Systems using CAS12 crispr nucleases for sequence-specific, DNA-independent detection of ssrna

Abstract

The present disclosure relates to one-RNP or two-RNP systems and methods used to detect one to many to a massively multiplexed number of target nucleic acids of interest, including ssDNA, dsDNA and ssRNA, where the detection of ssRNA is independent of the presence of DNA. The RNP in the one-RNP system and the first RNP in the two-RNP system comprise a Casl2 nuclease and a guide RNA.

Description

TITLE: SYSTEMS USING CAS12 CRISPR NUCLEASES FOR SEQUENCE- SPECIFIC, DNA-INDEPENDENT DETECTION OF SSRNARELATED APPLICATIONS

[0001] This application claims priority to USSN 63 / 527,565, filed 18 July 2023, which is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to compositions of matter and assay methods used for DNA-independent detection and / or cleavage of one or more single-stranded RNA targets of interest in a sample with Casl2 CRISPR nucleases.BACKGROUND OF THE INVENTION

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

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

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

[0006] The present disclosure provides compositions of matter and methods for DNA- independent detection and / or cleavage of single-stranded RNA targets of interest using Casl2 CRISPR nucleases. Single-stranded RNA targets of interest can be detected using a one-RNP system or a two-RNP system as described below. The systems employ Casl2 nucleases to detect and / or cleave all of double-stranded DNA targets of interest, single-stranded DNA targets of interest, and single-stranded RNA targets of interest alone or in combination. The systems can be multiplexed by employing engineered gRNAs of different sequences complexed with the Casl2 nuclease in ribonucleoprotein complexes (RNPs). The Casl2 / gRNA complexes (RNPs) bind to complementary dsDNAs, ssDNAs and ssRNAs in a sequence specific manner, triggering trans-cleavage activity. That is, ssRNA can function in a similar way to ssDNA / dsDNA to unleash the non-specific single-ssDNA trans-cleavage activity of the Cas 12 nuclease and can do so without the presence of ssDNA and / or dsDNA. Therefore, the present invention encompasses binding to (i.e., specifically detecting) in a sequence-specific manner ssRNA targets of interest using an RNP comprising a Cas 12 nuclease and an engineered gRNA specific to the ssRNA target(s) of interest.

[0007] Thus, the present disclosure provides a method for detecting binding of a ssRNA target of interest in a sample to an engineered gRNA using a one-RNP system comprising the steps of: providing a reaction mixture comprising ribonucleoprotein complexes (RNPs) each comprising a Cas 12 nucleic acid-guided nuclease and an engineered gRNA, wherein the engineered gRNA comprises a sequence complementary to the ssRNA target of interest; and wherein binding of the engineered gRNA to the ssRNA target of interest activates trans-cleavage activity of the Cas 12 nucleic acid-guided nuclease; contacting the reaction mixture with the sample under conditions that allow the ssRNA target of interest in the sample to bind to the RNPs, wherein the sample may or may not comprise dsDNA or ssDNA; and detecting thecleavage products, thereby detecting binding of the ssRNA target of interest to the engineered gRNA in the sample.

[0008] Alternatively, the present disclosure provides a method for detecting binding of a ssRNA target of interest in a sample using a two-RNP system comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNPls) each comprising a Casl2 nucleic acid-guided nuclease and a first engineered gRNA, wherein the first engineered gRNA comprises a sequence complementary to the ssRNA target of interest; and wherein binding of the first engineered gRNA to the ssRNA target of interest activates trans-cleavage activity of the Casl2 nucleic acid- guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second engineered gRNA that is not complementary to the ssRNA target of interest; and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second engineered gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp; contacting the reaction mixture with the sample under conditions that allow the ssRNA target of interest in the sample to bind to RNP1, wherein the sample may or may not comprise dsDNA or ssDNA; wherein upon binding of the ssRNA target of interest RNP1 becomes active initiating trans- cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, and wherein the at least one unblocked nucleic acid molecule binds to an RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting binding of the ssRNA target of interest to the first engineered gRNA in the sample.

[0009] In some aspects of these embodiments, the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans- cleavage activity by the RNP1 and RNP2 (if present) to identify the presence of one or more nucleic acid targets of interest — including ssRNA — in the sample. In some aspects, the reporter moieties are not coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP1 and RNP2, a signal from the reporter moiety is detected and in some aspects of the two-RNP system the reporter moieties are coupledto the blocked nucleic acid molecules, and wherein upon cleavage by RNP1 and RNP2, a signal from the reporter moiety is detected.

[0010] In some aspects, the RNP in the one-RNP system or the RNP1 in the two-RNP system is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2h, Casl2i, or Casl2j nuclease, and in some aspects, the RNP in the one-RNP system or the RNP1 in the two- RNP system is a Casl2a nuclease.

[0011] In some aspects, the reaction mixture comprises about IfM to about 10 pM of the RNP or RNP1. In some aspects, the reaction mixture comprises a plurality of at least two different RNPs or RNPls, wherein different RNPs or RNPls comprise different engineered gRNA or gRNAl sequences. In some aspects, the reaction mixture comprises a plurality of at least 2 to 10000 different RNPs or RNPls; in some aspects, the reaction mixture comprises a plurality of at least 2 to 1000 different RNPs or RNPls; in some aspects, the reaction mixture comprises a plurality of at least 2 to 100 different RNPs or RNPls. In some aspects, the reaction mixture comprises a plurality of at least 2 to 10 different RNPs or RNPls.

[0012] In some aspects, there are ssRNA targets of interest present in the sample as well as ssDNA and / or dsDNA targets of interest present in the sample and there is an engineered gRNA specific for each ssRNA, ssDNA and / or dsDNA target of interest in an RNP or an engineered gRNAl specific for each ssRNA, ssDNA and / or dsDNA target of interest in an RNP1.

[0013] These aspects and other features and advantages of the invention are described below in more detail.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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:

[0015] FIG. 1 A is an overview of the general principles underlying detection of ssRNA by a one-RNP system.

[0016] FIG. IB is an overview of the general principles underlying detection of ssRNA by a two-RNP system.

[0017] FIG. 2A is a diagram showing the sequence of steps for detecting ssRNA using a one-RNP system.

[0018] FIG. 2B is a diagram showing the sequence of steps for detecting ssRNA using a two-RNP system.

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

[0020] FIG. 3 illustrates three embodiments of reporter moieties.

[0021] FIG. 4A - 4D are a series of four graphs showing relative fluorescence plotted against time for cleavage of ssRNA (including a specific ssRNA target and the specific ssRNA target with varying 5' deletions) by four different Casl2 enzymes.

[0022] FIG. 5A - 5D are a series of four graphs showing relative fluorescence plotted against time for cleavage of ssRNA (with a specific ssRNA target and the specific ssRNA target with an internal mismatch or a non-specific ssRNA target) by four different Casl2 enzymes.

[0023] It should be understood that the drawings are not to scale, and that like reference numbers refer to like features.DEFINITIONS

[0024] 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.

[0025] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as "left," "right," "top," "bottom," "front," "rear," "side," "height," "length," "width," "upper," "lower," "interior," "exterior," "inner," "outer" that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as"first," "second," "third," etc., merely identify one of a number of portions, components, steps, operations, functions, and / or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0026] 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).

[0027] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0028] 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).

[0029] 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 theinvention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

[0030] As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0031] 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 the second ribonucleoprotein (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 the RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules to RNP2, low Kd values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high Kd values are in the range of 100 nM - 100 pM (10 mM) and thus are about 105- to 1010-fold or higher as compared to low Kd values.

[0032] 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. 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.

[0033] As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that, in a two-RNP system, cannot bind to either RNP1 or to RNP2 to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to RNP2 to activate cis-cleavage of additional blocked nucleic acid molecules. In the two-RNP system described herein, the blocked nucleic acid molecules comprise 1) a first region recognized by and complementary to the gRNA2 in the RNP2; 2) one or more secondary regions not complementary to the first region and forming at least one loop; and 3) one or more third regions complementary to and hybridized to the first region forming at least one clamp.

[0034] The terms “Cas RNA-guided endonuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated nuclease that is an RNA-guided nuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein complex (RNP).

[0035] As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”, “cismediated endonuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence- specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule, by a nucleic acid-guided nuclease in an RNP. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

[0036] 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, nucleic acid includes a nucleotide sequence described as having a "percent complementarity" 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'.

[0037] 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.

[0038] A “control” is a reference standard of a known value or range of values.

[0039] The terms “guide nucleic acid” or “guide ribonucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) an engineered crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid- guided nuclease. The crRNA region of the gRNA is an engineered, customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementaritywith a target nucleic acid of interest (or unblocked nucleic acid molecule) 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. Herein, the gRNAs, gRNAls and gRNA2s are “engineered” to detect a target nucleic acid of interest or an unblocked nucleic acid.

[0040] 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 nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

[0041] The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity (homology) 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)).

[0042] As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, or “RNP” refer to a complex containing an engineered guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is complexed with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest (or unblocked nucleic acid molecule), guides the RNP to the target nucleic acid of interest or unblocked nucleic acid molecule and hybridizes to it. The hybridized target nucleic acid-gRNA or unblocked nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease.

[0043] As used herein, the terms "protein" and "polypeptide" are used interchangeably. Proteins may or may not be made up entirely of amino acids.

[0044] As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimen or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water, air, or sewer sample; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0045] The terms “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by an engineered gRNA sequence in vitro or in vivo, including dsDNA targets of interest, ssDNA targets of interest, and ssRNA targets of interest and mixtures thereof. The “target strand” of a dsDNA target nucleic acid of interest is the strand of the doublestranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or ssRNA 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, andit 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. Target nucleic acids of interest include single- stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as pre-mRNA, mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer.

[0046] As used herein, the terms “trans-cleavage”, “trans-nuclease activity”, “transmediated nuclease activity” and variations thereof refer to indiscriminate, non- sequence-specific cleavage of a target nucleic acid molecule by nucleic acid-guided nuclease which is triggered binding of a target nucleic acid of interest or unblocked nucleic acid molecule to an RNP.

[0047] 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, Casl2e, Casl2d, Casl2i, Casl2k, Casl2j, Cas 13a nucleases or naturally-occurring proteins, such as a Casl2a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and Eubacterium eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

[0048] The term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications-including chemical modifications-to one or more amino acids that do not involve amino acid substitutions, additions or deletions.DETAILED DESCRIPTION

[0049] The present disclosure provides systems and methods for detecting target nucleic acids of interest in a sample without the need for amplifying the target nucleic acids of interest. The systems and methods provide for massive multiplexing, high accuracy, low cost, and minimum workflow. Further, a Casl2 enzyme can be used to detect all of dsDNA, ssDNA and ssRNA targets of interest where detection of ssRNA targets of interest is DNA-independent.

[0050] The systems described herein comprise at least one ribonucleoprotein complex (RNP) comprising a Casl2 CRISPR nuclease and an engineered gRNA. The first system utilizes a single Casl2 -RNP, and, optionally, reporter moieties. The second system utilizes first and second RNPs, blocked nucleic acid molecules, and, optionally, reporter moieties. The systems are unique in that a single Casl2 CRISPR nuclease can be used to detect and bind to ssRNA, ssDNA and dsDNA in a sequence-specific manner triggering trans-cleavage activity of the Casl2 enzyme. That is, the unique aspect of the systems and methods described herein is that a Casl2 CRISPR nuclease (e.g., Casl2a) can be used to detect all of ssRNA, ssDNA and dsDNA targets of interest in a sample where detection of ssRNA targets of interest is DNA-independent.

[0051] The one-RNP system comprises an RNP a Casl2 CRISRP nuclease and with engineered gRNAs designed with complementarity to one or more ssRNA, ssDNA and dsDNA targets of interest (and mixtures of ssRNA, ssDNA and dsDNA targets of interest), and in many embodiments there may be several to many to a multiplexed number of different Casl2-RNPs specific for each target of interest.

[0052] The two-RNP system utilizes two different RNPs, RNP1 and RNP2. RNP1 in the two-RNP system is the functional equivalent of the RNP in the one-RNP system; that is, RNP1 comprises a Casl2 CRISPR nuclease and engineered gRNAs designed with complementarity to one or more ssRNA, ssDNA or dsDNA targets of interest. RNP2 functions to boost a signal produced by the binding of a target nucleic acid(s) of interest to RNP1. The blocked nucleic acid molecules in the two-RNP system keep the RNP2s “locked” unless and until a target nucleic acid of interest binds to and activates RNP1. By “locked” it is meant that the blocked nucleic acid molecules are designed in such a way that they are largely blocked from interacting with the RNP2s; therefore, both RNP1 and RNP2 remain largely inactive (i.e., “locked”) unless and until a target nucleic acid of interest activates an RNP1. Details of the two-RNP system are described in USPNs 11,639,520; 11,702,686; 11,884,921; and 11,820,983.

[0053] The methods described herein comprise the steps of providing system components — RNP in the one-RNP system — and RNP1, RNP2 and blocked nucleic acids in the two-RNP system, contacting the system components with a sample where the sample may or may not comprise either ssDNA or dsDNA, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample.

[0054] Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of such diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. However, some modes of nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) rely on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for qPCR assays is due to the fact that there is a significant lag phase early in the amplification process where fluorescence above background cannot be detected. That is, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified.

[0055] FIG. 1A provides a simplified diagram demonstrating a method (100) of a one- RNP system for detecting (i.e., binding to) ssRNA, ssDNA and / or dsDNA targets of interest. The one-RNP system is initiated when the target nucleic acid of interest (104) (ssRNA, ssDNA and / or dsDNA) binds to and activates a pre-assembled ribonucleoprotein complex (RNP) (102). The ribonucleoprotein complex (RNP) comprises an engineered guide RNA (gRNA) and a Casl2 CRISPR nuclease, where the gRNA is complexed with the Casl2 CRISPR nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and binds to it. Typically, preassembled RNPs are employed in the reaction mixture - as opposed to separate nucleic acid-guided nucleases and gRNAs - to facilitate rapid binding and detection of the target nucleic acid(s) of interest.

[0056] ‘ ‘Activation” of the RNP (102 105) in the context of the systems herein refers to activating trans-cleavage activity of the Casl2 CRISPR nuclease in the RNP (105) triggered by the target nucleic acid of interest binding to the Casl2 CRISPR nuclease in RNP. Again, trans-cleavage is non-specific, multi-turnover cleavage of, i.e., ssDNAin the context of Casl2. This trans-cleavage activity triggers unquenching of ssDNA reporter moieties in the reaction mixture. Each activated RNP (105) unquenches more reporter moieties (112). The reporter moieties (112) shown here are synthetic ssDNA molecules linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5' end and a quencher on the 3' end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). The binding of a target nucleic acid of interest (104) to RNP (105) thus unquenches reporter moieties (118) and generates a signal (120).

[0057] FIG. IB provides a simplified diagram demonstrating a method (150) of a two- RNP system for detecting (i.e., by binding to) ssRNA, ssDNA and / or dsDNA targets of interest. The two-RNP system is initiated when the target nucleic acid of interest (154) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (152). The first ribonucleoprotein complex (RNP1) comprises a guide RNA (gRNA) and a Casl2 CRISPR nuclease, where the gRNA is complexed with the Casl2 CRISPR nuclease. The gRNA (here, gRNAl), which includes a sequence complementary to the target nucleic acid of interest, guides RNP1 to the target nucleic acid of interest and binds to it. As described above, typically preassembled RNP complexes (here, RNP1 and RNP2) are employed in the reaction mixture - as opposed to separate nucleic acid- guided nucleases and gRNAs - to facilitate rapid binding and detection of the target nucleic acid(s) of interest.

[0058] ‘ ‘Activation” of RNP1 (152 156) in the context of the systems herein refers to activating trans-cleavage activity of the Casl2 CRISPR nuclease in RNP1 (156) by the target nucleic acid of interest binding to the gRNA in RNP1, which initiates nonspecific, multi-turnover trans-cleavage. This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (158) by unblocking the blocked nucleic acid molecules, which are described in detail below. Each newly activated RNP2 (160) activates more RNP2s (158 160), which in turn cleave reporter moieties(162). As described above in relation to the one-RNP system, the reporter moieties (162) may be a synthetic molecule linked or conjugated to a quencher (164) and a fluorophore (166) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5' end and a quencher on the 3' end. The quencher (164) and fluorophore (166) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties may also be incorporated intoblocked nucleic acid molecules and are described in greater detail below in relation toFIG. 3.

[0059] As more RNP2s are activated (158 — 160), more trans-cleavage activity is activated and more reporter moieties are unquenched; thus, the binding of a target nucleic acid of interest (154) to RNP1 (152) initiates what becomes a cascade of signal production (170) which increases exponentially. The two-RNP system thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event. As described below in relation to FIG. 3, the reporter moieties (162) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease in the two-RNP system, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules (i.e., the target molecules for the RNP2).Target Nucleic Acids of Interest

[0060] The target nucleic acids of interest may be a dsDNA, ssDNA, RNA, or cDNA molecule or chimeras thereof and mixtures thereof. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques. The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep (and other livestock), and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

[0061] In some embodiments, the target nucleic acids of interest are from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals. As a non-limiting example, the target nucleic acids of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobactercloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and / or Gardnerella vaginalis.

[0062] As a non-limiting example, the target nucleic acids of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A / Hl, influenza A / H3, influenza A / Hl-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and / or human parvovirus B19 (B19V).

[0063] Also, as a non-limiting example, the target nucleic acids of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and / or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis, Bonamia exitiosa, Bonamia ostreae, Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Marteilia refringens, Perkinsus marinus, Perkinsus olseni, Theileria annulata, Theileria equi, Theileria parva, Tritrichomonas foetus, Trypanosoma brucei, Trypanosoma congolense, Trypanosoma equiperdum, Trypanosoma evansi and, Trypanosoma vivax.

[0064] Additionally, the target nucleic acids of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses, mycoplasma and other bovine mastitis pathogens, Clostridium perfringens, Campylobacter sp., Salmonella sp.,Pospirivoidae, Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasma sp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, or Botryotinis fuckleina.

[0065] In some embodiments, other target nucleic acids of interest may be for non- infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g, trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia and for tracking therapeutic / treatment efficacy. Other target nucleic acids of interest and samples are described herein. Target nucleic acids of interest may include engineered biologies, including cells such as chimeric antigen receptor T (CAR-T) cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing.

[0066] The RNP systems described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Further testing may be used to identify the specific member of the pool, if warranted.

[0067] Again, the unique aspect of the systems and methods described herein is that a Casl2 CRISPR nuclease (e.g., Casl2a) can be used to detect and bind to all of ssRNA, ssDNA and dsDNA targets of interest and can bind to and / or cleave ssRNA in the absence of dsDNA and / or ssDNA (i.e, the binding and / or cleavage of ssRNA is DNA- independent).Nucleic Acid-Guided Nucleases

[0068] The one-RNP systems and two-RNP systems described herein comprise nucleic acid-guided nucleases in the reaction mixture, either provided as a protein or, preferably, in a pre-assembled RNP. In the systems and methods herein the RNP in the one-RNP system and the RNP1 in the two-RNP system comprise a Casl2 CRISPR nuclease. The nucleic acid-guided nuclease in RNP2 in the two-RNP system embodiment can be any nucleic acid-guided nuclease having both cis- and transendonuclease activity, and if RNP2 comprises a Cas 12 CRISPR nuclease, the samenucleic acid-guided nuclease may be used for both RNP1 and RNP2. Alternatively, in the two-RNP system different nucleic acid nucleases may be used in RNP1 and RNP2; that is, RNP1 comprises a Casl2 CRISPR nuclease, and RNP2 comprises a different Casl2 CRISPR nuclease or a non-Casl2 CRISPR nuclease such as a Casl3 nuclease. Note that in both systems trans-cleavage activity is not triggered unless and until there is binding of a target nucleic acid to the Casl2 CRISPR nuclease in RNP in the one- RNP system or RNP1 in the two-RNP system. Nucleic acid-guided nucleases with properties described herein are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).

[0069] In prior art systems, the type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Casl2a, Casl4a, or Casl3) was utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Casl3a or Casl2g) was utilized if the target nucleic acid of interest is an RNA molecule. However, it has been observed that ssRNA is also detected and bound in a sequence-specific manner by Cas 12 CRISPR nucleases. Exemplary Casl2 CRISPR nucleases include, but are not limited to, Casl2a (e.g., AsCasl2a, EbCasl2a), Casl2b, Casl2c, Casl2d, Casl2e, Casl2g, Casl2h, Casl2i, and Casl2j. For RNP2, any of these Casl2 CRISPR nucleases may be used, as may Cas4, Casl3a (EbaCasl3, EbuCasl3, EwaCasl3), Casl3b (e.g., CccaCasl3b, PsmCasl3b), and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity.Guide RNA (gRNA)

[0070] The present disclosure detects a target nucleic acid of interest via a reaction mixture containing guide RNAs (gRNAs) incorporated into an RNP complex. In the one-RNP system, there is at least one gRNA present to detect a target nucleic acid of interest (ssRNA, ssDNA or dsDNA or mixtures thereof) and in many embodiments, there are several to many to a multiplexed number of different gRNAs present to detect several to many to a multiplexed number of target nucleic acids of interest (ssRNA,ssDNA and / or dsDNA or mixtures thereof). In the two-RNP system, there are at least two gRNAs present, one gRNA to detect a target nucleic acid of interest (ssRNA, ssDNA and / or dsDNA) (gRNAl in RNP1) and one gRNA to detect an unblocked nucleic acid molecule (gRNA2 in RNP2), and in many embodiments, there are several to many to a multiplexed number of different gRNAl s present to detect several to many to a multiplexed number of target nucleic acids of interest (ssRNA, ssDNA and / or dsDNA or mixtures thereof) in the RNPls and at least one species of gRNA2 present to detect the unblocked nucleic acid molecules. Suitable gRNAs include at least one crRNA region to enable specificity in every reaction.

[0071] Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementarity to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

[0072] The gRNA of RNP in the one-RNP system and the gRNA (gRNAl) in RNP1 in the two-RNP system is capable of complexing with the Casl2 CRISPR nuclease to detect and bind a target nucleic acid(s) of interest (i.e., a dsDNA, ssDNA and / or ssRNA). The gRNA (gRNA2) in RNP2 in the two-RNP system is capable of complexing with the nucleic acid-guided nuclease of RNP2 to detect and bind an unblocked nucleic acid molecule. Once any of these gRNAs bind the target nucleic acids of interest or unblocked nucleic acid molecule, trans-cleavage by the of ssDNAs in the reaction mixture via non-sequence-specific trans-cleavage activity takes place. The gRNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest or an unblocked blocked nucleic acid molecule. Target nucleic acids of interest may include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest.

[0073] In any of the foregoing embodiments, the gRNA may be a modified or non- naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and / or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and / or internucleoside linkage, such as a 2'-O-methyl (2'-0-Me) modifiednucleoside, a 2'-fluoro (2'-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.Ribonucleoprotein (RNP) Complex

[0074] As described above, although the one-RNP and two-RNP “reaction mixtures” may comprise separate nucleic acid-guided nucleases and gRNAs, both the one-RNP system and the two-RNP system preferably comprise preassembled RNPs in the reaction mixture, allowing for faster detection kinetics. As described herein, the one- RNP system employs one type of RNP, which comprises a Casl2 nuclease and a gRNA configured to detect a target nucleic acid of choice (or, most often, many different target nucleic acids of choice), including ssRNAs. In some embodiments, the reaction mixture contains about 1 fM to about 10 pM of a given RNP, or about 1 pM to about 1 pM of a given RNP, or about 10 pM to about 500 pM of a given RNP. In some embodiments the reaction mixture contains about 6 x 104to about 6 x 1012complexes per microliter (pl) of a given RNP, or about 6 x 106to about 6 x 1010complexes per microliter (pl) of a given RNP. By “given RNP” it is meant that for a multiplexed one- RNA system used to detect targets A, B, C and D, there will be about 1 fM to about 10 pM of the RNP to detect target A, about 1 fM to about 10 pM of the RNP to detect target B, about 1 fM to about 10 pM of the RNP to detect target C, and about 1 fM to about 10 pM of the RNP to detect target D.

[0075] The two-RNP system employs at least two types of RNP complexes, RNP1 and RNP2, each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 comprises a Casl2 nuclease and RNP2 may comprise the same Casl2 nuclease, a different Casl2 nuclease, or may comprise a non-Casl2 nuclease; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 pM of a given RNP1, or about 1 pM to about 1 pM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6 x 104to about 6 x 1012complexes per microliter (pl) of a given RNP1, or about 6 x 106to about 6 x 1010complexes per microliter (pl) of a given RNP1. By “given RNP1” it is meant that if, for example, the two-RNP system is performed to detect targets A, B, C and D, there will be about 1 fM to about 10 pM of the RNP1 to detect target A, about 1 fM to about 10 pM of the RNP1 to detect target B, about 1 fM to about 10 pM of theRNP1 to detect target C, and about 1 fM to about 10 M of the RNP1 to detect target D. In some embodiments, the reaction mixture contains about 1 fM to about 500 p M of RNP2s, or about 1 pM to about 250 pM of RNP2s, or about 10 pM to about 100 pM of RNP2s. In some embodiments the reaction mixture contains about 6 x 104to about 6 x 1012complexes per microliter (pl) of RNP2s or about 6 x 106to about 6 x 1012complexes per microliter (pl) of RNP2s.

[0076] In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNPs (in the one-RNP system) or RNPls (in the two-RNP system); e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 RNPs or RNPls; where different RNPs or RNPls comprise a Cas 12 nuclease and different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique Cas 12 nuclease-gRNA for the purpose of detecting more than one target nucleic acid of interest.

[0077] In any of the foregoing embodiments, the gRNA of RNP or RNP1 may be homologous or heterologous, relative to the gRNA of other RNP(s) or RNPl(s) present in the reaction mixture. A homologous mixture of RNP or RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP or RNP1 gRNAs has a number of gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and / or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty- four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such aheterologous mixture of RNP or RNP1 gRNAs in a single reaction enables multiplex testing.

[0078] As a first non-limiting example of a heterologous mixture of RNP or RNP1 gRNAs, the reaction mixture may contain: a number of RNPs or RNPls having a gRNA targeting parainfluenza virus 1 ; a number of RNPs or RNPls having a gRNA targeting human metapneumo virus; a number of RNPs or RNPls having a gRNA targeting human rhinovirus; a number of RNPs or RNPls having a gRNA targeting human enterovirus; a number of RNPs or RNPls having a gRNA targeting respiratory syncytial virus; and a number of RNPs or RNPls having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP or RNP1 gRNAs, the reaction mixture may contain: a number of RNPs or RNPls containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.l, B.1.617.2, BA.l, BA.2, BA.2.12.1, BAA, and BA.5 and subvariants thereof. Because Casl2 can detect dsDNA, ssDNA and ssRNA, Casl2 is used in RNP1 to detect any of the aforementioned viruses and bacterial, fungal or other targets simultaneously. The nucleic acid-guided nuclease in different RNPls may all be the same Casl2 nuclease or the Casl2 nucleases in the different RNPs or RNPls may be different. Using different nucleic acid-guided nucleases may make sense due to PAM preferences of the nucleic acid-guided nuclease.

[0079] As another non-limiting example of a heterologous mixture of RNP or RNP1 gRNAs, the reaction mixture may contain RNPs or RNPls targeting two or more target nucleic acids of interest from, e.g., organisms that infect vineyards, such as Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, and Botryotinis fuckleina.Blocked Nucleic Acid Molecules

[0080] In the two-RNP system, RNP1 and RNP2, along with blocked nucleic acids, are used. A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures. Such blocked nucleic acids typically have a low binding affinity, or high dissociation constant (Kd) in relation to binding to RNP2 and may be referred to herein as a high Kd nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules to RNP2 have low Kd values ranging from about 100 fM to about 1 aM or lower (e.g., 100 zM). High Kd values range from 100 nM to about lOmM; thus, high Kd values are about 105-, 106-, 107-, 108-, 109- to 1010-fold or higher as compared to low Kd values. Of course, the ideal blocked nucleic acid molecule would have an “infinite Kd.”

[0081] The blocked nucleic acid molecules (high Kd molecules) described herein can be converted into unblocked nucleic acid molecules (low Kd molecules - also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNPls and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in the RNP2 than does the blocked nucleic acid molecule.

[0082] Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to further cleaving of blocked nucleic acid molecules to produce more unblocked nucleic acid molecules able to bind to and activate more RNP2s, resulting in a positive feedback loop.

[0083] In embodiments where blocked nucleic acid molecules are linear and / or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to the gRNA of RNP2 (i.e., gRNA2), and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments comprising a, e.g., hairpin loop) or on separate nucleic acid molecules (e.g., for double strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-endonuclease activity of RNP2.

[0084] 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). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex thereby initiating cis- or trans-cleavage.

[0085] In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5' to the PAM sequence, or partial PAM sequence. Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.

[0086] Nucleotide mismatches can be introduced in structures containing double strand segments to reduce the melting temperature (Tm) of the segment such that once the loop is cleaved, the double strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. In other words, the number of hybridized bases can be less than or equal to the length of each double strand segment and vary based on number of mismatches introduced. For exemplary blocked nucleic acid molecule structures, formulas and sequences, see USPNs 11,639,520; 11,702,686; 11,884,921; and 11,820,983.

[0087] In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 3, mechanisms depicted at center and bottom.)

[0088] Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and / or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non- naturally occurring nucleoside, nucleotide, and / or internucleoside linkage, such as a 2'- O-methyl (2'-0-Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, and aphosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.Optional Reporter Moieties

[0089] The RNP in the one-RNP system and the RNP1 in the two-RNP system bind to and detect a target nucleic acid of interest, including ssRNA targets of interest. In some embodiments, binding of the ssRNA targets of interest (and other targets of interest in a multiplexed system) is detected via a signal generated in the reaction mixture by a reporter moiety.

[0090] Depending on the type of reporter moiety used, trans- and / or cis-cleavage by the nucleic acid-guided nuclease in RNP, RNP1 or RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone (e.g., not bound to blocked nucleic acid molecules) reporter moieties may generate (shown at bottom in FIGs. 2A and 2B). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal; thus, when the reporter moiety is a separate molecule, the reporter moieties are activated quickly by the trans-cleavage activity. The reporter moiety can comprise DNA, RNA, a chimera of DNA and RNA, or an oligonucleotide with modified nucleic acids. The reporter moiety also can comprise both single- and double-stranded portions.

[0091] In alternative embodiments, the reporter moiety in a two-RNP system may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule and conversion to an unblocked nucleic acid molecule may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown at FIG. 3, center). The signal is generated as the blocked nucleic acid molecule is unblocked, whether quickly or slowly. In yet another embodiment of a two-RNP system, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence (shown at FIG. 3, bottom). In this case, activation of RNP2 by cis- (target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively or in addition, the reporter moiety may be bound to the gRNA.

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

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

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

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

[0096] In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2'-O- methyl (2'-0-Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.Nucleic Acid Modifications

[0097] For the blocked nucleic acid molecules and / or reporter moieties, the molecules or one or more nucleotides in the molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acids, gRNAs, and / or reporter moieties described herein are introduced to optimize the molecule’s biophysical properties (e.g., increasing endonuclease resistance, increasing or decreasing binding efficiency, and / or increasing thermal stability). Modificationstypically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and / or alternative internucleoside linkages.

[0098] For example, one or more of the system components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl 1-C=C-CH ?) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. The blocked nucleic acid molecules, gRNAs and / or reporter moieties may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in USPN 3,687,808; Kroschwitz, ed., The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

[0099] In addition to or as an alternative to nucleoside modifications, the one-RNP or two-RNP system components may comprise 2' sugar modifications, including 2'-O- methyl (2’-0-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2- methoxyethyl) or 2'-M0E), 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMA0E, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O- CH2OCH2N(CH3)2. Other possible 2'-modifications that can modify the nucleic acid molecules described herein may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'- 5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0100] Finally, modifications to the one-RNP or two-RNP system components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.Detection of ssRNA Using a One-RNP System

[0101] FIG. 1A, described above, depicts the one-RNP system generally. A specific embodiment of the one-RNP system is depicted in FIG. 2A and described in detail below. The one-RNP system method (100) in FIG. 2 A begins with providing the one-RNP system component RNP (201). RNP (201) comprises a gRNA specific for a ssRNA target of interest (or ssDNA target of interest or dsDNA target of interest or mixtures of two or all three of these) and a Casl2 nuclease (e.g., Casl2a, Casl2b, Casl2c, Casl2d). Only one “type” of RNP (201) is shown (i.e., an RNP that detects the same target nucleic acid of interest).

[0102] In a first step, a sample comprising a ssRNA target of interest (204) is added to the one-RNP system reaction mixture. The ssRNA target of interest (204) combines with and activates RNP (201 202). Once the ssRNA target of interest(204) binds to RNP (201 202), trans -cleavage activity of the Casl2 nuclease in theRNP is triggered non-specifically cutting other nucleic acids present in the reaction mixture, specifically, reporter moieties.

[0103] FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1A, the reporter moieties are also subject to trans-cleavage by activated RNP (203). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases as the trans-cleavage activity of the Casl2 nuclease in the RNP cleaves reporter moieties (209).Detection of ssRNA Using a Two-RNP System

[0104] FIG. IB, described above, depicts the two-RNP system generally. A specific embodiment of the two-RNP system utilizing blocked nucleic acids is depicted in FIG. 2B and described in detail below. Like the two-RNP system shown in FIG. IB, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method (220) in FIG. 2B begins with providing the one-RNP system components RNP1 (221), RNP2 (222) and blocked nucleic acid molecules (223). RNP1 (221) comprises a gRNA specific for a ssRNA target of interest (or ssDNA target of interest or dsDNA target of interest or mixtures of two or all threeof these) and a Casl2 nuclease and RNP2 (222) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease which may be the same Casl2 nuclease in RNP1 or a different nucleic acid-guided nuclease. What is key, however, is that the nucleic acid-guided nuclease in RNP2 can be activated to have trans-cleavage activity following binding of an unblocked nucleic acid molecule to RNP2. Here there is only one RNP1 shown.

[0105] In a first step, a sample comprising a ssRNA target of interest (224) is added to the two-RNP system reaction mixture. The ssRNA target of interest (224) combines with and activates RNP1 (221— > 225) but does not interact with or activate RNP2 (222). Once the ssRNA target of interest binds to RNP1, the trans-cleavage by RNP1 is triggered or activated, and non-specific trans-cleavage of other nucleic acids present in the reaction mixture is initiated, including the blocked nucleic acid molecules (223). At least one of the blocked nucleic acid molecules (223) becomes an unblocked nucleic acid molecule (226) when the blocking moiety (227) is removed. “Blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and / or structural modifications.

[0106] Once at least one of the blocked nucleic acid molecules (223) is unblocked, the unblocked nucleic acid molecule (226) can then interact with and activate an RNP2 (222 228). Because the nucleic acid-guided nucleases in theRNPls (225) and RNP2s (228) have both cis- and trans-cleavage activity, more blocked nucleic acid molecules (223) become unblocked nucleic acid molecules (226) via the trans-cleavage activity of the activated RNP2, which triggers activation of more RNP2s (228) and more trans-cleavage activity in a cascade.

[0107] FIG. 2B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (229) comprise a quencher (230) and a fluorophore (231) linked by a nucleic acid sequence. As described above in relation to FIG. IB, the reporter moieties are also subject to trans-cleavage by activated RNP1 (225) and RNP2 (228). The intact reporter moieties (229) become activated reporter moieties (232) when the quencher (230) is separated from the fluorophore (231), emitting a fluorescent signal (233). Signal strength increases rapidly as more blocked nucleic acid molecules (223) become unblocked nucleic acid molecules (226) triggering cis-cleavage activation of more RNP2s (228) and thus more trans-cleavage activity of the reporter moieties (229). Again, here the reporter moieties are shown as separate molecules from the blockednucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 3.

[0108] FIG. 2C is a diagram showing an exemplary blocked nucleic acid molecule (250) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (250) comprising a target strand (252) may contain partial hybridization with a complementary non-target strand nucleic acid molecule (254) containing unhybridized and cleavable secondary loop structures (256) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNPls or, primarily, RNP2s, generates short strand nucleotide sequences (258) which, because of the short length and low melting temperature Tm, can dehybridize at room temperature (e.g., 15°-25°C), thereby unblocking the blocked nucleic acid molecule (250) to create an unblocked nucleic acid molecule (260), enabling the internalization of the unblocked nucleic acid molecule (260) (target strand) into an RNP2, leading to RNP2 activation. For exemplary blocked nucleic acid molecule structures, formulas and sequences, see USPNs 11,639,520; 11,702,686; 11,884,921; and 11,820,983.Reporter Moiety Configurations

[0109] FIG. 3 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 3 at top shows the mechanism discussed in relation to FIGs. 1A and IB and 2A and 2B. In this embodiment, a reporter moiety (309) is a separate molecule from the blocked nucleic acid molecules present in the reaction mixture. Reporter moiety (309) comprises a quencher (310) and a fluorophore (311). An activated reporter moiety (312) emits a signal from the fluorophore (311) once it has been physically separated from the quencher (310). Again, if the reporter moiety is a separate molecule that is not activated as part of the blocked nucleic acid molecule (or blocked primer molecule), then activation kinetics of the reporter will be more rapid; however, if activation of the reporter moiety is coupled to unblocking of the blocked nucleic acid molecules (or blocked primer molecules), activation kinetics will be slower.

[0110] FIG. 3 at center shows a blocked nucleic acid molecule (303), which is also a reporter moiety. In addition to quencher (310) and fluorophore (311), a blocking moiety (307) can be seen (see also blocked nucleic acid molecules 233 in FIG. 2B).Blocked nucleic acid molecule / reporter moiety (303) comprises a quencher (310) and a fluorophore (311). In this embodiment of the two-RNP system, when the blocked nucleic acid molecule (303) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (306) also becomes an activated reporter moiety with fluorophore (311) separated from quencher (310). Note both the blocking moiety (307) and the quencher (310) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked.

[0111] FIG. 3 at the bottom shows that cis-cleavage of an unblocked nucleic acid or a synthesized activation molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (308), unblocked nucleic acid molecule (361), quencher (310), and fluorophore (311) forming an activated RNP2 with the unblocked nucleic acid / reporter moiety intact (360). Cis-cleavage of the unblocked nucleic acid / reporter moiety (361) results in an activated RNP2 with the reporter moiety activated (362), comprising the activated RNP2 (308), the unblocked nucleic acid molecule with the reporter moiety activated (363), quencher (310) and fluorophore (311).Applications

[0112] The present disclosure describes one-RNP and two-RNP systems for detecting one or more target nucleic acids of interest in a sample where the RNP that binds the target nucleic acid of interest (i.e., RNP in the one-RNP system and RNP1 in the two-RNP system) comprises a Casl2 nuclease and can detect and / or cleave dsDNA, ssDNA, and / or ssRNA, including detection and / or cleavage of ssRNAs independent of the presence of DNA (either ssDNA or dsDNA). The one-RNP and two-RNP systems allow for massive multiplexing and minimum workflow yet provide accurate results at low cost. Moreover, the various embodiments of the one-RNP and two-RNP systems are notable in that, with the exception of the gRNA in RNP and RNP1, the system components can stay the same no matter what target nucleic acid(s) of interest are being detected. Further, both RNP and RNP1 are easily reprogrammed.

[0113] Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals,cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single-celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus.

[0114] For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

[0115] In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in USPN 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those. In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HC1. Suitable methods are contemplated in USPN 8,809,519 and USPN 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and / or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain celldebris free supernatant before applying the reagents. Suitable methods are contemplated in USPN 8,809,519, among other references. In still other embodiments,minimal processing may include performing DNA / RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

[0116] Table 1 below lists exemplary commercial sample processing kits, and Table 2 below lists point of care processing techniques.TABLE 1: Exemplary Commercial Sample and Nucleic Acid Processing KitsTABLE 2: Point of Care Sample Processing Techniques

[0117] Other applications include detection of biomarkers; for example, detection of alpha-synuclein in cerebrospinal fluid to monitor progression of Parkinson’s Disease; detection of sickle cell gene sequences in peripheral blood; and detection of donor-derived gene sequences in peripheral blood of transplant patients. In other applications, environmental monitoring may be performed with either the one- RNP or two-RNP systems, as can monitoring disease in livestock. See, e.g., USSN 18 / 078,031, filed 08 December 2022.

[0118] The components of the one-RNP and two-RNP systems may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemictesting sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: for the on-RNP system, RNPs and reporter moieties; and for the two-RNP system, RNPls, RNP2s, blocked nucleic acid molecules and reporter moieties. Note that there may be several to many to a multiplexed number of different RNP or RNP1 species, each for a different target nucleic acid of interest.

[0119] Any of the kits contemplated herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and / or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.EXAMPLES

[0120] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.Example I: RNP Formation

[0121] For RNP complex formation, 500 nM of LbCasl2a nuclease protein was incubated with 500 nM of a target specific gRNA in IX Buffer (lOmM Tris-HCl pH 8.0, lOOpg / mL BSA, 10 mM MgCh, 50 mM NaCl and 0.2 U / pL RNase inhibitor) at 37°C for 30 minutes and at room temperature for 30 minutes. The total reaction volume was 2pL. Other ratios of LbCasl2a nuclease to gRNAs were tested, including 1:1, 1:2and 1:5. The incubation temperature can range from 20°C - 37°C, and the incubation time can range from 10 minutes to 1.5 hours.Example II: Blocked Nucleic Acid Molecule Formation

[0122] Ramp cooling: For formation of the secondary structure of blocked nucleic acids, 2.5p M of a blocked nucleic acid molecule (any of Formulas I - IV) was mixed in a T50 buffer (20mM Tris HC1, 50mM NaCl) with lOmM MgCL for a total volume of 50, uL. The reaction was heated to 95°C at 1.6 °C / second and incubated at 95 °C for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37°C at 0.015°C / second to form the desired secondary structure.

[0123] Snap cooling: For formation of the secondary structure of blocked nucleic acids, 2.5,u M of a blocked nucleic acid molecule (any of Formulas I - IV) was mixed in a T50 buffer (20mM Tris HC1, 50mM NaCl) with lOmM MgCL for a total volume of 50, uL. The reaction was heated to 95°C at 1.6 °C / second and incubated at 95 °C for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.

[0124] Snap cooling on ice: For formation of the secondary structure of blocked nucleic acids, 2.5,uM of a blocked nucleic acid molecule (any of Formulas I - IV) was mixed in a T50 buffer (20mM Tris HC1, 50mM NaCl) with lOmM MgCh for a total volume of 50pL. The reaction was heated to 95°C at 1.6 °C / second and incubated at 95°C for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.Example III: Reporter Moiety Formation

[0125] The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-10 bases in length (e.g., with sequences of TTATT, TTTATTT, ATT AT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5' and 3' ends, respectively. In one example using a Casl2a cascade, the fluorophore was FAM-6, and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In another example using a Casl3cascade, the reporter moieties were single stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).Example IV: ssRNA Activation of Cast 2 Trans-cleavage

[0126] Trans-activity reaction: 25 nM of the RNP formed in Example I was incubated with 100 nM ssRNA and 500 nM reporter. The cleavage reaction buffer contained 10 mM MgCh, 20 mM NaCl, lOOpg / mL BSA, and 0.2 U / pL RNase inhibitor in 10 mM Tris (pH 8) at 25°C.

[0127] FIGs. 4 A - 4D are a series of four graphs showing relative fluorescence plotted against time for cleavage of ssRNA by four different Casl2 enzymes: LbCasl2a, AsCasl2a, a Casl2a variant SMA, and a Casl2a with the PAM-interacting domain deleted. In addition, the gRNA and three different ssRNA target sequences tested are listed. “Specific ssRNA” denotes a SPECIFIC sequence; “ssRNA w / 5base missing” denotes the SPECIFIC sequence with five 5' nucleotides deleted; “ssRNA w / lObase missing” denotes the SPECIFIC sequence with ten 5' nucleotides deleted. Note that both LbCasl2a and AsCasl2a are activated by the SPECIFIC sequence ssRNA sequence but were not activated by either the 5-base or 10-base deletion ssRNAs. Neither an SMA variant Casl2a nuclease or the PID-deletion variant Casl2a nuclease were activated by the SPECIFIC sequence ssRNA or either the 5- or 10-base deletion ssRNAs.

[0128] FIGs. 5A - 5D are a series of four graphs showing relative fluorescence plotted against time for cleavage of ssRNA by four different Casl2 enzymes: EbCasl2a, AsCasl2a, a Casl2a variant SMA, and a Casl2a with the PAM-interacting domain deleted. As with FIG. 4, the gRNA and ssRNA target sequences tested are listed. “Specific ssRNA” denotes a SPECIFIC sequence (the same sequence used to generate the data in FIGs. 4A - 4D); “single mismatch” denotes the SPECIFIC sequence with a single base change from the SPECIFIC sequence ssRNA; and nonspecific ssRNA denotes the SPECIFIC sequence having a random sequence. Note that both EbCasl2a and AsCasl2a are activated by the specific ssRNA sequence and the ssRNA with a single mismatch (albeit at a much slower rate) but were not activated by the non-specific ssRNA. Neither the SMA variant Casl2a or the PID-deletion Casl2a were activated by the SPECIFIC sequence ssRNA or the single mismatch.

[0129] While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures.

Claims

We Claim:

1. A method for detecting binding of a ssRNA target of interest in a sample that may or may not comprise DNA using a two-RNP system comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNPls) each comprising a Casl2 nucleic acid- guided nuclease and a first engineered gRNA, wherein the first engineered gRNA comprises a sequence complementary to the ssRNA target of interest; and wherein binding of the first engineered gRNA to the ssRNA target of interest activates trans-cleavage activity of the Casl2 nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid- guided nuclease and a second engineered gRNA that is not complementary to the ssRNA target of interest; and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second engineered gRNA; contacting the reaction mixture with the sample under conditions that allow the ssRNA target of interest in the sample to bind to engineered RNP1; wherein upon binding of the ssRNA target of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, wherein the at least one unblocked nucleic acid molecule binds to an RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting binding of the ssRNA target of interest to the first gRNA in the sample.

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

3. The method of claim 2, wherein the reporter moieties are not coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

4. The method of claim 2, wherein the reporter moieties are coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

5. The method of claim 2, wherein the reporter moieties comprise FRET pairs.

6. The method of claim 5, wherein the FRET pairs are selected from EDANS / fluorescein, lAEDANS / fluorescein, fluorescein / tetramethylrhodamine, fluorescein / Cy 5, IEDANS / DABCYL, fluorescein / QSY-7, fluorescein / LC Red 640, fluorescein / Cy 5.5, Texas Red / DABCYL, BODIPY / DABCYL, Lucifer yellow / DABCYL, coumarin / DABCYL, and fluorescein / LC Red 705.

7. The method of claim 1, wherein the RNP1 comprises a Casl2a nuclease.

8. The method of claim 1, wherein the reaction mixture comprises a plurality of at least two different RNPls, wherein different RNPls comprise different engineered gRNAl sequences.

9. The method of claim 8, wherein the reaction mixture comprises about IfM to about 10 pM of each different RNP1.

10. The method of claim 8, wherein the reaction mixture comprises a plurality of at least 2 to 10 different engineered RNPs.

11. The method of claim 10, wherein the reaction mixture comprises about IfM to about 10 pM of each different RNP1.

12. The method of claim 10, wherein the reaction mixture comprises a plurality of at least 2 to 20 different engineered RNPs.

13. The method of claim 12, wherein the reaction mixture comprises about IfM to about 10 pM of each different RNP1.

14. The method of claim 8, wherein the reaction mixture comprises a plurality of at least 2 to 40 different engineered RNPs.

15. The method of claim 14, wherein the reaction mixture comprises about IfM to about 10 pM of each different RNP1.

16. The method of claim 1, wherein there are ssDNA and / or dsDNA targets of interest in the sample as well as the ssRNA targets and wherein there is an engineered gRNA specific for each ssRNA, ssDNA and / or dsDNA target of interest in an RNP.

17. The method of claim 1, wherein a Kd of the blocked nucleic acid molecules binding to RNP2 ranges from about 100 nM to about 100 pM.

18. The method of claim 1, wherein a Kd of the at least one unblocked nucleic acid molecule binding to RNP2 ranges from about 100 fM to about 1 aM or lower.

19. The method of claim 1, wherein the nucleic acid-guided nuclease in RNP2 is selected from Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2g, Casl2h, Casl2i, Casl2j, Cas4, Casl3a, and Casl3b.

20. The method of claim 1 , wherein the ssRNA target of interest is from a viral sample.

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