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WO2025128819A1 - Codage à barres cinétique programmable pour détection d'arn multiplexé - Google Patents

Codage à barres cinétique programmable pour détection d'arn multiplexé Download PDF

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WO2025128819A1
WO2025128819A1 PCT/US2024/059767 US2024059767W WO2025128819A1 WO 2025128819 A1 WO2025128819 A1 WO 2025128819A1 US 2024059767 W US2024059767 W US 2024059767W WO 2025128819 A1 WO2025128819 A1 WO 2025128819A1
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rna
droplets
crrna
linker
effector
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Daniel A. Fletcher
Sungmin Son
Carlos Ng PITTI
Melanie Ott
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J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
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J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • BACKGROUND PCR-based assays are currently the gold standard for RNA detection, as they can achieve high sensitivity ( ⁇ 1 copy/ ⁇ L) 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).
  • 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.
  • direct detection with LbuCas13a measured down to ⁇ 200 copies/ ⁇ L in 30 minutes with 3 crRNAs (Fozouni et al., 2021) and ⁇ 63 copies/ ⁇ L in 2 hours with 8 crRNAs (Liu et al., 2021).
  • 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.
  • ddPCR droplet digital PCR
  • 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.
  • the Cas13 signal accumulation rate is equivalent to that of a bulk reaction containing 10 5 copies/ ⁇ L of target RNA (see, e.g., FIG.1A).
  • assay mixtures that include a population of droplets ranging in diameter from at least 10 to 60 ⁇ m, 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.
  • 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.
  • 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.
  • the igRNA with aptamers as part of the linker-effector region that bind to a ligand.
  • the igRNA with an aptamer effector modifies the activity of the Cas enzyme without the aptamer ligand.
  • 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.
  • 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.
  • 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.
  • 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. 1E graphically illustrate the fluorescent signal over time in three positive droplets (the green lines) and one background droplet (the grey line).
  • Guide RNA crRNA 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.
  • 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.
  • 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 10 4 copies/ ⁇ L 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 mm 2 (mean ⁇ SD of three replicates).
  • FIG.2D graphically illustrates the number of positive droplets quantified for different crRNA combinations after adding 100 copies/ ⁇ L 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.
  • 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.
  • FIG. 3A-3Q illustrate crRNA-dependent heterogenous Cas13a activities.
  • FIG. 3A graphically illustrates the slope of a bulk Cas13a reaction containing 3.5 x 10 4 copies/ ⁇ L 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.
  • FIG.3B graphically illustrates the number of positive droplets for a droplet Cas13a reaction with 3.5 x 10 4 copies/ ⁇ L 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 10 4 copies/ ⁇ L of SARS-CoV-2 RNA described for FIG.3B.
  • 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).
  • the one hundred individual trajectories were monitored from droplets ranging from 30 to 36 ⁇ m 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 ⁇ m 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.
  • 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.
  • FIGs. 3J-3M the distributions of key Cas13a kinetic parameters are represented as the box and whisker plots including outliers.
  • 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.
  • Slope Average slope
  • RMSD Root-mean-square-deviation
  • 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.
  • 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.
  • 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.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.
  • FIG. 4I graphically illustrates identification of the SARS- CoV-2 B.1.427 variant from clinical samples using the kinetic-barcoding methods.
  • 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. 4K graphically illustrates the p-values of increasing numbers of signal trajectories over time. The 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).
  • 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.
  • crRNAs target-specific CRISPR guide RNAs
  • 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.
  • 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 ⁇ m.
  • 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.
  • RNP ribonucleoprotein
  • 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.
  • 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.
  • RNP ribonucleoprotein
  • 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.
  • 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.
  • 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.
  • a Kalman filter e.g. in MATLAB
  • 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.
  • 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 (T init ), 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 T init to 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.
  • 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.
  • 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)).
  • 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).
  • PEG and other water-soluble polymers
  • PNA peptide nucleic acid
  • GAA glycol nucleic acid
  • TAA therose nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • XNA xeno nucleic acids
  • 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.
  • 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.
  • 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).
  • the nuclease activity that digests part of the effector is an activated (Cas) ribonucleoprotein complex.
  • the effector can hybridize to another sequence (e.g., the crRNA or target) and disrupt the active structure-conformation of the ribonucleoprotein complex.
  • the effector can be decoupled from the linker and crRNA.
  • 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.
  • ssDNA linker and effectors are resistant to cleavage by Cas13
  • RNA linker and effectors are resistant to cleavage by Cas12.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA or DNA that is capable of binding to a specific molecule with high affinity and specificity
  • RNA or DNA that is capable of binding to a specific molecule with high affinity and specificity
  • Tuerk et al. Science 249, 505-10 (1990)
  • 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.
  • 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.
  • 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.
  • aptamer molecules can be used as an effector using the methods and compositions described herein.
  • 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.
  • small organic molecules like dopamine, theophylline, sulforhodamine B, and cellobiose are used as ligands in the isolation of aptamers.
  • a dual enzyme system can be used to turn off the negative modulation produced by the igRNA (effector and linker) on kinetics.
  • 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.
  • RNA 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.
  • the samples can be any biological fluid or tissue from any virus, fungus, plant or animal that is suspected of having an RNA.
  • RNA types that can be evaluated in the methods include mRNAs, genomic RNAs, tRNAs, rRNAs, microRNAs, and combinations thereof.
  • 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.
  • RNA Ribonucleoproteins
  • RNP ribonucleoprotein
  • crRNA CRISPR guide RNA
  • the Cas nuclease can be one or more Cas12 or Cas13 (some previously known as C2c2) nucleases.
  • C2c2 Cas12 or Cas13
  • the Cas nucleases employed bind and cleave RNA substrates, rather than DNA substrates, to which Cas9 can bind.
  • the Cas nucleases can be from a variety of organisms and can have sequence variations.
  • 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.
  • a Leptotrichia wadei Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 71; NCBI accession no. WP_036059678.1).
  • 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 2
  • a Herbinix hemicellulosilytica Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 72; NCBI accession no. WP_103203632.1).
  • a Leptotrichia buccalis Cas13a endonuclease can be used that has the following sequence (SEQ ID NO: 73; NCBI accession no. WP_015770004.1).
  • 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 A
  • 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.
  • 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.
  • the Cas nuclease cleaves the RNA reporter, the fluorophore is separated from the associated quencher, such that a fluorescence signal becomes detectable.
  • RNA reporter is the RNaseAlert (IDT).
  • IDTT RNaseAlert
  • 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.
  • TIRF Total internal reflection fluorescence
  • 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.
  • the sample RNA can then be added.
  • the sample RNA acts as an 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.
  • 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.
  • RNP Cas nuclease/crRNA ribonucleoprotein
  • 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.
  • aliquots e.g., 5-50 microliters
  • 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.
  • reaction mixture-oil-surfactant combination can be emulsified to generate droplets ranging in diameter from at least 10 to 60 ⁇ m. In some cases, the size range is a narrower size range of about 20 to 50 ⁇ m.
  • 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.
  • a shallow flow cell can be used to minimize signal/droplet overlap.
  • 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 10 ⁇ m to about 60 ⁇ m apart.
  • 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 10 ⁇ m to about 60 ⁇ m thick e.g., about 20 ⁇ m thick
  • Such a flow cell can contain about 10 ⁇ 1 to about 60 ⁇ l fluid, where the droplets are free to move around in the fluid.
  • homogenous droplets may be formed using microfluidic devices.
  • sized microwells made by injection molding or other methods may be used in place of droplets for the kinetic barcoding assay.
  • 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%.
  • 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.
  • the igRNA with an aptamer effector modifies the activity of the Cas enzyme without the aptamer ligand.
  • 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.
  • the ribonucleoprotein complex with the aptamer effector can be used to detect the ligand that binds to the aptamer.
  • 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.
  • 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.
  • 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.
  • Example 1 Methods This Example illustrates some of the materials and methods used in developing the invention.
  • 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.
  • 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.
  • IDTT single-stranded DNA oligonucleotide template
  • NEB HiScribe T7 Quick High Yield RNA Synthesis Kit
  • 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 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1 GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 1
  • the 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/ ⁇ L Murine RNase Inhibitor (NEB, Cat# M0314), 0.1 vol% IGEPAL 630 (Fisher, Cat# ICN 19859650), and varying amounts of target RNA.
  • Fluorescence values were normalized by the values obtained from reactions containing only reporter and buffer.
  • Droplet formation To emulsify a Cas13a reaction mix, 20 ⁇ L of an aqueous mix was combined with 100 ⁇ L 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 ⁇ L pipet tip (VWR GL2023-016-2 / BK-2024-049-2 / 3730.228WO1 Cat # 37001-532).
  • the electronic pipette was used to mix 110 ⁇ L 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.
  • Flow cell for droplet imaging The sample flow cell was prepared by sandwiching double-sided tape (about 20 ⁇ m 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/cm 2 488-nm excitation with 500ms exposure time and 2x2 camera binning.
  • 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.
  • 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.
  • 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.
  • RNA reporter [ ⁇ ] was 400nM and ⁇ was estimated to be larger than 1 ⁇ M (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.
  • 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.
  • the inventors corrected for the photobleaching.
  • each trajectory point-by-point was corrected for photobleaching.
  • 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.
  • instantaneous slopes were calculated by dividing signal changes between frames by the frame interval and removing single outliers exhibiting high positive or negative slopes.
  • 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.
  • 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.
  • SVM Supported Vector Machine
  • 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).
  • 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.
  • the small droplet volume employed accelerates signal accumulation of the direct Cas13 reaction.
  • the Cas13 signal accumulation rate is equivalent to that of a bulk reaction containing 10 5 copies/ ⁇ L of target RNA.
  • 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 ⁇ m 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/ ⁇ L 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).
  • crRNA 4 SEQ ID NO: 4
  • a fluorophore-quencher pair tethered by RNA (reporter) and monitoring the reaction of positive droplets over time (FIG. 1D).
  • crRNA 4 the SARS-CoV-2 N gene
  • FET fluorophore-quencher pair tethered by 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).
  • the number of positive droplets remained significantly higher than the no-target control for the samples containing twenty (20) target copies/ ⁇ L or more (FIG.2E).
  • the direct detection limit of detection was lower than 1 copy/ ⁇ L 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • RMSD Root-mean-square-deviation
  • 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).
  • 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).
  • 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.
  • 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).
  • the Cas13a direct detection droplet assay can be used in situations where extremely low viral loads are present.
  • 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.
  • 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.
  • 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).
  • 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)
  • 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.
  • 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.
  • 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.
  • 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)
  • SARS-CoV-2 with 8A Effector-Linker-crRNA
  • SARS-CoV-2 delta with TT Effector-Linker-crRNA
  • SARS-CoV-2 omicron with crRNA, no Effector-Linker
  • 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.
  • 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.
  • 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.
  • FIG. 7 shows the results with the different igRNAs.
  • 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.
  • An assay mixture comprising a population of droplets ranging in diameter from at least 10 to 60 ⁇ m, 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 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).
  • 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 at least one target RNA comprises a coronavirus RNA.
  • the at least one target RNA comprises a mRNA for a disease marker.
  • the at least one target RNA comprises a microRNA.
  • the at least one reporter RNA comprises at least one fluorophore and at least one fluorescence quencher. 12.
  • 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.
  • 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.
  • the at least one target RNA comprises a viral RNA, a prokaryotic RNA, or a eukaryotic mRNA.
  • 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.
  • the at least one target RNA comprises a coronavirus RNA. 28.
  • 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.
  • 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.

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Abstract

Telle que décrite dans la description, la détection d'ARN avec une sensibilité élevée et une spécificité multiplexée peut être obtenue avec des temps de détection courts en encapsulant la réaction de Cas dans des gouttelettes et en surveillant la cinétique enzymatique par fluorescence.
PCT/US2024/059767 2023-12-12 2024-12-12 Codage à barres cinétique programmable pour détection d'arn multiplexé Pending WO2025128819A1 (fr)

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Citations (2)

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US20220073987A1 (en) * 2018-11-14 2022-03-10 The Broad Institute, Inc. Crispr system based droplet diagnostic systems and methods
WO2023278834A1 (fr) * 2021-07-02 2023-01-05 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Codage à code à barres cinétique pour améliorer la spécificité de réactions de crispr/cas

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220073987A1 (en) * 2018-11-14 2022-03-10 The Broad Institute, Inc. Crispr system based droplet diagnostic systems and methods
WO2023278834A1 (fr) * 2021-07-02 2023-01-05 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Codage à code à barres cinétique pour améliorer la spécificité de réactions de crispr/cas

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