Programmable kinetic barcoding for multiplexed RNA detectiion

Abstract

As described herein, RNA detection with high sensitivity and multiplexed specificity can be achieved with short detection times by encapsulating the Cas reaction in droplets and monitoring enzyme kinetics fluorescently.

Description

[0001]GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 PROGRAMMABLE KINETIC BARCODING FOR MULTIPLEXED RNA DETECTIION PRIORITY This application claims the benefit of the filing date of U.S. provisional application No. 63 / 608,933, filed on December 12, 2023, the disclosures of which is incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on November 30, 2024, is named 3730228WO1.xml and is 101,960 bytes in size. BACKGROUND PCR-based assays are currently the gold standard for RNA detection, as they can achieve high sensitivity (~1 copy / μL) with assay times under 2 hours. CRISPR-Cas13, a type VI CRISPR system, offers an alternate way of quantifying RNA by using its RNA-activated RNase activity to cleave a fluorescent reporter upon guide RNA-directed binding of a target RNA (East-Seletsky et al., 2016). Though Cas13 can be combined with reverse transcription, amplification, and transcription to increase sensitivity (Gootenberg et al., 2017), direct detection of RNA with Cas13 avoids the limitations of those steps and can achieve modest sensitivity by combining multiple crRNAs recognizing different regions of the target RNA. For the SAR-CoV-2 genome, direct detection with LbuCas13a measured down to ~200 copies / μL in 30 minutes with 3 crRNAs (Fozouni et al., 2021) and ~63 copies / μL in 2 hours with 8 crRNAs (Liu et al., 2021). However, PCR-level sensitivity has not yet been achieved with direct Cas13 detection, and approaches for identifying which of multiple virus variants are present in a single sample are limited (Jiao et al., 2021). Current uses of Cas13 and Cas12 nucleases for diagnostic applications also rely on bulk reactions that produce fluorescent signals in the presence of target RNA or DNA, which means the kinetics of individual Cas-guide-target complexes cannot be observed, only the bulk combination. As a result, bulk assays are not suitable for detecting variants. SUMMARY What is needed is a direct Cas-13 detection approach that enables amplification-free, purification-free multi-biomarker assays in a short period, e.g., less than about 15 minutes, to allow better, faster and cheaper diagnostic assays. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 As described herein, RNA detection with high sensitivity and multiplexed specificity can be achieved with short detection times by encapsulating the Cas reaction in droplets and monitoring enzyme kinetics fluorescently. Like droplet digital PCR (ddPCR) (Hindson et al., 2011), droplet detection of RNA enables quantification of the absolute amount of target RNA based on the number of positive droplets. Unlike ddPCR, the small droplet volumes used in the methods described herein accelerates signal accumulation of the direct Cas13 reaction and thereby shortens the time required to determine a positive or negative result. For example, when a single target RNA is encapsulated in a droplet with a volume of about 10 picoliters, the Cas13 signal accumulation rate is equivalent to that of a bulk reaction containing 105copies / μL of target RNA (see, e.g., FIG.1A). Described herein are assay mixtures that include a population of droplets ranging in diameter from at least 10 to 60 μm, the population comprising a test droplet subpopulation comprising at least one ribonucleoprotein complex, plus at least one reporter RNA, plus at least one target RNA. The ribonucleoprotein complex can include a Cas nuclease and a CRISPR guide RNA (crRNA). Upon binding of the ribonucleoprotein complex (via the crRNA), the ribonucleoprotein complex cleaves Reporter RNAs, to release a detectable signal. Also described herein are methods that can involve (a) combining a sample with at least one type of ribonucleoprotein complex and at least one type of reporter RNA to form a reaction mixture; (b) mixing the reaction mixture with oil and surfactant to form an emulsion comprising water-in-oil droplets, wherein at least some of the droplets encapsulate all components of the reaction mixture; (c) removing excess oil from the droplets; (d) selecting at least 1 droplet, or at least 3 droplets, or at least 10 droplets that emit fluorescence as positive droplets for monitoring; and (e) monitoring the fluorescence of the positive droplets over time or measuring the endpoint fluorescence relative to a defined start time for the reaction. Also described herein are methods and compositions for modifying the activity of a Cas enzyme with a linker-effector. The linker can be made with nucleic acid (e.g., RNA and / or DNA), other polymer (e.g., PEG or other hydrophilic polymer), or combinations of the foregoing. The linker connects the guide RNA with the effector and can constrain the position of the effector sequence in relation to an appropriate Cas enzyme. The linker sequence can be designed to provide flexibility, modulate interactions with the protein surface, and may include monomer modifications and other chemical changes to impart desired properties to the linker, e.g., preventing cleavage through the guide maturation process or from an activated Cas enzyme. The effector can also be nucleic acid (e.g., RNA and / or DNA), other polymer (e.g., PEG), or combinations of the foregoing. The effector can interfere with the trans-cleavage GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 activity of the Cas enzyme when in contact with the Cas enzyme, either directly or through allosteric effects, and modulation of the trans-cleavage activity can be altered by interaction of the effector sequence with other molecules that change the interaction between the effector and the Cas enzyme. The effector can be complementary to another sequence, and the effect caused by hybridization of the effector to the other sequence can be overcome by nuclease activity. Described herein are methods and compositions for detecting a desired target using a linker-effector with a guide RNA or crRNA and an appropriate Cas enzyme. The combination of a guide RNA with a linker-effect extension is referred to as an interfering guide RNA or igRNA. Such detection methods and compositions include a multiplex embodiment in which multiple different linker-effectors can be used to produce multiple different kinetic rates upon activation of Cas by the binding of an igRNA (linker-effector-crRNA) to an appropriate target. These different kinetic rates can be combined with reporters that produce light of different wavelengths, together with Cas enzymes with different crRNA preferences, to increase the number of targets that can be detected in a multiplex embodiment. Also described here are igRNA with aptamers as part of the linker-effector region that bind to a ligand. In one embodiment, the igRNA with an aptamer effector modifies the activity of the Cas enzyme without the aptamer ligand. For example, the complement target for the crRNA of the igRNA can be hybridized to the complement target but the ribonucleoprotein complex is inhibited by the aptamer effector in the absence of ligand. When the aptamer binds to its ligand, the conformation of the aptamer effector is altered, and the activity of the Cas enzyme is increased so that the Cas enzyme can cleave the reporter RNA. In another embodiment, binding of the aptamer to a molecule of interest could position the effector region to reduce Cas enzyme activity. Binding molecules other than aptamers may be used to achieve similar effects of positioning an effector region to increase or decrease Cas enzyme activity. DESCRIPTION OF THE DRAWINGS FIGS.1A-1N illustrate rapid detection of the single-molecule Cas13a reaction within heterogeneous droplet. FIG. 1A is a schematic illustrating an increased signal accumulation rate for a single Cas13 confined in decreasing volumes (red: activated Cas13a, white: inactive Cas13a). Each droplet contains hundreds of thousand copies of Cas13a RNP and millions of quenched RNA reporter. Only the droplet possessing one or more target RNA will acquire signal. FIG.1B is a schematic illustrating a droplet Cas13a assay method. A Cas13a reaction including one or more guide RNAs and target RNAs are mixed with an oil (HFE 7500 including 2 wt % Perfluoro-PEG surfactant) and emulsified by repeated pipette mixing at a constant speed for 2 minutes. The emulsified reaction is typically incubated for 15 minutes in 37˚C, and GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 the reaction is optionally quenched on ice. Subsequently, the emulsion is loaded into a custom flow cell and imaged with a fluorescent microscope. The complete assay takes 20 minutes. FIG.1C graphically illustrates the size distribution of droplets, which is reduced by 0.1 wt % IGEPAL in the Cas13a mix. The red line indicates the mean distribution of droplet size in the presence of 0.1 vol % IGEPAL. The black line indicates droplets in the absence of IGEPAL. The shadows indicate the S.D. from 5 independent droplet preparations. FIG.1D shows bright field (left) and fluorescent images of Cas13a reaction taken with a 20X objective lens. Time (T) = 0, 10, and 20 minutes since the beginning of imaging. Scale bar = 65 μm. FIG. 1E graphically illustrate the fluorescent signal over time in three positive droplets (the green lines) and one background droplet (the grey line). The signal is the mean fluorescent intensity change within a droplet normalized by the initial signal after background subtraction. Images are acquired every 30 seconds and corrected for the photobleaching (see Example 1). FIG. 1F graphically illustrates the single Cas13a turnover frequency measured from individual droplets containing crRNA 4 (SEQ ID NO: 4) and SARS-CoV-2 RNA (N = 478 droplets). Guide RNA crRNA 4 (SEQ ID NO: 4) targets the N gene of SARS-CoV-2 RNA. The box and whisker plot in the right panel indicates the median, the lower and upper quartiles, and the minimum and the maximum values. FIG. 1G graphically illustrates the signal-per-droplet with increasing incubation times is represented as the box and whisker plot. The signal is normalized by the median in 5 minutes timepoint. N > 800 droplets are used in all four timepoints. FIG. 1H graphically illustrates the number of positive droplets detected with increasing incubation times.1 x 104copies / μL of SARS-CoV-2 RNA were added to a bulk reaction prior to droplet formation and droplets are incubated for a specified time. Data are represented as mean ± SD of three technical replicates. P-values are determined from a two-tailed Student’s t-test: ns = not significant. FIG.1I shows an image of an automatic multi-channel pipettor (an 8-channel pipette; Integra biosciences, Part # 4623), which was used to generate emulsions. Sample of about 110 μL were mixed with the pipettor for 150 repetitions at the maximum speed (speed 10) to emulsify droplets to a narrow size range. The emulsion so formed was either directly loaded into a flow cell for time course imaging or incubated in a heating block at 37˚C before being transferred and imaged. FIG.1J is a schematic illustrating confocal imaging of a droplet at its midplane. FIG. 1K graphically illustrates the reaction velocities (change of cleaved reporter) in differently sized droplets. FIG.1L graphically illustrates the turnover frequency, as measured by total change of cleaved reporter in droplets with different diameters. FIG.1M graphically illustrates identification of positive reactions in droplet assays in reaction times of GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 15 minutes using a 4X / 0.20NA microscope objective. FIG. 1N graphically illustrates identification of positive reactions in droplet assays at various reaction time. FIGS.2A-2H illustrate the detection sensitivity of droplet Cas13a assay using crRNA combinations. FIG. 2A is a schematic illustrating two potential results of Cas13 droplet reactions that use two different crRNAs simultaneously: (1) Complete loading – if the whole N gene segment is loaded to one droplet containing crRNAs targeting two different regions of N gene, the signal will accumulate twice as fast as a droplet containing one copy of target RNA; or (2) Fragmented loading – if one N gene is fragmented to two halves and loaded into two separate droplets, the number of positive droplet will be doubled while the signal of individual ones remain identical to the droplet containing one copy of target RNA. FIG.2B graphically illustrates the distribution of signal-per-droplet for a Cas13a reaction as shown in a box and whisker plot. N > 250 for all three conditions. The Cas13a reaction included 2.5 x 104copies / μL of in vitro transcribed (IVT) N gene in a droplet assay mixture. A control Cas13a assay included no target RNA. The droplet assays included the following guide RNAs: only crRNA2 (SEQ ID NO: 2), only crRNA 4 (SEQ ID NO: 4), or both crRNA2 and crRNA4. Droplets were quantified after 1 hour of reaction incubation. FIG. 2C graphically illustrates the data for the assay described for FIG.2B as number of positive droplets per mm2(mean ± SD of three replicates). FIG. 2D graphically illustrates the number of positive droplets quantified for different crRNA combinations after adding 100 copies / μL of externally quantified SARS-CoV-2 RNA (BEI Resources). Each reaction was incubated for 15 minutes, and droplet images were taken with the 4X objective lens. Data are represented as mean ± SD of three replicates. FIG.2E graphically illustrates the number of positive droplets quantified for a series of dilutions of externally quantified SARS-CoV-2 RNA. Each reaction was incubated for 15 minutes. Data are represented as mean ± SD of three replicates. P-values were determined based on a two-tailed Student’s t-test: ns = not significant, *p < 0.05, ** p < 0.005, ***p < 0.001. FIG. 2F graphically illustrates the signal from individual assays using either crRNA 2 (SEQ ID NO: 2) or crRNA 4 (SEQ ID NO: 4). FIG.2G graphically illustrates that the activity of Cas13a remains constant even when only a small fraction of total RNPs in a droplet contained crRNA matching the target. FIG.2H graphically illustrates that the limit of detection was not improved when the assay reaction was incubated for 30 minutes instead of 15 minutes. SARS-CoV-2 RNA was used as the target. FIGS. 3A-3Q illustrate crRNA-dependent heterogenous Cas13a activities. FIG. 3A graphically illustrates the slope of a bulk Cas13a reaction containing 3.5 x 104copies / μL of SARS-CoV-2 RNAs using crRNAs that target different regions of the N gene (crRNA 4, GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 crRNA11A, crRNA12A). A control assay had no target RNA. The slope was determined by performing simple linear regression of data from each replicate (N = 3) individually. Data are represented as mean ± SD. FIG.3B graphically illustrates the number of positive droplets for a droplet Cas13a reaction with 3.5 x 104copies / μL of SARS-CoV-2 RNA after 30 minutes of incubation. A control assay had no SARS-CoV-2 RNA. Data are represented as mean ± SD of three replicates. For the RNP only condition, one measurement for each crRNA is merged. The crRNAs employed were crRNA 4, crRNA11A, crRNA12A. FIG.3C graphically illustrates the signal per droplet when different crRNAs were used in the droplet Cas13a reaction with 3.5 x 104copies / μL of SARS-CoV-2 RNA described for FIG.3B. Data are represented as box and whisker plots marking the median, the lower and upper quartiles, and the minimum and the maximum values. The signals were normalized by the median of crRNA 4. The crRNAs employed were crRNA 4, crRNA11A, crRNA12A. N > 1800 droplets were used in all three conditions. FIG.3D graphically illustrates signal trajectories over time for droplet assays for detecting SARS-CoV-2 RNA using crRNA 4 (SEQ ID NO: 4). FIG.3E graphically illustrates signal trajectories over time for droplet assays for detecting SARS-CoV-2 RNA using crRNA 11A (SEQ ID NO: 36). FIG.3F graphically illustrates signal trajectories over time for droplet assays for detecting SARS-CoV-2 RNA using crRNA 12A (SEQ ID NO: 37). For FIG.3D-3F the one hundred individual trajectories were monitored from droplets ranging from 30 to 36 μm size (show as the grey lines) along with arbitrarily selected, representative trajectories (the red lines). Signals were measured every 30 seconds for each trajectory. Data from two replicate runs were combined for each crRNA. FIG.3G illustrates time trajectories of the rare positive droplets from the Cas13a reactions without any target RNA. Thirty-one individual trajectories were measured in droplets ranging from 30 to 36 μm size, from two replicate runs. FIG.3H graphically illustrates the slope over time for droplet assays, illustrating the analytical strategy for individual Cas13a signal trajectories. The blue curve is an example trajectory obtained with crRNA 12A. The average slope (Slope (avg)), time from target addition to the initiation of enzyme activity (Tinit), and the Root-mean-square-deviation (RMSD) are determined by performing simple linear regression to the raw signal. Slopefast and Slopeslow correspond to the fast and slow periods determined as shown in FIG.3H-3I. FIG.3I illustrates the calculation of the instantaneous slopes by taking the time-derivative of the raw signal (the blue histogram) and its probability distribution as fitted with either a single-distribution or via binary-gaussian distributions (the red line). For data that favors the binary distribution, the Slopefast, Slopeslow, % Fast, and % Slow were determined from the mean and the proportion of each gaussian peak. FIG.3J graphically illustrates the normalized slope of droplet assay signals for GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 different crRNA represented as the box and whisker plots including outliers. The crRNAs employed were crRNA 4, crRNA11A, crRNA12A. FIG. 3K graphically illustrates the percentage of “fast” slope droplets for assays using different crRNAs. The crRNAs employed were crRNA 4, crRNA11A, crRNA12A. FIG.3L graphically illustrates the root-mean-square- deviation (RMSD) of signals from droplet assays using different crRNAs. FIG.3M graphically illustrates the time from target addition to the initiation of enzyme activity (Tinit) for droplet assays using different crRNAs. The crRNAs employed were crRNA 4, crRNA11A, crRNA12A. For FIGs. 3J-3M the distributions of key Cas13a kinetic parameters are represented as the box and whisker plots including outliers. Individual 30-minutes-long trajectories from droplets of arbitrary size is used after their signal is normalized for droplet size. N>250 for all conditions. P-values are determined from a two-tailed Student’s t-test: ns = not significant, *p < 0.05, ***p < 0.001. FIG. 3N graphically illustrates the average slope (Slope (avg)), time from target addition to the initiation of enzyme activity (Tinit), and the Root-mean-square-deviation (RMSD) for droplet assays using crRNA 4 (SEQ ID NO: 4) at low concentrations with SARS-CoV-2 RNA. FIG. 3O graphically illustrates average slope (Slope (avg)), time from target addition to the initiation of enzyme activity (Tinit), and the Root-mean-square-deviation (RMSD) for droplet assays using crRNA 12 (SEQ ID NO: 12) at high concentrations. FIG. 3P is a schematic illustrating recognition of target by RNP containing a Cas nuclease and a guide crRNA that upon binding the target activates the nuclease to cleave a reporter RNA, which generates the signal during the droplet assay. The two graphs illustrate signal trajectories over time for crRNA 4 (middle) and crRNA 12 (right) droplet assays. FIG. 3Q graphically illustrates average slope (Slope (avg)), time from target addition to the initiation of enzyme activity (Tinit), and the Root-mean-square-deviation (RMSD) for droplet assays using crRNA 2 (SEQ ID NO: 2) and a full-length SARS-CoV-2 RNA target. FIGS. 4A-4N illustrate the kinetic-barcoding methods for multiplexed detection of virus. FIG. 4A is a schematic diagram illustrating the kinetic-barcoding method for simultaneous detection of two different viruses. FIG.4B is a schematic diagram illustrating the kinetic-barcoding method for simultaneous detection of two different variants. The kinetic- barcoding method detects unique Cas13a kinetic signatures for specific combinations of crRNA guides and target RNAs. FIG. 4C shows representative graphs illustrating single Cas13a reaction trajectories when human coronavirus strain NL 63 (HCoV-NL 63) RNA was targeted by crRNA 7 or when SARS-CoV-2 RNA was targeted by crRNA 12. The signals were total fluorescence change in a droplet, which remains invariant regardless of droplet size. The GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 dotted red line shows a linear fit. FIG. 4D graphically illustrates the distribution between HCoV and SARS-CoV-2 of slope and RMSD values for individual Cas13a signal trajectories in droplet assays. The slope and RMSD values were determined from individual 30 minutes- long trajectories (N = 488). The RMSD values were first normalized by the mean signal of the same trajectory and then normalized to 0 to 1. The slope values were normalized to 0 to 1. FIG. 4E graphically illustrates identification of HCoV or SARS-CoV-2 based on the kinetic parameters of individual Cas13 reactions. Varying numbers of 30-minutes-long Cas13a trajectories were randomly selected from each condition and the difference between two groups was quantified as p-values based on a two-tailed Student’s t-test. FIG.4F shows representative graphs illustrating single Cas13a reaction trajectories using an RNA target that included the wild type SARS-CoV-2 S gene or an RNA target that included the D614G mutation in the SARS-CoV-2 S gene. The dotted red line shows a linear fit. FIG.4G graphically illustrates the distribution between slope values and RMSD values of individual Cas13a signal trajectories of targets having either the wild type SARS-CoV-2 S gene or the D614G mutant SARS-CoV-2 S gene (N = 208). FIG.4H graphically illustrates identification of wild type SARS-CoV-2 or the D614G mutant SARS-CoV-2 strain based on the kinetic parameters of individual Cas13 reactions. Varying numbers of 30-minutes-long Cas13a trajectories were randomly selected from each condition and the difference between two groups was quantified as p-values based on a two-tailed Student’s t-test. FIG. 4I graphically illustrates identification of the SARS- CoV-2 B.1.427 variant from clinical samples using the kinetic-barcoding methods. The average of slope or RMSD distribution was obtained by randomly selecting ten positive trajectories from many trajectories measured for each sample (the blue dots are WT (N=26) and the magenta dots are B.1.427 (N=86)). The blue and magenta squares are example values for each sample. The black dotted line indicates the slope threshold separating the WT from B.1.427 data. FIG.4J graphically illustrates the detection specificity of kinetic barcoding. The accuracy was determined from FIG. 4I). FIG. 4K graphically illustrates the p-values of increasing numbers of signal trajectories over time. The measurement interval was 30 seconds. Although extending the measurement time improved classification, measurement times longer than 10 minutes did not provide any improvement. FIG.4L graphically illustrates the p-values of increasing numbers of signal trajectories over time, where images were acquired every 3 minutes for 30 minutes instead of every 30 seconds for 10 minutes as shown for FIG.4K. The total measurement time was 30 minutes. FIG. 4M graphically illustrates the p-values of increasing numbers of signal trajectories for the SARS-CoV-2 D614G mutant RNA over time, illustrating the difference in the average slopes of 30 or more signal trajectories. The GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 measurement interval was 30 seconds. These data illustrate that the D614G mutant RNA could be distinguished from the wild type RNA within 5 minutes. FIG. 4N graphically illustrates RMSD vs slope values for a series of patient samples previously shown to be infected with SARS-CoV-2 (i.e., exhibiting Ct values of 15 to 20 in PCR testing). Positive trajectories (N=15 to 350) were measured among the droplets in the assays. Although individual trajectories from each sample exhibited heterogenous slopes and RMSDs, the slopes measured from the WT were significantly lower than those measured from the B.1.427 mutant (FIG.4I and 4N). FIGS.5A-5D illustrate the detection of four different viral targets in a single reaction. FIG.5A shows a histogram of signal slope normalized for droplet size. FIG.5B shows raw signal-time trajectories normalized for droplet size. FIG. 5C shows the prediction of target virus based on signal slope distribution. FIG. 5D shows prediction of target virus based on signal slope distribution. FIGS.6A-6C illustrate the detection of three different strains of SARS-CoV-2. FIG. 6A shows the design of the igRNA. FIG.6B shows the clinical sample analysis scheme. FIG. 6C shows the results for clinical samples. FIG.7 illustrates the impact of length for the linker-effector. DETAILED DESCRIPTION Described herein are methods, kits, and compositions for detecting RNA targets and other molecular targets using droplet assays. The droplets in the assays contain target-specific CRISPR guide RNAs (crRNAs) within Cas nuclease-crRNA ribonucleoprotein complexes that will cleave reporter RNA upon binding a target RNA, thereby generating fluorescence within the droplets that contain the target RNA. Not all of the droplets contain the target RNA. Hence, the number of fluorescent droplets can be a measure of the concentration of target RNA in a sample. Moreover, experiments described herein show that fluorescence generated by droplet- based Cas nuclease enzymatic activity is not continuous but exhibits variable kinetics. The droplets are designed to encapsulate just a single target RNA. As demonstrated herein, the kinetics of fluorescence production by a particular droplet is a signature that uniquely identifies the target RNA. Because the droplets are designed to include a single RNA target, and the kinetics of fluorescence by many droplets can simultaneously be monitored, droplet-based Cas nuclease-crRNA assay procedures can be multiplexed to detect multiple target RNAs in a population of droplets. When multiple crRNAs are used, they are used at equal concentrations so that a mixture of Cas nuclease-crRNA ribonucleoprotein complexes has approximately equal numbers of each type of crRNA-containing complexes. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 As demonstrated herein, sometimes the Cas enzyme is actively cleaving the reporter RNA and producing fluorescence, and sometimes the Cas enzyme is not actively cleaving the reporter RNA, and therefore not producing fluorescence. These stochastic changes were observed, for example, when the Cas protein / guide RNA was in the presence of targets with point mutations or different viral strains. The results show that the kinetics of the reaction are characteristic of the specific combination of Cas13, guide RNA, and target RNA. This means that by following the generation of a fluorescent signal from a single target molecule, the kinetics can be observed, and the presence of a specific variant or mutant nucleic acid can be determined based on known enzymatic rates for different targets of interest. This method is referred to herein as ‘kinetic barcoding.’ Assay mixtures are therefore described herein that can include a population of droplets. The mean diameter of the droplets can range from at least 10 to 60 μm. The droplet population including a test droplet subpopulation comprising at least one ribonucleoprotein (RNP) complex, plus at least one reporter RNA, plus at least one target RNA. In some cases, the population can include droplets that do not include one or more of a ribonucleoprotein complex, a reporter RNA, or a target RNA; these droplets can be used as control droplets. For example, the control droplets can be used to define background levels of fluorescence. Also described herein are methods for detecting and / or identifying an RNA. The methods can include (a) contacting a sample with at least one type of ribonucleoprotein (RNP) complex and at least one type of reporter RNA to form a reaction mixture; (b) mixing the reaction mixture with oil and surfactant to form an emulsion comprising droplets, where at least some of the droplets encapsulate an aqueous solution comprising the reaction mixture; (c) removing excess oil from the droplets; (d) selecting at least 1 droplet, or at least 3 droplets, or at least 10 droplets that emit fluorescence as positive droplets for monitoring; and (e) monitoring the fluorescence of the positive droplets over time. The ribonucleoprotein (RNP) complex includes a Cas nuclease and a CRISPR guide RNA (crRNA). The Cas nuclease cleaves a reporter RNA when the RNP binds to its target via the crRNA. The kinetics of positive droplet fluorescence relates to the accessibility of the RNP for its target. Hence, selection of a crRNA affects the kinetics of fluorescence production within positive droplets. For example, the location of the crRNA binding site on the target RNA, or the presence of sequence mismatches can affect the kinetics of a positive droplet’s fluorescence. Kinetics GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 The kinetics of fluorescence signals by droplets can be monitored by observing droplet fluorescence over time, for example by taking images of the droplet(s) at selected intervals. Droplets need not be monitored continuously, but droplets may move during the reaction time. Individual droplets must be distinguished and identified from one imaging interval to the next in order to quantify short-timescale variations in enzyme kinetics. Droplets can be identified by the track of their motion, for example, using a Kalman filter (e.g. in MATLAB) to predict the track's location in each image frame and to determine the likelihood that each detection within a series of image frames is being assigned to a particular tracked droplet. Only the droplets showing continuous trajectories in time and magnitude are selected for downstream analysis. In some cases, images can be obtained after excitation of the fluorescent dye at intervals, for example, of 1 second to 5 minutes. In some cases, the images are obtained at intervals of 2 seconds to 4 minutes, or at intervals of 3 seconds to 3 minutes, or at intervals of 5 seconds to 1 minute. For example, in some of the experiments described herein, sixteen field- of-views (FOV) were acquired every 30 seconds for the time course of imaging and 36 field- of views were acquired for the endpoint imaging. Several kinetic parameters can be used as ‘kinetic barcodes’ for identifying droplets and the targets encapsulated by those droplets. Individual signal trajectories can be evaluated by determining the slope of signal over time (slope), the time from target addition to the initiation of enzyme activity (Tinit), and the root-mean-square-deviation (RMSD) from signal time trajectories by linear regression. Because some time was used to prepare the reaction mixtures and the droplet, a constant set-up time can be added to Tinitto reflect the time from droplet formation until the beginning of timed imaging. In addition, the time periods during which droplet’s fluorescence signal increases quickly or slowly can be noted, and the percent ‘slopefast’ and ‘slopeslow’ parameters therefrom. For example, the slopefast and slopeslow parameters can be determined as a fraction or percent of time spent in each period, using a normal gaussian pdf (bell-curve) to obtain the instantaneous slope distribution. The slope, Tinit, RMSD, slopefast, and slopeslow parameters are all kinetic parameters that individually or in combination can be used as a kinetic barcode that uniquely defines which crRNA / target combination is present within a particular droplet, or a particular subpopulation of droplets. At a minimum, only average slope is needed to use kinetic barcoding to determine which of a set of known kinetic rates associated with specific targets are present in a droplet. In this case, all droplets can be assumed to start with the same background fluorescence, and only a single endpoint measurement is needed to estimate the slope. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Linker-Effector A linker-effector is attached to the 5’ end or 3’ end of the guide RNA for the purpose of modifying the activity of a Cas enzyme (e.g., any Cas or Cas-like enzyme (e.g., having the ability to cleave a reporter molecule)). The linker can be made of a polynucleotide (e.g., RNA and / or DNA), or another polymer (e.g., polypeptide, or other polymers), or combinations of any of the foregoing. Other polymers that can be used in the linker include, for example, PEG (and other water-soluble polymers), peptide nucleic acid (PNA), glycol nucleic acid (GNA), therose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA), xeno nucleic acids (XNA), phosphorodiamidate morpholino oligomer (PMO), intrinsically disordered proteins (IDP). The linker can be 1 to 100 or more nucleotides in length (about 1- 68 nanometers), preferably 8 nucleotides in length (5 nanometers) when there is no secondary structure. The linker connects the guide RNA with the effector sequence and can constrain the position of the effector sequence so that it modifies the Cas enzyme activity, e.g., by interfering with the trans-cleavage activity of the Cas enzyme or allosterically by interacting with another part of the enzyme that modifies trans-cleavage activity. The linker sequence can be designed to provide flexibility, modulate interactions with the protein surface, and may include nucleotide modifications and other chemical changes to prevent cleavage through the guide maturation process. The linker joins the crRNA with the effector, allowing it to be properly located to interfere with the function of or the trans activity of the Cas enzyme. It can have the length to allow the effector to reach the HEPN domain on the Cas enzyme. The linker can also act as a hinge to bring the effector closer to the HEPN domain of the Cas enzyme. The effector can be a polynucleotide (e.g., RNA and / or DNA), or another polymer (e.g., polypeptide, or other polymers), or combinations of any of the foregoing. Other polymers that can be used in the effector include, for example, PEG, peptide nucleic acid (PNA), glycol nucleic acid (GNA), therose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA), xeno nucleic acids (XNA), phosphorodiamidate morpholino oligomer (PMO), intrinsically disordered proteins (IDP). The effector can be, for example, an aptamer, a ribozyme, a deoxyribozyme, a target for hybridization, or a target for cleavage by another enzyme, such as a Cas enzyme. The effector can be 1 to 100 or more nucleotides in length (about 0.676 nanometers per nucleotide with no secondary structure). The length of the effector can vary, and longer effectors usually result in stronger kinetic (slope) hindrance. The effector can interfere with the trans-cleavage activity of the Cas enzyme when in contact with the Cas enzyme, and modulation of the trans-cleavage activity can be altered by interaction of the effector sequence with other molecules that change the contact between the effector and GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 the Cas enzyme. Interactions that change the contact between the effector and the Cas enzyme can include (1) binding of the effector to a target molecule (e.g., protein, metabolite, polysaccharide, lipid, ligand, etc.), (2) hybridization of the effector to DNA or RNA, or (3) cleavage of the effector by another enzyme, such as in the cleavage by Cas12 of a DNA effector blocking Cas13. The effector can interfere with the trans cleavage activity of the Cas enzyme by interacting with the Cas’ HEPN domain, reducing the speed of the reaction. The effector can also inhibit the ribonucleoprotein complex by interfering with the interaction of the crRNA with target nucleic acid. For example, the effector can be complementary to part of the crRNA so as to block the crRNA from interacting with target nucleic acid. Hybridization forms a structure that interferes with formation of an active ribonucleoprotein complex (e.g., the hybridized structure could contain loops that interfere. This inhibition can be overcome by nuclease activity that digests the effector to remove the obstructing structures (e.g., the loops). In an aspect, the nuclease activity that digests part of the effector is an activated (Cas) ribonucleoprotein complex. In an aspect, the effector can hybridize to another sequence (e.g., the crRNA or target) and disrupt the active structure-conformation of the ribonucleoprotein complex. In this aspect, the effector can be decoupled from the linker and crRNA. In this aspect, the hybridization of the effector can be disrupted by a nuclease, a competitive binding nucleic acid, etc. This allows multiplexing using a combination of guides for different targets with different reactions speeds (different linker-effectors resulting in different changes to the kinetic rate of cleavage by Cas), allowing multiplex detection based on either the slope or the end-point fluorescence. The effector can, for example, modify the Cas enzyme by a) sterically not allowing the Cas HEPN domain to meet its intended target (the reporter) which is RNA for Cas13 and ssDNA for Cas12; b) causing an actual conformational change of the Cas enzyme resulting in ablation not only of trans activity but also its affinity for the specific target; c) wrapping around the Cas enzyme; and / or d) interacting with the reporter molecule. Since many Cas13 (and Cas12) enzymes can process their own crRNA from longer fragments, the linker (and the effector) can be designed to be resistant to that cleavage. For example, ssDNA linker and effectors are resistant to cleavage by Cas13, and RNA linker and effectors are resistant to cleavage by Cas12. Further examples of cleavage resistant linkers and / or effectors can be made from XNAs, PMO, or PEG. The activity and / or the tertiary structure of the Cas enzymes, linker, and effectors can be modulated by changing the salt concentrations of the buffers, particularly K+, Na+, Mg2+, or Mn2+. The linker and the effector do not have to be made of the same type of nucleic acid (or polymer) backbone. A linker can be made of one type of polymer and the effector of another type. The linker and / or GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 effector can be made of a mix of types of polymers, and the individual linker and effector can be of mixed types of polymers. The crRNA-linker-effector structure can be called an igRNA or interfering guide RNA. The linker-effector can also be used to suppress detection of specific sequences or targets, such as those that could cause false-positives, by designing sequences that specifically target and suppress sequences or other targets that are similar to but different from the desired detection sequence or target. Such linker-effectors can be polynucleotides (e.g., RNA and / or DNA), or other polymers (e.g., polypeptide, or other polymers), or combinations of any of the foregoing. Guide RNAs can tolerate up to 2 mismatches and still activate a Cas complex. Hence, a single-mismatched non-target can lead to non-specific activation. To inhibit this off- target signal, we can make igRNA to specifically target 1-nucleotide mismatched or multiple mismatched off-targets. While on kinetic modulation we only wanted an effector to slightly decrease the trans activity of the Cas, here we want to completely abolish it by designing a stronger effector. This could be achieved by making it longer (longer effectors can decrease activity more), using an aptamer, using the synthetic nucleic acid polymers listed above (as they would be refractory to degradation by the Cas itself), or any combinations of the foregoing. The linker can also be modified the same way. This would be two separate complexes: a) Cas – crRNA for the desired target (with active trans activity on the Cas), either with or without a linker-effector to modulate kinetics as part of a kinetic barcoding assay, and b) Cas – igRNA for the undesired off targets (with inhibited trans activity on the Cas). Both the crRNA and the igRNA will be perfectly matched to their respective targets. Since we have a specific igRNA aiming at the off-target, the off-target will not be available to the regular crRNA aiming at the desired target. The linker-effectors can be a single chain polynucleotide that includes a guide RNA or can be formed by hybridization between one or more sequences that includes a guide RNA and one or more sequences that includes a linker and / or an effector. Aptamers that can be used in the linker-effectors include, for example, RNA or DNA that is capable of binding to a specific molecule with high affinity and specificity (Ellington et al., Nature 346, 818-22 (1990); Tuerk et al., Science 249, 505-10 (1990); and Methods in Molecular Biology, Nucleic Acid Aptamers: selection, characterization and application, Humana Press 2016 (ed. Günter Mayer), which are hereby incorporated by reference in their entirety for all purposes). Aptamers are short, structured, nucleic acid sequences that can change conformation (e.g., upon binding to a target) to disrupt or activate the function of, in this case, the HEPN domain of Cas 13 or Cas12 enzymes. They can themselves potentially GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 consists structurally of any of the nucleic acids mentioned above: DNA, RNA, LNA, PNA, GNA, TNA, PMO, CeNA, FAMA, SNA, L-aTNA, or PEG. Exemplary targets that can bind to an aptamer include, without limitation, small molecules, such as drugs, metabolites, intermediates, cofactors, transition state analogs, ions, metals, nucleic acids, and toxins. In some aspects, aptamers may also bind natural and synthetic polymers, including proteins, peptides, nucleic acids, polysaccharides, glycoproteins, hormones, receptors and cell surfaces such as cell walls and cell membranes. In some aspects, the binding of a ligand to an aptamer causes or favors a conformational change in the effector and alters the conformation of the effector changing its interaction with a Cas enzyme. Aptamers can be made that bind to a wide variety of molecules. Each of these aptamer molecules can be used as an effector using the methods and compositions described herein. In some aspects, organic molecules, nucleotides, amino acids, polypeptides, target features on cell surfaces, ions, metals, salts, saccharides, are used as ligands for making an aptamer that can specifically bind to the respective ligand. In some aspects, small organic molecules like dopamine, theophylline, sulforhodamine B, and cellobiose are used as ligands in the isolation of aptamers. For a review of aptamers that recognize small molecules, see Famulok, Science 9:324-9 (1999), which is hereby incorporated by reference in its entirety for all purposes. A dual enzyme system can be used to turn off the negative modulation produced by the igRNA (effector and linker) on kinetics. For example, a Cas13 system where the linker and effector are ssDNA, Cas12 can be used to remove the linker-effector and liberate the crRNA to increase the speed of the reaction again. The linker-effectors can make 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different kinetic rates that can be detected in the droplet format described herein. If the different kinetic rates are combined with colors or bar code strategies, the number of samples that can be detected from one sample increases by several orders of magnitude. The kinetic barcoding reactions can also be carried out in microwells in place of droplets. Samples A variety of samples can be evaluated to ascertain whether one or more RNA molecules are present. The source of the samples can be any biological material. For example, the samples can be any biological fluid or tissue from any virus, fungus, plant or animal that is suspected of having an RNA. Examples of RNA types that can be evaluated in the methods include mRNAs, genomic RNAs, tRNAs, rRNAs, microRNAs, and combinations thereof. In some cases the RNA is a viral RNA, a mRNA marker for disease, a rRNA that could define what GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 type of organism may be present in a sample, a microRNA that may silence gene function, or any other type of RNA. In some cases, it may not be known whether the biological sample contains RNA. However, such biological samples can still be tested using the methods described herein. To obtain potential RNA from biological samples, the samples can be subjected to lysis, RNA extraction, inhibition of RNase(s), storage until testing is initiated, or other manipulations. In general, such manipulations are used to purify and preserve the RNA so that accurate kinetic barcoding can be performed. Ribonucleoproteins As described herein, samples that are tested to determine the presence and / or type of a particular RNA are incubated with a ribonucleoprotein (RNP) complex that includes a Cas nuclease and a CRISPR guide RNA (crRNA). The Cas nuclease can be one or more Cas12 or Cas13 (some previously known as C2c2) nucleases. When a crRNA is present, the Cas nucleases employed bind and cleave RNA substrates, rather than DNA substrates, to which Cas9 can bind. In some cases, the Cas nucleases can be from a variety of organisms and can have sequence variations. For example, the Cas proteins can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any of the foregoing Cas 13 sequences from: Leptotrichia wadei, Leptotrichia buccalis, Rhodobacter capsulatus, Herbinix hemicellulosilytica, Leptotrichia buccalis (Lbu), Listeria seeligeri, Paludibacter propionicigenes, Lachnospiraceae bacterium, [Eubacterium] rectale, Listeria newyorkensis, Clostridium aminophilum, and / or Leptotrichia shahii. For example, a Leptotrichia wadei Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 71; NCBI accession no. WP_036059678.1). 1 MKITKIDGVS HYKKQDKGIL KKKWKDLDER KQREKIEARY 41 NKQIESKIYK EFFRLKNKKR IEKEEDQNIK SLYFFIKELY 81 LNEKNEEWEL KNINLEILDD KERVIKGYKF KEDVYFFKEG 121 YKEYYLRILF NNLIEKVQNE NREKVRKNKE FLDLKEIFKK 161 YKNRKIDLLL KSINNNKINL EYKKENVNEE IYGINPTNDR 201 EMTFYELLKE IIEKKDEQKS ILEEKLDNFD ITNFLENIEK 241 IFNEETEINI IKGKVLNELR EYIKEKEENN SDNKLKQIYN 281 LELKKYIENN FSYKKQKSKS KNGKNDYLYL NFLKKIMFIE 321 EVDEKKEINK EKFKNKINSN FKNLFVQHIL DYGKLLYYKE 361 NDEYIKNTGQ LETKDLEYIK TKETLIRKMA VLVSFAANSY 401 YNLFGRVSGD ILGTEVVKSS KTNVIKVGSH IFKEKMLNYF 441 FDFEIFDANK IVEILESISY SIYNVRNGVG HFNKLILGKY 481 KKKDINTNKR IEEDLNNNEE IKGYFIKKRG EIERKVKEKF 521 LSNNLQYYYS KEKIENYFEV YEFEILKRKI PFAPNFKRII GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 561 KKGEDLFNNK NNKKYEYFKN FDKNSAEEKK EFLKTRNFLL 601 KELYYNNFYK EFLSKKEEFE KIVLEVKEEK KSRGNINNKK 641 SGVSFQSIDD YDTKINISDY IASIHKKEME RVEKYNEEKQ 681 KDTAKYIRDF VEEIFLTGFI NYLEKDKRLH FLKEEFSILC 721 NNNNNVVDFN ININEEKIKE FLKENDSKTL NLYLFFNMID 761 SKRISEFRNE LVKYKQFTKK RLDEEKEFLG IKIELYETLI 801 EFVILTREKL DTKKSEEIDA WLVDKLYVKD SNEYKEYEEI 841 LKLFVDEKIL SSKEAPYYAT DNKTPILLSN FEKTRKYGTQ 881 SFLSEIQSNY KYSKVEKENI EDYNKKEEIE QKKKSNIEKL 921 QDLKVELHKK WEQNKITEKE IEKYNNTTRK INEYNYLKNK 961 EELQNVYLLH EMLSDLLARN VAFFNKWERD FKFIVIAIKQ 1001 FLRENDKEKV NEFLNPPDNS KGKKVYFSVS KYKNTVENID 1041 GIHKNFMNLI FLNNKFMNRK IDKMNCAIWV YFRNYIAHFL 1081 HLHTKNEKIS LISQMNLLIK LFSYDKKVQN HILKSTKTLL 1121 EKYNIQINFE ISNDKNEVFK YKIKNRLYSK KGKMLGKNNK 1161LENEFLE NVKAMLEYSE Other sequences for Leptotrichia wadei Cas13a endonucleases are also available, such as those NCBI accession nos. BBM46759.1, BBM48616.1, BBM48974.1, BBM48975.1, and WP_021746003.1. In another example, a Herbinix hemicellulosilytica Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 72; NCBI accession no. WP_103203632.1). 1 MKLTRRRISG NSVDQKITAA FYRDMSQGLL YYDSEDNDCT 41 DKVIESMDFE RSWRGRILKN GEDDKNPFYM FVKGLVGSND 81 KIVCEPIDVD SDPDNLDILI NKNLTGFGRN LKAPDSNDTL 121 ENLIRKIQAG IPEEEVLPEL KKIKEMIQKD IVNRKEQLLK 161 SIKNNRIPFS LEGSKLVPST KKMKWLFKLI DVPNKTFNEK 201 MLEKYWEIYD YDKLKANITN RLDKTDKKAR SISRAVSEEL 241 REYHKNLRTN YNRFVSGDRP AAGLDNGGSA KYNPDKEEFL 281 LFLKEVEQYF KKYFPVKSKH SNKSKDKSLV DKYKNYCSYK 321 VVKKEVNRSI INQLVAGLIQ QGKLLYYFYY NDTWQEDFLN 361 SYGLSYIQVE EAFKKSVMTS LSWGINRLTS FFIDDSNTVK 401 FDDITTKKAK EAIESNYFNK LRTCSRMQDH FKEKLAFFYP 441 VYVKDKKDRP DDDIENLIVL VKNAIESVSY LRNRTFHFKE 481 SSLLELLKEL DDKNSGQNKI DYSVAAEFIK RDIENLYDVF 521 REQIRSLGIA EYYKADMISD CFKTCGLEFA LYSPKNSLMP 561 AFKNVYKRGA NLNKAYIRDK GPKETGDQGQ NSYKALEEYR 601 ELTWYIEVKN NDQSYNAYKN LLQLIYYHAF LPEVRENEAL 641 ITDFINRTKE WNRKETEERL NTKNNKKHKN FDENDDITVN 681 TYRYESIPDY QGESLDDYLK VLQRKQMARA KEVNEKEEGN 721 NNYIQFIRDV VVWAFGAYLE NKLKNYKNEL QPPLSKENIG 761 LNDTLKELFP EEKVKSPFNI KCRFSISTFI DNKGKSTDNT 801 SAEAVKTDGK EDEKDKKNIK RKDLLCFYLF LRLLDENEIC 841 KLQHQFIKYR CSLKERRFPG NRTKLEKETE LLAELEELME 881 LVRFTMPSIP EISAKAESGY DTMIKKYFKD FIEKKVFKNP 921 KTSNLYYHSD SKTPVTRKYM ALLMRSAPLH LYKDIFKGYY 961 LITKKECLEY IKLSNIIKDY QNSLNELHEQ LERIKLKSEK 1001 QNGKDSLYLD KKDFYKVKEY VENLEQVARY KHLQHKINFE GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1041 SLYRIFRIHV DIAARMVGYT QDWERDMHFL FKALVYNGVL 1081 EERRFEAIFN NNDDNNDGRI VKKIQNNLNN KNRELVSMLC 1121 WNKKLNKNEF GAIIWKRNPI AHLNHFTQTE QNSKSSLESL 1161 INSLRILLAY DRKRQNAVTK TINDLLLNDY HIRIKWEGRV 1201 DEGQIYFNIK EKEDIENEPI IHLKHLHKKD CYIYKNSYMF 1241 DKQKEWICNG IKEEVYDKSI LKCIGNLFKF DYEDKNKSSA 1281 NPKHT However, in some cases the Cas13 proteins with the SEQ ID NO: 72 sequence are not used. In another example, a Leptotrichia buccalis Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 73; NCBI accession no. WP_015770004.1). 1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM 41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL 81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE 121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE 161 NNIEKVEGKS KRNIIYDYYR ESAKRDAYVS NVKEAFDKLY 201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF 241 AKIIYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK 281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI 321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI 361 ATSDFIARNR QNEAFLRNII GVSSVAYFSL RNILETENEN 401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN 441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL 481 ELEGKDIFAF KNIAPSEISK KMFQNEINEK KLKLKIFRQL 521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPSFTKLY 561 SRIDDLKNSL GIYWKTPKTN DDNKTKEIID AQIYLLKNIY 601 YGEFLNYFMS NNGNFFEISK EIIELNKNDK RNLKTGFYKL 641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF 681 IQKIFLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE 721 FDKFLKKYEQ NNNIKIPYEI NEFLREIKLG NILKYTERLN 761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI 801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK 841 FDTNKIYFDG ENIIKHRAFY NIKKYGMLNL LEKIADKAGY 881 KISIEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF 921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI 961 LHRLVGYTSI WERDLRFRLK GEFPENQYIE EIFNFENKKN 1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ 1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK 1081 LKNAVMKSVV DILKEYGFVA TFKIGADKKI GIQTLESEKI 1121 VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN However, in some cases the Cas13 proteins with the SEQ ID NO: 73 sequence are not used. In another example, a Leptotrichia seeligeri Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 74; NCBI accession no. WP_012985477.1). GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1 MWISIKTLIH HLGVLFFCDY MYNRREKKII EVKTMRITKV 41 EVDRKKVLIS RDKNGGKLVY ENEMQDNTEQ IMHHKKSSFY 81 KSVVNKTICR PEQKQMKKLV HGLLQENSQE KIKVSDVTKL 121 NISNFLNHRF KKSLYYFPEN SPDKSEEYRI EINLSQLLED 161 SLKKQQGTFI CWESFSKDME LYINWAENYI SSKTKLIKKS 201 IRNNRIQSTE SRSGQLMDRY MKDILNKNKP FDIQSVSEKY 241 QLEKLTSALK ATFKEAKKND KEINYKLKST LQNHERQIIE 281 ELKENSELNQ FNIEIRKHLE TYFPIKKTNR KVGDIRNLEI 321 GEIQKIVNHR LKNKIVQRIL QEGKLASYEI ESTVNSNSLQ 361 KIKIEEAFAL KFINACLFAS NNLRNMVYPV CKKDILMIGE 401 FKNSFKEIKH KKFIRQWSQF FSQEITVDDI ELASWGLRGA 441 IAPIRNEIIH LKKHSWKKFF NNPTFKVKKS KIINGKTKDV 481 TSEFLYKETL FKDYFYSELD SVPELIINKM ESSKILDYYS 521 SDQLNQVFTI PNFELSLLTS AVPFAPSFKR VYLKGFDYQN 561 QDEAQPDYNL KLNIYNEKAF NSEAFQAQYS LFKMVYYQVF 601 LPQFTTNNDL FKSSVDFILT LNKERKGYAK AFQDIRKMNK 641 DEKPSEYMSY IQSQLMLYQK KQEEKEKINH FEKFINQVFI 681 KGFNSFIEKN RLTYICHPTK NTVPENDNIE IPFHTDMDDS 721 NIAFWLMCKL LDAKQLSELR NEMIKFSCSL QSTEEISTFT 761 KAREVIGLAL LNGEKGCNDW KELFDDKEAW KKNMSLYVSE 801 ELLQSLPYTQ EDGQTPVINR SIDLVKKYGT ETILEKLFSS 841 SDDYKVSAKD IAKLHEYDVT EKIAQQESLH KQWIEKPGLA 881 RDSAWTKKYQ NVINDISNYQ WAKTKVELTQ VRHLHQLTID 921 LLSRLAGYMS IADRDFQFSS NYILERENSE YRVTSWILLS 961 ENKNKNKYND YELYNLKNAS IKVSSKNDPQ LKVDLKQLRL 1001 TLEYLELFDN RLKEKRNNIS HFNYLNGQLG NSILELFDDA 1041 RDVLSYDRKL KNAVSKSLKE ILSSHGMEVT FKPLYQTNHH 1081 LKIDKLQPKK IHHLGEKSTV SSNQVSNEYC QLVRTLLTMK For example, a Paludibacter propionicigenes Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 75; NCBI accession no. WP_013443710.1). 1 MRVSKVKVKD GGKDKMVLVH RKTTGAQLVY SGQPVSNETS 41 NILPEKKRQS FDLSTLNKTI IKFDTAKKQK LNVDQYKIVE 81 KIFKYPKQEL PKQIKAEEIL PFLNHKFQEP VKYWKNGKEE 121 SFNLTLLIVE AVQAQDKRKL QPYYDWKTWY IQTKSDLLKK 161 SIENNRIDLT ENLSKRKKAL LAWETEFTAS GSIDLTHYHK 201 VYMTDVLCKM LQDVKPLTDD KGKINTNAYH RGLKKALQNH 241 QPAIFGTREV PNEANRADNQ LSIYHLEVVK YLEHYFPIKT 281 SKRRNTADDI AHYLKAQTLK TTIEKQLVNA IRANIIQQGK 321 TNHHELKADT TSNDLIRIKT NEAFVLNLTG TCAFAANNIR 361 NMVDNEQTND ILGKGDFIKS LLKDNTNSQL YSFFFGEGLS 401 TNKAEKETQL WGIRGAVQQI RNNVNHYKKD ALKTVFNISN 441 FENPTITDPK QQTNYADTIY KARFINELEK IPEAFAQQLK 481 TGGAVSYYTI ENLKSLLTTF QFSLCRSTIP FAPGFKKVFN 521 GGINYQNAKQ DESFYELMLE QYLRKENFAE ESYNARYFML 561 KLIYNNLFLP GFTTDRKAFA DSVGFVQMQN KKQAEKVNPR 601 KKEAYAFEAV RPMTAADSIA DYMAYVQSEL MQEQNKKEEK 641 VAEETRINFE KFVLQVFIKG FDSFLRAKEF DFVQMPQPQL 681 TATASNQQKA DKLNQLEASI TADCKLTPQY AKADDATHIA GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 721 FYVFCKLLDA AHLSNLRNEL IKFRESVNEF KFHHLLEIIE 761 ICLLSADVVP TDYRDLYSSE ADCLARLRPF IEQGADITNW 801 SDLFVQSDKH SPVIHANIEL SVKYGTTKLL EQIINKDTQF 841 KTTEANFTAW NTAQKSIEQL IKQREDHHEQ WVKAKNADDK 881 EKQERKREKS NFAQKFIEKH GDDYLDICDY INTYNWLDNK 921 MHFVHLNRLH GLTIELLGRM AGFVALFDRD FQFFDEQQIA 961 DEFKLHGFVN LHSIDKKLNE VPTKKIKEIY DIRNKIIQIN 1001 GNKINESVRA NLIQFISSKR NYYNNAFLHV SNDEIKEKQM 1041 YDIRNHIAHF NYLTKDAADF SLIDLINELR ELLHYDRKLK 1081 NAVSKAFIDL FDKHGMILKL KLNADHKLKV ESLEPKKIYH 1121 LGSSAKDKPE YQYCTNQVMM AYCNMCRSLL EMKK For example, a Lachnospiraceae bacterium Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 76; NCBI accession no. WP_022785443.1). 1 MKISKVREEN RGAKLTVNAK TAVVSENRSQ EGILYNDPSR 41 YGKSRKNDED RDRYIESRLK SSGKLYRIFN EDKNKRETDE 81 LQWFLSEIVK KINRRNGLVL SDMLSVDDRA FEKAFEKYAE 121 LSYTNRRNKV SGSPAFETCG VDAATAERLK GIISETNFIN 161 RIKNNIDNKV SEDIIDRIIA KYLKKSLCRE RVKRGLKKLL 201 MNAFDLPYSD PDIDVQRDFI DYVLEDFYHV RAKSQVSRSI 241 KNMNMPVQPE GDGKFAITVS KGGTESGNKR SAEKEAFKKF 281 LSDYASLDER VRDDMLRRMR RLVVLYFYGS DDSKLSDVNE 321 KFDVWEDHAA RRVDNREFIK LPLENKLANG KTDKDAERIR 361 KNTVKELYRN QNIGCYRQAV KAVEEDNNGR YFDDKMLNMF 401 FIHRIEYGVE KIYANLKQVT EFKARTGYLS EKIWKDLINY 441 ISIKYIAMGK AVYNYAMDEL NASDKKEIEL GKISEEYLSG 481 ISSFDYELIK AEEMLQRETA VYVAFAARHL SSQTVELDSE 521 NSDFLLLKPK GTMDKNDKNK LASNNILNFL KDKETLRDTI 561 LQYFGGHSLW TDFPFDKYLA GGKDDVDFLT DLKDVIYSMR 601 NDSFHYATEN HNNGKWNKEL ISAMFEHETE RMTVVMKDKF 641 YSNNLPMFYK NDDLKKLLID LYKDNVERAS QVPSFNKVFV 681 RKNFPALVRD KDNLGIELDL KADADKGENE LKFYNALYYM 721 FKEIYYNAFL NDKNVRERFI TKATKVADNY DRNKERNLKD 761 RIKSAGSDEK KKLREQLQNY IAENDFGQRI KNIVQVNPDY 801 TLAQICQLIM TEYNQQNNGC MQKKSAARKD INKDSYQHYK 841 MLLLVNLRKA FLEFIKENYA FVLKPYKHDL CDKADFVPDF 881 AKYVKPYAGL ISRVAGSSEL QKWYIVSRFL SPAQANHMLG 921 FLHSYKQYVW DIYRRASETG TEINHSIAED KIAGVDITDV 961 DAVIDLSVKL CGTISSEISD YFKDDEVYAE YISSYLDFEY 1001 DGGNYKDSLN RFCNSDAVND QKVALYYDGE HPKLNRNIIL 1041 SKLYGERRFL EKITDRVSRS DIVEYYKLKK ETSQYQTKGI 1081 FDSEDEQKNI KKFQEMKNIV EFRDLMDYSE IADELQGQLI 1121 NWIYLRERDL MNFQLGYHYA CLNNDSNKQA TYVTLDYQGK 1161 KNRKINGAIL YQICAMYING LPLYYVDKDS SEWTVSDGKE 1201 STGAKIGEFY RYAKSFENTS DCYASGLEIF ENISEHDNIT 1241 ELRNYIEHFR YYSSFDRSFL GIYSEVFDRF FTYDLKYRKN 1281 VPTILYNILL QHFVNVRFEF VSGKKMIGID KKDRKIAKEK 1321 ECARITIREK NGVYSEQFTY KLKNGTVYVD ARDKRYLQSI 1361 IRLLFYPEKV NMDEMIEVKE KKKPSDNNTG KGYSKRDRQQ GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1401 DRKEYDKYKE KKKKEGNFLS GMGGNINWDE INAQLKN For example, a Leptotrichia shahii Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 77; NCBI accession no. BBM39911.1). 1 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN YDGNKYILNI 41 NENNNKEKID NNKFIRKYIN YKKNDNILKE FTRKFHAGNI 81 LFKLKGKEGI IRIENNDDFL ETEEVVLYIE AYGKSEKLKA 121 LGITKKKIID EAIRQGITKD DKKIEIKRQE NEEEIEIDIR 161 DEYTNKTLND CSIILRIIEN DELETKKSIY EIFKNINMSL 201 YKIIEKIIEN ETEKVFENRY YEEHLREKLL KDDKIDVILT 241 NFMEIREKIK SNLEILGFVK FYLNVGGDKK KSKNKKMLVE 281 KILNINVDLT VEDIADFVIK ELEFWNITKR IEKVKKVNNE 321 FLEKRRNRTY IKSYVLLDKH EKFKIERENK KDKIVKFFVE 361 NIKNNSIKEK IEKILAEFKI DELIKKLEKE LKKGNCDTEI 401 FGIFKKHYKV NFDSKKFSKK SDEEKELYKI IYRYLKGRIE 441 KILVNEQKVR LKKMEKIEIE KILNESILSE KILKRVKQYT 481 LEHIMYLGKL RHNDIDMTTV NTDDFSRLHA KEELDLELIT 521 FFASTNMELN KIFSRENINN DENIDFFGGD REKNYVLDKK 561 ILNSKIKIIR DLDFIDNKNN ITNNFIRKFT KIGTNERNRI 601 LHAISKERDL QGTQDDYNKV INIIQNLKIS DEEVSKALNL 641 DVVFKDKKNI ITKINDIKIS EENNNDIKYL PSFSKVLPEI 681 LNLYRNNPKN EPFDTIETEK IVLNALIYVN KELYKKLILE 721 DDLEENESKN IFLQELKKTL GNIDEIDENI IENYYKNAQI 761 SASKGNNKAI KKYQKKVIEC YIGYLRKNYE ELFDFSDFKM 801 NIQEIKKQIK DINDNKTYER ITVKTSDKTI VINDDFEYII 841 SIFALLNSNA VINKIRNRFF ATSVWLNTSE YQNIIDILDE 881 IMQLNTLRNE CITENWNLNL EEFIQKMKEI EKDFDDFKIQ 921 TKKEIFNNYY EDIKNNILTE FKDDINGCDV LEKKLEKIVI 961 FDDETKFEID KKSNILQDEQ RKLSNINKKD LKKKVDQYIK 1001 DKDQEIKSKI LCRIIFNSDF LKKYKKEIDN LIEDMESENE 1041 NKFQEIYYPK ERKNELYIYK KNLFLNIGNP NFDKIYGLIS 1081 NDIKMADAKF LFNIDGKNIR KNKISEIDAI LKNLNDKLNG 1121 YSKEYKEKYI KKLKENDDFF AKNIQNKNYK SFEKDYNRVS 1161 EYKKIRDLVE FNYLNKIESY LIDINWKLAI QMARFERDMH 1201 YIVNGLRELG IIKLSGYNTG ISRAYPKRNG SDGFYTTTAY 1241 YKFFDEESYK KFEKICYGFG IDLSENSEIN KPENESIRNY 1281 ISHFYIVRNP FADYSIAEQI DRVSNLLSYS TRYNNSTYAS 1321 VFEVFKKDVN LDYDELKKKF KLIGNNDILE RLMKPKKVSV 1361LELESYNSDY IKNLIIELLT KIENTNDTL In another example, a Leptotrichia buccalis C-1013-b Cas13a endonuclease can have the following sequence (SEQ ID NO: 78; NCBI accession no. C7NBY4; AltName LbuC2c2). 1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM 41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL 81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE 121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 161 NNIEKVEGKS KRNIIYDYYR ESAKRDAYVS NVKEAFDKLY 201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF 241 AKIIYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK 281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI 321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI 361 ATSDFIARNR QNEAFLRNII GVSSVAYFSL RNILETENEN 401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN 441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL 481 ELEGKDIFAF KNIAPSEISK KMFQNEINEK KLKLKIFRQL 521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPSFTKLY 561 SRIDDLKNSL GIYWKTPKTN DDNKTKEIID AQIYLLKNIY 601 YGEFLNYFMS NNGNFFEISK EIIELNKNDK RNLKTGFYKL 641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF 681 IQKIFLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE 721 FDKFLKKYEQ NNNIKIPYEI NEFLREIKLG NILKYTERLN 761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI 801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK 841 FDTNKIYFDG ENIIKHRAFY NIKKYGMLNL LEKIADKAGY 881 KISIEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF 921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI 961 LHRLVGYTSI WERDLRFRLK GEFPENQYIE EIFNFENKKN 1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ 1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK 1081 LKNAVMKSVV DILKEYGFVA TFKIGADKKI GIQTLESEKI 1121VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN In some cases, a modified Cas13 protein can be used. Such a modified Cas 13 protein can have increased in vivo endonuclease activity compared to a corresponding unmodified Cas13 protein. The modified Cas13 proteins, which can increase sensitivity of detecting at least one reporter RNA by about 10-fold to 100-fold are useful, for example, in the methods, kits, systems and devices described herein. The inventors have evaluated the kinetics of other Cas13a and Cas13b proteins. Such work indicates that in some cases Cas13b works faster in the SARS-CoV-2 RNA detection assay than Cas13a. For example, a Cas13b from Prevotella buccae can be used in the SARS-CoV-2 RNA detection methods, compositions and devices. A sequence for a Prevotella buccae Cas13b protein (NCBI accession no. WP_004343973.1) is shown below as SEQ ID NO: 79. 1 MQKQDKLFVD RKKNAIFAFP KYITIMENKE KPEPIYYELT 41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG 81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEMTNSK 121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS 161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE 201 NIDMQRDFTH LNRKKQVGRT KNIIDSPNFH YHFADKEGNM GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN 281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY 321 ERLREKDRES FKVPFDIFSD DYNAEEEPFK NTLVRHQDRF 361 PYFVLRYFDL NEIFEQLRFQ IDLGTYHFSI YNKRIGDEDE 401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ 441 EPYISKTAPH YHLENEKIGI KFCSAHNNLF PSLQTDKTCN 481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE 521 SADKVEGIIR KEISNIYAIY DAFANNEINS IADLTRRLQN 561 TNILQGHLPK QMISILKGRQ KDMGKEAERK IGEMIDDTQR 601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ 641 PVQKDQNNIP INNSKANSTE YRMLQRALAL FGSENFRLKA 681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK 721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL 761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP 801 LYFAEEYKDN VQPFYDYPFN IGNRLKPKKR QFLDKKERVE 841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK 881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE 921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET 961 ETKVLKQGNF KALVKDRRLN GLFSFAETTD LNLEEHPISK 1001 LSVDLELIKY QTTRISIFEM TLGLEKKLID KYSTLPTDSF 1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD 1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI 1121 EKSENKN Such a Prevotella buccae Cas13b protein can have a Km (Michaelis constant) substrate concentration of about 20 micromoles and a Kcat of about 987 / second (see, e.g., Slaymaker et al. Cell Rep 26 (13): 3741-3751 (2019)). Another Prevotella buccae Cas13b protein (NCBI accession no. WP_004343581.1) that can be used in the SARS-CoV-2 RNA detection methods, compositions and devices has the sequence shown below as SEQ ID NO: 80. 1 MQKQDKLFVD RKKNAIFAFP KYITIMENQE KPEPIYYELT 41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMD 81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK 121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS 161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE 201 NIDMQRDFTH LNRKKQVGRT KNIIDSPNFH YHFADKEGNM 241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN 281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY 321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF 361 PYFVLRYFDL NEIFEQLRFQ IDLGTYHFSI YNKRIGDEDE 401 VRHLTHHLYG FARIQDFAQQ NQPEVWRKLV KDLDYFEASQ 441 EPYIPKTAPH YHLENEKIGI KFCSTHNNLF PSLKTEKTCN 481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE 521 SADKVEGIIR KEISNIYAIY DAFANGEINS IADLTCRLQK 561 TNILQGHLPK QMISILEGRQ KDMEKEAERK IGEMIDDTQR 601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ 641 PVQKDQNNIP INNSKANSTE YRMLQRALAL FGSENFRLKA 681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL 761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP 801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE 841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK 881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE 921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET 961 ETKVLKQGNF KVLAKDRRLN GLLSFAETTD IDLEKNPITK 1001 LSVDHELIKY QTTRISIFEM TLGLEKKLIN KYPTLPTDSF 1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD 1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI 1121 EKSENKN An example of a Bergeyella zoohelcum Cas13b (R1177A) mutant sequence (NCBI accession no.6AAY_A) is shown below as SEQ ID NO: 81. 1 XENKTSLGNN IYYNPFKPQD KSYFAGYFNA AXENTDSVFR 41 ELGKRLKGKE YTSENFFDAI FKENISLVEY ERYVKLLSDY 81 FPXARLLDKK EVPIKERKEN FKKNFKGIIK AVRDLRNFYT 121 HKEHGEVEIT DEIFGVLDEX LKSTVLTVKK KKVKTDKTKE 161 ILKKSIEKQL DILCQKKLEY LRDTARKIEE KRRNQRERGE 201 KELVAPFKYS DKRDDLIAAI YNDAFDVYID KKKDSLKESS 241 KAKYNTKSDP QQEEGDLKIP ISKNGVVFLL SLFLTKQEIH 281 AFKSKIAGFK ATVIDEATVS EATVSHGKNS ICFXATHEIF 321 SHLAYKKLKR KVRTAEINYG EAENAEQLSV YAKETLXXQX 361 LDELSKVPDV VYQNLSEDVQ KTFIEDWNEY LKENNGDVGT 401 XEEEQVIHPV IRKRYEDKFN YFAIRFLDEF AQFPTLRFQV 441 HLGNYLHDSR PKENLISDRR IKEKITVFGR LSELEHKKAL 481 FIKNTETNED REHYWEIFPN PNYDFPKENI SVNDKDFPIA 521 GSILDREKQP VAGKIGIKVK LLNQQYVSEV DKAVKAHQLK 561 QRKASKPSIQ NIIEEIVPIN ESNPKEAIVF GGQPTAYLSX 601 NDIHSILYEF FDKWEKKKEK LEKKGEKELR KEIGKELEKK 641 IVGKIQAQIQ QIIDKDTNAK ILKPYQDGNS TAIDKEKLIK 681 DLKQEQNILQ KLKDEQTVRE KEYNDFIAYQ DKNREINKVR 721 DRNHKQYLKD NLKRKYPEAP ARKEVLYYRE KGKVAVWLAN 761 DIKRFXPTDF KNEWKGEQHS LLQKSLAYYE QCKEELKNLL 801 PEKVFQHLPF KLGGYFQQKY LYQFYTCYLD KRLEYISGLV 841 QQAENFKSEN KVFKKVENEC FKFLKKQNYT HKELDARVQS 881 ILGYPIFLER GFXDEKPTII KGKTFKGNEA LFADWFRYYK 921 EYQNFQTFYD TENYPLVELE KKQADRKRKT KIYQQKKNDV 961 FTLLXAKHIF KSVFKQDSID QFSLEDLYQS REERLGNQER 1001 ARQTGERNTN YIWNKTVDLK LCDGKITVEN VKLKNVGDFI 1041 KYEYDQRVQA FLKYEENIEW QAFLIKESKE EENYPYVVER 1081 EIEQYEKVRR EELLKEVHLI EEYILEKVKD KEILKKGDNQ 1121 NFKYYILNGL LKQLKNEDVE SYKVFNLNTE PEDVNINQLK 1161 QEATDLEQKA FVLTYIANKF AHNQLPKKEF WDYCQEKYGK 1201 IEKEKTYAEY FAEVFKKEKE ALIKLEHHHH HH GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Another example of a Cas13b protein sequence from Prevotella sp. MSX73 (NCBI accession no. WP_007412163.1) that can be used in the SARS-CoV-2 RNA detection methods, compositions and devices has is shown below as SEQ ID NO: 82. 1 MQKQDKLFVD RKKNAIFAFP KYITIMENQE KPEPIYYELT 41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG 81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK 121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS 161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE 201 NIDMQRDFTH LNRKKQVGRT KNIIDSPNFH YHFADKEGNM 241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN 281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY 321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF 361 PYFVLRYFDL NEIFEQLRFQ IDLGTYHFSI YNKRIGDEDE 401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ 441 EPYISKTAPH YHLENEKIGI KFCSTHNNLF PSLKREKTCN 481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE 521 SADKVEGIIR KEISNIYAIY DAFANNEINS IADLTCRLQK 561 TNILQGHLPK QMISILEGRQ KDMEKEAERK IGEMIDDTQR 601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVSDMMRFQ 641 PVQKDTNNAP INNSKANSTE YRMLQHALAL FGSESSRLKA 681 YFRQMNLVGN ANPHPFLAET QWEHQTNILS FYRNYLEARK 721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL 761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP 801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE 841 LWQKNKELFK NYPSEKNKTD LAYLDFLSWK KFERELRLIK 881 NQDIVTWLMF KELFKTTTVE GLKIGEIHLR DIDTNTANEE 921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET 961 ETKVLKQGNF KVLAKDRRLN GLLSFAETTD IDLEKNPITK 1001 LSVDYELIKY QTTRISIFEM TLGLEKKLID KYSTLPTDSF 1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD 1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI 1121 EKSENKN Hence, the sample can be incubated with at least one CRISPR RNA (crRNA) and at least one Cas13 protein. The Cas13 protein can, for example, be a Cas13a protein, Cas13b protein, or a combination thereof. Pre-incubation of the crRNA and Cas13 protein without the sample can facilitate RNA detection, so that the crRNA and the Cas13 protein can form a complex. For example, the Cas13 and crRNA are incubated for a period of time to form the inactive complex. In some cases, the Cas13 and crRNA complexes are formed by incubating together at 37 ºC for 30 minutes, 1 hour, or 2 hours (for example, 0.5 to 2 hours) to form an inactive complex. The inactive complex can then be incubated with the reporter RNA. Reporter RNA GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 The methods and compositions described herein for detecting and / or identifying an RNA can involve incubating a mixture having a sample suspected of containing RNA, a Cas13 protein, at least one CRISPR RNA (crRNA), and a reporter RNA for a period of time to form reporter RNA cleavage products that may be present in the mixture and detecting a level of any such reporter RNA cleavage products with a detector. The detector can be a fluorescence detector. The reporter RNA can, for example, be at least one quenched-fluorescent RNA reporter. Such quenched-fluorescent RNA reporter can optimize fluorescence detection. The quenched- fluorescent RNA reporters include an RNA oligonucleotide with both a fluorophore and a quencher of the fluorophore. The quencher decreases or eliminates the fluorescence of the fluorophore. When the Cas nuclease cleaves the RNA reporter, the fluorophore is separated from the associated quencher, such that a fluorescence signal becomes detectable. One example of such a fluorophore quencher–labelled RNA reporter is the RNaseAlert (IDT). RNaseAlert was developed to detect RNase contaminations in a laboratory, and the substrate sequence is optimized for RNase A species. Another approach is to use lateral flow strips to detect a FAM-biotin reporter that, when cleaved by a Cas nuclease, is detected by anti- FAM antibody-gold nanoparticle conjugates on the strip. Although this allows for instrument- free detection, it requires 90–120 minutes for readout, compared to under 30 minutes for most fluorescence-based assays (Gootenberg et al. Science.360(6387):439–44 (April 2018)). The sequence of the reporter RNA can be optimized for Cas nuclease cleavage. Different Cas nuclease homologs can have different sequence preferences at the cleavage site. In some cases, Cas13 preferentially exerts RNase cleavage activity at exposed uridine sites or adenosine sites. There are also secondary preferences for highly active homologs. The fluorophores used for the fluorophore quencher–labelled RNA reporters can include Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof. The fluorophores used for the fluorophore quencher–labelled RNA reporters can include Dabcyl, QSY 7, QSY 9, QSY 21, QSY 35, Iowa Black Quencher (IDT), or a combination thereof. Many quencher moieties are available, for example, from ThermoFisher Scientific. Various mechanisms and devices can be employed to detect fluorescence. Some mechanism or devices can be used to help eliminate background fluorescence. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to-noise ratio, and consequently, the resolution of signal from the RNA cleavage products of interest. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Total internal reflection fluorescence (TIRF) enables very low background fluorescence and single molecule sensitivity with a sufficiently sensitive camera. In some cases, a reporter RNA can be present while the crRNA and the Cas protein form a complex. However, in other cases, the reporter RNA can be added after the crRNA and the Cas protein already form a complex. Also, after formation of the crRNA / Cas complex, the sample RNA can then be added. The sample RNA acts as an activating RNA. Once activated by the activating RNA, the crRNA / Cas complex becomes a non-specific RNase to produce RNA cleavage products that can be detected using a reporter RNA, for example, a short quenched-fluorescent RNA. Cas13 / crRNA complexes that are activated by an RNA sample cleave RNA both in cis and in trans. When cleaving in cis, for example, the activated complex can cleave the sample RNA. When cleaving in trans, the activated complex can cleave the reporter RNA, thereby releasing a signal such as the fluorophore from the reporter RNA. Cleavage of a reporter with electrical activity, e.g. a gold nanoparticle tethered by RNA to a conducting surface, could be used in an electrochemical detection assay. Droplets Droplets are formed by emulsifying an aqueous reaction mixture with an oil and a surfactant to form water-in-oil droplets. Droplets containing a target RNA with the Cas nuclease / crRNA ribonucleoprotein (RNP) complex and a reporter RNA can emit fluorescence when the RNP complex binds to the target RNA. The droplets can be formed by agitating an oil with a surfactant. The oil and surfactant are selected to provide sufficient droplet stability and to allow visualization of fluorescence within the droplets. Droplets need not be separated from debris such as excess oil and / or surfactant prior to fluorescence monitoring. However, in some cases, background fluorescence can be reduced by separation of the droplets from the emulsion materials. A variety of methods can be used for separating the droplets from such debris. For example, the emulsion mixture can be centrifuged, and the oil removed from the bottom of the tube. To emulsify a Cas13a reaction mix, aliquots (e.g., 5-50 microliters) of an aqueous reaction mixture are combined with an excess amount of oil supplemented with a surfactant (e.g., 75-300 microliters). The oil can be HFE-7500 oil and the surfactant can be PEG-PFPE amphiphilic block copolymer surfactant (e.g., 008-Fluorosurfactant, RAN Biotechnologies). The oil can contain about 1%-5% (w / w) surfactant. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Such a reaction mixture-oil-surfactant combination can be emulsified to generate droplets ranging in diameter from at least 10 to 60 μm. In some cases, the size range is a narrower size range of about 20 to 50 μm. The fluorescence of droplets can be directly monitored. For example, the emulsion containing the droplets can be directly loaded into a flow cell for time course imaging. In some cases, the emulsion or the separated droplets are incubated in a heating block at 37˚C before being imaged. Although the fluorescence of droplets can be monitored in a variety of ways, in some cases the droplets are in thin layer of fluid to minimize signal overlap between overlapping droplets. A shallow flow cell can be used to minimize signal / droplet overlap. For example, such flow cells can each include two hydrophobic surfaces with sufficient space between the two surfaces for a single droplet to move about. At least one of the hydrophobic surfaces is transparent (often both are transparent) so that light can be introduced into the flow cell chamber to excite the fluorescent dye(s) of the reporter RNA, and the fluorescence emitted can be detected. The two hydrophobic surfaces can be spaced about 10μm to about 60μm apart. For example, one hydrophobic surface of the flow cell can be an acrylic slide (75mm x 25mm x 2mm) while the other hydrophobic surface is a siliconized coverslip (22mm x 22mm x 0.22mm). A spacer that is about 10μm to about 60μm thick (e.g., about 20μm thick) can be used to seal the edges of the coverslip to the slide. Such a flow cell can contain about 10μ1 to about 60μl fluid, where the droplets are free to move around in the fluid. Instead of emulsions that lead to heterogeneous droplets, homogenous droplets may be formed using microfluidic devices. Similarly sized microwells made by injection molding or other methods may be used in place of droplets for the kinetic barcoding assay. Uses The methods and compositions described herein can be used for diagnostic tests of polypeptides, metabolites, DNA, RNA, polysaccharides, lipids, etc. The linker-effector modifications can also be used for multiplex detection schemes as different linker-effectors can be used to increase the number of different kinetic rates that can be multiplexed in the detection methods disclosed herein. Using different linker-effectors we can create many different kinetic rates for multiplex detection which optionally can be combined with different wavelength detection (e.g., different dyes). Reactions with a difference in kinetic rate of ~20% have been distinguished in droplets using the methods described herein, though we expect the limit of detection for Kinetic Barcoding to be differences in slope of less than 10%. Thus, using linker- GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 effectors one can multiplex at least 10 different signals / targets in one reaction with one wavelength (e.g., color of dye). Linker-effectors have been used for multiplex detection of RSV, influenza, and coronavirus variants. Linker-effectors can be used in the methods and composition herein to increase specificity of detection by suppression of false-positive, off-target detection. Hybridizing effectors as described above can be used to increase signal from an activated ribonucleoprotein complex. The activated ribonucleoprotein complex can cleave the hybridized effector so that the structure and / or conformation that inhibits Cas activity is removed and the cleaved- hybridized effector now activates this nucleoprotein complex increasing signal output. In an aspect, the igRNA with an aptamer effector modifies the activity of the Cas enzyme without the aptamer ligand. For example, the complement target for the crRNA of the igRNA can be hybridized to the igRNA in the ribonucleoprotein complex but the complex is inhibited by the aptamer effector in the absence of ligand. When the aptamer binds to its ligand, the conformation of the aptamer effector is altered, and the activity of the Cas enzyme is increased so that the Cas enzyme can cleave the reporter RNA. In this aspect, the ribonucleoprotein complex with the aptamer effector can be used to detect the ligand that binds to the aptamer. As discussed above, the ligand for the aptamer can be a small molecule (e.g., drug, metabolite, intermediate, cofactor, transition state analog, ion, metal, nucleic acid, or toxin), biological molecule (e.g., protein, polypeptide, polysaccharide, lipids, polynucleotide), or other molecules. Alternatively, the complement target for the crRNA of the igRNA can be hybridized to the igRNA in the ribonucleoprotein complex and the aptamer-effector can inhibit the complex when it is complexed to the aptamer ligand. In an aspect, the aptamer-effector and the igRNA can be used to detect the ligand of the aptamer and the target sequence of the igRNA. Such a dual detection mode can incease confidence of detection as the target sequence and an associated ligand must be detected for a positive test. The following Examples describe some of the materials and experiments used in the develop of the invention. Appendix A included herewith may provide additional information. Example 1: Methods This Example illustrates some of the materials and methods used in developing the invention. Protein purification Protein purification was performed as described by Fozouni et al. (2020). Briefly, the LbauCas13a expression vector was used, which included a codon-optimized Cas13a genomic GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 sequence, an N-terminal His6-MBP-TEV cleavage site sequence, and a T7 promoter binding sequence (Addgene Plasmid #83482). The protein was expressed in Rosetta 2 (DE3) pLysS E. coli cells in Terrific broth at 16°C overnight. Soluble His6-MBP-TEV-Cas13a was isolated over metal ion affinity chromatography and the His6-MBP tag was cleaved with TEV protease at 4°C overnight. Cleaved Cas13a was loaded onto a HiTrap SP column (GE Healthcare) and eluted over a linear KCl (0.25-1.0M) gradient. Cas13a-containing fractions were further purified via size-exclusion chromatography on a S200 column (GE Healthcare) in gel filtration buffer (20 mM HEPES-K pH 7.0, 200 mM KCl, 10% glycerol, 1 mM TCEP) and were subsequently flash frozen for storage at -80°C. Preparation of SARS-CoV-2 RNA segments In vitro RNA transcription was performed as described by Fozouni et al. (2020). The SARS-CoV-2 N gene, S gene (WT), and S gene with the D614G mutation were transcribed from a single-stranded DNA oligonucleotide template (IDT) using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB) following manufacturer’s recommendations. Template DNA was removed by addition of DNase I (NEB), and in vitro transcribed RNA was subsequently purified using RNA STAT-60 (AMSBIO) and the Direct-Zol RNA MiniPrep Kit (Zymo Research). RNA concentration was quantified by Nanodrop and RNA copy numbers were calculated using the transcript lengths and concentrations. Preparation of virus full genomic RNA Full genomic viral RNAs were purified as described by Fozouni et al. (2020). Isolate USAWA1 / 2020 of SARS-CoV-2 (BEI Resources) was propagated in Vero CCL-81 cells. Isolate Amsterdam I of HCoV-NL63 (NR-470, BEI Resources) was propagated in Huh7.5.1- ACE2 cells. All viral cultures used in a Biosafety Level 3 laboratory. RNA was extracted from the viral supernatant via RNA STAT-60 (AMSBIO) and the Direct-Zol RNA MiniPrep Kit (Zymo Research). crRNA design CRISPR RNA guides (crRNAs) were designed and validated for SARS-CoV-2. Fifteen crRNAs were first designed with 20-nt spacers corresponding to SARS-CoV-2 genome. Additional crRNAs were later designed. Each crRNA included a crRNA stem that was derived from a bacterial sequence, while the spacer sequence is derived from the SARS-CoV-2 genome (reverse complement). See Table 1A-1B (below) for examples of crRNA sequences. Table 1A: Examples of SARS-CoV-2 crRNA Sequences GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Table 1B: crRNAs used to Generate the Data in the Figures GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1The oligonucleotide is consisted of DNA (underlined) followed by RNA. The 20-nt oligonucleotides complementary to the crRNA spacer sequence is indicated in bold. Bulk Cas13a nuclease assays LbuCas13a-crRNA RNP complexes were first preassembled at 133nM equimolar concentrations for 15 minutes at room temperature and then diluted to 25 nM LbuCas13a in cleavage buffer (20 mM HEPES-Na pH 6.8, 50 mM KCl, 5 mM MgCl2, and 5% glycerol) in the presence of 400 nM of reporter RNA (5’-Alexa488rUrUrUrUrU-IowaBlack FQ-3’; SEQ ID NO: 66), 1 U / μL Murine RNase Inhibitor (NEB, Cat# M0314), 0.1 vol% IGEPAL 630 (Fisher, Cat# ICN 19859650), and varying amounts of target RNA. For reactions using more than one crRNA, multiple guides were combined at equal concentrations and subsequently the total crRNA mix was assembled with Cas13 at 133nM equimolar concentration. Twenty-five nM (25nM) of RNP complex were used unless specified otherwise. The reaction mix was measured either in bulk or as droplets following emulsification (see droplet formation). For the bulk Cas13a assay, the reaction mix was loaded into a 0.2mL eight-tube strip (Fisher Cat# 14- 222-251) and incubated in a compact fluorescence detector (Axxin, T16-ISO) for 1 hour at 37°C with fluorescence measurements taken every about 30 seconds (FAM channel, gain 20). Fluorescence values were normalized by the values obtained from reactions containing only reporter and buffer. Droplet formation To emulsify a Cas13a reaction mix, 20 μL of an aqueous mix was combined with 100 μL of HFE-7500 oil supplemented with 2% (w / w) PEG-PFPE amphiphilic block copolymer surfactant (008-Fluorosurfactant, RAN Biotechnologies) in a 0.2 mL eight tube-strip. The oil / aqueous mix was emulsified by repeated pipetting without any manual handling using an electronic 8-channel pipette (Integra biosciences, Part # 4623) with a 200 μL pipet tip (VWR GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Cat # 37001-532). The electronic pipette was used to mix 110 μL of sample volume for 150 repetitions at the maximum speed (speed 10) to emulsify droplets to a narrow size range. The emulsion was either directly loaded into a flow cell for time course imaging or incubated in a heating block at 37˚C before being transferred and imaged. In both cases, the emulsion was quickly separated by spinning in a speed-controlled mini-centrifuge (about 50 rpm) for 10 seconds, the oil was completely removed from the bottom of the tube, and the emulsion was transferred into a custom flow cell after several cycles of gentle manual mixing. Flow cell for droplet imaging The sample flow cell was prepared by sandwiching double-sided tape (about 20μm thick, 3M Cat# 9457) between an acrylic slide (75mm x 25mm x 2mm, laser cut from a 2mm- thick acrylic plate) and a siliconized coverslip (22mm x 22mm x 0.22mm, Hampton research Cat# 500829). Both surfaces were hydrophobic, promoting thin layers of oil between the droplets and the two surfaces. Siliconized coverslips were rinsed with isopropanol to remove any auto-fluorescent debris (20 minutes sonication) and spin dried prior to assembly. Fifteen microliters of sample emulsion were loaded into the flow cell by capillary action, after which the inlet and outlet were sealed with Valap sealant. Microscopy and data acquisition Droplet imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) equipped with a Yokogawa CSU-X spinning disk. A 488-nm solid state laser (ILE-400 multimode fiber with BCU, Andor Technologies) was used to excite the RNA fluorescent probe. The fluorescence light was spectrally filtered with an emission 535 / 40nm filter (Chroma Technology) and imaged using an sCMOS camera (Zyla 4.2, Andor Technologies). A 20x water-immersion objective (CFI Apo LWD Lambda S, NA 0.95) was used with the Perfect Focus System to monitor droplets during the course of reaction and / or to accurately quantify fluorescence signals at reaction endpoints. Images were acquired through Micro-Manager under X W / cm2488-nm excitation with 500ms exposure time and 2x2 camera binning. Typically, sixteen field-of-views (FOV) are acquired every 30 seconds for the time course of imaging and 36 field-of views were acquired for the endpoint imaging. A 4x objective (CFI Plan Apo Lambda, NA 0.20) was used for the high-throughput droplet imaging at reaction endpoints. Thirty-six FOVs were acquired under xx W / cm2 excitation with 3s exposure time without camera binning. Image analysis – droplet detection A custom MATLAB (Mathworks R2020b) script was used to detect positive droplets and quantify fluorescence signals. First, the grayscale images were converted to binary images GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 based on a locally adaptive threshold. The threshold was defined generously at this stage to select all the positive droplets and potentially some negative droplets or debris. Second, connected droplets were separated by watershed transform. Third, individual droplets were identified by looking for circular continuous regions and droplet parameters such as radius, circularity. The fluorescence signals were then quantified in two different ways: the mean fluorescence signal of a droplet reflecting the density of cleaved reporter; and the total fluorescence signal reflecting the total amount of cleaved reporter within a droplet. Lastly, positive droplets were chosen based on their circularity and total fluorescence signal by applying a threshold that were consistently used throughout the experiments. Image analysis – droplet tracking in time course images To quantify signal accumulation in the same droplet over time, droplets were associated with their motion over time as estimated by a Kalman filter in MATLAB. The filter was used to predict the track's location in each frame and to determine the likelihood of each detection within a frame being assigned to a particular track. Only the droplets showing continuous trajectories in time and magnitude are selected for downstream analysis. Comparison of single Cas13a reaction with enzyme kinetics The Cas13a reaction was analyzed with a single crRNA (Fig.1F) using the Michaelis Menten enzyme kinetics model with the quasi-steady-state approximation: where ^^ is the reaction rate, [^^0] is ternary Cas13a, [^^] is the RNA reporter, ^^^^at and ^^^^ are the catalytic rate constant and the Michaelis constant. When low substrate concentration was used ([^^] << ^^^^), because the RNA reporter [^^] was 400nM and ^^^^ was estimated to be larger than 1μM (Slaymaker et al., 2019) the equation simplified to: where ^^ / [^^0] was turnover frequency, or the reciprocal of the mean waiting time <1 / t> in the single molecule Michaelis-Menten framework (Min et al., 2005). The ^^ / [^^0] turnover frequency could be obtained from FIG.1K and 1L after converting the fluorescence signal to molar concentration of cleaved reporter based on a calibration. Data analysis – Cas13a time trajectories The raw signal was processed in a series of steps prior to analysis. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 First, the raw signal was corrected for the global signal fluctuation, which arises from a slight drift in z-focus even with the Perfect Focus System. The global signal was characterized from the background droplets and was identified from the histogram of pixel values. In particular, the global signal was divided from the positive droplet signal in each image frame. Second, the inventors corrected for the photobleaching. The signal decay rate was characterized from more than 200 Cas13a curves exhibiting negative slopes and positive initial signals. Photobleaching was modeled as a linear function of initial signal based on the observed linear relationship between the decay rate versus the initial signal (R2= 0.87). Using this model, each trajectory point-by-point was corrected for photobleaching. Third, the trajectories were filtered with a weak Savitzky-Golay filter (order 5, frame length 9) to remove the high frequency measurement noise while preserving overall structure of the curve. Lastly, instantaneous slopes were calculated by dividing signal changes between frames by the frame interval and removing single outliers exhibiting high positive or negative slopes. To characterize key parameters of Cas13 kinetics, individual trajectories were analyzed in two different domains. First, the slope, time from target addition to the initiation of enzyme activity (Tinit), and RMSD were determined from signal time trajectories by linear regression. Because Tinit indicates time since droplet reaction, a constant time (12.5 minutes) was added that reflected the time from Cas13 droplet formation until the beginning of time course imaging. Second, the slopefast, slopeslow, and a fraction spent in each period were determined by fitting a gaussian pdf to the instantaneous slope distribution. The model qualities were compared between the single versus binary gaussian pdfs using Akaike's Information Criterion (AIC) to determine whether a trajectory exhibits two different periods of slope of not. Data analysis – kinetic barcoding The slope and RMSD of individual signal trajectories were used to compare Cas13a reactions between different target-crRNAs. Binary classification of trajectories was first performed based on the Supported Vector Machine (SVM) in MATLAB. For this, 200 to 400 signal trajectories in each condition we collected, and two or more independent experiments per condition were performed to prevent bias. The trajectories were converted into a 2D array consisting of the slope and RMSD and the array was divided into a training and a validation set. An algorithm was then trained using the training set with the known answers (i.e. target- crRNA condition) and the validation set was classified. The accuracy of identifying individual trajectories was 75% for HCoV-NL63 RNA vs SARS-CoV-2 RNA, and 73% for wild type versus D614G RNA (the D614G RNA was from a SARS-CoV-2 strain having a D614G GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 mutation in its Spike protein). To access significance between two groups of trajectories, a two- tailed Student’s t-test was employed to the predicted class and reported p-values. Example 2: High Sensitivity, High Specificity Multiplex RNA detection This Example demonstrates that RNA detection with high sensitivity and multiplexed specificity can be achieved in short detection times by encapsulating the Cas13 reaction in droplets and monitoring enzyme kinetics fluorescently. The methods described herein enable quantification of the absolute amount of target RNA based on the number of positive droplets. However, the small droplet volume employed accelerates signal accumulation of the direct Cas13 reaction. When a single target RNA is encapsulated in a droplet with a volume of approximately 10 picoliters as illustrated in FIG. 1A, the Cas13 signal accumulation rate is equivalent to that of a bulk reaction containing 105copies / μL of target RNA. To rapidly generate millions of droplets with volumes of about 10 pL, reaction mixtures containing LbuCas13a were emulsified in an excess volume of an oil / surfactant / detergent mixture as described in Example 1. The resulting droplets were imaged on an inverted fluorescence microscope (FIG.1B, 1I and 1J). Millions of droplets ranging from 10 to 40 μm diameter were formed after 2 minutes of pipetting with an automatic multi-channel pipettor (Fig.1C, 1I). Imaging the droplets allowed normalization of the fluorescence signal by droplet size (Byrnes et al., 2018) and avoided the need for slower and more complex systems to generate uniform droplet sizes. The Cas13 droplet assay was validated by forming droplets containing 10,000 copies / μL of SARS-CoV-2 RNA, along with LbuCas13a, crRNA targeting the SARS-CoV-2 N gene (crRNA 4, SEQ ID NO: 4) and a fluorophore-quencher pair tethered by RNA (reporter) and monitoring the reaction of positive droplets over time (FIG. 1D). At this target concentration, about 7% droplets contain the target RNA, with the vast majority of those containing only a single copy. The signal accumulation rate in droplets was inversely proportional to droplet size (FIG. 1K), with smaller droplets increasing faster than larger droplets. As shown in FIG.1E, a 9-fold increase in signal was observed for 23 μm droplets compared to a 3-fold increase in signal for 42 μm droplets. Measurements showed that a single LbuCas13a can cleave 471 ± 47 copies of reporter every second in the presence of 400nM reporter, indicating that the Kcat / KM is 1.2 x 109M-1s-1, which is two orders-of-magnitude higher than that measured for LbCas12a (Chen et al., 2018). These results are also consistent with those measured for LbuCas13a based on a bulk GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 assay (Shan et al., 2019). Notably, the absolute trans-cleavage rate of a single LbuCas13 remains consistent regardless of droplet size (FIG.1F). Longer incubation times resulted in linear increases in the average signals per droplet (FIG.1G). All the positive reactions could be correctly identified in reaction times as little as 5 minutes with a 20X / 0.95NA objective (FIG. 1H) and 15 minutes using a 4X / 0.20NA microscope objective (FIG.1M-1N). Guide combinations were tested to determine whether more signal could be obtained per target RNA and whether the detection time would be reduced in the Cas13a droplet assays. In vitro transcribed (IVT) target RNA corresponding to the N gene of SARS-CoV-2 (nucleotide positions 28274–29531) was used in droplets containing crRNA 2 (SEQ ID NO: 2), crRNA 4 (SEQ ID NO: 4), or both crRNAs as illustrated in FIG.2A. The number of the positive droplets and the quantity of signal from the positive droplets was measured. Surprisingly, although crRNAs 2 and 4 generated similar signals when used individually (FIG.2F) and might be expected to double the signal when both are present, the signal per droplet was not significantly different when using two crRNAs than when using just one crRNA (FIG.2B). Instead, the number of positive droplets almost doubled when droplets contained both crRNA 2 and crRNA 4 compared to just one of the crRNAs (FIG.2C). These data indicate that the N gene in vitro transcribed RNA was fragmented through cis-cleavage upon initiation of the reaction prior to droplet formation, causing the regions targeted by different crRNAs to be loaded into separate droplets (FIG. 2A). These results indicate that multiple crRNAs activate independent Cas13a reactions in different droplets. Increased guide combinations were evaluated in the droplet assay mixtures to ascertain whether they affect the number of detectable (positive) droplets. Twenty-six crRNAs were made by the inventors that targeted different regions of SARSCoV-2 genome, and that individually produced strong Cas13 signals (Table 1). As shown in FIG.2D, adding additional types of crRNAs while keeping the total RNP concentration to 25nM, increased the number of positive droplets. The activity of Cas13a remain constant even when only a small fraction of total RNPs in a droplet contained crRNA matching the target (FIG.2G). These data indicate that a large number of crRNAs (i.e.50 or more) can be combined to maximize the number of independent Cas13a reactions from the same target RNA. Droplet-based assays are fundamentally limited by the false-positive rate in the absence of target reactions, hence the generation of multiple positive droplets per target RNA can increase sensitivity of the assay. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 To further evaluate the sensitivity of the Cas13a droplet assay with guide combinations, serial dilutions were made of precisely tittered SARS-CoV-2 genomic RNA obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). The number of positive droplets in each dilution was quantified using either a single crRNA or all 26 crRNAs, using thirty-six images per condition (~160,000 droplets) after 15 minutes of reaction incubation (FIG.2E). For the single crRNA (crRNA 4, SEQ ID NO: 4), the number of positive droplets remained significantly higher than the no-target control for the samples containing twenty (20) target copies / μL or more (FIG.2E). For the combination of twenty-six (SEQ ID NO: 1-26) crRNAs, the direct detection limit of detection was lower than 1 copy / μL target, comparable to the sensitivity of PCR. This limit of detection was not improved if the assay reaction was incubated for 30 minutes instead of 15 minutes (FIG.2H). The fast Cas13a kinetics achievable in droplets depended on the crRNA and its target. For example, as illustrated in FIG. 3A, two crRNAs targeting a different segment of SARS- CoV-2 N gene., crRNA 11A and crRNA 12A (SEQ ID NOs: 36 and 37), exhibited significantly slower rates in a bulk reaction than crRNA 2 or 4 (SEQ ID NO: 2 or 4). For this reason, selection of crRNAs that support efficient Cas13 activity is critical for Cas13-based molecular diagnostics, though how different guide crRNAs affect the activity of Cas13 is not well understood (Wessels et al., 2020). The droplet assay was harnessed to study Cas13a enzymatic activity in the presence of single guide crRNAs, and hence single targets. As shown in FIG. 3B, while the number of positive droplets was reduced for guide crRNA 11A and 12A (SEQ ID NO: 36 and 37), the reduction in droplet count was significantly less than the change observed in bulk reaction (compare FIG.3B with FIG.3A). On the other hand, as shown in FIG.3C, the signal in each positive droplet was significantly reduced for crRNA 11A and 12A (SEQ ID NO: 36 and 37), compared to crRNA 4 (SEQ ID NO: 4). To further understand these differences, individual reaction trajectories were examined within positive droplets, where the reaction trajectories were reported as the change in fluorescence for each 30 second measurement time point. Interestingly, individual crRNA:Cas13a assays exhibited rich kinetic behaviors that were crRNA-dependent. As shown in FIG. 3D-3F, different endpoint signals resulted when using different guide crRNAs. The slope, shape, and x-intercept of the individual trajectories varied widely in droplets depending on the crRNA. In some cases, the trajectories exhibited striking stochastic behaviors, exhibiting periods of no signal increase followed by periods of rapid signal increase. However, the majority of positive droplets observed for all three crRNAs exhibited slopes significantly GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 higher (FIGS. 3D-3F) than the rare positive slopes exhibited for populations of RNP-only droplets (FIG. 3G). These data indicate that the specific combination of crRNA and target significantly affects Cas13a enzymatic activity even when a single target is present. To quantify differences in Cas13a kinetics within droplets, individual signal trajectories were characterized by their average slope, Root-mean-square-deviation (RMSD), and time from target addition to the initiation of enzyme activity (Tinit) (FIG. 3H). By calculating the instantaneous slopes at each point in the trajectory and fitting the slope distribution to a Gaussian curve, the inventors found that the trajectories exhibiting two different slopes: one ‘fast’ when fluorescence is increasing and one ‘slow’ when fluorescence is not increasing (FIG. 3I). Interestingly, even though the average slopes differed significantly for the different crRNAs (SEQ ID NO: 4, 36, and 37), the instantaneous ‘fast’ slopes were constant across all three crRNAs (FIG.3J). Consistent with this, crRNA 12A (SEQ ID NO: 37), which exhibited the lowest average slope of the three guide crRNAs, exhibited extended slow periods (FIG. 3K) and increased levels of signal fluctuation (FIG. 3L). In addition, the Tinit varied significantly among the three crRNAs evaluated. For example, crRNA 11A (SEQ ID NO: 36) exhibited the slowest Tinit of all (FIG.3M), resulting in reduced signal at the reaction endpoint (FIG.3C). To test if the stochastic behavior of the Cas13a reaction was caused by the unbinding of crRNA from Cas13a, the RNP concentration was changed to be below or above the Kd of crRNA-Cas13a. However, the stochastic behaviors remained unaltered (FIG. 3N and 3O) despite the changes in RNP concentration. In fact, the kinetic features remain qualitatively the same for both crRNA 4 (SEQ ID NO: 4) and crRNA 12A (SEQ ID NO: 37) even for droplets containing only single copies of each of the three Cas13a components: the Cas13a, the crRNA, and the target (FIG.3P). In contrast, when the SARS-CoV-2 RNA target was replaced with a 20-nucleotide fragment complementary to the crRNA12A spacer sequence (i.e. crRNA12C; SEQ ID NO: 83), the stochastic behavior of the reaction was no longer observed and Tinit was significantly shortened (FIG. 3Q). These data indicate that the reduced Cas13a activity observed for crRNA 12A (SEQ ID NO: 37) was caused by local or global folding of target RNA. Based on the distinct kinetic signatures observed for the different crRNA and target combinations, the inventors hypothesized that specific crRNA-target pairs could be identified based on their signal trajectories. As illustrated in FIG. 4A-4B, distinct crRNA:target kinetic signatures provide a method for multiplexed detection of different RNA viruses or different virus variants in a single droplet when using one fluorescent reporter. To test this hypothesis, GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 referred to herein as ‘kinetic barcoding,’ a crRNA was first combined with a common cold virus NL-63 (crRNA 63; SEQ ID NO: 61) and a second crRNA targeting SARS-CoV-2 (crRNA 12A; SEQ ID NO: 37). These two crRNA were chosen because they individually exhibit different kinetic signatures (FIG. 4C). Thirty-minute trajectories were collected from hundreds of droplets containing either NL63 or SARS-CoV-2 RNA. The droplets also contained Cas13a and both crRNAs. The two groups of trajectories were clearly distinguishable based on their average slopes and Root-mean-square-deviation (RMSD) (FIG.4D). To determine how clearly the NL63 RNA and the SARS-CoV-2 RNA can be distinguished, a subset of trajectories was randomly sampled, and their differences were compared by performing Student’s t-test on their binary classification result using the methods described in Example 1 (see FIG.4E). Increasing the number of trajectories and extending the measurement time improved classification (FIG.4K), though measurement times longer than 10 minutes did not provide any improvement. Overall, these data indicate that NL-63 and SARS-CoV-2 can be distinguish within 10 minutes provided that 20 or more trajectories are measured. Similar results are achieved when images were acquired every 3 minutes for 30 minutes instead of every 30 seconds for 10 minutes (FIG.4L). Next, the kinetic barcoding methods were evaluated to determine whether a mutant viral strain could be differentiated from the wild-type strain. One crRNA was used that targeted the variable region of SARS-CoV-2 S-protein and the signal trajectories generated from the in vitro transcribed wild type S gene were compared to the trajectories from the in vitro transcribed S gene harboring the D614G mutation. The D614G mutation is shared by all SARS- CoV-2 variants (CDC, 2020). As shown in FIG.4F, although both wild type and mutant signal trajectories are smooth (i.e. they exhibit low RMSD), the average slopes obtained with the mutant target were significantly lower than that of WT (see also FIG.4G). Using the difference in the average slopes of 30 or more signal trajectories, the D614G mutant RNA could be distinguished from the wild type RNA within 5 minutes (FIG.4M). The California SARS-CoV-2 variant (B.1.427 / B.1.429; Epsilon) was tested using the kinetic barcoding method to confirm its utility when with a clinical sample. The California SARS-CoV-2 variant (B.1.427 / B.1.429) harbors a unique S13I mutation and exhibits increased transmissibility and reduced neutralization by convalescent and post-vaccination sera (CDC, 2020). A crRNA targeting the region encompassing S13I mutation in SARS-CoV-2 S-protein was used that matched the mutant sequence. Viral RNA extracted from cultured viruses as well as RNA from patient samples was evaluated, where the RNA was known to have either the GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 wild type or the B.1.427 sequence. The patient samples exhibited Ct values of 15 to 20 in PCR testing and provided 15 to 350 positive trajectories among the droplets measured. Although individual trajectories from each sample exhibited heterogenous slopes and RMSDs, the slopes measured from the WT were significantly lower than those measured from the B.1.427 mutant (FIG.4I and 4N). To test if the B.1.427 / B.1.429 mutant strain could be correctly identified when only 10 individual trajectories are collected, 10 trajectories were randomly evaluated from each sample. As shown in FIG. 4I-4J, regardless of the choice of 10 trajectories, the average of slope distribution clearly distinguished between the WT and B.1.427 RNA, with a detection accuracy of about 99%. Overall, the data described herein demonstrate that a droplet-based Cas13 direct detection assay can achieve PCR-level sensitivity and can simultaneously distinguish different RNA targets based on their reaction kinetics. Because a crRNA can be diluted by 50 times or more without compromising its performance in the droplet-based assay, many different types of crRNAs can used within a droplet to further enhance detection sensitivity to lower than 1 copies / μL. At this sensitivity, the Cas13a direct detection droplet assay can be used in situations where extremely low viral loads are present. For example, the droplet cases Cas assay can be used for environmental samples, cancer miRNAs, latent HIV virus, as well as for different SARS-CoV-2 variants without the limitations and potential loss of RNA due to sample purification, reverse transcription, or amplification. The LbuCas13 was also found to be an efficient, diffusion-limited enzyme whose kinetics are controlled by the specific combination of crRNA and the target. The distribution of single Cas13 RNP’s activity was homogenous for crRNAs supporting high activities (FIG. 1F), suggesting that the active conformation of Cas13a RNP is stable over time. However, we found that certain crRNAs can switch off Cas13a activity for more than a minute. The observation that the stochastic signal is reduced when a short RNA fragment is presented instead of the full virus RNA (FIG.3Q) indicates that the target RNA’s local or global structure plays a role in the kinetics of Cas13a RNP. The data indicated that the effects of enzyme conformational switching (Liu et al., 2017) do not play a significant role. On the other hand, RNA mismatches between a crRNA and its target can reduce the slope of reaction without introducing the stochastic activity switching (FIG.4G-4H), indicating that multiple mechanisms can result in diverse Cas13a kinetics. Based on those kinetic signatures, the droplet methods were able to determine which virus or variant was present in a given droplet. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Digital assays are useful at enhancing the sensitivity and quantitative performance in ddPCR (Hindson et al., 2013; McDermott et al., 2013), protein detection (Rissin et al., 2010), and recently CRISPR-Cas-based nucleic acid detection (Ackerman et al., 2020; Shinoda et al., 2021; Tian et al., 2021; Yue et al., 2021). While some detection assays use existing ddPCR technologies, amplification-free Cas13a assays require smaller droplets (~10pL) than ddPCR (~900pL (Pinheiro et al., 2012)) to achieve useful signal amplification. The droplet-based Cas13a direct detection assay with kinetic barcoding described herein enable rapid and sensitive molecular diagnostics for multiple RNA viruses and RNA biomarkers. Example 3: Multiplex Detection with Different Linker-Effectors Multiplex measurement of 4 different viruses was performed using modified crRNAs containing different DNA effectors to produce different reaction kinetics for each virus. The detected viruses were HCoV NL63, SC2 delta, SC2 wt, and IAV H3N2. The linker-effector used with the target specific crRNA for each virus were: o HCoV-NL63 (with crRNA, no Effector-Linker) o SARS-CoV-2 delta (with AT Effector-Linker-crRNA) o SARS-CoV-2 (with TT Effector-Linker-crRNA) o IAV H3N2 (with TTTT Effector-Linker-crRNA) These igRNAs were mixed with target nucleic acids and Cas13 in a droplet assay as described above. The results are shown in FIG.5A to FIG.5D. FIG.5A shows the measurement of the four different viruses with modified crRNAs containing different DNA linker-effectors. Total N from two replicate measurements were 169, 406, 128, and 285 for HCoV NL63, SC2 delta, SC2 wt, and IAV H3N2. Signal slope is normalized for droplet size and its distribution is shown in histogram. FIG.5B shows raw signal time-trajectories for the four DNA-modified crRNAs targeting HCov NL63, SC2 wt, SC2 delta, and IAV H3N2. The signal is normalized for droplet size and its initial value is fixed as 0.15 representative trajectories are shown for each virus. FIG.5C shows prediction of target viruses based on the signal slope distribution. From more than 100 trajectories obtained for each sample in two replicate experiments, 30 trajectories are randomly selected (representative distribution shown in the inset and in D) and the virus compositions in the sample are predicted from the subset. The bar graph shows the mean and standard distribution of target predictions from 100 repeated samplings. FIG. 5D shows prediction of target viruses based on the signal slope distribution. Total 268, 272, 518, 157, 119 trajectories were obtained for HCoV, SC2 wt, IAV, HCoV+SC2wt, and SC2wt+IAV, GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 respectively. Among those, 30 trajectories are randomly selected from each sample (representative distribution shown on the left) and the target virus compositions are predicted from the subset. The bar graph on the right shows the mean and standard distribution of target predictions from 100 repeated sampling. Example 4: Multiplex Detection of Different SARS-CoV-2 Isolates Multiplex measurement of three different viruses was performed using modified crRNAs containing different DNA effectors to produce different reaction kinetics for each virus. The detected viruses were wt Sars-CoV-2, delta variant SARS-CoV-2, and omicron variant SARS-CoV-2. The linker-effector used with the target specific crRNA for each virus were: o SARS-CoV-2 (with 8A Effector-Linker-crRNA) o SARS-CoV-2 delta (with TT Effector-Linker-crRNA) o SARS-CoV-2 omicron (with crRNA, no Effector-Linker) These igRNAs were mixed with target nucleic acids and Cas13 in a droplet assay as described above. The results are shown in FIG. 6A to FIG. 6C. FIG.6A shows the design of crRNAs for multiplexed SC2 variant detection (top). Droplet Cas13a reaction is incubated with an individual variant’s synthetic RNA to obtain the standard curve (bottom). The dotted line indicates the kernel function of slope distribution. FIG.6B shows the clinical sample analysis scheme. The kernel function of clinical sample is compared with the standard curves obtained in A. The one that provides the smallest difference, measured in terms of RMSE between two curves, is selected as the target. FIG. 6C shows results for clinical samples and the kinetic barcoding prediction result. The Ct-values are obtained from the N-gene. For SC2 delta virus, RNA isolated from a cell was used (BEI Resources) instead of patient sample. The grey shading indicates incorrect predictions. Example 5: Effect of Linker-Effector Length The effect of length of the linker-effector was studies in this Example. Linker-Effectors with varying lengths of Ts or 12 As are incorporated into an igRNA, and the reaction rate is measured. The linkers were fixed at 8 nubcleotides, whereas the effectors length and sequence were varied to create the variable effects on Cas enzymes resulting in variabilities of kinetic slopes and endpoints. o TTTTTTTTTT (2 EFFECTOR + 8 linker). (2T). (SEQ ID NO: 84) o TTTTTTTTTTTC (4 EFFECTOR + 8 linker). (4T). (SEQ ID NO: 85) o TTTTTTTTCAGATC (5 EFFECTOR + 8 linker). (5T). (SEQ ID NO: 86) GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 o TTTTTTTTTTTTTC (6 EFFECTOR + 8 linker). (6T). (SEQ ID NO: 87) o TTTTTTTTTTTTTTT (7 EFFECTOR + 8 linker). (7T). (SEQ ID NO: 88) o TTTTTTTTTTTTTTTC (8 EFFECTOR + 8 linker). (8T). (SEQ ID NO: 89) o TTTTTTTTTTTTTTTTTC (10 EFFECTOR + 8 linker). (10T). (SEQ ID NO: 90) o TTTTTTTTTTTTTTTTTTTC (12 EFFECTOR + 8 linker). (12T). (SEQ ID NO: 91) o AAAAAAAAAACAGATC (8 EFFECTOR + 8 linker). (8A). (SEQ ID NO: 92) o AAAAAAAAAAAAAAAAAAAA (12 EFFECTOR + 8 linker). (12A). (SEQ ID NO: 93) o ATTTCAGATC (2 EFFECTOR + 8 linker). (AT). (SEQ ID NO: 94) o TTTTCAGATC (2 EFFECTOR + 8 linker). (TT). (SEQ ID NO: 95) o TTTTTTCAGATC (4 EFFECTOR + 8 linker). (TTTT). (SEQ ID NO: 96) FIG. 7 shows the results with the different igRNAs. The slope is normalized by the slope measured with unmodified crRNA4. Data are represented as mean ± SD of three replicates. Bibliography Ackerman, C.M., et al., 2020. Massively multiplexed nucleic acid detection with Cas13. Nature 1–6. doi.org / 10.1038 / s41586-020-2279-8 Bar-Even, A., et al., 2011. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry 50, 4402–4410. doi.org / 10.1021 / bi2002289 Byrnes, S.A., et al., 2018. Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction. Anal. 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Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol.28, 595– 599. doi.org / 10.1038 / nbt.1641 Samson, R., Deutch, J.M., 1978. Diffusion‐controlled reaction rate to a buried active site. J. Chem. Phys.68, 285–290. doi.org / 10.1063 / 1.435494 Shan, Y., et al., 2019. High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR / Cas13a trans-Cleavage Activity. Anal. Chem.91, 5278–5285. doi.org / 10.1021 / acs.analchem.9b00073 Shinoda, H., et al., 2021. Amplification-free RNA detection with CRISPR–Cas13. Commun. Biol.4, 1–7. doi.org / 10.1038 / s42003-021-02001-8 Slaymaker, I.M., et al., 2019. High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage. Cell Rep.26, 3741-3751.e5. doi.org / 10.1016 / j.celrep.2019.02.094 Tambe, A., et al., 2018. RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a. Cell Rep.24, 1025–1036. doi.org / 10.1016 / j.celrep.2018.06.105 Tian, T., et al., 2021. An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics. ACS Nano 15, 1167–1178. doi.org / 10.1021 / acsnano.0c08165 Wessels, H.-H., et al., 2020. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol.38, 722–727. doi.org / 10.1038 / s41587-020-0456-9 Yue, H., et al., 2021. Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level. Nano Lett. doi.org / 10.1021 / acs.nanolett.1c00715 All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. The following statements provide a summary of some aspects of the inventive nucleic acids and methods described herein. Statements: GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1. An assay mixture comprising a population of droplets ranging in diameter from at least 10 to 60 μm, the population comprising a test droplet subpopulation comprising at least one ribonucleoprotein complex, plus at least one reporter RNA, plus at least one target RNA. 2. The assay mixture of statement 1, wherein the at least one ribonucleoprotein complex comprises at least one Cas nuclease and at least one CRISPR guide RNA (crRNA). 3. The assay mixture of statement 2, wherein the Cas nuclease is a Cas13 nuclease, a Cas12 nuclease, or a combination of Cas13 nucleases and Cas12 nucleases. 4. The assay mixture of statement 2 or 3, wherein the at least one CRISPR guide RNA (crRNA) binds to at least one of the target RNA(s). 5. The assay mixture of any one of statements 1-4, wherein the at least one target RNA comprises a viral RNA, a prokaryotic RNA, or a eukaryotic mRNA. 6. The assay mixture of any one of statements 1-5, wherein the at least one target RNA comprises sequence that hybridizes to a wild type target RNA sequence. 7. The assay mixture of any one of statements 1-5, wherein the at least one target RNA comprises sequence that hybridizes to a variant or mutant target RNA sequence. 8. The assay mixture of any one of statements 1-7, wherein the at least one target RNA comprises a coronavirus RNA. 9. The assay mixture of any one of statements 1-7, wherein the at least one target RNA comprises a mRNA for a disease marker. 10. The assay mixture of any one of statements 1-7, wherein the at least one target RNA comprises a microRNA. 11. The assay mixture of any one of statements 1-10, wherein the at least one reporter RNA comprises at least one fluorophore and at least one fluorescence quencher. 12. The assay mixture of statement 11, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof. 13. The assay mixture of any one of statements 1-12, wherein the population of droplets ranges in diameter from 20 to 60 μm. 14. The assay mixture of any one of statements 1-13, more than one ribonucleoprotein complex, each comprising a different CRISPR guide RNA (crRNA). 15. The assay mixture of any one of statements 2-14, wherein the at least one CRISPR guide RNA (crRNA) has a SEQ ID NO:1-70 or 83 sequence. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 16. A method comprising (a) contacting a sample with at least one type of ribonucleoprotein complex and at least one type of reporter RNA to form a reaction mixture; (b) mixing the reaction mixture with oil and surfactant to form an emulsion comprising water-in-oil droplets, where at least some of the droplets encapsulate all components of the reaction mixture; (c) removing excess oil from the droplets; (d) selecting at least 1 droplet, or at least 3 droplets, or at least 10 droplets that emit fluorescence as positive droplets for monitoring; and (e) monitoring the fluorescence of the positive droplets over time. 17. The method of statement 14, further comprising determining one or more of the following kinetic parametes: a slope of signal over time (slope), a time from target addition to the initiation of enzyme activity (Tinit), a root-mean-square-deviation (RMSD) from signal time trajectories by linear regression for one or more of the positive droplets. 18. The method of statement 14 or 15, further comprising determining a slopefast parameter for one or more of the positive droplets, where the slopefast parameter comprises a percent of time where a fluorescence slope is steep. 19. The method of statement 14, 15 or 16, further comprising determining a slopeslow parameter for one or more of the positive droplets, where the slopeslow parameter comprises a percent of time where a fluorescence slope over time is shallow. 20. The method of any one of statements 14-17, further comprising identifying what target RNA(s) are present the sample. 21. The method of any one of statements 14-18, wherein the at least one ribonucleoprotein complex comprises at least one Cas nuclease and at least one CRISPR guide RNA (crRNA). 22. The method of any one of statements 14-19, wherein the Cas nuclease is a Cas13 nuclease, a Cas12 nuclease, or a combination of Cas13 nucleases and Cas12 nucleases. 23. The method of any one of statements 14-20, wherein the at least one CRISPR guide RNA (crRNA) binds to at least one of the target RNA(s). 24. The method of any one of statements 14-21, wherein the at least one target RNA comprises a viral RNA, a prokaryotic RNA, or a eukaryotic mRNA. 25. The method of any one of statements 14-22, wherein the at least one target RNA comprises sequence that hybridizes to a wild type target RNA sequence. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 26. The method of any one of statements 14-23, wherein the at least one target RNA comprises sequence that hybridizes to a variant or mutant target RNA sequence. 27. The method of any one of statements 14-24, wherein the at least one target RNA comprises a coronavirus RNA. 28. The method of any one of statements 14-25, wherein the at least one target RNA comprises a mRNA for a disease marker. 29. The method of any one of statements 14-26, wherein the at least one target RNA comprises a microRNA. 30. The method of any one of statements 14-27, wherein the at least one reporter RNA comprises at least one fluorophore and at least one fluorescence quencher. 31. The method of statement 28, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof. 32. The method of any one of statements 14-29, wherein the population of droplets ranges in diameter from 20 to 60 μm. 33. The method of any one of statements 16-32, comprising more than one ribonucleoprotein complex, each comprising a different CRISPR guide RNA (crRNA). 34. The method of any one of statements 14-29, wherein the at least one CRISPR guide RNA (crRNA) has a SEQ ID NO:1-70 or 83 sequence. The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a protein” or “a cell” includes a plurality of such nucleic acids, proteins, or cells (for example, a solution or dried preparation of nucleic acids or expression cassettes, a solution of proteins, or a population of cells), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 WHAT IS CLAIMED:

1. An assay mixture comprising a population of droplets ranging in diameter from at least 10 to 60 μm, the population comprising a test droplet subpopulation comprising at least one ribonucleoprotein complex, at least one reporter RNA, and at least one target RNA.

2. A method comprising (a) contacting a sample with at least one type of ribonucleoprotein complex and at least one type of reporter RNA to form a reaction mixture; (b) mixing the reaction mixture with oil and surfactant to form an emulsion comprising water-in-oil droplets, where at least some of the droplets encapsulate all components of the reaction mixture; (c) removing excess oil from the droplets; (d) selecting at least 1 droplet, or at least 3 droplets, or at least 10 droplets that emit fluorescence as positive droplets for monitoring; and (e) monitoring the fluorescence of the positive droplets over time.

3. A method comprising (a) contacting a sample with at least one type of ribonucleoprotein complex and at least one type of reporter RNA to form a reaction mixture, wherein the ribonucleoprotein complex comprises at least one type of igRNA that modifies an activity of the ribonucleoprotein complex; (b) mixing the reaction mixture with oil and surfactant to form an emulsion comprising water-in-oil droplets, where at least some of the droplets encapsulate all components of the reaction mixture; (c) removing excess oil from the droplets; (d) selecting at least 1 droplet, or at least 3 droplets, or at least 10 droplets that emit fluorescence as positive droplets for monitoring; and (e) monitoring the fluorescence of the positive droplets over time.

4. The method of claim 3, wherein the ribonucleoprotein complex comprises a Cas enzyme and the igRNA modifies the activity of the Cas enzyme.

5. The method of claim 3, wherein the igRNA is comprised of a nucleic acid, an amino acid, a hydrophilic polymer, or a combination thereof.

6. The method of claim 5, wherein the igRNA comprises a DNA or a RNA.

7. The method of claim 4, wherein the igRNA comprises a DNA and the Cas enzyme is a Cas13.GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 8. The method of claim 4, wherein the igRNA comprises a RNA and the Cas enzyme is a Cas 12.

9. The method of claim 3, wherein the modified activity of the ribonucleoprotein complex is a rate at which RNA cleavage products are formed from a reporter RNA.

10. The method of claim 9, wherein the rate at which RNA cleavage products are formed is reduced.

11. The method of claim 3, wherein the igRNA is comprised of a linker, an effector, and a crRNA.

12. The method of claim 11, wherein the linker has a length of 1 to 68 nanometers.

13. The method of claim 12, wherein the linker has a length of 5 nanometers.

14. The method of claim 13, wherein the linker is comprised of eight nucleotides.

15. The method of claim 11, wherein the effector comprises a nucleic acid.

16. The method of claim 15, wherein the effector comprises an aptamer that binds to a ligand.

17. The method of claim 16, wherein the ligand for the aptamer is a drug, a metabolite, an intermediate, a cofactor, a transition state analog, an ion, a metal, a nucleic acid, or a toxin.

18. The method of claim 16, wherein the ligand for the aptamer is a protein, a peptide, a nucleic acid, a polysaccharide, a glycoprotein, a hormone, or a receptor.

19. The method of claim 16, wherein a complementary target for the crRNA is added, and wherein the effector inhibits the activity of the ribonucleoprotein complex in the absence of the ligand for the aptamer.GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 20. The method of claim 19, wherein the binding of ligand by the aptamer modifies an activity of the ribonucleoprotein complex.

21. The method of claim 20, wherein the modified activity of the ribonucleoprotein complex is a rate at which RNA cleavage products are formed from a reporter RNA.

22. A composition comprising an igRNA comprised of a linker, an effector, and a crRNA.

23. The composition of claim 22, wherein the linker has a length of 1 to 68 nanometers.

24. The composition of claim 23, wherein the linker has a length of 5 nanometers.

25. The composition of claim 24, wherein the linker is comprised of eight nucleotides.

26. The composition of claim 22, wherein the effector comprises a nucleic acid.

27. The composition of claim 26, wherein the effector comprises an aptamer that binds a ligand.

28. The composition of claim 27, wherein the ligand for the aptamer is a drug, a metabolite, an intermediate, a cofactor, a transition state analog, an ion, a metal, a nucleic acid, or a toxin.

29. The composition of claim 27, wherein the ligand for the aptamer is a protein, a peptide, a nucleic acid, a polysaccharide, a glycoprotein, a hormone, or a receptor.

30. The composition of claim 26, further comprising a Cas enzyme and a target polynucleotide for the igRNA.

31. The composition of claim 30, further comprising a ligand for an aptamer, wherein the binding of ligand by the aptamer modifies an activity of the Cas enzyme.GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 32. The composition of claim 31, wherein the modified activity of the ribonucleoprotein complex is a rate at which RNA cleavage products are formed from a reporter RNA.

33. A method of detecting a target polynucleotide in a biological sample comprising: a) contacting the biological sample with: i) a labeled detector RNA; ii) a Cas 13 enzyme that cleaves the labeled detector RNA; and iii) a guide RNA (igRNA) comprising a linker, an effector, and a crRNA that hybridizes with the target polynucleotide, wherein the igRNA binds the Cas 13 enzyme; and b) measuring a detectable signal produced by the cleavage of the labeled detector RNA.

34. The method of claim 33, wherein the linker has a length of 1 to 68 nanometers.

35. The method of claim 34, wherein the linker has a length of 5 nanometers.

36. The method of claim 35, wherein the linker is comprised of eight nucleotides.

37. The method of claim 33, wherein the effector comprises a nucleic acid.

38. The method of claim 37, wherein the effector comprises an aptamer that binds a ligand.

39. The method of claim 33, wherein the labeled detector RNA comprises a fluorescence- emitting dye pair or a quencher / fluor pair.

40. The method of claim 33, wherein the measuring is carried out in a multiwell plate.

41. The method of claim 33, wherein the labeled detector RNA comprises a gold nanoparticle tethered by the RNA to a conducting surface.

42. The method of claim 33, further comprising a plurality of guide RNA’s (igRNA’s) having different linker-effectors that can be used to produce multiple different kinetic rates upon activation of the Cas 13 enzyme by the binding of the plurality of igRNA’s to a plurality of target polynucleotides.GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 43. The method of claim 41, further comprising a plurality of labeled detector RNA’s that produce light of different wavelengths to increase the number of target polynucleotides that can be detected in a multiplex assay.

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