Enhanced CAS12 cascade activation

Abstract

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods allow one to control reaction kinetics of a cascade assay to prevent false positives in the absence of a target nucleic acid of interest yet enhance signal production in the presence of a target nucleic acid of interest.

Description

TITLE: ENHANCED CAS12 CASCADE ACTIVATIONRELATED CASES

[0001] This application claims priority to U.S. Ser. Nos. 63 / 661,589, filed 19 June 2024; 63 / 665,028, filed 27 June 2024; 63 / 686,919, filed 26 August 2024; and 63 / 750,231, filed 27 January 2025, all of which are incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods allow one to control reaction kinetics of a cascade assay to prevent false positives in the absence of a target nucleic acid of interest yet enhance signal production in the presence of a target nucleic acid 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 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 assays that allow very rapid and accurate detection of nucleic acids yet minimize false positive signals are therefore needed for timelydiagnosis and treatment of disease, to identify toxins in consumables and the environment, as well as 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 compositions of matter and assay methods to detect target nucleic acids of interest where reaction kinetics of the assay can be controlled via molecular design of at least one of the reaction components. The “nucleic acid-guided nuclease cascade assays” or “signal boost cascade assays” or “cascade assays” described herein comprise two different ribonucleoprotein complexes, blocked nucleic acid molecules — here, cither blocked target strand displacement molecules or blocked primer extension molecules — where a polymerase and deoxyribonucleotides are included in the reaction mixture. The blocked nucleic acid molecules keep one of the ribonucleoprotein complexes — the second ribonucleoprotein complexes (i.e., RNP2s) — “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex — the first ribonucleoprotein complexes (i.e., RNPls). In the context of the cascade assays, “locked” means that the blocked nucleic acid molecules are designed in such a way that they are largely blocked from interacting with second ribonucleoprotein complexes; therefore, the second ribonucleoprotein complexes remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex.

[0007] The combination of trans-cleavage activity of the first ribonucleoprotein complex and the activity of the polymerase unblocks the blocked nucleic acid molecules thereby enhancing signal production in the cascade assay. The present nucleic acid-guided nuclease cascade assay can detect one to many to a large number of target nucleic acids ofinterest (in this case, 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. A particularly advantageous feature of the cascade assay generally is that, with the exception of the guide nucleic acid(s) in RNPl(s), the cascade assay components can be the same in each assay no matter what target nucleic acid(s) of interest is being detected.

[0008] In the present disclosure, the target nucleic acids may be RNA-based or DNA-based nucleic acids; thus, the nucleic acid-guided nuclease in RNP1 may be a DNA-cleaving nucleic acid-guided nuclease or an RNA-cleaving nucleic acid-guided nuclease. However, the RNP2s herein comprise a DNA-cleaving nucleic acid-guided nuclease. If RNP1 comprises a DNA-cleaving nucleic acid-guided nuclease, the blocked target strand displacement molecules or blocked primer extension molecules may comprise all deoxyribonucleotides; however, if one or more of the RNPls comprises an RNA-cleaving nucleic acid-guided nuclease, the blocked target strand displacement molecules or blocked primer extension molecules must comprise at least one ribonucleotide in the region of the blocked target strand displacement molecule or blocked primer extension molecule that is cleaved to unblock the molecule.

[0009] Thus, there is provided in a first embodiment a method for identifying a target nucleic acid of interest in a sample using a signal boost 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 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 wherein the second nucleic acid-guided nuclease is a DNA-cleaving nucleic acid-guided nuclease and where the second nucleic acid-guided nuclease exhibits both cisand trans-cleavage activity; a plurality of blocked target strand displacement molecules each comprising a non-target strand and a non-target strand complement, wherein the nontarget strand complement comprises from 5' to 3': a clamp region complementary to a portion of the non-target strand, a single- strand loop not complementary to a portion of thenon-target strand, and a target strand segment complementary to a portion of the non-target strand and complementary to the second gRNA; a polymerase comprising polymerase activity and optional 3' — > 5' exonuclease activity; and dNTPs; 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 the single- strand loop of at least one of the plurality of the blocked target strand displacement molecules resulting in 3 ' and 5' cleaved loop regions; allowing the polymerase to remove single-strand nucleotides from the 3' cleaved loop region and to extend the clamp region thereby displacing the target strand segment, wherein the displaced target strand segment binds to an RNP2 initiating trans-cleavage of the single-strand loop of at least one additional blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

[0010] In some aspects of this first embodiment, the blocked nucleic acid molecule typically ranges from 60 bp to 300 bp in length, the non-target strand ranges from 30 bp to 150 bp in length; and the non-target strand complement ranges from 10 bp to 60 bp in length, with the target strand segment ranging from 10 bp to 60 bp in length and the singlestrand loop ranging from 4 bp to 20 bp in length. In some aspects of this first embodiment, the polymerase comprises 3' — > 5' exonuclease activity, and in other aspects, the polymerase lacks 3' — > 5' exonuclease activity.

[0011] A second embodiment provides a method for identifying a target nucleic acid of interest in a sample using a signal boost 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 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, wherein the second nucleic acid- guided nuclease is a DNA-cleaving nucleic acid-guided nuclease and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; a plurality of blocked primer extension molecules each comprising a non-target strand and a targetstrand; wherein the non-target strand comprises from 5' to 3': a target strand segment template with homology to the second gRNA and a clamp region complementary to a region of the target strand, and wherein the target strand comprises from 5' to 3': a clamp region complementary to the clamp region of the non-target strand, a single-strand region not complementary to a portion of the non-target strand, and one or more 3' terminal blocking moieties; a DNA 3' phosphatase; a DNA polymerase comprising polymerase activity and 3' — > 5' exonuclease activity; and dNTPs; 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 the single-strand region of the target strand of at least one of the plurality of the blocked primer extension molecules thereby unblocking the at least one blocked primer extension molecule by removing the 3' terminal blocking moiety; allowing the DNA 3' phosphatase to convert the 3' terminal 2', 3'-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group; removing the single-strand nucleotides from the 3' cleaved single-strand region of the target strand; allowing the DNA polymerase to extend the clamp region of the target strand by copying the target strand segment template of the non-target strand thereby providing a target strand segment capable of binding the second gRNA and activating an RNP2 initiating trans- cleavage of the single-strand region of at least one additional blocked primer extension molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

[0012] In some aspects of this second embodiment, the polymerase comprises 3' — > 5' exonuclease activity, and in other aspects, the polymerase lacks 3' — > 5' exonuclease activity.

[0013] A third embodiment provides a method for identifying a target nucleic acid of interest in a sample using a signal boost 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 trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) each comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest, wherein the second nucleic acid- guidcd nuclease is an RNA-clcaving nucleic acid-guidcd nuclease and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; a plurality of blocked primer extension molecules each comprising a target strand and a non-target strand; wherein the target strand comprises from 5' to 3': a single-strand region that serves as a target for activating the second gRNA; a region that will pair with the non-target strand; and a terminal region comprising one or more 3' terminal blocking moieties; and wherein the non-target strand comprises from 5' to 3': a region that will pair with the target strand; and a cleavage flap comprising one or more 3' terminal blocking moieties; a DNA 3' phosphatase; a DNA polymerase; a sequence-specific nickase; and dNTPs; 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 the cleavage flap region of the non-target strand of at least one of the plurality of the blocked primer extension molecules thereby unblocking the at least one blocked primer extension molecule by removing the 3' terminal blocking moiety; allowing the DNA 3' phosphatase to convert the 3' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group; removing the single-strand nucleotides from the 3' cleaved single-strand region of the target strand; allowing the DNA polymerase to create an extension product by extending the paired region of the target and non-target strand by copying the target for activating the second gRNA; allowing the nickase to nick the extension product 5' of the target for activating the second gRNA; allowing the DNA to synthesize another copy of the target for activating the second gRNA, thereby displacing the first copy of the target for activating the second gRNA; wherein the first copy of the target sequence is capable of binding the second gRNA and activating an RNP2 initiating trans-cleavage of the cleavage flap region of at least one additional blocked primer extension molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.In some aspects of the first, second and third embodiments, the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

[0014] In some aspects of the first, second and third embodiments, the non-target strand and target strand arc connected to one another by a single- strand hairpin loop between the 3' end of the non-target strand and the 5' end of the target strand.

[0015] These aspects and other features and advantages of the invention are described below in more detail.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

[0017] FIG. 1 is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

[0018] FIG. 2A is a diagram showing the sequence of steps in an exemplary prior art cascade assay utilizing blocked nucleic acids.

[0019] FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

[0020] FIG. 3 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.

[0021] FIG. 4A is a simplified illustration of an exemplary blocked target strand displacement molecule useful in certain of the reaction mixtures and methods described herein.

[0022] FIG. 4B is a simplified illustration of polymerase-induced target strand displacement for unblocking blocked target strand displacement molecules.

[0023] FIG. 5 is a diagram showing the sequence of steps in an exemplary prior art signal boost cascade assay utilizing circular blocked primer molecules and linear template molecules.

[0024] FIG. 6 is a simplified illustration of an exemplary blocked primer extension molecule suitable for use with an RNP2 comprising a Casl2a nucleic acid-guided nuclease and an example sequence therefor.

[0025] FIG. 7A is a simplified illustration of a primer extension method of the cascade assay utilizing an exemplary blocked primer extension molecule such as that shown in FIG.6.

[0026] FIG. 7B is a simplified illustration of a primer extension method of the cascade using a blocked primer extension molecule such as that shown in FIG. 6, a DNA polymerase, and a sequence- specific nickase.

[0027] It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.DEFINITIONS

[0028] 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 ail 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.

[0029] All of the functionalities described in connection with one embodiment of the compositions and / or methods described herein are intended to be applicable to the additional 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.

[0030] 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.

[0031] 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).

[0032] 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 value in 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.

[0033] 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).

[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 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.

[0035] 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).

[0036] 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 Ka (which in the context of the present disclosure refers to blocked nucleic acid 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 unblocked nucleic acid molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules 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.

[0037] 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.

[0038] As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second ribonuclcoprotcin complex (RNP) (i.c., 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 blocked nucleic acid molecules are blocked target strand displacement molecules or blocked primer extension molecules and the unblocked nucleic acid molecules are unblocked target strand displacement molecules or unblocked primer extension molecules.

[0039] 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.

[0040] 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 of interest, including an unblocked nucleic acid molecule, 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.

[0041] 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'; andthe nucleotide sequence 3'-ATCGAT-5' is 100% complementary to a region of the nucleotide sequence 5'-GCTAGCTAG-3'.

[0042] 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.

[0043] A “control” is a reference standard of a known value or range of values.

[0044] 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.

[0045] “Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and / or intemucleoside 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.

[0046] As used herein, a “partition” is an isolate region (e.g., a feature surrounded by an interstitial region) or an isolate depression (e.g., a well) on a substrate, or a droplet. Partitions are used, in relation to the present disclosure, to separate a plurality of ribonucleoprotein complexes (RNP Is) comprising different guide nucleic acids (gRNA Is) into compartments (e.g., separate wells, features, or droplets). Partitions may be disposed upon a detection substrate.

[0047] The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide orpolypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that arc 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)).

[0048] 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, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. 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 which exhibits target-activated trans-cleavage activity, 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 unblocked nucleic acid, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease. In the context of the present disclosure, RNP2 comprises a DNA-cleaving nucleic acid-guided nuclease which exhibits cis- and trans-cleavage activity.

[0049] As used herein, the terms "protein" and "polypeptide" are used interchangeably. Proteins may or may not be made up entirely of amino acids.

[0050] 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.

[0051] The terms "target DNA 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 in vivo. The “target strand” of a target nucleic acid of interest is the strand of a double-stranded 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.

[0052] 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.

[0053] 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 and target nucleic acid interaction. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single tum-over cis-cleavage event.

[0054] 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, Francisella tularensis 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. Type VI CRISPR / Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR / Cas effector nucleases which specifically target and cleave RNA molecules instead of DNA molecules, and include Casl3a, Casl3b, Casl3c and Casl3d.

[0055] 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 variantthat 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.

[0056] 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

[0057] The present disclosure provides compositions of matter and signal boost 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 massive multiplexing, high fidelity, low background, high signal-to-noise ratios, low cost, minimum workflow, with results at ambient temperatures.

[0058] The cascade assays described herein comprise first and second ribonucleoprotein complexes, blocked nucleic acid molecules (here, blocked target strand displacement molecules and blocked primer extension molecules), and polymerases that optionally exhibit 3' exonuclease activity. The blocked nucleic acid 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 nucleic acid molecules are designed in such a way that they are largely blocked from interacting with 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 a “switch”, where the switch aids in the unblocking of the blocked nucleic acid molecules allowing for use of a more robust blocked nucleic acid molecule configuration to minimize false positive signals. The methods comprise the steps of providing cascade assay components, contacting the 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.

[0059] 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 amplification process 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.

[0060] 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 mechanism comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked nucleic acid molecules (blocked target strand displacement molecules and blocked primer extension molecules), reporter moieties, and a polymerase that exhibits 3' exonuclease activity along with dNTPs. 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.

[0061] The improvement to the signal amplification or signal boost cascade assay described herein is drawn to being able to employ a tightly “locked” blocked target strand displacement molecule or blocked primer extension molecule that can be unblocked via a combination of trans-cleavage activity by a nucleic acid-guided nuclease in the ribonucleoprotein complexes in the reaction mixture and a polymerase that exhibits 3' — >5' exonuclease activity. This combination of enzymatic activity unblocks the blocked nucleic acid molecules by either displacing or exposing a portion of the blocked nucleic acid molecules that can be copied by the polymerase thereby providing a complement to the crRNA portion of the gRNA in RNP2 (i.e., gRNA2), which can then bind to and activate RNP2 as described in detail below.

[0062] 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 and isothermal DNA amplification affect the rapidity of currently available nucleic acid-guided nuclease detection systems. Once the ribonucleoprotein complex is activated after completion of cis-cleavage, trans-cleavage activity of the reporter moieties that are initially quenched is generated. However, the turnover (Kcat) of, e.g., activated Casl2a complex is 17 / sec and 3 / sec for dsDNA and ssDNA targets, respectively. 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.

[0063] 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.

[0064] ‘ ‘Activation” of RNP1 (106) in the context of the cascade assay refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) via binding of a target nucleic acid to the gRNA in RNP1, where trans-cleavage is indiscriminate leadingto non-sequence- specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-clcavagc activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) via blocked nucleic acid 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 nucleic acid molecules — which also affects the kinetics of the cascade assay reaction — and are described in greater detail below.

[0065] 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 arc 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 nucleic acid molecules.Target Nucleic Acids of Interest

[0066] The target nucleic acid(s) 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 limitedto, 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, industrial sites and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

[0067] 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.

[0068] 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 metapneumovirus, 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).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] While the methods described herein do not require the target nucleic acid(s) of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid(s) 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 the biological sample with the composition. Alternatively, RNA target nucleic acids of interest can be detected directly via RNA-specific nucleic acid nucleases such as Casl3a or Casl2g.Nucleic Acid-Guided Nucleases

[0074] 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. Any nucleic acid-guided nuclease having target-activated 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-clcavagc activity is not triggered unless and until sequence-specific binding interactions occur between the target nucleic acid and the gRNA in RNP1 or an unblocked nucleic acid molecule and the gRNA in RNP2. 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 HEPN nuclease domain or HEPN-like nuclease 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.paiis-saclay.fr).

[0075] 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 Casl2a, Casl4a, 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 arc not limited to, Cas RNA-guided DNA endonucleases, such as Cas3, Casl2a (e.g., AsCasl2a, LbCasl2a), Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2h, Casl2i, and Casl2j; 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 exhibiting collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as a Casl2a, Casl3a, or Casl4a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.

[0076] In the present disclosure, the target nucleic acids may be RNA-based or DNA-based nucleic acids; thus, the nucleic acid-guided nuclease in RNP1 may be a DNA-cleaving nucleic acid-guided nuclease or an RNA-cleaving nucleic acid-guided nuclease. However, the RNP2s herein comprise a DNA-cleaving nucleic acid-guided nuclease. If RNP1 comprises a DNA-cleaving nucleic acid-guided nuclease, the blocked target strand displacement molecules or blocked primer extension molecules may comprise all deoxyribonucleotides; however, if one or more of the RNPls comprises an RNA-cleaving nucleic acid-guided nuclease, the blocked target strand displacement molecules or blocked primer extension molecules must comprise at least one ribonucleotide in the region of the blocked target strand displacement molecule or blocked primer extension molecule that is cleaved to unblock the molecule.Guide RNA (gRNA)

[0077] 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 RNP1 is specific to a target nucleic acid of interest and the gRNA of RNP2 is specific to the target strand (i.e., the region complementary to the crRNA region) of an unblocked nucleic acid (described in detail below), here, an unblocked target strand displacement molecule or an unblocked primerextension molecule. Like the nucleic acid-guided nuclease, the gRNA in most embodiments is provided in the cascade assay reaction mixture in a prcasscmblcd 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.

[0078] The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP1 to perform cis-cleavage of a target nucleic acid of interest (i.e., a DNA or RNA), 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 unblocked target strand displacement molecules or unblocked primer extension molecules 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 doublestranded break either inside or outside the protospacer region of the target nucleic acid of interest.

[0079] 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 intemucleoside 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

[0080] 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 more rapid 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 contains about 1 fM to about 500 pM 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.

[0081] In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNPls (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 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 RNPls), where different RNPls 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 sourcesor more than one RNP1 may be present for targeting more than one target nucleic acid of interest from a single organism or condition.

[0082] 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 RNP1 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 twenty heterologous 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.

[0083] 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 rhinovirus; 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.1.617.2, BA.l , BA.2, BA.2.12.1 , BA.4, and BA.5 and subvariants thereof.

[0084] 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, Uncinula necator, Botrytis cincerea, Plasmopara viticola, and Botryotinis fuckleina.Reporter Moieties

[0085] 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 nucleic acid 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. 2A). 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 (particularly useful if one or more target nucleic acids is RNA-based and one or more RNPls comprise RNA-cleaving nucleic acid-guided nucleases), or an oligonucleotide with modified nucleic acids. The reporter moiety also can comprise both single- and doublestranded portions.

[0086] In alternative embodiments, the reporter moiety may be bound to the blocked target strand displacement molecule or blocked primer extension molecule, where trans-cleavage of the blocked target strand displacement molecule or blocked primer extension molecule and conversion to an unblocked target strand displacement molecule or unblocked primer extension 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 the blocked nucleic acid molecule is unblocked, whether quickly or slowly.In yet another embodiment, both stand-alone reporters and blocked nucleic acid molecule comprising reporter moictics may be utilized.

[0087] 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.

[0088] 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.

[0089] 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 canbe 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.

[0090] 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, fhiorescein / 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. Useful quenchers include, but are not limited to, DABCYL, QSY™ (succinimidyl ester) 7 and QSY™ (succinimidyl ester) 33.

[0091] 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.Nucleic Acid Modifications

[0092] For any of the nucleic acid molecules described herein (e.g., blocked target strand displacement molecules, blocked primer extension molecules, gRNAs, and / or reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked target strand displacement molecules, blocked primer extension 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 are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and / or alternative internucleoside linkages.

[0093] For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 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-thiothyminc and 2-thiocytosinc, 5-halouracil and cytosine, 5- propynyl (-C=C-CH3) 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 and8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3- deazaadenine. The nucleic acid molecules described herein (i.e., blocked nucleic acid 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.

[0094] In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2' sugar modifications, including 2'-O-mcthyl (2’-0-Mc), 2'- methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-M0E), 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 nucleic acid 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.

[0095] Finally, modifications to the cascade assay components may comprise intemucleoside modifications such as phosphorothioates, phosphorodithioates, 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, thionoalkylphosphotriesters, 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

[0096] Before getting to the details relating to aiding the unblocking of the blocking nucleic acid molecules via polymerase-induced target strand displacement in the cascade assay, understanding the cascade assay itself is key. FIG. 1, described above, depicts the cascade assay generally. A specific prior art embodiment of the cascade assay utilizing blocked nucleic acids is depicted in FIG. 2A and described in detail below. In this embodiment, 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. 2A begins with providing the cascade assay components RNP1 (201) (only one RNP1 is shown), RNP2 (202) and blocked nucleic acid 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 unblocked nucleic acid molecule or a Cas 13a for an RNA 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 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 gRNA target interaction within the RNP. Note, too, that a single RNP1 is shown here. In practice, several to many cascade assays will be performed in parallel with different RNP Is in separate partitions.

[0097] 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, RNP1 initiates 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.

[0098] 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 RNPls (205) and RNP2s (208) exhibit 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. 2A 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.

[0099] FIG. 2B is a diagram showing an exemplary prior art blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules 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 singlestranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (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 (228) 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 (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.The Signal Boosting Cascade Assay Employing Polymerase-Induced Target Strand Displacement for Unblocking Blocked Nucleic Acid Molecules

[0100] 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 the blocked nucleic acid molecules. Non-specific unblocking of the blocked nucleic acid molecules leads to non-specific activation of RNP2 in the absence of a target nucleic acid of interest.

[0101] FIG. 3 at top is a simplified diagram of the “correct” pathway for unblocking the blocked nucleic acid molecule, where the unblocking is due to trans-cleavage of singlestrand regions of a blocked nucleic acid molecule leading to activation of RNP2 (see FIG. 2B). FIG. 3 at bottom illustrates a “failure” pathway, where the unblocking of the blocked nucleic acid molecule is due not to trans-cleavage 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.

[0102] 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., the 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 RNPI trans-cleavage activity has not been activated, yet RNP2 becomes activated leading to a false positive signal. A solution, as described herein, involves designing the blocked nucleic acid molecule 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 RNPI — thereby circumventing the failure pathway — but then to “liberate” the target strand segment of the blocked nucleic acid molecule once the single-strand loop of the blocked nucleic acid molecule has been trans-cleaved.

[0103] In the polymerase-induced strand displacement method embodiment described herein, once trans-clcavagc activity nicks the single-strand loop of the blocked target strand displacement molecule adjacent to and 5' of the target strand segment of the blocked target strand displacement molecule, the 3' — > 5' exonuclease or “proofreading” activity of the polymerase, if present, “chews back” the single-strand 3' portion of the nicked loop region. After the single-stranded portion of the nicked region is eliminated, the polymerase extends the double-strand region 5' 3', displacing the target strand segment thereby making the target strand segment available for binding to gRNA2 with subsequent activation of RNP2. As described in reference to FIG. 4B below, the target strand segments of the blocked target strand displacement molecules resist displacement due to the high melting temperature of the “clamp” or double-stranded regions of the blocked target strand displacement molecules until nicking of the single-strand loop of the target strand occurs via trans-cleavage activity followed by the exonuclease and polymerase activity of the polymerase. In addition to length and / or GC content of the clamp to increase Tm, LNAs may be used as well. In alternative embodiments, polymerases lacking 3' — 5' exonuclease or “proofreading” activity may be employed, and a separate exonuclease may be used. In yet another alternative, an exonuclease may be eliminated entirely and the single- strand 3' portion of the nicked loop region may be eliminated by trans-cleavage activity of the activated RNPs.

[0104] FIG. 4A is an illustration of an exemplary blocked target strand displacement molecule (400) employed in the polymerase-induced target strand displacement embodiments described herein. The blocked target strand displacement molecule (400) comprises a non-target strand (402) and a non-target strand complement (406) coupled by a hairpin loop (404). The non-target strand complement (406) comprises from 5' to 3' 5' of the hairpin loop: a region (405) between the hairpin loop (404) and singlestrand internal loop (410); and a target strand segment (408) (bolded line). The target strand segment (408) is the portion of the blocked target strand displacement molecule (400) that is complementary to the crRNA region of the gRNA2 which binds to gRNA2 and subsequently activates RNP2.

[0105] The blocked target strand displacement molecule (400) typically ranges from 60 bp to 300 bp in length, with the non-target strand (402) ranging from 30 bp to 150bp in length; the hairpin loop (404), if present, typically ranging from 4 bp to 20 bp in length; and the non-target strand complement (406) ranging from 10 bp to 60 bp in length, with the target strand segment (408) ranging from 10 bp to 60 bp in length and the internal single-strand loop (410) ranging from 4 bp to 20 bp in length. Hairpin loop (404) is optional, as instead of a single molecule the blocked target strand displacement molecule may comprise two separate hybridized oligonucleotides. The region (405) of the non-target strand complement (406) 5' of the internal single-strand loop (410) may comprise a “clamp” or high Tmdouble-strand region to aid in keeping a “lock” on the blocked target strand displacement molecule. Alternatively or in addition, a portion of the target strand segment (408) (bolded line) toward the 3' end may comprise a “clamp” as well. Additionally, to prevent trans-cleavage of the hairpin loop, the nucleotides of the hairpin loop may comprise locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2'-O- methyl (2'-0-Me) modified nucleosides, 2'-lluoro (2'-F) modified nucleoside, and / or a phosphorothioate (PS) bonds that resist cleavage. Additionally, the loop may be kept small, such as, e.g., between 1 and 10 nucleotides, which prevents cleavage due to the tight curvature.

[0106] The composition of the blocked target strand displacement molecule (400) employed in the polymerase-induced target strand displacement methods will depend upon the nucleic acid-guided nuclease employed in RNP1 and RNP2. If, for example, one or more of the RNPls comprise an RNA-cleaving nucleic acid-guided nuclease (such as, e.g., Casl3a), at least two adjacent nucleotides in the single-strand internal loop (410) will be ribonucleotides to allow for trans-cleavage by RNP1. Keep in mind that the target nucleic acids of interest may include both RNA and DNA target nucleic acids, such as, e.g., a mix of target nucleic acids of interest from RNA viruses and bacterial DNA. If, for example, all RNPls comprise an RNA-cleaving nucleic acid-guided nuclease that targets RNA, many nucleotides in the single-strand internal loop (410) may be ribonucleotides to allow for trans-cleavage by RNP1. As contemplated herein, the nucleic acid-guided nuclease employed in RNP2 is a DNA-cleaving nucleic acid-guided nuclease that targets DNA — such as, e.g., Casl2a — thus, nucleotides in the target strand segment (408) will comprise deoxyribonucleotides, as the activator for gRNA2 in RNP2 and some of the nucleotides in the single-strand internal loop (410) will be deoxyribonucleotides to allow for transcleavage by RNP2. The other portions of the blocked target strand displacement molecule (400) — namely the non-targct strand (402), hairpin loop (404) and a portion of the nontarget strand complement (406) — may comprise deoxyribonucleotides or ribonucleotides, preferably modified or “protected” to prevent erroneous trans-cleavage.

[0107] FIG. 4B is an illustration showing the steps involved in polymerase-induced target strand displacement. In step 1, the single- strand loop 5' of the target strand segment (bolded line) on the non-target strand complement in the blocked target strand displacement molecule is nicked by the trans-cleavage of activated RNP1, resulting in a nicked blocked target strand displacement molecule. As noted above, the blocked target strand displacement molecules often are primarily DNA molecules; however, if the nucleic acid-guided nuclease in RNP1 is an RNA-cleaving nucleic acid-guided nuclease — or if one RNP1 in a mix of RNPls comprises an RNA-cleaving nucleic acid-guided nuclease — at least two adjacent nucleotides in the single-strand internal loop will be ribonucleotides to allow for trans-cleavage by RNP1.

[0108] In step 2, the 3' — > 5' exonuclease or “proofreading” activity of the polymerase present in the reaction mixture “chews back” or removes the unhybridized single-strand DNA from the cleaved loop that is left at the 3' end of the double-strand portion of the non-target strand complement proximal to the hairpin loop (indicated by the dashed line along loop fragment). Once the single- strand DNA is removed, the polymerase begins to synthesize a copy of the non-target strand (indicated by the dashed line from double-strand region), displacing the target strand segment as the synthesis takes place. The action of the polymerase results in a polymerization product — which here is a hairpin construct comprising a non-target strand and a non-target strand complement but without an internal loop structure — and the displaced unblocked target strand displacement molecule. At step 3, the displaced unblocked target strand displacement molecule is free and binds to and activates RNP2, thus triggering the trans-cleavage activity of RNP2, which nicks the single-strand loop portion of more blocked target strand displacement molecules in step 4.

[0109] The polymerases used in the present reaction mixtures and methods allow for use of strongly “locked” blocked target strand displacement molecules to prevent nonspecific unblocking but serve as a “key” to release the lock upon nicking of the single-strand loop 5' of the target strand segment as described above. Preferably, the polymerase used comprises both 5 ' — > 3' DNA polymerase activity (or RNA 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. What is key is that the target strand segment (408 in FIG. 4A) — the “target” for RNP2” — be displaced from the blocked target strand displacement molecules and made available for complexing with RNP2.The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

[0110] Also as known in the art, a cascade assay may employ blocked primer molecules as described 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. Blocked primer molecules include a sequence complementary to a primer binding region (PBR) on a template molecule. A PBR serves as a nucleotide sequence for primer hybridization followed by primer extension by a polymerase. The unblocked primer molecule can bind to a template molecule at the PBR and copy the template molecule via polymerization by a polymerase.

[0111] An exemplary embodiment of the cascade assay that utilizes blocked primer molecules is depicted in FIG. 5. As with the embodiment of the cascade assay shown in FIG. 2A, keep in mind that in the multiplexed cascade assays described herein, the single reaction method depicted in FIG. 5 is performed in many reactions in separate partitions in parallel, wherein each partition comprises different RNPls. In the embodiment using blocked nucleic acid molecules described above in relation to FIG. 2A, activation of RNP1 by binding of X number of nucleotides of the target nucleic acid molecule initiates trans-cleavage of the blocked nucleic acid molecules which were used to activate RNP2 - that is, the unblocked nucleic acid molecules arc a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primer molecules, activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing activating molecules that are the target sequence for the gRNA in RNP2.

[0112] FIG. 5 is a diagram showing the sequence of steps in an exemplary cascade assay (500) involving circular’ blocked primer molecules and linear' template molecules. At left of FIG. 5 is a cascade assay reaction mix comprising 1) RNPls (501) (only one RNP1 is shown); 2) RNP2s (502); 3) linear template molecules (530) (which is the nontarget strand); 4) a circular blocked primer molecule (534) (i.e., a high Ka molecule); and 5) a polymerase (538), such as a 029 polymerase. The linear template molecule (530) (non-target strand) comprises a PAM sequence (531), a primer binding region (PBR) (532) and, optionally, a nucleoside modification (533) to protect the linear template molecule (530) from 3' — 5' exonuclease activity. Blocked primer molecule (534) comprises a cleavable region (535) and a complement (536) to the PBR (532) on the linear template molecule (530).

[0113] Upon addition of a sample comprising a target nucleic acid of interest (504) (capable of complexing with the gRNA in RNP1 (501)), the target nucleic acid of interest (504) is bound with and activates RNP1 (505) but does not interact with or activate RNP2 (502). Once activated, RNP1 cuts the target nucleic acid of interest (504) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (534). The circular blocked primer molecule (534) (i.e., a high Ka molecule, where high Ka relates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (544) (a low Ka molecule, where low Ka relates to binding to RNP2), which has the region (536) complementary to the PBR (532) on the linear template molecule (530) and can bind to the linear template molecule (530) (top right of FIG. 5).

[0114] Once the unblocked linear primer molecule (544) and the linear template molecule (530) are hybridized (i.e., hybridized at the PBR (532) of the linear template molecule (530) and the PBR complement (536) on the unblocked linear primer molecule(544)), 3' — > 5' exonuclease activity of the polymerase (538) removes the unhybridized single- stranded DNA at the end of the unblocked primer molecule (544) and the polymerase (538) can copy the linear template molecule (530) to produce a synthesized activating molecule (546) which is a complement of the non-target strand, and is therefore the target strand (middle center of FIG. 5). The synthesized activating molecule (546) is capable of activating RNP2 (502 — > 508). As described above, because the nucleic acid- guided nuclease in the RNP2 (508) complex exhibits (that is, possesses) both cis- and transcleavage activity, more blocked primer molecules (534) become unblocked primer molecules (544) triggering activation of more RNP2s (508) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.

[0115] FIG. 5 at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (509) comprise a quencher (510) and a fluorophore (511). As described above in relation to FIGs. 1 and 2 A, the reporter moieties are also subject to trans-cleavage by activated RNP1 (505) and RNP2 (508). The intact reporter moieties (509) become activated reporter moieties (512) when the quencher (510) is separated from the fluorophore (511), and the fluorophore emits a fluorescent signal (513). Signal strength increases rapidly as more blocked primer molecules (534) become unblocked primer molecules (544) generating synthesized activating molecules (546) and triggering activation of more RNP2 (508) complexes and more trans-cleavage activity of the reporter moieties (509). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed. Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.The Signal Boosting Cascade Assay Employing Blocked Primer Extension Molecules

[0116] FIG. 6 is a simplified illustration of an exemplary blocked primer extension molecule (600) useful in cascade assays where the RNP2 comprises a DNA-cleaving nucleic acid-guided nuclease such as Casl2a. Blocked primer extension molecule (600) comprises at the 5' end a single-strand region of the non-target strand that will serve as the target strand segment template (603); an optional single-strand region of the non-target strand (607); a paired region of the non-target strand (606); an optional hairpin loop (601); a paired region of the target strand (605); an unpaired region of the target strand (604) that serves as a cleavage substrate for trans-cleavage by nucleic acid-guided nucleases; and a region with one or more blocking moieties (602).

[0117] Note that there are four single- strand regions of this exemplary blocked primer extension molecule (600), including the region of the non-target strand that will serve as the target strand segment template (603), which is adjacent to the optional singlestrand region of the non-target strand (607), the single-strand hairpin loop (601), and the unpaired region of the target strand (604); however, it is preferred that only the unpaired region of the target strand (604) be susceptible to trans-cleavage activity. Thus, to prevent trans-cleavage of the region of the non-target strand that will serve as the target strand segment template (603), adjacent region (607), and the hairpin loop (601), the nucleotides of these single- strand regions may comprise locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2'-O-methyl (2'-0-Me) modified nucleosides, 2'-fluoro (2'-F) modified nucleoside, phosphorothioate (PS) bonds, and / or other molecular modifications that serve to resist cleavage. Also, hairpin loop (601) is optional, as instead of a single molecule the blocked nucleic acid molecule may comprise two separate hybridized oligonucleotides.

[0118] Blocking moiety (602) comprises a moiety that prevents 3' — > 5' exonuclease activity of the polymerase before trans-cleavage of the blocking moiety (602) in the unpaired region of the target strand (604) and prevents the polymerase from extending the 3' end of the blocked primer extension molecule (600). Blocking moieties include inverted nucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2'-O-methyl (2'-0-Me) modified nucleosides, 2'-fluoro (2'-F) modified nucleoside, and / or a phosphorothioate (PS) bonds that resist cleavage; thus, blocking moieties (602) includeessentially any moiety that removes the 3' -OH group of the 3' terminal deoxynucleotide of the blocked primer extension molecule (600).

[0119] The blocked primer extension molecule (600) typically ranges from 50 bp to 300 bp in length; with the target strand segment template (603) ranging from 10 bp to 100 bp in length; the single-strand region of the non-target strand (607) ranging from 0 bp to 100 bp in length; the paired region of the non-target strand (606) and paired region of the target strand (505) ranging from 10 bp to 100 bp in length; the hairpin loop (601), if present, ranging from 2 bp to 40 bp in length; and the unpaired region of the target strand (604) ranging from 2 bp to 100 bp in length. FIG. 6 at bottom also provides an exemplary sequence for a blocked primer extension molecule where the RNP2 comprises a DNA- cleaving nucleic acid-guided nuclease, where asterisks denote phosphorothioate modified nucleotides and “3InvdT” denotes an inverted dT, which creates a 3'-3' linkage between the last two nucleotides at the 3 ' end of the blocked primer extension molecule, inhibiting exonuclease activity and preventing polymerization.

[0120] FIG. 7A is a simplified illustration of the primer extension method utilizing, e.g., the exemplary blocked primer extension molecule shown in FIG. 6 where the RNP2 comprises a DNA-cleaving nucleic acid-guided nuclease, which is a unique variation of the blocked primer method described above in relation to FIG. 5. In FIG. 7A, in step 1 trans-cleavage activity of RNP1 is triggered upon the binding of a target nucleic acid of interest to RNP1, which can then cleave the single-strand “flap” region of the blocked primer extension molecule 5' of the blocking moiety (shown here as a terminal circle). In this embodiment, a 3' phosphatase is 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 then be “chewed back” via the optional 3' — > 5' exonuclease or “proofreading” activity of the DNA polymerase present in the reaction mixture to reveal the 3' end of the double-strand region comprising the complementary regions of the non- target strand and the target strand. Removal of the flap can also occur via repeated trans- cleavage of all the flap nucleotides without 3' — > 5' exonuclease. Once the single-strand DNA is removed, the DNA polymerase begins to synthesize a copy of the target strand template region of the non-target strand, where this copy is the target for RNP2. At step 3, the polymerization product comprising the target for RNP2 is available for binding to andactivating the RNP2, which triggers the trans-cleavage activity of RNP2 removing the blocking moiety of more blocked primer extension molecules in step 4, cycling back to step 2 in a cascade.

[0121] The DNA polymerases used in the present reaction mixtures and methods allow for use of strongly “locked” blocked primer extension 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. 3' phosphatases of use in this embodiment include T4 polynucleotide kinase-phosphatase (T4 PNKp), the DNA 3’ phosphastase of Sacchromyces cerevisiae, human polynucleotide kinase / 3 'phosphatase.

[0122] FIG. 7B is a simplified illustration of a primer extension method of the cascade using a blocked primer extension molecule, a DNA polymerase, and a sequencespecific nicking endonuclease. In step 1 of FIG. 7B, trans-cleavage activity of RNP1 is triggered upon the binding of a target nucleic acid of interest to RNP1, which can then cleave the ribonucleotides in a single-strand “cleavage flap” region of a blocked primer extension molecule 5' of a blocking moiety (shown here as a terminal circle). As described above, if one or more of the RNP Is comprise DNA-cleaving nucleic acid-guided nucleases, the single-strand “cleavage flap” region may comprise only deoxy ribonucleotides;however, if one or more of the RNPl s comprise an RNA-cleaving nucleic acid-guided nuclease, the single-strand “cleavage flap” region may comprise both ribonucleotides and deoxyribonucleotides, since RNA-cleaving nucleic acid-guided nucleases preferentially trans-cleave ribonucleotides.

[0123] Once the blocked primer extension molecule is unblocked by removal of the blocking moiety, in step 2 three events occur. First, a 3' phosphatase is employed to convert the 3' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group. Second, the 3' 5' exonuclease “proofreading” activity of theDNA polymerase present in the reaction mixture “chews back” or removes the unhybridized single-strand DNA cleavage flap that is left at the 3' end of the double-strand region and serves as the primer region for the DNA polymerase. Finally, the 5' — 3' polymerase activity of the DNA polymerase then synthesizes the target strand segment for gRNA2 (indicated by bolded line). The sequence-specific nickase can then nick the primer extension molecule 5' of the synthesized target strand segment, and the DNA polymerase can synthesize another copy of the target strand segment, displacing the first synthesized target strand segment. In step 3, both the displaced target strand segment and the repaired extension product may serve as targets for gRNA2 and thus activators for RNP2 trans- cleavage. Further, the repaired extension products can continue to serve as templates for synthesis of additional target strand segments until they are “consumed” by RNP2 ciscleav age, essentially super-ch arging the cascade further increasing the rate of exponential signal amplification. In step 4, activating RNP2 triggers the trans-cleavage activity of RNP2, removing the blocking moiety of more blocked primer extension molecules 4, cycling back to step 2 in a cascade.Applications of the Cascade Assay

[0124] The present disclosure describes cascade assays for detecting one or more target nucleic acids of interest in a sample. The cascade assays allow for massive multiplexing and minimum workflow yet provide accurate results at low cost. Moreover, the various 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.

[0125] Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but arc 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.

[0126] 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).

[0127] 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 (see, e.g., Example I below). In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidineisothiocyanate 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.

[0128] 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

[0129] 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 ribonuclcoprotcin complexes (RNPls), second ribonuclcoprotcin complexes (RNP2s), blocked nucleic acid molecules, a polymerase having 3' exonuclease activity, dNTPs, 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 nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans- cleavage of the blocked nucleic acid molecule may result in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. However, inclusion of the polymerase having 3' exonuclease activity improves performance of the assay by allowing for use of blocked nucleic acid molecules with tight “locks” to avoid false positives yet aids in the unblocking of the blocked nucleic acid molecules. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and / or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.

[0130] Any of the kits described herein may further include a sample collection device, c.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

[0131] 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

[0132] 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 cellsand the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that arc mixed (e.g., vortcxcd) 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).

[0133] 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 and chemicals 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.

[0134] 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 bindingcolumns for solid phase extraction as described above, are required to further purify the nucleic acids.

[0135] 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.

[0136] Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in Dl / distilled 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 cellular membrane. 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 ail.

[0137] 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 desirednucleic 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.

[0138] 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.

[0139] 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

[0140] 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.

[0141] Enzymes were diluted on ice in reaction buffer, as were guide RNAs. RNP1 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: Blocked Nucleic Acid Molecule Formation

[0142] Blocked targets (i.c., blocked nucleic acid molecules) containing ribonucleotides were stored dehydrated and required resuspension in buffer at pH 7.5 at 100 pM concentration. Blocked targets comprising only DNA were frozen and thawed for use, then suspended in lx STE buffer (Sigma, St. Louis MO, US). 25 pL of each target was dispensed into a PCR strip tube and placed into a BioRad (Hercules CA, USA) thermocycler, protocol Hyb0325). After the protocol ended (~ 40 minutes), the hybridized blocked targets were stored overnight at room temperature. After ~ 16 hours at room temperature, the blocked targets were stored at -20°C. For RNA blocked targets, after hybridization, the hybridized targets were stored at room temperature for 1 hour, then stored at -80°C.Example IV: Reporter Moiety Formation

[0143] The reporter moieties used in the reactions herein were single- stranded DNA oligonucleotides 5-10 bases in length (c.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).Example V: Cascade Assay

[0144] 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.

[0145] 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 andsystems 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 signal boost 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 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 wherein the second nucleic acid-guided nuclease is a DNA-cleaving nucleic acid-guided nuclease and where the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; a plurality of blocked target strand displacement molecules each comprising a nontarget strand and a non-target strand complement, wherein the non-target strand complement comprises from 5' to 3': a clamp region complementary to a portion of the non-target strand, a single- strand loop not complementary to a portion of the non-target strand, and a target strand segment complementary to a portion of the non-target strand and complementary to the second gRNA; a polymerase comprising polymerase activity and optional 3' — > 5' exonuclease activity; and dNTPs; 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 the single-strand loop of at least one of the plurality of the blocked target strand displacement molecules resulting in 3' and 5' cleaved loop regions; allowing the polymerase to remove single- strand nucleotides from the 3' cleaved loop region and to extend the clamp region thereby displacing the target strand segment,wherein the displaced target strand segment binds to an RNP2 initiating trans-cleavage of the single-strand loop of at least one additional blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

2. The method of claim 1, wherein the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

3. The method of claim 1, wherein the non-target strand and target strand are connected to one another by a single-strand hairpin loop between the 3' end of the non-target strand and the 5' end of the target strand.

4. The method of claim 3, wherein the blocked nucleic acid molecule typically ranges from 60 bp to 300 bp in length, the non-target strand ranges from 30 bp to 150 bp in length; and the non-target strand complement ranges from 10 bp to 60 bp in length, with the target strand segment ranging from 10 bp to 60 bp in length and the single- strand loop ranging from 4 bp to 20 bp in length.

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

6. The method of claim 1, wherein the polymerase lacks 3' — > 5' exonuclease activity.

7. A method for identifying a target nucleic acid of interest in a sample using a signal boost 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 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, wherein the second nucleic acid-guided nuclease is a DNA-cleaving nucleic acid-guided nuclease and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity;a plurality of blocked primer extension molecules each comprising a non-target strand and a target strand; wherein the non-target strand comprises from 5' to 3': a target strand segment template with homology to the second gRNA and a clamp region complementary to a region of the target strand, and wherein the target strand comprises from 5' to 3': a clamp region complementary to the clamp region of the non-target strand, a single- strand region not complementary to a portion of the non-target strand, and one or more 3' terminal blocking moieties; a DNA 3' phosphatase; a DNA polymerase comprising polymerase activity and 3' — > 5' exonuclease activity; and dNTPs; 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 transcleavage of the single- strand region of the target strand of at least one of the plurality of the blocked primer extension molecules thereby unblocking the at least one blocked primer extension molecule by removing the 3' terminal blocking moiety; allowing the DNA 3' phosphatase to convert the 3 ' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group; removing the singlestrand nucleotides from the 3' cleaved single-strand region of the target strand; allowing the DNA polymerase to extend the clamp region of the target strand by copying the target strand segment template of the non-target strand thereby providing a target strand segment capable of binding the second gRNA and activating an RNP2 initiating trans-cleavage of the single-strand region of at least one additional blocked primer extension molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

8. The method of claim 7, wherein the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

9. The method of claim 7, wherein the non-target strand and target strand are connected to one another by a single-strand hairpin loop between the 3' end of the non-target strand and the 5' end of the target strand.

10. A method for identifying a target nucleic acid of interest in a sample using a signal boost 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 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, wherein the second nucleic acid-guided nuclease is an RNA-cleaving nucleic acid-guided nuclease and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; a plurality of blocked primer extension molecules each comprising a target strand and a non-target strand; wherein the target strand comprises from 5' to 3': a singlestrand region that serves as a target for activating the second gRNA; a region that will pair with the non-target strand; and a terminal region comprising one or more 3' terminal blocking moieties; and wherein the non-target strand comprises from 5' to 3': a region that will pair with the target strand; and a cleavage flap comprising one or more 3' terminal blocking moieties; a DNA 3' phosphatase; a DNA polymerase; a sequence- specific nickase; and dNTPs; 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 the cleavage flap region of the non-target strand of at least one of theplurality of the blocked primer extension molecules thereby unblocking the at least one blocked primer extension molecule by removing the 3' terminal blocking moiety; allowing the DNA 3' phosphatase to convert the 3' terminal 2', 3 '-cyclic phosphate resulting from trans-cleavage of the blocking moiety to a 3' OH group; removing the single-strand nucleotides from the 3' cleaved single-strand region of the target strand; allowing the DNA polymerase to create an extension product by extending the paired region of the target and non-target strand by copying the target for activating the second gRNA; allowing the nickase to nick the extension product 5' of the target for activating the second gRNA; allowing the DNA to synthesize another copy of the target for activating the second gRNA, thereby displacing the first copy of the target for activating the second gRNA; wherein the first copy of the target sequence is capable of binding the second gRNA and activating an RNP2 initiating trans-cleavage of the cleavage flap region of at least one additional blocked primer extension molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

11. The method of claim 10, wherein the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

12. The method of claim 10, wherein the non-target strand and target strand are connected to one another by a single-strand hairpin loop between the 3' end of the non-target strand and the 5' end of the target strand.

13. The method of claim 10, wherein the polymerase comprises 3 ' — 5 ' exonuclease activity.

14. The method of claim 10, wherein the polymerase lacks 3' — >• 5' exonuclease activity.

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