Blocked PAM-distal target regions

Abstract

The present disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest with enhanced fidelity. The cascade assays" or "signal boost assays comprise two different ribonucleoprotein complexes (RNPs) — RNP1 and RNP2 — and RNP2 activator molecules comprising a P AM-proximal region, which may be blocked in some embodiments and a blocked P AM-distal region. The blocked P AM-distal region molecules keep the second ribonucleoprotein complex or "RNP2" "locked" unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex.

Description

TITLE: BLOCKED PAM-DISTAL TARGET REGIONSRELATED CASES

[0001] This International PCT application claims priority to U.S. Ser. No. 63 / 603,614, filed 28 November 2023 and U.S. Ser. No. 63 / 566,188, filed 15 March 2024, both of which are incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to compositions of matter and assay systems used to detect one or more target nucleic acids of interest in a sample with high fidelity, low background and high signal-to-noise ratios. The assay systems provide signal amplification upon detection of target nucleic acids without amplification of the target nucleic acids.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 such as single nucleotide polymorphisms (SNPs) 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.

[0005] Improved assays that allow very rapid and accurate detection of nucleic acids — particularly at ambient temperature — are therefore needed for timely diagnosis andATTORNEY DOCKET NUMBER: VB029PCT treatment of disease, to identify toxins in consumables and the environment, as well as other applications.SUMMARY OF THE INVENTION

[0006] 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.

[0007] The present disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest with enhanced fidelity. The “nucleic acid-guided nuclease signal boost assays”, “cascade assays” or “signal boost assays” described herein comprise two different ribonucleoprotein complexes (RNPs) — RNP1 and RNP2 — and divided or split RNP2 activator molecules comprising a PAM-proximal region (i.e., protospacer adjacent motif-proximal region), which may be blocked in some embodiments and a blocked PAM-distal region (i.e., protospacer adjacent motif-distal region). The blocked PAM-distal region molecules keep the second ribonucleoprotein complex or “RNP2” “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex or “RNP1.” In the context of the signal boost assays, “locked” means that blocked PAM-distal region molecules are designed in such a way that they are largely prevented or blocked from interacting with the RNP2s until the blocked PAM-distal region molecules are unblocked; therefore, the RNP2s remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates an RNP1 which triggers unblocking of the blocked PAM-distal region molecules.

[0008] There are two general embodiments of RNP2s employed with the blocked PAM- distal region molecules. In one embodiment, the RNP2 comprises 1) a nucleic acid-guided nuclease, such as a Casl2a or Cas 13a nuclease; 2) a gRNA; and 3) a portion of the nontarget strand, a portion of the target strand 3' of the PAM, the PAM, and a PAM-proximal region of the RNP2 activator. That is, the RNP2 is missing only the PAM-distal region ofATTORNEY DOCKET NUMBER: VB029PCT the activator, which is necessary for trans -cleavage activity of the nucleic acid-guided nuclease to be initiated (see, e.g., FIGs. 3A and 3B and the descriptions thereof). Note, however, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

[0009] In an alternative embodiment, the RNP2 comprises 1) a nucleic acid-guided nuclease, such as a Casl2a or Cas 13a nuclease; 2) a gRNA; and 3) a portion of the nontarget strand of the activator, a portion of the target strand 3' of the PAM site, and the PAM site; however, this alternative embodiment does not include the PAM-proximal region; instead, both the PAM-proximal region of the activator and the PAM-distal region of the activator must separately complex with the RNP2 for nuclease activity of the nucleic acid- guided nuclease to be initiated (see, e.g., FIGs. 3C and 3D). Note, however, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

[0010] Physically dividing the RNP2 activator molecule into PAM-proximal regions and PAM-distal regions and blocking the PAM-distal regions reduces the likelihood that nonspecific triggering of the RNP2s will occur. Non-specific unwinding results from enzyme- mediated unwinding of blocked nucleic acid molecules triggered by the presence of the PAM-proximal region; however, since the PAM-proximal region is not present in the blocked nucleic acid molecule (FIGs. 3A and 3B) or is present separate from the PAM- distal region (FIGs. 3C and 3D), enzyme-mediated unwinding does not trigger transcleavage activity because trans-cleavage activity of the nucleic acid-guided nuclease in RNP2 cannot be initiated unless both the PAM-proximal and PAM-distal regions are bound to gRNA2 of RNP2. Thus, by physically dividing the PAM-proximal and PAM-distal regions on the RNP2 activator, the enzyme-mediated unwinding and trans-cleavage activities of the nucleic acid-guided nuclease in RNP2 are decoupled.

[0011] There is thus presented in one embodiment a blocked PAM-distal region construct comprising: a first portion comprising a PAM-distal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising aATTORNEY DOCKET NUMBER: VB029PCT first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion.

[0012] In some aspects, the PAM-distal region of the first portion is adjacent to a second loop region, a second flanking region is adjacent to the second loop region, and the second portion comprises a fourth region not hybridized to the second loop region of the first portion, and a fifth region hybridized to the second flanking region of the first portion.

[0013] In some aspects, the PAM-distal region is at least 6 nucleotides in length.

[0014] In yet another embodiment there is provided a blocked combination PAM-proximal region / P AM-distal region construct comprising: a first portion comprising a PAM-distal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a PAM-proximal region of the RNP activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the PAM-proximal region of the first portion.

[0015] In some aspects of this embodiment, the lengths of the PAM-proximal and PAM- distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

[0016] In yet another embodiment there is 1) a blocked PAM-distal region construct comprising: a first portion comprising a PAM-distal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; and 2) a separate blocked PAM-proximate region construct comprising: a third portion comprising a PAM-proximate region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-proximate region is adjacent to a second loop region and the second loop region is adjacent to a second flanking region; and a fourth portion comprising a fourth region hybridized to the PAM-proximateATTORNEY DOCKET NUMBER: VB029PCT region of the third portion, a fifth region not hybridized to the second loop region of the third portion, and a sixth region hybridized to the second flanking region of the third portion.

[0017] In some aspects, the PAM-distal region and / or the PAM-proximal region lies between loops where the loops are further flanked by a flanking region.

[0018] In some aspects, the PAM-distal region is at least 6 nucleotides in length and in some aspects, the PAM-proximal region is at least 6 nucleotides in length, and in some aspects, the lengths of the PAM-distal and PAM-proximal regions of the RNP2 activator together total at least 20 nucleotides.

[0019] Also provided is an embodiment of a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, the PAM, and a PAM-proximal region; and a plurality of blocked PAM-distal region constructs, wherein each of the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM- distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM- distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion.

[0020] An alternative embodiment of a reaction mixture comprises: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; aATTORNEY DOCKET NUMBER: VB029PCT second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, and the PAM; and a plurality of blocked combination PAM-proximal region / P AM-distal region constructs each comprising: a first portion comprising a PAM- distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a loop region and the first loop region is adjacent to a PAM-proximal region of the RNP2 activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the loop region of the first portion, and a third region hybridized to the PAM-proximal region of the first portion.

[0021] Yet another alternative embodiment of a reaction mixture comprises: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, and the PAM; a plurality of blocked PAM-distal region constructs each comprising: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a loop region and the first loop region is adjacent to a first flanking region of the RNP2 activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; and a plurality of blocked PAM-proximal region constructs comprising: a third portion comprising a PAM-proximate region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-proximate region is adjacent to a second loop region and the second loop region is adjacent to a second flanking region; and a fourth portion comprising a fourth region hybridized to the PAM-proximate region of the third portion, a fifth region not hybridized to the second loop region of the third portion, and a sixth region hybridizedATTORNEY DOCKET NUMBER: VB029PCT to the second flanking region of the third portion.

[0022] In some aspects of these embodiments, the lengths of the PAM-proximal and PAM- distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

[0023] Also provided is a method for detecting a nucleic acid target of interest in a sample comprising the steps of: providing a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid- guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid- guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, the PAM, and a PAM-proximal region; and a plurality of blocked PAM-distal region constructs, wherein the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; and contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to the RNP1, wherein: upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked PAM-distal region constructs, thereby producing at least one unblocked PAM-distal region molecule that can complex with the PAM-proximal region of the RNP2 activator; and upon binding of the at least one unblocked blocked PAM-distal region molecule to the PAM- proximal region of the RNP2 activator, the RNP2 becomes active trans-cleaving at least one more of the blocked PAM-distal region constructs; allowing the cascade to continue; and detecting the activated RNP2s, thereby detecting the target nucleic acid of interest inATTORNEY DOCKET NUMBER: VB029PCT the sample.

[0024] An alternative embodiment of the method provides a method for detecting a nucleic acid target of interest in a sample comprising the steps of: providing a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, and the PAM; and a plurality of blocked combination P AM- proximal region / P AM-distal region construct comprising: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a P AM-proximal region of the RNP2 activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the P AM-proximal region of the first portion; and contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to the RNP1, wherein: upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans -cleaving at least one of the blocked combination PAM-proximal region / P AM-distal region constructs, thereby producing at least one unblocked PAM-proximal region molecule and at least one unblocked PAM-distal region molecule that can complex with the region 3' to the PAM- proximal region of the RNP2 activator in the RNP2; and upon binding of the at least one unblocked PAM-proximal region molecule and at least one unblocked PAM-distal region molecule to the PAM-proximal region 3' to the PAM-proximal region of the RNP2 activator, the RNP2 becomes active trans-cleaving at least one more of the blocked combination PAM-proximal region / P AM-distal region constructs; allowing the cascade to continue; and detecting the RNP2s, thereby detecting the target nucleic acid of interest in the sample.ATTORNEY DOCKET NUMBER: VB029PCT

[0025] Note, however, that in any of these embodiments the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

[0026] These aspects and other features and advantages of the invention are described below in more detail.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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:

[0028] FIG. 1 is a simplified diagram of the cascade assay described in detail in USPNs 11,693,520; 11,702,686; 11,821,025; 11,970,730; and 11,987,8394.

[0029] FIG. 2 is a simplified diagram of the “correct” pathway for unblocking the blocked nucleic acid molecule, where the unblocking is due to trans-cleavage of a blocked nucleic acid molecule, leading to activation of RNP2 and the “failure” pathway for unblocking the blocked nucleic acid molecule, where the unblocking is due not to trans- cleavage of the blocked nucleic acid molecule, but instead is due to erroneous enzyme- mediated unwinding of the blocked nucleic acid molecule.

[0030] FIGs. 3A - 3D are diagrams showing four different exemplary embodiments for splitting or dividing the RNP2 activators of the cascade assay into PAM-proximal regions and PAM-distal regions where at least the PAM-distal region molecules are blocked and, when unblocked, can activate RNP2.

[0031] FIG. 4A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked PAM-distal region constructs and RNP2s comprising a nucleic acid-guided nuclease, a gRNA and a PAM-proximal region of an RNP2 activator.

[0032] FIG. 4B is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked combination PAM-proximal region / P AM-distal region constructs and RNP2s comprising a nucleic acid-guided nuclease and a gRNA without the PAM-proximal region of the RNP2 activator.ATTORNEY DOCKET NUMBER: VB029PCT

[0033] FIG. 5 shows exemplary blocked PAM-distal region constructs, a blocked PAM- proximal region construct, and a blocked combination PAM-proximal region / P AM-distal region construct.

[0034] It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.DEFINITIONS

[0035] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

[0036] 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.

[0037] 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 orientationATTORNEY DOCKET NUMBER: VB029PCT 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.

[0038] 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).

[0039] 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 limits, ranges excluding either or both of those included limits are also included in the invention.

[0040] 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).

[0041] As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated orATTORNEY DOCKET NUMBER: VB029PCT otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0042] As used herein, the terms “binding affinity” or “dissociation constant” or “Kd” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high Kd (which in the context of the present disclosure refers to blocked nucleic acid molecules binding to 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, low Kd values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kd values are in the range of 100 nM - 100 pM (10 mM) and thus are about 105- to 1010-fold or higher as compared to low Kd values.

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

[0044] As used herein, the terms “blocked nucleic acid molecule” or “blocked nucleic acid” refers to nucleic acid molecules that cannot bind to the first or second RNP complex 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. In the context of the present disclosure, the blocked nucleic acid molecules may be “blocked PAM-distal region constructs” comprising “blocked PAM-distal regions” or “blocked PAM-distal region molecules” of RNP2 activator molecules. In addition, the blocked nucleic acid molecules may be “blocked PAM-proximal region constructs” comprising “blocked PAM-proximal regions” or “blocked PAM-proximal region molecules” of RNP2 activator molecules. In some embodiments, the blocked nucleic acid molecules comprise both a blocked PAM-ATTORNEY DOCKET NUMBER: VB029PCT distal region and a blocked PAM-proximal region in a “blocked combination PAM- proximal region / P AM-distal region construct.”

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

[0046] As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guided nuclease activity”, “cis-mediated nucleic acid-guided nuclease 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.

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

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

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

[0050] The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1 ) a crRNA region or guide sequence capable of hybridizing toATTORNEY DOCKET NUMBER: VB029PCT 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 (or activator in the case of RNP2) to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid.

[0051] “Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In 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 internucleoside linkages. In another embodiment, a modified protein (e.g., a modified or variant nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

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

[0053] As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. In the cascade assays described herein, a firstATTORNEY DOCKET NUMBER: VB029PCT ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, casl2a or casl4a for a DNA target nucleic acid, or casl3a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification comprises a second nucleic acid-guided nuclease; a second guide RNA specific to an RNP2 activator molecule; and a portion of a non-target strand the RNP2 activator, a portion of the target strand 3' to the PAM, the PAM, and, in some embodiments, a PAM-proximal region of the RNP2 activator molecule. In other embodiments, the second ribonucleoprotein complex (RNP2) for signal amplification comprises a second nucleic acid-guided nuclease; a second guide RNA specific to an RNP2 activator molecule; and a portion of a non-target strand the RNP2 activator, a portion of the target strand 3' to the PAM; and the PAM but does not comprise the PAM-proximal region. Note, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

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

[0055] 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, 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 specimens or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water (from both natural and treatment sites), air, or sewer samples; 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.ATTORNEY DOCKET NUMBER: VB029PCT

[0056] 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 (here, gRNAl in RNP1) in vitro or in vivo. 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 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.

[0057] As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guided nuclease activity”, “trans-mediated nucleic acid-guided nuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non- sequence-specific cleavage of a target nucleic acid molecule by a nucleic acid-guided nuclease (such as by a Casl2, Casl3, and Casl4) which is triggered by binding of N nucleotides of a target nucleic acid molecule to a gRNA. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved once initiated.

[0058] 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), Cas 13a nucleases or naturally-occurring proteins, such as a Cas 12a isolated from, for example, Francisella tidarensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID:ATTORNEY DOCKET NUMBER: VB029PCT15139718), and [Eubacterium eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

[0059] The term "variant" in the context of the present disclosure 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 one or more amino acid residues (e.g., substitutions, additions, and / or deletions). Variants include modifications - including chemical modifications - to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

[0060] 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

[0061] 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 in some less than thirty minutes.

[0062] The cascade assays described herein comprise first and second ribonucleoprotein complexes (RNPls and RNP2s) and at least blocked PAM-distal region molecules, as well as reporter moieties. The blocked PAM-distal region molecules keep the second ribonucleoprotein complexes (RNP2s) “locked” unless and until a target nucleic acid of interest activates a first ribonucleoprotein complex (RNP1). By “locked” it is meant that the blocked PAM-distal region molecules (and, in some embodiments, blocked PAM- proximal region molecules) are designed in such a way that they are largely blocked from interacting with the RNP2s; therefore, both RNP1 and RNP2 remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates an RNP1. In anATTORNEY DOCKET NUMBER: VB029PCT improvement over the signal boost assays described in USPNs 11,693,520; 11,702,686; 11,821,025; 11 / 970,730; and 11 / 987,839, the blocked PAM-distal region molecules further assure the RNP2s remain locked. 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.

[0063] Early and accurate detection and determination of infections and diseases is crucial for appropriate prevention strategies, accurate testing, confirmation, and further diagnosis and treatment. Nucleic acid-guided nucleases, such as the Casl2a endonuclease, can be utilized as diagnostic tools for the detection of target nucleic acids associated with diseases. However, currently available state-of-the-art CRISPR Casl2a-based nucleic acid detection relies on DNA amplification before using Casl2a enzymes, which significantly hinders the ability to perform rapid point-of-care testing. This is due to the fact that target-specific activation of Casl2a enzymes, referred herein as cis-cleavage, is a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of target nucleic acid copies in the sample. Once a ribonucleoprotein (RNP) complex is activated after completion of cis-cleavage, the RNP starts rapid non-specific trans-endonuclease activity. Some currently available methods use trans-cleavage to cleave fluorescent reporters that are initially quenched to generate a signal, thereby indicating the presence of a cis-cleavage event-the target nucleic acid. However, the Kcat of 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 60 minutes. Hence, all current technologies rely on DNA amplification to first generate billions of target copies to activate a proportional number of nucleic acid- guided nucleases to generate a detectable signal in 30-60 minutes. There is a need in the field to detect target nucleic acids (e.g., bacterial, viral, and fungal nucleic acid molecules) at a much faster rate for more efficient testing.

[0064] The improvements to the cascade assay described herein result from preventing undesired premature unwinding of blocked PAM-distal region constructs in a reaction mix by a second ribonucleoprotein complex (RNP2) before the blocked PAM-distal region constructs are unblocked via trans-cleavage by a first ribonucleoprotein complex (RNP1) as the result of binding of a target nucleic acid. Preventing undesired unwinding leads toATTORNEY DOCKET NUMBER: VB029PCT increased efficiency, reduced background, and increased signal-to-noise ratio in the cascade assay. That is, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, in the RNP2s) and the blocked PAM- distal region molecules in the cascade assay such that only blocked PAM-distal region molecules that become unblocked due to trans-cleavage activity react with the nucleic acid- guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions, decreases false positive signals. That is, the nucleic acid-guided nucleases are focused on desired interactions which then lead to immediate signal amplification or boost in the cascade assay.

[0065] The present disclosure describes RNP2 activator molecules comprising two regions: a PAM-proximal region of the activator and a PAM-distal region of the activator. The “RNP2 activator molecule” is the “target molecule” for the RNP2; that is, the RNP2 activator molecule has sequence complementarity with gRNA2 — the gRNA in RNP2 — where the binding of the RNP2 activator molecule to the gRNA2 initiates both cis- and trans-cleavage activity of RNP2. The present disclosure describes embodiments of the cascade assay that can be separated into two “buckets” as described in detail below; however, a theme underlying both cascade assay “buckets” is that the RNP2 activator molecule is physically divided into the PAM-proximal region and the PAM-distal region, and at least the PAM-distal region is a blocked nucleic acid molecule. In the present compositions and methods, the PAM-proximal regions and PAM-distal regions are divided into portions where the two portions, when joined, mimic a full-length activator and total at least 20 nucleotides in length, where the shortest portion — either the PAM-proximal region or the PAM-distal region — is at least 6 nucleotides in length. Thus, the number of nucleotides in the PAM-proximal region may be 14, 13, 12, 11, 10, 9, 8, 7, or 6 or more nucleotides and the corresponding number of nucleotides in the PAM-distal region may be 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more nucleotides for a total of 20 nucleotides or more once the two regions of the split or divided activator are combined.

[0066] The cascade assays provide signal amplification upon detection of the target nucleic acid(s) thereby affording rapid and accurate detection of one or more target nucleic acids in about 30 minutes or less. Signal amplification utilizes two RNPs and reporter molecules able to reach attomolar (aM) detection (or lower) limits without the need to amplify theATTORNEY DOCKET NUMBER: VB029PCT target, thus circumventing the complications of false positives produced from primerdimerization, which usually occur in DNA amplification-based technologies when multiple primer sets are included in a single reaction. Moreover, since sequence-specific gRNAs are internalized into nucleic acid-guided nucleases to form preassembled RNPs, the disclosed methods further allow for accurate multiplex screening of a panel of target nucleic acids.

[0067] FIG. 1 is a simplified diagram of the basic cascade assay reaction described in USPNs 11,693,520; 11,702,686; 11,821,025; 11 / 970,730; and 11,987,839 using blocked nucleic acid molecules as described in these references. FIG. 1 at left (step 1) illustrates a first preassembled ribonucleoprotein complex (RNP1) where RNP1 comprises a first nucleic acid-guided nuclease and a first guide RNA (gRNA) specific to a target nucleic acid of interest. The first nucleic acid-guided nuclease may be, for example, Casl2a or Casl4a for a DNA target, or Casl3a for an RNA target. Also seen at left is a target nucleic acid (RNA or DNA). When the target nucleic acid binds to the first gRNA in RNP1, RNP1 is activated and cis-cleavage of the target nucleic acid occurs, initiating trans-cleavage of other nucleic acids in the sample. Moving right (step 2), a “locked” RNP2 complex is seen, where the locked RNP2 complex comprises a second nucleic acid-guided nuclease and a second guide RNA (gRNA).

[0068] In addition to RNP1, RNP2 and the target nucleic acid, also present in the reaction mixture are blocked nucleic acid molecules. Blocked nucleic acid molecules are nucleic acid molecules that cannot bind to either the RNP 1 or RNP2 complexes to activate cis- or trans-cleavage. The blocked nucleic acid molecules do not bind to RNP1 due to sequence incompatibility with the first gRNA, and although the blocked nucleic acid molecules do possess sequence compatibility with the gRNA in RNP2 (i.e., gRNA2), the blocked nucleic acid molecules have been configured so that they cannot act as a substrate for RNP2 processing until they are unblocked (or are poor substrates for RNP2 until they are unblocked). Thus, the blocked nucleic acid molecules serve as gatekeepers for preventing errant activation of RNP2. Only upon binding of the target nucleic acid to RNP1 and the triggering trans-cleavage activity of RNP1 are the blocked nucleic acid molecules unblocked - by providing single-stranded nucleic acids that are sequence compatible with the gRNA in RNP2. The unblocked single-stranded nucleic acids thenATTORNEY DOCKET NUMBER: VB029PCT activate RNP2. The activated RNP2 complexes trigger further trans-cleavage, and more blocked nucleic acid molecules are converted to unblocked nucleic acid molecules which then activate more RNP2 complexes, providing exponential cleavage of blocked nucleic acid molecules and RNP2 formation and activation.

[0069] Also present in the reactions are reporter molecules. Here as in the methods presented herein, the reporter molecules are illustrated as separate from the RNP2 complex. The reporter molecule may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (FAM) on the 5' end and a minor groove binder (MGB) and a quencher on the 3' end. The reporter and quencher can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Signal generation, however, may occur through different mechanisms, as described below. Other detectable moieties, labels or reporters can also be used to detect a target nucleic acid. Reporter molecules can be labeled in a variety of ways, including the direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, colorimetric moiety and the like.

[0070] Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs and the like. 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, phycoerythrin and the like. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, cholinesterases and the like. Identifiable markers also include radioactive compounds such as125I,35S,14C, or3H. The trans- cleavage triggered by the activation of RNP2 complexes in the cascade cleaves the fluorescent reporters that are initially quenched to generate a signal in step 3. In step 4, exponential signal generation is achieved in only a few minutes.ATTORNEY DOCKET NUMBER: VB029PCT

[0071] FIG. 2 is a simplified diagram of a cascade assay system using one embodiment of a blocked nucleic acid molecule where the RNP2 activator molecule is not physically divided into the PAM-proximal region and the PAM-distal region. Instead, the entire RNP2 activator molecule is incorporated into the blocked nucleic acid molecule. In this example, the “correct” pathway for unblocking the blocked nucleic acid molecule is shown at top. When target nucleic acids (not shown) bind to the first gRNA in RNP1 (not shown), RNP1 is activated and cis-cleavage of the target nucleic acid occurs and transcleavage of other nucleic acids in the sample is also initiated, including the blocked nucleic acid molecules. In addition to RNP1, there is also an RNP2 complex comprising a second nucleic acid-guided nuclease and a second guide RNA (gRNA).

[0072] One embodiment of a blocked nucleic acid molecule is shown in FIG. 2 at left (the double stranded nucleic acid with a loop structure). Again, blocked nucleic acid molecules are nucleic acid molecules that in theory cannot bind to either the RNP1 or RNP2 complexes to activate cis- or trans-cleavage. The blocked nucleic acid molecules do not bind to RNP1 due to sequence incompatibility with the first gRNA and due to the configuration of the blocked nucleic acid molecules, which are, e.g., “bulky” (here, with a loop) and thus prevent internalization into the RNP 1 complex. The blocked nucleic acid molecules should not bind to RNP2 either, because even though there is sequence compatibility with the second gRNA the configuration of the blocked nucleic acid molecules should prevent binding and internalization into the RNP2 complex.

[0073] In the reaction shown at the top of FIG. 2, only upon binding of the target nucleic acid to RNP1 (again, not shown) and triggering trans-cleavage activity should the blocked nucleic acid molecules be unblocked — here, by cleavage of the loop structures in the non-target strand of the blocked nucleic acid molecules — thereby generating singlestranded target nucleic acids that are sequence compatible with the gRNA in RNP2, thus available to activate RNP2.

[0074] The reaction shown at bottom of FIG. 2 is a “failure” pathway. In this scenario, the unblocking is due not to trans-cleavage of the blocked nucleic acid molecule, but instead is due to enzyme-mediated unwinding of the blocked nucleic acid molecule by the nucleic acid-guided nuclease in RNP2. The unwinding of the blocked nucleic acid molecule is triggered by the presence of the PAM in the blocked nucleic acid molecule (if present) andATTORNEY DOCKET NUMBER: VB029PCT sequence compatibility with the PAM-proximal region of the blocked nucleic acid molecule (i.e., the activator or target molecule for RNP2) which leads to double-stranded DNA unpairing of the blocked nucleic acid molecule, R-loop formation, and subsequent activation of RNP2, including trans-cleavage activity and subsequent signal generation. In this failure mode, a target nucleic acid is not present, yet RNP2 is activated leading to a false positive. However, as described herein, physically dividing the RNP2 activator molecule into a PAM-proximal region and a PAM-distal region and blocking at least the PAM-distal region reduces the likelihood that enzyme-mediated unwinding of the blocked PAM-distal region molecules will occur. This reduction occurs because enzyme-mediated unwinding is triggered by the sequence of the PAM-proximal region, but trans-cleavage cannot be initiated unless both the PAM-proximal and PAM-distal regions are bound to gRNA2 of RNP2. Thus, by physically dividing the PAM-proximal and PAM-distal regions on the RNP2 activator, the enzyme-mediated unwinding and trans-cleavage activities of the nucleic acid-guided nuclease in RNP2 are decoupled.Blocked PAM-Distal Targets and Blocked PAM-Proximal Targets

[0075] FIGs. 3A - 3D show four exemplary embodiments for splitting the RNP2 activator molecule into PAM-proximal and PAM-distal regions where at least the PAM-distal region is a blocked PAM-distal region molecule in a PAM-distal region construct. Typically, a full-length activator is approximately 20 nucleotides in length, and it has been shown that an activator (i.e., a gRNA-target DNA interaction) longer than 14 nucleotides is necessary to initiate trans-cleavage of Casl2a (see Stella, et al, Cell, 175: 1856-71 (2018)). It also has been shown that as the length of the PAM-distal region of the activator is decreased, trans- cleavage activity decreases until it is knocked out completely; however, trans-cleavage activity can be restored by adding back the PAM-distal region (see, Rananaware, et al., Nature Communications, 14:549 (2023)).

[0076] As discussed above, in the present compositions and methods, the PAM-proximal regions and PAM-distal regions are divided into portions where the two portions, when joined, mimic a full-length activator and total at least 20 nucleotides in length, where the shortest portion — either the PAM-proximal region or the PAM-distal region — is at least 6 nucleotides in length. Thus, the number of nucleotides in the PAM-proximal region mayATTORNEY DOCKET NUMBER: VB029PCT be 14, 13, 12, 11, 10, 9, 8, 7, or 6 or more nucleotides and the corresponding number of nucleotides in the PAM-distal region may be 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more nucleotides for a total of 20 nucleotides or more when the two regions of the split or divided activator are combined.

[0077] FIG. 3 A shows method 300 where present is an RNP2 (307) comprising 1) a nucleic acid-guided nuclease (301), typically a Type V Cas nuclease such as Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasY (Casl2d), or Cas 13a; 2) a gRNA (302); and 3) a partial RNP2 activator (303) comprising a portion of a nontarget strand (304), a portion of the target strand 3' of the PAM (340), the PAM (314) and a portion of the target strand comprising a PAM-proximal region (305p). Also seen is a blocked PAM-distal region construct (311) comprising a PAM-distal region (305d) of the RNP2 activator molecule, two loop regions (309), and two flanking regions (310) partially hybridized to a partially complementary region (306) of the blocked PAM-distal region construct (311).

[0078] Upon binding of a target nucleic acid of interest (not seen) to RNP1 (also not seen) triggering trans-cleavage activity, the single-strand loop regions (309) of the blocked PAM-distal region construct (311) are cleaved producing three short portions including the PAM-distal region (305d) which, because of the short length and low melting temperature Tm, can dehybridize at room temperature (e.g., 15°-25°C) from the partially complementary region (306), thereby unblocking the blocked PAM-distal region construct (311) to create an unblocked PAM-distal region molecule (305d). The unblocked PAM- distal region molecule (305d) is now free to bind to the gRNA (302) of RNP2 (307) adjacent to the PAM-proximal region (305p), thereby forming a complete RNP2 activator (313), having both a PAM-proximal region (305p) and a PAM-distal region (305d), which can now trigger trans-cleavage activity of the fully-activated RNP2 (308). Note, however, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

[0079] FIG. 3B shows method (315) where, like in FIG. 3A, present is an RNP2 (307) comprising 1) a nucleic acid-guided nuclease (301), typically a Type V Cas nuclease such as Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasYATTORNEY DOCKET NUMBER: VB029PCT(Casl2d), or Cas 13a; 2) a gRNA (302); and 3) a partial RNP2 activator (303) comprising a portion of a non-target strand (304), a portion of the target strand 3' of the PAM (340), the PAM (314) and a portion of the target strand comprising a PAM-proximal region (305p). In addition, a blocked PAM-distal region construct (312) is seen comprising a PAM-distal region (305d) of the RNP2 activator molecule, one loop region (309), and one flanking region (310) partially hybridized to a partially complementary region (306) of the blocked PAM-distal region construct (311).

[0080] Upon binding of a target nucleic acid of interest (not seen) to RNP1 (also not seen) triggering trans-cleavage activity, the single-strand loop region (309) of the blocked PAM- distal region construct (311 ) is cleaved producing two short portions including the PAM- distal region (305d), and which, because of the short length and low melting temperature Tm, dehybridizes from the partially complementary region (306) thereby unblocking the blocked PAM-distal region construct (312) to create an unblocked PAM-distal region molecule (305d). The unblocked PAM-distal region molecule (305d) is now free to bind to the gRNA (302) of RNP2 (307) adjacent to the PAM-proximal region (305p), thereby forming a complete RNP2 activator (313), which can now trigger trans-cleavage activity of the fully-activated RNP2 (308). Note, however, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.

[0081] FIG. 3C shows method (320) where present is an RNP2 (327) comprising 1) a nucleic acid-guided nuclease (301), typically a Type V Cas nuclease such as Cas 12a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasY (Casl2d), or Cas 13a; 2) a gRNA (322); and 3) a partial RNP2 activator (323) comprising a portion of a nontarget strand (324), a portion of the target strand 3' of the PAM (340), and the PAM (330); however, unlike FIGs. 3A and 3B, the PAM-proximal region of the RNP2 activator is not present in the RNP2 (327). Also present is a blocked combination PAM-proximal region / P AM-distal region construct (321) comprising a PAM-proximal region (325p) of the RNP2 activator molecule, a PAM-distal region (325d) of the RNP2 activator molecule, one loop region (329) separating the PAM-proximal (325p) and PAM-distal region (325d) regions, and a partially complementary region (326) of the blocked combination PAM- proximal region / P AM-distal region construct (330).ATTORNEY DOCKET NUMBER: VB029PCT

[0082] Upon binding of a target nucleic acid of interest (not seen) to RNP1 (also not seen) triggering trans-cleavage activity, the single-strand loop region (329) of the blocked combination PAM-proximal region / P AM-distal region construct (330) is cleaved producing two portions, one comprising the PAM-proximate region molecule (325p) and one comprising the PAM-distal region molecule (325d) which, because of the short length and low melting temperature Tmof both portions, dehybridize from the partially complementary region (326) thereby unblocking the blocked combination PAM-proximal region / P AM-distal region construct (330) to create both an unblocked PAM-proximal region molecule (325p) and an unblocked PAM-distal region molecule (325d). The unblocked PAM-proximal region molecule (325p) and unblocked PAM-distal region molecule (325d) are now free to bind to the gRNA (322) adjacent to the PAM (330) of the target strand of the RNP2 activator (323) creating a complete RNP2 activator (333), which can now trigger trans-cleavage activity of the fully-activated RNP2 (328). Note that in this embodiment it is possible to include the PAM with the blocked PAM-proximal region in the blocked combination PAM-proximal region / P AM-distal region construct; however, in most embodiments it is preferable to separate the PAM from the PAM-proximal region to decrease the likelihood of premature or non-specific unwinding of the blocked PAM- proximal region of the blocked combination PAM-proximal region / PAM-distal region construct.

[0083] FIG. 3D shows method (350) where present is an RNP2 (327) comprising 1) a nucleic acid-guided nuclease (301), typically a Type V Cas nuclease such as Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasY (Casl2d), or Cas 13a; 2) a gRNA (322); and 3) a partial RNP2 activator (323) comprising a portion of a nontarget strand (324), a portion of the target strand 3' of the PAM (340), and the PAM (330); however, unlike FIGs. 3A and 3B and like FIG. 3C, the PAM-proximal region of the RNP2 activator is not present in the RNP2 (327). Also present are a blocked PAM-proximal region construct (332) and a blocked PAM-distal region construct (312). The PAM- proximal region construct (332) comprises a PAM-proximal region (325p) of the RNP2 activator molecule, one loop region (329) separating the PAM-proximal region (325p) from a flanking region (310), and a partially complementary region (326) of the blocked PAM-proximal region construct (332). The PAM-distal region construct (312) comprisesATTORNEY DOCKET NUMBER: VB029PCT a PAM-distal region (305d) of the RNP2 activator molecule, one loop region (309) separating the PAM-distal region (305p) from a flanking region (310), and a partially complementary region (306) of the blocked PAM-distal region construct (312).

[0084] Upon binding of a target nucleic acid of interest (not seen) to RNP1 (also not seen) triggering trans-cleavage activity, the single-strand loop region (329) of the blocked PAM- proximal region construct (330) and the single-strand loop region (309) of the blocked PAM-distal region construct are cleaved producing the PAM-proximate region molecule (325p) and the PAM-distal region molecule (305d), both of which, because of their short length and low melting temperature Tm, dehybridize from the partially complementary regions (326 and 306, respectively) thereby unblocking the blocked PAM-proximal region (325p) and PAM-distal region (305d) to create both an unblocked PAM-proximal region molecule (325p) and an unblocked PAM-distal region molecule (305d). The unblocked PAM-proximal region molecule (325p) and unblocked PAM-distal region molecule (305d) are now free to bind to the gRNA (322) adjacent to the PAM (330) of the target strand of the RNP2 activator (323) creating a complete RNP2 activator (343), which can now trigger trans-cleavage activity of the fully-activated RNP2 (338). Again, note that in this embodiment it is possible to include the PAM with the blocked PAM-proximal region in the blocked combination PAM-proximal region / PAM-distal region construct; however, in most embodiments it is preferable to separate the PAM from the PAM-proximal region to decrease the likelihood of premature or non-specific unwinding of the blocked PAM- proximal region of the blocked combination PAM-proximal region / PAM-distal region construct. Note, however, that the PAM site is optional; thus, in some embodiments, the portion of the target strand 3' of the PAM corresponds to the portion of the target strand 3' of the position where the PAM would typically be located.The Signal Boosting Cascade Assay Employing Blocked PAM-Distal Region Constructs or Blocked Combination PAM-Proximal Region / PAM-Distal Region Constructs

[0085] FIG. 1, described above, depicts the cascade assay generally. An exemplary embodiment of the cascade assay (400) utilizing blocked PAM-distal region constructs is depicted in FIG. 4A and described in detail below. In this embodiment, blocked PAM-ATTORNEY DOCKET NUMBER: VB029PCT distal region constructs are used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 4A begins with providing the cascade assay components RNP1 (401), RNP2 (402), and blocked PAM-distal region constructs (403). RNP1 (401) comprises 1) a gRNA specific for a target nucleic acid of interest (not separately labeled); and 2) 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) (also not separately labeled). RNP2 (402) comprises 1) a gRNA specific for an unblocked nucleic acid molecule (not separately labeled); 2) 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) (also not separately labeled); and 3) a partial RNP2 activator (406p) comprising a portion of a non-target strand, a portion of the target strand 3' of the PAM, the PAM, and a portion of the target strand comprising a PAM-proximal region (where these individual features are not separately labeled, but see FIGs. 3A and 3B). The nucleic acid-guided nucleases in RNP1 (401) and RNP2 (402) can be the same or different depending on the type — i.e., DNA vs. RNA — of target nucleic acid of interest and unblocked PAM-distal region molecules that are to be detected by RNP1 and RNP2, respectively. What is key, however, is that trans-cleavage activity can be triggered in the nucleic acid-guided nucleases in RNP1 and RNP2 following binding of the target nucleic acid of interest and the unblocked PAM-distal region molecule, respectively.

[0086] In a first step, a sample comprising a target nucleic acid of interest (404) is added to the cascade assay reaction mixture. The target nucleic acid of interest (404) combines with and activates RNP1 (405) but does not interact with or activate RNP2 (402). Once activated, RNP1 cuts the target nucleic acid of interest (404) via sequence- specific ciscleav age, and the binding also activates non-specific trans-cleavage by RNP1 (405) of other nucleic acids present in the reaction mixture, including the blocked PAM-distal region constructs (403). At least one of the blocked PAM-distal region constructs (403) becomes unblocked, producing an unblocked PAM-distal region molecule (406d) when the blocking moiety (407) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and / or structural modifications.ATTORNEY DOCKET NUMBER: VB029PCT

[0087] Once at least one of the blocked PAM-distal region constructs (403) is unblocked, the unblocked PAM-distal region molecule (406d) can then combine with the portion of the RNP2 activator comprising the PAM and PAM-proximal region (406p) of the RNP2 activator to form an intact RNP2 activator (406 (406p + 406d = 406)), which then can interact with and activate an RNP2 (402— >408). Because the nucleic acid-guided nucleases in the RNPls (405) and RNP2s (408) have both cis- and trans-cleavage activity, more blocked PAM-distal region constructs (403) become unblocked and produce more unblocked PAM-distal region molecules (406d) which can combine with the portions of the RNP2 activator (406p) in other RNP2s (402) comprising the PAM-proximal regions of the RNP2 activator to form more intact RNP2 activators (406), which then activate these RNP2s (408) triggering more trans-cleavage activity in a cascade.

[0088] FIG. 4A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (409) comprise a quencher (410) and a fluorophore (411) 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 (405) and RNP2 (408). The intact reporter moieties (409) become activated reporter moieties (412) when the quencher (410) is separated from the fluorophore (411), emitting a fluorescent signal (413). Signal strength increases rapidly as more blocked nucleic acid molecules (403) become unblocked nucleic acid molecules (406) triggering cis-cleavage activation of more RNP2s (408) and thus more trans-cleavage activity of the reporter moieties (409). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed. One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNAl), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

[0089] A second exemplary embodiment of the cascade assay (450) utilizing blocked combination PAM-proximal region / P AM-distal region constructs is depicted in FIG. 4B and described in detail below. In this embodiment, blocked combination PAM-proximal region / P AM-distal region constructs (470) are used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 4B begins with providing the cascade assay components RNP1 (451), RNP2 (452), and blockedATTORNEY DOCKET NUMBER: VB029PCT combination PAM-proximal region / P AM-distal region constructs (470). RNP1 (451) comprises 1) a gRNA specific for a target nucleic acid of interest (not separately labeled); and 2) 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) (also not separately labeled). RNP2 (452) comprises 1) a gRNA specific for an unblocked nucleic acid molecule (not separately labeled); 2) 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) (also not separately labeled); and 3) a partial RNP2 activator comprising a portion of a non-target strand, a portion of the target strand 3' of the PAM, and the PAM, but not a portion of the target strand comprising a PAM-proximal region (this partial RNP2 activator is not shown). The nucleic acid-guided nucleases in RNP1 (451) and RNP2 (452) can be the same or different depending on the type — i.e., DNA vs. RNA — of target nucleic acid of interest and unblocked PAM-distal region molecules are to be detected by RNP 1 and RNP2, respectively. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following binding of the target nucleic acid of interest and the unblocked PAM-distal region molecule, respectively. The blocked combination PAM-proximal region / P AM- distal region constructs (470) comprise a PAM-proximal region (47 Ip) of the RNP2 activator and a PAM-distal region (47 Id) of the RNP2 activator and a blocking moiety (472).

[0090] In a first step, a sample comprising a target nucleic acid of interest (454) is added to the cascade assay reaction mixture. The target nucleic acid of interest (454) combines with and activates RNP1 (455) but does not interact with or activate RNP2 (452). Once activated, RNP1 cuts the target nucleic acid of interest (454) via sequence- specific ciscleav age, and the binding also activates non-specific trans-cleavage by RNP1 (451) of other nucleic acids present in the reaction mixture, including the blocked combination PAM-proximal region / P AM-distal region constructs (470). At least one of the blocked combination PAM-proximal region / P AM-distal region constructs (470) becomes unblocked, producing an unblocked PAM-proximal region molecule (47 Ip) and an unblocked PAM-distal region molecule (47 Id) when the blocking moiety (472) isATTORNEY DOCKET NUMBER: VB029PCT removed. Again, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and / or structural modifications.

[0091] Once at least one of the blocked combination PAM-proximal region / P AM-distal region constructs (470) is unblocked, the unblocked PAM-proximal region molecule (47 Ip) and the PAM-distal region molecule (47 Id) can then combine to form the intact RNP2 activator (471 (47 Ip + 47 Id = 471)), which then can interact with and activate an RNP2 (452— >458). Because the nucleic acid-guided nucleases in the RNPls (455) and RNP2s (458) have both cis- and trans-cleavage activity, more blocked combination PAM- proximal region / P AM-distal region constructs (470) become unblocked and produce unblocked PAM-proximal region molecules (47 Ip) and PAM-distal region molecules (47 Id). These unblocked PAM-proximal region molecules (47 Ip) and PAM-distal region molecules (47 Id) combine to form intact RNP2 activators (471), triggering activation of more RNP2s (458) and more trans-cleavage activity in a cascade.

[0092] FIG. 4B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (409) comprise a quencher (410) and a fluorophore (411) 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 (455) and RNP2 (408). The intact reporter moieties (459) become activated reporter moieties (462) when the quencher (460) is separated from the fluorophore (461), emitting a fluorescent signal (463). Signal strength increases rapidly as more blocked combination PAM-proximal region / P AM-distal region constructs (470) become unblocked triggering cis-cleavage activation of more RNP2s (458) and thus more trans-cleavage activity of the reporter moieties (459). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed. One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNAl), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

[0093] The embodiment of the cascade assay depicted in FIG. 3D in detail would look much like that shown in FIG. 4B except that instead of blocked combination PAM- proximal region / P AM-distal region constructs (470), there would be two different constructs — blocked PAM-distal region constructs and blocked PAM-proximal regionATTORNEY DOCKET NUMBER: VB029PCT constructs — where at least one of each of both constructs must become unblocked and combine with the portion of the RNP2 activator comprising the PAM to activate RNP2.Blocked PAM-Distal Region Constructs and Blocked Combination PAM-Proximal Region / PAM-Distal Region Constructs

[0094] A blocked PAM-distal region construct or blocked combination PAM-proximal region / P AM-distal region construct may be single-stranded or double-stranded and typically contains a partially hybridized nucleic acid sequence forming one or more cleavable secondary loop structures. Blocked PAM-distal regions and blocked PAM- proximal regions have a low binding affinity, or high dissociation constant (Kd) in relation to binding to RNP2 compared to unblocked PAM-distal regions and unblocked PAM- proximal regions and may be referred to herein as a high Kd nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked PAM-distal regions or blocked or unblocked PAM-proximal regions, low Kd values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kd values are in the range of 100 nM to about 10-100 mM and thus are about 105-, 106-, 107-, 108-, 109- to 1010-fold or higher as compared to low Kd values.

[0095] The blocked nucleic acid molecules (i.e., blocked PAM-distal regions and blocked PAM-proximal regions) (high Kd molecules) described herein can be converted into unblocked nucleic acid molecules (i.e., unblocked PAM-distal regions and unblocked PAM-proximal regions) (low Kd molecules - also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNPls and RNP2s). Once the unblocked nucleic acid molecules — either unblocked PAM-distal regions in the embodiment where the PAM proximal regions are provided as part of RNP2 or both the unblocked PAM-distal regions and unblocked PAM-proximal regions in the embodiment where the PAM proximal regions are not provided as part of RNP2 — is bound to RNP2, RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop or cascade.

[0096] The blocked PAM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs may be single-stranded (ss) or double-stranded (ds)ATTORNEY DOCKET NUMBER: VB029PCT and will contain a first nucleotide sequence and a second nucleotide sequence where the first nucleotide sequence and second nucleotide sequence have at least partial complementarity (see FIGs. 3A - 3D and FIG. 5). The first nucleotide sequence has a region of sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. Because the first nucleotide sequence is complementary to both the gRNA and portions of the second sequence of the blocked PAM-distal region constructs, blocked P AM-proximal region constructs, or blocked combination P AM- proximal region / P AM-distal region constructs, the gRNA in RNP2 (gRNA2) and second sequence of the blocked PAM-distal region constructs or blocked combination PAM- proximal region / P AM-distal region constructs and the gRNA2 share some degree of sequence homology. The first and second nucleotide sequences of a blocked PAM-distal region construct, blocked PAM-proximal region construct or blocked combination PAM- proximal region / P AM-distal region construct may be on the same nucleic acid molecule (e.g., for single-strand embodiments, see FIG. 5 at (iii) and (v)) or on separate nucleic acid molecules (e.g., for double-strand embodiments, see FIG. 5 at (i), (ii), (iv) and (vi)). Transcleavage (e.g., via RNP1 or RNP2) of the loops of the first nucleotide sequence converts the blocked PAM-distal region constructs, blocked PAM-proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs to a singlestrand unblocked PAM-distal region, unblocked PAM-proximal region, or separate unblocked PAM-proximal region and unblocked PAM-distal region. The unblocked PAM-proximal regions and PAM-distal regions contain only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-cleavage activity of RNP2.

[0097] The second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops) in the first nucleotide sequence. In some embodiments, the blocked PAM-proximal region may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence; however, in some embodiments, a PAM sequence is not present.

[0098] FIG. 5 depicts six exemplary blocked split activators 500. (I) depicts a PAM-distal region (501), two flanking regions (503), two loops (502) and a region (505)ATTORNEY DOCKET NUMBER: VB029PCT complementary to the flanking regions and PAM-distal region. (II) depicts a blocked combination PAM -proximal region / P AM-distal region construct comprising a PAM-distal region (501), a PAM-proximal region (504), a flanking region (503), two loops (502) and a region (505) complementary to the flanking region, PAM-proximal region and PAM- distal region. (Ill) depicts a single-molecule embodiment with a PAM-distal region (501), a flanking region (503), one internal loop (502), one hairpin loop (506) and a region (505) complementary to the flanking region and PAM-distal region. (IV) depicts a separate PAM-proximal region construct (comprising a PAM-proximal region (504), two flanking regions (503), two loops (502) and a region (505) complementary to the flanking regions and PAM-proximal region) and a PAM-distal region construct (comprising a PAM-distal region (501), two flanking regions (503), two loops (502) and a region (505) complementary to the flanking regions and PAM-distal region). (V) depicts a singlemolecule embodiment of a blocked combination PAM-proximal / P AM-distal region construct with a PAM-proximal region (504), a flanking region (503), one internal loop (502), one hairpin loop (506) and a region (505) complementary to the flanking region and PAM-distal region. (VI) depicts a blocked combination PAM-proximal region / P AM-distal region construct comprising two PAM-distal regions (501), two PAM-proximal regions (504), three loops (502) and a region (505) complementary to the two PAM-proximal regions and two PAM-distal regions.

[0099] Note that in the case of the construct shown in (VI), more than one RNP2 may be activated upon the complete unblocking of this construct. Accordingly, if one were to utilize RNP2s comprising PAM-proximal regions and blocked PAM-distal region constructs comprising, e.g., four PAM-distal regions (501), theoretically the complete unblocking of one blocked construct could activate four RNPs. The six exemplary blocked split activators depicted in FIG. 5 are exemplary only and it should be apparent to one of ordinary skill in the art given the present disclosure that variations on these constructs are also within the scope of the disclosure.

[0100] Nucleotide mismatches may be introduced into the regions of blocked PAM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs containing the double-strand segments to reduce the melting temperature (Tm) of the segment such that once the at least one loop is cleaved, the double-strand segment isATTORNEY DOCKET NUMBER: VB029PCT unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary.

[0101] In any of the foregoing embodiments, the blocked P AM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs of the disclosure may further contain a reporter moiety attached thereto such that cleavage of the blocked PAM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs releases a signal from the reporter moiety.

[0102] Also, in any of the foregoing embodiments, the blocked PAM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs may comprise a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked PAM-distal region constructs or blocked combination PAM- proximal region / P AM-distal region constructs of the disclosure may comprise a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and / or a peptide nucleic acid (PNA). The blocked PAM-distal region constructs, blocked PAM-proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs may comprise a modified or non-naturally occurring nucleoside, nucleotide, and / or internucleoside linkage, such as a 2'-O-methyl (2'-0-Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.

[0103] In some embodiments, the blocked PAM-distal region constructs or blocked combination PAM-proximal region / P AM-distal region constructs provided herein may be configured as circular DNAs, RNAs or chimeric (DNA-RNA) molecules, and the blocked PAM-distal region constructs, blocked PAM-proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs may include different base compositions. For the circular design of blocked PAM-distal region constructs, blocked PAM-proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs, the 5 ' and 3 ' ends are covalently linked together. This configuration makes internalization of the blocked PAM-distal region constructs, blocked PAM-proximal region constructs, or blocked combination PAM-proximal region / P AM- distal region constructs into RNP2 - and subsequent RNP2 activation - sterically unfavorable, thereby blocking the progression of the cascade assay. Thus, RNP2 activationATTORNEY DOCKET NUMBER: VB029PCT(e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked P AM- distal region constructs, blocked P AM-proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs followed by linearization and internalization of the unblocked PAM-distal regions, unblocked PAM-proximal regions or unblocked PAM-proximal regions + unblocked PAM-distal regions into RNP2.

[0104] In some embodiments, the blocked PAM-distal region constructs, blocked PAM- proximal region constructs, or blocked combination PAM-proximal region / P AM-distal region constructs are topologically circular molecules with 5' and 3' portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tmcauses the structure to effectively behave as a circular molecule even though the 5' and 3' ends are not covalently linked. The 5' and 3' ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.

[0105] While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. §112,(|[6.

Claims

ATTORNEY DOCKET NUMBER: VB029PCTWe claim:

1. A reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, the PAM and a P AM-proximal region; and a plurality of blocked PAM-distal region constructs, wherein the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion.

2. The reaction mixture of claim 1 , wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

3. The reaction mixture of claim 1 , further comprising reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP1 and / or the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

4. The reaction mixture of claim 3, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.ATTORNEY DOCKET NUMBER: VB029PCT5. The reaction mixture of claim 1, wherein the portion of the RNP2 activator does not comprise a PAM sequence.

6. The reaction mixture of claim 1, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2h, Casl2i, Casl2j, Casl3a, and Casl3b.

7. The reaction mixture of claim 1, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease that is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease.

8. The reaction mixture of claim 1 , wherein the first nucleic acid-guided nuclease is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease.

9. A reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, and the PAM; and a plurality of blocked combination PAM-proximal region / P AM-distal region construct comprising: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a loop region and the first loop region is adjacent to a PAM-proximal region of the RNP2 activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the loop region of the first portion, and a third region hybridized to the PAM-proximal region of the first portion.ATTORNEY DOCKET NUMBER: VB029PCT10. The reaction mixture of claim 9, wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

11. The reaction mixture of claim 9, further comprising reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP1 and / or the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

12. The reaction mixture of claim 11, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

13. The reaction mixture of claim 9, wherein the portion of the RNP2 activator does not comprise a PAM sequence.

14. The reaction mixture of claim 9, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2h, Casl2i, Casl2j, Casl3a, and Casl3b.

15. The reaction mixture of claim 9, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease that is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease.

16. The reaction mixture of claim 9, wherein the first nucleic acid-guided nuclease is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease.

17. A reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, and the PAM;ATTORNEY DOCKET NUMBER: VB029PCT a plurality of blocked PAM-distal region constructs, wherein the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; and a plurality of blocked PAM-proximal region constructs, wherein the blocked PAM- proximal region constructs comprise: a third portion comprising a PAM- proximal region of the RNP2 activator, wherein the PAM- proximal region is adjacent to a second loop region and the second loop region is adjacent to a second flanking region; and a fourth portion comprising a fourth region hybridized to the PAM-proximal region of the third portion, a fifth region not hybridized to the second loop region of the third portion, and a sixth region hybridized to the second flanking region of the third portion.

18. The reaction mixture of claim 17, wherein the lengths of the PAM-proximal and PAM- distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

19. The reaction mixture of claim 17, further comprising reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP1 and / or the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

20. The reaction mixture of claim 19, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

21. The reaction mixture of claim 17, wherein the portion of the RNP2 activator does not comprise a PAM sequence.ATTORNEY DOCKET NUMBER: VB029PCT22. The reaction mixture of claim 17, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2h, Casl2i, Casl2j, Casl3a, and Casl3b.

23. The reaction mixture of claim 17, wherein the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease that is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease.

24. The reaction mixture of claim 17, wherein the first nucleic acid-guided nuclease is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease.

25. A blocked PAM-distal region construct comprising: a first portion comprising a PAM-distal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion.

26. The blocked PAM-distal region construct of claim 1 , wherein the PAM-distal region of the first portion is adjacent to a second loop region, a second flanking region is adjacent to the second loop region, and the second portion comprises a fourth region not hybridized to the second loop region of the first portion, and a fifth region hybridized to the second flanking region of the first portion.

27. The blocked PAM-distal region construct of claim 25, wherein the PAM-distal region is at least 6 nucleotides in length.

28. A blocked combination PAM-proximal region / PAM-distal region construct comprising: a first portion comprising a PAM-distal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-distal region is adjacent to a loop region and the first loop region is adjacent to a PAM-proximal region of the RNP activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the loop region of the first portion, and a third region hybridized to the PAM-proximal region of the first portion.

29. The blocked combination PAM-proximal region / PAM-distal region construct of claim 28, wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activatorATTORNEY DOCKET NUMBER: VB029PCT together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

30. A blocked PAM-proximal region construct comprising: a third portion comprising a PAM-proximal region of a ribonucleoprotein complex (RNP) activator, wherein the PAM-proximal region is adjacent to a second loop region and the second loop region is adjacent to a second flanking region; and a fourth portion comprising a fourth region hybridized to the PAM- proximal region of the third portion, a fifth region not hybridized to the second loop region of the third portion, and a sixth region hybridized to the second flanking region of the third portion.

31. A composition of matter comprising the blocked PAM-distal region construct of claim 25 and the blocked PAM-proximal region construct of claim 30.

32. A method for detecting a nucleic acid target of interest in a sample comprising the steps of: providing a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and transcleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis- cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, the PAM and the PAM-proximal region; and a plurality of blocked PAM-distal region constructs, wherein the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; andATTORNEY DOCKET NUMBER: VB029PCT a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; and contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to the RNP1, wherein: upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked PAM-distal region constructs, thereby producing at least one unblocked PAM-distal region molecule that can complex with the PAM-proximal region of the RNP2 activator; and upon binding of the at least one unblocked blocked PAM-distal region molecule to the PAM-proximal region of the RNP2 activator, the RNP2 becomes active trans-cleaving at least one more of the blocked PAM-distal region constructs; allowing the cascade to continue; and detecting the activated RNP2s, thereby detecting the target nucleic acid of interest in the sample.

33. The method of claim 32, wherein the portion of the RNP2 activator does not comprise a PAM sequence.

34. The method of claim 32, wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

35. A method for detecting a nucleic acid target of interest in a sample comprising the steps of: providing a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and transcleavage activity;ATTORNEY DOCKET NUMBER: VB029PCT a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis- cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM; and the PAM; and a plurality of blocked combination PAM -proximal region / P AM-distal region construct comprising: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a loop region and the first loop region is adjacent to a PAM -proximal region of the RNP2 activator; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the loop region of the first portion, and a third region hybridized to the PAM-proximal region of the first portion; and contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to the RNP1, wherein: upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked combination PAM-proximal region / P AM-distal region constructs, thereby producing at least one unblocked PAM-proximal region molecule and at least one unblocked PAM-distal region molecule that can complex with the region 3' to the PAM-proximal region of the RNP2 activator in the RNP2; and upon binding of the at least one unblocked PAM-proximal region molecule and at least one unblocked PAM-distal region molecule 5' of the PAM of the RNP2 activator, the RNP2 becomes active trans-cleaving at least one more of the blocked combination PAM-proximal region / P AM-distal region constructs; allowing the cascade to continue; and detecting the RNP2s, thereby detecting the target nucleic acid of interest in the sample.

36. The method of claim 35, wherein the portion of the RNP2 activator does not comprise a PAM sequence.ATTORNEY DOCKET NUMBER: VB029PCT37. The method of claim 35, wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

38. A method for detecting a nucleic acid target of interest in a sample comprising the steps of: providing a reaction mixture comprising: a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and transcleavage activity; a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease; a second gRNA that is not complementary to the target nucleic acid of interest, wherein the second nucleic acid-guided nuclease exhibits both cis- cleavage activity and trans-cleavage activity; and a portion of an RNP2 activator comprising a non-target strand region, a region 3' to a PAM, the PAM and the PAM-proximal region; a plurality of blocked PAM-distal region constructs, wherein the blocked PAM-distal region constructs comprise: a first portion comprising a PAM-distal region of the RNP2 activator, wherein the PAM-distal region is adjacent to a first loop region and the first loop region is adjacent to a first flanking region; and a second portion comprising a first region hybridized to the PAM-distal region of the first portion, a second region not hybridized to the first loop region of the first portion, and a third region hybridized to the first flanking region of the first portion; a plurality of blocked PAM-proximal region constructs, wherein the blocked PAM- proximal region constructs comprise:ATTORNEY DOCKET NUMBER: VB029PCT a third portion comprising a PAM-proximal region of the RNP2 activator, wherein the PAM-proximal region is adjacent to a second loop region and the second loop region is adj acent to a second flanking region; and a fourth portion comprising a fourth region hybridized to the PAM-proximal region of the fourth portion, a fifth region not hybridized to the second loop region of the third portion, and a sixth region hybridized to the second flanking region of the third portion; and contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to the RNP1, wherein: upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked PAM-distal region constructs and at least one of the blocked PAM-proximal region constructs, thereby producing at least one unblocked PAM-distal region molecule and at least one unblocked PAM-proximal region molecule that can bind 5' of the PAM of the RNP2 activator; and upon binding of the at least one unblocked PAM-proximal region molecule and at least one unblocked PAM-distal region molecule 5' of the PAM of the RNP2 activator, the RNP2 becomes active trans-cleaving at least one more of the blocked PAM-distal region constructs; allowing the cascade to continue; and detecting the activated RNP2s, thereby detecting the target nucleic acid of interest in the sample.

39. The method of claim 38, wherein the portion of the RNP2 activator does not comprise a PAM sequence.

40. The method of claim 38, wherein the lengths of the PAM-proximal and PAM-distal regions of the RNP activator together total at least 20 nucleotides, and wherein the length of each of the PAM-proximal and PAM-distal regions of the RNP activator is at least 6 nucleotides in length.

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