WO2024040112A2 - Signal amplification assays for nucleic acid detection - Google Patents
Signal amplification assays for nucleic acid detection Download PDFInfo
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- WO2024040112A2 WO2024040112A2 PCT/US2023/072302 US2023072302W WO2024040112A2 WO 2024040112 A2 WO2024040112 A2 WO 2024040112A2 US 2023072302 W US2023072302 W US 2023072302W WO 2024040112 A2 WO2024040112 A2 WO 2024040112A2
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- Prior art keywords
- nucleic acid
- cleavage
- target nucleic
- programmable nuclease
- reporter
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- Diagnostic assays can detect ailments at the point of need through the use of diagnostic devices. Diagnostic tests that detect the presence or absence of nucleic acids often have an advantage in sensitivity and specificity over other test methods, such as those based on antigen detection of the pathogenic organism. In some cases, while antibody/antigen tests may have the ability to directly assay a subject’s immune response to a pathogenic organism, these systems can have sensitivity and specificity challenges related to the response time and the variabilities of antibodies/antigens produced by the subject’s body. However, nucleic acidbased detection systems are not without limitations to detection sensitivity.
- the present disclosure provides a system for detecting a target nucleic acid.
- the system comprises a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter.
- the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template;
- the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript;
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate;
- the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product;
- the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circular
- the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- the target nucleic acid comprises RNA.
- the linear transcription template is single-stranded.
- the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils).
- the RNA polymerase is a T7 RNA polymerase.
- the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the RNA polymerase is an SP6 RNA polymerase.
- the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- the programmable nuclease is a type VI CRISPR/Cas effector protein.
- the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e).
- the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
- the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated complex.
- the detectable cleavage product comprises the quantum dot.
- the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
- the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
- the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the programmable nuclease, a different non-naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter; (b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and (c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
- the present disclosure provides a system for detecting a target nucleic acid.
- the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter.
- the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid
- the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template
- the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid
- the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated upon binding the RNA transcript
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate
- the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) clea
- the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- the target nucleic acid comprises RNA.
- the linear transcription template is single-stranded.
- the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils).
- the RNA polymerase is a T7 RNA polymerase.
- the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the RNA polymerase is an SP6 RNA polymerase.
- the first programmable nuclease, the second programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- the first or second programmable nuclease is a type VI CRISPR/Cas effector protein.
- the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e).
- the first programmable nuclease and the second programmable nuclease are the same.
- the first programmable nuclease and the second programmable nuclease are different.
- the first non- naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same.
- the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different.
- the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
- the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated first or second complex.
- the detectable cleavage product comprises the quantum dot.
- the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
- the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
- the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the first programmable nuclease, a different first non-naturally occurring guide nucleic acid, the same or different linear transcription template, the RNA polymerase, the ligase, the second programmable nuclease, the same or different second non-naturally occurring guide nucleic acid, and the reporter; (b) each different first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of a different target nucleic acid; and (c) each different transcription template, if different, is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
- the present disclosure provides a method for detecting a target nucleic acid in a sample using a system disclosed herein.
- the method comprises (a) contacting the system with the sample; and (b) detecting the detectable cleavage product.
- the present disclosure provides a method for detecting a target nucleic acid in a sample in a single reaction volume. In another aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample in two or more reaction volumes (e.g., a two-pot assay).
- the method comprises: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non- naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease; (b) cleaving a linear transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product; (c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template; (d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript; (e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a second non- naturally occurring guide nucleic acid that hybridizes to a portion of the RNA transcript, thereby activating the second programmable nucleas
- the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template.
- the target nucleic acid comprises RNA.
- the linear transcription template is single-stranded.
- the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides (e.g., at least two uracils).
- the DNA-dependent RNA polymerase is a T7 RNA polymerase.
- the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the DNA-dependent RNA polymerase is an SP6 RNA polymerase.
- the first programmable nuclease, second programmable nuclease, DNA- dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein.
- the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e).
- the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume.
- the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
- the method further comprises repeating the method in parallel in each of a plurality of single reaction volumes (or, in the case of a two-pot assay, in each of a plurality of dual reaction volumes), wherein each of the plurality of single reaction volumes comprises a different first non-naturally occurring guide nucleic acid.
- each of the plurality of single reaction volumes comprises a different linear transcription template.
- the present disclosure provides a reporter complex comprising a quantum dot conjugated to a plurality of reporter oligonucleotides.
- each of the reporter oligonucleotides comprises a secondary fluorophore or a quencher.
- FIG. 1 is a graphical representation of fluorescence-based signal amplification for two circular ssDNAs (“Dumbbell 7” and “Dumbbell 12”) which each contain the antisense sequence for a Casl3 crRNA target.
- the “Trigger” condition includes a second oligonucleotide used to double strand a portion of the circular ssDNA template.
- the top line in both Dumbbell 7 and Dumbbell 12 graphs represent the “No trigger” condition
- the second, slightly lower line represents the “Trigger” condition
- the third line along the x-axis represents the “No NTPs”, meaning no functional RCT was included in the reaction condition.
- FIG. 2 is a graphical representation demonstrating signal generation capacity of “Dumbbell 7” and Dumbbell 12” ssDNAs in an RCT/DETECTR assay using the ThermoFisher Q5 thermocycler for heating and fluorescence detection.
- the sensitivity of the signal generation was in the pM range for the circular ssDNA template under the conditions tested.
- FIG. 3 is a graphical representation of the capacity for a T7 RNA polymerase (RNAP) to initiate an RCT/DETECTR reaction and fluorescence emission using either a promoterless circular (top line in both graphs) or linear (bottom line in both graphs) template.
- RNAP T7 RNA polymerase
- FIG. 4 is a graphical representation of a one-pot RCT/DETECTR reaction in the presence of either additives betaine (left) or DMSO (right).
- FIG. 5 is a graphical representation of the fluorescence emission for one-pot RCT/DETECTR assays with T7 RNAP, T3 RNAP (bottom data set), or SP6 RNAP, in the presence of circular (solid lines) and linear (dashed lines) templates.
- FIG. 6 is a gel image for an assay demonstrating the cleavage activity of activated Cast 3 on poly-Uracil tracts that are internal to a larger DNA oligonucleotide.
- the columns show illustrative results for: 1) FAM-oligo only; 2) FAM-oligo duplex with no bulge, target, and programmable nuclease complex; 3) FAM-oligo duplex with a 5’-UU-3’ (2U) bulge, programmable nuclease complex, and no target control (NTC); 4) FAM-oligo duplex with a 2U bulge, target, and programmable nuclease complex; 5) FAM-oligo duplex with 5’- UUUU-3’ (5U) bulge, programmable nuclease complex, and NTC; 6) FAM-oligo duplex with 5U bulge, target, and programmable nuclease complex; 7) FAM-oligo duplex with 5’- UUUU
- FIG. 7 is a schematic representation of an exemplary RCT/DETECTR signal amplification assay.
- the presence of the target nucleic acid activates the programmable nuclease complex (e.g., a programmable nuclease in complex with a guide nucleic acid), which then cleaves the blocking end of a linear template.
- the programmable nuclease complex e.g., a programmable nuclease in complex with a guide nucleic acid
- This allows for circularization through ligation, producing a functional template on which the RNA Polymerase performs RCT, in turn generating a targetable RNA product for a programmable nuclease (e.g., a Type VI Cas protein, which may be the same programmable nuclease complex or a different programmable nuclease complex).
- the programmable nuclease complex detects this targetable RNA product and cleaves more blocked templates alongside non-target reporters.
- FIGS. 8A-8B show schematic representations of quantum dot reporter according to embodiments of type I (FIG. 8A) and type II (FIG. 8B) quantum dot reporters.
- the illustrated type I quantum dot reporter in FIG. 8 A is conjugated to a reporter oligonucleotide comprising a single stranded nucleic acid which is conjugated to a fluorescence quencher moiety.
- programmable nuclease-based cleavage of the reporter oligonucleotide releases the quencher moiety and results in increased fluorescence.
- a reporter oligonucleotide comprising a single stranded nucleic acid which is conjugated to a fluorescent moiety (e.g., a secondary fluorophore) for use as a fluorescence resonance energy transfer (FRET) donoracceptor pair.
- a fluorescent moiety e.g., a secondary fluorophore
- FRET fluorescence resonance energy transfer
- programmable nuclease-based cleavage of the reporter oligonucleotide releases the fluorescent moiety and results in a color shift.
- the fluorescent moieties conjugated to each nucleic acid of the reporter oligonucleotide can be of a single type, or multiple types to allow for a multiplex assay with a readout of different color shifts for different targets.
- the fluorescent moiety could also be another quantum dot.
- FIG. 9 is a flow diagram illustrating a signal amplification process, in accordance with some embodiments.
- the intermediate cleavage target is a transcription template, as disclosed herein.
- FIG. 10 is an illustrative graphical representation of the capacity for a ligase to initiate an RCT/DETECTR reaction and fluorescence emission using an unblocked linear template.
- the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/— 10% of the specified value. In embodiments, about means the specified value. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.
- thermostable and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.
- percent identity refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment.
- an amino acid sequence is X% identical to SEQ ID NO: Y refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two.
- computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.
- a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay.
- a programmable nuclease-based detection e.g., DETECTR
- sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, and programmable nuclease- based detection (e.g., DETECTR) assays are carried out in a single volume.
- amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region).
- Readout of the detection (e.g., DETECTR) assay may occur in the single volume or in a second volume.
- the product of the one-pot DETECTR reaction e.g., a cleaved detection moiety comprising an enzyme
- another volume e.g., a volume comprising an enzyme substrate
- a “two-pot” reaction refers to a reaction in which more than one reaction occurs in two (or more) volumes alongside a programmable nuclease-based detection (e.g., DETECTR) assay.
- a programmable nuclease-based detection e.g., DETECTR
- sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof are carried out in a first volume and programmable nuclease-based detection (e.g., DETECTR) assays are carried out in a second volume.
- a two-pot assay may refer to detection of a target nucleic acid in a sample by a programmable nuclease (e.g., DETECTR)-based reaction in a first volume and signal amplification in a second volume.
- a programmable nuclease-based reaction occurs in a first volume whereby the first programmable nuclease and a first guide nucleic acid are activated in the presence of a target nucleic acid in a sample.
- the first programmable nuclease cleaves an intermediate cleavage target, and optionally a reporter.
- the cleaved intermediate cleavage target is transferred from the first volume to a second volume which contains signal amplification reagents (e.g., a second programmable nuclease, a second guide nucleic acid configured to recognize the cleaved intermediate cleavage target, a reporter a ligase, a polymerase, etc.).
- the signal amplification reaction may generate a detectable signal in the second volume which is indicative of the presence of the target nucleic acid in the sample.
- HotPot refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously.
- a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).
- nucleic acid amplification and “amplifying a nucleic acid,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.
- nucleic acid refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T/U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid.
- the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.
- the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end.
- Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
- cleavage assay refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid.
- the cleavage activity may be cis-cleavage activity.
- the cleavage activity may be trans-cleavage activity.
- Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions.
- DETECTR DNA endonuclease targeted CRISPR trans reporter
- detection of programmable nuclease-based reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.
- detecttable signal refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.
- detecting a nucleic acid and its grammatical equivalents, as used herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.
- effector protein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid.
- the complex comprises multiple effector proteins.
- the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid.
- the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid.
- effector protein refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule).
- the effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).
- guide nucleic acid refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein.
- the first sequence may be referred to herein as a spacer sequence.
- the second sequence may be referred to herein as a repeat sequence.
- the first sequence is located 5’ of the second nucleotide sequence.
- the first sequence is located 3’ of the second nucleotide sequence.
- nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid.
- a modification e.g., chemical modification, nucleotide sequence, or amino acid sequence
- compositions or systems described herein refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system.
- a composition may include an effector protein and a guide nucleic acid that do not naturally occur together.
- an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.
- PAM protospacer adjacent motif
- reporter and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.
- sample generally refers to something comprising a target nucleic acid.
- the sample is a biological sample, such as a biological fluid or tissue sample.
- the sample is an environmental sample.
- the sample may be a biological sample or environmental sample that is modified or manipulated.
- samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.
- target nucleic acid refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein.
- a target nucleic acid may comprise RNA, DNA, or a combination thereof.
- a target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).
- target sequence refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
- the present disclosure provides a system for detecting a target nucleic acid.
- the system comprises a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter.
- the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template;
- the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript;
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate;
- the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product;
- the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template;
- the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter.
- the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid
- the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template
- the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid
- the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated upon binding the RNA transcript
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate
- the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) cleave the reporter
- the first programmable nuclease and the second programmable nuclease are the same. In some embodiments, the first programmable nuclease and the second programmable nuclease are different. In some embodiments, the first non- naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different.
- the system further comprises a reverse transcriptase (RT).
- RT reverse transcriptase
- the RT is effective to reverse-transcribe a target nucleic acid that is an RNA to form a target cDNA.
- the target cDNA may function in place of or as an alternative to the target nucleic acid RNA in forming the activated complex with the programmable nuclease and non-naturally-occurring guide nucleic acid.
- the RT is effective to reverse transcribe the RNA transcript of the circularized template to form a transcript cDNA.
- the transcript cDNA functions in place of or as an alternative to the RNA transcript in forming an activated complex with the programmable nuclease and the non-naturally-occurring guide nucleic acid. Accordingly, the choice of programmable nuclease is not necessarily limited by whether the target nucleic acid is DNA or RNA, or by the RNA-nature of the RNA transcript. Accordingly, programmable nucleases that are most effectively activated by a target DNA are contemplated for use in embodiments of the systems and methods disclosed herein.
- the target can comprise, for example, a target sequence, a target molecule, or a target nucleic acid.
- a target can be referred to interchangeably as a target sequence, target molecule, or target nucleic acid.
- a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling process as described elsewhere herein).
- the target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any human, plant, animal, virus, bacteria, or microbe of interest.
- the systems, devices, apparatuses, methods, and compositions provided herein can be used to perform rapid tests in a single integrated system.
- the single integrated system may be a reusable unit or a disposable unit.
- the target can be, for example, a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm or other agents or organisms responsible for and/or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like).
- the target nucleic acid can be a nucleic acid, or a portion thereof.
- the target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample.
- the target nucleic acid can be a portion of an RNA or DNA from any organism in the sample.
- the target may comprise, for example, a biological sequence.
- the biological sequence can comprise a nucleic acid sequence or an amino acid sequence.
- the target may be associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.
- the target can be associated with one or more pathogenic viruses, pathogenic bacteria, pathogenic worms, pathogenic fungi, or cancer cells.
- the pathogenic viruses are selected from the group consisting of respiratory viruses, adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, SARS-CoV, SARS-CoV-2, MERS, gastrointestinal viruses, noroviruses, rotaviruses, astroviruses, exanthematous viruses, hepatic viral diseases, cutaneous viral diseases, herpes, hemorrhagic viral diseases, Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, neurologic viruses, polio, viral meningitis, viral encephalitis, rabies, sexually transmitted viruses, HIV, HPV, immunodeficiency viruses, influenza virus, dengue virus, West Nile virus, herpes virus, yellow fever virus, Hepatitis Virus C, Hepatitis Virus A, Hepatitis Virus B, and papillomavirus.
- SARS severe
- the target can be indicative of a respiratory disorder or respiratory pathogen.
- the respiratory disorder or respiratory pathogen is selected from the group consisting of SARS-CoV-2 and corresponding variants, human coronavirus hCoV (e g., 229E, NL63, OC43, HKU1), MERS-CoV, (MERS), SARS-CoV (SARS), Flu A, Flu B, RSV, Rhinovirus, Strep A, and TB.
- the target is indicative of a sexually transmitted infection (STI) or infection related to a woman’s health.
- the STI or infection related to a woman’s health is selected from the group consisting of CT, NG, MG, TV, HPV, Candida, B. Vaginosis Syphilis and UTI.
- the target comprises a single nucleotide polymorphism (SNP).
- the SNP is indicative of NASH disorder or Alpha- 1 disorder.
- the target is a blood borne pathogen selected from the group consisting of HIV, HBV, HCV, and/or Zika.
- the target is indicative of H. Pylori, C. Difficile, Norovirus, HSV, and/or Meningitis.
- the present disclosure provides a system for detecting a target nucleic acid.
- the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter.
- the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid
- the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template
- the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid
- the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated upon binding the RNA transcript
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate
- the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) clea
- the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- the target nucleic acid comprises RNA.
- the linear transcription template is single-stranded.
- the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils).
- the RNA polymerase is a T7 RNA polymerase.
- the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the RNA polymerase is an SP6 RNA polymerase.
- the first programmable nuclease, the second programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- the first or second programmable nuclease is a type VI CRISPR/Cas effector protein.
- the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e).
- the first programmable nuclease and the second programmable nuclease are the same.
- the first programmable nuclease and the second programmable nuclease are different.
- the first non- naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same.
- the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different.
- the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
- the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated first or second complex.
- the detectable cleavage product comprises the quantum dot.
- the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
- the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
- the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the first programmable nuclease, a different first non-naturally occurring guide nucleic acid, the same or different linear transcription template, the RNA polymerase, the ligase, the second programmable nuclease, the same or different second non-naturally occurring guide nucleic acid, and the reporter; (b) each different first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of a different target nucleic acid; and (c) each different transcription template, if different, is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
- one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter molecule.
- a programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter molecule.
- reporter refers to a reagent comprising a polynucleotide, wherein cleavage of the polynucleotide results in a change in a signal.
- the reporter may comprise a fluorescent label joined to a quencher by a short polynucleotide sequence.
- the reporter may comprise a detection moiety (e.g., a fluorescent label) immobilized to a surface by a short polynucleotide sequence, cleavage of which releases the detection moiety and results in a decrease in signal from the detection moiety at the surface.
- a detection moiety e.g., a fluorescent label
- Alternative detection moieties and arrangements for producing a change in signal upon cleavage of the polynucleotide portion of the reporter are possible, and illustrative examples are described herein.
- the polynucleotide of the reporter can comprise DNA, RNA, modified nucleotides, or a combination of two or more of these.
- the programmable nuclease can be, in some nonlimiting embodiments, an RNA-activated programmable RNA nuclease.
- the programmable nucleases disclosed herein can bind to a target DNA to initiate trans cleavage of a reporter.
- the programmable nuclease can be a DNA-activated programmable RNA nuclease.
- a programmable nuclease as described herein can be activated by a target RNA or a target DNA.
- the programmable nuclease may comprise a Cas enzyme which can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave one or more reporters (e.g., RNA reporter molecules).
- the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters.
- a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter.
- the programmable nuclease can comprise a DNA-activated programmable DNA nuclease.
- the programmable nuclease can comprise an enzyme.
- the enzyme may be, for example, a Cas enzyme.
- the Cas enzyme is selected from the group consisting of a Cas 12, Cas 12a, Cas 13, Cas 14, Cas 14a, and CasPhi.
- CRISPR/Cas enzymes are programmable nucleases that can be used to implement the methods and systems disclosed herein.
- CRISPR/Cas enzymes can include any of the known Classes and Types of CRISPR/Cas enzymes.
- Programmable nucleases disclosed herein can include, for instance, Class 1 CRISPR/Cas enzymes, such as the Type I, Type IV, or Type III CRISPR/Cas enzymes.
- Programmable nucleases disclosed herein can also include, for example, the Class 2 CRISPR/Cas enzymes, such as the Type II, Type V, and Type VI CRISPR/Cas enzymes.
- the Type V CRISPR/Cas enzyme can comprise a programmable Cast 2 nuclease.
- Type V CRISPR/Cas enzymes e.g., a Cast 2 or Cast 4
- a Casl2 nuclease of the present disclosure can cleave a nucleic acid via a single catalytic RuvC domain.
- the RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Casl2 nuclease can further comprise a recognition, or “REC” lobe.
- a programmable Cast 2 nuclease can be a Cast 2a protein, a Cast 2b protein, Cast 2c protein, Cast 2d protein, a Casl2e protein, a Casl2f, a Cast 2g, a Casl2h, a Casl2i, a Casl2j, or a Cast 2k.
- the programmable nuclease can be a Cast 3 enzyme.
- the Cast 3 enzyme can be a Cast 3 a, a Cast 3b, a Cast 3 c, a Cast 3d, a Casl3e, or a Casl3f.
- the programmable nuclease can be Mad7 or Mad2.
- the programmable nuclease can be a Casl2 enzyme as described elsewhere herein.
- the Cast 2 can be a Cast 2a, a Cast 2b, a Cast 2c, a Cast 2d, a Casl2e, a Casl2f, a Cast 2g, a Casl2h, a Casl2i, a Casl2j, or a Cast 2k.
- the Cast 2 can be a Cast 2 variant (SEQ ID NO: 17), which is a specific protein variant within the Casl2 protein family/classification).
- the programmable nuclease can be Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ.
- the Csml can also be also called smCmsl, miCmsl, obCmsl, or suCmsl.
- Casl3a can also be called C2c2.
- CasZ can also be called Casl4a, Cast 4b, Cast 4c, Casl4d, Casl4e, Casl4f, Cast 4g, or Casl4h.
- the programmable nuclease can be a type V CRISPR-Cas system. In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system.
- the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system.
- the programmable nuclease can be from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rea), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp.
- Psm Capnocytophaga canimorsus
- Ca Lachnospiraceae bacterium
- Bzo Bergeyella zoohelcum
- Prevotella intermedia Pin
- Prevotella buccae Pbu
- Alistipes sp. Asp
- Riemerella anatipestifer Ran
- Prevotella aurantiaca Pau
- Prevotella saccharolytica Psa
- Pin2 Capnocytophaga canimorsus
- Porphyromonas gulae Pgu
- Prevotella sp Prevotella sp.
- the Casl3 is at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, or LshCasl3a.
- the trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid.
- the trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid.
- the target nucleic acid can be RNA or DNA
- programmable nucleases comprise a Type V CRISPR/Cas protein.
- Type V CRISPR/Cas proteins comprise nucleic acid cleavage activity.
- Type V CRISPR/Cas proteins cleave or nick single-stranded nucleic acids, double stranded nucleic acids, or a combination thereof.
- Type V CRISPR/Cas proteins cleave single-stranded nucleic acids.
- Type V CRISPR/Cas proteins cleave double-stranded nucleic acids.
- Type V CRISPR/Cas proteins nick double-stranded nucleic acids.
- guide RNAs of Type V CRISPR/Cas proteins hybridize to ssDNA or dsDNA. However, the trans cleavage activity of Type V CRISPR/Cas protein is typically directed towards ssDNA.
- the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain.
- a catalytically inactive domain of a Type V CRISPR/Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild type nuclease domain of the Type V CRISPR/Cas protein. Said mutations may be present within a cleaving or active site of the nuclease.
- the Type V CRISPR/Cas protein may be a Cast 4 protein.
- the Cas 14 protein may be a Casl4a.l protein (SEQ ID NO: 3).
- the Casl4a.l protein SEQ ID NO: 3
- l protein may comprise a sequence of:
- the Casl4 protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any exemplary sequence described herein. In some cases, the Casl4 protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any exemplary sequence described herein.
- the Casl4 protein may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any exemplary sequence described herein.
- the Type V CRISPR/Cas protein can be modified (also referred to as an engineered protein).
- a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS).
- the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF.
- Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell.
- the Type V CRISPR/Cas protein is codon optimized for a human cell.
- the Type V CRISPR/Cas protein comprises a Casl4 protein.
- Casl4 proteins may comprise a bilobed structure with distinct amino-terminal and carboxyterminal domains.
- the amino- and carboxy-terminal domains may be connected by a flexible linker.
- the flexible linker may affect the relative conformations of the amino- and carboxyl- terminal domains.
- the flexible linker may be short, for example less than 10 amino acids, less than 8 amino acids, less than 6 amino acids, less than 5 amino acids, or less than 4 amino acids in length.
- the flexible linker may be sufficiently long to enable different conformations of the amino- and carboxy-terminal domains among two Cast 4 proteins of a Cast 4 dimer complex (e.g., the relative orientations of the amino- and carboxy-terminal domains differ between two Casl4 proteins of a Casl4 homodimer complex).
- the linker domain may comprise a mutation which affects the relative conformations of the amino- and carboxyl-terminal domains.
- the linker may comprise a mutation which affects Casl4 dimerization.
- a linker mutation may enhance the stability of a Cast 4 dimer.
- the amino-terminal domain of a Cast 4 protein comprises a wedge domain, a recognition domain, a zinc finger domain, or any combination thereof.
- the wedge domain may comprise a multi-strand P-barrel structure.
- a multi-strand P-barrel structure may comprise an oligonucleotide/oligosaccharide-binding fold that is structurally comparable to those of some Cast 2 proteins.
- the recognition domain and the zinc finger domain may each (individually or collectively) be inserted between P-barrel strands of the wedge domain.
- the recognition domain may comprise a 4-a-helix structure, structurally comparable but shorter than those found in some Casl2 proteins.
- the recognition domain may comprise a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex.
- a REC lobe may comprise a binding affinity for a PAM sequence in the target nucleic acid.
- the amino-terminal may comprise a wedge domain, a recognition domain, and a zinc finger domain.
- the carboxy -terminal may comprise a RuvC domain, a zinc finger domain, or any combination thereof.
- the carboxy -terminal may comprise one RuvC and one zinc finger domain.
- Casl4 proteins may comprise a RuvC domain or a partial RuvC domain.
- the RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the Cast 4 protein.
- a partial RuvC domain does not have any substrate binding activity or catalytic activity on its own.
- a Casl4 protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity.
- a Casl4 may include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Cast 4 protein, but form a RuvC domain once the protein is produced and folds.
- a Casl4 protein may comprise a linker loop connecting a carboxy terminal domain of the Cast 4 protein with the amino terminal domain of the Cas 14 protein, and wherein the carboxy terminal domain comprises one or more RuvC domains and the amino terminal domain comprises a recognition domain.
- Casl4 proteins may comprise a zinc finger domain.
- a carboxy terminal domain of a Casl4 protein comprises a zinc finger domain.
- an amino terminal domain of a Cas 14 protein comprises a zinc finger domain.
- the amino terminal domain comprises a wedge domain (e.g., a multi-P-barrel wedge structure), a zinc finger domain, or any combination thereof.
- the carboxy terminal domain comprises the RuvC domains and a zinc finger domain, and the amino terminal domain comprises a recognition domain, a wedge domain, and a zinc finger domain.
- Cas 14 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing.
- a Cas 14 protein may be less likely to adsorb to a surface or another biological species due to its small size. The smaller nature of these proteins also allows for them to be more easily packaged as a reagent in a system or assay, and delivered with higher efficiency as compared to other larger Cas proteins.
- a Casl4 protein is 400 to 800 amino acid residues long, 400 to 600 amino acid residues long, 440 to 580 amino acid residues long, 460 to 560 amino acid residues long, 460 to 540 amino acid residues long, 460 to 500 amino acid residues long, 400 to 500 amino acid residues long, or 500 to 600 amino acid residues long.
- a Casl4 protein is less than about 550 amino acid residues long. In some cases, a Casl4 protein is less than about 500 amino acid residues long.
- a Cas protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid.
- a Cas protein is capable of catalyzing non-sequence-specific cleavage of a single stranded nucleic acid.
- a Cas protein is activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity is also referred to as “collateral” or “transcollateral” cleavage.
- Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated programmable nuclease, such as trans cleavage of a reporter comprising a nucleic acid and a detection moiety.
- a programmable nuclease as disclosed herein is an RNA- activated programmable RNA nuclease.
- a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease.
- a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR/Cas enzyme (e.g., a Casl3 nuclease).
- Casl3a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cast 3a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cast 3a for trans cleavage of an RNA reporter.
- An RNA reporter can be an RNA-based reporter molecule.
- the Casl3a recognizes and detects ssDNA to initiate transcleavage of RNA reporters.
- Multiple Casl3a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA.
- Lbu-Casl3a and Lwa- Casl3a can both be activated to transcollaterally cleave RNA reporters by target DNA.
- Type VI CRISPR/Cas enzyme e.g., a Cas 13 nuclease, such as Cas 13 a
- Cas 13 nuclease such as Cas 13 a
- DNA-activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values.
- target ssDNA detection by Casl3 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2.
- target RNA detection by Casl3 can exhibit high cleavage activity of pH values from 7.9 to 8.2.
- a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease can have DNA targeting preferences that are distinct from its RNA targeting preferences.
- the optimal ssDNA targets for Cast 3a have different properties than optimal RNA targets for Cast 3 a.
- gRNA performance on ssDNA can not necessarily correlate with the performance of the same gRNAs on RNA.
- gRNAs can perform at a high level regardless of target nucleotide identity at a 3’ position on a target RNA sequence.
- gRNAs can perform at a high level in the absence of a G at a 3’ position on a target ssDNA sequence.
- target DNA detected by Cast 3 disclosed herein can be directly taken from organisms or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein.
- Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA- activated programmable RNA nuclease, such as Casl3a can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection.
- a programmable nuclease is used in a detection reaction.
- the detection reaction comprises the steps of (a) forming an activated complex comprising a programmable nuclease, a guide nucleic acid, and a target nucleic acid (or an amplicon thereof), thereby activating the programmable nuclease, and (b) cleaving reporters with the activated programmable nuclease.
- steps (a) and (b) are performed at an incubation temperature, such as a temperature of about 25 °C to about 80 °C, about 30 °C to about 70 °C, or about 37 °C to about 65 °C.
- the incubation temperature is about 37 °C. In some embodiments, the incubation temperature is maintained for about 10 minutes to about 2 hours, about 20 minutes to about 90 minutes, or about 25 minutes to about 60 minutes. In some embodiments, the incubation temperature is maintained for a duration of about 30 minutes. In some embodiments, reporter signal is
- reporter signal is measured at the end of the incubation.
- compositions and systems comprising at least one of an engineered programmable nuclease and an engineered guide nucleic acid, which may simply be referred to herein as a programmable nuclease and a guide nucleic acid, respectively.
- an engineered programmable nuclease and an engineered guide nucleic acid can refer to programmable nucleases and guide nucleic acids, respectively, that are not found in nature.
- systems and compositions comprise at least one non-naturally occurring component.
- compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid.
- compositions and systems comprise at least two components that do not naturally occur together.
- compositions and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together.
- composition and systems may comprise a guide nucleic acid and a programmable nuclease that do not naturally occur together.
- a programmable nuclease or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes programmable nucleases and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.
- the guide nucleic acid may comprise a non-natural nucleobase sequence.
- the non-natural sequence is a nucleobase sequence that is not found in nature.
- the non-natural sequence may comprise a portion of a naturally occurring sequence, wherein the portion of the naturally occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence.
- the guide nucleic acid may comprise two naturally occurring sequences arranged in an order or proximity that is not observed in nature.
- compositions and systems comprise a ribonucleotide complex comprising a CRISPR/Cas effector protein and a guide nucleic acid that do not occur together in nature.
- Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together.
- an engineered guide nucleic acid may comprise a sequence of a naturally occurring repeat region and a spacer region that is complementary to a naturally occurring eukaryotic sequence.
- the engineered guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism.
- An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different.
- the guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid.
- an engineered guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA coupled by a linker sequence.
- compositions and systems described herein comprise an engineered Cas protein that is similar to a naturally occurring Cas protein.
- the engineered Cas protein may lack a portion of the naturally occurring Cas protein.
- the Cas protein may comprise a mutation relative to the naturally-occurring Cas protein, wherein the mutation is not found in nature.
- the Cas protein may also comprise at least one additional amino acid relative to the naturally-occurring Cas protein.
- the Cas protein may comprise an addition of a nuclear localization signal relative to the natural occurring Cas protein.
- the nucleotide sequence encoding the Cas protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.
- compositions and systems provided herein can comprise a multi-vector system for encoding any programmable nuclease or guide nucleic acid described herein, wherein the guide nucleic acid and the programmable nuclease are encoded by the same or different vectors.
- the engineered guide nucleic acid and the engineered programmable nuclease can be encoded by different vectors of the system.
- thermostable programmable nucleases a programmable nuclease is referred to as an effector protein.
- An effector protein may be thermostable.
- known effector proteins e.g., Casl2 nucleases
- a thermostable protein may have enzymatic activity, stability, or folding comparable to those at 37 °C.
- the trans cleavage activity (e.g., the maximum trans cleavage rate as measured by fluorescent signal generation) of an effector protein in a trans cleavage assay at 40 °C may be at least 50% of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%,
- the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C).
- the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
- the trans cleavage activity may be measured against a negative control in a trans cleavage assay.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid.
- the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25- fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
- one or more programmable nucleases may be used to detect one or more targets (e.g., one or more target nucleic acids).
- a programmable nuclease is capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, the transcript of an RNA polymerase, and/or a cDNA of any of these.
- a programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence.
- the programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and can non-specifically degrade a non-target nucleic acid in its environment.
- the programmable nuclease has trans cleavage activity once activated.
- a programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease or Cas effector protein).
- a guide nucleic acid (e.g., crRNA) and Cas protein can form a CRISPR enzyme (also referred to, interchangeably, as a Cas complex, Cas probe, or CRISPR probe).
- one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule).
- a programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter.
- the programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease.
- the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter.
- a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease.
- a programmable nuclease as described herein can be activated by a target RNA or a target DNA.
- a programmable nuclease e.g., a Cas enzyme
- the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters.
- a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA- activated programmable DNA nuclease.
- the programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity.
- Trans cleavage activity can be non-specific cleavage of nearby singlestranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporter nucleic acids comprising a detection moiety.
- the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal.
- the reporter and/or the detection moiety can be immobilized on a support medium.
- the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid.
- the detection moiety binds to a capture molecule on the support medium to be immobilized.
- the detectable signal can be visualized on the support medium to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a SNP associated with a disease, cancer, or genetic disorder.
- a programmable nuclease can be any enzyme that can be or has been designed, modified, or engineered by human contribution so that the enzyme targets or cleaves a nucleic acid in a sequence-specific manner.
- Programmable nucleases can include, for example, zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and/or RNA-guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl.
- Programmable nucleases can also include, for example, PfAgo and/or NgAgo.
- the programmable nuclease is a Cas nuclease.
- the programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments.
- the programmable nuclease is a Type V Cas protein.
- a Type V Cas effector protein comprises a RuvC domain, but lacks an HNH domain.
- the RuvC domain of the Type V Cas effector protein comprises three patrial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains).
- the three RuvC subdomains are located within the C-terminal half of the Type V Cas effector protein.
- none of the RuvC subdomains are located at the N terminus of the protein.
- the RuvC subdomains are contiguous.
- the RuvC subdomains are not contiguous with respect to the primary amino acid sequence of the Type V Cas protein, but form a ruvC domain once the protein is produced and folds. In some instances, there are zero to about 50 amino acids between the first and second RuvC subdomains. In some instances, there are zero to about 50 amino acids between the second and third RuvC subdomains.
- the Cas effector is a Casl4 effector.
- the Casl4 effector is a Casl4a, Casl4a.l (SEQ ID NO: 3), Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, or Casl4u effector.
- the Cas effector is a CasPhi (also referred to herein as a Cas ) effector.
- the Cas effector is a Casl2 effector.
- the Casl2 effector is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, or Casl2j effector.
- the Type V Cas protein is a Cas protein.
- a Cas protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid.
- a programmable Cas nuclease may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable Cas nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.
- the programmable nuclease is a Type VI Cas protein.
- the Type VI Cas protein is a programmable Casl3 nuclease.
- the general architecture of a Cas 13 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains.
- the HEPN domains each comprise aR-X4-H motif. Shared features across Cast 3 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase.
- programmable Cast 3 nucleases also consistent with the present disclosure include Cast 3 nucleases comprising mutations in the HEPN domain that enhance the Cast 3 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains.
- Programmable Cast 3 nucleases consistent with the present disclosure also Casl3 nucleases comprising catalytic components.
- the Cas effector is a Cas 13 effector.
- the Cast 3 effector is a Cas 13 a, a Cas 13b, a Cas 13c, a Cas 13d, or a Cas 13e effector protein.
- the programmable nuclease comprises a Casl2 protein, wherein the Cas 12 enzyme binds and cleaves double stranded DNA and single stranded DNA.
- programmable nuclease comprises a Cas 13 protein, wherein the Cas 13 enzyme binds and cleaves single stranded RNA.
- programmable nuclease comprises a Cas 14 protein, wherein the Cas 14 enzyme binds and cleaves both double stranded DNA and single stranded DNA.
- Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity.
- programmable nucleases described herein comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-71.
- programmable nucleases described herein may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-71.
- programmable nucleases described herein comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID Nos: 1-71.
- Table 1 Amino Acid Sequences of Exemplary Programmable Nucleases
- effector proteins disclosed herein are engineered proteins.
- Engineered proteins are not identical to a naturally-occurring protein.
- Engineered proteins may provide enhanced nuclease or nickase activity as compared to a naturally occurring nuclease or nickase.
- An engineered protein may comprise a modified form of a wild type counterpart protein.
- effector proteins comprise at least one amino acid change (e.g., deletion, insertion, or substitution) that enhances or reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart.
- the methods and compositions of the present disclosure are compatible with any type of programmable nuclease that is human-engineered or naturally occurring.
- the programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof.
- a programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest.
- the programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated.
- CRISPR-based programmable nuclease In any instances or embodiments where a CRISPR-based programmable nuclease is described or used, it is recognized herein that any other type of programmable nuclease can be used in addition to or in substitution of such CRISPR-based programmable nuclease.
- the methods and compositions of the present disclosure are compatible with a plurality of programmable nucleases, including any of the programmable nucleases described herein.
- the system can comprise a plurality of programmable nuclease probes comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids.
- the plurality of programmable nuclease probes can be the same.
- the plurality of programmable nuclease probes can be different.
- the plurality of programmable nuclease probes can comprise different programmable nucleases and/or different guide nucleic acids associated with the programmable nucleases.
- the system may comprise a first programmable nuclease that forms an activated complex with a target nucleic acid, and a second programmable nuclease that forms an activated complex with an RNA transcript produced by an RNA polymerase in the system.
- trans cleavage activity can be initiated by one or more activated programmable nucleases, including, for instance, trans cleavage of one or more reporters (e.g., reporter nucleic acids) comprising a detection moiety.
- reporters e.g., reporter nucleic acids
- the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal.
- the reporter molecules described herein can comprise, in some non-limiting examples, RNA.
- the reporter molecules can comprise ssDNA.
- a reporter molecule can comprise: RNA, dsDNA, one or more modified nucleotides, and/or any combination thereof.
- the reporter molecules can comprise at least one nucleic acid and a detectable moiety.
- a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect a target such as dsDNA and, further, can specifically trans-cleave ssDNA reporters.
- a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect a target such as RNA and, further, can specifically trans-cleave RNA reporters.
- the detection of the target nucleic acid in the sample can indicate the presence of the sequence in the sample and can provide information about the presence or absence of a nucleic acid sequence in a sample, e.g., as a diagnostic for disease.
- Cleavage of a protein-nucleic acid can produce a signal.
- the signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample.
- cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
- Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample.
- the sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
- optical sensors e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies
- SPR surface plasmon resonance
- interferometric sensors or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
- the systems, devices, apparatuses, methods, and compositions of the present disclosure may be used to analyze one or more samples to detect a presence or an absence of one or more targets as described elsewhere herein.
- the one or more samples can be taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine.
- the sample is taken from nematodes, protozoans, helminths, or malarial parasites.
- the sample comprises nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell.
- the sample comprises nucleic acids expressed from a cell.
- the sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein.
- the target sequence is a portion of a nucleic acid.
- a nucleic acid can be from a genomic locus, a viral locus, a transcribed mRNA, or a reverse transcribed cDNA.
- a nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length.
- a nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length.
- a nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from
- a nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
- the target nucleic acid can be reverse complementary to a guide nucleic acid.
- at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.
- the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample.
- the target sequence in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample.
- the target sequence in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample.
- the target sequence in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample.
- the target sequence in some cases, is a portion of a nucleic acid from sepsis, in the sample.
- These diseases can include but are not limited to respiratory viruses (e.g., SARS-CoV-2 (i.e., a virus that causes CO VID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus/Enterovirus, Influenza A, Influenza A/Hl, Influenza A/H3, Influenza A/Hl-2009, Influenza B, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus) and respiratory bacteria (e.g.
- respiratory viruses e.g., SARS-CoV-2 (i.e., a virus that causes CO VID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus
- Bordetella parapertussis Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae).
- Other viruses include human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis.
- Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites.
- Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms.
- Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.
- pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii.
- Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and Candida albicans.
- Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g., the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox/shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B, C, D, E); cutaneous viral diseases (e.g., warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum); hemmorhagic viral diseases (e.g.
- respiratory viruses e.g.
- Ebola Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever
- neurologic viruses e.g., polio, viral meningitis, viral encephalitis, rabies
- sexually transmitted viruses e.g., HIV, HPV, and the like
- immunodeficiency virus e.g., HIV
- influenza virus dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like.
- Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus anthracis, Bortadella pertussis, Burkholderia cepacia, Corynebacterium diphtheriae, Coxiella burnetii, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella longbeachae, Legionella pneumophila, Leptospira interrogans, Moraxella catarrhalis, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria elongate, Neisseria gonorrhoeae, Parechovirus, Pneumococcus, Pneumocystis jirovecii, Crypto
- the target nucleic acid may comprise a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample.
- the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis.
- HCV human immunodeficiency virus
- HPV human papillomavirus
- chlamydia gonorrhea
- syphilis syphilis
- trichomoniasis sexually transmitted infection
- malaria Dengue fever
- Ebola chikungunya
- leishmaniasis leishmaniasis
- Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites.
- Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms.
- Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.
- pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii.
- Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
- Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like.
- immunodeficiency virus e.g., HIV
- influenza virus dengue; West Nile virus
- herpes virus yellow fever virus
- Hepatitis Virus C Hepatitis Virus A
- Hepatitis Virus B Hepatitis Virus B
- papillomavirus papillomavirus
- Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Streptococcus pyogenes, Streptococcus salivarius, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory
- the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.
- the sample used for cancer testing or cancer risk testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
- the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle.
- the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer.
- the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus.
- viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV-1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma).
- Epstein-Barr virus e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma
- the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever.
- the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, M
- the sample used for genetic disorder testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
- the genetic disorder is hemophilia, sickle cell anemia, P-thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis.
- the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder.
- the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of CFTR, FMRI, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL, ACAT1, AC0X1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASPA, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL, CH
- the sample used for phenotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
- the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a phenotypic trait.
- the sample used for genotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
- the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a genotype.
- the sample used for ancestral testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein.
- the target nucleic acid segment in some cases, is a portion of a nucleic acid from a gene associated with a geographic region of origin or ethnic group.
- the sample can be used for identifying a disease status.
- a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject.
- the disease can be a cancer or genetic disorder.
- a method comprises obtaining a serum sample from a subject; and identifying a disease status of the subject.
- the disease status is prostate disease status.
- the device can be configured for asymptomatic, pre- symptomatic and/or symptomatic diagnostic applications, irrespective of immunity.
- the device can be configured to perform one or more serological assays on a sample (e.g., a sample comprising blood).
- the sample can be used to identify a mutation in a target nucleic acid of a plant or of a bacteria, virus, or microbe associated with a plant or soil.
- the devices and methods of the present disclosure can be used to identify a mutation of a target nucleic acid that affects the expression of a gene.
- a mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene.
- the mutation is a single nucleotide mutation.
- the systems, devices, apparatuses, and methods disclosed herein may be used to detect a presence or an absence of one or more targets in one or more samples.
- the one or more samples can comprise one or more target sequences or nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein.
- a sample can be taken from any place where a nucleic acid can be found.
- Samples can be taken from an individual/human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest.
- a biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal , cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions/mucus, vaginal secretions/mucus, anal secretion/mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, any combination thereof.
- tissue fluid interstitial
- a sample can be an aspirate of a bodily fluid from an animal (e.g., human, animals, livestock, pet, etc.) or plant.
- a tissue sample can be from any tissue that can be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like).
- a tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure.
- a sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and/or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.).
- a sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid/ water, or soil.
- a sample from an environment can be from soil, air, or water.
- the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest.
- the raw sample is applied to the detection system.
- the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system.
- the sample is contained in no more than about 200 nanoliters (nL). In some cases, the sample is contained in about 200 nL. In some cases, the sample is contained in a volume that is greater than about 200 nL and less than about 20 microliters (pL).
- the sample is contained in no more than 20 pl. In some cases, the sample is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 pl, or any of value from 1 pl to 500 pl.
- the sample is contained in from 1 pL to 500 pL, from 10 pL to 500 pL, from 50 pL to 500 pL, from 100 pL to 500 pL, from 200 pL to 500 pL, from 300 pL to 500 pL, from 400 pL to 500 pL, from 1 pL to 200 pL, from 10 pL to 200 pL, from 50 pL to 200 pL, from 100 pL to 200 pL, from 1 pL to 100 pL, from 10 pL to 100 pL, from 50 pL to 100 pL, from 1 pL to 50 pL, from 10 pL to 50 pL, from 1 pL to 20 pL, from 10 pL to 20 pL, or from 1 pL to 10 pL. Sometimes, the sample is contained in more than 500 pl.
- the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine.
- the sample is taken from nematodes, protozoans, helminths, or malarial parasites.
- the sample may comprise nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell.
- the sample may comprise nucleic acids expressed from a cell.
- the sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein.
- the target sequence is a portion of a nucleic acid.
- a nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA.
- a nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length.
- a nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length.
- a nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from 35 to 50, or from 45 to 50 nucleotides in length.
- a nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length.
- the target nucleic acid can be reverse complementary to a guide nucleic acid.
- At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.
- the target nucleic acid is a single stranded nucleic acid.
- the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents.
- the target nucleic acid can be RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples.
- the target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA).
- the target nucleic acid is mRNA.
- the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
- the target nucleic acid is transcribed from a gene as described herein.
- target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids.
- the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids.
- the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to 8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to 6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to 8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids.
- the method detects target nucleic acid present at least at one copy per 10 1 non-target nucleic acids, 10 2 non-target nucleic acids, 10 3 non-target nucleic acids, 10 4 nontarget nucleic acids, 10 5 non-target nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or 10 10 non-target nucleic acids.
- a number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations.
- the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations.
- the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 10 1 non-target nucleic acids, 10 2 non- target nucleic acids, 10 3 non-target nucleic acids, 10 4 non-target nucleic acids, 10 5 non-target nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or IO 10 non-target nucleic acids.
- the target nucleic acid populations can be present at different concentrations or amounts in the sample.
- the sample may be separated into a plurality of droplets, aliquots, or subsamples.
- One or more targets e.g., nucleic acids, biomolecules, etc.
- the plurality of droplets, aliquots, or subsamples may be contained within the plurality of droplets, aliquots, or subsamples.
- the targets may be amplified before detection occurs.
- the system comprises a nucleic acid detection device.
- a nucleic acid detection device comprises a chamber or subsystem for amplifying the targets.
- the nucleic acid detection devices can be configured to amplify the target sequences or target nucleic acids contained within the plurality of droplets by individually processing each of the plurality of droplets (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below).
- the plurality of droplets can undergo separate thermocycling or isothermal processes.
- the thermocycling or isothermal processes can occur simultaneously. In other cases, the thermocycling or isothermal processes can occur at different times for each droplet.
- the nucleic acid detection devices can be further configured to remix the droplets, aliquots, or subsamples after the target nucleic acids in each of the droplets undergo amplification.
- the nucleic acid detection device can be configured to provide the remixed sample comprising the droplets, aliquots, or subsamples to a detection chamber of the device.
- the detection chamber can be configured to direct the remixed droplets, aliquots, or sub samples to a plurality of programmable nuclease probes (described in greater detail below).
- the detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples along one or more fluid flow paths such that the remixed droplets, aliquots, or subsamples are positioned adjacent to and/or in contact with the one or more programmable nuclease probes. In some cases, the detection chamber can be configured to recirculate or recycle the remixed droplets, aliquots, or subsamples such that the remixed droplets, aliquots, or subsamples are repeatedly placed in contact with one or more programmable nuclease probes.
- the nucleic acid detection device can comprise a single integrated system that is configured to perform sample collection, sample processing, droplet generation, droplet processing (e.g., amplification of target nucleic acids in droplets), droplet remixing, and/or circulation of the remixed droplets within a detection chamber so that at least a portion of the remixed droplets is placed in contact with one or more programmable nuclease probes coupled to the detection chamber.
- the nucleic acid detection devices of the present disclosure can be disposable devices configured to perform one or more rapid single reaction or multi -reach on tests to detect a presence and/or an absence of one or more target sequences or target nucleic acids.
- the present disclosure provides exemplary methods for programmable nuclease-based detection.
- the method can comprise collecting a sample.
- the sample can comprise any type of sample as described herein.
- the method can comprise preparing the sample.
- Sample preparation can comprise one or more sample preparation steps. The one or more sample preparation steps can be performed in any suitable order.
- the one or more sample preparation steps can comprise physical filtration of non-target materials using a macro filter, nucleic acid purification, lysis, heat inactivation, or adding one or more enzymes or reagents to prepare the sample for target detection.
- the method can comprise generating one or more droplets, aliquots, or subsamples from the sample.
- the one or more droplets, aliquots, or subsamples can correspond to a volumetric portion of the sample.
- the sample can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more droplets, aliquots, or subsamples.
- the sample is not divided into subsamples.
- the method can comprise amplifying one or more targets within each droplet, aliquot, or subsample.
- Amplification of the one or more targets within each droplet can be performed in parallel and/or simultaneously for each droplet. Dividing the sample into a plurality of droplets can enhance a speed and/or an efficiency of the amplification process (e.g., a thermocycling process) since the droplets comprise a smaller volume of material than the bulk sample introduced.
- Amplifying the one or more targets within each individual droplet can also permit effective amplification of various target nucleic acids that cannot be amplified as efficiently in a bulk sample containing the various target nucleic acids if the bulk sample were to undergo a singular amplification process.
- amplification is performed on the bulk sample without first dividing the sample into subsamples.
- the method can further comprise using one or more CRISPR-based or programmable nuclease-based probes (as described elsewhere herein) to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample.
- targets e.g., target sequences or target nucleic acids
- the sample can be divided into a plurality of droplets, aliquots, or subsamples to facilitate sample preparation and to enhance the detection capabilities of the devices of the present disclosure.
- the sample is not divided into subsamples.
- the target nucleic acids are not amplified before combining with a programmable nuclease in a detection reaction.
- the sample can be provided manually to a nucleic acid detection device.
- a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device.
- the sample can be provided via an automated syringe.
- the automated syringe can be configured to control a flow rate at which the sample is provided to the nucleic acid detection device.
- the automated syringe can be configured to control a volume of the sample that is provided to the nucleic acid detection device over a predetermined period.
- the sample can be provided directly to the nucleic acid detection device.
- a swab sample can be inserted into a sample chamber on the nucleic acid detection device.
- the sample can be prepared before one or more targets are detected within the sample.
- the sample preparation steps described herein can process a crude sample to generate a pure or purer sample.
- Sample preparation can one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or eluting.
- sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, and/or base/acid lysis.
- nucleic acid purification can be performed on the sample. Purification can comprise disrupting a biological matrix of a cell to release nucleic acids, denaturing structural proteins associated with the nucleic acids (nucleoproteins), inactivating nucleases that can degrade the isolated product (RNase and/or DNase), and/or removing contaminants (e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and/or other cellular debris).
- nucleoproteins denaturing structural proteins associated with the nucleic acids
- RNase and/or DNase inactivating nucleases that can degrade the isolated product
- contaminants e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and/or other cellular debris.
- lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme).
- a protease e.g., a Proteinase K or PK enzyme.
- Exemplary proteases include serine proteases (e.g., Proteinase K, Savinase®, trypsin, Protamex®, etc.), metalloproteinases (e.g., MMP-3, etc.), cysteine proteases (e.g, cathepsin B, papin, etc.), threonine proteases, aspartic proteases (e.g., renin, pepsin, cathepsin D, etc.), glutamic proteases, asparagine peptide lyases, or the like.
- serine proteases e.g., Proteinase K, Savinase®, trypsin, Protam
- a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease.
- Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA.
- One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate.2H20, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M P-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used.
- Alkaline buffers can also be used for cells with hard shells, particularly for environmental samples.
- Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers.
- Cell lysis can also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. In some cases, depending on the type of sample, nanoscale barbs, nanowires, acoustic generators, integrated lasers, integrated heaters, and/or microcapillary probes can be used to perform lysis.
- heat inactivation can be performed on the sample.
- a processed/lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g, a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, viral proteins, etc.).
- a heating element integrated into the nucleic acid detection device can be used for heatinactivation. The heating element can be powered by a battery or another source of thermal or electric energy that is integrated with the nucleic acid detection device.
- enzyme inactivation can be performed on the sample.
- a processed/lysed sample can undergo enzyme inactivation to inhibit or inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, etc.).
- a solution of reagents can be used to inactivate one or more enzymes present in the sample. Enzyme inactivation can occur before, during, or after lysis, when lysis is performed.
- an RNase inhibitor may be included as a lysis reagent to inhibit native RNases within the sample (which might otherwise impair target and/or reporter detection downstream).
- RNase inhibitors include RNAse Inhibitor, Murine (NEB), Rnaseln Plus (Promega), Protector Rnase Inhibitor (Roche), Superasein (Ambion), RiboLock (Thermo), Ribosafe (Bioline), or the like.
- a protease inhibitor can be applied to the lysed sample to inactivate the protease prior to contacting the sample nucleic acids to the programmable nuclease.
- protease inhibitors include AEBSF, antipain, aprotinin, bestatin, chymostatin, EDTA, leupeptin, pepstatin A, phosphoramidon, PMSF, soybean trypsin inhibitor, TPCK, or the like.
- enzyme inactivation may occur before, during, after, or instead of heat inactivation.
- a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme.
- the target nucleic acid within a purified sample can be amplified.
- amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and/or polymerase chain reaction (PCR).
- LAMP loop mediated amplification
- RPA isothermal recombinase polymerase amplification
- PCR polymerase chain reaction
- Droplet digitization or droplet generation can comprise splitting a volume of the sample into multiple droplets, aliquots, or subsamples.
- the sample can have a volume that ranges from about 10 microliters to about 500 microliters.
- the plurality of droplets, aliquots, or subsamples can have a volume that ranges from about 0.01 microliters to about 100 microliters.
- the plurality of droplets, aliquots, or subsamples can have a same or substantially similar volume. In some cases, the plurality of droplets, aliquots, or subsamples can have different volumes.
- the droplets, aliquots, or subsamples can be generated using a physical filter or the one or more movable mechanisms described above.
- each droplet of the sample can undergo one or more sample preparation steps (e.g., nucleic acid purification, lysis, heat inactivation, amplification, etc.) independently and/or in parallel while the droplets are physically constrained or thermally isolated.
- sample preparation steps e.g., nucleic acid purification, lysis, heat inactivation, amplification, etc.
- Such nucleic acid amplification of the sample can improve at least one of a sensitivity, specificity, or accuracy of the detection of the target nucleic acid.
- the reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase.
- the nucleic acid amplification can be transcription mediated amplification (TMA).
- TMA transcription mediated amplification
- Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA).
- HDA helicase dependent amplification
- cHDA circular helicase dependent amplification
- SDA strand displacement amplification
- the nucleic acid amplification can be recombinase polymerase amplification (RPA).
- the nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR).
- Nucleic acid amplification is, in some cases, by rolling circle transcription (RCT), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequencebased amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).
- RCT rolling circle transcription
- LCR ligase chain reaction
- SPIA simple method amplifying RNA targets
- SPIA single primer isothermal amplification
- MDA multiple displacement amplification
- NASBA nucleic acid sequencebased amplification
- HIP hinge-initiated primer-dependent amplification of nucleic acids
- NEAR nick
- the nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification is performed for from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to 40, or from 25 to 35 minutes. Sometimes, the nucleic acid amplification is performed for from 5 to 60, from 10 to 60, from 15 to 60, from 30 to 60, from 45 to 60, from 1 to 45, from 5 to 45, from 10 to 45, from 30 to 45, from 1 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 1 to 15, from 5 to 15, or from 10 to 15 minutes.
- nucleic acid amplification can comprise thermocycling of the sample.
- Thermocycling can be carried out in a vessel, for one or more droplets of the sample in parallel, and/or independently in separate locations. This can be accomplished by methods such as (1) by holding droplets stationary in locations where a heating element is in close proximity to the droplet on one of the droplet sides and a heat sink element is in close proximity to the other side of the droplet, or (2) flowing the droplet through zones in a fluid channel where heat flows across it from a heating source to a heat sink.
- one or more resistive heating elements can be used to perform thermocycling.
- the nucleic acid amplification reaction is performed at a temperature of around 20-45°C.
- the nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
- the nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
- the nucleic acid amplification reaction is performed at a temperature of from 20°C to 45°C, from 25°C to 40°C, from 30°C to 40°C, or from 35°C to 40°C.
- the nucleic acid amplification reaction is performed at a temperature of from 45°C to 65°C, from 50°C to 65°C, from 55°C to 65°C, or from 60°C to 65°C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 20 °C to 45 °C, from 25 °C to 45 °C, from 30 °C to 45 °C, from 35 °C to 45 °C, from 40 °C to 45 °C, from 20 °C to 37 °C, from 25 °C to 37 °C, from 30 °C to 37 °C, from 35 °C to 37 °C, from 20 °C to 30 °C, from 25 °C to 30 °C, from 20 °C to 25 °C, or from about 22 °C to 25 °C.
- the nucleic acid amplification reaction can be performed at a temperature that ranges from about 40 °C to 65 °C, from 45 °C to 65 °C, from 50 °C to 65 °C, from 55 °C to 65 °C, from 60 °C to 65 °C, from 40 °C to 60 °C, from 45 °C to 60 °C, from 50 °C to 60 °C, from 55 °C to 60 °C, from 40 °C to 55 °C, from 45 °C to 55 °C, from 50 °C to 55 °C, from 40 °C to 50 °C, or from about 45 °C to 50 °C.
- thermocycling comprises a plurality of cycles, wherein each cycle comprises denaturation at a first temperature and primer extension by a polymerase at a second temperature that is lower than the first temperature.
- each cycle is about or less than about 20 seconds in duration (e.g., about or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 seconds in duration).
- each cycle is less than 15 seconds in duration.
- each cycle is less than 10 seconds in duration.
- the plurality of cycles are about 2 seconds to about 20 seconds in duration, about 3 seconds to about 10 seconds in duration, or about 5 seconds in duration.
- the cycles are about 4 seconds in duration.
- the denaturation step is about 0.5 to about 5 seconds in duration, about 1 to about 3 seconds in duration, or about 2 seconds in duration. In some embodiments, the denaturation is about 1 second in duration. In some embodiments, the first temperature is about 90°C to about 99°C, about 94°C to about 98°C, or about 95°C. In some embodiments, the first temperature is about 98°C. In some embodiments, the primer extension step is about 1 to about 15 seconds in duration, about 2 to about 10 seconds in duration, or about 5 seconds in duration. In some embodiments, the primer extension step is about 4 seconds in duration.
- the second temperature is about 45 °C to about 75 °C, about 50 °C to about 70 °C, or about 55 °C to about 65 °C. In some embodiments, the second temperature is about 55 °C. In some embodiments, each cycle comprises denaturation at the first temperature for about 1 second and primer extension at the second temperature for about 3 seconds. In some embodiments, the plurality of cycles comprises about or at least about 20 cycles (e.g., about or more than about 25, 30, 35, 40, or 45 cycles). In some embodiments, the plurality of cycles comprises about 20 cycles to about 50 cycles, or about 30 cycles to about 45 cycles. In some embodiments, the plurality of cycles comprises about 45 cycles.
- the plurality of cycles is preceded by an initial denaturation step at the first temperature that is longer in duration that the durations of the individual denaturation steps in each of the cycles.
- the initial denaturation step is about 10 seconds to about 120 seconds in duration, about 15 seconds to about 60 seconds in duration, or about 20 seconds to about 50 seconds in duration.
- the initial denaturation step is about 30 seconds in duration.
- the total duration of the amplification by thermocycling is about 1 minute to about 20 minutes, about 2 minutes to about 15 minutes, or about 3 minutes to about 10 minutes. In some embodiments, the total duration of the amplification by thermocycling is less than about 10 minutes. In some embodiments, the total duration of the amplification by thermocycling is about 5 minutes.
- the target nucleic acid(s) can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA) of the programmable nuclease (e.g., CRISPR enzyme).
- This amplification can be PCR amplification or isothermal amplification.
- This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA.
- the reagents for nucleic acid amplification can comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase.
- SSB single-stranded DNA binding
- the nucleic acid amplification can be transcription mediated amplification (TMA).
- TMA transcription mediated amplification
- Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA).
- HDA helicase dependent amplification
- cHDA circular helicase dependent amplification
- SDA strand displacement amplification
- the nucleic acid amplification can be recombinase polymerase amplification (RPA).
- RPA recombinase polymerase amplification
- the nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR).
- LAMP loop mediated amplification
- EXPAR exponential amplification reaction
- Nucleic acid amplification is, in some cases, by rolling circle transcription (RCT), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).
- the nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes.
- the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 °C.
- the nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
- the nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
- the nucleic acids described and referred to herein can comprise a plurality of nucleotides.
- a nucleotide may form a base pair with another nucleotide (e.g., as in a complementary strand or internal base pairing of a hairpin structure).
- a base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds.
- Nucleobases can comprise adenine, guanine, cytosine, thymine, and/or uracil.
- the nucleic acids described and referred to herein can comprise different nucleotides.
- the nucleic acids described and referred to herein can comprise one or more modified nucleotides.
- the one or more modified nucleotides can be produced when one or more nucleotides undergo a chemical modification leading to new bases.
- the one or more modified nucleotides can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7- Methylguanine, 7-Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5- Methylcytidine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5- carboxylcytosine (5caC).
- the target nucleic acid is a single stranded nucleic acid.
- the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents.
- the target nucleic acid can be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples.
- the target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA).
- the target nucleic acid is mRNA.
- the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
- the target nucleic acid is transcribed from a gene as described herein.
- target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population.
- the sample has at least 2 target nucleic acids.
- the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids.
- the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids.
- the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to 8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to 6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to 8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids.
- the method detects target nucleic acid present at least at one copy per 10 1 non-target nucleic acids, 10 2 non-target nucleic acids, 10 3 non-target nucleic acids, 10 4 nontarget nucleic acids, 10 5 non-target nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or 10 10 non-target nucleic acids.
- a number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations.
- the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations. In some cases, the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations.
- the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 10 1 non-target nucleic acids, 10 2 nontarget nucleic acids, 10 3 non-target nucleic acids, 10 4 non-target nucleic acids, 10 5 non-target nucleic acids, 10 6 non-target nucleic acids, 10 7 non-target nucleic acids, 10 8 non-target nucleic acids, 10 9 non-target nucleic acids, or 10 10 non-target nucleic acids.
- the target nucleic acid populations can be present at different concentrations or amounts in the sample.
- the systems and methods of the present disclosure may be implemented using one or more programmable nuclease probes.
- the one or more programmable nuclease probes may be used to detect one or more targets in one or more samples.
- the one or more targets and the one or more samples may comprise any target or sample described above.
- the one or more programmable nuclease probes may be placed in a detection chamber of a device or apparatus.
- the one or more programmable nuclease probes may be immobilized to a surface of a device or an apparatus.
- the one or more programmable nuclease probes may not or need not be immobilized to a surface of a device or an apparatus.
- the programmable nuclease probe can comprise a guide nucleic acid that is complexed with or capable of being complexed with a programmable nuclease.
- the programmable nuclease can comprise any type of programmable nuclease as described herein.
- the programmable nuclease probe may comprise a guide nucleic acid complexed with an enzyme.
- the enzyme may be, in some instances, a CRISPR enzyme.
- the programmable nuclease probe can comprise a programmable nuclease and/or a guide nucleic acid.
- the guide nucleic acid can bind to a target nucleic acid, as described in greater detail below.
- the device can be configured to generate an electropotential gradient or to provide heat energy to one or more regions proximal to the programmable nuclease probe, to enhance targeting preci sion/accuracy.
- the guide nucleic acid-enzyme complex may include, in some cases, a reporter.
- a reporter e.g., a CRISPR probe
- the binding event can trigger a trans-cut that (i) releases the reporter into a detectable region or (ii) changes or modifies (e.g., physically or chemically) the reporter.
- Detection mechanisms can involve interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.
- the reporter of the programmable nuclease probe can initiate a signal amplification reaction with another molecular species after the complementary binding induced trans-cutting.
- species can be a reactive solid or gel matrix, or other reactive entity to generate an amplified signal during detection.
- the signal amplification reaction can be physical or chemical in nature.
- the released reporter, X after a complementary binding induced trans-cut, can initiate an interaction and/or a reaction with another entity, Y, to produce an amplified or modified signal.
- entities can comprise a molecular species, a solid, a gel, or other entities.
- the signal amplification interaction can be a physical or chemical reaction.
- the interaction involves free-radical, anionic, cationic or coordination polymerization reactions.
- the cut reporter can trigger aggregation, or agglutination, of molecules, cells, or nanoparticles.
- the cut reporter can interact with a semiconductor material.
- the chemical or physical change caused by the interaction is detected by optical detection means such as interferometry, surface plasmon resonance, reflectivity or other.
- the chemical or physical change caused by the interaction is detected by potentiometric, amperometric, field effect transistor, or other electronic means.
- the signal amplification reaction comprises a transcription step.
- a first programmable nuclease and a non-naturally occurring guide nucleic acid form a complex that is activated upon binding the target nucleic acid.
- the activated complex cleaves a linear transcription template to remove a terminal blocking moiety, which allows the ends of the transcription template to be ligated together to form a circular template.
- RNA transcript that serves as a further target nucleic acid for a second programmable nuclease (the same programmable nuclease as the first programmable nuclease, a programmable nuclease of the same type, or a programmable nuclease of a different type).
- a second programmable nuclease the same programmable nuclease as the first programmable nuclease, a programmable nuclease of the same type, or a programmable nuclease of a different type.
- the activated first and second programmable nucleases may both cleave reporters, providing a signal amplification as compared to relying solely on direct activation of first programmable nucleases by target nucleic acid in the sample.
- the plurality of programmable nuclease probes as described herein can be arranged in various configurations.
- the plurality of programmable nuclease probes can be arranged in a lateral configuration.
- the plurality of programmable nuclease probes can be arranged in a circular configuration such that each programmable nuclease probe is equidistant from a common center point.
- the plurality of programmable nuclease probes can be distributed with a same separation distance or spacing between the programmable nuclease probes.
- a first programmable nuclease probe and a second programmable nuclease probe can be separated by a first separation distance or spacing
- a third programmable nuclease probe and a fourth programmable nuclease probe can be separated by a second separation distance or spacing that is different than the first separation distance or spacing
- target nucleic acid amplicons can be detected by immobilized programmable nuclease probes, such as, for example, programmable nuclease guide nucleic acid probes (e.g., a CRISPR probe).
- immobilized programmable nuclease probes such as, for example, programmable nuclease guide nucleic acid probes (e.g., a CRISPR probe).
- a cutting event can occur that releases a reporter or generates a signal that is then detected by a sensor.
- the guide nucleic acid of the programmable nuclease or CRISPR probe can be immobilized adjacent to a bottom surface of the chamber.
- the CRISPR enzyme will cut and release a reporter molecule which will then be sensed or detected by a sensor/detector. Since the specific guide RNA of the immobilized programmable nuclease or CRISPR probe can be spatially registered, multiplexed detection can be achieved. In some cases, where one sensor corresponds to one immobilized probe, electrical detection can be used. Other methods of detection can also be used, such as optical imaging, surface plasmon resonance (SPR), and/or interferometric sensing.
- SPR surface plasmon resonance
- the programmable nuclease probe (e.g., a CRISPR probe) can be immobilized to an immobilization matrix.
- the interior side of the immobilization matrix may be exposed to an inside wall of a circulation chamber of a detection system, device or apparatus.
- the guide nucleic acid or guide RNA can be exposed to target amplicons inside the circulation chamber.
- the reporter can be in proximity to or oriented towards an “exterior” side of the immobilization matrix.
- the exterior side of the immobilization matrix can be in proximity to a detection region.
- the detection region may correspond to a region from which a detectable signal can originate.
- the detectable signal may indicate the presence or the absence of one or more targets of interest.
- the binding event between the guide nucleic acid and a target nucleic acid can trigger a trans-cut that (i) releases the reporter into a detectable region or (ii) physically or chemically changes the reporter.
- Detection mechanisms for detecting the reporter or any detectable signals generated by the reporter can involve, for instance, interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.
- the programmable nuclease, guide nucleic acid, reporter, or a combination thereof can be immobilized to a device surface (e.g., by a linkage).
- the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, an interaction (e.g., a covalent or noncovalent bond) between members of a binding pair (e.g., streptavidin and biotin), an amide bond, or any combination thereof.
- the linkage may be the same or different for each species.
- the guide nucleic acid may be immobilized to the surface by a single-stranded linker polynucleotide
- the reporters may be immobilized by the interaction between a first member of a binding pair on the reporters and a second member of a binding pair on the surface.
- binding pair refers to a first and a second moiety that have a specific binding affinity for each other.
- a binding pair has a dissociation constant Kd of less than or equal to about: 10' 8 mol/L, 10' 9 mol/L, IO' 10 mol/L, 10' 11 mol/L, 10' 12 mol/L, 10' 13 mol/L, 10' 14 mol/L, 10' 15 mol/L, or ranges including two of these values as endpoints.
- Non limiting examples of binding pairs include an antibody or an antigen-binding portion thereof and an antigen (e.g., fluorescein, digoxin, digoxigenin); a biotin (bio) moiety and an avidin (or streptavidin) moiety; a dinitrophenol (DNP) and an anti- DNP antibody; a hapten and an anti hapten; folate and a folate binding protein; vitamin B12 and an intrinsic factor; a carbohydrate and a lectin or carbohydrate receptor; a polysaccharide and a polysaccharide binding moiety; a lectin and a receptor; a ligand and a receptor; a drug and a drug receptor; complementary chemical reactive groups (e.g., sulfhydryl/maleimide, thiol/maleimide, sulfhydryl/haloacetyl derivative, amine/epoxy, amine/isotriocyanate, amine/succinimidyl este
- the linkage utilizes non-specific absorption.
- the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface.
- the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface.
- the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5’ end of the guide nucleic acid and the surface.
- the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3’ end of the guide nucleic acid and the surface.
- target nucleic acid amplicons are detected by immobilized programmable nuclease probes, such as, for example, CRISPR CAS guide RNA probes (referred to as CRISPR probe).
- CRISPR probe immobilized programmable nuclease probes
- a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe e.g., an immobilized CRISPR CAS / guide RNA complex
- a cutting event will occur that release a reporter that is then detected by a sensor.
- one or more guide nucleic acids are used in conjunction with systems, compositions, and methods disclosed herein to carry out highly efficient, rapid, and accurate reactions for detecting whether a target (e.g., a nucleic acid) is present in a sample.
- the guide nucleic acid can bind to a single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein.
- the guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment.
- the guide nucleic acid can bind to a single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein.
- the guide nucleic acid can be complementary to one or more target nucleic acids. Often the guide nucleic acid binds specifically to the target nucleic acid.
- the target nucleic acid may be a RNA, DNA, or synthetic nucleic acids.
- a guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid.
- a guide nucleic acid can be a crRNA.
- a guide nucleic acid may comprise a crRNA and tracrRNA.
- the guide nucleic acid can bind specifically to the target nucleic acid.
- the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids.
- the target nucleic acid can be designed and made to provide desired functions.
- the targeting region of a guide nucleic acid is 20 nucleotides in length.
- the targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
- the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
- the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to
- the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable, or to bind specifically.
- the guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid.
- the guide nucleic acid in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid.
- the guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
- the guide nucleic acid in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
- the guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest.
- the guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV18.
- guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay.
- the pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein.
- the pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein.
- the tiling for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling comprises gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential.
- a method for detecting a target nucleic acid comprises contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some reporters (e.g., detector nucleic acids) of a population of reporters.
- Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that can be caused by multiple organisms.
- a guide nucleic acid may be complexed with a programmable nuclease in order to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample.
- the programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments.
- the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid.
- the programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity.
- Trans cleavage activity can be non-specific cleavage of nearby single- stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety.
- the detection moiety can be released from the detector nucleic acid and can generate a signal.
- a signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage.
- the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage.
- the detectable signal can be immobilized on a support medium for detection.
- the programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid.
- the CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme.
- a guide nucleic acid can be a CRISPR RNA (crRNA).
- a guide nucleic acid comprises a crRNA and a trans-activating crRNA (tracrRNA).
- the programmable nuclease used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and reporters.
- the programmable nuclease may comprise any of the programmable nucleases described or referenced elsewhere herein.
- the present disclosure provides reagents comprising one or more programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment or portion.
- a programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence.
- the programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and degrades non-specifically nucleic acid in its environment.
- the programmable nuclease has trans cleavage activity once activated.
- a programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease).
- a crRNA and Cas protein can form a CRISPR enzyme.
- the programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments.
- the detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein.
- Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as a Type VI protein, with a DNA reporter and an RNA reporter can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively.
- RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing.
- Methods for the generation of ssDNA for DNA-activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion.
- DNA- activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein.
- target ssDNA detection by Casl3a can be employed in a nucleic acid detection device as disclosed herein.
- the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid.
- the programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid, which can initiate trans cleavage activity.
- the trans cut or trans cleavage can cut and/or release a reporter molecule.
- the trans cut or trans cleavage can produce an analog of a target, which can be directly detected.
- Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of reporters comprising a cleavable nucleic acid and a detection moiety.
- the detection moiety can be released from the reporter and can generate a signal.
- the detection moiety can be immobilized on a support medium for detection. The signal can be visualized to assess whether a target nucleic acid is present or absent.
- Reporters which can be referred to interchangeably as reporter molecules, or detector molecules (e.g., detector nucleic acids), can be used in conjunction with the compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, etc.) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample.
- the reporter can be suspended in solution or immobilized on a surface.
- the reporter can be immobilized on the surface of a chamber in a device as disclosed herein.
- the reporter can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber.
- the reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal.
- the detectable signal can correspond to a release of one or more elements (X).
- the release of the one or more elements (X) can initiate a reaction with another element (Y) when the element (Y) is in the presence of the element (X).
- the reaction between the element (Y) and the element (X) can initiate a chemical chain reaction in a solid phase material.
- Such a chemical chain reaction can produce one or more physical or chemical changes. In some cases, the physical or chemical changes can be optically detected.
- one or more cascade amplification reactions can occur to further amplify the signal before sensing or detection.
- the reporter can comprise a single stranded detector nucleic acid comprising a detection moiety.
- the reporters described herein can be, for example, RNA reporters.
- the RNA reporters can comprise at least one ribonucleic acid and a detectable moiety.
- a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters.
- the detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.
- cleavage of a reporter can produce a signal.
- the signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample.
- cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
- Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample.
- the sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
- optical sensors e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies
- SPR surface plasmon resonance
- interferometric sensors or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
- the reporter may comprise a single-stranded nucleic acid comprising deoxyribonucleotides.
- the reporter may comprise a single-stranded nucleic acid comprising ribonucleotides.
- the reporter can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide.
- the reporter is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site.
- the reporter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position.
- the reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position.
- the reporter comprises from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position.
- the ribonucleotide residues are continuous.
- the ribonucleotide residues are interspersed in between non-ribonucleotide residues.
- the reporter has only ribonucleotide residues. In some cases, the reporter has only deoxyribonucleotide residues.
- the reporter comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the reporter comprises synthetic nucleotides. In some cases, the reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the reporter is 5-20, 5-15, 5-10, 7- 20, 7-15, or 7-10 nucleotides in length.
- the reporter is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length.
- the reporter comprises at least one uracil ribonucleotide. In some cases, the reporter comprises at least two uracil ribonucleotides.
- the reporter has only uracil ribonucleotides. In some cases, the reporter comprises at least one adenine ribonucleotide. In some cases, the reporter comprises at least two adenine ribonucleotides. In some cases, the reporter has only adenine ribonucleotides. In some cases, the reporter comprises at least one cytosine ribonucleotide. In some cases, the reporter comprises at least two cytosine ribonucleotides. In some cases, the reporter comprises at least one guanine ribonucleotide. In some cases, the reporter comprises at least two guanine ribonucleotides.
- a reporter can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof.
- the reporter is from 5 to 12 nucleotides in length.
- the reporter is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
- the reporter is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
- a reporter nucleic acid can be 5, 8, or 10 nucleotides in length.
- a reporter nucleic acid can be 10 nucleotides in length.
- the single stranded reporter nucleic acid can comprise a detection moiety capable of generating a first detectable signal.
- the reporter comprises a protein capable of generating a signal.
- a signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- a detection moiety is on one side of the cleavage site.
- a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety.
- the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some cases, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the reporter. Sometimes the detection moiety is at the 3’ terminus of the reporter. In some cases, the detection moiety is at the 5’ terminus of the reporter. In some cases, the quenching moiety is at the 3’ terminus of the reporter. In some cases, the single-stranded reporter nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal.
- the single-stranded reporter nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal.
- the present invention there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of single-stranded reporter nucleic acids capable of generating a detectable signal.
- multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of reporters capable of generating a detectable signal.
- the present invention there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of reporters capable of generating a detectable signal.
- a detection moiety can be an infrared fluorophore.
- a detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm.
- a detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the detection moiety emits fluorescence at a wavelength of 700 nm or higher. In other cases, the detection moiety emits fluorescence at about 660 nm or about 670 nm.
- the detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm.
- a detection moiety can be a fluorophore that emits a fluorescence in the same range as 6- Fluorescein, IRDye 700, TYE 665, Alex Fluor, or ATTO TM 633 (NHS Ester).
- a detection moiety can be fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester).
- a detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).
- a detection moiety can be fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).
- Any of the detection moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the detection moieties listed.
- a detection moiety can be chosen for use based on the type of sample to be tested. For example, a detection moiety that is an infrared fluorophore is used with a urine sample. As another example, SEQ ID NO: 1 with a fluorophore that emits around 520 nm is used for testing in non-urine samples, and SEQ ID NO: 8 with a fluorophore that emits a fluorescence around 700 nm is used for testing in urine samples.
- a quenching moiety can be chosen based on its ability to quench the detection moiety.
- a quenching moiety can be a non-fluorescent fluorescence quencher.
- a quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm.
- a quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher.
- the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety quenches a detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm.
- a quenching moiety can quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester).
- a quenching moiety can be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher.
- a quenching moiety can quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies).
- a quenching moiety can be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.
- the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety comprises an infrared (IR) dye. In some cases, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a polypeptide. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.
- FRET fluorescence resonance energy transfer
- detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
- the conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer.
- the surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety or another affinity molecule of the cleaved detector molecule as described herein.
- the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample.
- a signal is generated upon cleavage of the reporter by the programmable nuclease.
- the signal changes upon cleavage of the reporter by the programmable nuclease.
- a signal may be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease.
- a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
- a detection moiety can be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- a reporter sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid.
- a calorimetric signal is heat produced after cleavage of the reporters.
- a calorimetric signal is heat absorbed after cleavage of the reporters.
- a potentiometric signal is electrical potential produced after cleavage of the reporters.
- An amperometric signal can be movement of electrons produced after the cleavage of a reporter.
- the signal is an optical signal, such as a colorimetric signal or a fluorescence signal.
- An optical signal is, for example, a light output produced after the cleavage of the reporters.
- an optical signal is a change in light absorbance between before and after the cleavage of reporters.
- a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
- the protein-nucleic acid is an enzyme-nucleic acid.
- the enzyme may be sterically hindered when present as in the enzyme-nucleic acid, but then functional upon cleavage from the nucleic acid.
- the enzyme is an enzyme that produces a reaction with a substrate.
- An enzyme can be invertase.
- the substrate of invertase is sucrose and DNS reagent.
- the protein-nucleic acid is a substrate-nucleic acid.
- the substrate is a substrate that produces a reaction with an enzyme.
- a protein-nucleic acid may be attached to a solid support.
- the solid support for example, may be a surface.
- a surface can be an electrode.
- the solid support is a bead.
- the bead is a magnetic bead.
- the protein is liberated from the solid and interacts with other mixtures.
- the protein is an enzyme, and upon cleavage of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected.
- the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.
- the reporter comprises a nucleic acid conjugated to an affinity molecule and the affinity molecule conjugated to the fluorophore (e.g., nucleic acid - affinity molecule - fluorophore) or the nucleic acid conjugated to the fluorophore and the fluorophore conjugated to the affinity molecule (e.g., nucleic acid - fluorophore - affinity molecule).
- a linker conjugates the nucleic acid to the affinity molecule.
- a linker conjugates the affinity molecule to the fluorophore.
- a linker conjugates the nucleic acid to the fluorophore.
- a linker can be any suitable linker known in the art.
- the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule.
- “directly conjugated” indicates that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other.
- a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore - no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore.
- the affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.
- the reporter comprises a substrate-nucleic acid.
- the substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal.
- the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity.
- the detection moiety comprises a fluorescent label.
- a reporter comprising fluorescent moiety may further comprise a FRET acceptor, such as another fluorescent label or a quencher.
- the detection moiety comprises a quencher.
- the reporter comprises a quantum dot.
- the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher, and the nucleic acid cleavage substrate is a substrate for the activated complex.
- cleavage of the reporter produces a detectable cleavage product comprising the detection moiety (e.g., a quantum dot).
- the cleavage of the reporter and/or the release of a detection moiety may generate a detectable signal.
- the signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the generation of the detectable signal from the release of the detection moiety can indicate that cleavage by the programmable nuclease has occurred and that the sample contains one or more target nucleic acids.
- a detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the reporter can be a protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid.
- a calorimetric signal is heat produced after cleavage of the reporter.
- a calorimetric signal is heat absorbed after cleavage of the reporter.
- a potentiometric signal for example, is electrical potential produced after cleavage of the reporter.
- An amperometric signal can be movement of electrons produced after the cleavage of the reporter.
- the signal is an optical signal, such as a colorimetric signal or a fluorescence signal.
- An optical signal is, for example, a light output produced after the cleavage of the reporter.
- an optical signal is a change in light absorbance between before and after the cleavage of the reporter.
- a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
- Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
- the signal can be measured using one or more sensors integrated with the device or operatively coupled to the device.
- the detection methods disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample.
- a signal is generated upon cleavage of the reporter by the programmable nuclease.
- the signal changes upon cleavage of the reporter by the programmable nuclease.
- a signal can be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease.
- a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
- the programmable nuclease may or may not be immobilized as described elsewhere herein.
- the signal can comprise a colorimetric signal or a signal visible by eye.
- the signal is fluorescent, electrical, chemical, electrochemical, or magnetic.
- a signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the detectable signal is a colorimetric signal or a signal visible by eye.
- the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic.
- the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid.
- the system can detect more than one type of target nucleic acid, wherein the system may comprise more than one type of guide nucleic acid and more than one type of reporter.
- the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.
- the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes.
- real-time detection of targets can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.
- the detectable signals can be detected and analyzed in various ways.
- the detectable signals can be detected using an imaging device.
- the imaging device can comprise a digital camera, such a digital camera on a mobile device.
- the mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein.
- the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope.
- a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope.
- the presently disclosed devices, systems, kits, and methods for detecting the presence of a target nucleic acid in a sample may be used to generate and detect signals indicative of the presence or absence of the target nucleic acid in the sample.
- the generation of a signal indicative of the presence or absence of the target nucleic acid in the sample can enable highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample.
- detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
- detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
- the conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer.
- the surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety or another affinity molecule of the cleaved detector molecule as described herein.
- the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or measuring a signal indicating that the target nucleic acid is absent in a sample.
- a signal is generated upon cleavage of the reporter by the programmable nuclease.
- the signal changes upon cleavage of the reporter by the programmable nuclease.
- a signal may be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease.
- a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
- the reporter e.g., a single stranded reporter
- the reporter can comprise a detection moiety capable of generating a first detectable signal.
- the reporter comprises a protein capable of generating a signal.
- a signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid.
- a detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the reporter can be a protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid.
- a calorimetric signal is heat produced after cleavage of the reporters (e.g., detector nucleic acids).
- a calorimetric signal is heat absorbed after cleavage of the reporters.
- a potentiometric signal for example, is electrical potential produced after cleavage of the reporters.
- An amperometric signal can be movement of electrons produced after the cleavage of the reporter.
- the signal is an optical signal, such as a colorimetric signal or a fluorescence signal.
- An optical signal is, for example, a light output produced after the cleavage of the reporters.
- an optical signal is a change in light absorbance between before and after the cleavage of reporters.
- a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
- Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease.
- the signal can be measured using one or more sensors integrated with the device or operatively coupled to the device.
- the detecting steps disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample.
- a signal is generated upon cleavage of the reporter by the programmable nuclease.
- the signal changes upon cleavage of the reporter by the programmable nuclease.
- a signal can be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease.
- a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
- the signal can comprise a colorimetric signal or a signal visible by eye.
- the signal is fluorescent, electrical, chemical, electrochemical, or magnetic.
- a signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
- the detectable signal is a colorimetric signal or a signal visible by eye.
- the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic.
- the first detection signal is generated by binding of the detection moiety to a capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid.
- the system can detect more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter.
- the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.
- the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes.
- real-time detection of the Cas reaction can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.
- the detectable signals can be detected and analyzed in various ways.
- the detectable signals can be detected using an imaging device.
- the imaging device can comprise a digital camera, such a digital camera on a mobile device.
- the mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein.
- the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).
- a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).
- detecting a target nucleic acid comprises a transcription-based signal amplification reaction.
- a first programmable nuclease and a non-naturally occurring guide nucleic acid form a complex that is activated upon binding the target nucleic acid.
- the activated complex trans-cleaves the linear transcription template to remove a terminal blocking moiety therefrom, which allows the ends of the transcription template to be ligated together by a ligase to form a circular template for signal amplification by a second programmable nuclease complex (which may be the same or different from the first programmable nuclease complex).
- FIG. 7 provides an illustration of an exemplary process for the formation of the circular template through activation of a first programmable nuclease in the presence of a target nucleic acid.
- the activated first programmable nuclease cleaves the blocking end of a linear transcription template, thereby enabling circularization through ligation by a ligase and producing a functional circular template on which an RNA polymerase can act.
- Transcription of the circular template by a DNA-dependent RNA polymerase generates an RNA transcript that serves as a further target nucleic acid for a second programmable nuclease- guide nucleic acid complex.
- the second programmable nuclease may be the same programmable nuclease as the first programmable nuclease, a programmable nuclease of the same type, or a programmable nuclease of a different type.
- the second guide nucleic acid may target the same target nucleic acid sequence as the first guide nucleic acid or a different target nucleic acid sequence (e.g., the first guide nucleic acid may recognize a first target nucleic acid in a sample and the second guide nucleic acid may recognize at least a portion of the circular template/RNA transcript).
- many second programmable nucleases are activated by the initial activation event of the first programmable nuclease.
- the activated first and second programmable nucleases may both cleave reporters, providing a signal amplification as compared to relying solely on direct activation of first programmable nucleases by target nucleic acid in the sample.
- blocking moi eties effective to prevent ligation between the ends of a linear transcription template may be used.
- Non-limiting examples of blocking moi eties include an RNA nucleotide, a dideoxy nucleotide (dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- ligases each having characterized reaction conditions, are available, and include, without limitation NAD+-dependent ligases including tRNA ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia coli DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), thermostable ligase, Ampligase thermostable DNA ligase, VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, and novel ligases discovered by bioprospecting; ATP-dependent ligases including T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase IV, and novel ligases discovered by bioprospecting; GTP-dependent ligases including RtcB ligase,
- the ligation reaction includes the presence of a bridge oligonucleotide.
- the bridge oligonucleotide stabilizes the two ends of the linear transcription template in proximity to one another by hybridizing to both, thereby creating a local region of double-stranded nucleic acid at the ends to be joined.
- the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- the terminal blocking moiety prevents extension of the linear transcription template by a polymerase.
- the linear transcription template comprises a single-stranded nucleic acid comprising deoxyribonucleotides. In some cases, the linear transcription template comprises a cleavage site for the activated programmable nuclease, where cleavage at the cleavage site releases the blocking moiety. In some embodiments, the linear transcription template is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the linear transcription template comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position.
- the linear transcription template comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position.
- the reporter comprises from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position.
- the ribonucleotide residues are continuous.
- the ribonucleotide residues are interspersed in between non-ribonucleotide residues.
- the cleavage site in the linear transcription template is from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length.
- the linear transcription template comprises at least one uracil ribonucleotide. In some cases, the linear transcription template comprises at least two uracil ribonucleotides. In some cases, the linear transcription template comprises at least one adenine ribonucleotide. In some cases, the linear transcription template comprises at least two adenine ribonucleotides.
- the first and/or second programmable nuclease preferentially cleaves uracil ribonucleotide residues over adenine ribonucleotide residues. In some embodiments, the first and/or second programmable nuclease preferentially cleaves adenine ribonucleotide residues over uracil ribonucleotide residues.
- the linear transcription template is about or at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In some embodiments, the linear transcription template is about 40 to about 100 nucleotides in length. In some embodiments, the linear transcription template is about 50 to about 75 nucleotides in length. In some embodiments, the linear transcription template is about 60 nucleotides in length.
- the circularized nucleic acid becomes a template for rolling-circle transcription by a DNA-dependent RNA polymerase.
- transcription by the RNA polymerase continues multiple times around the same circular template, creating an RNA transcript comprising repeating units complementary to the circular template.
- DNA-dependent RNA polymerases are available.
- DNA dependent RNA polymerases synthesize multiple RNA copies from a DNA template.
- the DNA template comprises a promoter recognized by the RNA polymerase.
- the DNA template does not comprise a promoter recognized by the RNA polymerase.
- Non-limiting examples of RNA polymerases are polymerases from E.
- the RNA polymerase is a T7 RNA polymerase.
- the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the RNA polymerase is an SP6 RNA polymerase.
- the circular template encodes an RNA transcript comprising the sequence of the target nucleic acid that is recognized by the non-naturally- occurring guide nucleic acid, such that a single non-naturally-occurring guide nucleic acid is effective to both recognize the presence of the target nucleic acid in the sample, and the transcripts of the RNA transcription reaction.
- the circular template encodes an RNA transcript that is recognized by a different non-naturally-occurring guide nucleic acid, the sequence of which is different from the non-naturally-occurring guide nucleic acid that recognizes the target nucleic acid.
- the reaction may comprise two non- naturally-occurring guide nucleic acid: one that recognizes the target nucleic acid, and one that recognizes the RNA transcript.
- the programmable nuclease that forms a complex with the first non-naturally-occurring guide nucleic acid and the programmable nuclease that forms a complex with the second non-naturally-occurring guide nucleic acid may be the same or different.
- the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C (e.g., about 22 °C to about 39 °C, about 23 °C to about 38 °C, or about 25 °C to about 37 °C). In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 25 °C. In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 37°C.
- the transcription step enhances the sensitivity of the detection reaction, lowering the threshold for detection of a target nucleic acid in a sample and/or increasing the speed of the reaction for a given amount of target nucleic acid present.
- Time to completion may be measured by the consumption of a reagent.
- a singlebuffer comprising detection reagents may consume (e.g., subject to transcollateral cleavage) at least 50 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- a single-buffer comprising detection reagents may consume at least 20 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- a single-buffer comprising detection reagents may consume at least 10 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- a single-buffer comprising detection reagents may consume at least 5 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- a single-buffer comprising detection reagents may consume at least 1 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- a single-buffer comprising detection reagents may consume at least 500 pM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
- at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system.
- at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system.
- At least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some embodiments, at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
- the system comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the programmable nuclease, a different non- naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter; (b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and (c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
- the initial target sequence or resulting signal of the sample can be amplified using quantum dots (QDs).
- the reporter comprises a nucleic acid capable of being trans-cleaved by the programmable nuclease, as described herein.
- the nucleic acid of the reporter is a substrate for the activated programmable nuclease complex and is conjugated to (i) a QD and (ii) either a fluorescent moiety (e.g., a secondary fluorophore) or a quencher.
- a fluorescent moiety e.g., a secondary fluorophore
- the latter further comprises the QD.
- QDs are semiconductor nanoparticles with unique physical and optical characteristics.
- the quantum dots are composed of a Cadmium- Selenium semiconductor.
- QDs are also available as different Cadmium based semiconductors (e.g., Cadmium-Tellurium) and Zinc based semiconductors (e.g., Zinc-Sulfur or Zinc- Selenium).
- QDs can have an inner core of one semiconductor material, a shell made of another semiconductor material, and a coating that allows for further conjugation to other chemicals, including biomolecules.
- QDs vary in size, with the size of the nanoparticle dictating the emission spectra of the molecule.
- the excitation spectra may be shared among QDs of a variety of sizes.
- a single excitation light can yield a readout of many different colors by using different sized QDs.
- QDs have broad excitation spectra, are capable of large Stokes shifts, have long emission lifetimes, have narrow emission spectra, and are generally brighter than their organic fluorophore counterparts.
- QDs are capable of increased sensitivity in molecular diagnostics in accordance with the present disclosure.
- a reporter may comprise a first nucleic acid linker coupled to a quantum dot on a first end thereof and a quencher on a second end thereof. Cleavage of the first nucleic acid linker by an activated programmable nuclease releases the quencher and generates a detectable cleavage product comprising the quantum dot. In this example, the detectable cleavage product has increased fluorescence compared to an uncleaved reporter.
- the reporter comprises a second nucleic acid linker coupled to the quantum dot on a first end thereof and a quencher on a second end thereof.
- the first nucleic acid linker and the second nucleic acid linker may be the same (as shown in the left panel of FIG. 8A) or different (as shown in the right panel of FIG. 8A). Different first and second nucleic acid linkers may enable multiplexing for different targets with different programmable nuclease cleavage preferences.
- the first nucleic acid linker comprises one or more uracil ribonucleotides and the second nucleic acid linker comprises one or more adenine ribonucleotides when a first programmable nuclease preferentially cleaves uracils and a second programmable nuclease preferentially cleaves adenines.
- the first nucleic acid linker comprises ribonucleotides and the second nucleic acid linker comprises deoxyribonucleotides when a first programmable nuclease preferentially cleaves RNA and a second programmable nuclease preferentially cleaves DNA.
- Different levels of fluorescence and/or different color shifts may be used to distinguish between the presence of the first, second, or both target nucleic acids in the sample.
- a reporter may comprise a first nucleic acid linker coupled to a quantum dot on a first end thereof and a secondary fluorophore on a second end thereof.
- the reporter comprises a second nucleic acid linker coupled to the quantum dot on a first end thereof and a same or different secondary fluorophore on a second end thereof.
- the first nucleic acid linker and the second nucleic acid linker may be the same (as shown in the left panel of FIG. 8B) or different (as shown in the right panel of FIG. 8B).
- first and second nucleic acid linkers may enable multiplexing for different targets with different programmable nuclease cleavage preferences.
- the first nucleic acid linker comprises one or more uracil ribonucleotides and the second nucleic acid linker comprises one or more adenine ribonucleotides when a first programmable nuclease preferentially cleaves uracils and a second programmable nuclease preferentially cleaves adenines.
- the first nucleic acid linker comprises ribonucleotides and the second nucleic acid linker comprises deoxyribonucleotides when a first programmable nuclease preferentially cleaves RNA and a second programmable nuclease preferentially cleaves DNA.
- Different wavelength shifts may be used to distinguish between the presence of the first, second, or both target nucleic acids in the sample.
- any of the systems described herein may be used to detect one or more target nucleic acids in a sample.
- detecting the one or more target nucleic acids may comprise one or more of the following steps: sample collection, sample extraction, sample lysis, protein degradation, nucleic acid extraction, nucleic acid purification, nucleic acid concentration, waste removal, nucleic acid elution, nucleic acid amplification, a programmable nuclease-based detection reaction, target detection, and/or reporter detection, or any combination thereof.
- the present disclosure provides a method for detecting a target nucleic acid in a sample using a system disclosed herein, in accordance with any of the various aspects.
- the method comprises (a) contacting the system with the sample; and (b) detecting the detectable cleavage product.
- the present disclosure provides a method for detecting a target nucleic acid in a sample in a single reaction volume.
- the method comprises: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease; (b) cleaving a linear transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product; (c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template; (d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript; (e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a
- Non-limiting examples of target nucleic acids, programmable nucleases, non-naturally occurring guide nucleic acids, linear transcription templates, ligases, blocking moieties, RNA polymerases, and reporters are provided herein, including with regard to aspects of the systems described herein, and may suitably be employed in the methods described herein.
- the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template.
- the target nucleic acid comprises RNA.
- the linear transcription template is single-stranded.
- the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl.
- cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides (e.g., at least two uracils).
- the DNA-dependent RNA polymerase is a T7 RNA polymerase.
- the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- the DNA-dependent RNA polymerase is an SP6 RNA polymerase.
- the first programmable nuclease, second programmable nuclease, DNA- dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein.
- the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e).
- the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume.
- the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
- the method further comprises repeating the method in parallel in each of a plurality of single reaction volumes, wherein each of the plurality of single reaction volumes comprises a different first non-naturally occurring guide nucleic acid. In some embodiments, each of the plurality of single reaction volumes comprises a different linear transcription template.
- FIG. 7 illustrates a schematic of an RCT/DETECTR assay, in accordance with some embodiments.
- the Cas protein becomes activated in the presence of the target nucleic acid, and upon activation cleaves the blocking end of a linear transcription template. This enables circularization through ligation, producing a functional template in which an RNA Polymerase performs RCT, generating an RNA product that can be targeted. Casl3 detects this product and cleaves more of the linear transcription template with a blocking end alongside fluorophore quencher probes.
- FIG. 9 is a flow diagram illustrating a signal amplification process, in accordance with some embodiments.
- the intermediate cleavage target is a transcription template, as disclosed herein.
- an activated first complex comprising a first programmable nuclease, a first guide nucleic acid, and a target nucleic acid
- the first guide nucleic acid may comprise a sequence that hybridizes to a segment of a target nucleic acid in a sample.
- the target nucleic acid in the sample may be referred to as the primary target nucleic acid.
- transcollateral cleavage activity of the first programmable nuclease may be activated.
- the activated first programmable nuclease may cleave a reporter to generate a cleavage product and detectable signal as described herein.
- the activated first complex may cleave an intermediate cleavage target.
- the intermediate cleavage target may comprise a capped or blocked transcription template as described herein.
- the activated first complex may transcleave a cleavage substrate of a transcription template to release a terminal blocking moiety that blocks ligation by a ligase.
- a signal amplification reaction is set off in the presence of the cleaved intermediate cleavage target.
- the ends of the cleaved intermediate cleavage target having released its terminal blocking moiety, may be ligated to form a circularize template.
- the circularized nucleic acid becomes a template for rolling-circle transcription by a DNA-dependent RNA polymerase.
- transcription by the RNA polymerase continues multiple times around the same circular template, creating an RNA transcript comprising repeating units complementary to the circular template.
- an activated second complex comprising a second programmable nuclease, a second guide nucleic acid, and a second target nucleic acid
- the second guide nucleic acid may comprise a sequence that hybridizes to a segment of the RNA transcript generated in step 3.
- the RNA transcript may be referred to as the secondary target nucleic acid in the reaction.
- transcollateral cleavage activity of the second programmable nuclease may be activated.
- the activated second complex may cleave a reporter to generate a cleavage product as described herein.
- a signal of the detectable cleavage product is detected as described herein.
- the first programmable nuclease and the second programmable nuclease may be the same programmable nuclease. In some instances, the first programmable nuclease and the second programmable nuclease may be different. In some instances, the first programmable nuclease and the second programmable nuclease may have the same trans-cleavage preferences (e.g., cleave the same type of reporters). In some instances, the first programmable nuclease and the second programmable nuclease may have different trans-cleavage preferences (e.g., cleave different types of reporters).
- the first guide nucleic acid and the second guide nucleic acid may be the same (e.g., both the first and second guide nucleic acids may comprise a sequence that hybridizes to (i) a segment of the primary target nucleic acid, and (ii) a segment of the secondary target nucleic acid). In some instances, the first guide nucleic acid and the second guide nucleic acid may be different.
- steps described with reference to FIG. 9 above show a method of signal amplification of a programmable nuclease-based detection assay
- the steps may be completed in a different order. Some of the steps may be completed concurrently with one another in the same reaction volume. Some of the steps may be completed subsequent to one another in different reaction volumes. Steps may be added or deleted. Some of the steps may comprise sub-steps. Many of the steps may be repeated as often as necessary to complete the signal amplification reaction.
- Embodiment 1 A system for detecting a target nucleic acid, the system comprising a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter; wherein:
- the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template;
- the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript;
- the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate;
- the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product;
- the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template
- the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript.
- Embodiment 2 The system of Embodiment 1, further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- the bridge oligonucleotide comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template;
- ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
- Embodiment s The system of Embodiment 1 or 2, wherein the target nucleic acid comprises RNA.
- Embodiment 4 The system of any one of Embodiments 1-3, wherein the linear transcription template is single-stranded.
- Embodiment 5 The system of any one of Embodiments 1-4, wherein the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
- Embodiment 6 The system of any one of Embodiments 1-5, wherein the cleavage substrate comprises one or more RNA nucleotides.
- Embodiment 7 The system of Embodiment 6, wherein the one or more
- RNA nucleotides of the cleavage substrate comprises at least two uracils.
- Embodiment 8 The system of any one of Embodiments 1-7, wherein the
- RNA polymerase is a T7 RNA polymerase.
- Embodiment 9 The system of Embodiment 8, wherein the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- Embodiment 10 The system of any one of Embodiments 1-7, wherein the RNA polymerase is an SP6 RNA polymerase.
- Embodiment 11 The system of any one of Embodiments 1-10, wherein the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- Embodiment 12 The system of any one of Embodiments 1-11, wherein the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- Embodiment 13 The system of any one of Embodiments 1-12, wherein the programmable nuclease is a type VI CRISPR/Cas effector protein.
- Embodiment 14 The system of Embodiment 13, wherein the type VI
- CRISPR/Cas effector protein is a Casl3 protein.
- Embodiment 15 The system of Embodiment 14, wherein the Cast 3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
- Embodiment 16 The system of any one of Embodiments 1-15, wherein
- Embodiment 17 The system of any one of Embodiments 1-15, wherein the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
- Embodiment 18 The system of any one of Embodiments 1-17, wherein the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated complex.
- Embodiment 19 The system of Embodiment 18, wherein the detectable cleavage product comprises the quantum dot.
- Embodiment 20 The system of any one of Embodiments 1-19, wherein the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
- Embodiment 21 The system of Embodiment 20, wherein the detection moiety comprises a fluorescent label, a quencher, or an enzyme.
- Embodiment 22 The system of Embodiment 21, wherein the detection moiety comprises an enzyme that catalyzes a colorimetric reaction.
- Embodiment 23 The system of any one of Embodiments 1-22, further comprising a plurality of reaction volumes, wherein
- each reaction volume comprises the programmable nuclease, a different non- naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter;
- each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template;
- each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
- Embodiment 24 A method for detecting a target nucleic acid in a sample, the method comprising:
- Embodiment 25 A method for detecting a target nucleic acid in a sample, the method comprising the following steps in a single reaction volume:
- RNA transcript (d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript
- Embodiment 26 The method of Embodiment 25, wherein (i) the first programmable nuclease and the second programmable nuclease are the same, and/or (ii) the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same.
- Embodiment 27 The method of Embodiment 25 or 26, wherein the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template.
- Embodiment 28 The method of any one of Embodiments 25-27, wherein the target nucleic acid comprises RNA.
- Embodiment 29 The method of any one of Embodiments 25-28, wherein the linear transcription template is single-stranded.
- Embodiment 30 The method of any one of Embodiments 25-29, wherein the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
- Embodiment 31 The method of any one of Embodiments 25-30, wherein cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides.
- Embodiment 32 The method of Embodiment 31, wherein the one or more
- RNA nucleotides of the cleavage substrate comprises at least two uracils.
- Embodiment 33 The method of any one of Embodiments 25-32, wherein the DNA-dependent RNA polymerase is a T7 RNA polymerase.
- Embodiment 34 The method of Embodiment 33, wherein the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
- Embodiment 35 The method of any one of Embodiments 25-32, wherein the DNA-dependent RNA polymerase is an SP6 RNA polymerase.
- Embodiment 36 The method of any one of Embodiments 25-35, wherein the first programmable nuclease, second programmable nuclease, DNA-dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
- Embodiment 37 The method of any one of Embodiments 25-36, wherein the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
- Embodiment 38 The method of any one of Embodiments 25-37, wherein the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein.
- Embodiment 39 The method of Embodiment 38, wherein the type VI CRISPR/Cas effector protein is a Casl3 protein.
- Embodiment 40 The method of Embodiment 39, wherein the Casl3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
- Embodiment 41 The method of any one of Embodiments 25-40, wherein
- Embodiment 42 The method of any one of Embodiments 25-41, wherein the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume.
- Embodiment 43 The method of any one of Embodiments 25-42, wherein the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
- Embodiment 44 The method of any one of Embodiments 25-43, further comprising repeating the method in parallel in each of a plurality of single reaction volumes, wherein each of the plurality of single reaction volumes comprises a different first non- naturally occurring guide nucleic acid.
- Embodiment 45 The method of Embodiment 44, wherein each of the plurality of single reaction volumes comprises a different linear transcription template.
- Embodiment 46 A reporter complex comprising a quantum dot conjugated to a plurality of reporter oligonucleotides, wherein each of the reporter oligonucleotides comprises a secondary fluorophore or a quencher.
- Example 1 Low level detection of target nucleic acids using RCT and DETECTR
- RCT rolling circle transcription
- a Cas protein such as Casl3, can control RNA cleavage internally to a larger nucleotide, and the RCT/DETECTR assay described herein detects low levels of target nucleic acid RNA using an RNA polymerase in combination with a programmable nuclease (FIG. 7).
- RCT and DETECTR can be performed in the same reaction volume
- RCT and DETECTR were performed in a single reaction, without requiring a T7 promoter sequence or a double-stranded nature for the T7 RNAPs to initiate transcription (FIG. 1).
- a Cas 13a variant SEQ ID NO: 21
- the lx concentration of proteins was 40 nM and the final concentration of guide nucleic acid was 40 nM.
- T7 polymerase master mix comprising T7 buffer, T7 RNAP, and 25mM NTPs or OmM NTPs “no NTP” condition
- trigger oligonucleotide or water comprising 1 ul of trigger oligonucleotide or water
- RCT master mix comprising IsoAmp Buffer (NEB) and dumbbell 7 or dumbbell 12 as the circular template, 40nM final concentration
- FQ-RNA reporter 200 nM final concentration
- FIG. 1 is a graphical representation of fluorescence-based signal amplification for two circular ssDNAs (“Dumbbell 7” and “Dumbbell 12”) which each contain the antisense sequence for a Casl3 crRNA target.
- the “Trigger” condition includes a second oligonucleotide used to double strand a portion of the circular ssDNA template.
- the top line in both Dumbbell 7 and Dumbbell 12 graphs represent the “No trigger” condition
- the second, slightly lower line represents the “Trigger” condition
- the third line along the x-axis represents the “No NTPs”, meaning no functional RCT was included in the reaction condition.
- dumbbell 7 the LOD of the RCT/DETECTR reaction was about 100 pM of targetable circular template under the conditions tested.
- dumbbell 12 the LOD of the RCT/DETECTR reaction was about 10 pM of targetable circular template under the conditions tested.
- T7 RNA polymerase can initiate RCT/DETECTR with both circular and linear templates
- the one-pot RCT/DETECTR assay was performed as described above 40 nM final concentration of Dumbbell 7 target in linear or circularized form. While T7 transcription from a circular template was stronger, both linear and circular templates showed efficacy in initiating the RCT/DETECTR reaction in the absence of the T7 promoter under the conditions tested (see FIG. 3).
- Test reactions in the one-pot RCT/DETECTR assays were conducted with different RNAPs including SP6, which have not been used previously for running RCT (FIG. 5).
- the one-pot RCT/DETECTR reactions were run as described above with 40nM T7 RNAP, T3 RNAP, or SP6 as the RNAP for RCT and either circular (solid lines) or linearized (dashed lines) Dumbbell 12 target. SP6 showed the best separation between circular and linear templates under the conditions tested.
- a Cas 13a variant (SEQ ID NO: 21) was pre-complexed with a guide RNA at 37°C in MBufferl.
- the lx concentration of proteins was 5 nM and the final concentration of guide nucleic acid was 5 nM.
- Duplexes were prepared by annealing the bulge strand to the target strand. 5 ul of the complexing reaction was combined with 10 uL of the duplex, or 8 uL of duplex and 2 uL of target, and incubated for 30 minutes at 37°C before being run on a PAGE gel. In all cases, the final concentration of FAM-oligo duplex was about luM.
- FIG. 6 shows a gel image for an assay demonstrating the cleavage activity of the activated Cas 13 variant on poly-uracil tracts that are internal to a larger DNA oligonucleotide.
- the columns show illustrative results for: 1) FAM-oligo only; 2) FAM-oligo duplex with no bulge (TGAGCGAGGACTGCAGCGTAGACG), target, and programmable nuclease complex; 3) FAM-oligo duplex with a 5’-UU-3’ (2U) bulge
- TGAGCGAGGACTrUrUGCAGCGTAGACG programmable nuclease complex
- NTC no target control
- 5) FAM-oligo duplex with 5’-UUUU-3’ (5U) bulge TGAGCGAGGACTrUrUrUrUGCAGCGTAGACG
- programmable nuclease complex TGAGCGAGGACTrUrUrUrUGCAGCGTAGACG
- NTC no target control
- T4 ligase can initiate an RCT/DETECTR with unblocked linear transcription template.
- FIG. 10 is an illustrative graphical representation of the capacity for a ligase to initiate an RCT/DETECTR reaction and fluorescence emission using an unblocked linear template. No signal was generated in reactions lacking either ligase or the circular template.
- QDs Quantum Dots
- a nucleic acid fluorophore or nucleic acickquencher as nanoparticle-based reporters
- the sensitivity is increased in one of two capacities. First, the QDs themselves provide signal amplification. Second, each QD provides an increased number of “reporter” molecules.
- the development of the reporters for the DETECTR assay is based on utilizing QDs as cores, with a layer of oligonucleotides conjugated to the shell of the QD from one end, and the other end is conjugated to either (i) a fluorescence quencher molecule, such that when the oligonucleotide is cleaved in a DETECTR assay, there is an increase in fluorescence; or (ii) other fluorophores, such that when the oligonucleotide is cleaved in a DETECTR assay, there is a shift in color that can be differentiated in a separate detection channel.
- a fluorescence quencher molecule such that when the oligonucleotide is cleaved in a DETECTR assay, there is an increase in fluorescence
- other fluorophores such that when the oligonucleotide is cleaved in a DETECTR assay, there is a shift in
- FIGS. 8A-8B provide illustrative examples of QD-based reporters.
- a first reporter type is depicted, wherein the QD is conjugated to one end of a single stranded oligonucleotide, and the other end is conjugated to the aforementioned fluorescence quencher molecule.
- the oligonucleotide can be any type of nucleic acid, and the QD can be covered in a single oligonucleotide type (left) or a mixture of different oligonucleotides (right).
- FIG. 8B a second reporter type is depicted, wherein the end of the single stranded oligonucleotide opposite the QD is conjugated to a secondary fluorophore for use as a FRET donor-acceptor pair instead of a fluorescence quencher molecule.
- Cas nuclease cleavage of the linker oligonucleotide results in a color shift instead of an increased fluorescence
- the linker again can be any type of nucleic acid and the QD can be covered in a single type (left) or mixture of types of oligonucleotides (right).
- the fluorophores conjugated to each oligonucleotide can be of a single type or multiple types, enabling a multiplexing assay with a readout of different color shifts.
- the secondary fluorophore can also be another QD.
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Abstract
The present disclosure provides systems, compositions, and methods for the detection of nucleic acids. In some embodiments, a system for detecting a target nucleic acid comprises a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a reporter, and one or more compositions for amplifying signal from an activated programmable nuclease. In some embodiments, signal amplification comprises formation of a circularized transcription template by the activated programmable nuclease, and transcription of the circularized transcription template by an RNA polymerase. Methods for using systems disclosed herein are also provided.
Description
SIGNAL AMPLIFICATION ASSAYS FOR NUCLEIC ACID DETECTION
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/371,629, filed on August 16, 2022, which is incorporated herein by reference in its entirety for all purposes.
SEQUENCE LISTING
[0002] The Sequence Listing titled 203477-753601_SL.xml, which was created on August 15, 2023 and is 155,592 bytes in size, is hereby incorporated by reference in its entirety.
BACKGROUND
[0003] The detection of ailments, especially at the early stages of disease or infection, may provide guidance on treatment or intervention to reduce the progression or transmission of the ailment. Diagnostic assays can detect ailments at the point of need through the use of diagnostic devices. Diagnostic tests that detect the presence or absence of nucleic acids often have an advantage in sensitivity and specificity over other test methods, such as those based on antigen detection of the pathogenic organism. In some cases, while antibody/antigen tests may have the ability to directly assay a subject’s immune response to a pathogenic organism, these systems can have sensitivity and specificity challenges related to the response time and the variabilities of antibodies/antigens produced by the subject’s body. However, nucleic acidbased detection systems are not without limitations to detection sensitivity.
SUMMARY
[0004] In view of the foregoing, there is a need for improved nucleic acid-bases detection systems. In various aspects, the present disclosure provides methods and compositions that address this need, and provide further advantages as well.
[0005] In one aspect, the present disclosure provides a system for detecting a target nucleic acid. In some embodiments, the system comprises a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter. In some embodiments of the system: (a) the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template; (b) the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript; (c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate; (d) the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product; (e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template; and (f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript. [0006] In some embodiments, the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the linear transcription template is single-stranded. In some embodiments, the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. In some embodiments, the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils). In some embodiments, the RNA polymerase is a T7 RNA polymerase. In some embodiments where the RNA polymerase is a T7 RNA polymerase, the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the RNA polymerase is an SP6 RNA polymerase. In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C. In some embodiments, the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length. In some embodiments, the programmable nuclease is a type VI CRISPR/Cas effector protein.
In some embodiments, the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e). In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid. In some embodiments, the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated complex. In some embodiments, the detectable cleavage product comprises the quantum dot. In some embodiments, the detectable cleavage product comprises a portion of the reporter comprising a detection moiety. In some embodiments, the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
[0007] In some embodiments, the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the programmable nuclease, a different non-naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter; (b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and (c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
[0008] In one aspect, the present disclosure provides a system for detecting a target nucleic acid. In some embodiments, the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter. In some embodiments of the system: (a) the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid,
and the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template; (b) the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid, and the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated upon binding the RNA transcript; (c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate; (d) the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) cleave the reporter to produce a detectable cleavage product; (e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template; (f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript; and (g) the activated second complex is effective to (i) cleave the cleavage substrate, and (ii) cleave the reporter to produce a detectable cleavage product.
[0009] In some embodiments, the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the linear transcription template is single-stranded. In some embodiments, the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. In some embodiments, the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils). In some embodiments, the RNA polymerase is a T7 RNA polymerase. In some embodiments where the RNA polymerase is a T7 RNA polymerase, the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the RNA polymerase is an SP6 RNA polymerase. In some embodiments, the first programmable nuclease, the second programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C. In some embodiments, the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length. In some embodiments, the first or second
programmable nuclease is a type VI CRISPR/Cas effector protein. In some embodiments, the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e). In some embodiments, the first programmable nuclease and the second programmable nuclease are the same. In some embodiments, the first programmable nuclease and the second programmable nuclease are different. In some embodiments, the first non- naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different. In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid. In some embodiments, the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated first or second complex. In some embodiments, the detectable cleavage product comprises the quantum dot. In some embodiments, the detectable cleavage product comprises a portion of the reporter comprising a detection moiety. In some embodiments, the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
[00010] In some embodiments, the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the first programmable nuclease, a different first non-naturally occurring guide nucleic acid, the same or different linear transcription template, the RNA polymerase, the ligase, the second programmable nuclease, the same or different second non-naturally occurring guide nucleic acid, and the reporter; (b) each different first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of a different target nucleic acid; and (c) each different transcription template, if different, is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
[00011] In one aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample using a system disclosed herein. In some embodiments, the method comprises (a) contacting the system with the sample; and (b) detecting the detectable cleavage product.
[00012] In one aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample in a single reaction volume. In another aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample in two or more reaction volumes (e.g., a two-pot assay). In some embodiments, the method comprises: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non- naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease; (b) cleaving a linear transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product; (c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template; (d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript; (e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a second non- naturally occurring guide nucleic acid that hybridizes to a portion of the RNA transcript, thereby activating the second programmable nuclease; (f) cleaving reporters with the activated first or second programmable nuclease to produce detectable cleavage products; and (g) detecting the detectable cleavage products.
[00013] In some embodiments of the method, (i) the first programmable nuclease and the second programmable nuclease are the same, and/or (ii) the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the linear transcription template is single-stranded. In some embodiments,
[00014] In some embodiments, the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. In some embodiments, cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or
more RNA nucleotides (e.g., at least two uracils). In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase. In some embodiments where the DNA-dependent RNA polymerase is a T7 RNA polymerase, the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the DNA-dependent RNA polymerase is an SP6 RNA polymerase. In some embodiments, the first programmable nuclease, second programmable nuclease, DNA- dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C. In some embodiments, the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length. In some embodiments, the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein. In some embodiments, the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e). In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume. In some embodiments, the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume. In some embodiments, the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
[00015] In some embodiments, the method further comprises repeating the method in parallel in each of a plurality of single reaction volumes (or, in the case of a two-pot assay, in each of a plurality of dual reaction volumes), wherein each of the plurality of single reaction volumes comprises a different first non-naturally occurring guide nucleic acid. In some embodiments, each of the plurality of single reaction volumes comprises a different linear transcription template.
[00016] In one aspect, the present disclosure provides a reporter complex comprising a
quantum dot conjugated to a plurality of reporter oligonucleotides. In some embodiments, each of the reporter oligonucleotides comprises a secondary fluorophore or a quencher.
BRIEF DESCRIPTION OF THE DRAWINGS
[00017] FIG. 1 is a graphical representation of fluorescence-based signal amplification for two circular ssDNAs (“Dumbbell 7” and “Dumbbell 12”) which each contain the antisense sequence for a Casl3 crRNA target. The “Trigger” condition includes a second oligonucleotide used to double strand a portion of the circular ssDNA template. The top line in both Dumbbell 7 and Dumbbell 12 graphs represent the “No trigger” condition, the second, slightly lower line represents the “Trigger” condition, and the third line along the x-axis represents the “No NTPs”, meaning no functional RCT was included in the reaction condition.
[00018] FIG. 2 is a graphical representation demonstrating signal generation capacity of “Dumbbell 7” and Dumbbell 12” ssDNAs in an RCT/DETECTR assay using the ThermoFisher Q5 thermocycler for heating and fluorescence detection. The sensitivity of the signal generation was in the pM range for the circular ssDNA template under the conditions tested.
[00019] FIG. 3 is a graphical representation of the capacity for a T7 RNA polymerase (RNAP) to initiate an RCT/DETECTR reaction and fluorescence emission using either a promoterless circular (top line in both graphs) or linear (bottom line in both graphs) template.
[00020] FIG. 4 is a graphical representation of a one-pot RCT/DETECTR reaction in the presence of either additives betaine (left) or DMSO (right).
[00021] FIG. 5 is a graphical representation of the fluorescence emission for one-pot RCT/DETECTR assays with T7 RNAP, T3 RNAP (bottom data set), or SP6 RNAP, in the presence of circular (solid lines) and linear (dashed lines) templates.
[00022] FIG. 6 is a gel image for an assay demonstrating the cleavage activity of activated Cast 3 on poly-Uracil tracts that are internal to a larger DNA oligonucleotide. The columns show illustrative results for: 1) FAM-oligo only; 2) FAM-oligo duplex with no bulge, target, and programmable nuclease complex; 3) FAM-oligo duplex with a 5’-UU-3’ (2U) bulge, programmable nuclease complex, and no target control (NTC); 4) FAM-oligo duplex with a 2U bulge, target, and programmable nuclease complex; 5) FAM-oligo duplex with 5’- UUUUU-3’ (5U) bulge, programmable nuclease complex, and NTC; 6) FAM-oligo duplex with 5U bulge, target, and programmable nuclease complex; 7) FAM-oligo duplex with 5’-
UUAUU-3’ (2UA2U) bulge, programmable nuclease complex, and NTC; 8) FAM-oligo duplex with 2UA2U bulge, target, and programmable nuclease complex; 9) FAM-oligo duplex with 5’-TUUAUUT-3’ (T2UA2UT) bulge, programmable nuclease complex, and NTC; 10) FAM-oligo duplex with T2UA2UT bulge, target, and programmable nuclease complex; 11) FAM-oligo duplex with 5’-TTUAUTT-3’ (2TUAU2T) bulge, programmable nuclease complex, and NTC; and 12) FAM-oligo duplex with 2TUAU2T, target, and programmable nuclease complex.
[00023] FIG. 7 is a schematic representation of an exemplary RCT/DETECTR signal amplification assay. The presence of the target nucleic acid activates the programmable nuclease complex (e.g., a programmable nuclease in complex with a guide nucleic acid), which then cleaves the blocking end of a linear template. This allows for circularization through ligation, producing a functional template on which the RNA Polymerase performs RCT, in turn generating a targetable RNA product for a programmable nuclease (e.g., a Type VI Cas protein, which may be the same programmable nuclease complex or a different programmable nuclease complex). The programmable nuclease complex then detects this targetable RNA product and cleaves more blocked templates alongside non-target reporters.
[00024] FIGS. 8A-8B show schematic representations of quantum dot reporter according to embodiments of type I (FIG. 8A) and type II (FIG. 8B) quantum dot reporters. The illustrated type I quantum dot reporter in FIG. 8 A is conjugated to a reporter oligonucleotide comprising a single stranded nucleic acid which is conjugated to a fluorescence quencher moiety. In this case, programmable nuclease-based cleavage of the reporter oligonucleotide releases the quencher moiety and results in increased fluorescence. The illustrated type II quantum dot reporter in FIG. 8B is conjugated to a reporter oligonucleotide comprising a single stranded nucleic acid which is conjugated to a fluorescent moiety (e.g., a secondary fluorophore) for use as a fluorescence resonance energy transfer (FRET) donoracceptor pair. In this case, programmable nuclease-based cleavage of the reporter oligonucleotide releases the fluorescent moiety and results in a color shift. The fluorescent moieties conjugated to each nucleic acid of the reporter oligonucleotide can be of a single type, or multiple types to allow for a multiplex assay with a readout of different color shifts for different targets. In some embodiments, the fluorescent moiety could also be another quantum dot.
[00025] FIG. 9 is a flow diagram illustrating a signal amplification process, in accordance with some embodiments. In some embodiments, the intermediate cleavage target is a transcription template, as disclosed herein.
[00026] FIG. 10 is an illustrative graphical representation of the capacity for a ligase to initiate an RCT/DETECTR reaction and fluorescence emission using an unblocked linear template.
DETAILED DESCRIPTION
[00027] All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference in their entireties.
[00028] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those of skill in the art. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
[00029] As used herein, the singular terms “a”, “an”, and “the” include the plural reference unless the context clearly indicates otherwise.
[00030] Reference throughout this specification to, for example, “one embodiment”, “an embodiment”, “another embodiment”, “a particular embodiment”, “a related embodiment”, “a certain embodiment”, “an additional embodiment”, or “a further embodiment” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00031] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using
measurements generally acceptable in the art. In embodiments, about means a range extending to +/— 10% of the specified value. In embodiments, about means the specified value. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.
[00032] As used herein, the terms “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.
[00033] As used herein, the terms “percent identity,” “% identity,” and “% identical” refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two. Generally, computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): l l-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt l):387-95). For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.
[00034] As used herein, a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay. For example, in a one-pot assay, sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, and programmable nuclease- based detection (e.g., DETECTR) assays (optionally including signal amplification) are carried out in a single volume. In some embodiments, amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region). Readout of the
detection (e.g., DETECTR) assay may occur in the single volume or in a second volume. For example, the product of the one-pot DETECTR reaction (e.g., a cleaved detection moiety comprising an enzyme) may be transferred to another volume (e.g., a volume comprising an enzyme substrate) for signal generation and indirect detection of reporter cleavage by a sensor or detector (or by eye in the case of a colorimetric signal).
[00035] As used herein, a “two-pot” reaction refers to a reaction in which more than one reaction occurs in two (or more) volumes alongside a programmable nuclease-based detection (e.g., DETECTR) assay. For example, in a two-pot assay, sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, are carried out in a first volume and programmable nuclease-based detection (e.g., DETECTR) assays are carried out in a second volume. When signal amplification is included as part of the programmable nuclease-based detection assay, a two-pot assay may refer to detection of a target nucleic acid in a sample by a programmable nuclease (e.g., DETECTR)-based reaction in a first volume and signal amplification in a second volume. For example, in some embodiments, a first programmable nuclease-based reaction occurs in a first volume whereby the first programmable nuclease and a first guide nucleic acid are activated in the presence of a target nucleic acid in a sample. Upon activation, the first programmable nuclease cleaves an intermediate cleavage target, and optionally a reporter. Upon completion of the first programmable nuclease reaction, the cleaved intermediate cleavage target is transferred from the first volume to a second volume which contains signal amplification reagents (e.g., a second programmable nuclease, a second guide nucleic acid configured to recognize the cleaved intermediate cleavage target, a reporter a ligase, a polymerase, etc.). The signal amplification reaction may generate a detectable signal in the second volume which is indicative of the presence of the target nucleic acid in the sample.
[00036] As used herein, “HotPot” refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously. In many embodiments, a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).
[00037] The terms, “nucleic acid amplification” and “amplifying a nucleic acid,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.
[00038] The term, “complementary,” as used herein with reference to a nucleic acid
refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T/U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
[00039] The term, “cleavage assay,” as used herein refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some cases, the cleavage activity may be cis-cleavage activity. In some cases, the cleavage activity may be trans-cleavage activity.
[00040] Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes (e.g., CRISPR-Cas enzymes) are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions. As used herein, detection of programmable nuclease-based reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.
[00041] The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.
[00042] The term, “detecting a nucleic acid” and its grammatical equivalents, as used herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.
[00043] The term, “effector protein,” as used herein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. In some embodiments, the complex comprises multiple effector proteins. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies
the target nucleic acid. A non-limiting example of modifying a target nucleic acid is cleaving (hydrolysis) of a phosphodiester bond. Additional examples of modifying target nucleic acids are described herein and throughout. In some embodiments, the term, “effector protein” refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule). The effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).
[00044] The term, “guide nucleic acid,” as used herein refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence. In some embodiments, the first sequence is located 5’ of the second nucleotide sequence. In some embodiments, the first sequence is located 3’ of the second nucleotide sequence.
[00045] The terms, “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of human intervention. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.
[00046] The term, “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given effector protein may not require a PAM sequence being present in a
target nucleic acid for the effector protein to modify the target nucleic acid.
[00047] The terms, “reporter” and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.
[00048] The term, “sample,” as used herein generally refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.
[00049] The term, “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).
[00050] The term, “target sequence,” as used herein when used in reference to a target nucleic acid refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
[00051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[00052] SYSTEMS
[00053] In one aspect, the present disclosure provides a system for detecting a target nucleic acid. In some embodiments, the system comprises a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter. In some embodiments, (a) the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template; (b) the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript; (c) the
transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate; (d) the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product; (e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template; and (f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript.
[00054] In some embodiments, the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter. In some embodiments, (a) the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid, and the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template; (b) the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid, and the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated upon binding the RNA transcript; (c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate; (d) the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) cleave the reporter to produce a detectable cleavage product; (e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template; (f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript; and (g) the activated second complex is effective to (i) cleave the cleavage substrate, and (ii) cleave the reporter to produce a detectable cleavage product. In some embodiments, the first programmable nuclease and the second programmable nuclease are the same. In some embodiments, the first programmable nuclease and the second programmable nuclease are different. In some embodiments, the first non- naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different.
[00055] In some embodiments, the system further comprises a reverse transcriptase (RT). In some embodiments, the RT is effective to reverse-transcribe a target nucleic acid that
is an RNA to form a target cDNA. The target cDNA may function in place of or as an alternative to the target nucleic acid RNA in forming the activated complex with the programmable nuclease and non-naturally-occurring guide nucleic acid. In some embodiments, the RT is effective to reverse transcribe the RNA transcript of the circularized template to form a transcript cDNA. In some embodiments, the transcript cDNA functions in place of or as an alternative to the RNA transcript in forming an activated complex with the programmable nuclease and the non-naturally-occurring guide nucleic acid. Accordingly, the choice of programmable nuclease is not necessarily limited by whether the target nucleic acid is DNA or RNA, or by the RNA-nature of the RNA transcript. Accordingly, programmable nucleases that are most effectively activated by a target DNA are contemplated for use in embodiments of the systems and methods disclosed herein.
[00056] The target can comprise, for example, a target sequence, a target molecule, or a target nucleic acid. As used herein, a target can be referred to interchangeably as a target sequence, target molecule, or target nucleic acid. Further, a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling process as described elsewhere herein). The target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any human, plant, animal, virus, bacteria, or microbe of interest. The systems, devices, apparatuses, methods, and compositions provided herein can be used to perform rapid tests in a single integrated system. The single integrated system may be a reusable unit or a disposable unit.
[00057] The target can be, for example, a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm or other agents or organisms responsible for and/or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like). The target nucleic acid can be a nucleic acid, or a portion thereof. The target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample. The target nucleic acid can be a portion of an RNA or DNA from any organism in the sample.
[00058] The target may comprise, for example, a biological sequence. The biological sequence can comprise a nucleic acid sequence or an amino acid sequence. In some embodiments, the target may be associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest. [00059] In some cases, the target can be associated with one or more pathogenic viruses, pathogenic bacteria, pathogenic worms, pathogenic fungi, or cancer cells. In some embodiments, the pathogenic viruses are selected from the group consisting of respiratory
viruses, adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, SARS-CoV, SARS-CoV-2, MERS, gastrointestinal viruses, noroviruses, rotaviruses, astroviruses, exanthematous viruses, hepatic viral diseases, cutaneous viral diseases, herpes, hemorrhagic viral diseases, Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, neurologic viruses, polio, viral meningitis, viral encephalitis, rabies, sexually transmitted viruses, HIV, HPV, immunodeficiency viruses, influenza virus, dengue virus, West Nile virus, herpes virus, yellow fever virus, Hepatitis Virus C, Hepatitis Virus A, Hepatitis Virus B, and papillomavirus. Other examples of viruses include Orthopoxviruses, such as smallpox, cowpox, horsepox, camelpox, and monkeypox. In some embodiments, the virus to be detected is a monkeypox virus.
[00060] In some cases, the target can be indicative of a respiratory disorder or respiratory pathogen. In some embodiments, the respiratory disorder or respiratory pathogen is selected from the group consisting of SARS-CoV-2 and corresponding variants, human coronavirus hCoV (e g., 229E, NL63, OC43, HKU1), MERS-CoV, (MERS), SARS-CoV (SARS), Flu A, Flu B, RSV, Rhinovirus, Strep A, and TB.
[00061] In some embodiments, the target is indicative of a sexually transmitted infection (STI) or infection related to a woman’s health. In some embodiments, the STI or infection related to a woman’s health is selected from the group consisting of CT, NG, MG, TV, HPV, Candida, B. Vaginosis Syphilis and UTI. In some embodiments, the target comprises a single nucleotide polymorphism (SNP). In some embodiments, the SNP is indicative of NASH disorder or Alpha- 1 disorder. In some embodiments, the target is a blood borne pathogen selected from the group consisting of HIV, HBV, HCV, and/or Zika. In some embodiments, the target is indicative of H. Pylori, C. Difficile, Norovirus, HSV, and/or Meningitis.
[00062] In one aspect, the present disclosure provides a system for detecting a target nucleic acid. In some embodiments, the system comprises a first programmable nuclease, a first non-naturally occurring guide nucleic acid, a second programmable nuclease, a second non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter. In some embodiments of the system: (a) the first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid, and the second non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of an RNA transcript of the transcription template; (b) the first programmable nuclease and the first non-naturally-occurring guide nucleic acid form a first complex that is activated upon binding the target nucleic acid, and the second programmable nuclease and the second non-naturally occurring guide nucleic acid form a second complex that is activated
upon binding the RNA transcript; (c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate; (d) the activated first complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and optionally (ii) cleave the reporter to produce a detectable cleavage product; (e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template; (f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript; and (g) the activated second complex is effective to (i) cleave the cleavage substrate, and (ii) cleave the reporter to produce a detectable cleavage product.
[00063] In some embodiments, the system further comprises a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the linear transcription template is single-stranded. In some embodiments, the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. In some embodiments, the cleavage substrate comprises one or more RNA nucleotides (e.g., at least two uracils). In some embodiments, the RNA polymerase is a T7 RNA polymerase. In some embodiments where the RNA polymerase is a T7 RNA polymerase, the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the RNA polymerase is an SP6 RNA polymerase. In some embodiments, the first programmable nuclease, the second programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C. In some embodiments, the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length. In some embodiments, the first or second programmable nuclease is a type VI CRISPR/Cas effector protein. In some embodiments, the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e). In some embodiments, the first programmable nuclease and the second programmable nuclease are the same. In some embodiments, the first programmable nuclease and the second programmable nuclease are different. In some embodiments, the first non-
naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are different. In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid. In some embodiments, the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated first or second complex. In some embodiments, the detectable cleavage product comprises the quantum dot. In some embodiments, the detectable cleavage product comprises a portion of the reporter comprising a detection moiety. In some embodiments, the detection moiety comprises a fluorescent label, a quencher, or an enzyme (e.g., an enzyme that catalyzes a colorimetric reaction).
[00064] In some embodiments, the system further comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the first programmable nuclease, a different first non-naturally occurring guide nucleic acid, the same or different linear transcription template, the RNA polymerase, the ligase, the second programmable nuclease, the same or different second non-naturally occurring guide nucleic acid, and the reporter; (b) each different first non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of a different target nucleic acid; and (c) each different transcription template, if different, is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
[00065] Programmable Nuclease
[00066] In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter molecule. A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter molecule. In general, the term “reporter” as used in this
context refers to a reagent comprising a polynucleotide, wherein cleavage of the polynucleotide results in a change in a signal. For example, the reporter may comprise a fluorescent label joined to a quencher by a short polynucleotide sequence. Little to no fluorescence is detectable from the fluorescent label when joined to the quencher. However, upon cleavage of the polynucleotide, the fluorescent label is separated from the quencher, resulting in a significant and detectable increase in fluorescent signal upon excitation of the label. As a further example, the reporter may comprise a detection moiety (e.g., a fluorescent label) immobilized to a surface by a short polynucleotide sequence, cleavage of which releases the detection moiety and results in a decrease in signal from the detection moiety at the surface. Alternative detection moieties and arrangements for producing a change in signal upon cleavage of the polynucleotide portion of the reporter are possible, and illustrative examples are described herein. The polynucleotide of the reporter can comprise DNA, RNA, modified nucleotides, or a combination of two or more of these. The programmable nuclease can be, in some nonlimiting embodiments, an RNA-activated programmable RNA nuclease. In some instances, the programmable nucleases disclosed herein can bind to a target DNA to initiate trans cleavage of a reporter. In some cases, the programmable nuclease can be a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, the programmable nuclease may comprise a Cas enzyme which can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave one or more reporters (e.g., RNA reporter molecules). In some embodiments, the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter. In some non-limiting embodiments, the programmable nuclease can comprise a DNA-activated programmable DNA nuclease.
[00067] In some embodiments, the programmable nuclease can comprise an enzyme. The enzyme may be, for example, a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group consisting of a Cas 12, Cas 12a, Cas 13, Cas 14, Cas 14a, and CasPhi.
[00068] Several programmable nucleases are consistent with the systems and methods of the present disclosure. For example, CRISPR/Cas enzymes are programmable nucleases that can be used to implement the methods and systems disclosed herein. CRISPR/Cas enzymes can include any of the known Classes and Types of CRISPR/Cas enzymes. Programmable nucleases disclosed herein can include, for instance, Class 1 CRISPR/Cas enzymes, such as the Type I, Type IV, or Type III CRISPR/Cas enzymes. Programmable nucleases disclosed herein
can also include, for example, the Class 2 CRISPR/Cas enzymes, such as the Type II, Type V, and Type VI CRISPR/Cas enzymes.
[00069] In some embodiments, the Type V CRISPR/Cas enzyme can comprise a programmable Cast 2 nuclease. Type V CRISPR/Cas enzymes (e.g., a Cast 2 or Cast 4) may lack an HNH domain. A Casl2 nuclease of the present disclosure can cleave a nucleic acid via a single catalytic RuvC domain. The RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Casl2 nuclease can further comprise a recognition, or “REC” lobe. The REC and NUC lobes are connected by a bridge helix and the Cast 2 proteins additionally include two domains for PAM recognition termed the PAM interacting (PI) domain and the wedge (WED) domain. In some instances, a programmable Cast 2 nuclease can be a Cast 2a protein, a Cast 2b protein, Cast 2c protein, Cast 2d protein, a Casl2e protein, a Casl2f, a Cast 2g, a Casl2h, a Casl2i, a Casl2j, or a Cast 2k.
[00070] In some embodiments, the programmable nuclease can be a Cast 3 enzyme. Sometimes the Cast 3 enzyme can be a Cast 3 a, a Cast 3b, a Cast 3 c, a Cast 3d, a Casl3e, or a Casl3f. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be a Casl2 enzyme as described elsewhere herein. Sometimes the Cast 2 can be a Cast 2a, a Cast 2b, a Cast 2c, a Cast 2d, a Casl2e, a Casl2f, a Cast 2g, a Casl2h, a Casl2i, a Casl2j, or a Cast 2k. In some cases, the Cast 2 can be a Cast 2 variant (SEQ ID NO: 17), which is a specific protein variant within the Casl2 protein family/classification). In some cases, the programmable nuclease can be Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ. Sometimes, the Csml can also be also called smCmsl, miCmsl, obCmsl, or suCmsl. Sometimes Casl3a can also be called C2c2. Sometimes CasZ can also be called Casl4a, Cast 4b, Cast 4c, Casl4d, Casl4e, Casl4f, Cast 4g, or Casl4h. Sometimes, the programmable nuclease can be a type V CRISPR-Cas system. In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. Sometimes the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system. In some cases, the programmable nuclease can be from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rea), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm), Capnocytophaga canimorsus (Cea, Lachnospiraceae bacterium (Lba), Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotella buccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran), Prevotella
aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotella intermedia (Pin2), Capnocytophaga canimorsus (Cea), Porphyromonas gulae (Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotella intermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius (Ls), or Thermus thermophilus (Tt). Sometimes the Casl3 is at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, or LshCasl3a.
[00071] The trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid. The trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid. The target nucleic acid can be RNA or DNA
[00072] In some instances, programmable nucleases comprise a Type V CRISPR/Cas protein. In some instances, Type V CRISPR/Cas proteins comprise nucleic acid cleavage activity. In some instances, Type V CRISPR/Cas proteins cleave or nick single-stranded nucleic acids, double stranded nucleic acids, or a combination thereof. In some cases, Type V CRISPR/Cas proteins cleave single-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins cleave double-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins nick double-stranded nucleic acids. Typically, guide RNAs of Type V CRISPR/Cas proteins hybridize to ssDNA or dsDNA. However, the trans cleavage activity of Type V CRISPR/Cas protein is typically directed towards ssDNA.
[00073] In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. A catalytically inactive domain of a Type V CRISPR/Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild type nuclease domain of the Type V CRISPR/Cas protein. Said mutations may be present within a cleaving or active site of the nuclease.
[00074] In some embodiments, the Type V CRISPR/Cas protein may be a Cast 4 protein.
The Cas 14 protein may be a Casl4a.l protein (SEQ ID NO: 3). In one example, the
Casl4a. l protein may comprise a sequence of:
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVA AYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYN QSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLK ELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDK YRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYI EVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKSPLVCAINNAFSRYSIS
DNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWA CEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGI EIRI<VAPNNTSI<TCSI<CGHLNNYFNFEYRI<I<NI<FPHFI<CEI<CNFI<ENADYNAALNIS NPKLKSTKEEP (SEQ ID NO: 3). In some cases, the Casl4 protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any exemplary sequence described herein. In some cases, the Casl4 protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any exemplary sequence described herein. In some cases, the Casl4 protein may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any exemplary sequence described herein.
[00075] In some instances, the Type V CRISPR/Cas protein can be modified (also referred to as an engineered protein). For example, a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). In some cases, the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF. Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR/Cas protein is codon optimized for a human cell.
[00076] In some instances, the Type V CRISPR/Cas protein comprises a Casl4 protein. Casl4 proteins may comprise a bilobed structure with distinct amino-terminal and carboxyterminal domains. The amino- and carboxy-terminal domains may be connected by a flexible linker. The flexible linker may affect the relative conformations of the amino- and carboxyl- terminal domains. The flexible linker may be short, for example less than 10 amino acids, less than 8 amino acids, less than 6 amino acids, less than 5 amino acids, or less than 4 amino acids in length. The flexible linker may be sufficiently long to enable different conformations of the amino- and carboxy-terminal domains among two Cast 4 proteins of a Cast 4 dimer complex (e.g., the relative orientations of the amino- and carboxy-terminal domains differ between two Casl4 proteins of a Casl4 homodimer complex). The linker domain may comprise a mutation which affects the relative conformations of the amino- and carboxyl-terminal domains. The linker may comprise a mutation which affects Casl4 dimerization. For example, a linker mutation may enhance the stability of a Cast 4 dimer.
[00077] In some instances, the amino-terminal domain of a Cast 4 protein comprises a wedge domain, a recognition domain, a zinc finger domain, or any combination thereof. The wedge domain may comprise a multi-strand P-barrel structure. A multi-strand P-barrel structure may comprise an oligonucleotide/oligosaccharide-binding fold that is structurally comparable to those of some Cast 2 proteins. The recognition domain and the zinc finger domain may each (individually or collectively) be inserted between P-barrel strands of the wedge domain. The recognition domain may comprise a 4-a-helix structure, structurally comparable but shorter than those found in some Casl2 proteins. The recognition domain may comprise a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some cases, a REC lobe may comprise a binding affinity for a PAM sequence in the target nucleic acid. The amino-terminal may comprise a wedge domain, a recognition domain, and a zinc finger domain. The carboxy -terminal may comprise a RuvC domain, a zinc finger domain, or any combination thereof. The carboxy -terminal may comprise one RuvC and one zinc finger domain.
[00078] Casl4 proteins may comprise a RuvC domain or a partial RuvC domain. The RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the Cast 4 protein. In some instances, a partial RuvC domain does not have any substrate binding activity or catalytic activity on its own. A Casl4 protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, a Casl4 may include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Cast 4 protein, but form a RuvC domain once the protein is produced and folds. A Casl4 protein may comprise a linker loop connecting a carboxy terminal domain of the Cast 4 protein with the amino terminal domain of the Cas 14 protein, and wherein the carboxy terminal domain comprises one or more RuvC domains and the amino terminal domain comprises a recognition domain.
[00079] Casl4 proteins may comprise a zinc finger domain. In some instances, a carboxy terminal domain of a Casl4 protein comprises a zinc finger domain. In some instances, an amino terminal domain of a Cas 14 protein comprises a zinc finger domain. In some instances, the amino terminal domain comprises a wedge domain (e.g., a multi-P-barrel wedge structure), a zinc finger domain, or any combination thereof. In some cases, the carboxy terminal domain comprises the RuvC domains and a zinc finger domain, and the amino terminal domain comprises a recognition domain, a wedge domain, and a zinc finger domain.
[00080] Cas 14 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing. For instance, a Cas 14 protein may be less likely to adsorb to a surface or another biological species due to its small size. The smaller nature of these proteins also allows for them to be more easily packaged as a reagent in a system or assay, and delivered with higher efficiency as compared to other larger Cas proteins. In some cases, a Casl4 protein is 400 to 800 amino acid residues long, 400 to 600 amino acid residues long, 440 to 580 amino acid residues long, 460 to 560 amino acid residues long, 460 to 540 amino acid residues long, 460 to 500 amino acid residues long, 400 to 500 amino acid residues long, or 500 to 600 amino acid residues long. In some cases, a Casl4 protein is less than about 550 amino acid residues long. In some cases, a Casl4 protein is less than about 500 amino acid residues long.
[00081] In some instances, a Cas protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid. In some instances, a Cas protein is capable of catalyzing non-sequence-specific cleavage of a single stranded nucleic acid. In some cases, a Cas protein is activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity is also referred to as “collateral” or “transcollateral” cleavage. Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated programmable nuclease, such as trans cleavage of a reporter comprising a nucleic acid and a detection moiety.
[00082] In some embodiments, a programmable nuclease as disclosed herein is an RNA- activated programmable RNA nuclease. In some embodiments, a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR/Cas enzyme (e.g., a Casl3 nuclease). For example, Casl3a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cast 3a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cast 3a for trans cleavage of an RNA reporter. An RNA reporter can be an RNA-based reporter molecule. In some embodiments, the Casl3a recognizes and detects ssDNA to initiate transcleavage of RNA reporters. Multiple Casl3a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA. For example, Lbu-Casl3a and Lwa- Casl3a can both be activated to transcollaterally cleave RNA reporters by target DNA. Thus, Type VI CRISPR/Cas enzyme (e.g., a Cas 13 nuclease, such as Cas 13 a) can be DNA-activated
programmable RNA nucleases, and therefore can be used to detect a target DNA using the methods as described herein. DNA-activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values. For example, target ssDNA detection by Casl3 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2. In contrast, target RNA detection by Casl3 can exhibit high cleavage activity of pH values from 7.9 to 8.2. In some embodiments, a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease, can have DNA targeting preferences that are distinct from its RNA targeting preferences. For example, the optimal ssDNA targets for Cast 3a have different properties than optimal RNA targets for Cast 3 a. As one example, gRNA performance on ssDNA can not necessarily correlate with the performance of the same gRNAs on RNA. As another example, gRNAs can perform at a high level regardless of target nucleotide identity at a 3’ position on a target RNA sequence. In some embodiments, gRNAs can perform at a high level in the absence of a G at a 3’ position on a target ssDNA sequence. Furthermore, target DNA detected by Cast 3 disclosed herein can be directly taken from organisms or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein. Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA- activated programmable RNA nuclease, such as Casl3a, can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection.
[00083] In some embodiments, a programmable nuclease is used in a detection reaction. In some embodiments, the detection reaction comprises the steps of (a) forming an activated complex comprising a programmable nuclease, a guide nucleic acid, and a target nucleic acid (or an amplicon thereof), thereby activating the programmable nuclease, and (b) cleaving reporters with the activated programmable nuclease. In some embodiments, steps (a) and (b) are performed at an incubation temperature, such as a temperature of about 25 °C to about 80 °C, about 30 °C to about 70 °C, or about 37 °C to about 65 °C. In some embodiments, the incubation temperature is about 37 °C. In some embodiments, the incubation temperature is maintained for about 10 minutes to about 2 hours, about 20 minutes to about 90 minutes, or about 25 minutes to about 60 minutes. In some embodiments, the incubation temperature is maintained for a duration of about 30 minutes. In some embodiments, reporter signal is
- l-
monitored during the incubation. In some embodiments, reporter signal is measured at the end of the incubation.
[00084] Engineered programmable nuclease probes
[00085] Disclosed herein are non-naturally occurring compositions and systems comprising at least one of an engineered programmable nuclease and an engineered guide nucleic acid, which may simply be referred to herein as a programmable nuclease and a guide nucleic acid, respectively. In general, an engineered programmable nuclease and an engineered guide nucleic acid can refer to programmable nucleases and guide nucleic acids, respectively, that are not found in nature. In some instances, systems and compositions comprise at least one non-naturally occurring component. For example, compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some instances, compositions and systems comprise at least two components that do not naturally occur together. For example, compositions and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together. Also, by way of example, composition and systems may comprise a guide nucleic acid and a programmable nuclease that do not naturally occur together. Conversely, and for clarity, a programmable nuclease or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes programmable nucleases and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.
[00086] In some instances, the guide nucleic acid may comprise a non-natural nucleobase sequence. In some instances, the non-natural sequence is a nucleobase sequence that is not found in nature. The non-natural sequence may comprise a portion of a naturally occurring sequence, wherein the portion of the naturally occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some instances, the guide nucleic acid may comprise two naturally occurring sequences arranged in an order or proximity that is not observed in nature. In some instances, compositions and systems comprise a ribonucleotide complex comprising a CRISPR/Cas effector protein and a guide nucleic acid that do not occur together in nature. Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, an engineered guide nucleic acid may comprise a sequence of a naturally occurring repeat region and a spacer region that is complementary to a naturally occurring eukaryotic sequence. The engineered guide nucleic acid may comprise a sequence of a repeat region that occurs naturally
in an organism and a spacer region that does not occur naturally in that organism. An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. For example, an engineered guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA coupled by a linker sequence.
[00087] In some instances, compositions and systems described herein comprise an engineered Cas protein that is similar to a naturally occurring Cas protein. The engineered Cas protein may lack a portion of the naturally occurring Cas protein. The Cas protein may comprise a mutation relative to the naturally-occurring Cas protein, wherein the mutation is not found in nature. The Cas protein may also comprise at least one additional amino acid relative to the naturally-occurring Cas protein. For example, the Cas protein may comprise an addition of a nuclear localization signal relative to the natural occurring Cas protein. In certain embodiments, the nucleotide sequence encoding the Cas protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.
[00088] In some instances, compositions and systems provided herein can comprise a multi-vector system for encoding any programmable nuclease or guide nucleic acid described herein, wherein the guide nucleic acid and the programmable nuclease are encoded by the same or different vectors. In some embodiments, the engineered guide nucleic acid and the engineered programmable nuclease can be encoded by different vectors of the system.
[00089] Thermostable programmable nuclease
[00090] Described herein are various embodiments of thermostable programmable nucleases. In some embodiments, a programmable nuclease is referred to as an effector protein. An effector protein may be thermostable. In some instances, known effector proteins (e.g., Casl2 nucleases) are relatively thermo-sensitive and only exhibit activity (e.g., cis and/or trans cleavage) sufficient to produce a detectable signal in a diagnostic assay at temperatures less than 40° C, and optimally at about 37° C. A thermostable protein may have enzymatic activity, stability, or folding comparable to those at 37 °C. In some instances, the trans cleavage activity (e.g., the maximum trans cleavage rate as measured by fluorescent signal generation) of an effector protein in a trans cleavage assay at 40 °C may be at least 50% of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that
at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00091] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00092] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00093] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00094] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans
cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00095] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.
[00096] In some instances, the trans cleavage activity may be measured against a negative control in a trans cleavage assay. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least
40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative
control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25- fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.
[00097] Additional Examples of Programmable Nucleases
[00098] As described elsewhere herein, one or more programmable nucleases may be used to detect one or more targets (e.g., one or more target nucleic acids). In some cases, a programmable nuclease is capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, the transcript of an RNA polymerase, and/or a cDNA of any of these. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and can non-specifically degrade a non-target nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred
to, interchangeably, as a Cas nuclease or Cas effector protein). A guide nucleic acid (e.g., crRNA) and Cas protein can form a CRISPR enzyme (also referred to, interchangeably, as a Cas complex, Cas probe, or CRISPR probe).
[00099] In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA- activated programmable DNA nuclease.
[000100] The programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby singlestranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporter nucleic acids comprising a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal. The reporter and/or the detection moiety can be immobilized on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimes the detection moiety binds to a capture molecule on the support medium to be immobilized. The detectable signal can be visualized on the support medium to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a SNP associated with a disease, cancer, or genetic disorder.
[000101] A programmable nuclease can be any enzyme that can be or has been designed, modified, or engineered by human contribution so that the enzyme targets or cleaves a nucleic
acid in a sequence-specific manner. Programmable nucleases can include, for example, zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and/or RNA-guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl. Programmable nucleases can also include, for example, PfAgo and/or NgAgo. In certain preferred embodiments, the programmable nuclease is a Cas nuclease. The programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments.
[000102] In some instances, the programmable nuclease is a Type V Cas protein. In general, a Type V Cas effector protein comprises a RuvC domain, but lacks an HNH domain. In most instances, the RuvC domain of the Type V Cas effector protein comprises three patrial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains). In some instances, the three RuvC subdomains are located within the C-terminal half of the Type V Cas effector protein. In some instances, none of the RuvC subdomains are located at the N terminus of the protein. In some instances, the RuvC subdomains are contiguous. In some instances, the RuvC subdomains are not contiguous with respect to the primary amino acid sequence of the Type V Cas protein, but form a ruvC domain once the protein is produced and folds. In some instances, there are zero to about 50 amino acids between the first and second RuvC subdomains. In some instances, there are zero to about 50 amino acids between the second and third RuvC subdomains. In some instances, the Cas effector is a Casl4 effector. In some instances, the Casl4 effector is a Casl4a, Casl4a.l (SEQ ID NO: 3), Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, or Casl4u effector. In some instances, the Cas effector is a CasPhi (also referred to herein as a Cas ) effector. In some instances, the Cas effector is a Casl2 effector. In some instances, the Casl2 effector is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, or Casl2j effector.
[000103] In some instances, the Type V Cas protein is a Cas protein. A Cas protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable Cas nuclease may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable Cas nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.
[000104] In some instances, the programmable nuclease is a Type VI Cas protein. In some embodiments, the Type VI Cas protein is a programmable Casl3 nuclease. The general architecture of a Cas 13 protein includes an N-terminal domain and two HEPN (higher
eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains. The HEPN domains each comprise aR-X4-H motif. Shared features across Cast 3 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase. Thus, two activatable HEPN domains are characteristic of a programmable Cast 3 nuclease of the present disclosure. However, programmable Cast 3 nucleases also consistent with the present disclosure include Cast 3 nucleases comprising mutations in the HEPN domain that enhance the Cast 3 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains. Programmable Cast 3 nucleases consistent with the present disclosure also Casl3 nucleases comprising catalytic components. In some instances, the Cas effector is a Cas 13 effector. In some instances, the Cast 3 effector is a Cas 13 a, a Cas 13b, a Cas 13c, a Cas 13d, or a Cas 13e effector protein.
[000105] In some embodiments, the programmable nuclease comprises a Casl2 protein, wherein the Cas 12 enzyme binds and cleaves double stranded DNA and single stranded DNA. In some embodiments, programmable nuclease comprises a Cas 13 protein, wherein the Cas 13 enzyme binds and cleaves single stranded RNA. In some embodiments, programmable nuclease comprises a Cas 14 protein, wherein the Cas 14 enzyme binds and cleaves both double stranded DNA and single stranded DNA.
[000106] Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity. In some instances, programmable nucleases described herein comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-71. In some instances, programmable nucleases described herein may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-71. In some instances, programmable nucleases described herein comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID Nos: 1-71.
[000107] Table 1 : Amino Acid Sequences of Exemplary Programmable Nucleases
[000108] In some instances, effector proteins disclosed herein are engineered proteins. Engineered proteins are not identical to a naturally-occurring protein. Engineered proteins may provide enhanced nuclease or nickase activity as compared to a naturally occurring nuclease or nickase. An engineered protein may comprise a modified form of a wild type counterpart protein. In some instances, effector proteins comprise at least one amino acid change (e.g., deletion, insertion, or substitution) that enhances or reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart.
[000109] The methods and compositions of the present disclosure are compatible with any type of programmable nuclease that is human-engineered or naturally occurring. The programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof. A programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest. The programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated. In any instances or embodiments where a CRISPR-based programmable nuclease is described or used, it is recognized herein that any other type of programmable nuclease can be used in addition to or in substitution of such CRISPR-based programmable nuclease.
[000110] The methods and compositions of the present disclosure are compatible with a plurality of programmable nucleases, including any of the programmable nucleases described herein. The system can comprise a plurality of programmable nuclease probes comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids. The plurality of programmable nuclease probes can be the same. Alternatively, the plurality of programmable nuclease probes can be different. For example, the plurality of programmable nuclease probes can comprise different programmable nucleases and/or different guide nucleic acids associated with the programmable nucleases. For example, the system may comprise a first programmable nuclease that forms an activated complex with a target nucleic acid, and a second programmable nuclease that forms an activated complex with an RNA transcript produced by an RNA polymerase in the system.
[000111] As described above, trans cleavage activity can be initiated by one or more activated programmable nucleases, including, for instance, trans cleavage of one or more reporters (e.g., reporter nucleic acids) comprising a detection moiety. Once the one or more reporters are cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal. The reporter molecules described herein can comprise, in some non-limiting examples, RNA. In some embodiments, the reporter molecules can comprise ssDNA. In some embodiments, a reporter molecule can comprise: RNA, dsDNA, one or more modified nucleotides, and/or any combination thereof. The reporter molecules can comprise at least one nucleic acid and a detectable moiety. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect a target such as dsDNA and, further, can specifically trans-cleave ssDNA reporters. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect a target such as RNA and, further, can specifically trans-cleave RNA reporters. The detection of the target nucleic acid in the sample can indicate the presence of the sequence in the sample and can provide information about the presence or absence of a nucleic acid sequence in a sample, e.g., as a diagnostic for disease.
[000112] Cleavage of a protein-nucleic acid can produce a signal. The signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some cases, cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the
sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
[000113] Sample
[000114] The systems, devices, apparatuses, methods, and compositions of the present disclosure may be used to analyze one or more samples to detect a presence or an absence of one or more targets as described elsewhere herein. In some instances, the one or more samples can be taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some instances, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some cases, the sample comprises nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some cases, the sample comprises nucleic acids expressed from a cell.
[000115] The sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. In some cases, the target sequence is a portion of a nucleic acid. A nucleic acid can be from a genomic locus, a viral locus, a transcribed mRNA, or a reverse transcribed cDNA. A nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. A nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length. A nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from
35 to 50, or from 45 to 50 nucleotides in length. A nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The target nucleic acid can be reverse complementary to a guide nucleic acid. In some cases, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.
[000116] In some cases, the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from sepsis, in the sample. These diseases can include but are not limited to respiratory viruses (e.g., SARS-CoV-2 (i.e., a virus that causes CO VID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus/Enterovirus, Influenza A, Influenza A/Hl, Influenza A/H3, Influenza A/Hl-2009, Influenza B, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus) and respiratory bacteria (e.g. Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae). Other viruses include human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and Candida albicans. Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g., the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox/shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B,
C, D, E); cutaneous viral diseases (e.g., warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum); hemmorhagic viral diseases (e.g. Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever); neurologic viruses (e.g., polio, viral meningitis, viral encephalitis, rabies), sexually transmitted viruses (e.g., HIV, HPV, and the like), immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus anthracis, Bortadella pertussis, Burkholderia cepacia, Corynebacterium diphtheriae, Coxiella burnetii, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella longbeachae, Legionella pneumophila, Leptospira interrogans, Moraxella catarrhalis, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria elongate, Neisseria gonorrhoeae, Parechovirus, Pneumococcus, Pneumocystis jirovecii, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. Vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Enterobacter cloacae, Kiebsiella aerogenes, Proteus vulgaris, Serratia macesens, Enterococcus faecalis, Enterococcus faecium, Streptococcus intermdius, Streptococcus pneumoniae, and Streptococcus pyogenes. Often the target nucleic acid may comprise a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample. In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus
in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Streptococcus pyogenes, Streptococcus salivarius, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and AL pneumoniae. In some cases, the target sequence is a portion of a nucleic acid from a genomic
locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.
[000117] In some cases, the sample used for cancer testing or cancer risk testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some cases, the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus. Some non-limiting examples of viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV-1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma). One skilled in the art will recognize that viruses can cause or contribute to other types of cancers. In some cases, the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLDI, POLE, POTI, PRKAR1A, PTCHI, PTEN, RAD50, RAD51C, RAD51D, RBI, RECQL4, RET, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1, STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.
[000118] In some cases, the sample used for genetic disorder testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the
reagents described herein. In some embodiments, the genetic disorder is hemophilia, sickle cell anemia, P-thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of CFTR, FMRI, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL, ACAT1, AC0X1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASPA, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CNGB3, COL27A1, COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CTNS, CTSK, CYBA, CYBB, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLRE1C, DHCR7, DHDDS, DLD, DMD, DNAH5, DNAI1, DNAI2, DYSF, EDA, EIF2B5, EMD, ERCC6, ERCC8, ESCO2, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F9, FAH, FAM161A, FANCA, FANCC, FANCG, FH, FKRP, FKTN, G6PC, GAA, GALC, GALK1, GALT, GAMT, GBA, GBE1, GCDH, GFM1, GJB1, GJB2, GLA, GLB1, GLDC, GLE1, GNE, GNPTAB, GNPTG, GNS, GRHPR, HADHA, HAX1, HBA1„ HBA2, HBB, HEXA, HEXB, HGSNAT, HLCS, HMGCL, H0GA1, HPS1, HPS3, HSD17B4, HSD3B2, HYAL1, HYLS1, IDS, IDUA, IKBKAP, IL2RG, IVD, KCNJ11, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAP1, LHX3, LIFR, LIPA, L0XHD1, LPL, LRPPRC, MAN2B1, MC0LN1, MED17, MESP2, MFSD8, MKS1, MLC1, MMAA, MMAB, MMACHC, MMADHC, MPI, MPL, MPV17, MTHFR, MTM1, MTRR, MTTP, MUT, MY07A, NAGLU, NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NPC1, NPC2, NPHS1, NPHS2, NR2E3, NTRK1, OAT, OP A3, OTC, PAH, PC, PCCA, PCCB, PCDH15, PDHA1, PDHB, PEX1, PEX10, PEX12, PEX2, PEX6, PEX7, PFKM, PHGDH, PKHD1, PMM2, P0MGNT1, PPT1, PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12, RMRP, RPE65, RPGRIP1L, RSI, RTEL1, SACS, SAMHD1, SEPSECS, SGCA, SGCB, SGCG, SGSH, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15, SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7, SMARCAL1, SMPD1, STAR, SUMF1, TAT, TCIRG1, TECPR2, TFR2,
TGM1, TH, TMEM216, TPP1, TRMU, TSFM, TTP A, TYMP, USH1C, USH2A, VPS 13 A, VPS13B, VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.
[000119] In some cases, the sample used for phenotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a phenotypic trait.
[000120] In some cases, the sample used for genotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a genotype.
[000121] In some cases, the sample used for ancestral testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a geographic region of origin or ethnic group.
[000122] In some embodiments, the sample can be used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. The disease can be a cancer or genetic disorder. Sometimes, a method comprises obtaining a serum sample from a subject; and identifying a disease status of the subject. Often, the disease status is prostate disease status. In any of the embodiments described herein, the device can be configured for asymptomatic, pre- symptomatic and/or symptomatic diagnostic applications, irrespective of immunity. In any of the embodiments described herein, the device can be configured to perform one or more serological assays on a sample (e.g., a sample comprising blood).
[000123] In some embodiments, the sample can be used to identify a mutation in a target nucleic acid of a plant or of a bacteria, virus, or microbe associated with a plant or soil. The devices and methods of the present disclosure can be used to identify a mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Often, the mutation is a single nucleotide mutation.
[000124] The systems, devices, apparatuses, and methods disclosed herein may be used to detect a presence or an absence of one or more targets in one or more samples. The one or more
samples can comprise one or more target sequences or nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample can be taken from any place where a nucleic acid can be found. Samples can be taken from an individual/human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest. A biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal , cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions/mucus, vaginal secretions/mucus, anal secretion/mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, any combination thereof. A sample can be an aspirate of a bodily fluid from an animal (e.g., human, animals, livestock, pet, etc.) or plant. A tissue sample can be from any tissue that can be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like). A tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure. A sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and/or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.). A sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid/ water, or soil. A sample from an environment can be from soil, air, or water. In some instances, the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. In some instances, the raw sample is applied to the detection system. In some instances, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system. In some cases, the sample is contained in no more than about 200 nanoliters (nL). In some cases, the sample is contained in about 200 nL. In some cases, the sample is contained in a volume that is greater than about 200 nL and less than about 20 microliters (pL). In some cases, the sample is contained in no more than 20 pl. In some cases, the sample is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 pl, or any of value from 1 pl to 500 pl. In some cases, the sample is contained in from 1 pL to 500 pL, from 10 pL to 500 pL, from 50 pL to 500 pL, from 100 pL to 500 pL, from 200 pL to 500 pL, from 300 pL to 500 pL, from 400 pL to 500 pL, from 1 pL to 200 pL, from 10 pL to 200 pL, from 50 pL to 200 pL, from 100 pL to 200 pL, from 1 pL to 100 pL, from 10 pL to 100 pL, from 50 pL to 100 pL,
from 1 pL to 50 pL, from 10 pL to 50 pL, from 1 pL to 20 pL, from 10 pL to 20 pL, or from 1 pL to 10 pL. Sometimes, the sample is contained in more than 500 pl.
[000125] In some instances, the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some instances, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some cases, the sample may comprise nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some cases, the sample may comprise nucleic acids expressed from a cell.
[000126] The sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. In some cases, the target sequence is a portion of a nucleic acid. A nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA. A nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. A nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length. A nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from 35 to 50, or from 45 to 50 nucleotides in length. A nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The target nucleic acid can be reverse complementary to a guide nucleic acid. In some cases, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.
[000127] In some instances, the target nucleic acid is a single stranded nucleic acid. Alternatively, or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents. The target nucleic acid can be RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA). In some cases, the target nucleic acid is mRNA. In some cases, the target nucleic acid is from a virus, a parasite,
or a bacterium described herein. In some cases, the target nucleic acid is transcribed from a gene as described herein.
[000128] A number of target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids. In some cases, the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids. In some cases, the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to 8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to 6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to 8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 nontarget nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids.
[000129] A number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations. In some cases, the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations. In some cases, the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 101 non-target nucleic acids, 102 non- target nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target
nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or IO10 non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.
[000130] Sample preparation
[000131] In some cases, the sample may be separated into a plurality of droplets, aliquots, or subsamples. One or more targets (e.g., nucleic acids, biomolecules, etc.) may be contained within the plurality of droplets, aliquots, or subsamples.
[000132] In some cases, the targets may be amplified before detection occurs. In some cases, the system comprises a nucleic acid detection device. In some embodiments, a nucleic acid detection device comprises a chamber or subsystem for amplifying the targets. In some cases, the nucleic acid detection devices can be configured to amplify the target sequences or target nucleic acids contained within the plurality of droplets by individually processing each of the plurality of droplets (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below). In some cases, the plurality of droplets can undergo separate thermocycling or isothermal processes. In some cases, the thermocycling or isothermal processes can occur simultaneously. In other cases, the thermocycling or isothermal processes can occur at different times for each droplet.
[000133] In some cases, the nucleic acid detection devices can be further configured to remix the droplets, aliquots, or subsamples after the target nucleic acids in each of the droplets undergo amplification. The nucleic acid detection device can be configured to provide the remixed sample comprising the droplets, aliquots, or subsamples to a detection chamber of the device. The detection chamber can be configured to direct the remixed droplets, aliquots, or sub samples to a plurality of programmable nuclease probes (described in greater detail below). In some cases, the detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples along one or more fluid flow paths such that the remixed droplets, aliquots, or subsamples are positioned adjacent to and/or in contact with the one or more programmable nuclease probes. In some cases, the detection chamber can be configured to recirculate or recycle the remixed droplets, aliquots, or subsamples such that the remixed droplets, aliquots, or subsamples are repeatedly placed in contact with one or more programmable nuclease probes.
[000134] In any of the embodiments described herein, the nucleic acid detection device can comprise a single integrated system that is configured to perform sample collection, sample processing, droplet generation, droplet processing (e.g., amplification of target nucleic acids in droplets), droplet remixing, and/or circulation of the remixed droplets within a detection
chamber so that at least a portion of the remixed droplets is placed in contact with one or more programmable nuclease probes coupled to the detection chamber. The nucleic acid detection devices of the present disclosure can be disposable devices configured to perform one or more rapid single reaction or multi -reach on tests to detect a presence and/or an absence of one or more target sequences or target nucleic acids.
[000135] In one aspect, the present disclosure provides exemplary methods for programmable nuclease-based detection. The method can comprise collecting a sample. The sample can comprise any type of sample as described herein. The method can comprise preparing the sample. Sample preparation can comprise one or more sample preparation steps. The one or more sample preparation steps can be performed in any suitable order.
[000136] In some cases, the one or more sample preparation steps can comprise physical filtration of non-target materials using a macro filter, nucleic acid purification, lysis, heat inactivation, or adding one or more enzymes or reagents to prepare the sample for target detection.
[000137] In some cases, the method can comprise generating one or more droplets, aliquots, or subsamples from the sample. The one or more droplets, aliquots, or subsamples can correspond to a volumetric portion of the sample. The sample can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more droplets, aliquots, or subsamples. In some embodiments, the sample is not divided into subsamples.
[000138] In some cases, the method can comprise amplifying one or more targets within each droplet, aliquot, or subsample. Amplification of the one or more targets within each droplet can be performed in parallel and/or simultaneously for each droplet. Dividing the sample into a plurality of droplets can enhance a speed and/or an efficiency of the amplification process (e.g., a thermocycling process) since the droplets comprise a smaller volume of material than the bulk sample introduced. Amplifying the one or more targets within each individual droplet can also permit effective amplification of various target nucleic acids that cannot be amplified as efficiently in a bulk sample containing the various target nucleic acids if the bulk sample were to undergo a singular amplification process. In some embodiments, amplification is performed on the bulk sample without first dividing the sample into subsamples.
[000139] In some cases, the method can further comprise using one or more CRISPR-based or programmable nuclease-based probes (as described elsewhere herein) to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample. In some cases, the sample can be divided into a plurality of droplets, aliquots, or subsamples to facilitate sample
preparation and to enhance the detection capabilities of the devices of the present disclosure. In some cases, the sample is not divided into subsamples. In some embodiments, the target nucleic acids are not amplified before combining with a programmable nuclease in a detection reaction.
[000140] In some embodiments, the sample can be provided manually to a nucleic acid detection device. For example, a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device. In other embodiments, the sample can be provided via an automated syringe. The automated syringe can be configured to control a flow rate at which the sample is provided to the nucleic acid detection device. The automated syringe can be configured to control a volume of the sample that is provided to the nucleic acid detection device over a predetermined period. In some embodiments, the sample can be provided directly to the nucleic acid detection device. For example, a swab sample can be inserted into a sample chamber on the nucleic acid detection device.
[000141] The sample can be prepared before one or more targets are detected within the sample. The sample preparation steps described herein can process a crude sample to generate a pure or purer sample. Sample preparation can one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or eluting. In certain instances, sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, and/or base/acid lysis.
[000142] In some embodiments, nucleic acid purification can be performed on the sample. Purification can comprise disrupting a biological matrix of a cell to release nucleic acids, denaturing structural proteins associated with the nucleic acids (nucleoproteins), inactivating nucleases that can degrade the isolated product (RNase and/or DNase), and/or removing contaminants (e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and/or other cellular debris).
[000143] In some embodiments, lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme). Exemplary proteases include serine proteases (e.g., Proteinase K, Savinase®, trypsin, Protamex®, etc.), metalloproteinases (e.g., MMP-3, etc.), cysteine proteases (e.g, cathepsin B, papin, etc.), threonine proteases, aspartic proteases (e.g., renin, pepsin, cathepsin D, etc.), glutamic proteases, asparagine peptide lyases, or the like. In some cases, a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease. Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt
the three-dimensional structure in macromolecules such as proteins, DNA, or RNA. One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate.2H20, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M P-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used. Alkaline buffers can also be used for cells with hard shells, particularly for environmental samples. Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers. Cell lysis can also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. In some cases, depending on the type of sample, nanoscale barbs, nanowires, acoustic generators, integrated lasers, integrated heaters, and/or microcapillary probes can be used to perform lysis.
[000144] In certain instances, heat inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g, a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, viral proteins, etc.). In some cases, a heating element integrated into the nucleic acid detection device can be used for heatinactivation. The heating element can be powered by a battery or another source of thermal or electric energy that is integrated with the nucleic acid detection device.
[000145] In certain instances, enzyme inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo enzyme inactivation to inhibit or inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, etc.). In some cases, a solution of reagents can be used to inactivate one or more enzymes present in the sample. Enzyme inactivation can occur before, during, or after lysis, when lysis is performed. For example, an RNase inhibitor may be included as a lysis reagent to inhibit native RNases within the sample (which might otherwise impair target and/or reporter detection downstream). Exemplary RNase inhibitors include RNAse Inhibitor, Murine (NEB), Rnaseln Plus (Promega), Protector Rnase Inhibitor (Roche), Superasein (Ambion), RiboLock (Thermo), Ribosafe (Bioline), or the like. Alternatively, or in combination, when a protease is used for sample lysis, a protease inhibitor can be applied to the lysed sample to inactivate the protease prior to contacting the sample nucleic acids to the programmable nuclease. Additional application of heat may not be required to inhibit the protease (e.g., proteinase K) sufficiently to prevent additional activity of the protease (which could potentially impair programmable nuclease activity downstream, in some embodiments). Exemplary protease inhibitors include
AEBSF, antipain, aprotinin, bestatin, chymostatin, EDTA, leupeptin, pepstatin A, phosphoramidon, PMSF, soybean trypsin inhibitor, TPCK, or the like. In some instances, enzyme inactivation may occur before, during, after, or instead of heat inactivation.
[000146] In some cases, a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme. The target nucleic acid within a purified sample can be amplified. In some instances, amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and/or polymerase chain reaction (PCR).
[000147] In some instances, digital droplet amplification can be used. Droplet digitization or droplet generation can comprise splitting a volume of the sample into multiple droplets, aliquots, or subsamples. The sample can have a volume that ranges from about 10 microliters to about 500 microliters. The plurality of droplets, aliquots, or subsamples can have a volume that ranges from about 0.01 microliters to about 100 microliters. The plurality of droplets, aliquots, or subsamples can have a same or substantially similar volume. In some cases, the plurality of droplets, aliquots, or subsamples can have different volumes. In some cases, the droplets, aliquots, or subsamples can be generated using a physical filter or the one or more movable mechanisms described above. In some cases, each droplet of the sample can undergo one or more sample preparation steps (e.g., nucleic acid purification, lysis, heat inactivation, amplification, etc.) independently and/or in parallel while the droplets are physically constrained or thermally isolated. Such nucleic acid amplification of the sample can improve at least one of a sensitivity, specificity, or accuracy of the detection of the target nucleic acid.
[000148] The reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA). In additional cases, nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle transcription (RCT), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequencebased amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement
amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification is performed for from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to 40, or from 25 to 35 minutes. Sometimes, the nucleic acid amplification is performed for from 5 to 60, from 10 to 60, from 15 to 60, from 30 to 60, from 45 to 60, from 1 to 45, from 5 to 45, from 10 to 45, from 30 to 45, from 1 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 1 to 15, from 5 to 15, or from 10 to 15 minutes.
[000149] In some embodiments, nucleic acid amplification can comprise thermocycling of the sample. Thermocycling can be carried out in a vessel, for one or more droplets of the sample in parallel, and/or independently in separate locations. This can be accomplished by methods such as (1) by holding droplets stationary in locations where a heating element is in close proximity to the droplet on one of the droplet sides and a heat sink element is in close proximity to the other side of the droplet, or (2) flowing the droplet through zones in a fluid channel where heat flows across it from a heating source to a heat sink. In some cases, one or more resistive heating elements can be used to perform thermocycling. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 20°C to 45°C, from 25°C to 40°C, from 30°C to 40°C, or from 35°C to 40°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 45°C to 65°C, from 50°C to 65°C, from 55°C to 65°C, or from 60°C to 65°C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 20 °C to 45 °C, from 25 °C to 45 °C, from 30 °C to 45 °C, from 35 °C to 45 °C, from 40 °C to 45 °C, from 20 °C to 37 °C, from 25 °C to 37 °C, from 30 °C to 37 °C, from 35 °C to 37 °C, from 20 °C to 30 °C, from 25 °C to 30 °C, from 20 °C to 25 °C, or from about 22 °C to 25 °C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 40 °C to 65 °C, from 45 °C to 65 °C, from 50 °C to 65 °C, from 55 °C to 65 °C, from 60 °C to 65 °C, from 40 °C to 60 °C, from 45 °C to 60 °C, from 50 °C to 60 °C, from 55 °C to 60 °C, from 40 °C to 55 °C, from 45 °C to 55 °C, from 50 °C to 55 °C, from 40 °C to 50 °C, or from about 45 °C to 50 °C.
[000150] In some embodiments, thermocycling comprises a plurality of cycles, wherein each cycle comprises denaturation at a first temperature and primer extension by a polymerase at a
second temperature that is lower than the first temperature. In some embodiments, each cycle is about or less than about 20 seconds in duration (e.g., about or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 seconds in duration). In some embodiments, each cycle is less than 15 seconds in duration. In some embodiments, each cycle is less than 10 seconds in duration. In some embodiments, the plurality of cycles are about 2 seconds to about 20 seconds in duration, about 3 seconds to about 10 seconds in duration, or about 5 seconds in duration. In some embodiments, the cycles are about 4 seconds in duration. In some embodiments, the denaturation step is about 0.5 to about 5 seconds in duration, about 1 to about 3 seconds in duration, or about 2 seconds in duration. In some embodiments, the denaturation is about 1 second in duration. In some embodiments, the first temperature is about 90°C to about 99°C, about 94°C to about 98°C, or about 95°C. In some embodiments, the first temperature is about 98°C. In some embodiments, the primer extension step is about 1 to about 15 seconds in duration, about 2 to about 10 seconds in duration, or about 5 seconds in duration. In some embodiments, the primer extension step is about 4 seconds in duration. In some embodiments, the second temperature is about 45 °C to about 75 °C, about 50 °C to about 70 °C, or about 55 °C to about 65 °C. In some embodiments, the second temperature is about 55 °C. In some embodiments, each cycle comprises denaturation at the first temperature for about 1 second and primer extension at the second temperature for about 3 seconds. In some embodiments, the plurality of cycles comprises about or at least about 20 cycles (e.g., about or more than about 25, 30, 35, 40, or 45 cycles). In some embodiments, the plurality of cycles comprises about 20 cycles to about 50 cycles, or about 30 cycles to about 45 cycles. In some embodiments, the plurality of cycles comprises about 45 cycles. In some embodiments, the plurality of cycles is preceded by an initial denaturation step at the first temperature that is longer in duration that the durations of the individual denaturation steps in each of the cycles. In some embodiments, the initial denaturation step is about 10 seconds to about 120 seconds in duration, about 15 seconds to about 60 seconds in duration, or about 20 seconds to about 50 seconds in duration. In some embodiments, the initial denaturation step is about 30 seconds in duration. In some embodiments, the total duration of the amplification by thermocycling is about 1 minute to about 20 minutes, about 2 minutes to about 15 minutes, or about 3 minutes to about 10 minutes. In some embodiments, the total duration of the amplification by thermocycling is less than about 10 minutes. In some embodiments, the total duration of the amplification by thermocycling is about 5 minutes.
[000151] In some non-limiting embodiments, the target nucleic acid(s) can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA) of the programmable nuclease
(e.g., CRISPR enzyme). This amplification can be PCR amplification or isothermal amplification. This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA. The reagents for nucleic acid amplification can comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA). In additional cases, nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle transcription (RCT), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 °C The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.
[000152] Nucleic Acids
[000153] The nucleic acids described and referred to herein (including, for instance, guide nucleic acids, reporter nucleic acids, and/or target nucleic acids) can comprise a plurality of nucleotides. A nucleotide may form a base pair with another nucleotide (e.g., as in a complementary strand or internal base pairing of a hairpin structure). A base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds. Nucleobases can comprise adenine, guanine, cytosine, thymine, and/or uracil. In some cases, the nucleic acids described and referred to herein can comprise different nucleotides. In some cases, the nucleic acids described and referred to herein can comprise one or more modified nucleotides. The one or more modified nucleotides can be produced when one or more
nucleotides undergo a chemical modification leading to new bases. The one or more modified nucleotides can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7- Methylguanine, 7-Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5- Methylcytidine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5- carboxylcytosine (5caC).
[000154] In some instances, the target nucleic acid is a single stranded nucleic acid. Alternatively, or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents. The target nucleic acid can be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA). In some cases, the target nucleic acid is mRNA. In some cases, the target nucleic acid is from a virus, a parasite, or a bacterium described herein. In some cases, the target nucleic acid is transcribed from a gene as described herein.
[000155] A number of target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids. In some cases, the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids. In some cases, the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to 8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to 6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to 8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 nontarget nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids.
[000156] A number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations. In some cases, the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations. In some cases, the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 101 non-target nucleic acids, 102 nontarget nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.
[000157] Programmable Nuclease Probes
[000158] In some cases, the systems and methods of the present disclosure may be implemented using one or more programmable nuclease probes. The one or more programmable nuclease probes may be used to detect one or more targets in one or more samples. The one or more targets and the one or more samples may comprise any target or sample described above. In some cases, the one or more programmable nuclease probes may be placed in a detection chamber of a device or apparatus. In some cases, the one or more programmable nuclease probes may be immobilized to a surface of a device or an apparatus. In other cases, the one or more programmable nuclease probes may not or need not be immobilized to a surface of a device or an apparatus.
[000159] In some embodiments, the programmable nuclease probe can comprise a guide nucleic acid that is complexed with or capable of being complexed with a programmable nuclease. The programmable nuclease can comprise any type of programmable nuclease as described herein. In some cases, the programmable nuclease probe may comprise a guide nucleic acid complexed with an enzyme. The enzyme may be, in some instances, a CRISPR enzyme.
[000160] The programmable nuclease probe can comprise a programmable nuclease and/or a guide nucleic acid. The guide nucleic acid can bind to a target nucleic acid, as described in
greater detail below. In some cases, to minimize off-target binding (which can slow down detection or inhibit accurate detection), the device can be configured to generate an electropotential gradient or to provide heat energy to one or more regions proximal to the programmable nuclease probe, to enhance targeting preci sion/accuracy.
[000161] The guide nucleic acid-enzyme complex may include, in some cases, a reporter. When one or more targets bind to the programmable nuclease probe (e.g., a CRISPR probe), the binding event can trigger a trans-cut that (i) releases the reporter into a detectable region or (ii) changes or modifies (e.g., physically or chemically) the reporter. Detection mechanisms can involve interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.
[000162] In certain instances, the reporter of the programmable nuclease probe can initiate a signal amplification reaction with another molecular species after the complementary binding induced trans-cutting. Such species can be a reactive solid or gel matrix, or other reactive entity to generate an amplified signal during detection. The signal amplification reaction can be physical or chemical in nature. In some embodiments, after a complementary binding induced trans-cut, the released reporter, X, can initiate an interaction and/or a reaction with another entity, Y, to produce an amplified or modified signal. Such entities can comprise a molecular species, a solid, a gel, or other entities. The signal amplification interaction can be a physical or chemical reaction. In some embodiments, the interaction involves free-radical, anionic, cationic or coordination polymerization reactions. In other embodiments the cut reporter can trigger aggregation, or agglutination, of molecules, cells, or nanoparticles. In some instances, the cut reporter can interact with a semiconductor material. In some embodiments, the chemical or physical change caused by the interaction is detected by optical detection means such as interferometry, surface plasmon resonance, reflectivity or other. In other embodiments, the chemical or physical change caused by the interaction is detected by potentiometric, amperometric, field effect transistor, or other electronic means.
[000163] In some embodiments, the signal amplification reaction comprises a transcription step. For example, a first programmable nuclease and a non-naturally occurring guide nucleic acid form a complex that is activated upon binding the target nucleic acid. The activated complex cleaves a linear transcription template to remove a terminal blocking moiety, which allows the ends of the transcription template to be ligated together to form a circular template. Transcription of the circular template by a DNA-dependent RNA polymerase generates an RNA transcript that serves as a further target nucleic acid for a second programmable nuclease (the same programmable nuclease as the first programmable nuclease, a programmable
nuclease of the same type, or a programmable nuclease of a different type). In this way, many second programmable nucleases are activated by the initial activation event of the first programmable nuclease. The activated first and second programmable nucleases may both cleave reporters, providing a signal amplification as compared to relying solely on direct activation of first programmable nucleases by target nucleic acid in the sample.
[000164] The plurality of programmable nuclease probes as described herein can be arranged in various configurations. For example, the plurality of programmable nuclease probes can be arranged in a lateral configuration. Alternatively, the plurality of programmable nuclease probes can be arranged in a circular configuration such that each programmable nuclease probe is equidistant from a common center point. In some cases, the plurality of programmable nuclease probes can be distributed with a same separation distance or spacing between the programmable nuclease probes. In other cases, a first programmable nuclease probe and a second programmable nuclease probe can be separated by a first separation distance or spacing, and a third programmable nuclease probe and a fourth programmable nuclease probe can be separated by a second separation distance or spacing that is different than the first separation distance or spacing.
[000165] Probe Immobilization
[000166] In some embodiments, target nucleic acid amplicons can be detected by immobilized programmable nuclease probes, such as, for example, programmable nuclease guide nucleic acid probes (e.g., a CRISPR probe). Upon a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe (e.g., an immobilized programmable nuclease/guide nucleic acid complex) a cutting event can occur that releases a reporter or generates a signal that is then detected by a sensor.
[000167] In some embodiments, the guide nucleic acid of the programmable nuclease or CRISPR probe can be immobilized adjacent to a bottom surface of the chamber. When a complementary interaction between the probe and the target occurs, the CRISPR enzyme will cut and release a reporter molecule which will then be sensed or detected by a sensor/detector. Since the specific guide RNA of the immobilized programmable nuclease or CRISPR probe can be spatially registered, multiplexed detection can be achieved. In some cases, where one sensor corresponds to one immobilized probe, electrical detection can be used. Other methods of detection can also be used, such as optical imaging, surface plasmon resonance (SPR), and/or interferometric sensing.
[000168] In some embodiments, the programmable nuclease probe (e.g., a CRISPR probe) can be immobilized to an immobilization matrix. In some cases, the interior side of the
immobilization matrix may be exposed to an inside wall of a circulation chamber of a detection system, device or apparatus. The guide nucleic acid or guide RNA can be exposed to target amplicons inside the circulation chamber. The reporter can be in proximity to or oriented towards an “exterior” side of the immobilization matrix. The exterior side of the immobilization matrix can be in proximity to a detection region. The detection region may correspond to a region from which a detectable signal can originate. The detectable signal may indicate the presence or the absence of one or more targets of interest. The binding event between the guide nucleic acid and a target nucleic acid can trigger a trans-cut that (i) releases the reporter into a detectable region or (ii) physically or chemically changes the reporter. Detection mechanisms for detecting the reporter or any detectable signals generated by the reporter can involve, for instance, interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.
[000169] In some embodiments, the programmable nuclease, guide nucleic acid, reporter, or a combination thereof can be immobilized to a device surface (e.g., by a linkage). In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, an interaction (e.g., a covalent or noncovalent bond) between members of a binding pair (e.g., streptavidin and biotin), an amide bond, or any combination thereof. In embodiments where more than one element is immobilized to the surface (e.g., reporters and guide nucleic acid, programmable nuclease and reporters, or all three), the linkage may be the same or different for each species. For example, the guide nucleic acid may be immobilized to the surface by a single-stranded linker polynucleotide, and the reporters may be immobilized by the interaction between a first member of a binding pair on the reporters and a second member of a binding pair on the surface. In general, the term “binding pair” refers to a first and a second moiety that have a specific binding affinity for each other. In some embodiments, a binding pair has a dissociation constant Kd of less than or equal to about: 10'8 mol/L, 10'9 mol/L, IO'10 mol/L, 10'11 mol/L, 10'12 mol/L, 10'13 mol/L, 10'14 mol/L, 10'15 mol/L, or ranges including two of these values as endpoints. Non limiting examples of binding pairs include an antibody or an antigen-binding portion thereof and an antigen (e.g., fluorescein, digoxin, digoxigenin); a biotin (bio) moiety and an avidin (or streptavidin) moiety; a dinitrophenol (DNP) and an anti- DNP antibody; a hapten and an anti hapten; folate and a folate binding protein; vitamin B12 and an intrinsic factor; a carbohydrate and a lectin or carbohydrate receptor; a polysaccharide and a polysaccharide binding moiety; a lectin and a receptor; a ligand and a receptor; a drug and a drug receptor; complementary chemical reactive groups (e.g., sulfhydryl/maleimide, thiol/maleimide, sulfhydryl/haloacetyl derivative, amine/epoxy, amine/isotriocyanate,
amine/succinimidyl ester, and amine/sulfonyl halides); an antibody (e.g., IgG) and protein A or protein G; a toxin and a toxin receptor; and an enzyme substrate and an enzyme. In some embodiments, the binding pair comprises biotin and either of avidin or streptavidin.
[000170] In some embodiments, the linkage utilizes non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5’ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3’ end of the guide nucleic acid and the surface.
[000171] In some embodiments, target nucleic acid amplicons are detected by immobilized programmable nuclease probes, such as, for example, CRISPR CAS guide RNA probes (referred to as CRISPR probe). Upon a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe (e.g., an immobilized CRISPR CAS / guide RNA complex) a cutting event will occur that release a reporter that is then detected by a sensor.
[000172] Guide nucleic acid
[000173] In some embodiments, one or more guide nucleic acids are used in conjunction with systems, compositions, and methods disclosed herein to carry out highly efficient, rapid, and accurate reactions for detecting whether a target (e.g., a nucleic acid) is present in a sample. The guide nucleic acid can bind to a single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid can bind to a single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid can be complementary to one or more target nucleic acids. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide
nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable, or to bind specifically.
[000174] The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one
uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
[000175] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling comprises gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid comprises contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some reporters (e.g., detector nucleic acids) of a population of reporters. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that can be caused by multiple organisms.
[000176] As discussed herein, a guide nucleic acid may be complexed with a programmable nuclease in order to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments. The programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-
stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The detectable signal can be immobilized on a support medium for detection. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA (crRNA). Sometimes, a guide nucleic acid comprises a crRNA and a trans-activating crRNA (tracrRNA).
[000177] In some embodiments, the programmable nuclease used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and reporters. The programmable nuclease may comprise any of the programmable nucleases described or referenced elsewhere herein.
[000178] In another aspect, the present disclosure provides reagents comprising one or more programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment or portion. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and degrades non-specifically nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease). A crRNA and Cas protein can form a CRISPR enzyme. The programmable nuclease can be any of a variety of suitable programmable nucleases, including the non-limiting examples of which that are disclosed herein with regard to any of the various aspects and embodiments.
[000179] The detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein. Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as
a Type VI protein, with a DNA reporter and an RNA reporter, can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively. Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing. Methods for the generation of ssDNA for DNA-activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion. Thus, DNA- activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein. For example, target ssDNA detection by Casl3a can be employed in a nucleic acid detection device as disclosed herein.
[000180] In any of the embodiments described herein, the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid, which can initiate trans cleavage activity. In some cases, the trans cut or trans cleavage can cut and/or release a reporter molecule. In other cases, the trans cut or trans cleavage can produce an analog of a target, which can be directly detected. Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of reporters comprising a cleavable nucleic acid and a detection moiety. Once the nucleic acid of the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released from the reporter and can generate a signal. The detection moiety can be immobilized on a support medium for detection. The signal can be visualized to assess whether a target nucleic acid is present or absent.
[000181] Reporters
[000182] Reporters, which can be referred to interchangeably as reporter molecules, or detector molecules (e.g., detector nucleic acids), can be used in conjunction with the compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, etc.) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The reporter can be suspended in solution or immobilized on a surface. For example, the reporter can be immobilized on the surface of a chamber in a device as disclosed herein. In some cases, the reporter can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet
placed below the chamber. The reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal. The detectable signal can correspond to a release of one or more elements (X). The release of the one or more elements (X) can initiate a reaction with another element (Y) when the element (Y) is in the presence of the element (X). The reaction between the element (Y) and the element (X) can initiate a chemical chain reaction in a solid phase material. Such a chemical chain reaction can produce one or more physical or chemical changes. In some cases, the physical or chemical changes can be optically detected. In some embodiments, one or more cascade amplification reactions can occur to further amplify the signal before sensing or detection. There can be a single point of attachment between the reporter molecule and the element (X). Cutting the single point of attachment can release a macro molecule (X), which can undergo a series of reactions based on the macro molecule (X) itself. In any of the embodiments described herein, the reporter can comprise a single stranded detector nucleic acid comprising a detection moiety.
[000183] The reporters described herein can be, for example, RNA reporters. The RNA reporters can comprise at least one ribonucleic acid and a detectable moiety. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters. The detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.
[000184] As described elsewhere herein, cleavage of a reporter (e.g., a protein-nucleic acid) can produce a signal. The signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some cases, cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
[000185] In some cases, the reporter may comprise a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the reporter may comprise a single-stranded nucleic acid comprising ribonucleotides. The reporter can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the reporter is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the reporter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the reporter comprises from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the reporter has only ribonucleotide residues. In some cases, the reporter has only deoxyribonucleotide residues. In some cases, the reporter comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the reporter comprises synthetic nucleotides. In some cases, the reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the reporter is 5-20, 5-15, 5-10, 7- 20, 7-15, or 7-10 nucleotides in length. In some cases, the reporter is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the reporter comprises at least one uracil ribonucleotide. In some cases, the reporter comprises at least two uracil ribonucleotides. Sometimes the reporter has only uracil ribonucleotides. In some cases, the reporter comprises at least one adenine ribonucleotide. In some cases, the reporter comprises at least two adenine ribonucleotides. In some cases, the reporter has only adenine ribonucleotides. In some cases, the reporter comprises at least one cytosine ribonucleotide. In some cases, the reporter comprises at least two cytosine ribonucleotides. In some cases, the reporter comprises at least one guanine ribonucleotide. In some cases, the reporter comprises at least two guanine ribonucleotides. A reporter can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the reporter is from 5 to 12 nucleotides in length. In some cases, the reporter is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the reporter is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Casl3, a reporter nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a reporter nucleic acid can be 10 nucleotides in length.
[000186] The single stranded reporter nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the reporter comprises a protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, a detection moiety is on one side of the cleavage site. Optionally, a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some cases, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some cases, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the reporter. Sometimes the detection moiety is at the 3’ terminus of the reporter. In some cases, the detection moiety is at the 5’ terminus of the reporter. In some cases, the quenching moiety is at the 3’ terminus of the reporter. In some cases, the single-stranded reporter nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the single-stranded reporter nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal. Optionally, there can be more than one population of single-stranded reporter nucleic acids. In some cases, there can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or greater than 50, or any number spanned by the range of this list of different populations of single-stranded reporter nucleic acids capable of generating a detectable signal. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of single-stranded reporter nucleic acids capable of generating a detectable signal. In some cases there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of single-stranded reporter nucleic acids capable of generating a detectable signal.
[000187] In certain aspects of this disclosure, multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of reporters capable of generating a detectable signal. In some cases there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of reporters capable of generating a detectable signal.
/56-FAM/: 5' 6-Fluorescein (Integrated DNA Technologies)
/3IABkFQ/: 3' Iowa Black FQ (Integrated DNA Technologies)
/5IRD700/: 5' IRDye 700 (Integrated DNA Technologies)
/5TYE665/: 5' TYE 665 (Integrated DNA Technologies)
/5Alex594N/: 5' Alexa Fluor 594 (NHS Ester) (Integrated DNA Technologies)
/5ATTO633N/: 5' ATTO TM 633 (NHS Ester) (Integrated DNA Technologies)
/3IRQC1N/: 3' IRDye QC-1 Quencher (Li-Cor)
/3IAbRQSp/: 3' Iowa Black RQ (Integrated DNA Technologies) rU: uracil ribonucleotide rG: guanine ribonucleotide
*This Table refers to the detection moiety and quencher moiety as their tradenames and their source is identified. However, alternatives, generics, or non-tradename moieties with similar function from other sources can also be used.
[000188] A detection moiety can be an infrared fluorophore. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the detection moiety emits fluorescence at a wavelength of 700 nm or higher. In other cases, the detection moiety emits fluorescence at about 660 nm or about 670 nm. In some cases, the detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6- Fluorescein, IRDye 700, TYE 665, Alex Fluor, or ATTO TM 633 (NHS Ester). A detection moiety can be fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A detection moiety can be fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Any of the detection moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the detection moieties listed.
[000189] A detection moiety can be chosen for use based on the type of sample to be tested. For example, a detection moiety that is an infrared fluorophore is used with a urine sample. As another example, SEQ ID NO: 1 with a fluorophore that emits around 520 nm is used for testing in non-urine samples, and SEQ ID NO: 8 with a fluorophore that emits a fluorescence around 700 nm is used for testing in urine samples.
[000190] A quenching moiety can be chosen based on its ability to quench the detection moiety. A quenching moiety can be a non-fluorescent fluorescence quencher. A quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. A quenching moiety can quench a detection moiety that emits fluorescence in the
range of from 500 nm and 720 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In other cases, the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety quenches a detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A quenching moiety can quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A quenching moiety can be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. A quenching moiety can quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A quenching moiety can be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.
[000191] The generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. In some cases, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety comprises an infrared (IR) dye. In some cases, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a polypeptide. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.
[000192] Alternatively, or in combination, in some embodiments, detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer. The surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety
or another affinity molecule of the cleaved detector molecule as described herein. Thus, the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal may be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
[000193] A detection moiety can be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A reporter, sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the reporters. Sometimes, a calorimetric signal is heat absorbed after cleavage of the reporters. A potentiometric signal, for example, is electrical potential produced after cleavage of the reporters. An amperometric signal can be movement of electrons produced after the cleavage of a reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the reporters. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of reporters. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
[000194] Often, the protein-nucleic acid is an enzyme-nucleic acid. The enzyme may be sterically hindered when present as in the enzyme-nucleic acid, but then functional upon cleavage from the nucleic acid. Often, the enzyme is an enzyme that produces a reaction with a substrate. An enzyme can be invertase. Often, the substrate of invertase is sucrose and DNS reagent.
[000195] Sometimes the protein-nucleic acid is a substrate-nucleic acid. Often the substrate is a substrate that produces a reaction with an enzyme.
[000196] A protein-nucleic acid may be attached to a solid support. The solid support, for example, may be a surface. A surface can be an electrode. Sometimes the solid support is a bead. Often the bead is a magnetic bead. Upon cleavage, the protein is liberated from the solid and interacts with other mixtures. For example, the protein is an enzyme, and upon cleavage
of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.
[000197] In some embodiments, the reporter comprises a nucleic acid conjugated to an affinity molecule and the affinity molecule conjugated to the fluorophore (e.g., nucleic acid - affinity molecule - fluorophore) or the nucleic acid conjugated to the fluorophore and the fluorophore conjugated to the affinity molecule (e.g., nucleic acid - fluorophore - affinity molecule). In some embodiments, a linker conjugates the nucleic acid to the affinity molecule. In some embodiments, a linker conjugates the affinity molecule to the fluorophore. In some embodiments, a linker conjugates the nucleic acid to the fluorophore. A linker can be any suitable linker known in the art. In some embodiments, the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule. In this context, “directly conjugated” indicates that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other. For example, if a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore - no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore. The affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.
[000198] In some cases, the reporter comprises a substrate-nucleic acid. The substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal. Often, the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity.
[000199] In some embodiments, the detection moiety comprises a fluorescent label. A reporter comprising fluorescent moiety may further comprise a FRET acceptor, such as another fluorescent label or a quencher. In some embodiments, the detection moiety comprises a quencher. In some embodiments, the reporter comprises a quantum dot. In some
embodiments, the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher, and the nucleic acid cleavage substrate is a substrate for the activated complex. In some embodiments, cleavage of the reporter produces a detectable cleavage product comprising the detection moiety (e.g., a quantum dot).
[000200] Detectable Signal
[000201] As described elsewhere herein, the cleavage of the reporter and/or the release of a detection moiety may generate a detectable signal. The signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. The generation of the detectable signal from the release of the detection moiety can indicate that cleavage by the programmable nuclease has occurred and that the sample contains one or more target nucleic acids. A detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal.
[000202] In some cases, the reporter can be a protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the reporter. Sometimes, a calorimetric signal is heat absorbed after cleavage of the reporter. A potentiometric signal, for example, is electrical potential produced after cleavage of the reporter. An amperometric signal can be movement of electrons produced after the cleavage of the reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the reporter. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of the reporter. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
[000203] Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The signal can be measured using one or more sensors integrated with the device or operatively coupled to the device. Thus, the detection methods disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the reporter by the programmable nuclease. In other embodiments, the signal
changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal can be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease. The programmable nuclease may or may not be immobilized as described elsewhere herein.
[000204] In some cases, the signal can comprise a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system can detect more than one type of target nucleic acid, wherein the system may comprise more than one type of guide nucleic acid and more than one type of reporter. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.
[000205] In some cases, the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes. In some cases, real-time detection of targets can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.
[000206] In some cases, the detectable signals can be detected and analyzed in various ways. For example, the detectable signals can be detected using an imaging device. The imaging device can comprise a digital camera, such a digital camera on a mobile device. The mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein. In some embodiments, the colorimetric, fluorescence, amperometric, potentiometric, or another
electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope.
[000207] The presently disclosed devices, systems, kits, and methods for detecting the presence of a target nucleic acid in a sample may be used to generate and detect signals indicative of the presence or absence of the target nucleic acid in the sample. The generation of a signal indicative of the presence or absence of the target nucleic acid in the sample can enable highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample.
[000208] As disclosed herein, in some embodiments, detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. Alternatively, or in combination, in some embodiments, detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer. The surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety or another affinity molecule of the cleaved detector molecule as described herein. Thus, the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal may be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
[000209] As described above, the reporter (e.g., a single stranded reporter) can comprise a detection moiety capable of generating a first detectable signal. Sometimes the reporter comprises a protein capable of generating a signal. A signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. The generation of the detectable signal from the release of the detection moiety indicates
that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. A detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the reporter can be a protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the reporters (e.g., detector nucleic acids). Sometimes, a calorimetric signal is heat absorbed after cleavage of the reporters. A potentiometric signal, for example, is electrical potential produced after cleavage of the reporters. An amperometric signal can be movement of electrons produced after the cleavage of the reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the reporters. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of reporters. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.
[000210] Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The signal can be measured using one or more sensors integrated with the device or operatively coupled to the device. Thus, the detecting steps disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal can be present in the absence of reporter cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
[000211] In some cases, the signal can comprise a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to a capture molecule in the
detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system can detect more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.
[000212] In some cases, the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes. In some cases, real-time detection of the Cas reaction can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.
[000213] In some cases, the detectable signals can be detected and analyzed in various ways. For example, the detectable signals can be detected using an imaging device. The imaging device can comprise a digital camera, such a digital camera on a mobile device. The mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein. In some embodiments, the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).
[000214] Transcription-Based Signal Amplification
[000215] In some embodiments of the systems, compositions, devices, and methods disclosed herein, detecting a target nucleic acid comprises a transcription-based signal amplification reaction. For example, a first programmable nuclease and a non-naturally occurring guide nucleic acid form a complex that is activated upon binding the target nucleic acid. The activated complex trans-cleaves the linear transcription template to remove a terminal blocking moiety therefrom, which allows the ends of the transcription template to be ligated together by a ligase to form a circular template for signal amplification by a second programmable nuclease complex (which may be the same or different from the first programmable nuclease complex).
FIG. 7 provides an illustration of an exemplary process for the formation of the circular template through activation of a first programmable nuclease in the presence of a target nucleic acid. Upon hybridization of a guide nucleic acid of the first programmable nuclease complex to the target nucleic acid, the activated first programmable nuclease cleaves the blocking end of a linear transcription template, thereby enabling circularization through ligation by a ligase and producing a functional circular template on which an RNA polymerase can act. Transcription of the circular template by a DNA-dependent RNA polymerase generates an RNA transcript that serves as a further target nucleic acid for a second programmable nuclease- guide nucleic acid complex. The second programmable nuclease may be the same programmable nuclease as the first programmable nuclease, a programmable nuclease of the same type, or a programmable nuclease of a different type. The second guide nucleic acid may target the same target nucleic acid sequence as the first guide nucleic acid or a different target nucleic acid sequence (e.g., the first guide nucleic acid may recognize a first target nucleic acid in a sample and the second guide nucleic acid may recognize at least a portion of the circular template/RNA transcript). In this way, many second programmable nucleases are activated by the initial activation event of the first programmable nuclease. The activated first and second programmable nucleases may both cleave reporters, providing a signal amplification as compared to relying solely on direct activation of first programmable nucleases by target nucleic acid in the sample.
[000216] Any of a variety of blocking moi eties effective to prevent ligation between the ends of a linear transcription template may be used. Non-limiting examples of blocking moi eties include an RNA nucleotide, a dideoxy nucleotide (dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. Once the blocking moiety is removed from the linear transcription template, the two ends thereof become active substrates for the ligase. Multiple ligases, each having characterized reaction conditions, are available, and include, without limitation NAD+-dependent ligases including tRNA ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia coli DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), thermostable ligase, Ampligase thermostable DNA ligase, VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, and novel ligases discovered by bioprospecting; ATP-dependent ligases including T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase IV, and novel ligases discovered by bioprospecting; GTP-dependent ligases including RtcB ligase, and novel ligases discovered by bioprospecting; and wild- type, mutant isoforms, and genetically engineered variants thereof.
[000217] In some embodiments, the ligation reaction includes the presence of a bridge oligonucleotide. In general, the bridge oligonucleotide stabilizes the two ends of the linear transcription template in proximity to one another by hybridizing to both, thereby creating a local region of double-stranded nucleic acid at the ends to be joined. In some embodiments, the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety. In some embodiments, the terminal blocking moiety prevents extension of the linear transcription template by a polymerase.
[000218] In some embodiments, the linear transcription template comprises a single-stranded nucleic acid comprising deoxyribonucleotides. In some cases, the linear transcription template comprises a cleavage site for the activated programmable nuclease, where cleavage at the cleavage site releases the blocking moiety. In some embodiments, the linear transcription template is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the linear transcription template comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the linear transcription template comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the reporter comprises from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some embodiments, the cleavage site in the linear transcription template is from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the linear transcription template comprises at least one uracil ribonucleotide. In some cases, the linear transcription template comprises at least two uracil ribonucleotides. In some cases, the linear transcription template comprises at least one adenine ribonucleotide. In some cases, the linear transcription template comprises at least two adenine ribonucleotides. In some embodiments, the first and/or second programmable nuclease preferentially cleaves uracil ribonucleotide residues over adenine ribonucleotide residues. In some embodiments, the first and/or second programmable nuclease preferentially cleaves adenine ribonucleotide residues over uracil ribonucleotide residues.
[000219] In some embodiments, the linear transcription template is about or at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In some embodiments, the linear transcription template is about 40 to about 100 nucleotides in length. In some embodiments, the linear transcription template is about 50 to about 75 nucleotides in length. In some embodiments, the linear transcription template is about 60 nucleotides in length.
[000220] Once the ends are ligated, the circularized nucleic acid becomes a template for rolling-circle transcription by a DNA-dependent RNA polymerase. In other words, transcription by the RNA polymerase continues multiple times around the same circular template, creating an RNA transcript comprising repeating units complementary to the circular template. A variety of suitable DNA-dependent RNA polymerases are available. DNA dependent RNA polymerases synthesize multiple RNA copies from a DNA template. In some embodiments, the DNA template comprises a promoter recognized by the RNA polymerase. In some embodiments, the DNA template does not comprise a promoter recognized by the RNA polymerase. Non-limiting examples of RNA polymerases are polymerases from E. coli and bacteriophages T7, T3 and SP6. In some embodiments, the RNA polymerase is a T7 RNA polymerase. In some embodiments, where the RNA polymerase is a T7 RNA polymerase, the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the RNA polymerase is an SP6 RNA polymerase.
[000221] In some embodiments, the circular template encodes an RNA transcript comprising the sequence of the target nucleic acid that is recognized by the non-naturally- occurring guide nucleic acid, such that a single non-naturally-occurring guide nucleic acid is effective to both recognize the presence of the target nucleic acid in the sample, and the transcripts of the RNA transcription reaction. In some embodiments, the circular template encodes an RNA transcript that is recognized by a different non-naturally-occurring guide nucleic acid, the sequence of which is different from the non-naturally-occurring guide nucleic acid that recognizes the target nucleic acid. Where the circular template encodes an RNA that does not comprise a sequence of the target nucleic acid, the reaction may comprise two non- naturally-occurring guide nucleic acid: one that recognizes the target nucleic acid, and one that recognizes the RNA transcript. In reactions that comprise two different non-naturally- occurring guide nucleic acid, the programmable nuclease that forms a complex with the first non-naturally-occurring guide nucleic acid and the programmable nuclease that forms a complex with the second non-naturally-occurring guide nucleic acid may be the same or
different.
[000222] In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C (e.g., about 22 °C to about 39 °C, about 23 °C to about 38 °C, or about 25 °C to about 37 °C). In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 25 °C. In some embodiments, the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 37°C.
[000223] In some embodiments, the transcription step enhances the sensitivity of the detection reaction, lowering the threshold for detection of a target nucleic acid in a sample and/or increasing the speed of the reaction for a given amount of target nucleic acid present. Time to completion may be measured by the consumption of a reagent. For example, a singlebuffer comprising detection reagents may consume (e.g., subject to transcollateral cleavage) at least 50 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising detection reagents may consume at least 20 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising detection reagents may consume at least 10 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising detection reagents may consume at least 5 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising detection reagents may consume at least 1 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising detection reagents may consume at least 500 pM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.
[000224] In some embodiments, at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some embodiments, at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some embodiments, at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some embodiments, at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
[000225] In some embodiments, the system comprises a plurality of reaction volumes, wherein (a) each reaction volume comprises the programmable nuclease, a different non- naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter; (b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and (c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
Quantum Dots
[000226] In some embodiments, the initial target sequence or resulting signal of the sample can be amplified using quantum dots (QDs). In these embodiments, the reporter comprises a nucleic acid capable of being trans-cleaved by the programmable nuclease, as described herein. The nucleic acid of the reporter is a substrate for the activated programmable nuclease complex and is conjugated to (i) a QD and (ii) either a fluorescent moiety (e.g., a secondary fluorophore) or a quencher. Additionally, when the reporter is cleaved to form the detectable cleavage product, the latter further comprises the QD.
[000227] QDs are semiconductor nanoparticles with unique physical and optical characteristics. In some embodiments, the quantum dots are composed of a Cadmium- Selenium semiconductor. QDs are also available as different Cadmium based semiconductors (e.g., Cadmium-Tellurium) and Zinc based semiconductors (e.g., Zinc-Sulfur or Zinc-
Selenium). QDs can have an inner core of one semiconductor material, a shell made of another semiconductor material, and a coating that allows for further conjugation to other chemicals, including biomolecules. QDs vary in size, with the size of the nanoparticle dictating the emission spectra of the molecule. Yet, due to the properties of semiconductors, the excitation spectra may be shared among QDs of a variety of sizes. Thus, a single excitation light can yield a readout of many different colors by using different sized QDs. QDs have broad excitation spectra, are capable of large Stokes shifts, have long emission lifetimes, have narrow emission spectra, and are generally brighter than their organic fluorophore counterparts. Thus, QDs are capable of increased sensitivity in molecular diagnostics in accordance with the present disclosure.
[000228] In an exemplary embodiment, as shown in FIG. 8A, a reporter may comprise a first nucleic acid linker coupled to a quantum dot on a first end thereof and a quencher on a second end thereof. Cleavage of the first nucleic acid linker by an activated programmable nuclease releases the quencher and generates a detectable cleavage product comprising the quantum dot. In this example, the detectable cleavage product has increased fluorescence compared to an uncleaved reporter. In some embodiments, the reporter comprises a second nucleic acid linker coupled to the quantum dot on a first end thereof and a quencher on a second end thereof. The first nucleic acid linker and the second nucleic acid linker may be the same (as shown in the left panel of FIG. 8A) or different (as shown in the right panel of FIG. 8A). Different first and second nucleic acid linkers may enable multiplexing for different targets with different programmable nuclease cleavage preferences. For example, the first nucleic acid linker comprises one or more uracil ribonucleotides and the second nucleic acid linker comprises one or more adenine ribonucleotides when a first programmable nuclease preferentially cleaves uracils and a second programmable nuclease preferentially cleaves adenines. In another example, the first nucleic acid linker comprises ribonucleotides and the second nucleic acid linker comprises deoxyribonucleotides when a first programmable nuclease preferentially cleaves RNA and a second programmable nuclease preferentially cleaves DNA. Different levels of fluorescence and/or different color shifts may be used to distinguish between the presence of the first, second, or both target nucleic acids in the sample. [000229] In another exemplary embodiment, as shown in FIG. 8B, a reporter may comprise a first nucleic acid linker coupled to a quantum dot on a first end thereof and a secondary fluorophore on a second end thereof. Cleavage of the first nucleic acid linker by an activated programmable nuclease releases the secondary fluorophore and generates a detectable cleavage product comprising the quantum dot. In this example, the detectable
cleavage product has a change in fluorescence wavelength compared to an uncleaved reporter. In some embodiments, the reporter comprises a second nucleic acid linker coupled to the quantum dot on a first end thereof and a same or different secondary fluorophore on a second end thereof. The first nucleic acid linker and the second nucleic acid linker may be the same (as shown in the left panel of FIG. 8B) or different (as shown in the right panel of FIG. 8B). Different first and second nucleic acid linkers may enable multiplexing for different targets with different programmable nuclease cleavage preferences. For example, the first nucleic acid linker comprises one or more uracil ribonucleotides and the second nucleic acid linker comprises one or more adenine ribonucleotides when a first programmable nuclease preferentially cleaves uracils and a second programmable nuclease preferentially cleaves adenines. In another example, the first nucleic acid linker comprises ribonucleotides and the second nucleic acid linker comprises deoxyribonucleotides when a first programmable nuclease preferentially cleaves RNA and a second programmable nuclease preferentially cleaves DNA. Different wavelength shifts may be used to distinguish between the presence of the first, second, or both target nucleic acids in the sample.
METHODS
[000230] Any of the systems described herein (which may comprise any of the compositions, components, or devices described herein) may be used to detect one or more target nucleic acids in a sample. In some embodiments, detecting the one or more target nucleic acids may comprise one or more of the following steps: sample collection, sample extraction, sample lysis, protein degradation, nucleic acid extraction, nucleic acid purification, nucleic acid concentration, waste removal, nucleic acid elution, nucleic acid amplification, a programmable nuclease-based detection reaction, target detection, and/or reporter detection, or any combination thereof.
[000231] In one aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample using a system disclosed herein, in accordance with any of the various aspects. In some embodiments, the method comprises (a) contacting the system with the sample; and (b) detecting the detectable cleavage product.
[000232] In one aspect, the present disclosure provides a method for detecting a target nucleic acid in a sample in a single reaction volume. In some embodiments, the method comprises: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease; (b) cleaving a linear
transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product; (c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template; (d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript; (e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a second non-naturally occurring guide nucleic acid that hybridizes to a portion of the RNA transcript, thereby activating the second programmable nuclease; (f) cleaving reporters with the activated first or second programmable nuclease to produce detectable cleavage products; and (g) detecting the detectable cleavage products. Non-limiting examples of target nucleic acids, programmable nucleases, non-naturally occurring guide nucleic acids, linear transcription templates, ligases, blocking moieties, RNA polymerases, and reporters are provided herein, including with regard to aspects of the systems described herein, and may suitably be employed in the methods described herein.
[000233] In some embodiments of the method, (i) the first programmable nuclease and the second programmable nuclease are the same, and/or (ii) the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same. In some embodiments, the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the linear transcription template is single-stranded. In some embodiments,
[000234] In some embodiments, the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide (e.g., dideoxy cytosine), a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl. In some embodiments, cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides (e.g., at least two uracils). In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase. In some embodiments where the DNA-dependent RNA polymerase is a T7 RNA polymerase, the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77). In some embodiments, the DNA-dependent RNA polymerase is an SP6 RNA polymerase. In some embodiments, the first programmable nuclease, second programmable nuclease, DNA- dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about
40 °C. In some embodiments, the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length. In some embodiments, the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein. In some embodiments, the type VI CRISPR/Cas effector protein is a Casl3 protein (e.g., Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e). In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume. In some embodiments, the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume. In some embodiments, the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
[000235] In some embodiments, the method further comprises repeating the method in parallel in each of a plurality of single reaction volumes, wherein each of the plurality of single reaction volumes comprises a different first non-naturally occurring guide nucleic acid. In some embodiments, each of the plurality of single reaction volumes comprises a different linear transcription template.
[000236] FIG. 7 illustrates a schematic of an RCT/DETECTR assay, in accordance with some embodiments. Here, the Cas protein becomes activated in the presence of the target nucleic acid, and upon activation cleaves the blocking end of a linear transcription template. This enables circularization through ligation, producing a functional template in which an RNA Polymerase performs RCT, generating an RNA product that can be targeted. Casl3 detects this product and cleaves more of the linear transcription template with a blocking end alongside fluorophore quencher probes. The entire RCT/DETECTR assay may take place in a single volume (e.g., a one-pot assay) or in two or more volumes (e.g., a two-pot assay) as described herein.
[000237] FIG. 9 is a flow diagram illustrating a signal amplification process, in accordance with some embodiments. In some embodiments, the intermediate cleavage target is a transcription template, as disclosed herein.
[000238] In a first step, an activated first complex comprising a first programmable nuclease, a first guide nucleic acid, and a target nucleic acid may be formed. For example, the first guide nucleic acid may comprise a sequence that hybridizes to a segment of a target nucleic acid in a sample. For the sake of clarity, the target nucleic acid in the sample may be referred to as the primary target nucleic acid. Upon hybridization of the first guide nucleic acid to the primary target nucleic acid, transcollateral cleavage activity of the first programmable nuclease may be activated. Optionally, the activated first programmable nuclease may cleave a reporter to generate a cleavage product and detectable signal as described herein.
[000239] In a second step, the activated first complex may cleave an intermediate cleavage target. For example, the intermediate cleavage target may comprise a capped or blocked transcription template as described herein. The activated first complex may transcleave a cleavage substrate of a transcription template to release a terminal blocking moiety that blocks ligation by a ligase.
[000240] In a third step, a signal amplification reaction is set off in the presence of the cleaved intermediate cleavage target. For example, the ends of the cleaved intermediate cleavage target, having released its terminal blocking moiety, may be ligated to form a circularize template. Once the ends are ligated, the circularized nucleic acid becomes a template for rolling-circle transcription by a DNA-dependent RNA polymerase. In other words, transcription by the RNA polymerase continues multiple times around the same circular template, creating an RNA transcript comprising repeating units complementary to the circular template.
[000241] In a fourth step, an activated second complex comprising a second programmable nuclease, a second guide nucleic acid, and a second target nucleic acid may be formed. For example, the second guide nucleic acid may comprise a sequence that hybridizes to a segment of the RNA transcript generated in step 3. For the sake of clarity, the RNA transcript may be referred to as the secondary target nucleic acid in the reaction. Upon hybridization of the second guide nucleic acid to the secondary target nucleic acid, transcollateral cleavage activity of the second programmable nuclease may be activated.
[000242] In a fifth step, the activated second complex may cleave a reporter to generate a cleavage product as described herein.
[000243] In a sixth step, a signal of the detectable cleavage product is detected as
described herein.
[000244] In some instances, the first programmable nuclease and the second programmable nuclease may be the same programmable nuclease. In some instances, the first programmable nuclease and the second programmable nuclease may be different. In some instances, the first programmable nuclease and the second programmable nuclease may have the same trans-cleavage preferences (e.g., cleave the same type of reporters). In some instances, the first programmable nuclease and the second programmable nuclease may have different trans-cleavage preferences (e.g., cleave different types of reporters).
[000245] In some instances, the first guide nucleic acid and the second guide nucleic acid may be the same (e.g., both the first and second guide nucleic acids may comprise a sequence that hybridizes to (i) a segment of the primary target nucleic acid, and (ii) a segment of the secondary target nucleic acid). In some instances, the first guide nucleic acid and the second guide nucleic acid may be different.
[000246] Although the steps described with reference to FIG. 9 above show a method of signal amplification of a programmable nuclease-based detection assay, one of ordinary skill in the art will recognize many variations based on the teachings described herein. The steps may be completed in a different order. Some of the steps may be completed concurrently with one another in the same reaction volume. Some of the steps may be completed subsequent to one another in different reaction volumes. Steps may be added or deleted. Some of the steps may comprise sub-steps. Many of the steps may be repeated as often as necessary to complete the signal amplification reaction.
ILLUSTRATIVE EMBODIMENTS
[000247] The present disclosure provides the following illustrative embodiments.
[000248] Embodiment 1. A system for detecting a target nucleic acid, the system comprising a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter; wherein:
(a) the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template;
(b) the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript;
(c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate;
(d) the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product;
(e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template;
(f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript.
[000249] Embodiment 2. The system of Embodiment 1, further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
[000250] Embodiment s. The system of Embodiment 1 or 2, wherein the target nucleic acid comprises RNA.
[000251] Embodiment 4. The system of any one of Embodiments 1-3, wherein the linear transcription template is single-stranded.
[000252] Embodiment 5. The system of any one of Embodiments 1-4, wherein the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
[000253] Embodiment 6. The system of any one of Embodiments 1-5, wherein the cleavage substrate comprises one or more RNA nucleotides.
[000254] Embodiment 7. The system of Embodiment 6, wherein the one or more
RNA nucleotides of the cleavage substrate comprises at least two uracils.
[000255] Embodiment 8. The system of any one of Embodiments 1-7, wherein the
RNA polymerase is a T7 RNA polymerase.
[000256] Embodiment 9. The system of Embodiment 8, wherein the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
[000257] Embodiment 10. The system of any one of Embodiments 1-7, wherein the RNA polymerase is an SP6 RNA polymerase.
[000258] Embodiment 11. The system of any one of Embodiments 1-10, wherein the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
[000259] Embodiment 12. The system of any one of Embodiments 1-11, wherein the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
[000260] Embodiment 13. The system of any one of Embodiments 1-12, wherein the programmable nuclease is a type VI CRISPR/Cas effector protein.
[000261] Embodiment 14. The system of Embodiment 13, wherein the type VI
CRISPR/Cas effector protein is a Casl3 protein.
[000262] Embodiment 15. The system of Embodiment 14, wherein the Cast 3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
[000263] Embodiment 16. The system of any one of Embodiments 1-15, wherein
(a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system.
[000264] Embodiment 17. The system of any one of Embodiments 1-15, wherein the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
[000265] Embodiment 18. The system of any one of Embodiments 1-17, wherein the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated complex.
[000266] Embodiment 19. The system of Embodiment 18, wherein the detectable cleavage product comprises the quantum dot.
[000267] Embodiment 20. The system of any one of Embodiments 1-19, wherein the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
[000268] Embodiment 21. The system of Embodiment 20, wherein the detection moiety comprises a fluorescent label, a quencher, or an enzyme.
[000269] Embodiment 22. The system of Embodiment 21, wherein the detection moiety comprises an enzyme that catalyzes a colorimetric reaction.
[000270] Embodiment 23. The system of any one of Embodiments 1-22, further comprising a plurality of reaction volumes, wherein
(a) each reaction volume comprises the programmable nuclease, a different non- naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter;
(b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and
(c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
[000271] Embodiment 24. A method for detecting a target nucleic acid in a sample, the method comprising:
(a) contacting the system of any one of Embodiments 1-23 with the sample; and
(b) detecting the detectable cleavage product.
[000272] Embodiment 25. A method for detecting a target nucleic acid in a sample, the method comprising the following steps in a single reaction volume:
(a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease;
(b) cleaving a linear transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product;
(c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template;
(d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript;
(e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a second non-naturally occurring guide nucleic acid that hybridizes to a portion of the RNA transcript, thereby activating the second programmable nuclease;
(f) cleaving reporters with the activated first or second programmable nuclease to produce detectable cleavage products; and
(g) detecting the detectable cleavage products.
[000273] Embodiment 26. The method of Embodiment 25, wherein (i) the first programmable nuclease and the second programmable nuclease are the same, and/or (ii) the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same.
[000274] Embodiment 27. The method of Embodiment 25 or 26, wherein the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template.
[000275] Embodiment 28. The method of any one of Embodiments 25-27, wherein the target nucleic acid comprises RNA.
[000276] Embodiment 29. The method of any one of Embodiments 25-28, wherein the linear transcription template is single-stranded.
[000277] Embodiment 30. The method of any one of Embodiments 25-29, wherein the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
[000278] Embodiment 31. The method of any one of Embodiments 25-30, wherein cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides.
[000279] Embodiment 32. The method of Embodiment 31, wherein the one or more
RNA nucleotides of the cleavage substrate comprises at least two uracils.
[000280] Embodiment 33. The method of any one of Embodiments 25-32, wherein the DNA-dependent RNA polymerase is a T7 RNA polymerase.
[000281] Embodiment 34. The method of Embodiment 33, wherein the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
[000282] Embodiment 35. The method of any one of Embodiments 25-32, wherein the DNA-dependent RNA polymerase is an SP6 RNA polymerase.
[000283] Embodiment 36. The method of any one of Embodiments 25-35, wherein
the first programmable nuclease, second programmable nuclease, DNA-dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
[000284] Embodiment 37. The method of any one of Embodiments 25-36, wherein the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
[000285] Embodiment 38. The method of any one of Embodiments 25-37, wherein the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein.
[000286] Embodiment 39. The method of Embodiment 38, wherein the type VI CRISPR/Cas effector protein is a Casl3 protein.
[000287] Embodiment 40. The method of Embodiment 39, wherein the Casl3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
[000288] Embodiment 41. The method of any one of Embodiments 25-40, wherein
(a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume.
[000289] Embodiment 42. The method of any one of Embodiments 25-41, wherein the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume.
[000290] Embodiment 43. The method of any one of Embodiments 25-42, wherein the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
[000291] Embodiment 44. The method of any one of Embodiments 25-43, further comprising repeating the method in parallel in each of a plurality of single reaction volumes, wherein each of the plurality of single reaction volumes comprises a different first non- naturally occurring guide nucleic acid.
[000292] Embodiment 45. The method of Embodiment 44, wherein each of the plurality of single reaction volumes comprises a different linear transcription template.
[000293] Embodiment 46. A reporter complex comprising a quantum dot conjugated to a plurality of reporter oligonucleotides, wherein each of the reporter oligonucleotides comprises a secondary fluorophore or a quencher.
EXAMPLES
[000294] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1: Low level detection of target nucleic acids using RCT and DETECTR
[000295] A modular assay using rolling circle transcription (RCT) was developed to detect target nucleic acids of interest present at low levels in a sample in a programmable nuclease-based detection assay. In general, RCT can have specific RNAPs initiate transcription of a circular ssDNA probe in a sequence independent manner. By changing the guide nucleic acid alone, CRISPR-driven target nucleic acid detection can be conducted for a variety of different target nucleic acids with relative ease. Rapid detection via an isothermal device for heat modulation requirements were incorporated in this example. A Cas protein, such as Casl3, can control RNA cleavage internally to a larger nucleotide, and the RCT/DETECTR assay described herein detects low levels of target nucleic acid RNA using an RNA polymerase in combination with a programmable nuclease (FIG. 7).
[000296] RCT and DETECTR can be performed in the same reaction volume
[000297] RCT and DETECTR were performed in a single reaction, without requiring a T7 promoter sequence or a double-stranded nature for the T7 RNAPs to initiate transcription (FIG. 1). Briefly, a Cas 13a variant (SEQ ID NO: 21) was pre-complexed with its guide RNA at 37°C in IsoAmp (acetate) Buffer (NEB). The lx concentration of proteins was 40 nM and the final concentration of guide nucleic acid was 40 nM. 5 ul of the complexing reaction was combined with 5ul of T7 polymerase master mix (comprising T7 buffer, T7 RNAP, and 25mM NTPs or OmM NTPs “no NTP” condition), 1 ul of trigger oligonucleotide or water, and 9 ul of RCT master mix (comprising IsoAmp Buffer (NEB) and dumbbell 7 or dumbbell 12 as the
circular template, 40nM final concentration), and an FQ-RNA reporter (200 nM final concentration). For each reaction, RCT/DETECTR was run at 37°C for 60 minutes. Trans cleavage activity of the Cast 3a variant was detected by fluorescence signal upon cleavage of the FQ reporter in the RCT/DETECTR reaction.
[000298] FIG. 1 is a graphical representation of fluorescence-based signal amplification for two circular ssDNAs (“Dumbbell 7” and “Dumbbell 12”) which each contain the antisense sequence for a Casl3 crRNA target. The “Trigger” condition includes a second oligonucleotide used to double strand a portion of the circular ssDNA template. The top line in both Dumbbell 7 and Dumbbell 12 graphs represent the “No trigger” condition, the second, slightly lower line represents the “Trigger” condition, and the third line along the x-axis represents the “No NTPs”, meaning no functional RCT was included in the reaction condition. Strong fluorescence signal was observed in both the Trigger and No Trigger conditions for both dumbbell targets, indicating that RCT and DETECTR can be performed in the same reaction volume and that the presence of a double-stranding trigger oligonucleotide was unnecessary for T7-based transcription under the conditions tested. Interestingly, it was surprising to find that the circular ssDNA template did not require a T7 promoter sequence or a double stranded nature in order for the T7 RNAP to initiate transcription.
[000299] Limit of detection of circular template in a one-pot RCT/DETECTR reaction [000300] The one-pot RCT/DETECTR assay was performed as described above with varying concentrations (InM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, or 0 fM) of Dumbbell 7 or 12 “target” added to the reaction mix. Results were evaluated using a Q5 thermocycler to establish detection sensitivities of the RCT/DETECTR assay for the circular template in order to determine how much of a linear transcription would need to be unblocked and ligated in order to generate a distinguishable signal. Illustrative results are shown in FIG. 2. For dumbbell 7, the LOD of the RCT/DETECTR reaction was about 100 pM of targetable circular template under the conditions tested. For dumbbell 12, the LOD of the RCT/DETECTR reaction was about 10 pM of targetable circular template under the conditions tested.
[000301] T7 RNA polymerase can initiate RCT/DETECTR with both circular and linear templates
[000302] The one-pot RCT/DETECTR assay was performed as described above 40 nM final concentration of Dumbbell 7 target in linear or circularized form. While T7 transcription from a circular template was stronger, both linear and circular templates showed efficacy in initiating the RCT/DETECTR reaction in the absence of the T7 promoter under the conditions
tested (see FIG. 3).
[000303] Effects of additives on RCT/DETECTR activity
[000304] Various additives, including betaine and DMSO, were included in a one-pot RCT/DETECTR assay with 40 nM circular Dumbbell 7 target, performed as described above, and were demonstrated to still produce quantitatively analyzable results under the conditions tested (FIG. 4).
[000305] RCT/DETECTR with different RNAPs
[000306] Test reactions in the one-pot RCT/DETECTR assays were conducted with different RNAPs including SP6, which have not been used previously for running RCT (FIG. 5). The one-pot RCT/DETECTR reactions were run as described above with 40nM T7 RNAP, T3 RNAP, or SP6 as the RNAP for RCT and either circular (solid lines) or linearized (dashed lines) Dumbbell 12 target. SP6 showed the best separation between circular and linear templates under the conditions tested.
[000307] Cast 3a cleaves internal RNA nucleotides
[000308] An assay for assessing Cas 13 -based cleavage of RNA internal to a larger oligonucleotide was also developed in order to assess the ability of Cas 13 nucleases to cleave specific RNA nucleotide(s) in a hybrid DNA/RNA oligonucleotide. For a Cas 13 variant (SEQ ID NO: 21), various oligonucleotides having uracil and/or adenine residue(s) internal to a DNA oligo, with the RNA residues forming a bulge in the DNA duplex, were tested to determine whether a Cas 13 could be used to initiate the RCT/DETECTR reaction described in FIG. 7. Briefly, a Cas 13a variant (SEQ ID NO: 21) was pre-complexed with a guide RNA at 37°C in MBufferl. The lx concentration of proteins was 5 nM and the final concentration of guide nucleic acid was 5 nM. Duplexes were prepared by annealing the bulge strand to the target strand. 5 ul of the complexing reaction was combined with 10 uL of the duplex, or 8 uL of duplex and 2 uL of target, and incubated for 30 minutes at 37°C before being run on a PAGE gel. In all cases, the final concentration of FAM-oligo duplex was about luM.
[000309] FIG. 6 shows a gel image for an assay demonstrating the cleavage activity of the activated Cas 13 variant on poly-uracil tracts that are internal to a larger DNA oligonucleotide. The columns show illustrative results for: 1) FAM-oligo only; 2) FAM-oligo duplex with no bulge (TGAGCGAGGACTGCAGCGTAGACG), target, and programmable nuclease complex; 3) FAM-oligo duplex with a 5’-UU-3’ (2U) bulge
(TGAGCGAGGACTrUrUGCAGCGTAGACG), programmable nuclease complex, and no target control (NTC); 4) FAM-oligo duplex with a 2U bulge, target, and programmable nuclease complex; 5) FAM-oligo duplex with 5’-UUUUU-3’ (5U) bulge
(TGAGCGAGGACTrUrUrUrUrUGCAGCGTAGACG), programmable nuclease complex, and NTC; 6) FAM-oligo duplex with 5U bulge, target, and programmable nuclease complex;
7) FAM-oligo duplex with 5’-UUAUU-3’ (2UA2U) bulge
(TGAGCGAGGACTrUrUrArUrUGCAGCGTAGACG), programmable nuclease complex, and NTC; 8) FAM-oligo duplex with 2UA2U bulge, target, and programmable nuclease complex; 9) FAM-oligo duplex with 5’-TUUAUUT-3’ (T2UA2UT) bulge (TGAGCGAGGACTTrUrUrArUrUTGCAGCGTAGACG), programmable nuclease complex, and NTC; 10) FAM-oligo duplex with T2UA2UT bulge, target, and programmable nuclease complex; (11) FAM-oligo duplex with 5’-TTUAUTT-3’ (2TUAU2T) bulge (TGAGCGAGGACTTrUrUrArUrUTGCAGCGTAGACG), programmable nuclease complex, and NTC; and (12) FAM-oligo duplex with 2TUAU2T, +target, and programmable nuclease complex. It was found that the Cast 3 variant was able to cleave poly-uracil tracts contained within a DNA duplex, and that a tract as short as 2 uracils could be cleaved, albeit at a lower efficiency than larger uracil tracts under the conditions tested. Casl3 is therefore an attractive candidate for us in an RCT/DETECTR assay as described in FIG. 7 to unblock an RNA-capped linear transcription template and activate the RCT/DETECTR reaction upon recognition of a target sequence as described herein. Additionally, design of an RCT template having an RNA-based blocker for use in the method described in FIG. 7 may therefore be optimized by adjusting the length and/or composition of the RNA blocker.
[000310] T4 ligase can initiate an RCT/DETECTR with unblocked linear transcription template.
[000311] An assay for assessing the ability of a ligase to initiate an RCT/DETECTR signal amplification reaction comprising subsequent transcription and reporter cleavage was performed. Briefly, a Cast 3a variant (SEQ ID NO: 5) was pre-complexed with its guide RNA at 37°C in IsoAmp (acetate) Buffer (NEB). The lx concentration of proteins was 40 nM and the final concentration of guide nucleic acid was 40 nM. 5 ul of the complexing reaction was combined with 5ul of T7 polymerase master mix (comprising T7 buffer, T7 RNAP, and 25mM NTPs), and 10 ul of RCT master mix (comprising IsoAmp Buffer (NEB), T4 PNK, T4 ligase or no T4 ligase, unblocked dumbbell 7 as the circular template (500fM or OfM), and an FQ- RNA reporter (200 nM final concentration). For each reaction, RCT/DETECTR was run at 37°C for 60 minutes. Trans cleavage activity of the Casl3a variant was detected by fluorescence signal upon cleavage of the FQ reporter in the RCT/DETECTR reaction.
[000312] FIG. 10 is an illustrative graphical representation of the capacity for a ligase to initiate an RCT/DETECTR reaction and fluorescence emission using an unblocked linear
template. No signal was generated in reactions lacking either ligase or the circular template.
Example 2: Quantum Dot reporters for fluorescent readout of DETECTR
[000313] An assay using Quantum Dots (QDs) together with a nucleic acid: fluorophore or nucleic acickquencher as nanoparticle-based reporters was developed to increase the sensitivity of direct target detection. The sensitivity is increased in one of two capacities. First, the QDs themselves provide signal amplification. Second, each QD provides an increased number of “reporter” molecules.
[000314] The development of the reporters for the DETECTR assay is based on utilizing QDs as cores, with a layer of oligonucleotides conjugated to the shell of the QD from one end, and the other end is conjugated to either (i) a fluorescence quencher molecule, such that when the oligonucleotide is cleaved in a DETECTR assay, there is an increase in fluorescence; or (ii) other fluorophores, such that when the oligonucleotide is cleaved in a DETECTR assay, there is a shift in color that can be differentiated in a separate detection channel. The conjugation of nucleic acid to the QD can be either DNA, RNA, or a combination of both to facilitate multiplexing of different Cas activities. Different combinations of QDs, oligonucleotides, fluorophores, and quenchers enable several possibilities for a multiplexing DETECTR system. [000315] FIGS. 8A-8B provide illustrative examples of QD-based reporters. In FIG. 8A, a first reporter type is depicted, wherein the QD is conjugated to one end of a single stranded oligonucleotide, and the other end is conjugated to the aforementioned fluorescence quencher molecule. Here, Cas nuclease cleavage of the linker oligonucleotide results in increased fluorescence. The oligonucleotide can be any type of nucleic acid, and the QD can be covered in a single oligonucleotide type (left) or a mixture of different oligonucleotides (right). In FIG. 8B, a second reporter type is depicted, wherein the end of the single stranded oligonucleotide opposite the QD is conjugated to a secondary fluorophore for use as a FRET donor-acceptor pair instead of a fluorescence quencher molecule. Here, Cas nuclease cleavage of the linker oligonucleotide results in a color shift instead of an increased fluorescence, though the linker again can be any type of nucleic acid and the QD can be covered in a single type (left) or mixture of types of oligonucleotides (right). Further, the fluorophores conjugated to each oligonucleotide can be of a single type or multiple types, enabling a multiplexing assay with a readout of different color shifts. The secondary fluorophore can also be another QD.
[000316] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are
provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein can be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims
1. A system for detecting a target nucleic acid, the system comprising a reaction volume comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, a transcription template, an RNA polymerase, a ligase, and a reporter; wherein:
(a) the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of the target nucleic acid, and (ii) a segment of an RNA transcript of the transcription template;
(b) the programmable nuclease and the non-naturally-occurring guide nucleic acid form a complex that is activated upon binding (i) the target nucleic acid, or (ii) the RNA transcript;
(c) the transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate;
(d) the activated complex is effective to (i) cleave the cleavage substrate and release the terminal blocking moiety, and (ii) cleave the reporter to produce a detectable cleavage product;
(e) the ligase is effective to ligate ends of the linear transcription template upon release of the terminal blocking moiety to form a circularized template;
(f) the RNA polymerase is a DNA-dependent RNA polymerase that is effective to transcribe the circularized template to form the RNA transcript.
2. The system of claim 1, further comprising a bridge oligonucleotide, wherein the bridge oligonucleotide (i) comprises a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template; (ii) comprises a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template; and (iii) is effective to stabilize the ends of the linear transcription template in proximity to each other upon release of the terminal blocking moiety.
3. The system of claim 1, wherein the target nucleic acid comprises RNA.
4. The system of claim 1, wherein the linear transcription template is singlestranded.
5. The system of claim 1, wherein the terminal blocking moiety comprises an RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
6. The system of claim 1, wherein the cleavage substrate comprises one or more RNA nucleotides.
7. The system of claim 6, wherein the one or more RNA nucleotides of the cleavage substrate comprises at least two uracils.
8. The system of claim 1, wherein the RNA polymerase is a T7 RNA polymerase.
9. The system of claim 8, wherein the linear transcription template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
10. The system of claim 1, wherein the RNA polymerase is an SP6 RNA polymerase.
11. The system of claim 1, wherein the programmable nuclease, RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
12. The system of claim 1, wherein the linear transcription template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
13. The system of claim 1, wherein the programmable nuclease is a type VI CRISPR/Cas effector protein.
14. The system of claim 13, wherein the type VI CRISPR/Cas effector protein is a Cast 3 protein.
15. The system of claim 14, wherein the Casl3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
16. The system of claim 1, wherein (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target
nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system.
17. The system of claim 1, wherein the system is effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid.
18. The system of claim 1, wherein the reporter comprises a nucleic acid cleavage substrate conjugated to (i) a quantum dot and (ii) a secondary fluor ophore or a quencher; wherein the nucleic acid cleavage substrate is a substrate for the activated complex.
19. The system of claim 18, wherein the detectable cleavage product comprises the quantum dot.
20. The system of claim 1, wherein the detectable cleavage product comprises a portion of the reporter comprising a detection moiety.
21. The system of claim 20, wherein the detection moiety comprises a fluorescent label, a quencher, or an enzyme.
22. The system of claim 21, wherein the detection moiety comprises an enzyme that catalyzes a colorimetric reaction.
23. The system of claim 1, further comprising a plurality of reaction volumes, wherein
(a) each reaction volume comprises the programmable nuclease, a different non-naturally occurring guide nucleic acid, a different linear transcription template, the RNA polymerase, the ligase, and the reporter;
(b) each different non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to (i) a segment of a different target nucleic acid, and (ii) a segment of an RNA transcript of the corresponding transcription template; and
(c) each different transcription template is a linear polynucleotide comprising DNA, a terminal blocking moiety that blocks ligation by the ligase, and a cleavage substrate for the programmable nuclease.
24. A method for detecting a target nucleic acid in a sample, the method comprising:
(a) contacting the system of any one of claims 1-23 with the sample; and
(b) detecting the detectable cleavage product.
25. A method for detecting a target nucleic acid in a sample, the method comprising the following steps in a single reaction volume:
(a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the first programmable nuclease;
(b) cleaving a linear transcription template with the activated first programmable nuclease to release a terminal blocking moiety that blocks formation of a ligation product;
(c) ligating ends of the cleaved linear transcription template with a ligase to form a circularized template;
(d) transcribing the circularized template with a DNA-dependent RNA polymerase to form an RNA transcript;
(e) forming a second complex comprising the RNA transcript, a second programmable nuclease, and a second non-naturally occurring guide nucleic acid that hybridizes to a portion of the RNA transcript, thereby activating the second programmable nuclease;
(f) cleaving reporters with the activated first or second programmable nuclease to produce detectable cleavage products; and
(g) detecting the detectable cleavage products.
26. The method of claim 25, wherein (i) the first programmable nuclease and the second programmable nuclease are the same, and/or (ii) the first non-naturally occurring guide nucleic acid and the second non-naturally occurring guide nucleic acid are the same.
27. The method of claim 25, wherein the ligating further comprises stabilizing the ends of the cleaved linear transcription template in proximity to each other by hybridization
to a bridge oligonucleotide, and further wherein the bridge oligonucleotide comprises (i) a 5’ sequence that is complementary to a 5’ sequence of the linear transcription template, and (ii) a 3’ sequence that is complementary to a 3’ sequence of the linear transcription template.
28. The method of claim 25, wherein the target nucleic acid comprises RNA.
29. The method of claim 25, wherein the linear transcription template is singlestranded.
30. The method of claim 25, wherein the terminal blocking moiety comprises an
RNA nucleotide, a dideoxy nucleotide, a 5’ terminal nucleotide lacking a 5’ phosphate, or a 3’ terminal nucleotide lacking a 3’ hydroxyl; optionally wherein the dideoxy nucleotide is a dideoxy cytosine.
31. The method of claim 25, wherein cleaving a linear transcription template comprises cleavage at a cleavage substrate comprising one or more RNA nucleotides.
32. The method of claim 31, wherein the one or more RNA nucleotides of the cleavage substrate comprises at least two uracils.
33. The method of claim 25, wherein the DNA-dependent RNA polymerase is a T7 RNA polymerase.
34. The method of claim 33, wherein the circularized template does not comprise a T7 promoter sequence of TAATACGACTCACTATAG (SEQ ID NO: 77).
35. The method of claim 25, wherein the DNA-dependent RNA polymerase is an SP6 RNA polymerase.
36. The method of claim 25, wherein the first programmable nuclease, second programmable nuclease, DNA-dependent RNA polymerase, and ligase are all active at a temperature of about 20 °C to about 40 °C.
37. The method of claim 25, wherein the circularized template is (i) about 40 to about 100 nucleotides in length, (ii) about 50 to about 75 nucleotides in length, or (iii) about 60 nucleotides in length.
38. The method of claim 25, wherein the first programmable nuclease and/or the second programmable nuclease is a type VI CRISPR/Cas effector protein.
39. The method of claim 38, wherein the type VI CRISPR/Cas effector protein is a Cast 3 protein.
40. The method of claim 39, wherein the Casl3 protein comprises Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.
41. The method of claim 25, wherein (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the single reaction volume; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the single reaction volume.
42. The method of claim 25, wherein the detectable cleavage products are detectable in less than 45 minutes after adding the sample to the single reaction volume.
43. The method of claim 25, wherein the reporters comprise nucleic acid cleavage substrates conjugated to (i) a quantum dot and (ii) a secondary fluorophore or a quencher; wherein the nucleic acid cleavage substrates are substrates for the activated first or second programmable nuclease.
44. The method of any one of claims 25-43, further comprising repeating the method in parallel in each of a plurality of single reaction volumes, wherein each of the plurality of single reaction volumes comprises a different first non-naturally occurring guide nucleic acid.
45. The method of claim 44, wherein each of the plurality of single reaction volumes comprises a different linear transcription template.
46. A reporter complex comprising a quantum dot conjugated to a plurality of reporter oligonucleotides, wherein each of the reporter oligonucleotides comprises a secondary fluorophore or a quencher.
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