absence of trans-cleavage activity triggered by the binding of a target nucleic acid of interest to RNP1 — thereby circumventing the failure pathway — but to then synthesize RNP2 activator transcript products from the unblocked RNP2 activator molecule. Once a blocking moiety on the blocked RNP2 activator molecule hasbeen removed by the trans-cleavage activity of a nucleic acid-guided nuclease and a singlestrand region is removed by exonuclease activity of a polymerase, a template strand — the complement of and target for gRNA2 — is then transcribed by an RNA polymerase to produce RNP2 activator transcript products.

[0112] Generally in the TNT cascade methods herein, the blocked RNP2 activator molecule becomes unblocked and provides a template for synthesis for the target for RNP2 by: 1) removing a blocking moiety on the blocked RNP2 activator molecule via trans- cleavage activity from RNP1 triggered by the binding of a target nucleic acid of interest to RNP1 ; 2) removing a single- strand region on the now unblocked RNP2 activator molecule via 3' — > 5' exonuclease activity of a DNA polymerase or repeated trans-cleavage in the absence of 3' — > 5' exonuclease; 3) synthesizing via the DNA polymerase a second strand of a T7 RNA polymerase promoter and an RNP2 activator template; and 4) synthesizing, via an RNA polymerase, a multitude of RNP2 activator transcript products from the RNP2 activator template on the unblocked RNP2 activator molecule.

[0113] FIG. 5A is a simplified illustration of an exemplary thermonuclear cascade with T7 RNA polymerase (“TNT”) detection and signal amplification cascade system, showing the RNP1 system — the “detection” system — on the left and the RNP2 system — the “sign amplification” system — on the right. This embodiment is TNT1, the first iteration of the TNT assay. As described above in relation to the cascade systems described in FIGs. 1 and 2, RNP1 is designed to detect a target nucleic acid of interest, if present, and in doing so both the cis- and trans-cleavage activity of the RNP1 CRISPR enzyme (here, Cas 13) is activated. The trans-cleavage activity of RNP1 unblocks a blocked RNP2 activator molecule, removing a blocking moiety (here, shown as a terminal circle) from the 3' end of a cleavage flap on the RNP2 activator molecule. A 3' phosphatase is then employed to convert the 3' terminal 2', 3'-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group. The 3' OH group and flap can be “chewed back” via exonuclease activity of a DNA polymerase where the double strand region serves as an extension substrate for the 5' — > 3' polymerase activity of the DNA polymerase to synthesize a double-strand DNA extension product. This extension product comprises a T7 RNA polymerase promoter and the template for the RNA target for gRNA2. The RNA polymerase transcribes RNP2 activator transcription products from the template, which canthen activate cis- and trans-cleavage activity of the RNP2 CRISPR enzyme (here, also Cas 13), which in turn can unblock additional blocked RNP2 activator molecules. Note that T7 RNA polymerase is recited here; however, one of ordinary skill in the art given the present disclosure should appreciate that other RNA polymerases could be utilized, such as T3, SP6, Syn5, VSW-3, or phi6 RNA polymerases, or others as described above.

[0114] FIG. 5B is a simplified illustration the exemplary TNT detection and signal amplification cascade system of FIG. 5 A (TNT1) and one point of failure that reduces the effectiveness of this embodiment of the TNT cascade system. FIG. 5B, like FIG. 5 A, begins with an RNP1 designed to detect a target nucleic acid of interest, if present. In doing so, both the cis- and trans-cleavage activity of the RNP1 CRISPR enzyme (here again, Cas 13) is activated, thereby unblocking a blocked RNP2 activator molecule by removing a blocking moiety (here, shown as a terminal circle) from the 3' end of a cleavage flap on the RNP2 activator molecule. A 3' phosphatase then converts the 3' terminal 2', 3'- cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group. The 3' OH group and flap can be “chewed back” via exonuclease activity of a DNA polymerase and the double strand region then serves as an extension substrate for the 5' — > 3' polymerase activity of the DNA polymerase. A double-strand DNA extension product is synthesized comprising a T7 RNA polymerase promoter and the template for the RNA target for gRNA2. Using the T7 RNA promoter, the RNA polymerase then transcribes RNP2 activator transcription products from the template, which then bind to RNP2 and activate cis- and trans-cleavage activity of the RNP2 CRISPR enzyme (here, also Cas 13). The trans-cleavage activity of RNP2 can, in turn, unblock additional blocked RNP2 activator molecules.

[0115] It has, however, been determined empirically that not only are the RNP2 activator transcription products transcribed, but non-canonical transcripts are also synthesized by the T7 RNA polymerase in the absence of RNP1 activation and unblocking of the blocked RNP2 activator molecule, which causes background. In the context of the present disclosure, “non-canonical transcription” refers to T7 RNA polymerase transcription that is not driven by a dsDNA promoter. Here, non-canonical transcription includes the transcription of the blocked template strand of the blocked RNP2 activator molecule, the transcription of the blocked or unblocked non-template strand of the RNP2activator molecule, and transcription of free-floating gRNA and other RNAs in the reaction mix. It is postulated that this non-canonical transcription is due to the high concentration of T7 RNA polymerase and blocked RNP2 activator molecules in an in vitro reaction such as this.

[0116] FIG. 6A is another illustration of the TNT detection and signal amplification cascade system shown in FIG. 5A (“TNT1”). In step 1 of FIG. 6A, the trans-cleavage activity of RNP1 triggers the unblocking of the blocked RNP2 activator molecule 5' of the blocking moiety (shown here as a terminal circle). The blocking moiety serves two purposes. First, it prevents 3' exonuclease activity in the absence of trans-cleavage of the single-strand “cleavage flap.” Second, it prevents the DNA polymerase from extending the template in the absence of trans-cleavage of the cleavage flap.

[0117] As for the cleavage flap, this cleavage flap region of the blocked RNP2 activator molecule may comprise ribonucleotides and / or deoxynucleotides. If RNP1 comprises a DNA-cleaving nucleic acid-guided nuclease and RNA2 comprises an RNA- cleaving nucleic acid-guided nuclease (in the context of the TNT assays, the nucleic acid- guided nuclease in RNP2 is a RNA-cleaving nucleic acid nuclease), the single-strand “cleavage flap” region may comprise both ribonucleotides and deoxyribonucleotides, since DNA-cleaving nucleic acid-guided nucleases preferentially trans-cleave deoxyribonucleotides; however, if RNP1 comprises an RNA-cleaving nucleic acid-guided nuclease, the single-strand “cleavage flap” region may comprise only ribonucleotides, since RNA-cleaving nucleic acid-guided nucleases preferentially trans-cleave ribonucleotides and here, both RNP1 and RNP2 comprise an RNA-cleaving nucleic acid- guided nuclease. After the 3' blocking moiety is removed, a 3' phosphatase — such as polynucleotide kinase — then converts the 3' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group.

[0118] Once the blocked RNP2 activator molecule is unblocked by removal of the blocking moiety, in step 2 two events occur. First, the 3' — > 5' exonuclease “proofreading” activity of the DNA polymerase present in the reaction mixture “chews back” or removes what is left of the single-strand DNA cleavage flap at the 3' end of the template strand of the unblocked RNP2 activator molecule, where the double-strand region can then serve as the primer region for the DNA polymerase. Second, the 5' — > 3' polymerase activity of theDNA polymerase then synthesizes the sense strand of the T7 RNA polymerase promoter and the complement of the non-template strand (i.e., the template strand). Once the T7 promoter is synthesized, the T7 RNA polymerase can use the double strand polymerization product to synthesize RNP2 activator transcription products. At step 3, the RNP2 activator transcription products then activate the trans-cleavage activity of the Casl3 nuclease in RNP2, which removes the blocking moiety of more blocked RNP2 activator molecules in step 4, cycling back to step 2 in a cascade.

[0119] Note that the unblocked RNP2 activator molecules can continue to serve as templates for the production of more RNP2 activator transcription products (i.e., the RNA targets for RNP2) given the synthesized double-strand T7 promoters and the existence of RNA polymerase and rNTPs in the reaction mix. Thus, not only does the unblocking of the blocked RNP2 activator molecules activate RNP2 to unblock more blocked RNP2 activator molecules, but the unblocked RNP2 activator molecules continue to generate RNP2 activator transcription products for RNP2 as long as RNA polymerase and rNTPs are present in the reaction mix, essentially super-charging the cascade further increasing the rate of exponential signal amplification.

[0120] The DNA polymerases used in the present reaction mixtures and methods allow for use of strongly “locked” blocked RNP2 activator molecules to prevent nonspecific unblocking but serve as a “key” to release the lock upon nicking of the singlestrand loop 5' of the blocking moiety and synthesizing the target strand segment as described above. Preferably, the polymerase used comprises both 5' — > 3' DNA polymerase activity and 3' — > 5' exonuclease activity but lacks 5' — > 3' exonuclease activity. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich MA., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5' — > 3' DNA polymerase activity and 3' — > 5' exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or any active portion or variant thereof. Also, a 3' — > 5' exonuclease can be separately used if the polymerase lacks this activity, or a 3'— > 5' exonuclease or the activity thereof can be eliminated altogether as the trans-cleavage activity will eventually cleave all single-strand nucleotides in the flap; however, reaction kinetics are improved if a 3' — > 5' exonuclease is employed.

[0121] FIG. 6B is a simplified illustration of an exemplary blocked activator molecule suitable for use with the exemplary TNT1 detection and signal amplification cascade system in FIG. 6A and an example sequence for each strand therefor. The blocked RNP2 activator molecule comprises on the strand at left, from the 5' end a single-strand region that is a template for the T7 RNA polymerase promoter; a paired region comprising the RNP2 activator non-template strand; and a single-strand region terminating at its 3' end with a blocking moiety (note that this single-strand region is not a loop; however, including a loop is optional). The blocked RNP2 activator molecule comprises on the strand at right, from the 5' end a paired region comprising the RNP2 activator template strand; an unpaired region cleavage flap that serves as a cleavage substrate for trans-cleavage by the nucleic acid-guided nucleases in RNP1 and RNP2; and finally at the 3' end, one or more blocking moieties (here, denoted as an inverted T).

[0122] Note that there are three single-strand regions of this exemplary blocked RNP2 activator molecule, including the region of the single-strand region that serves as the template for the T7 RNA polymerase promoter, the single-strand region terminating at its 3' end with the one or more blocking moieties (which again, optionally could be a hairpin loop), and the cleavage flap also terminating at its 3' end with one or more blocking moieties. These single-strand regions are vulnerable to non-specific cleavage by nucleases that may be present in the reaction mix via., e.g., the sample comprising the target nucleic acids. The blocking moieties incorporated on both of the 3' ends of the blocked RNP2 activator molecule are present — as described above — to prevent 3' exonuclease activity and erroneous extension. In addition to the blocking moieties, one to several modified nucleotides, such as, e.g., phosphorothioate-modified (“PS”) nucleotides, may be added in the single-strand regions as well to prevent non-specific cleavage. In addition to phosphoro thioate modifications, the nucleotides of these single- strand regions may comprise locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2'-O-methyl (2'-O- Me) modified nucleosides, and / or 2'-fluoro (2'-F) modified nucleosides and / or other modifications that resist cleavage. The paired region of the now unblocked RNP2 activatormolecule should be of sufficient length to serve as a primer for extension by DNA polymerase and subsequent transcription from the T7 promoter as well as possess a Tmhigh enough to prevent melting at the reaction temperature.

[0123] FIG. 6B also provides an exemplary sequence for each strand of a blocked RNP2 activator molecule where RNP2 comprises an RNA-cleaving nucleic acid-guided nuclease, where asterisks denote phosphorothioate modified nucleotides, and where the two “3InvdT” denote inverted dTs which create a 3'-3' linkage between the last two nucleotides at the 3' ends of the blocked RNP2 activator molecule.

[0124] FIG. 6B also states one point of failure that reduces the effectiveness of the TNT1 embodiment of the TNT cascade system. First, the presence of the complete nontemplate strand in the blocked RNP2 activator molecules appears to be able to activate RNP2 without unblocking, particularly in an in vitro system where the blocked RNP2 activator molecules are at a high relative concentration. Second — as discussed above — it has been determined empirically that not only are the RNP2 activator transcription products transcribed, but non-canonical transcripts are also synthesized by the T7 RNA polymerase. Again, “non-canonical transcription” refers to T7 RNA polymerase transcription that is not driven by a dsDNA promoter. In this TNT1 embodiment, for example, non-canonical transcription includes the transcription of the blocked template strand of the blocked RNP2 activator molecule and the transcription of both the blocked and unblocked non-template strand of the RNP2 activator molecule. Non-canonical transcription of the template strand sequence — which is complementary to the Casl3 RNP2 guide — then erroneously activates RNP2.

[0125] FIG. 7A is a simplified illustration of an alternative exemplary TNT detection and signal amplification cascade system (termed “TNT2”). The TNT2 embodiment differs from the TNT1 embodiment by designing the blocked RNP2 activator molecule such that it does not include a sequence complementary to gRNA2 (i.e., the gRNA component of RNP2); however, for the most part the two embodiments are very similar. It was thought that removing the sequence that is complementary to gRNA2 and appeared to be triggering activation of RNP2 would address the issue of premature activation of RNP2. Looking at FIG. 7A, in step 1 the trans-cleavage activity of RNP1 triggers the unblocking of the blocked RNP2 activator molecule 5' of the blocking moiety(shown here as a terminal circle). Again, the blocking moiety prevents both 3 ' exonuclease activity and erroneous extension.

[0126] Note that in this TNT2 embodiment, the blocked RNP2 activator molecule does not comprise a sequence that is complementary to the gRNA2 in RNP2; instead, it is the later extension by the DNA polymerase that creates the double-strand DNA T7 RNA polymerase promoter and the complement (i.e., target) of gRNA2. As described in relation to FIG. 6A, if RNP1 comprises a DNA-cleaving nucleic acid-guided nuclease, the singlestrand “cleavage flap” region may comprise both ribonucleotides and deoxyribonucleotides, since DNA-cleaving nucleic acid-guided nucleases preferentially trans-cleave deoxyribonucleo tides; however, if RNP1 comprises an RNA-cleaving nucleic acid-guided nuclease, the single-strand “cleavage flap” region may comprise only ribonucleotides, since RNA-cleaving nucleic acid-guided nucleases preferentially trans- cleave ribonucleotides (again, in these TNT embodiments, RNP2 comprises an RNA- cleaving nucleic acid-guided nuclease). Also in step 1, once the blocking moiety is removed a 3' phosphatase converts the 3' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group.

[0127] Once the blocked RNP2 activator molecule is unblocked by removal of the blocking moiety, in step 2 two events occur. First, the 3' — > 5' exonuclease “proofreading” activity of the DNA polymerase present in the reaction mixture “chews back” or removes what is left of the single-strand DNA cleavage flap at the 3' end of the template strand of the unblocked RNP2 activator molecule, where the double-strand region can then serve as the primer region for the DNA polymerase. Second, the 5' — > 3' polymerase activity of the DNA polymerase then synthesizes the sense strand of the T7 RNA polymerase promoter and the complement of the non-template strand (i.e., the template strand and gRNA2 target). As with the TNT1 embodiment, once the T7 promoter is synthesized, the T7 RNA polymerase can use the double-strand polymerization product to synthesize RNP2 activator transcription products. At step 3, the RNP2 activator transcription products then activate the trans-cleavage activity of the Casl3 nuclease in RNP2, which removes the blocking moiety of more blocked RNP2 activator molecules in step 4, cycling back to step 2 in a cascade. Again, note that the unblocked RNP2 activator molecules can continue to serve as templates for the production of more RNP2 activator transcription products (i.e., theRNA targets for RNP2) given the synthesized double-strand T7 promoters and the existence of RNA polymerase and rNTPs in the reaction mix, as not only does the unblocking of the blocked RNP2 activator molecules activate RNP2 to unblock more blocked RNP2 activator molecules, but the unblocked RNP2 activator molecules continue to generate RNP2 activator transcription products for RNP2.

[0128] FIG. 7B is a simplified illustration of an exemplary blocked activator molecule suitable for use with the exemplary TNT2 detection and signal amplification cascade embodiment illustrated in FIG. 7A including an example sequence for each strand. The blocked RNP2 activator molecule comprises on the strand at left (the “antisense strand”), from the 5' end: a single-strand region that is the reverse complement of the T7 RNA polymerase promoter and a reverse complement of the RNP2 activator; a paired region which is essentially inert; and a single-strand region terminating at its 3' end with a blocking moiety (note that this single-strand region is not a loop; however, including a loop is optional). The blocked RNP2 activator molecule comprises on the strand at right (“the sense strand” which will be created by extension), from the 5' end: a paired region which is essentially inert; an unpaired region cleavage flap that serves as a cleavage substrate for trans-cleavage by the nucleic acid-guided nucleases in RNP1 and RNP2; and finally at the 3' end, one or more blocking moieties (here, denoted as an inverted T).

[0129] As with the blocked RNP2 activator molecule in FIG. 6B, there are three single-strand regions of this exemplary blocked RNP2 activator molecule, including the region of the single-strand region that is the reverse complement of the T7 RNA polymerase promoter and the template for the RNP2 activator, the single-strand region terminating at its 3' end with the one or more blocking moieties (which again, optionally could be a hairpin loop), and the cleavage flap also terminating at its 3' end with one or more blocking moieties. Assuming the nucleic acid-guided nuclease in RNP1 is Casl3a, the cleavage flap will comprise at least one and preferably more adjacent ribonucleotides available for trans-cleavage. As discussed above, the 3' ends of the blocked RNP2 activator molecule comprise blocking moieties to prevent 3' exonuclease activity and erroneous extension. However, in addition to the blocking moieties, one to several modified nucleotides, such as, e.g., phosphorothioate-modified (“PS”) nucleotides, may be added in the single-strand regions as well. In addition to phosphorothioate modifications, thenucleotides of these single-strand regions may comprise locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2'-0-methyl (2'-0-Me) modified nucleosides, and / or 2'- fluoro (2'-F) modified nucleosides and / or other modifications that resist cleavage. The paired region of the RNP2 activator molecule illustrated in FIG. 7B — here, the pairing of the two sequences noted above as “inert” — should be of sufficient length to serve as a primer for extension by DNA polymerase and subsequent transcription from the T7 promoter as well as possess a Tmhigh enough to prevent melting at the reaction temperature.

[0130] Like FIG. 6B, FIG. 7B provides an exemplary sequence for each strand — here the “antisense strand” and “sense strand” of the blocked RNP2 activator molecule — where in the TNT embodiments described herein the RNP2 comprises an RNA-cleaving nucleic acid-guided nuclease, where asterisks denote phosphorothioate modified nucleotides and where the two “3InvdT” denote inverted dTs, which create a 3' -3' linkage between the last two nucleotides at the 3' ends of the blocked RNP2 activator molecule.

[0131] FIG. 7B, like FIG. 6B, also states one point of failure that reduces the effectiveness of the TNT2 embodiment of the TNT cascade system. Here, despite omitting the gRNA2 complement from the TNT2 blocked RNP2 activator molecule — the effect of which is that there is no double-strand RNP2 activator sequence present in the blocked RNP2 activator molecule — significant non-specific activation of RNP2 was observed. Thus, it was concluded that it is the non-canonical transcription and resulting transcripts synthesized by the T7 RNA polymerase that are the source of the non-specific activation.

[0132] FIG. 8 is a simplified illustration of yet another exemplary TNT detection and signal amplification cascade system (termed “TNT3”). As noted here, TNT3 is essentially identical to TNT1; however, in TNT3 only a partial template is provided in the blocked RNP2 activator molecule. In step 1 of FIG. 8, the trans-cleavage activity of RNP1 triggers the unblocking of the blocked RNP2 activator molecule 5' of the blocking moiety. Note that the blocked RNP2 activator comprises only a partial RNP2 activator template strand such that non-canonical transcription of this partial template would result in a truncated RNP2 activator transcript. Since partial RNP2 activators do not activate Casl3 well, non-canonical transcription of this region does not result in appreciable RNP2 activation. In addition, there is a partial non-template strand configured such that non-canonical transcription of this partial non-template strand results in a sequence essentially identical to a portion of gRNA2, which does not bind to and activate RNP2. Further, the transcription product of the partial non-template strand — although complementary to gRNA2 — is also only a partial RNP2 activator and thus a poor activator of gRNA2.

[0133] As for the cleavage flap, as described above if RNP 1 comprises a DNA- cleaving nucleic acid-guided nuclease, the single-strand “cleavage flap” region may comprise both ribonucleotides and deoxyribonucleotides; however, if RNP 1 comprises an RNA-cleaving nucleic acid-guided nuclease, the single-strand “cleavage flap” region may comprise only ribonucleotides. Once the blocking moiety is removed, a 3' phosphatase then converts the 3' terminal 2', 3'-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group.

[0134] Once the blocked RNP2 activator molecule is unblocked by removal of the blocking moiety and the 3' terminal phosphate is converted to a 3' -OH, in step 2 two events occur. First, the 3' — > 5' exonuclease “proofreading” activity of the DNA polymerase present in the reaction mixture “chews back” or removes what is left of the single-strand DNA cleavage flap at the 3' end of the template strand of the unblocked RNP2 activator molecule, which then serves as the primer region for the DNA polymerase. Second, the 5' — > 3' polymerase activity of the DNA polymerase then synthesizes the sense strand of the T7 RNA polymerase promoter and the complement of the non-template strand (i.e., the template strand). Once the T7 promoter is synthesized, the T7 RNA polymerase can use the double strand polymerization product to synthesize RNP2 activator transcription products. At step 3, the RNP2 activator transcription products activate the trans-cleavage activity of the Casl3 nuclease in RNP2, which removes the blocking moiety of more blocked RNP2 activator molecules in step 4, cycling back to step 2 in a cascade. Again, as with previous embodiments the unblocked RNP2 activator molecules can continue to serve as templates for the production of more RNP2 activator transcription products (i.e., the RNA targets for RNP2) given the synthesized double-strand T7 promoters and the existence of RNA polymerase and rNTPs in the reaction mix. Thus, the unblocking of the blocked RNP2 activator molecules activate RNP2 to unblock more blocked RNP2 activator molecules and the unblocked RNP2 activator molecules continue to generate RNP2 activator transcription products for RNP2.

[0135] FIG. 9A is a simplified illustration of an enhancement to the TNT3 embodiment employing three rNTPs and one drNTP, which lacks a 3'-OH group (rNTP Absent Sense Strand and with Transcript Arrest “RASTA”). As noted here, RASTA is essentially identical to TNT3 but with the added feature of providing a nucleotide in the partial non-template strand whose complement rNTP is either omitted from the reaction mix or whose complement is provided as a drNTP, lacking a 3'-OH group. The impact of a lack of a 3'-OH group is that as soon as a drNTP is incorporated, transcription is arrested due to chain termination — similar to the technique employed in Sanger sequencing. Alternatively, an rNTP comprising an alternative 3' nucleotide modification functionally equivalent to a drNTP, which lacks a 3'-OH group may be employed.

[0136] In step 1 of FIG. 9 A, the trans-cleavage activity of RNP1 triggers the unblocking of the blocked RNP2 activator molecule 5' of the blocking moiety. As in TNT3 illustrated in FIG. 8, note that the blocked RNP2 activator comprises only a partial RNP2 activator template strand such that non-canonical transcription of this partial template results in a truncated RNP2 activator transcript. In addition, there is a partial non-template strand configured such that non-canonical transcription of this partial non-template strand results in a partial gRNA2 sequence, which does not bind to and activate RNP2, and even transcription of this transcripts results in only a partial (e.g., truncated) RNP2 activator sequence.

[0137] The key to RASTA is that the partial template strand is designed to require only three rNTPs for transcription of the extended template; however, the partial non- template strand is designed to need all four rNTPs for non-canonical transcription. This approach can solve the problem of non-canonical transcription via two different approaches. The first approach involves engineering the template such that only three of four rNTPs are required to extend the template. If only the three rNTPs needed for transcription of the extended template are provided in the reaction mix — where the fourth rNTP is not — then non-canonical transcription of the partial non-template strand should be very inefficient. This approach is most effective an RNA polymerase that has been engineered to exhibit high fidelity is employed preferably in conjunction with using rNTPs that are very pure; that is, using rNTPs that are not contaminated with the fourth rNTP that should be excluded from the reaction mix. Second, rather than excluding the fourth rNTPfrom the reaction mix, the fourth rNTP can be provided as a drNTP, which acts as a chain terminator, thereby arresting transcription of the non-template strand. Requiring only three rNTPs for transcription of the extended template but requiring all four rNTPs for transcription of the non-template strand can essentially “shut down” transcription of the partial non-template strand.

[0138] As in the TNT1 , TNT2 and TNT3 embodiments described above, the singlestrand “cleavage flap” region may comprise both ribonucleotides and deoxyribonucleotides if RNP1 comprises a DNA-cleaving nucleic acid-guided nuclease and using only ribonucleotides if RNP1 comprises an RNA-cleaving nucleic acid-guided nuclease. After removal of the blocking moiety, a 3' phosphatase then converts the 3' terminal 2', 3'-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group.

[0139] As with the TNT1, TNT2 and TNT3 embodiments, once the blocked RNP2 activator molecule is unblocked by removal of the blocking moiety, in step 2 two events occur. First, the 3' — > 5' exonuclease “proofreading” activity of the DNA polymerase present in the reaction mixture “chews back” or removes what is left of the single-strand DNA cleavage flap at the 3' end of the template strand of the unblocked RNP2 activator molecule, which then serves as the primer region for the DNA polymerase. Second, the 5' — > 3' polymerase activity of the DNA polymerase then synthesizes the sense strand of the T7 RNA polymerase promoter and the complement of the non-template strand (i.e., the template strand). Once the T7 promoter is synthesized, the T7 RNA polymerase can use the double strand polymerization product to synthesize RNP2 activator transcription products. At step 3, the RNP2 activator transcription products activate the trans-cleavage activity of the Casl3 nuclease in RNP2, which removes the blocking moiety of more blocked RNP2 activator molecules in step 4, cycling back to step 2 in a cascade. The unblocked RNP2 activator molecules can continue to serve as templates for the production of more RNP2 activator transcription products given the synthesized double-strand T7 promoters and the existence of RNA polymerase and rNTPs in the reaction mix. Thus, the unblocking of the blocked RNP2 activator molecules activate the RNP2s to unblock more blocked RNP2 activator molecules and the unblocked RNP2 activator molecules continue to generate RNP2 activator transcription products for RNP2.

[0140] FIG. 9B shows an exemplary sequence for a RASTA blocked RNP2 activator molecule that was determined to be difficult to unblock. In this instance, a single U-U ribo-dinucleotide was incorporated into the cleavage flap. This single U-U locus would be the only preferred cleavage site for an RNP1 or RNP2 that comprises an RNA- cleaving nucleic acid-guided nuclease such as Casl3.

[0141] FIG. 9C shows a simplified illustration of an exemplary blocked RNP2 activator molecule suitable for use with the RASTA detection and signal amplification cascade system in FIG. 9A and provides an example sequence therefor. In this embodiment, not only is a unimolecular blocked RNP2 activator molecule employed, but a free single-strand non-template construct is provided in the reaction mix as well. It has been determined that the blocked unimolecular RNP2 activator molecule largely remains self-hybridized before trans-cleavage by RNP1 or RNP2 (see the bar graph in FIG. 9C); however, following trans-cleavage in the hairpin loop — resulting in the unimolecular RNP2 activator molecule being cleaved into two molecules — the template strand of the now unblocked RNP2 activator molecule will preferentially hybridize to the free singlestrand non-template construct as long as the conditions for hybridization are more favorable for the template strand of the now unblocked RNP2 activator molecule: free single-strand non-template construct duplex. For example, if the number of complementary bases available for hybridization in the unimolecular molecule is less than the number of complementary bases available for hybridization in the unblocked RNP2 activator molecule: free single-strand non-template construct duplex, the unblocked RNP2 activator molecule is more likely to form a duplex with the free single-strand non-template construct than with the now single-strand non-template strand resulting from the unimolecular molecule after cleavage.

[0142] FIG. 9D is a simplified illustration of an alternative to the exemplary blocked RNP2 activator molecule shown in FIG. 9C. The exemplary blocked RNP2 activator molecule shown in FIG. 9D is a “split activator” configuration, which splits the target sequence for gRNA2 into two regions separated by a linker. This split activator embodiment is also suitable for use with the RASTA detection and signal amplification cascade system in FIG. 9A. In this configuration, the activator for RNP2 is split in two, where the portions are connected by a linker. When the split portions are hybridized togRNA2, they form a “whole” activator. For example, the RNP2 activator molecule (the target for RNP2) may be, e.g., 21 nucleotides in length and be split 14 / 7. In another example, the target portion may vary from 20 to 30 nucleotides in length and be split, e.g., 14 / 6, 13 / 7, 12 / 8, 11 / 9, 10 / 10, 9 / 11, 8 / 12, 7 / 13, or 6 / 14; or 14 / 7, 13 / 8, 12 / 9, 11 / 10, 10 / 11, 9 / 12, 8 / 13, 7 / 14, or 6 / 15; or 14 / 8, 13 / 9, 12 / 10, 11 / 11, 10 / 12, 9 / 13, 8 / 14, 7 / 15, or 6 / 16; or 14 / 9, 13 / 10, 12 / 11, 11 / 12, 10 / 13, 9 / 14, 8 / 15, 7 / 16, or 6 / 17; or 14 / 10, 13 / 11, 12 / 12, 11 / 13, 10 / 14, 9 / 15, 8 / 16, 7 / 17, or 6 / 18; or 14 / 11, 13 / 12, 12 / 13, 11 / 14, 10 / 15, 9 / 16, 8 / 17, 7 / 18, or 6 / 19; or 14 / 12, 13 / 13, 12 / 14, 11 / 15, 10 / 16, 9 / 17, 8 / 18, 7 / 19, or 6 / 20; or 14 / 13, 13 / 14, 12 / 15, 11 / 16, 10 / 17, 9 / 18, 8 / 19, 7 / 20, or 6 / 21; or 14 / 14, 13 / 15, 12 / 16, 11 / 17, 10 / 18, 9 / 19, 8 / 20, 7 / 21, or 6 / 22; or 14 / 15, 13 / 16, 12 / 17, 11 / 18, 10 / 19, 9 / 20, 8 / 21, 7 / 22, or 6 / 23; or 14 / 16, 13 / 17, 12 / 18, 11 / 19, 10 / 20, 9 / 21, 8 / 22, 7 / 23, or 6 / 24.

[0143] Also in the split activator embodiment, the linker comprises nucleotides and is shown as A(is) with a 3' terminal blocking T ; however, A(is) is exemplary only. The linker that is employed should be a linker that is resistant to trans-cleavage by both the nucleic acid-guided nuclease in both RNP1 and RNP2. Thus, if both RNP1 and RNP2 comprise a RNA-cleaving nucleic acid-guided nuclease, a linker employing deoxyribonucleotides or a chemical linker should be employed. The length of the linker typically ranges from 15 to 50 nucleotides in length, or the equivalent thereof, or from 20 to 40 nucleotides in length. In certain embodiments, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. In addition to nucleic acid-based linkers, there are also peptide and nonpeptide linkers, and linkers are generally classified into three categories according to their structures. In addition to peptide linkers, there are PEG linkers, which are chemically functionalized polyethylene glycol (PEG) linkers and are attractive due to their aqueous solubility.

[0144] An advantage to the split activator configuration is that the linker provides nucleotides that can be used for cleavage by an RNA-cleaving nucleic acid-guided nuclease. That is, the linker provides flexibility for the choice and number of ribonucleotides included for a cleavage site without being constricted by the sequence ofthe template. In addition, the linker provides the primer region from which transcription is initiated regardless of which U-U bond is cleaved.Applications of the Cascade Assay

[0145] The present disclosure describes TNT cascade assays for detecting one or more target nucleic acids of interest in a sample. The TNT cascade assays allow for massive multiplexing and minimum workflow yet provide accurate results at low cost. Moreover, the presently described embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components can stay the same no matter what target nucleic acid(s) of interest are being detected and RNP1 is easily reprogrammed.

[0146] Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus.

[0147] For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint(for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

[0148] In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in USPN 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those. In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HC1. Suitable methods are contemplated in USPN 8,809,519 and USPN 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and / or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in USPN 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA / RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

[0149] Table 1 below lists exemplary commercial sample processing kits, and Table 2 below lists point of care processing techniques.TABLE 1: Exemplary Commercial Sample and Nucleic Acid Processing KitsTABLE 2: Point of Care Sample Processing Techniques

[0150] The components of the cascade assay may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemic testing sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNPls), second ribonucleoprotein complexes (RNP2s), blocked RNP2 activator molecules, a kinase, a DNA polymerase having 3' exonuclease activity, an RNA polymerase, dNTPs, rNTPs and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked RNP2 activator molecule comprises atemplate from which the RNP2 activator transcript products — the targets for gRNA2 in RNP2 — are synthesized. Inclusion of the DNA polymerase having 3' exonuclease activity and RNA polymerase improves performance of the assay by allowing for use of blocked RNP2 activator molecules with tight “locks” to avoid false positives yet aids in the amplification of the signal. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked RNP2 activator molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and / or are operably linked to the blocked RNP2 activator molecules and produce a detectable signal upon cleavage by RNP2.

[0151] Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and / or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.EXAMPLES

[0152] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.Example I: Preparation of Nucleic Acids of Interest

[0153] Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely-used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral / Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose, CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).

[0154] Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separate the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and / or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components andchemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non- specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.

[0155] Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90°C, however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.

[0156] Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.

[0157] Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in Dl / di stilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellularmembrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.

[0158] Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic -hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.

[0159] Another form of chemical lysis is the widely-used procedure of phenolchloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

[0160] Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.Example II: RNP Formation

[0161] Master reaction buffer mixes were prepared with NaCl, MgCh, TCEP, MnCh, BSA, and other components, as required, with at least 300 pL excess volume in sterile RNase-free tubes. Reaction diluent comprising ROX, reporter and target was prepared and kept at room temperature.

[0162] Enzymes were diluted on ice in reaction buffer, as were guide RNAs. RNP 1 formations were set up by adding water, buffer, enzyme and gRNAs together, mixing and then heating to 23°C for 30 minutes. After 30 minutes, the tubes were removed and kept at room temperature until ready to use. RNP1 formations were diluted (on ice) to final reaction concentrations. RNP2 formations were transferred wells in a 96-well plate and kept on ice at least 5 minutes before moving onto the final assembly step.Example III: Reporter Moiety Formation

[0163] The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-10 bases in length (e.g., with sequences of TTATT, TTTATTT, ATT AT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5' and 3' ends, respectively. In one example using a Casl2a cascade, the fluorophore was FAM-6, and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In another example using a Casl3 cascade, the reporter moieties were single stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).Example IV: Cascade Assay

[0164] Reaction plates were chilled to 4°C for at least 5 minutes and the reactions were assembled in a place on ice. RNP1, RNP2, targets and blocked targets were added to wells according to experimental design. QuantStudio (Thermo Fisher Scientific, Waltham MA, USA) was set up according to the manufacturer’s instructions and was pre-heated to the desired reaction temperature prior to the reaction and the reaction parameters were set and run according to a program.

[0165] While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures.

Claims

We claim:

1. A method for identifying a target nucleic acid of interest in a sample using a thermonuclear cascade with RNA polymerase assay comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNPls) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits gRNA-target hybridization dependent transcleavage activity; second ribonucleoprotein complexes (RNP2s) each comprising a second nucleic acid- guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest and comprises a crRNA of X nucleotides in length, where X = 12 to 35 nucleotides, wherein the crRNA is capable of hybridizing to an RNP2 activator transcript product, wherein the second nucleic acid-guided nuclease is an RNA-cleaving nucleic acid-guided nuclease that exhibits gRNA- target hybridization-dependent trans-cleavage activity; a plurality of blocked RNP2 split activator molecules each comprising a blocked partial transcription template and a non-template strand; wherein the blocked partial transcription template comprises from 5' to 3': a sequence of at least four nucleotides complementary to a 5' portion of the second gRNA, a first linker region having a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least two adjacent ribonucleotides, and a terminal region comprising a first 3' blocking moiety; and wherein the non-template stand comprises from 5' to 3': a sequence at least X minus 4 nucleotides in length having substantially a same nucleotide sequence as a 3' portion of the second gRNA, a sequence of nucleotides coding for one strand of an RNA polymerase promoter sequence, a second linker region having a length equivalent to at least 15 nucleotides with complementarity to the first linker region, and a terminal region comprising a second 3' blocking moiety; a 3' phosphatase;a DNA polymerase optionally comprising both polymerase and 3' — > 5' exonuclease activity; a RNA polymerase; dNTPs and rNTPs; and reporter moieties that produce a signal upon trans-cleavage by the first or second nucleic acid-guided nuclease; and contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample, if present, to bind to RNP1; wherein upon binding of the target nucleic acid of interest, RNP1 becomes active initiating trans- cleavage of a bond between the at least two adjacent nucleotides of the blocked partial transcription template thereby unblocking the at least one blocked RNP2 activator molecule by removing the first 3' terminal blocking moiety; allowing the DNA polymerase to remove single-strand nucleotides from the 3' cleaved single-strand region of the blocked partial transcription template, if present, and to extend the blocked partial transcription template by copying the sequence complementary to the 3' portion of the second gRNA of non-template stand and the RNA polymerase promoter sequence creating a polymerization product; allowing the RNA polymerase to use the polymerization product to transcribe RNP2 activator transcript products, wherein the RNP2 activator transcript products are capable of binding to the second gRNAs and activating trans-cleavage of the RNP2s thereby unblocking at least one additional blocked RNP2 activator molecule; and detecting a presence or absence of the signal from the reporter moieties.

2. The method of claim 1, wherein the crRNA of the second gRNA is 20-30 nucleotides in length.

3. The method of claim 2, wherein the blocked partial transcription template comprises a sequence of at least 6 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises a sequence at least X minus 6 nucleotides in length.

4. The method of claim 3, wherein the blocked partial transcription template comprises a sequence of at least 7 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises at least X minus 7 nucleotides in length.

5. The method of claim 4, wherein the blocked partial transcription template comprises a sequence of at least 10 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises at least X minus 10 nucleotides in length.

6. The method of claim 5, wherein the blocked partial transcription template comprises a sequence of at least 12 nucleotides complementary to a 5' portion of the second gRNA and the non-template stand comprises a sequence at least X minus 12 nucleotides in length.

7. The method of claim 1, wherein the first linker region has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least four adjacent ribonucleotides.

8. The method of claim 7, wherein the first linker region has a length equivalent to at least 20 nucleotides and wherein the first linker comprises at least four adjacent ribonucleotides.

9. The method of claim 7, wherein the first linker region has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least six adjacent ribonucleotides.

10. The method of claim 9, wherein the first linker region has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least eight adjacent ribonucleotides.

11. The method of claim 10, wherein the first linker region has a length equivalent to at least 15 nucleotides and wherein the first linker comprises at least ten adjacent ribonucleotides.

12. The method of claim 1, wherein the linker comprises only deoxyribonucleotides and ribonucleotides.

13. The method of claim 1 , wherein the reporter moieties are separate molecules from RNP1, RNP2 and the plurality of blocked RNP2 activator molecules.

14. The method of claim 1, wherein the blocked RNP2 activator molecules comprise the reporter moieties.

15. The method of claim 1, wherein the RNA polymerase is selected from T7 RNA polymerase, Enterobacteria phage T3 RNA polymerase), Salmonella phage SP6 RNA polymerase, Synechococcus phage Syn5 RNA polymerase, Pseudomonas phage VSW-3 RNA polymerase, Pseudomonas phage phi6 RNA polymerase, E. coll RNA polymerase containing subunit beta, human RNA polymerase I containing P0LR1A, human RNA polymerase II containing P0LR2A, Human RNA polymerase III containing P0LR3A, human mitochondrial RNA polymerase or chimeras thereof.

16. The method of claim 1, wherein the dNTPs and rNTPs may be modified dNTPs and rNTPs.

17. The method of claim 1 , wherein the DNA polymerase exhibits 3' — > 5' exonuclease activity.

18. The method of claim 1, wherein the DNA polymerase does not exhibit 3' — > 5' exonuclease activity.

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