Direct detection of RNA targets by CAS12a

Abstract

Methods, systems, and kits for detection of ribonucleic acid targets, comprising assembling a ribonucleoprotein by incubating a Cas12a enzyme with a guide ribonucleic acid in a detection buffer, reacting the ribonucleoprotein with a target ribonucleic acid, reacting the ribonucleoprotein with the target ribonucleic acid and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer, and detecting and recording a resulting signal.

Description

[0001] DIRECT DETECTION OF RNA TARGETS BY CAS12A

[0002] CROSS REFERENCE TO RELATED APPLICATIONS / PRIORITY

[0003] The present invention claims priority to United States Provisional Patent Application Number 63 / 517,030 filed August 1, 2023, which is incorporated by reference into the present disclosure as if fully restated herein. Any conflict between the incorporated material and the specific teachings of this disclosure shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this disclosure shall be resolved in favor of the latter.

[0004] INCORPORATION BY REFERENCE OF A SEQUENCE LISTING XML

[0005] A Sequence Listing is provided herewith as a Sequence Listing XML, GLOB- 2023106pct_SeqList.xml” created on Aug. 1, 2024 and having a size of 161 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0006] BACKGROUND

[0007] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -associated protein systems have emerged as powerful tools for nucleic acid detection and manipulation. These systems utilize RNA-guided nucleases to recognize and cleave specific DNA or RNA sequences. One such CRISPR-associated enzyme, Cas12a, has gained attention for its ability to cleave double-stranded DNA targets and subsequently exhibit non-specific single-stranded DNA cleavage activity.

[0008] The widely stated consensus in the literature holds that DNA but not RNA targets activate Cas12a. This prevailing view has persisted since the original description of DNA target-activated trans-cleavage by Cas12a (Chen et al., 2018), where it was reported that “ssRNA was neither capable of activating trans-cleavage nor susceptible to degradation by LbCas12a.” Consequently, existing employment of Cas12a in diagnostics is limited to the detection of DNA targets or RNA targets that have been converted to DNA. Traditionally, detecting RNA targets using Cas12a-based systemshas relied on an initial step of reverse transcription to convert RNA into DNA. This additional step introduces complexity to the detection process and may impact the overall sensitivity and specificity of the assay. Furthermore, the requirement for reverse transcription can increase the time and cost associated with RNA detection.

[0009] The development of methods for direct RNA detection without the need for reverse transcription could potentially simplify and improve nucleic acid detection assays. Such advancements could lead to more rapid, cost-effective, and sensitive detection of RNA targets, which is particularly valuable in scenarios requiring quick and accurate identification of pathogens or other RNA-based biomarkers.

[0010] As the field of nucleic acid detection continues to evolve, there is an ongoing need for improved methods that can overcome existing limitations and provide enhanced performance in terms of sensitivity, specificity, and ease of use. Advancements in this area could have significant implications for various applications, including point-of- care diagnostics, environmental monitoring, and basic research in molecular biology.

[0011] SUMMARY

[0012] Wherefore, it is an object of the present invention to overcome the above-mentioned shortcomings and drawbacks associated with the current technology. The present invention is directed systems, kits, and methods for detection of ribonucleic acid targets, comprising assembling a ribonucleoprotein by incubating a Cas12a enzyme with a guide ribonucleic acid in a detection buffer; reacting the ribonucleoprotein with a target ribonucleic acid; reacting the ribonucleoprotein with the target ribonucleic acid and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer; and detecting and recording a resulting signal. According to further embodiments, assembling the ribonucleoprotein comprises incubating 200 nM Cas12a enzyme with 100 nM of the guide ribonucleic acid for about 30 minutes at or around room temperature. According to further embodiments, the detection buffer comprises: 10 mM Tris pH 7.5; 10 mM MgCl2; 1 mM TCEP; 0.01% Igepal CA630; and 40 μg / ml bovine serum albumin. According to further embodiments, the method comprises diluting the ribonucleoprotein into the detection buffer to 4 nM. According to further embodiments, reacting the ribonucleoprotein with the target ribonucleic acid comprisesreacting at 2 nM for a length of time between 30 – 60 minutes at or around a temperature of 37°C. According to further embodiments, the trans-substrate is at a concentration of one of 50nM and 200 nM or substantially 100 nM. According to further embodiments, the polymer separating the quencher molecule and the reporter molecule comprises greater than five cytosine or thymidine residues. According to further embodiments, the polymer is at least 10 cytosine or thymidine residues long. According to further embodiments, the reporter molecule is selected from the group consisting of: a luciferase enzyme, a phosphatase enzyme, a cleavage enzyme, peptidase, protease, glycase, lipase, nuclease, endonuclease, restriction endonuclease, a Cas protein, horseradish peroxidase, beta galactosidase, glucose oxidase, α-amylase, β-amylase, a fluorescent protein, a fluorescent molecule, a luminescent molecule, a luminescent protein, beta-glucuronidase, a fluorescein dye, an Alexa Fluor, a quantum dot, a quantum nanodot, a restriction endonuclease, a heterocyclic small molecule, a carbocyclic small molecule, an aliphatic small molecule, a heteroaliphatic small molecule, an inorganic species, an organometallic species, a radioactive molecule, and a nanoparticle. According to further embodiments, detecting and recording the resulting signal comprises measuring fluorescence. According to further embodiments, detecting and recording the resulting signal comprises visualizing on a lateral flow readout. According to further embodiments, the target ribonucleic acid is a pathogen nucleic acid sequence. According to further embodiments, the target ribonucleic acid is a microRNA. According to further embodiments, the method comprises using the resulting signal to detect the presence of a human disease.

[0013] The presently disclosed invention further relates to kits for performing the detection of ribonucleic acid targets, comprising a Cas12a enzyme, a guide ribonucleic acid, a detection buffer, and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer. According to further embodiments, the detection buffer comprises 10 mM Tris pH 7.5, 10 mM MgCl2, 1 mM TCEP, 0.01% Igepal CA630, and 40 μg / ml bovine serum albumin. According to further embodiments, the polymer separating the quencher molecule and the reporter molecule comprises greater than five cytosine or thymidine residues.

[0014] The presently disclosed invention further relates to systems for performing the detection of ribonucleic acid targets, comprising a reaction chamber configured to maintain a temperature of about 37°C; a fluorescence detector; and a processor configured to record and analyze fluorescence signals. According to further embodiments, the system further comprises a display for presenting detection results. According to further embodiments, the system further comprises a lateral flow readout device.

[0015] The presently disclosed invention further relates to kits, systems, and methods for detection of ribonucleic acid targets, comprising assembling a ribonucleoprotein by incubating 200 nM Cas12a enzyme with 100 nM of a guide ribonucleic acid for about 30 minutes at or around room temperature (~22°C) in a detection buffer, wherein the detection buffer comprises 10 mM Tris pH 7.5; 10 mM MgCl2; 1 mM TCEP, 0.01% Igepal CA630; and 40 μg / ml bovine serum albumin; diluting the ribonucleoprotein into the detection buffer to 4 nM; immediately after dilution, reacting the ribonucleoprotein at 2 nM with a target ribonucleic acid for a length of time between 30 – 60 minutes and at or around a temperature of 37°C; reacting the ribonucleoprotein with the target ribonucleic acid and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer of greater than five cytosine or thymidine residues at a concentration of approximately 100 nM at or around a temperature of 37°C in the detection buffer; and detecting and recording a resulting signal.

[0016] Various objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings in which like numerals represent like components. The present invention may address one or more of the problems and deficiencies of the current technology discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate various embodiments of the invention and together with the general description of the invention given above and the detailed description of the drawings given below, serve to explain the principles of the invention. It is to be appreciated that the accompanying drawings are not necessarily to scale since the emphasis is instead placed on illustrating the principles of the invention. The invention will now be described, by way of example, with reference to the accompanying drawings in which:

[0019] Figs.1a and 1b are diagrams illustrating methods for detecting RNA targets by crRNA-Cas12a RNP cleavage, according to some embodiments;

[0020] Figs. 2a and 2b are diagrams illustrating methods for detecting DNA and RNA targets by trans-nucleolytic activities of crRNA-Cas12a RNP, according to some embodiments;

[0021] Fig. 3a – 3f are diagrams illustrating methods of detecting stoichiometric amounts of DNA and RNA targets using trans-nucleolytic activities of crRNA-Cas12a RNP, according to some embodiments;

[0022] Figs. 4a and 4b are diagrams illustrating Michaelis-Menten analysis of crRNA-Cas12a RNP activated by RNA target, according to some embodiments;

[0023] Figs. 5a – 5j are graphs illustrating time course of DNA and RNA target- activated trans-nucleolytic activity of crRNA-Cas12a RNP, according to some embodiments;

[0024] Fig. 6a and 6b are graphs illustrating the detection of SARS CoV-2 RNA target using trans-nucleolytic activity of crRNA-Cas12a RNP pools, and Fig 6c is a graph illustrating the detection non-specific DNA inhibits activation of crRNA-Cas12a RNP by ssDNA targets;

[0025] Figs. 7a and 7b are three graphs and a table depicting experimental results for the detection of a rate of cleavage for ribonucleic acid targets using Cas12a ribonucleoproteins, according to aspects of the present disclosure;

[0026] Figs. 8a - 8 is two sets of diagrams, a chart and a graph depicting a survey of trans‐cleavage by crRNA‐Cas12a RNP activated by long DNA targets;

[0027] Figs.9a – 9g. are a set of agarose electrophoretic gels stained with ethidium bromide, diagrams, three graphs, and a table showing experimental results for target cleavage by crRNA-Cas12a RNP;

[0028] Figs. 10a – 10j are a set of graphs and charts showing experimental results for trans‐nucleolytic activities of crRNA‐Cas12a RNP activated by DNA and RNA targets;

[0029] Figs. 11a – 11h illustrate a target‐induced activation of the crRNA‐Cas12a RNP trans‐nuclease;

[0030] Figs. 12a – 12e depict experimental results of low rates of target‐induced activation account for low activity of RNP‐A19;

[0031] Figs. 13a – 13f illustrate experimental results of determinants of crRNA‐ Cas12a RNP activation by dsDNA targets;

[0032] Figs. 14a-14c illustrate experimental results of determinants of crRNA‐ Cas12a RNP activation by ssDNA targets a system for analyzing activity of CRISPR- Cas12a ribonucleoproteins with targets;

[0033] Figs. 15a and 15b illustrate a schematic representation and experimental results of determinants of Cas12a activation and implications for assay performance;

[0034] Figs.16a – 16h depicts graphs showing trans-substrate cleavage by crRNA- Cas12a RNP activated by long DNA fragments;

[0035] Figs. 17a-17d depict experimental results showing kinetics of target DNA cleavage by crRNA‐Cas12a RNP;

[0036] Figs. 18a-18j show graphs of DNA and RNA target‐activated trans‐ nucleolytic activity of crRNA‐Cas12a RNP;

[0037] Figs. 19a – 19q depict graphs showing steady-state analysis of trans- substrate cleavage by crRNA-Cas12a RNP;

[0038] Figs. 20a-20h show graphs of activation and activity of crRNA‐Cas12a RNPs by short dsDNA targets;

[0039] Fig. 21a-21h depict graphs showing concentration dependence of crRNA‐ Cas12a RNP activation by short ds‐ and ssDNA targets;

[0040] Figs. 22a and 22b depict experimental results for activation of Cas12a ribonucleoproteins by DNA targets;

[0041] Figs. 23a and 23b show footprints of crRNA‐Cas12a RNP in HPV‐16;

[0042] Figs. 24a-24e depict activation of crRNA‐Cas12a RNP with long dsDNA and short ssDNA targets derived from Target‐E;

[0043] Fig. 25 illustrates a table summarizing Michaelis-Menten steady-state constants for trans-cleavage, according to aspects of the present disclosure.

[0044] Figs. 26a and 26b show a table of activation parameters of Cas12a RNP reacted with targets;

[0045] Fig. 27 illustrates a table summarizing trans-substrate cleavage data for Cas12a ribonucleoprotein complexes;

[0046] Fig.28 illustrates a system for trans-substrate cleavage activated by titration of Cas12a RNP;

[0047] Fig. 29 illustrates a system for characterizing activation and steady-state phases of trans-substrate cleavage, according to aspects of the present disclosure.

[0048] Figs. 30A and 30B illustrate a system for activating Cas12a ribonucleoprotein complexes specific to Target-A;

[0049] Figs. 31A -31F illustrate a system for analyzing determinants of Cas12a RNP activation by targets, according to aspects of the present disclosure.

[0050] Figs. 32A and 32B illustrate a system for trans-substrate cleavage by Cas12a RNP activated by targets;

[0051] Fig. 33 depicts a data table showing short synthetic nucleic acid sequences;

[0052] Fig. 34 depicts a data table showing long sequences of DNA targets; and

[0053] Fig. 35 illustrates a table containing information about plasmid DNA used in detection.

[0054] DETAILED DESCRIPTION

[0055] The present invention will be understood by reference to the following detailed description, which should be read in conjunction with the appended drawings. It is to be appreciated that the following detailed description of various embodiments is by way of example only and is not meant to limit, in any way, the scope of the present invention. In the summary above, in the following detailed description, in the claims below, and in the accompanying drawings, reference is made to particular features(including method steps) of the present invention. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features, not just those explicitly described. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and grammatical equivalents and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. are used herein to mean that other components, ingredients, steps, etc. are optionally present. For example, an article “comprising” (or “which comprises”) components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

[0056] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1. The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40% means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number),” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100mm. Where reference is made to a figure number that containing multiple drawings, such reference is interpreted as referring to all drawings in that figure. For example, as used in this disclosure, the statement “As shown in Fig. 8” means “As shown in Figs. 8a – 8d”, and “Fig. 11 demonstrates…” means “Figs. 11a – 11h demonstrate…”

[0057] As used herein, the term “ Nucleic acid “ may refer to a polymeric form of nucleic acids of any length or strandedness (double or single); either ribonucleotides (RNA) or deoxyribonucleotides (DNA); and hybrid molecules (comprising DNA and RNA). The disclosed nucleic acids may also include naturally occurring and synthetic or non- natural nucleobases. Natural nucleobases include adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U).

[0058] As used herein, the term “Cas enzyme” may refer to a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated enzyme / protein. The presently disclosed Cas proteins possess a nuclease activity that may be activated upon binding of a target sequence to a guide RNA bound by the Cas enzyme.

[0059] As used herein, the term “Cas12a” may refer to a CRISPR-associated effector enzyme commonly used in gene editing and diagnostic applications. When complexed with crRNA to form ribonucleoproteins, Cas12a cleaves specific dsDNA target sequences in cis to enable editing.

[0060] As used herein, the term “ribonucleoprotein” may refer to a complex comprising a ribonucleic acid molecule and at least one protein. In some aspects, the ribonucleoprotein may include a Cas12a enzyme and a guide ribonucleic acid.

[0061] As used herein, the term “guide ribonucleic acid” may refer to a ribonucleic acid molecule that is capable of binding to a Cas12a enzyme and directing the Cas12a enzyme to a specific target nucleic acid sequence. In some cases, the guide ribonucleic acid may comprise a spacer sequence complementary to a target sequence and a scaffold sequence that interacts with the Cas12a enzyme.

[0062] As used herein, the term “trans-substrate” may refer to a nucleic acid molecule that is cleaved by an activated Cas12a enzyme in a non-specific manner. In some aspects, the trans-substrate may contain a quencher molecule and a reporter molecule separated by a polymer of nucleotides.

[0063] As used herein, the term “quencher molecule” may refer to a molecule that is capableof absorbing the energy emitted by a reporter molecule, thereby reducing or eliminating the detectable signal from the reporter molecule when in close proximity.

[0064] As used herein, the term “reporter molecule” may refer to a molecule that is capable of producing a detectable signal. In some cases, the reporter molecule may be a fluorophore, a luminescent molecule, or an enzyme that produces a detectable product.

[0065] As used herein, the term “protospacer adjacent motif” or “PAM” may refer to a short DNA sequence adjacent to the target DNA sequence that is recognized by the Cas12a enzyme. The PAM sequence may be necessary for the Cas12a enzyme to bind to and cleave the target DNA.

[0066] As used herein, the term “limit of detection” or “LOD” may refer to the lowest concentration or amount of a target nucleic acid that can be reliably detected by the system or method described herein.

[0067] As used herein, the term “apparent turnover rate” or “kapp” may refer to the observed rate of product formation in an enzyme-catalyzed reaction under specific conditions. In some aspects, it may represent the rate of trans-substrate cleavage by an activated Cas12a enzyme.

[0068] The embodiments set forth the below represent the necessary information to enable those skilled in the art to practice the invention and illustrate the best mode of practicing the invention. For the measurements listed, embodiments including measurements plus or minus the measurement times 5%, 10%, 20%, 50% and 75% are also contemplated. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0069] The term “substantially” means that the property is within 80% of its desired value. In other embodiments, “substantially” means that the property is within 90% of its desired value. In other embodiments, “substantially” means that the property is within 95% of its desired value. In other embodiments, “substantially” means that the property is within 99% of its desired value. For example, the term “substantially complete” means that a process is at least 80% complete, for example. In other embodiments, the term“substantially complete” means that a process is at least 90% complete, for example. In other embodiments, the term “substantially complete” means that a process is at least 95% complete, for example. In other embodiments, the term “substantially complete” means that a process is at least 99% complete, for example.

[0070] The term “substantially” includes a value that is within 10% less than or greater than the indicated value. In certain embodiments, the value is within 5% less than or greater than of the indicated value. In certain embodiments, the value is within 2.5% less than or greater than of the indicated value. In certain embodiments, the value is within 1% less than or greater than of the indicated value. In certain embodiments, the value is within 0.5% less than or greater than of the indicated value.

[0071] The term “about” includes when value is within 10% of the indicated value. In certain embodiments, the value is within 5% of the indicated value. In certain embodiments, the value is within 2.5% of the indicated value. In certain embodiments, the value is within 1% of the indicated value. In certain embodiments, the value is within 0.5% of the indicated value.

[0072] In addition, the invention does not require that all the advantageous features and all the advantages of any of the embodiments need to be incorporated into every embodiment of the invention.

[0073] Turning now to Figs. 1a – 35, a brief description concerning the various components of the present invention will now be briefly discussed.

[0074] The RNA‐guided CRISPR‐associated (Cas) enzyme Cas12a cleaves specific double‐ stranded (ds‐) or single-stranded (ss‐) DNA targets (in cis), unleashing non‐specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, the inventors show that the formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer‐adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans‐activity. The inventors identify several activation determinants, including sequences of the PAM (for ds‐ but not ssDNA targets) and those within and outside the protospacer, DNA topology, target length, presence ofnon‐specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation with direct implications for Cas‐based diagnostics.

[0075] Clustered, regularly interspaced short palindromic repeats (CRISPR) systems provide bacteria with acquired immunity using RNA guides transcribed from CRISPR loci to destroy invading DNA and RNA. One CRISPR‐associated enzyme, Cas12a, cleaves specific double‐stranded DNA (dsDNA) targets downstream of protospacer adjacent motifs (PAM) in cis and non‐specific single‐stranded DNA (ssDNA) in trans, which has been coopted to generate detectable signals for diagnostic purposes. Though RNA can activate certain Cas12 family members, DNA but not RNA targets have been shown to activate LbCas12a. CRISPR RNA (crRNA) within the Cas12a ribonucleoprotein (RNP) hybridizes with the protospacer target strand (TS), displacing the non‐target strand (NTS). Formation of this R‐loop induces a large conformational change within Cas12a, leading to activation of endonucleolytic activity, resulting in sequential cleavage of the NTS and TS and acquisition of trans‐activity.

[0076] Since time to result is an important characteristic of any diagnostic, developers would benefit from a more comprehensive understanding of which target features rapidly activate the ability of Cas12a to generate signal. Besides the protospacer, target features responsible for Cas12a activation are poorly understood. Much focus has centered on the PAM for detection of dsDNA targets. The canonical TTTV PAM (see Fig. 8a) is most effectively recognized by LbCas12a, as this AT‐rich sequence forms a narrowed minor groove aligning with WED, REC1, and P1 domains to trigger allosteric transition to the catalytically active state. Noncanonical PAMs can activate Cas12a, but they stimulate less efficient enzymatic activity because structural alterations in both enzyme and target duplex reduce binding stability. Hence Cas12a applications commonly target protospacers downstream of canonical PAMs, though RNPs utilizing suboptimal PAMs increase sensitivity for detection of nucleic acid sequences generated by reverse transcription‐RPA by reducing amplification interference. Despite success in characterizing protospacer sequences for Cas‐based gene editing and general principles of optimal guide design, relatively little is known about other possible determinants of enzyme activation.

[0077] Wide variability in trans‐cleavage activities for different Cas12a RNPs is observed. As some have assumed that target‐induced Cas enzyme activation is not rate‐limiting, much attention has been given to understanding how catalytic efficiency impacts enzyme performance. However, the extent to which differences in assay performance arise from variation in enzyme activation remains untested. The inventors performed quantitative kinetic analysis of the steps leading to activation of the Cas12a nuclease and observed that acquisition of trans‐cleavage capacity is coupled to two activating steps, target binding and an allosteric transition. The inventors show that variability in trans‐activity by RNPs arises from both complex determinants of activation and steady‐ state cleavage of trans‐ substrate. These results provide a more complete understanding of the target features responsible for eliciting the Cas12a assay signal, offering insight into how to optimize the time to result.

[0078] This application discloses methods for CRISPR-associated (Cas) enzyme Cas12a activation by RNA targets through utilization of Cas12a ribonucleoproteins’ (RNPs) trans-cleavage properties. Such activation is previously uncharacterized, allowing for strategic exploitation of Cas12a in diagnostic applications with an RNA target.

[0079] This application discloses the assembly of RNPs capable of detecting RNA. Cas12a and guide are incubated at room temperature for 30 minutes. Subsequent incubation with RNA targets involves RNP concentrations at 10 nM or lower.

[0080] In preferred embodiments, this application involves the use of detection buffer that contains 10 mM Tris pH 7.5, 10 mM MgCl2, 1 mM TCEP, 0.01% Igepal CA630, and 40 μg / ml bovine serum albumin. The detection buffer concerns low salt concentration and the presence of a reductant such as TCEP, DTT, or BME, and detergent, which are the major drivers of detection results; however, other named buffer components are also important.

[0081] Some embodiments of this invention involve the use of trans-substrates containing biotin and a reporter molecule, such as Alexa488, separated by a polymer of greater than five cytosine or thymidine residues. Other embodiments involve the use of trans- substrates containing a quencher molecule, such as BHQ1, and a reporter molecule, separated by a polymer of greater than five cytosine or thymidine residues. In preferredembodiments of either of the above, the polymer separating the quencher molecule or the biotin and the reporter molecule is at least 10 cytosine or thymidine residues long.

[0082] In some embodiments, the reporter molecule may be one or more of a luciferase enzyme, including but not limited to NanoLuc®, firefly luciferase, and Renilla reniformis luciferase; a phosphatase enzyme such as alkaline phosphatase; other enzymes including but not limited to a cleavage enzyme, peptidase, protease, glycase, lipase, nuclease, endonuclease, and restriction endonuclease; a Cas protein; horseradish peroxidase; beta galactosidase; glucose oxidase, α- or β-amylase; a fluorescent protein including but not limited to green fluorescent protein and yellow fluorescent protein; a fluorescent molecule; a luminescent molecule; a luminescent protein; a protein; a fusion protein; beta-glucuronidase; fluorescein dyes; Alexa Fluors®; quantum dots; quantum nanodots; a restriction endonuclease such as EcoRI; a heterocyclic or carbocyclic small molecule; an aliphatic or heteroaliphatic small molecule; an inorganic species; an organometallic species; a radioactive molecule; and a nanoparticle such as a surface enhanced Raman spectroscopy (SERS) particle or a metal nanoparticle including but not limited to gold. Suitable reporter molecules may result in a signal that is detectable by one or more of Raman spectroscopy, fluorescence spectroscopy, spectroscopy, electrochemical methods, visual inspection (for example, color, turbidity), or surface Plasmon resonance. In embodiments wherein the reporter molecule is an enzyme, the signal may be colorimetric, fluorescent, or luminescent. In embodiments wherein the reporter molecule is a fluorophore, nanoparticle, or includes a dye molecule, the signal may be absorbance and / or emission of light at a particular wavelength. In some embodiments, the signal may be detected by visual inspection, microscope, or light detector.

[0083] In a preferred embodiment, RNP is prepared by incubating Cas12a with guide RNA. RNP is then reacted with a target RNA and a trans-substrate in the buffer described above. A resulting signal is generated from the reaction. The resulting signal is detected by measuring fluorescence, or visualizing on lateral flow readout. A detailed explanation of this process follows the steps below:

[0084] Step 1: RNP is assembled by incubating 200 nM Cas12a with 100 nM guide RNA for 30 minutes at room temperature (~22°C) in the detection buffer. RNP is diluted into the detection buffer to 4 nM immediately prior to reaction with target RNA.

[0085] Step 2: RNP at 2 nM is reacted with target RNA for 30 – 60 minutes at 37°C prior to reaction with trans-substrate.

[0086] Step 3: Target RNA-activated RNP is reacted with trans-substrate at 100 nM at 37°C and fluorescence is recorded continuously or after a desired period of time.

[0087] Turning now to the disclosed figures, Fig. 1 illustrates methods for detecting RNA target using crRNA-Cas12a RNP cleavage. In some embodiments, (see Fig. 1a) demonstrates measuring Cas12a RNP cis-cleavage of RNA target by Scheme RNP+FB-T, in which pre-assembled Cas12a RNP is reacted with dual-labeled target (FB-T) under single-turnover conditions (RNP in excess of target). Fluorophore (F) remaining in supernatant after removal of substrate via biotin (B) capture tag represents cleaved target. In some embodiments, (see Fig. 1b) demonstrate target cleavage using Scheme RNP+FB-T. Endpoints after 2.0 hr by 2.0 nM dual-labeled FB-RNA target with varying RNP-A2 at 37°C. Points represent mean (± SE) of background-corrected replicates (n = 3). Line and shaded 95% confidence interval show fit with hyperbolic equation to obtain kobs (± SE) equal to 0.0008 (± 0.0001) min-1.

[0088] Fig. 2 illustrates methods for detecting RNA targets using trans-nucleolytic activities of crRNA-Cas12a RNP. In some embodiments, (see Fig. 2a) demonstrate measuring cleavage of C10trans-substrates (S) by Cas12a RNP pre-reacted with unlabeled target (RNP·T) under multiple-turnover conditions (excess of trans-substrate over RNP), using FB-C10 tagged at opposite ends with fluorophore (F) and biotin (B) capture probe, enabling removal of uncleaved substrate (Scheme RNP·T+FB-S), or FQ-C10where capture tag is replaced with fluorescence quencher (Q) (Scheme RNP·T+FQ-S), enabling real-time detection. In some embodiments (see Fig. 2b), demonstrate trans- cleavage by RNP-A2 and -A19 using Scheme RNP·T+FQ-S with DNA and RNA targets. Cleavage of 100 nM trans-substrate FQ-C10 after 2.0 hr by 1.0 nM RNP incubated with 100 pM targets. Points represent mean (± SE) of replicates (n = 3). The horizontal dotted line represents the signal obtained in the absence of target. Both RNPs show activation by their cognate but not by non-cognate targets.

[0089] Fig. 3 illustrates methods for detecting stoichiometric amounts of RNA targets using trans-nucleolytic activity of crRNA-Cas12a RNP. In some embodiments (see Figs.3a- 3c), demonstrate cleavage of FB-C10 trans-substrate using Scheme RNP·T+FB-S after 1.0 min by 0.25 nM RNP-A2 titrated with ds- and ssDNA targets (see Fig.3a) and after 2.0 hr by 1.0 nM RNP titrated with RNA targets (see Fig. 3b). Points represent mean (± SE) of background-corrected replicates (n = 3). Lines show hyperbolic fit to obtain kapp(± SE) plotted in Fig. 3c. In some embodiments, see Figs. 3d-3f, demonstrate cleavage of FB-C10 trans-substrate using Scheme RNP·T+FB-S after 1.0 min by 0.25 nM RNP-A19 titrated with ds and ssDNA targets (see Fig. 3d) and after 10 min or 60 min by 1.0 nM RNP titrated with RNA targets (see Fig. 3e). Points represent mean (± SE) of background-corrected replicates (n = 3). Lines show hyperbolic fit to obtain kapp(± SE) plotted in Fig. 3f.

[0090] Fig. 4 illustrates a Michaelis-Menten analysis of crRNA-Cas12a RNP activated by RNA target. In some embodiments, the determination of Michaelis-Menten constants for FB-C10 trans-substrate cleavage uses Scheme RNP·T+FB-S for RNP-A19 activated by 0.5 nM RNA target. Some embodiments (see Fig. 4a) demonstrate a representative time course, where lines represent the linear fit of individual values to obtain V0for each concentration of trans-substrate (nM). In some embodiments, (see Fig. 4b) demonstrates substrate-dependence of activated enzyme-normalized V0, where points represent V0 / E0(± SE), and lines and shaded 95% confidence interval show hyperbolic fit to obtain kcat / KM= 2.1 (± 0.1) x 106M-1s-1, kcat= 0.61 (± 0.01) s-1, and KM= 285 (± 4) nM.

[0091] Fig. 5 illustrates the time course of DNA and RNA target-activated trans-nucleolytic activity of crRNA-Cas12a RNP. In some embodiments, trans-cleavage by RNP-A2 (see Figs. 5a–5d) or -A19 (see Figs. 5e–5h) uses Scheme RNP·T+FQ-S with titrated dsDNA (see Figs. 5a and 5e), ssDNA-t (see Figs. 5b and 5f) and RNA-t (see Figs. 5c and 5g) targets and 100 nM trans-substrate FQ-C10. Points represent mean (± SE) of replicates (n = 3). Signals obtained at 2.0 hr are plotted in Fig. 5d and 5h, where insets show analysis of signal at low target concentrations; solid lines show linear fit whose slopes provide estimates for kappwhen normalized to time. 2-hr LOD and kappvalues are summarized in Fig. 5j.

[0092] Fig.6 illustrates detection of SARS CoV-2 RNA target using trans-nucleolytic activity of crRNA-Cas12a RNP pools. In some embodiments, (see Fig.6a) demonstrates trans- cleavage by Cas12a lacking crRNA or assembled with 10 combined SARS CoV-2 N- gene-specific cRNA. Some embodiments (see Fig. 6b) demonstrate using Scheme RNP·T+FQ-S with 160 pM plasmid DNA encoding SARS CoV-2 N-gene or synthetic SARS CoV-2 RNA and 100 nM trans-substrate FQ-C10. Points represent mean (± SE) of replicates (n = 3).

[0093] Fig. 7 illustrates the system for detecting ribonucleic acid targets using the Cas12a enzyme involves the comparison of cleavage rates for different RNP-Target combinations, buffer conditions, and fluorescent substrates. The figure is divided into two panels labeled Fig.7a and Fig.7b. Panel Fig.7a shows three graphs displaying the rate of cleavage versus target concentration for three different RNP-Target combinations. Each graph compares results using two different buffer conditions (Buffer A and Buffer B) and two different fluorescent substrates (FQ-C10and FQ-T5). Panel Fig.7b presents a table summarizing the apparent turnover rates (kapp) calculated from the data in panel Fig. 7a for each RNP-Target, buffer, and substrate combination.

[0094] Fig. 8 illustrates a survey of trans‐cleavage by crRNA‐Cas12a RNP activated by long DNA targets. Fig. 8a shows a schematic of LbCas12a RNP bound to target. Base‐ specific interactions by side chains of residues in LbCas12a are designated by red arrows. PAMs are denoted by the four NTS bases at positions ‐4* to‐1* (5’ to 3’) immediately 5’ to the first protospacer nucleotide (position 1), highlighted in red. For single‐stranded targeting, the PAM is denoted by positions ‐4 to ‐1 (3’ to 5’) of the TS. Fig. 8b shows measuring cleavage of C10 trans‐substrates (S) by Cas12a RNP pre‐ reacted with unlabeled target (RNP∙T) under multiple‐turnover conditions (excess of trans‐substrate over RNP), using FB‐C10 tagged at opposite ends with fluorophore (F) and biotin (B) capture probe, enabling removal of uncleaved substrate (Scheme RNP∙T+FB‐S) or FQ‐C10where capture tag is replaced with fluorescence quencher (Q) (Scheme RNP∙T+FQ‐S). Fig. 8c shows trans‐substrate cleavage by RNPs targeting protospacer sequences embedded within DNA fragments 858–1378‐bp in length. Symbols represent kappcalculated from trans‐cleavage rates (see Figs. 16a‐16f) normalized to target concentration (see Fig.25). Symbols are color‐coded according toPAM sequence at NTS positions ‐3* and ‐2* flanking the protospacers recognized. Average coefficient of variation (SE / |value|) over all data was 11%. Significance thresholds, indicated by thick horizontal lines, represents the mean + 3SE of values measured for Cas12a in the absence of crRNA for each target. Fig. 8d shows representation of how both the rates of enzyme activation (kact) and steady‐state trans‐ cleavage (kss) can impact measurement of apparent substrate cleavage (kapp). Progress curves (thick solid lines) for three mock reactions (a–c) are displayed on the left, with heat maps of rates on the right. Steady‐state components are represented by dashed lines, whose slopes reflect kss, which, when extrapolated, intersect the abscissa at the characteristic activation time (1 / kact). In this example the time at which product is measured (circles) results in identical kappvalues for reactions b and c. As compared to reference reaction a, the reduction in kapp is solely a result of reduced kact and kss for reactions b and c, respectively.

[0095] Fig. 9 illustrates target‐cleavage by crRNA‐Cas12a RNP. Figs. 9a and 9b (Top) show cleavage of plasmid DNA by RNPs. Time course for cleavage of Target‐F (see Fig.9a) or Target‐A (see Fig. 9b) plasmids by representative RNPs at 25°C. RNPs were incubated with plasmid DNA, and at the indicated times, reactions were quenched and subjected to electrophoresis on a 1.5% agarose gel containing ethidium bromide followed by visualization. Migration of double‐stranded‐cleaved, linear (L) and uncleaved, supercoiled (S) forms of the DNA are indicated by arrowheads. (Bottom) Heat maps comparing observed rates of plasmid cis‐cleavage (kobs; see Fig. 17c) to trans‐cleavage activated by long DNA targets (kapp; see Fig.27). Other RNPs analyzed in Figs. 17a–17c. Fig. 9c shows measuring Cas12a RNP cis‐cleavage of target by Scheme RNP+FB‐T, in which pre‐assembled Cas12a RNP is reacted with dual‐labeled target (FB‐T) under single‐turnover conditions (RNP in excess of target). Fluorophore (F, green) remaining in supernatant after removal of substrate via biotin (B) capture tag represents cleaved target. Fig. 9d– Fig. 9g show target cleavage using Scheme RNP+FB‐T. Time course with 1.0 nM dual‐labeled FB‐dsDNA (see Fig. 9d) and FB‐ ssDNA targets (see Fig.9e) at 25°C, and endpoints after 2.0 hr by 2.0 nM dual‐labeled FB‐RNA target with varying RNP‐A2 at 37°C (see Fig. 9f). Points represent mean (± SE) of background‐corrected replicates (n = 2–3).Line and shaded 95% confidenceinterval show global fit of two pooled experiments with exponential Eq. 3 (see Fig. 9d and 9e) or fit with hyperbolic Eq. 4 (see Fig. 9f) to obtain kobs (see Fig. 9g). Similar rates for DNA target cleavage were obtained when tag positions were swapped (see Fig. 17d).

[0096] Fig. 10 illustrates trans‐nucleolytic activities of crRNA‐Cas12a RNP activated by DNA and RNA targets. Fig.10a and Fig.10b show cleavage of FB‐C10 trans‐substrate using Scheme RNP∙T+FB‐S after 1.0 min by 0.25 nM RNP titrated with ds‐ and ssDNA targets (see Fig. 10a) and after 2.0 hr by 1.0 nM RNP titrated with RNA targets (see Fig. 10b). Points represent mean (± SE) of background‐corrected replicates (n = 3). Lines show hyperbolic fit to obtain kapp. Fig. 10c and Fig. 10d show cleavage of FB‐ C10trans‐substrate by RNPs activated with their cognate ds‐ and ssDNA and RNA targets, from Fig. 10a, 10b, 18b, and 18c, where bars represent kapp (± SE). Fig. 10e and Fig. 10f show cleavage of FQ‐C10 trans‐substrate using Scheme RNP∙T+FQ‐S by RNPs activated with their cognate ds‐ and ssDNA‐t and RNA‐t targets after 2.0 hr (from Figs. 18d–18i). Points represent mean (± SE) of replicates (n = 3). Inset shows analysis of signal at low target concentrations, where solid line shows fit with linear Eq. 1, the slope of which provides estimates for kappwhen normalized to time. LOD and kapp values are summarized in Fig. 18j. Fig. 10g– Fig. 10j show determination of Michaelis‐Menten constants for FB‐C10 trans‐substrate cleavage using Scheme RNP∙T+FB‐S for RNP‐A2 activated by 10 pM DNA (see Figs. 10g and 10h) or by RNP‐A19 activated by 0.5 nM RNA target (see Figs.10i and 10j). Representative time courses for the indicated RNP‐target combinations (see Figs.10g and 10i), where lines represent linear fit of individual values to obtain V0 for each concentration of trans‐ substrate (nM). Substrate-dependence of activated enzyme‐normalized V0(see Fig.10h and Fig. 10j), where points represent V0 / E0 (± SE), and lines and shaded 95% confidence interval show hyperbolic fit to obtain kcat / KM and kcat (see Fig.25). Steady‐ state analysis for FQ‐C10trans‐substrate cleavage is shown in Fig. 19.

[0097] Fig.11 illustrates two‐step, target‐induced activation of the crRNA‐Cas12a RNP trans‐ nuclease. Fig.11a shows measuring the time course of target‐induced activation of the trans‐nuclease by Scheme RNP+T,FQ‐S under conditions for single‐turnover of target and multi‐turnover of trans‐substrate. Fig. 11b– Fig. 11d show activation of RNP‐A2trans‐nuclease by short dsDNA targets, revealing lag and steady‐state phases in cleavage of FQ‐C10 trans‐substrate. Components were reacted by Scheme RNP+T,FQ‐ S (see Fig. 11b) or RNP∙T+FQ‐S (see Fig. 11c). Points represent means (± SE) rom background‐corrected replicates (n = 3). Lines show fit to activation equation Eq. 7 to obtain rate constants (± SE) for activation of the nuclease kact and steady‐state rate of trans‐substrate turnover kss (see Fig. 11b) or fit to linear Eq. 5 (see Fig. 11c); bars (see Fig. 11d) summarize constants (see Fig. 19). Note the lag in the development of trans‐ nuclease activity (see Fig. 11b) is eliminated by pre‐reacting RNP with target (see Fig. 11c). Fig.11e– Fig.11h show the effect of RNP concentration on activation rates using Scheme RNP+T,FQ‐S for RNP‐A2 activated with a short ds‐ (see Figs. 11e and 11f) or ssDNA (see Figs.11g and 11h) targets. Points in time course (see Fig. 11e and 11g) represent means (± SE) from background‐corrected replicates (n = 3) of a representative experiment; lines show fit to activation equation Eq. 7 to obtain kact. Global fitting from multiple experiments, indicated by lines and shaded 95% confidence intervals, was performed with hyperbolic Eq. 8 over the full RNP concentration range (see Figs. 11f and 11h) to obtain RNP∙target dissociation constant (Kd) and net rates of the activation step (kact (max)) from asymptotes, and from ratios of the two, apparent second‐order RNP activation rates (kon (app)) representing products of intrinsic RNP‐target binding and forward isomerization rates, an upper limit for RNP‐ target binding. Global analysis over low RNP concentrations (insets) was performed with linear Eq.9 to yield independent estimates for kon (app)and koff (net)from slopes and y‐intercepts, respectively (see Fig. 26A).

[0098] Fig. 12 illustrates low rates of target‐induced activation account for low activity of RNP‐A19. Fig. 12a and Fig. 12b show the effect of RNP concentration on activation rates using Scheme RNP+T,FQ‐S for RNP‐A19 activated with its short ds‐DNA target, carried out as in Figs.11e–11h. Global fitting from multiple experiments, indicated by lines and shaded 95% confidence intervals, was performed with linear Eq. 9 over the full RNP concentration range (see Fig. 12b) to obtain kon (app) and koff (net) from slopes and y‐intercepts, respectively (see Fig.26B). Fig.12c shows the activation time courses for Cas12a RNP‐A2 and ‐A19 induced by their protospacers embedded in a long DNA target (1378‐bp Target‐A), employing Scheme RNP+T,FQ‐S or RNP∙T+FQ‐S withFQ‐C10trans‐ substrate at 37°C. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7 to obtain kact and kss for Scheme RNP+T,FQ‐S and fit to linear Eq. 5 to obtain kss for RNP∙T+FQ‐S (see Fig. 30B) plotted in heat maps along with kapp(see Fig. 27). Inset shows the same data plotted to longer times. Fig.12d‐ Fig.12e show the cleavage of FQ‐C10 trans‐substrate using Scheme RNP+T,FQ‐S by RNPs activated with their cognate short (40‐bp) and long (1378‐bp Target‐A) DNA targets after 2.0 hr (from Fig.31c‐31f). Points (see Fig. 12d) represent mean (± SE) of replicates (n = 3). LOD (± SE) was calculated at each time point (see Fig. 12e), indicating the more slowly activating RNP‐A19 achieves LOD at longer times compared to RNP‐A2.

[0099] Fig. 13 illustrates the determinants of crRNA‐Cas12a RNP activation by dsDNA targets. Fig.13a shows the activation time courses for RNPs induced by their respective protospacers embedded within a fixed target background sequence recognized by RNP‐ F3 (blue bars), employing Scheme RNP+T,FQ‐S and RNP∙T+FQ‐S with FQ‐C10trans‐ substrate. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7 to obtain kact for Scheme RNP+T,FQ‐S or to linear Eq.5 to obtain kssfor RNP∙T+FQ‐S (see Fig.31A) plotted in heat maps. Fig.13b shows the effect of single‐base substitutions at each of the four PAM positions on target‐induced activation as described in Fig.13a, yielding values (see Fig.31B) plotted in heat maps. Fig. 13c shows the effect of target sequence adjoining the PAM‐ protospacer on target‐induced RNP activation. Chimeric dsDNA targets were constructed from sequences of target rapidly activating RNP‐F3 (blue bars) or slowly activating RNP‐F25 (red), including native PAMs utilized by RNP‐F25 (TTTT) or ‐F3 (TTTC). Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7, yielding values (see Fig. 31C) plotted in heat map. Fig. 22a shows targets containing TTTT PAMs. Fig. 13d shows the effect of target context on RNP activation as described in Fig. 13a, yielding values (see Fig. 31D) plotted in heat maps, using protospacers embedded with 55‐bp duplexes (short) or 1299‐bp Target‐F (long). See Fig. 22b for other RNPs. Fig. 13e shows the effect of non‐specific DNA on activation of RNP‐A2 at 37°C as described in Fig. 13c, yielding values (see Fig. 20E) plotted in heat maps. RNPs were reacted with 50 pM 40‐bpdsDNA targets (blue symbols), 3.87‐kb non‐specific plasmid DNA (white) or equimolar mixtures of the two (red). See Fig. 20b for RNP‐A1. Fig. 13f shows the activation time courses for RNPs targeting different protospacers embedded within 3970‐bp plasmid DNA encoding Target‐A as described in Fig. 13c, yielding values (see Fig. 20F) plotted in heat maps. RNPs were reacted with supercoiled plasmid (S) or relaxed circular plasmid DNA (R) generated by nicking endonuclease digestion.

[0100] Fig. 14 illustrates the determinants of crRNA‐Cas12a RNP activation by ssDNA targets. Fig. 14a shows trans‐activities of RNPs in response to long ssDNA targets measured using Scheme RNP∙T+FQ‐S. (Top) Bars represent mean (± SE) of product released after 1.0 hr in replicates (n = 3) using 20 pM ssDNA target ss‐E(308) (green) and ss‐E(494) (blue) by RNPs, whose PAM sequences and intended specificity are indicated. (Bottom) Schematic of positions of protospacers (red) in ssDNA corresponding to 308‐nt ss‐E(308) (green, bottom strand) and 425‐nt ss‐E(425) (blue, top strand) from the 858‐bp Target‐E (grey, double strands). Note the lack of ssDNA target‐induced activation of RNP‐E27 and ‐E23, which target overlapping protospacers in target ss‐E(308), suggesting potential secondary structure interference. Fig. 14b shows the cleavage of FQ‐C10trans‐substrate by RNPs activated with their respective ssDNA targets or long dsDNA Target‐E, where bars represent kss (± SE) (see Fig. 24 and Fig.32A). RNP‐E26 and ‐27 utilize dipurine PAMs and exhibit little activity when activated by dsDNA targets, whereas RNP‐E1 and ‐E18 utilize dipyrimidine PAMs. Fig. 14c shows the activation time courses for RNPs induced by their respective short ssDNA targets, employing Scheme RNP+T,FQ‐S with FQ‐C10 trans‐substrate at 37°C. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7 to obtain activation parameters (see Fig. 32B) plotted in heat maps. RNP‐E18 was activated at a rate too fast to measure and was assigned a lower limit activation rate of 1.0 min‐1. Note the lack of requirement for purines in positions ‐2 and ‐3 of the TS PAM for ssDNA target‐induced activation.

[0101] Fig. 15 illustrates the determinants of Cas12a activation and implications for assay performance. Fig. 15a shows the depiction of determinants for activation of Cas12a RNP Cas12a nucleolytic activity by ds‐ and ssDNA targets described in the text. Fig. 15b shows the effect of activation time on assay performance for different scenariosthat impact catalytic efficiency. Heat maps represent the time required for product formation to exceed a 1% threshold. Modeling was performed with Eq. 10, which combines expressions for enzyme activation (see Eq.7) and steady‐state trans‐cleavage (see Eq. 6) using 100 nM trans‐substrate. A reference condition is represented in the upper left panel. Use of multiple pooled crRNA (upper right) increases apparent kcat. Competitive inhibitors of the trans‐cleavage reaction (lower left) increase KM. Reduction of temperature (lower right) reduces both kcatand KM(see Fig. 25).

[0102] Fig. 16. Illustrates trans‐substrate cleavage by crRNA‐Cas12a RNP activated by long DNA fragments. Fig. 16a– Fig. 16f show trans‐cleavage activities of representative low‐activity RNPs (those displaying kapp< 0.125 s‐1) activated with Target‐A (see Figs. 16a and 16b), ‐B (see Fig. 16c), ‐C (see Fig. 16d), ‐D (see Fig. 16e), and ‐E (see Fig. 16f), measured using Schemes RNP∙T+FB‐S (see Figs. 16a–16e) and RNP∙T+FQ‐S (see Fig. 16f). Points represent mean (± SE) of replicates (n = 3). Lines show fit with linear Eq. 1 to obtain slopes, which when normalized to assay time, yield apparent turnover rate kapp of trans‐substrate (see Fig.8C and Fig.27). “None” represents control assay carried out with Cas12a lacking added crRNA, the slopes of which were used to determine background thresholds. Fig. 16g shows trans‐cleavage activities of select high‐ and low‐activity RNPs in response to 10 pM of specific and non‐specific long dsDNA targets measured using Scheme RNP∙T+FB‐S. Bars represent mean (± SE) of product released after 1.0 hr in replicates (n = 3). Fig. 16h shows trans‐cleavage activities of select high‐ and low‐activity RNPs in response to 10 pM specific (blue) and non‐specific (red) long dsDNA targets measured using Scheme RNP∙T+FQ‐S. Bars represent mean (± SE) of product released after 1.0 hr in replicates (n = 3). RNPs are segregated by central dinucleotides of PAMs utilized.

[0103] Fig. 17 shows the kinetics of target DNA cleavage by crRNA‐Cas12a RNP. Fig. 17a– Fig. 17c show the quantification of time course for cleavage of plasmid encoding Target‐F (see Fig. 17a) or Target‐A (see Fig. 17b) by RNPs, as shown in Figs. 9a and 9b. Background‐corrected intensities of cleaved, linear forms of the DNA were quantified at each time point. Lines and shaded 95% confidence intervals show fit with exponential Eq. 3 to obtain kobs(see Fig. 17c) for RNPs that cleave at least 50% target by 30 minutes (F3, F5, F6, A2, and A4). A linear fit is shown for the remaining RNPs,and an upper limit for kobsis assigned as < 0.02 min‐1. Fig. 17d shows target cleavage using Scheme RNP+FB‐T with swapped probes. Time course of target cleavage using RNP‐A2 and 1.0 nM short dual‐labeled BF‐dsDNA and BF‐ssDNA targets, performed as in Fig. 9d and 9e. Points represent mean (± SE) of background‐corrected replicates (n = 3). Lines show fit with exponential Eq. 3 to obtain kobs (see Fig. 9g). Targets BF‐ dsDNA and BF‐ssDNA represent the same target sequences as used in Fig. 9d and 9e with fluorophore and capture probe positions swapped.

[0104] Fig.18 illustrates DNA and RNA target‐activated trans‐nucleolytic activity of crRNA‐ Cas12a RNP. Fig. 18a shows trans‐cleavage by RNP‐A2 and ‐A19 using Scheme RNP∙T+FQ‐S with DNA and RNA targets. Cleavage of 100 nM trans‐substrate FQ‐ C10after 2.0 hr by 1.0 nM RNP incubated with 100 pM targets. Points represent mean (± SE) of replicates (n = 3). The horizontal dotted line represents the signal obtained in the absence of target. Both RNPs show activation by their cognate but not by non‐ cognate targets. Figs. 18b and 18c show trans‐cleavage by RNP‐A19 using Scheme RNP∙T+FB‐S with DNA and RNA targets. Cleavage of 200 nM trans‐substrate FB‐C10 after 1.0 min by 0.25 nM RNP titrated with ds and ssDNA targets and 100 nM trans‐ substrate by 1.0 nM RNP titrated with ss target RNA (60 min) and ‐ss‐t (10 min), performed as in Figs. 18a and 18b. Points represent mean (± SE) of background‐ corrected replicates (n = 3). Lines show fit with hyperbolic Eq. 2 obtain kapp (see Fig. 26), results of which are plotted in Fig. 10d. Figs. 18d–18i show trans‐cleavage by RNP‐A2 (see Figs. 18d–18f) or ‐A19 (see Figs. 18g–18i) using Scheme RNP∙T+FQ‐S with titrated dsDNA (see Figs. 18d and 18g), ssDNA‐t (see Figs. 18e and 18h) and RNA‐t (see Figs. 18f and 18i) targets and 100 nM trans‐substrate FQ‐C10. Signals obtained at 2.0 hr are plotted in Figs. 10e and 10f. Fig.18j shows a summary of trans‐ cleavage of FQ‐C10 by RNPs activated by DNA and RNA targets, using values plotted in Figs. 10e and 10f.

[0105] Fig.19 illustrates a steady‐state analysis of trans‐substrate cleavage by crRNA‐Cas12a RNP. Determination of Michaelis‐Menten steady‐state constants for trans‐substrate cleavage using Schemes RNP∙T+FB‐S or RNP∙T+FQ‐S for Cas12a RNP activated by DNA and RNA targets. (Top) Time courses for cleavage of trans‐substrate FB‐C10(see Figs. 19a–19c) and FQ‐C10(see Figs. 19d–19q) by the indicated RNP‐targetcombinations at 37°C (see Figs. 19a–19j) or 25°C (see Figs. 19k–19q), where points represent mean (± SE) of background‐corrected replicates (n = 3); lines represent fit with linear Eq. 5 to obtain V0 for each trans‐substrate concentration. (Bottom) Substrate‐dependence of activated enzyme‐normalized V0, where points represent V0 / E0 (± SE), and lines and shaded 95% confidence interval show fit with hyperbolic Eq. 6 to obtain kcat / KM and kcat (see Fig. 25).

[0106] Fig. 20 illustrates activation of crRNA‐Cas12a RNPs by short dsDNA targets. Figs. 20a and 20b show activation time courses for Cas12a RNP‐A2 (see Fig.20a) and ‐A19 (see Fig. 20b) induced by their short cognate ds‐ and ssDNA targets, employing Scheme RNP+T,FQ‐S or RNP∙T+FQ‐S with FQ‐C10trans‐substrate. In representative experiments shown, points represent mean (± SE) of background‐corrected replicates (n= 3). Solid lines show fit to activation Eq. 7 to obtain kact and kss for Scheme RNP+T,FQ‐S and fit to linear Eq. 5 to obtain kss for RNP∙T+FQ‐S (see Fig. 30A) plotted in heat maps. Figs. 20c–20f show trans‐cleavage by RNP‐A2 (see Figs. 20c and 20d) or ‐A19 (see Figs. 20e and 20f) using Scheme RNP+T,FQ‐S at 37°C with titrated short (see Figs.20c and 20e) or long (see Figs.20d and 20f) dsDNA targets and 100 nM trans‐substrate FQ‐C10. Signals obtained at 2.0 hr are plotted in Fig.12d. LODs calculated at intervals are plotted in Fig.12e. Fig.20g shows activation time course for RNP‐A1 induced by its short dsDNA target, employing Scheme RNP+T,FQ‐S and RNP∙T+FQ‐S at 25°C. Points represent means (± SE) from background‐corrected replicates (n = 3). Lines show fit to activation equation Eq. 7 to obtain rate constants (± SE) for kact and kss (Scheme RNP+T,FQ‐S) or fit to linear Eq. 5 for kss (Scheme RNP∙T+FQ‐S) (see Fig. 30A). Fig. 20h shows the effect of non‐specific DNA on activation of RNPs using Scheme RNP+T,FQ‐S with FQ‐C10trans‐substrate at 37°C. RNP‐A1 was reacted with 50 pM 40‐bp DNA target (blue symbols), 3.87‐kb non‐ specific plasmid DNA (white) or equimolar mixtures of the two (red) as in Fig. 13e. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq.7 to values (see Fig.31E) plotted in heat maps (see Fig.13e).

[0107] Fig. 21 illustrates the concentration dependence of crRNA‐Cas12a RNP activation by short ds‐ and ssDNA targets, and the effect of RNP concentration on activation rates using Scheme RNP+T,FQ‐S for RNPs activated with short DNA targets. (Top) Pointsin time course represent means (± SE) from background‐ corrected replicates (n = 3) of a representative experiment; lines show fit to activation equation Eq. 7 to obtain kact. (Bottom) Global fitting from multiple experiments, indicated by lines and shaded 95% confidence intervals, was performed with hyperbolic Eq. 8 over the full RNP concentration range to obtain kact (max) and Kd or with linear Eq.9 limited to a low range of RNP from the same experiments (Insets) to obtain kon (app) and koff (net) from slopes and y‐intercepts, respectively (see Fig. 26).

[0108] Fig. 22 illustrates the activation of crRNA‐Cas12a RNP with long dsDNA and short ssDNA targets. Fig. 22a shows the effect of target sequence adjoining the PAM‐ protospacer on target‐induced activation of RNP‐F25, employing Scheme RNP+T,FQ‐ S with FQ‐C10trans‐substrate. Chimeric dsDNA targets were constructed as in Fig. 13c from sequences originating from the target that rapidly activates RNP‐F3 (blue) or slowly activates RNP‐F25 (red) and the TTTT PAM native to the target of RNP‐F25. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7 to obtain kact and kss. Fig. 22b shows the activation time courses for RNPs targeting different protospacers embedded in short, 55‐bp duplexes or a long DNA target (1299‐bp Target‐F) using Scheme RNP+T,FQ‐S and Scheme RNP∙T+FQ‐S with 20 pM target and FQ‐C10 trans‐substrate at 25°C. Points represent mean (± SE) of background‐corrected replicates (n = 3). Solid lines show fit to activation Eq. 7 to obtain kactfor Scheme RNP+T,FQ‐S or fit to linear Eq. 5 to obtain kssfor Scheme RNP∙T+FQ‐S, yielding values (see Fig. 31D) plotted in heat maps (see Fig. 13d).

[0109] Fig.23 illustrates footprints of crRNA‐Cas12a RNP in HPV‐16 and footprints for RNP binding within a region of Target‐E based on exonuclease mapping (16), indicating 20‐ bp TS protospacer (red), 11‐bp PAM‐proximal (yellow) and 5‐bp PAM‐distal (cyan) ends. Underlining indicates the central dipurines of the PAMs utilized by RNPs‐E24– 30 (see Fig. 23a) and the central dipyrimidines of PAMs utilized by RNP‐E18 and ‐ E23 (see Fig. 23b).

[0110] Fig. 24 shows activation of crRNA‐Cas12a RNP with long dsDNA and short ssDNA targets derived from Target‐E and time courses for cleavage of trans‐substrate FQ‐C10by RNPs activated by long, 858‐bp DNA Target‐E (blue symbols) or short, 40‐ntssDNA targets (red symbols). Solid lines and filled symbols represent linear ranges used to calculate kss (Fig. 32A) plotted in Fig.14b. RNPs tested include those utilizing dipurine (E25 and E27) and dipyrimidine (E1 and E19) PAMs.

[0111] Canonical PAMs are neither required nor predictive of high trans‐nuclease activity. The inventors surveyed activity of 120 LbCas12a RNPs designed against protospacers in 858–1378‐bp DNA targets corresponding to cDNA encoding portions (Targets‐A– D) of SARS‐CoV‐2, E6‐E7 oncogenes (Target‐E) from human papillomavirus (HPV), and IS2404 (Target‐F) of Mycobacterium ulcerans, causative agent of Buruli ulcer disease. RNPs are designated by protospacers against which they are designed: e.g., RNP‐A1 recognizes protospacer‐1 of Target‐A. Using trans‐substrate cleavage assays (Fig. 8b) the inventors measured product formation (Figs. 16a–16f) and calculated apparent turnover rates (kapp; Fig. 27), plotted with color‐coding indicating PAM utilization to highlight central dinucleotides of non‐canonical PAMs (Fig. 8c). Select high‐ and low‐activity RNPs exhibit no cross‐reactivity against unrelated targets (Figs. 16g and 16h) except for one (RNP‐A1).

[0112] Most RNPs utilizing canonical TTTV and non‐canonical dipyrimidine PAMs displayed higher activity than those utilizing dipurines PAMs, paralleling thymidine preference with cytosine acceptance in central PAM positions. Maximal turnover rates (0.6–2.6 s‐1) were achieved with dithymidine PAM‐utilizing RNPs, though there was no general rule RNPs utilizing canonical PAMs displayed the highest activities. Thus, canonical PAM utilization is neither required nor predictive of a high‐activity RNP, suggesting there are structural determinants of RNP activity besides the PAM. Across all targets, RNPs displayed trans‐ activities that varied by as much as two orders of magnitude, illustrating the complex influence of target sequence on trans‐cleavage. Because these kapp measurements of trans activity are derived from measurements at a single time point, differences could arise not only from variations in the ability of the fully activated enzyme to cleave substrate (kss), but also from the rate at which the enzyme develops the capacity to cleave substrate (kact) (Fig. 8d).

[0113] Rates of plasmid cleavage parallel trans‐activity. The inventors tested whether trans‐ cleavage activities for representative RNPs track with cis‐cleavage of supercoiled plasmids (see Figs. 9a and 9b). High‐activity Target‐F RNPs (F3, F5, and F6) cleavedplasmid rapidly and completely within 30 min, whereas lower‐activity RNPs (F16 and F25) effected only partial cleavage. Very low‐activity dipurine PAM‐utilizing RNP (F40) failed to cleave DNA. Similar results were seen for Target‐A RNPs, indicating high‐activity RNPs cleave targets more rapidly than lower‐activity ones. Given target cleavage by RNPs utilizing non‐canonical PAMs (A2, F5, and F6) can be on par with canonical PAM‐utilizing RNPs (F3 and A4), canonical PAMs are not required for rapid target cleavage, as observed elsewhere.

[0114] Cis‐ and trans‐cleavage properties of RNPs are activated by DNA and RNA targets. The inventors used 40‐bp targets to investigate enzyme activation further, focusing on high‐activity RNP‐A2, which utilizes non‐canonical PAM CTTC, comparing results to previous findings on RNPs utilizing canonical PAMs and to low‐ activity RNP‐A19, which utilizes non‐canonical PAM ACCA. Using Scheme RNP+FB‐T (see Fig. 9c), the inventors measured the cleavage of ds‐ and ssDNA targets by RNP‐A2 under single‐turnover conditions with dual-labeled targets (FB‐T). As before, since cleavage was too rapid at 37°C for accurate determination, time courses were recorded at 25°C, yielding a dsDNA TS cleavage rate kobs (0.69±0.10 min‐1; see Figs. 9d and 9g) comparable to that for canonical PAM‐utilizing RNP‐F4 (0.72 min‐1). Cleavage of ssDNA TS was indistinguishable (0.72±0.10 s‐1; see Figs. 9e and 9g), and similar rates were obtained when reporter and capture probe positions were swapped (see Fig. 9g).

[0115] Because RNA targets activate Cas12g1 and Cas12a2, and LbCas12a can cleave RNA in trans, the inventors tested whether Cas12a performs cis‐cleavage of RNA targets. Since cleavage was prohibitively slow at 25°C, reaction temperature was raised to 37°C, and target was titrated with RNP‐A2 (see Fig.9f). After 2.0 hr, less than 10% of target was cleaved, revealing a cleavage rate 860‐fold slower than for DNA (see Fig. 9g). Though this RNP binds target RNA with relatively high affinity, exhibiting near stoichiometric target cleavage below nanomolar concentrations, its low target cleavage rate provides further evidence the Cas12a RuvC nuclease is a preferred deoxyribonuclease.

[0116] The inventors measured trans‐cleavage by RNPs activated with short DNA and RNA targets, confirming target specificity of the RNPs (see Fig. 18a). For RNP‐A2, trans‐ activity increased stoichiometrically with titrated target (see Figs. 10a and 10b),indicating high‐affinity binding to both DNA and RNA targets with sub‐nanomolar apparent dissociation constants, confirming complete activation of RNP at levels dictated by target concentrations, a key assumption for steady‐state analysis. Both ds‐ and ssDNA targets induced similar trans‐activities (1.4–2.5 s‐1; see Figs.10a and 10c), higher than observed for protospacer embedded within a long dsDNA target (0.68±0.03 s‐1; see Fig.27). Further, removal of four nucleotides at the 5’ PAM‐distal end to mimic end products of target cleavage had no effect on ssDNA target potency. RNA target‐ induced trans‐activities were considerably lower, but 5’‐truncated RNA targets activated higher rates of trans-cleavage than their longer counterparts. A similar analysis performed using RNP‐A19 revealed despite exhibiting low trans‐activity in response to long dsDNA target (0.0126±0.0001 s‐1; see Fig. 27), it exhibits 160‐fold greater activity in response to short ds‐ or ssDNA targets (2.0–2.2 s‐1; see Fig.10d) and higher RNA target‐induced activity than RNP‐A2. Again, 5’‐truncated RNA targets resulted in considerably higher rates of trans‐cleavage than longer ones. Together, these results demonstrate that the protospacer context and target ribose backbone contribute to activating RNP trans‐cleavage. Further, activating RNP with RNA targets possessing bases beyond presumptive cleavage sites diminishes trans‐activity, suggesting an unprocessed target interferes with trans‐cleavage.

[0117] The inventors measured RNA target‐detection sensitivity using trans‐activity (see Figs. 10e and 10f). As for FB‐C10trans‐cleavage, LODs for synthetic DNA targets were well below picomolar concentrations (see Fig. 18j), 16 and 18 fM (RNP‐A2) and 161 and 116 fM (RNP‐A19) for ds‐ and ssDNA targets. Consistent with stoichiometric analysis, LOD for target RNA‐t by RNP‐A19 (880 fM) was considerably lower than that for RNP‐A2 owing to an 18‐fold higher rate of trans‐cleavage. These results demonstrate, like Cas12a2, Cas12a may be used for direct detection of RNA targets.

[0118] dsDNA targets containing non‐canonical PAMs and ssDNA can trigger the formation of high‐activity trans-nucleases. The inventors performed Michaelis‐Menten analysis to compare kinetic properties of DNA and RNA target‐activated nucleases (see Figs. 10g–10j and Fig. 25). First, using Scheme RNP∙T+FB‐S, the inventors measured FB‐ C10trans‐cleavage by RNP‐A2. As before (see Fig. 10c), ds‐ and ssDNA‐activated RNP exhibited similar turnover (2.8–2.9 s‐1) and catalytic efficiency (4.6–8.7x107M‐1s‐1), comparable to those for canonical PAM utilizing RNP‐F4 and ‐F2. For RNA target‐activated RNP‐A19, >20‐fold lower catalytic efficiency was measured, confirming the influence of the target ribose backbone in effecting trans‐cleavage.

[0119] Using Scheme RNP∙T+FQ‐S, the inventors then measured trans‐substrate FQ‐C10cleavage by various target-activated RNPs (see Fig. 25) at both 37°C and 25°C for comparison with FB‐C10 and DNA target cleavage rates. For dsDNA target‐activated RNPs, kcat / KMwas somewhat higher for FQ‐C10than for FB‐C10at 37°C, suggesting FQ‐C10 records a higher catalytic potential. As observed for FB‐C10, catalytic constants for ssDNA target‐activated RNP‐A2 and ‐A19 were indistinguishable from those for their respective dsDNA target‐activated RNPs. Though temperature reduction to 25°C had little effect on catalytic efficiency, turnover and KMdecreased for all RNP tested, indicating the impact on assay performance near room temperatures.

[0120] These results further confirm that RNPs utilizing non‐canonical PAMs, including those composed of dicytosines, and ssDNA can induce formation of equally high‐activity trans‐nucleases, indicating their catalytically active enzymes possess similar conformations and active sites of comparable chemical architectures. RNPs activated by short or long DNA targets, as exemplified by RNP‐A19, may exhibit widely disparate levels of trans‐activity, indicating targets possess complex determinants for RNP activation.

[0121] Defining activation and steady‐state phases of the trans‐nuclease. Having characterized steady‐state properties of Cas12a trans‐nuclease activated by ds‐ and ssDNA targets, the inventors used Scheme RNP+T,FQ‐S (see Fig. 11a) to investigate activation kinetics. The reaction of RNP‐A2 resulted in a cleavage time course composed of an exponential lag phase followed by a linear phase (see Fig. 11b). The lag phase, composed of a characteristic time on the order of minutes and independent of target concentrations, disappeared when RNP‐A2 was pre‐reacted with the target before incubation with trans‐substrate (see Figs. 11c and 11d). Linear phase slopes were directly proportional to target concentration and, when normalized to target concentrations serving as proxies for activated RNP, yielded steady‐state rates (kss) indistinguishable from each other and those of RNPs pre‐reacted with the target (see Fig. 11d). This steady‐state rate (0.6 s‐1; see Fig. 29) agrees closely with that (0.5 s‐1)predicted by Michaelis‐Menten analysis (see Fig. 24). Together, these results indicate the lag phase represents conversion of RNP to an active nuclease initiated by target binding, which precedes steady‐state trans‐cleavage.

[0122] Defining two steps in target‐induced enzyme activation. Holding the dsDNA target concentration constant and varying RNP concentration resulted in a hyperbolic increase in activation rate that approached a maximum (see Figs. 11e and 11f), suggesting the formation of activated nuclease by target proceeds via two steps. Assuming Step 1 represents rapidly equilibrating, reversible RNP‐target binding, Step 2 represents a slower, reversible first‐order conversion, described as an allosteric transition, of enzyme to an activated state. For RNP‐A2, the affinity of dsDNA target binding was in the low nanomolar range, and its activation plateaued at 0.60±0.09 min‐1, comparable to dsDNA TS cleavage (0.69±0.10 min‐1; see Fig. 9g), consistent with the inventors’ previous determination that activation of trans‐nucleolytic potential is directly coupled to slow cleavage of dsDNA target by Cas12a. The net inactivation rate (koff (net)) from extrapolated y‐intercepts could not be determined with high precision: visual inspection reveals that it is low, considerably smaller than kact (max), which is consistent with single‐ target turnover behavior of Cas12a.

[0123] Rapid activation of trans‐activity arises from high‐affinity binding and rapid conversion to activated enzyme. The inventors measured RNP‐target binding equilibria and allosteric transition rates for ssDNA target-activated RNPs (see Figs.11g.11h, and 26A), observing ssDNA target induces conversion of RNP‐A2 to activated enzyme at a rate (1.1±0.2 min‐1) greater than that observed for dsDNA target (0.60±0.09 min‐1) and slightly higher than ssDNA target cleavage (0.72±0.10 min‐1; Fig.9g). Removal of PAM‐distal nucleotides at 5’ ends of ssDNA targets had no effect on the activation rate. The affinity of binding to ssDNA targets was somewhat higher (Kd 1.1±0.4 and 1.4±0.7 nM) than dsDNA targets (3.4±1.1 nM), and, together with higher allosteric transition rates, hastened activation (kon (app)1.3±0.7 – 1.6±0.7x107M‐1s‐1) above that for dsDNA target (0.30±0.11x107M‐1s‐1). Likewise, RNP‐A19 showed high affinity for ssDNA targets (Kd 0.30±0.25 – 0.36±0.26 nM) and high allosteric transition rates (kact (max) 0.9±0.2 – 1.1±0.2 min‐1), leading to high activation rates (kon (app)5±4x107M‐1s‐1),indicating the faster activation of RNPs by ssDNA targets arises both from high target affinity and rapid conversion to activated enzyme.

[0124] Because activation of RNP‐A19 by dsDNA targets was slow and inefficient (see Figs. 20a and 20b), target and RNP concentrations were increased. Here, activation rates increased linearly without reaching saturation (see Figs. 12a and 12b), indicative of relatively low‐affinity binding (Kd > 20 nM). Nevertheless, estimates (koff 0.043±0.001 min‐1; see Fig.26B) suggest even these RNP‐target complexes are relatively long‐lived, with lifetimes of 23 minutes. Though estimates for kact (max) could not be determined, the apparent rate for activating RNP‐A19 by its dsDNA target (kon (app) 5.1±0.1x104M‐1s‐1) was 1000‐fold smaller than for ssDNA targets.

[0125] The inventors then compared time courses for activation of RNP‐A2 and ‐A19 by long DNA targets (see Figs.12c and 30B). As for short dsDNA, activation of RNP‐A19 with long DNA targets was slow, but even prolonged target pre‐activation failed to generate high‐activity RNP. Thus, though both RNP‐A2 and ‐A19 exhibit capacity for formation of high catalytic efficiency trans‐nucleases (see Fig. 25), rates at which their targets activate these RNPs differ considerably (see Fig. 26). This confirms at least some variation in RNP performance may be explained by timescales of enzyme activation. Such differences profoundly affect the rates at which limits of detection are achieved (see Figs. 12d‐12f).

[0126] Protospacer influences the rate of trans‐nuclease activation. The inventors determined the RNP concentration dependence of five additional RNPs activated by protospacers within short, 55‐bp dsDNA targets (see Fig.26), including four (F3, F47, F41, and F23) utilizing canonical PAMs and one (F25) utilizing non‐canonical PAM TTTT. As before, net inactivation rates were very small compared to maximal activation rates, which for all RNPs except F25 were comparable to RNP‐A2 activation by short dsDNA. These rates falling somewhat below those for ssDNA targets (1.1 min‐1) suggests the latter represents an upper limit for the enzyme at 25°C. As for RNP‐A2 (see above), dsDNA kact (max) for RNP‐F4 (0.81±0.14 min‐1) was similar to target cleavage (kobs 0.72 min‐1), again confirming that the appearance of trans‐activity is coupled to dsDNA target cleavage. In contrast, kact (max)for RNP‐F25 was at least 9‐ fold lower than the others, paralleling its slow cleavage of plasmid DNA target and lowtrans‐activity (see Fig. 9a), though as will be shown, utilization of a non‐canonical PAM only partly accounts for its slower activation. These results indicate that even for RNPs utilizing dithymidine PAMs, apparent rates of activation by short dsDNA targets can vary widely, mainly due to differences in allosteric transition rates.

[0127] To assess the protospacer contribution to RNP activation and trans‐cleavage, the inventors tested the activation of several RNPs with their protospacers embedded within a backbone of the target recognized by canonical PAM‐utilizing RNP‐F3 (see Fig. 13a), since this target rapidly activates RNP trans‐activity (see Fig. 26). The inventors chose protospacers for RNPs (F20, F25, F27, and F32) weakly activated by long DNA targets, with all but RNP‐ F25 utilizing canonical PAMs. Though steady‐ state rates of trans‐cleavage activated by chimeric targets were within a factor of three of that for RNP‐F3, rates for activating these RNPs were 5‐ to 20‐fold lower (see Fig. 31A), indicating protospacer sequences predominantly influence activation rates. Additionally, the inventors observed that RNP‐F25 is more rapidly activated by its protospacer in this chimeric target (kact 0.22±0.01 min‐1) than in its native context (0.079±0.008 min‐1; see Fig. 26), suggesting sequences immediately outside the protospacer also influence RNP activation, an effect the inventors investigate below.

[0128] PAM sequence influences the rate of trans‐nuclease activation. The inventors probed the influence of PAM sequence on RNP activation and trans‐cleavage by testing individual substitutions of the four nucleotides in the canonical TTTC PAM of RNP‐ F3 (see Fig.13b). Substitution at positions ‐1*, ‐2*, and ‐4* reduced the activation rate 9‐ to 67‐fold but had little effect on steady‐state rates of target‐induced trans‐cleavage, resulting in kss comparable to the original target‐RNP combination. In contrast, substitution at position ‐ 3* slowed the activation rate 18‐fold and resulted in reduced trans‐cleavage activity. Even after prolonged pre‐incubation of RNP with this target, steady‐state trans‐cleavage was reduced 20‐fold, suggesting this substitution might affect both enzyme activation and trans‐cleavage rates. Together, these results confirm that the four PAM bases each contribute to the determinants of RNP activation by dsDNA targets.

[0129] Target sequence flanking the 5’ end of protospacers influences the rate of trans‐ nuclease activation. Having observed that RNP‐F25 is more rapidly activated by itsprotospacer in a chimeric target than in its native context, the inventors probed the influence of target sequences immediately outside the protospacer and PAM (see Fig. 13c). Chimeric dsDNA targets containing the protospacer recognized by RNP‐F25 were constructed from flanking sequences of targets for RNP‐F3 and ‐F25. Converting non‐canonical TTTT PAM to a canonical TTTC sequence had little or no effect on the rate of RNP‐F25 activation. However, for chimeric targets, the presence of PAM‐distal sequences outside the protospacer, specifically the first 10‐ bp, of target‐F3 promoted more rapid target‐induced activation of RNP‐F25 than those containing native sequences of target‐F25. Rate enhancement by this F3‐sequence was 4‐ to 5‐fold for targets containing canonical PAMs (see Fig. 13c) and 2‐ to 4‐fold for non‐canonical PAMs (see Fig. 22b), indicating determinants of target‐induced RNP activation reside within the first 10‐bp immediately adjacent to the protospacer PAM‐distal end. Thus, the relatively slow activation of RNP‐F25 by its native target (see Fig. 26) arises from the combined determinants of protospacer and sequences immediately adjacent to its PAM‐distal end, not by its utilization of a non‐canonical PAM (see Figs.13a and 13c).

[0130] Protospacer context influences activation and steady‐state rates of trans‐cleavage. The inventors tested the effect of protospacer context on RNP activation by comparing several RNPs activated by protospacers embedded within 1299‐bp DNA fragments or 55‐bp duplexes (see Fig. 13d). Activation rates were slowed when protospacers were embedded within long DNA targets, as were steady‐state rates, for all but RNP‐25. Likewise, the addition of non‐specific plasmid DNA slowed rates of RNP activation by short dsDNA targets and reduced steady‐state trans‐cleavage (see Fig. 13e). These results suggest non‐specific DNA, provided in cis (on the same fragment as protospacers) or in trans (elsewhere), inhibits by slowing activation rates, possibly by providing sites for non‐productive encounters, thus reducing the apparent affinity of RNP‐target binding. Further, non‐specific DNA might additionally reduce trans‐ cleavage by competing for the binding of ssDNA trans‐substrates, consistent with previous findings that Cas12a can cleave dsDNA trans‐substrates, albeit poorly.

[0131] Target DNA supercoiling hastens trans‐nuclease activation, especially for non‐ canonical PAM‐utilizing RNPs. Target context was further tested by examining the effect of plasmid supercoiling on activation for RNPs of varying trans‐activities andPAM utilization (see Fig.13f). Paralleling the cleavage of plasmid DNA (see Figs. 9a and 9b), RNPs exhibiting high trans‐activity (A1, A2, A4, and A6) are more rapidly activated by plasmid DNA than RNPs of lower activity (A8 and A19) (see Fig. 13f). As for activation by long DNA targets (see Fig. 12c), RNP‐A19 was activated by plasmid considerably more slowly and less efficiently than RNP‐A2. Canonical PAM‐ utilizing RNP‐A6 and ‐A4 were more rapidly activated than all other RNPs, and supercoil relaxation by site‐specific nicking had little effect on their activation, decreasing rates by less than 2‐fold, but decreasing activation rates for non‐canonical PAM‐utilizing RNPs by as much as 8‐fold. These results indicate that target‐induced RNP activation rates are influenced by the supercoiling of the DNA in which cognate protospacers are embedded, as previously shown for target cleavage itself.

[0132] Determinants of trans‐nuclease activation by ssDNA targets. The inventors surveyed the trans‐activity of Target‐E RNPs activated by synthetic 308‐ and 494‐nt ssDNA targets (see Fig. 14a). Control RNPs targeting protospacers absent from both ssDNA targets failed to be activated by either ssDNA, and seven RNPs were activated by correct, but not incorrect targets. However, two RNPs (E23 and E27) failed to be activated by their expected target. These two RNPs target overlapping protospacers within the long ssDNA (see Figs.23a and 23b): RNP‐E23 utilizes a dipyrimidine PAM and is activated by dsDNA, whereas RNP-E27 utilizes a dipurine PAM and is not activated by dsDNA but can be activated by 40‐nt ssDNA (see Fig. 14b). Given the capacity of these two RNPs to form high‐activity trans‐nucleases, the inventors speculate that formation of secondary structure within or proximal to their protospacers in long ssDNA interferes with activation. In addition, since three RNPs (E24, E25, and E29) activated by ssDNA‐E(494) target protospacers flanked by dipyrimidines in positions ‐2 and ‐3 of the TS PAM and exhibit trans‐cleavage activities comparable to RNPs (E21 and E18) targeting protospacers flanked by TS PAMs containing dipurines (see Fig.14a), there is no requirement for dipurines in central positions of the TS PAM. This is as expected if activation by ssDNA targets is PAM‐indifferent. Since high activation rates and similar steady‐state trans‐activities are exhibited by dipurine‐ and dipyrimidine PAM‐utilizing RNPs (see Fig.14c), these results provide further evidence that dipurines in central positions of the PAM (TS positions ‐2 and ‐3) are not requiredfor rapid activation by ssDNA. Further, Michaelis‐Menten analysis reveals that ssDNA‐activated RNP‐E27 exhibits a catalytic efficiency on par with other RNPs (see Fig. 25).

[0133] These disclosed experimental results provide insight into Cas12a activation, indicating that in addition to the steady‐state properties of the mature enzyme, the timescale of enzyme activation is a critical determinant of RNP performance. Different RNP‐target combinations exhibit varying activation profiles and ds‐ and ssDNA targets possess different structural determinants for RNP activation besides the protospacer (see Fig. 15a).

[0134] These results indicate that two parameters define Cas12a assay performance: first, activation time, which depends upon RNP‐target affinity and a first‐order transition that ultimately limits activation; and second, steady‐state trans‐cleavage rate. Assumptions that activation is fast and the trans‐cleavage step is rate‐limiting justified focus on steady‐state properties of activated RNPs, measured using short synthetic dsDNA targets, as the sole determinant of assay sensitivity. However, several lines of evidence suggest the timescale of RNP activation plays a central role. First, the appearance of trans‐cleavage is temporally coupled to target cleavage, as expected, since both use the same catalytic site, which is on the order of minutes. Second, though different RNPs may cleave trans-substrates with comparable catalytic efficiencies, rates at which they are activated may vary as much as 10‐fold or more. Third, catalytic efficiencies can exceed 108M‐1s‐1and turnovers are on the order of seconds, considerably shorter than activation times. Therefore, enzyme activation is slow compared to trans‐substrate turnover, and a more complete picture of assay performance takes into account activation time, especially since protospacer context (i.e. form of the target encountered in the diagnostic assay) impacts both activation and steady‐state properties. Modeling reveals that both enzyme activation time and catalytic efficiency directly impact the time required for detecting targets (see Fig. 15b). Developers may better optimize assay performance by taking into account determinants of both properties and how assay conditions impact them.

[0135] Target‐induced conversion of RNP to an active nuclease involves multiple steps, including an initial PAM-mediated search for the correct protospacer and the ensuingallosteric transition accompanying changes in DNA structure. For short dsDNA, bimolecular rates representing upper limits for RNP‐target binding approach 107M‐1s‐1 or more, comparable to those for LbCas12a (1.3x107M‐1s‐1) and AsCas12a (~108M‐1s‐1). These are well below the diffusion limit (~109–1010M‐1s‐1), demonstrating the magnitude by which post‐binding steps diminish the productivity of the RNP‐target encounter. This encounter is rendered even more unproductive when protospacers are embedded within longer DNA targets or presented in non‐specific DNA backgrounds, as observed elsewhere, conditions that likely reflect those encountered in a diagnostic context. Though such non‐productive, transient RNP encounters with off‐target sites, which have been directly visualized, may be effectively overcome with non‐limiting RNP concentrations, the net rate remains limited by the first-order conversion to the activated enzyme.

[0136] The composition of the target determines both the coupling of target cleavage to trans‐ activity and catalytic efficiency. Trans‐cleavage appears slightly faster than ssDNA TS cleavage, suggesting for some ssDNA targets, trans‐cleavage might commence prior to TS cleavage, which would be consistent with previous observation that RNPs activated by non‐cleavable ssDNA targets retain high trans‐activity. For RNA targets, cleaved 1000‐fold more slowly than DNA counterparts, removing four nucleotides from 5’ ends to mimic cleaved TS fragments greatly enhances trans‐cleavage, suggesting intact portions of longer targets interfere with trans‐substrate accessibility. For dsDNA targets, trans‐activity appears at the same rates as TS cleavage, suggesting both events are tightly coupled and that full trans‐activity is unleashed only upon target cleavage and, by implication, dissociation of the PAM‐distal segment of the dsDNA target. This requirement might explain why sequences within 10 bases immediately adjacent to protospacer PAM‐proximal ends can affect RNP activation, given that the stability of base pairs and subsequent DNA distortions flanking the PAM‐distal region of the R‐loop is linked to TS cleavage. Since after NTS cleavage, TS cleavage commences only upon the formation of a transient but stable clamped state involving DNA sequences downstream of the R‐loop that serve to reorient the TS for cleavage, the formation of the intermediate state may be affected by this 10‐bp sequence.

[0137] The target ribosyl backbone influences the activated enzyme's rates of both target and trans‐substrate cleavage. As DNA target‐activated LbCas12a may cleave RNA in trans, the inventors observe cis‐ and trans‐cleavage from RNA targets—an activity not previously recorded for LbCas12a— possibly due to the use of shorter, less efficient reporters. RNA targets are cleaved at least 1000‐fold more slowly than DNA, which parallels the 100‐fold lower catalytic efficiency for ribosyl trans‐substrates and provides further evidence that LbCas12a is a preferred DNase. Since RNA target‐ activated LbCas12a also cleaves ssDNA trans‐substrates at reduced efficiencies, target backbone 2’ hydroxyls may additionally introduce allosteric changes in the activated nuclease or interfere with trans‐substrate access. Despite the potential for sub‐ picomolar LOD for RNA targets, a sensitivity that outperforms values for Cas12a2, LbCas12a may not be well‐suited for direct detection of long RNA targets, such as mRNA, without modification owing to RNA secondary structure and suppression of trans‐cleavage by target 5’ extensions. However, shorter RNA targets, such as microRNAs, might be amenable for detection. Other strategies for RNA detection by Cas12a have been devised, although sensitivity is not as great as observed here.

[0138] Protospacer sequences influence Cas12a activation rates and account for at least 20‐ fold variation in activation timescales, with lesser effect on steady‐state rates of trans‐ cleavage. Since Cas12a makes no direct sequence‐specific protospacer contacts except through the spacer, either sequence may indirectly (as secondary structure formation within crRNA) or directly influence multiple steps in activation, including R‐loop initiation and propagation and target cleavage. Since all R‐loop positions contribute to the rate of target binding to AsCas12a, and reversible propagation of R‐loop formation must pass through a late transition state for target cleavage, differences in transition state height or timing might modulate net rates of activation demonstrated by different protospacers.

[0139] DNA supercoiling affects LbCas12a activation, an effect more pronounced for lower- activity RNPs utilizing non‐canonical PAMs. This observation recapitulates the enhancement of LbCas12a target cleavage by negative DNA supercoiling brought on by faster R‐loop formation. Since upon DNA binding, LbCas12a undergoes a structural rearrangement that is responsive to the degree of helix distortion within the PAMduplex, supercoiling may accelerate RNP activation by stabilizing favorable conformations in either enzyme or PAM that increase binding affinity or accelerate DNA unwinding.

[0140] The PAM sequence is important for enzyme activation by ds‐ but not ssDNA or RNA. For a canonical PAM‐utilizing RNP targeting dsDNA, the inventors observe that individual substitution at each PAM position decreases enzyme activation rates, as observed elsewhere for cleavage of the target and activation of trans‐cleavage. The striking preference of LbCas12a for the canonical sequence TTTV sequence, both for cleavage of targets in vitro and for in vivo gene editing, arises from the high stability of the LbCas12a‐target complex: changes in PAM sequences decrease RNP affinity due to alterations in shape‐ and sequence‐specific interactions driving enzyme conformational changes. Rapid, PAM‐indifferent activation by ssDNA likely arises from greater target flexibility to affect the allosteric transition. Although the inventors observe that RNPs utilizing PAMs containing central dipurines are only weakly activated by dsDNA targets, RNPs may target protospacers with non‐canonical PAMs, as a canonical PAM is not absolutely required for cleavage of target or trans‐substrate. These results support the proposal that NYYN PAM sequences can be effectively used for in vitro applications relying on LbCas12a, expanding the repertoire of protospacers amenable to Cas-mediated detection.

[0141] These findings help account for the wide range of RNP trans‐cleavage activities observed and provide guidance in the design of rapid, Cas‐based diagnostics. RNPs displaying the greatest trans‐activities exhibit the highest rates of target‐induced activation and steady‐state trans‐cleavage, and a complex set of determinants influences both. The fastest activation rates are achievable at ~1 min‐1, necessitating at least 5 min for the development of full catalytic activity, which may surpass 108M‐1s‐1. Still, even this capacity may be limited by other factors, including protospacer context and the presence of non‐specific DNA. RNPs utilizing consensus PAMs may be able to achieve high activation rates owing to high-affinity target binding (Kd < 1 nM) and rapid R‐loop formation. Still, unfavorable determinants, including those within the protospacer and PAM‐distal segment, may suppress rates. Some deviations from the consensus PAM are tolerable, enabling fast activation owing to encounterswith favorable determinants, though others slow activation by reducing target‐binding affinity. For all RNPs, activation may be further modulated by other target‐imposed constraints, such as protospacer context, competing non‐specific DNA, and DNA topology, each of which must be considered when analyzing biological samples.

[0142] MATERIALS AND METHODS: Reagents. crRNA and RNA targets were purchased from Synthego. Single‐stranded oligonucleotides and HPLC‐purified oligonucleotide duplexes were purchased from Integrated DNA Technologies (IDT; Coralville, IA). Double‐stranded (gBlocks Gene Fragments) and single‐stranded (Megamer® ssDNA Fragment) DNA fragments were purchased from IDT and GenScript (Piscataway, NJ). Nucleic acids were quantified using UV spectrophotometry based on extinction coefficients provided by the manufacturer or, for dsDNA, calculated elsewhere (http: / / molbiotools.com / dnacalculator.html). Sequences of short and long synthetic nucleic acids are given in Figs. 33 and 34. Sources of plasmids are given in Fig. 35. Plasmid DNA was quantified using Qubit™ 1X dsDNA High Sensitivity Kit (cat. no. Q33230) purchased from Thermo Fisher Scientific (Waltham, MA). Recombinant Lba Cas12a (cat. no. M0653T), Nb.BtsI (cat. no. R0707), and 6X Purple Gel Loading Dye (cat. no. B7024S) were purchased from New England BioLabs (Ipswich, MA). Dynabeads™ MyOne™ Streptavidin C1 (cat. no. 65002) were purchased from ThermoFisher.

[0143] RNP assembly.200 nM Cas12a was incubated with 100 nM crRNA in 1X Cas12 assay buffer (10 mM Tris‐ Cl pH 7.5, 10 mM MgCl2, 1.0 mM Tris(2‐carboxyethyl)phosphine hydrochloride, 0.01% Igepal CA‐630, 40 µg / mL BSA) for at least 30 min at room temperature and diluted immediately before reaction with substrates. All subsequent cleavage reactions were conducted in 1X Cas12 assay buffer.

[0144] Trans‐substrate cleavage reactions. To measure trans‐cleavage using Scheme RNP∙T+FB‐S (see Fig. 8b), RNP and targets were pre‐reacted at 37°C for 30 min prior to reaction with dual fluorophore‐biotin capture tag‐labeled trans‐substrate FB‐C10in a final volume of 20 µL at 37°C. For endpoint measurements, at the desired times, unreacted trans‐substrate was removed by bead capture using 100 µL of 1.0 M NaCl, 10 mM Tris 7.5, 10 mM EDTA, 0.01% Tween‐2 containing 10 µg of streptavidin‐ conjugated magnetic beads, and products remaining in supernatants were quantified(see Fig.8c, 10a–10d, 16a–16e, 16g, 29b, and 29c). For the Michaelis‐Menten analysis, reactants were temperature-equilibrated prior to the reaction. Signals were corrected for background recorded in cleavage reactions containing RNP without target (see Figs. 10g–10j and 30a–30c). To measure trans‐cleavage using Scheme RNP∙T+FQ‐S (Fig. 8b), RNP and targets were pre‐reacted at 37°C for 30 min prior to reaction with dual fluorophore‐quencher tag‐labeled trans-substrate FQ‐C10 in a final volume of 20 µL at 37°C. For endpoint measurements, at the desired time, 5.0 uL of a stop solution consisting of 20 mM Tris 7.5, 100 mM EDTA, 0.01% Tween‐20.01% was added, and fluorescence was recorded (see Figs. 8c, 14a, 16f, and 16h). For Michaelis‐Menten analysis, reactants were temperature equilibrated for several minutes prior to mixing, after which reactions were continuously monitored, and signals were corrected for background recorded in cleavage reactions containing RNP without target (see Figs. 19d–19q). In other trans‐substrate cleavage reactions using FQ‐C10 trans‐substrate, RNP, target, and trans‐substrate were mixed at room temperature with (see Figs. 10e, 10f, 18a, and 18d–18i) or without (see Figs. 14b and 24) dedicated pre‐activation of RNP with the target, and then incubated at 37°C, and reaction products were monitored continuously.

[0145] Target‐cleavage reactions. For cleavage of plasmid DNA targets (see Figs. 9a, 9b, and 17a–17c), 5.0 nM RNP and 2.0 nM plasmid were equilibrated at the desired temperature, then mixed to initiate the reaction. At desired time points, 20 µL aliquots were removed, and reactions were terminated by mixing with 4.0 µL of 6X Gel Loading Dye to achieve 0.08% SDS and 10 mM EDTA (final). 10 µL was applied to 1.5% agarose / TAE gels containing 7.5 µg / mL ethidium bromide. After electrophoresis for 2.0 hr at 75V, gels were imaged by fluorescence with UV light illumination on a Gel Doc EZ Gel Documentation System (Bio‐Rad). Images were processed using Image Lab™ Software. For illustrative purposes, image displays were inverted. Background‐ subtracted band intensities were quantified using volume tools.

[0146] For cleavage of short ds and ssDNA targets (Figs. 9d–9g and 17d) using Scheme RNP+FB‐T (Fig.9c), RNP and dual fluorophore‐biotin capture tag‐labeled targets were equilibrated to 25°C then mixed to initiate reaction in a final volume of 100 µL. At desired time points, 10 µL aliquots were removed and mixed with 20 µL of stopsolution (90% formamide, 20 mM EDTA). Unreacted targets were removed by bead capture as described above. Signals were corrected for background recorded in target reactions containing enzymes lacking crRNA. For cleavage of RNA target (see Figs. 9f and 9g), RNP dual fluorophore-biotin-labeled targets were incubated for 2 hr at 37°C prior to removal of unreacted substrate by bead capture.

[0147] Trans‐nuclease activation reactions. To record the time course of trans‐nuclease activation (see Figs.11b–11h, 12, 13, 14c, 31, 32, and Fig.33) via Scheme RNP+T,FQ‐ S (see Fig.11a), temperature‐ equilibrated RNP and solutions containing the target and FQ‐C10 trans‐substrate were mixed to initiate the reaction. For the corresponding control experiments to measure trans‐cleavage by pre‐activated RNP (see Figs. 11c, 11d, 12a -12d, 20, and 22b) via Scheme RNP∙T+FQ‐S (see Fig. 8b), RNP and targets were pre‐reacted at 37°C for 30 min prior to reaction with trans‐substrate. In both schemes, reaction products were monitored continuously at the desired temperature. Where indicated, signals were corrected for background recorded in cleavage reactions containing RNP without the target. For testing the effect of DNA topology (see Fig. 13f), 1 µg of plasmid DNA was digested with 10 U Nb.BtsI in NEBuffer2 for 2.0 hr at 37°C, then the enzyme was heat‐inactivated for 20 min at 80°C. Control plasmid was treated similarly, except that nuclease was omitted and used as a source of supercoiled plasmid.

[0148] Fluorescence quantification of reaction products. Alexa488 content of samples was determined on a BioTeK Synergy H1 fluorescence microplate reader using λex490 nm and λem 525 nm. The concentration of the product was calculated by converting fluorescence values (RFU) to molarity by interpolation to linear standard curves. Uncleaved target and FB‐C10were used as standards for reactions employing substrate‐ capture. Cleaved substrate FQ‐C10 standard was prepared by digesting 1.0 µM of FQ‐ C10 with 1.0 nM of target‐activated RNP for 2.0 hrs at 37°C with cleavage reaction monitoring in real‐time to confirm that the reaction had progressed to completion.

[0149] DATA ANALYSIS. Trans‐substrate cleavage. Target‐dependence of product formation over low [Target] relative to RNP (see Figs. 8c, 10e, 10f (insets) and 16a– 16f) was fitted with the 2‐parameter linear equation: [Product] = k[Target] + Y^Eq. 1where k represents the rate of trans‐substrate cleavage and Y0is an offset; kapp, the apparent turnover rate, was calculated by dividing k by the assay duration. Target concentration‐dependence of product formation over stoichiometric ranges of [Target] (see Figs.10a, 10b, 18b, and 18c) was fitted with the 3‐ parameter hyperbolic equation: [Product]=Y ^[Target]max [Target]^K^^^ + Y0Eq. 2 ^where parameters are as 1, and the difference in Ymaxand Y0represents the net target‐normalized product formed, which, when divided by assay duration, represents kapp.

[0150] Target cleavage. For cleavage of plasmid targets (see Figs. 9a, 9b, and 17a–17c), time (t)‐dependence of cleaved product formation (background‐corrected band intensity) was fitted with the 3‐parameter single‐exponential equation: [Product] = (Y^− Y^)e(-kobst)+ Y^Eq. 3 where kobsis the rate Yfis the final product, and Y0is anoffset.

[0151] For cleavage of ds and ssDNA targets (see Figs. 9d, 9e, and 17d), time (t)‐dependence of product formation was globally fitted with Eq. 3 utilizing shared parameters from data collected in independent experiments. For cleavage of RNA target (see Fig. 9f), RNP‐dependence of product formation was fitted with the 3‐parameter hyperbolic equation: [Product] ^[RNP]^ + Eq. 4 where Ymaxis theformed at the endpoint, K1 / 2is the midpoint (approximating the apparent dissociation constant of the RNP‐target complex), and Y0 is an offset; kobs was calculated by dividing Ymax by the assay duration.

[0152] Steady‐state analysis of trans‐substrate cleavage. Time‐dependence of product formation was fitted with the 2‐parameter linear equation: [Product] = vt + Y0 Eq. 5where v is the reaction velocity, and Y0is an offset. For Michaelis‐Menten analysis (see Figs. 10g, 10i, 19), v represents the rate of the initial velocity (V0). V0 was normalized to the concentration of activated enzyme E0 approximated by the target concentration, which is justified given the high apparent affinity of RNP for the target, as revealed by stoichiometric analysis. The substrate (S)‐dependence of V0 / E0 (see Fisg. 10h, 10j, and 19) was fitted with Johnson’s 2‐parameter adaptation of the Michaelis‐Menten equation: ^^^= k!"#[!]$^ %&'[!]⁄ %()*, Eq. 6 to directly (kcat) and the specificity constant (kSP) equal tocatalytic efficiency (kcat / KM), which is treated as a single parameter. The Michaelis‐ Menten constant (KM) was calculated by dividing kcat / KM by kcat. This approach provides the same kinetic constants as those obtained by applying the conventional Michaelis‐Menten equation. Still, it provides more accurate values for catalytic efficiency by avoiding over-estimating errors when calculated from kcat and KM. Results from all Michaelis‐Menten analyses were congruent with consistency criteria.

[0153] In other experiments (see Figs.14b and 24), steady‐state phases of trans‐substrate cleavage reactions were identified and fitted with the 2‐parameter linear equation (see Eq. 5) to obtain reaction rates; in some cases, rates were normalized to the target concentration approximation of activated RNP to obtain kss.

[0154] Trans‐nuclease activation reactions. Time‐dependence of the product formation in experiments utilizing Scheme RNP+T, FQ‐S (Figs. 11b, 11e, and 11g, Figs. 12a and 12c, Fig. 13, Fig. 14c, Figs. 20a, 20b, 20g, and 20h, Fig. 21, and Fig. 22) was fitted with a 3‐parameter activation equation, described previously as a process leading to a steady‐ state: =k2#t+e(-k1t)-1+ Y07 where k2 isk1 is an apparent rate for RNP activation (kact), manifested as a lag in the time course, and Y0is an offset; ksswas calculated by dividing k2 by the target concentration approximation of activated RNP.For Scheme RNP∙T+FQ‐S (see Fig. 11c, 12c, 13a, 13b, 13d, 20a, 20b, 20g, and 22b), where no lag was apparent, data were fitted with Eq.5, and kss was similarly calculated.

[0155] For processes displaying saturation kinetics (see Figs. 11f, 11h, and 21), RNP‐ dependence of kactover wide ranges of [RNP] from multiple independent experiments was globally fitted with the 2‐parameter hyperbolic equation: kact= kact (max)#[RNP][RNP]^K-, Eq. 8

[0156] with shared where kact (max) represents themaximal activation rate, and Kdis the dissociation rate constant for RNP binding to target; the bimolecular rate of RNP activation (kon (app)) was calculated from kact (max) / Kd. RNP‐dependence of kact over low [RNP] (see Figs. 11f, 11h(insets), and 21) was likewise global fitted with the linear equation: kact= k. / (011)[RNP]+ koff(net)Eq. 9 with kon (app) is the bimolecularrate of RNP activation koff (net) is the net inactivation rate. For the slower process showing no signs of saturation kinetics (see Fig. 12b), RNP‐dependence of kactover wide ranges of RNP concentrations was globally fitted with Eq. 9.

[0157] Limits of detection. Analytical limits of detection (LOD) and 95% confidence intervals (see Fig. 12e and 18j) were calculated with a four‐parameter logistic fit of raw fluorescence values recorded after 2 hr (see Figs.18d–18i) or at intervals over 2 hr (see Figs. 20c‐20f).

[0158] Simulation of trans‐nuclease activation reactions. Time‐dependence of product formation was simulated (see Fig. 16c) by joining expressions for enzyme activation (see Eq. 7) and steady‐state trans‐cleavage (see Eq. 6): oduct =[target] k[ (-kactt)1 [Pr ]!] e!", #t+ - Eq. 10 The model usesof kcat= 1.0 s‐1, kSP= 108M‐1s‐1, and KM= 10 nM, representing those obtained at higher temperatures, and assumes 100 nM trans‐substrate. For each target concentration, kact was varied, and the time required to achieve 1% cleavage of trans‐substrate was determined. Steady‐state parameters were varied to reflect hypothetical changes brought about by the pooling of crRNA (10‐fold increase in kcat), the presence of competitive inhibitors of the trans‐cleavage reaction (10‐fold increase in KM), and reduced temperature (10‐fold decreases in both kcat and KM) (Fig. 25).

[0159] Any of RNA target detection method components described herein can be used together with a set of instructions, i.e., to form a kit. The kit may include instructions for use of the component as for detection and or diagnosis as described herein.

[0160] The invention illustratively disclosed herein suitably may explicitly be practiced in the absence of any element which is not specifically disclosed herein. While various embodiments of the present invention have been described in detail, it is apparent that various modifications and alterations of those embodiments will occur to and be readily apparent those skilled in the art. However, it is to be expressly understood that such modifications and alterations are within the scope and spirit of the present invention, as set forth in the appended claims. Further, the invention(s) described herein is capable of other embodiments and of being practiced or of being carried out in various other related ways. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items, while only the terms “consisting of” and “consisting only of” are to be construed in the limitative sense.

Claims

Wherefore, I / we claim:

1. A method for detection of ribonucleic acid targets, comprising: assembling a ribonucleoprotein by incubating a Cas12a enzyme with a guide ribonucleic acid in a detection buffer; reacting the ribonucleoprotein with a target ribonucleic acid; reacting the ribonucleoprotein with the target ribonucleic acid and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer; and detecting and recording a resulting signal.

2. The method of claim 1, wherein assembling the ribonucleoprotein comprises incubating 200 nM Cas12a enzyme with 100 nM of the guide ribonucleic acid for about 30 minutes at or around room temperature.

3. The method of claims 1 or 2, wherein the detection buffer comprises: 10 mM Tris pH 7.5; 10 mM MgCl2; 1 mM TCEP; 0.01% Igepal CA630; and 40 μg / ml bovine serum albumin.

4. The method of any of the preceding claims, further comprising diluting the ribonucleoprotein into the detection buffer to 4 nM.

5. The method of any of the preceding claims, wherein reacting the ribonucleoprotein with the target ribonucleic acid comprises reacting at 2 nM for a length of time between 30 – 60 minutes at or around a temperature of 37°C.

6. The method of any of the preceding claims, wherein the trans-substrate is at a concentration of one of 50 nM and 200 nM or substantially 100 nM.

7. The method of any of the preceding claims, wherein the polymer separating the quencher molecule and the reporter molecule comprises greater than five cytosine or thymidine residues.

8. The method of claim 7, wherein the polymer is at least 10 cytosine or thymidine residues long.

9. The method of any of the preceding claims, wherein the reporter molecule is selected from the group consisting of: a luciferase enzyme, a phosphatase enzyme, a cleavage enzyme, peptidase, protease, glycase, lipase, nuclease, endonuclease, restriction endonuclease, a Cas protein, horseradish peroxidase, beta galactosidase, glucose oxidase, α-amylase, β-amylase, a fluorescentprotein, a fluorescent molecule, a luminescent molecule, a luminescent protein, beta- glucuronidase, a fluorescein dye, an Alexa Fluor, a quantum dot, a quantum nanodot, a restriction endonuclease, a heterocyclic small molecule, a carbocyclic small molecule, an aliphatic small molecule, a heteroaliphatic small molecule, an inorganic species, an organometallic species, a radioactive molecule, and a nanoparticle.

10. The method of any of the preceding claims, wherein detecting and recording the resulting signal comprises measuring fluorescence.

11. The method of any of claims 1 to 9, wherein detecting and recording the resulting signal comprises visualizing on a lateral flow readout.

12. The method of any of the preceding claims, wherein the target ribonucleic acid is a pathogen nucleic acid sequence.

13. The method of any of claims 1 to 11, wherein the target ribonucleic acid is a microRNA.

14. The method of any of the preceding claims, further comprising using the resulting signal to detect the presence of a human disease.

15. A kit for performing the method of claim 1, comprising: a Cas12a enzyme; a guide ribonucleic acid; a detection buffer; and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer.

16. The kit of claim 15, wherein the detection buffer comprises 10 mM Tris pH 7.5, 10 mM MgCl2, 1 mM TCEP, 0.01% Igepal CA630, and 40 μg / ml bovine serum albumin.

17. The kit of claims 15 or 16, wherein the polymer separating the quencher molecule and the reporter molecule comprises greater than five cytosine or thymidine residues.

18. A system for performing the method of claim 1, comprising: a reaction chamber configured to maintain a temperature of about 37°C; a fluorescence detector; and a processor configured to record and analyze fluorescence signals.

19. The system of claim 18, further comprising a display for presenting detection results.

20. A system for performing the method of claim 1, comprising a lateral flow readout device.

21. A method for detection of ribonucleic acid targets, comprising:assembling a ribonucleoprotein by incubating 200 nM Cas12a enzyme with 100 nM of a guide ribonucleic acid for about 30 minutes at or around room temperature (~22°C) in a detection buffer, wherein the detection buffer comprises 10 mM Tris pH 7.5; 10 mM MgCl2; 1 mM TCEP, 0.01% Igepal CA630; and 40 μg / ml bovine serum albumin; diluting the ribonucleoprotein into the detection buffer to 4 nM; immediately after dilution, reacting the ribonucleoprotein at 2 nM with a target ribonucleic acid for a length of time between 30 – 60 minutes and at or around a temperature of 37°C; reacting the ribonucleoprotein with the target ribonucleic acid and a trans-substrate containing a quencher molecule and a reporter molecule separated by a polymer of greater than five cytosine or thymidine residues at a concentration of approximately 100 nM at or around a temperature of 37°C in the detection buffer; and detecting and recording a resulting signal.

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