Exonuclease-linked real-time endonuclease activity assay

Abstract

The present invention includes rapid in vitro methods for assessing the activity of endonucleases, and substrates therefor. Compositions, diagnostic methods, and kits are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 272,091, filed October 26, 2021.

[0002] The present invention relates to the field of nucleic acid modifying enzymes, and more particularly to the field of developing and testing active endonucleases.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH none.

[0004] Sequence Listing Placeholder. [Background technology]

[0005] Endonucleases are enzymes widely used in laboratory practice and the molecular diagnostics industry. The process of designing and isolating new and improved endonucleases requires a fast and simple method for assessing endonuclease activity. The most widely used endonuclease assays are cumbersome and have low throughput. Furthermore, commonly used methods are not quantitative. There is no established means to measure differences in activity between different sources, variants, or lots of endonucleases. Similarly, there is no easy or rapid means to evaluate different targets so that the specificity of an endonuclease can be accurately determined. Summary of the Invention [Problem to be solved by the invention]

[0006] Many current state-of-the-art endonuclease assays are unsuitable for use with crude cell lysates or partially purified protein preparations because host nucleases rapidly degrade target DNA. There is an unmet need for rapid endonuclease assays that function on crude lysates and semi-crude elution fractions from early steps in the purification process. Such assays would aid efforts to improve enzyme purification and production, accelerating the overall enzyme engineering process. [Means for solving the problem]

[0007] The present invention provides methods, compositions, and kits for assessing endonuclease activity. The present invention utilizes double-stranded end-protected nucleic acid substrates labeled with a reporter fluorophore and a quencher fluorophore. Only after the endonuclease cleaves the substrate can the exonuclease hydrolyze the substrate, allowing fluorescence to occur.

[0008] In one embodiment, the present invention provides a nucleic acid substrate for detecting endonuclease activity, comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure in at least one nucleic acid strand that inhibits exonucleolytic cleavage of the substrate; and a recognition sequence for the endonuclease. The structure that inhibits exonucleolytic cleavage of the substrate is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. The exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the acceptor fluorophore is a quencher fluorophore. The donor fluorophore and acceptor fluorophore can be located on the same strand of the substrate or on different strands of the substrate, separated by 1 to 12 nucleotides. In some embodiments, the substrate is single-stranded. In some embodiments, one of the donor and acceptor fluorophores is located at or near the 5' end of the substrate.In some embodiments, the donor fluorophore is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), coumarin dyes, Alexa Fluor dyes, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, Texas Red, and BODIPY® dyes. In some embodiments, the acceptor fluorophore is selected from the group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL (4-[[4-(dimethylamino)-phenyl]-azo]-benzoic acid), Cy5 and Cy5.5, anthraquinone dyes, nitrothiazole dyes, nitroimidazole dyes, LC-Red 610, LC-Red 640, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ, Iowa Black RQ, HEX (hexachloro-fluorescein), TET (tetrachloro-fluorescein), JOE (5'-dichloro-dimethoxy-fluorescein), BODIPY® dyes, Eclipse quenchers (4-[[2-chloro-4-nitro-phenyl]-azo]-aniline, BHQ-1 ([(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline), BHQ-2 ([(4-(1-nitro In some embodiments, the endonuclease is selected from the group consisting of exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease.In some embodiments, the endonuclease is a nucleic acid-guided endonuclease, such as a CRISPR class I (CASCADE) endonuclease. In some embodiments, the substrate comprises a protospacer adjacent motif (PAM). In some embodiments, the PAM consists of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR class II endonuclease. In some embodiments, the endonuclease is a CRISPR Cas9 endonuclease. In some embodiments, the endonuclease is a CRISPR Cas12a endonuclease. In some embodiments, the PAM consists of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3'. In some embodiments, the PAM consists of a sequence selected from 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region in a substrate. In some embodiments, the NATNA is capable of interacting with the endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides.In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, endoribonucleases selected from RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and restriction endonucleases. In some embodiments, the structure that inhibits exonucleolytic cleavage of the substrate is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more (five or more) phosphorothioate linkages. In some embodiments, the endonuclease is a deoxyribonuclease and the substrate contains DNA. In some embodiments, the endonuclease is a ribonuclease and the substrate contains RNA. In some embodiments, the ribonuclease is selected from a ribozyme, a hammerhead ribozyme, a DNAzyme, a PNAzyme, or an engineered endoribonuclease.

[0009] In one embodiment, the present invention is a composition for detecting endonuclease activity, comprising the above-described nucleic acid substrate, an exonuclease, and optionally an endonuclease. In some embodiments, the endonuclease is a nickase. In some embodiments, the exonuclease is selected from exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease. In some embodiments, the exonuclease is capable of initiating hydrolysis from a nick. In some embodiments, the exonuclease is selected from T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease III, and exonuclease Bal31. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3' 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide.In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of a substrate. In some embodiments, the NATNA is capable of interacting with an endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, an endoribonuclease selected from RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and a restriction endonuclease. In some embodiments, the structure that inhibits exonucleolytic cleavage of the substrate is selected from a hairpin, a strand overhang, and a nucleic acid modification, eg, one or more (five or more) phosphorothioate linkages.

[0010] In one embodiment, the present invention provides a method for detecting endonuclease activity, comprising contacting an endonuclease and an exonuclease with a reaction mixture containing a nucleic acid substrate, the nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure in at least one strand that inhibits exonucleolytic cleavage of the substrate; and a recognition sequence for the endonuclease; and measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates endonuclease activity. In some embodiments, the at least one structure that inhibits exonucleolytic cleavage of the substrate is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the reaction mixture is contacted with the endonuclease and the exonuclease simultaneously. In some embodiments, the reaction mixture is contacted with the endonuclease first, followed by the exonuclease. In some embodiments, no purification step is performed between contacting the reaction mixture with the endonuclease and contacting the reaction mixture with the exonuclease. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease.In some embodiments, the nucleic acid-guided endonuclease is a CRISPR class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3' 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of the substrate. In some embodiments, the NATNA is capable of interacting with an endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides. In some embodiments, the contacting comprises contacting a series of reaction mixtures comprising the same NATNA with a series of different endonucleases. In some embodiments, the contacting comprises contacting a series of reaction mixtures comprising the same endonuclease with a series of different NATNAs. In some embodiments, the contacting comprises contacting a series of reaction mixtures comprising the same components under different reaction conditions.In some embodiments, the contacting comprises contacting a series of reaction mixtures containing the same components with different isolates of the endonuclease. In some embodiments, the contacting comprises contacting a series of reaction mixtures containing the same endonuclease with a series of different nucleic acid substrates containing different sequences. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonucleases, Argonaute endonucleases, Arcus endonucleases, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and restriction endonucleases. In some embodiments, the structure that inhibits exonucleolytic cleavage of the substrate is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more (five or more) phosphorothioate linkages. In some embodiments, the nucleic acid substrate or endonuclease is in an unpurified form.

[0011] In one embodiment, the present invention provides a kit for detecting endonuclease activity, comprising: a nucleic acid substrate, the nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure in at least one strand that inhibits exonucleolytic cleavage of the substrate; and a recognition sequence for the endonuclease; and an exonuclease. In some embodiments, the at least one structure that inhibits exonucleolytic cleavage of the substrate is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the endonuclease tested is a nucleic acid-guided endonuclease. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'.In some embodiments, the nucleic acid-guided endonuclease is CRISPR Cas9 endonuclease or CRISPR Cas12a endonuclease, and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the kit further comprises a nucleic acid targeting nucleic acid (NATNA) capable of forming a complex with the endonuclease to be tested. In some embodiments, the NATNA is a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of the substrate. In some embodiments, NATNA can interact with endonucleases. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonucleases, Argonaute endonucleases, Arcus endonucleases, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and restriction endonucleases. In some embodiments, the structure that inhibits exonuclease cleavage of the substrate is selected from hairpins, strand overhangs, and nucleic acid modifications, such as one or more (five or more) phosphorothioate bonds.

[0012] In one embodiment, the present invention provides a device for detecting the activity of an endonuclease using a substrate as defined in claim 1, comprising: a reaction chamber for carrying out an enzymatic reaction and a fluorescence detector.

[0013] In one embodiment, the present invention provides a method for detecting the presence of a target nucleic acid in a sample, the method comprising: contacting the sample with a reaction mixture comprising an endonuclease, an exonuclease, and a nucleic acid probe capable of hybridizing to the target nucleic acid, the probe comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage of the probe by the exonuclease; and a recognition sequence for the endonuclease; and measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample. In some embodiments, the at least one structure that inhibits cleavage of the probe by the exonuclease is selected from a structure at each 3' end that inhibits cleavage by a 3'-5' exonuclease, a structure at each 5' end that inhibits cleavage by a 5'-3' exonuclease, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the target nucleic acid is selected from a sequence characteristic of a bacterium, a sequence characteristic of a virus, a sequence characteristic of a parasite, and a patient sequence characteristic of a disease or condition of the patient. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease.In some embodiments, the endonuclease is a CRISPR class I (CASCADE) endonuclease and the probe comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the probe comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide CRISPR guide RNAs. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of the probe. In some embodiments, the NATNA is capable of interacting with the endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides.In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and endoribonucleases selected from restriction endonucleases. In some embodiments, the structure that inhibits exonuclease cleavage of the probe is selected from hairpins, strand overhangs, and nucleic acid modifications, such as one or more (five or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude preparation of nucleic acid.

[0014] In one embodiment, the present invention provides a method for detecting the presence of two or more target nucleic acids in a sample, comprising contacting the sample with a reaction mixture comprising an endonuclease, an exonuclease, and two or more nucleic acid probes, each capable of hybridizing to two or more target nucleic acids, each probe comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage of the probe by the exonuclease; and a recognition sequence for the endonuclease; and measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample. In some embodiments, the two or more nucleic acid probes comprise at least one different fluorophore. In some embodiments, all of the two or more nucleic acid probes comprise the same fluorophore. In some embodiments, the at least one structure that inhibits cleavage of the probe by the exonuclease is selected from a structure at each 3' end that inhibits cleavage by a 3'-5' exonuclease, a structure at each 5' end that inhibits cleavage by a 5'-3' exonuclease, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the two or more target nucleic acids are selected from sequences characteristic of bacteria, sequences characteristic of viruses, sequences characteristic of parasites, and patient sequences characteristic of a patient's disease or condition. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease.In some embodiments, the endonuclease is a CRISPR class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, at least one NATNA is used for each of two or more probes, and each NATNA comprises a targeting region that can hybridize to a region of at least one probe.In some embodiments, the NATNA can interact with an endonuclease.In some embodiments, the NATNA comprises DNA and RNA nucleotides.In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and endoribonucleases selected from restriction endonucleases. In some embodiments, the structure that inhibits exonuclease cleavage of the probe is selected from hairpins, strand overhangs, and nucleic acid modifications, such as one or more (five or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude preparation of nucleic acid.

[0015] In one embodiment, the present invention provides a method for detecting the presence of a target nucleic acid in a sample, comprising: binding to the target nucleic acid in the sample: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits exonucleolytic cleavage; and a recognition sequence for the endonuclease to form a modified nucleic acid; and contacting the sample with the endonuclease and the exonuclease; measuring the fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample. In some embodiments, the at least one structure that inhibits exonucleolytic cleavage is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the binding is via ligation of an adapter comprising a donor fluorophore, an acceptor fluorophore, and a structure that inhibits cleavage by the exonuclease. In some embodiments, the target nucleic acid is amplified by PCR prior to the binding. In some embodiments, the binding is via one or more rounds of extension using an amplification primer that includes a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; a structure that inhibits exonucleolytic cleavage.In some embodiments, the target nucleic acid is selected from a sequence characteristic of bacteria, a sequence characteristic of viruses, a sequence characteristic of parasites, and a patient sequence characteristic of a disease or condition of the patient. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease. In some embodiments, the endonuclease is a CRISPR class I (CASCADE) endonuclease, and the target nucleic acid or modified target nucleic acid comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the target nucleic acid or modified target nucleic acid comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide CRISPR guide RNAs. In some embodiments, the NATNA comprises a crRNA or tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to the target nucleic acid or modified target nucleic acid. In some embodiments, the NATNA is capable of interacting with an endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides.In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and restriction endonucleases. In some embodiments, the structure that inhibits exonucleolytic cleavage is selected from hairpins, strand overhangs, and nucleic acid modifications, such as one or more (five or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude preparation of nucleic acid.

[0016] In one embodiment, the present invention provides a kit for performing a diagnostic procedure according to the method of claim 116, comprising: an endonuclease; and a nucleic acid probe capable of hybridizing to a target nucleic acid, the probe comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair, at least one structure that inhibits exonucleolytic cleavage of the probe; and a recognition sequence for the endonuclease; and measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample, the target nucleic acid being selected from a sequence characteristic of a bacterium, a sequence characteristic of a virus, a sequence characteristic of a parasite, and a patient sequence characteristic of a patient's disease or condition. In some embodiments, the at least one structure that inhibits exonucleolytic cleavage of the probe is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. In some embodiments, the kit further comprises an exonuclease selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease.In some embodiments, the nucleic acid-guided endonuclease is a CRISPR class I (CASCADE) endonuclease and the probe comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the probe comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease is a CRISPR endonuclease complexed with a nucleic acid targeting nucleic acid (NATNA), and the NATNA is a CRISPR guide RNA selected from single-guide and dual-guide CRISPR guide RNAs. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA is capable of interacting with the endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nuclease (ZFN), ZFN conjugated to Fok I, transcription activator-like effector nuclease (TALEN), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and restriction endonuclease.In some embodiments, the structure that inhibits exonucleolytic cleavage is selected from a hairpin, a strand overhang, and a nucleic acid modification, eg, one or more (five or more) phosphorothioate linkages.

[0017] In one embodiment, the present invention provides a kit for detecting the presence of a target nucleic acid in a sample, the kit comprising one or more oligonucleotides capable of binding to the target nucleic acid to form a modified nucleic acid, the oligonucleotides comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits exonucleolytic cleavage; the modified nucleic acid comprises a recognition sequence for an endonuclease and the endonuclease, and the target nucleic acid is selected from a sequence characteristic of a bacterium, a sequence characteristic of a virus, a sequence characteristic of a parasite, and a patient sequence characteristic of a patient's disease or condition. In some embodiments, the at least one structure that inhibits exonucleolytic cleavage is selected from a structure at each 3' end that inhibits 3'-5' exonucleolytic cleavage, a structure at each 5' end that inhibits 5'-3' exonucleolytic cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the binding is via ligation of an oligonucleotide to the target nucleic acid, and the kit optionally includes a ligase. In some embodiments, the binding is via one or more rounds of extension using oligonucleotides that act as amplification primers, and the kit optionally includes reagents for performing the amplification. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease.In some embodiments, the endonuclease is a CRISPR class I (CASCADE) endonuclease, and the target nucleic acid or modified target nucleic acid comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the target nucleic acid or modified target nucleic acid comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide CRISPR guide RNAs. In some embodiments, the NATNA comprises a crRNA or tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to the target nucleic acid or modified target nucleic acid. In some embodiments, the NATNA is capable of interacting with an endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides.In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, an endoribonuclease selected from RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and a restriction endonuclease. In some embodiments, the structure that inhibits exonucleolytic cleavage is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more (five or more) phosphorothioate linkages.

[0018] In one embodiment, the present invention provides a method for optimizing an endonuclease digestion reaction, comprising: preparing a series of reaction mixtures using an exonuclease and a nucleic acid substrate, the nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure in at least one strand that inhibits exonucleolytic cleavage of the substrate; and a recognition sequence for the endonuclease; contacting each of the series of reaction mixtures with a different amount of endonuclease; measuring the fluorescence emitted by the reaction mixtures, where the change in fluorescence indicates the activity of the endonuclease; and selecting the amount of endonuclease that results in the highest fluorescence of the reaction mixture or the highest rate of increase in fluorescence of the reaction mixture as the optimal endonuclease concentration. In some embodiments, the structures that inhibit exonucleolytic cleavage of the substrate are selected from structures at each 3' end that inhibit 3'-5' exonucleolytic cleavage, structures at each 5' end that inhibit 5'-3' exonucleolytic cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II. In some embodiments, the reaction mixture is contacted with the endonuclease and the exonuclease simultaneously. In some embodiments, the reaction mixture is contacted with the endonuclease first, followed by the exonuclease. In some embodiments, no purification step is performed between contacting the reaction mixture with the endonuclease and contacting the reaction mixture with the exonuclease.

[0019] In some embodiments, the endonuclease is a nucleic acid-guided endonuclease, such as a CRISPR class I (CASCADE) endonuclease, and the substrate is a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3', or a CRISPR Cas9 endonuclease or a CRISPR The substrate comprises a Cas12a endonuclease and includes a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0020] In some embodiments, the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of a substrate. In some embodiments, the NATNA is capable of interacting with the endonuclease. In some embodiments, the NATNA comprises DNA and RNA nucleotides.

[0021] In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P, Cas3, and Cas7-11, and an endoribonuclease, and a restriction endonuclease. In some embodiments, the structure that inhibits exonucleolytic cleavage of the substrate is selected from a hairpin, a strand overhang, and a nucleic acid modification. In some embodiments, the nucleic acid modification comprises one or more phosphorothioate linkages. In some embodiments, the nucleic acid modification comprises five or more phosphorothioate linkages.

[0022] In one embodiment, the present invention provides a method for optimizing a CRISPR endonuclease digestion reaction, comprising: preparing a series of reaction mixtures using a CRISPR endonuclease, an exonuclease, and a nucleic acid substrate, the nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; at least one structure in at least one strand that inhibits exonuclease cleavage of the substrate; and a recognition sequence for the endonuclease; contacting each of a series of nucleic acid targeting nucleic acids (NATNAs); measuring the fluorescence emitted by the reaction mixtures, where a change in fluorescence indicates the activity of the endonuclease; and selecting the NATNA that produces the highest fluorescence of the reaction mixture or the highest rate of increase in the fluorescence of the reaction mixture as the optimal NATNA. In some embodiments, the at least one structure that inhibits exonuclease cleavage of the substrate is selected from a structure at each 3' end that inhibits 3'-5' exonuclease cleavage, a structure at each 5' end that inhibits 5'-3' exonuclease cleavage, or both. In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exonuclease I, and exonuclease II. In some embodiments, the reaction mixture is contacted with the endonuclease and the exonuclease simultaneously. In some embodiments, the reaction mixture is contacted with the endonuclease first, followed by the exonuclease.

[0023] In some embodiments, no purification step is performed between contacting the reaction mixture with the endonuclease and contacting the reaction mixture with the exonuclease.

[0024] In some embodiments, the CRISPR endonuclease is a Class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In some embodiments, the CRISPR endonuclease is Cas9 endonuclease or Cas12a endonuclease, and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'. In some embodiments, the NATNA is a CRISPR guide RNA selected from single-guide and dual-guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a targeting region capable of hybridizing to a region of the substrate. In some embodiments, the NATNA comprises DNA and RNA nucleotides.

[0025] In some embodiments, the structure that inhibits exonuclease cleavage of the substrate is selected from a hairpin, a strand overhang, and a nucleic acid modification. In some embodiments, the nucleic acid modification comprises one or more phosphorothioate linkages. In some embodiments, the nucleic acid modification comprises five or more phosphorothioate linkages. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a diagram of a substrate and method according to the present invention. [Figure 2] FIG. 1 shows experimental validation of the utility of exonuclease-linked fluorescence detection (Example 2). [Figure 3] FIG. 1 shows cleavage of the substrate (FIG. 1) by Cas12a endonuclease (Example 3). [Figure 4] FIG. 1 shows experimental determination of the linear range of the assay for Cas12a RNP concentration (Example 4). [Figure 5] FIG. 1 shows experimental confirmation that exonucleases hydrolyze DNA, but Cas12a does not (Example 5). [Figure 6] FIG. 1 shows experimental determination of the linear range of the assay with respect to DNA substrate concentration (Example 6). [Figure 7] FIG. 1 is a detailed diagram of a FAM-labeled substrate for a CRISPR endonuclease. [Figure 8] FIG. 1 shows application of the exonuclease assay (Example 9) to the cleavage of a FAM-labeled substrate (FIG. 7) by Cas12a endonuclease. [Figure 9] FIG. 1 shows application of the exonuclease assay to the cleavage of TAMRA-labeled substrates by Cas12a endonuclease (Example 10). [Figure 10] 1 is a diagram of FAM-labeled substrates with different placement of the FAM fluorophore. [Figure 11] FIG. 11 shows the application of the exonuclease assay (Example 11) to the cleavage of various FAM-labeled substrates (FIG. 10) by Cas12a endonuclease. [Figure 12] FIG. 12 is a diagram summarizing the data shown in FIG. [Figure 13] FIG. 1 shows the rate of change in fluorescence of the reaction mixture (Example 11). [Figure 14]FIG. 1 shows titration of exonuclease in an assay applied to cleavage of non-target FAM substrate by Cas12a endonuclease (Example 12). [Figure 15] FIG. 1 shows application of the exonuclease assay to the cleavage of FAM-labeled substrates by Cas9 endonuclease (Example 13). [Figure 16] FIG. 1 shows the application of an exonuclease assay to the cleavage of a FAM-labeled substrate by a restriction endonuclease (Example 14). DETAILED DESCRIPTION OF THE INVENTION

[0027] definition The following definitions are provided to aid in the understanding of this disclosure.

[0028] The term "endonuclease" refers to an enzyme that catalyzes the hydrolysis of a phosphodiester bond between two nucleoside residues (neither of which is a terminal nucleoside residue) within a polynucleotide (DNA or RNA).

[0029] The term "exonuclease" refers to an enzyme that catalyzes the hydrolysis of the phosphodiester bond between the terminal and penultimate nucleoside residues in a polynucleotide (DNA or RNA). Exonucleases can be processive, i.e., capable of the stepwise removal of multiple nucleoside residues from the end of a nucleic acid chain.

[0030] The term "CRISPR repeat" or "CRISPR repeat sequence" refers to a minimal CRISPR repeat sequence.

[0031] The term "endoribonuclease" refers to an enzyme that catalyzes the hydrolysis of phosphodiester bonds in RNA. In some embodiments, the endoribonuclease can be a site-directed polypeptide. The endoribonuclease can be a member of a CRISPR system (e.g., type I, type II, type III). The endoribonuclease can refer to the RAMP (Repeat Associated Mysterious Protein) superfamily of proteins (e.g., Cas6, Cas6, Cas5 family). The endoribonuclease can also include RNase A, RNase H, RNase I, RNase III family members (e.g., Drosha, Dicer, RNase N), RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1, and RNase V.

[0032] The term "inhibit" refers to the ability of a chemical structure to partially or completely inhibit a chemical reaction. Those skilled in the art will understand that whether inhibition is partial or complete depends on the sensitivity of the detection method. The term "inhibit cleavage" with respect to a nuclease refers to the ability to detectably reduce the amount of cleavage products. The term "prevent cleavage" with respect to a nuclease refers to the ability to reduce the amount of cleavage products below the level of detection.

[0033] The term "NATNA" refers to a nucleic acid targeting nucleic acid. NATNAs can be part of a programmable endonuclease system, such as a CRISPR system. NATNAs can be composed of two nucleic acid targeting polynucleotides ("dual guides"), including a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). NATNAs can also be composed of an engineered single nucleic acid targeting polynucleotide ("single guide"), including a crRNA and a tracrRNA connected by a fusion region (linker). NATNAs can also be composed of a naturally occurring single guide (e.g., Cas12a guide RNA). The crRNA can include a targeting region and an activation region. The tracrRNA can include a region capable of hybridizing to the activation region of the crRNA. The term "targeting region" refers to a region capable of hybridizing to a sequence in a target nucleic acid. The term "activation region" refers to a region that interacts with a polypeptide, such as a CRISPR nuclease.

[0034] Nucleic acids labeled with a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher) are a popular type of probe or enzyme substrate. Fluorophore-labeled nucleic acids are a popular type of probe or enzyme substrate. Particularly popular are probes and substrates labeled with two fluorophores that form a FRET pair. Popular types of dual-labeled probes include Taqman and molecular beacon probes.

[0035] In the Taqman assay (U.S. Patent No. 5,210,015), a dual-labeled oligonucleotide probe hybridizes to the nascent amplification product during PCR. Fluorescence is detected when the 5'-3' exonuclease activity of DNA polymerase hydrolyzes the probe between the two fluorophores.

[0036] Molecular beacons are hairpin-shaped, dual-labeled probes that unravel when the probe binds to its target. This unraveling separates the FRET pair, allowing the fluorescence of the donor fluorophore to be detected. Tyagi S et al. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 14(3):303.

[0037] In vitro enzyme activity can be detected using a nucleic acid substrate labeled with two fluorophores capable of conditional fluorescence that form a FRET pair. Enzyme activity can indicate, for example, the presence of an infectious agent in a sample. The use of a probe labeled with a fluorophore and a quencher fluorophore to detect microbial contamination is described in U.S. Patent No. 10,663,459. According to this method, the presence of microorganisms in a sample results in cleavage of the probe by one or more nucleases (e.g., endonucleases or exonucleases). All possible cleavages result in the physical separation of the fluorophore and quencher and the emission of detectable fluorescence, indicating the presence of microorganisms in the sample.

[0038] A similar principle is used in U.S. Patent No. 10,653,800, in which an RNA substrate incorporates 2'-O-methyl modified pyrimidines, making it uniquely susceptible to mycoplasmal RNase. The substrate is labeled with a fluorophore and a quencher. The presence of mycoplasma in the sample results in digestion of the probe by mycoplasmal RNase and the emission of detectable fluorescence, indicating the presence of mycoplasma in the sample.

[0039] Double-stranded nucleic acid substrates labeled with fluorophores and quenchers have also been used to detect the specific editing activity of CRISPR endonucleases. For example, Smith et al. (2020) "Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates," Biochemistry 59:1474, describes the cleavage of such substrates by the trans-cleavage ("trans-shredding") activity of CRISPR Cas12a nucleases. Trans-shredding activity is triggered by binding of the Cas12a-crRNA complex to the double-stranded DNA target. Trans-shredding nuclease activity is directed toward any double-stranded DNA in the vicinity of the Cas12a-crRNA-target complex. Shredding of the fluorophore- and quencher-labeled substrate results in the emission of detectable fluorescence, indicating the formation of Cas12a-crRNA-target complexes in the sample.

[0040] CRISPR-Cas nuclease activity is typically measured by incubating ribonucleoprotein complexes (RNPs) with model substrates followed by separation of cleaved fragments from intact substrates by agarose gel electrophoresis. This approach is low-throughput and generally limited to end-point analysis, thus providing little or no information about reaction kinetics.

[0041] A fluorescence-based assay has also been described for Cas12a, but this assay only measures the nonspecific transactivity of the Cas12a enzyme (see Smith CW, Biochemistry. 2020, supra). When the Cas12a RNP binds to and cleaves the target sequence (cis activity), Cas12a is activated and nonspecifically degrades short segments of DNA (trans activity or "trans-shredding" activity). Measuring transactivity as an indicator of cis activity has several limitations. First, the precise correlation between cis activity and trans activity is unclear. Second, because the two reactions are performed by the same enzyme and cannot be separated, surrogate assays provide limited kinetic information on cis activity. Finally, many endonucleases do not exhibit transactivity, making this assay less generally applicable.

[0042] A fluorescent assay for measuring Cas9 activity was developed in 2018 (see Seamon et al., (2018), Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity, Anal. Chem., 2018, 90, 11, pp. 6913-6921). However, this assay relies on denaturation of the DNA substrate after endonuclease cleavage and therefore provides only end-point analysis, not real-time data.

[0043] The present invention overcomes these drawbacks by providing a convenient real-time assay for endonuclease activity. The present invention includes a simple, high-throughput, fluorescence-based assay as an evaluation and quality control tool.

[0044] The present invention further includes diagnostic assays for the specific activity of the endonuclease which indicate the presence of the diagnostic target in a sample.

[0045] In some embodiments, the present invention provides a substrate molecule for detecting and assessing endonuclease activity. As shown in Figure 1, panel A, in some embodiments, the substrate is a nucleic acid having at least one double-stranded region. In some embodiments, the substrate is single-stranded. In some embodiments, the substrate is a double-stranded nucleic acid comprising two nucleic acid strands that form a duplex through hybridization. In other embodiments, the substrate is a single nucleic acid strand that forms a secondary structure containing a double-stranded region, such as a hairpin, through hybridization. Those skilled in the art will recognize that hybridization and the formation of a double-stranded region do not require 100% complementarity along the entire length of the nucleic acid strand. Under suitable conditions defined by the ionic strength of the buffer and temperature, stable hybrids (double-stranded regions) can form between two nucleic acid strands with less than 100% complementarity, e.g., 90%, 80%, 75%, or less.

[0046] As further seen in Figure 1, the nucleic acid substrate comprises a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore) that form a fluorescence resonance energy transfer (FRET) pair.

[0047] Fluorophores and quenchers can be placed at various locations on a double-stranded nucleic acid substrate. Furthermore, multiple fluorophores can be used. As shown in Figure 10, fluorophores and quenchers can be placed on the target strand, the non-target strand, or both the target and non-target strands.

[0048] Fluorescence resonance energy transfer (FRET), also known as Förster (or Förster) resonance energy transfer, is the transfer of excitation energy from one molecule to another without fluorescence or reabsorption. A donor chromophore absorbs light at a specific wavelength and then enters an electronically excited state. The donor transfers the energy to the acceptor, which is elevated to an electronically excited state. The acceptor's electronic excited state then decays, resulting in the emission of detectable light. Because the acceptor reduces, or quenches, the fluorescence of the donor, the acceptor is sometimes called a quencher. The donor is sometimes called a reporter. In conventional FRET technology, both the donor and acceptor are fluorophores. The donor fluorophore absorbs light at a specific absorption wavelength, and the acceptor emits light at a specific emission wavelength that is longer than the absorption wavelength. FRET occurs when the donor and acceptor are close together (e.g., 1–10 nm). In some embodiments, the donor and acceptor fluorophores are spaced 0-12 nucleotides apart. The donor and acceptor fluorophores are located on the same strand of the substrate or on different (opposite) strands of the substrate. The donor, acceptor, or both can be located near the ends of the nucleic acid strand, such as the 5' or 3' ends. The donor and acceptor fluorophores can be located on the same strand or on opposite strands. Those skilled in the art will recognize various options for positioning the donor and acceptor fluorophores (or reporter fluorophores and quenchers) within a double-stranded substrate to achieve the desired proximity of the fluorophores (e.g., 1-10 nm).

[0049] Existing literature provides ample guidance for selecting appropriate reporter-quencher pairs capable of FRET. See, for example, U.S. Patent Nos. 5,538,848; 8,350,038; and 8,137,616. Generally, as recommended in U.S. Patent Application Publication No. US20060088855, the donor fluorophore should absorb in the 350-800 nm, preferably 350-600 nm or 500-750 nm range, and the donor-acceptor distance should be 10-100 Å. See also Pesce et al., eds., Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed. (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, ed., Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949);

[0050] Many donors, acceptors, and donor / acceptor pairs that exhibit FRET are commercially available. Popular donors include fluorescein dyes such as 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), and 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE). Other donors include coumarin dyes, dyes from the Alexa Fluor family, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, and Texas Red. Popular acceptors include rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA) and tetrapropano-6-carboxyrhodamine (ROX), cyanine dyes including DABSYL, DABCYL, Cy5, and Cy5.5, anthraquinones, nitrothiazoles, and nitroimidazole compounds. Additional acceptors include LC-Red 610, LC-Red 640, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ, and Iowa Black RQ, as well as the sulfonated cyanine dyes disclosed in U.S. Patent No. 6,027,709. Popular donor-acceptor combinations include fluorescein / rhodamine, particularly carboxyfluorescein / tetramethyl-rhodamine (FAM / TAMRA). TAMRA as a quencher can also be paired with donors such as HEX (hexachlorofluorescein), TET (tetrachlorofluorescein), JOE (5'-dichlorodimethoxyfluorescein), and cyanine dyes. Another donor / acceptor pair is disclosed in U.S. Patent No. 9,796,746, which consists of an oxidized carbaNADH-based first fluorophore and a second fluorophore excitable with light having a wavelength between 445 and 540 nm and an emission maximum above 560 nm.

[0051] Another group of fluorescent compounds are naphthylamines, which have an amino group at the alpha or beta position. Such naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, and 2-p-toluidinyl-6-naphthalenesulfonate. Other dyes include acridines such as 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacridine, and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, and pyrenes.

[0052] Another category of fluorophores are the BODIPY® dyes, described in U.S. Patent No. 5,994,063. "BODIPY®" is a class of modified, spectrally discriminatory fluorophores in which the parent heterocyclic molecule is a dipyrrometheneboron difluoride compound. Most BODIPY® fluorophores have an absorption maximum of about 450-700 and an emission maximum of about 450-700. Examples include BODIPY® 503 / 512-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 523 / 547 (4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 530 / 550 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 558 / 568 (4,4-difluoro-5-(2-thienyl)-4bora-3a,4a-diaza-s-indacene-3-propionic acid), Examples of suitable fluorocarbons include BODIPY® 564 / 570 (4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 576 / 589 (4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), and BODIPY® 581 / 591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

[0053] One type of quencher is the "dark quencher." These non-fluorescent acceptors allow for low background fluorescence, thus improving assay sensitivity. When a dark quencher is used, the donor fluorophore does not emit light until the quencher is removed from the vicinity of the donor. For example, if the donor and quencher are conjugated to an oligonucleotide, donor fluorescence can occur only when the quencher is removed by hydrolysis of the oligonucleotide by a nuclease. One example of a dark quencher is DABCYL (4-[[4-(dimethylamino)-phenyl]-azo]-benzoic acid), which quenches donor dyes in the 380-530 nm range. Another dark quencher is the eclipse quencher (4-[[2-chloro-4-nitro-phenyl]-azo]-aniline (available from Epoch Biosciences, Inc.), which has an absorption maximum at 530 nm and quenches efficiently across the spectrum from 520 to 670 nm. Yet another category of dark quenchers are black hole quenchers, such as BHQ-1 ([(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline) and BHQ-2 ([(4-(1-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline) (all available from Biosearch Technologies, Inc.).

[0054] Another type of quencher includes pyridinyl-isoquinoline-dione derivatives disclosed in U.S. Patent No. 8,350,038. These compounds feature low background signal and high quenching efficiency. Another category of quenchers is the non-fluorescent cyanine quencher compound linked to the base of nucleotide via a linker compound disclosed in U.S. Patent No. 6,348,596. Another category of quenchers is the weakly luminescent cyanine substituted with one or more heteroaromatic quenching moieties disclosed in U.S. Patent No. 8,093,411. These quenchers show little or no observable luminescence and effectively quench a wide range of luminescent compounds.

[0055] Methods for synthesizing oligonucleotides and covalently attaching fluorophores to nucleic acids are known in the art. See, e.g., U.S. Patent Nos. 3,996,345; 4,351,760; 4,757,141; 4,739,044; 4,997,928; 5,538,848; 5,188,934; 5,231,191; and 7,759,469; as well as Eckstein (ed.), Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15:5305-5321 (1987) (3' thiol groups on oligonucleotides); Sharma et al., Nucleic Acids Research, 19:3019 (1991) (3' sulfhydryls); Giusti et al., PCR Methods and See, for example, Agrawal et al., Tetrahedron Letters, 31:1543-1546 (1990) (linkage via phosphoramidate linkage); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5' mercapto group); Nelson et al., Nucleic Acids Research, 17:7187-7194 (1989) (3' amino group). Functional groups and linking moieties can be used to synthesize labeled nucleic acid probes. Typically, presynthesized fluorophore-labeled nucleotides are incorporated into oligonucleotides using standard phosphoramidite-based chemistry. By incorporating such nucleotides at desired positions within the oligonucleotide, donor and acceptor fluorophores can be incorporated at any internal or terminal position within the oligonucleotide. In presynthesized fluorophore-labeled nucleotides, the label may be attached, for example, to a functional group attached to the amino group of the base of the nucleotide. In other embodiments, the label is attached to a portion of the nucleotide via a linking moiety.For example, in some embodiments, the nucleotide base is modified to allow for conjugation to a label. For example, U.S. Patent No. 7,759,469 discloses substituted nitroindole nucleotides that can be conjugated to a fluorophore.

[0056] In some embodiments, the double-stranded nucleic acid substrate is about 10 to about 90 base pairs in length. For example, in the case of the endonuclease Cas12a, the substrate may be 35 to 90 base pairs in length, including a PAM (5 nt), a spacer (20 nt), and end-protection (5 nt at each end), for a total of 35 base pairs. Generally, endonucleases have recognition sequences of various sizes and structures. Therefore, the optimal length of the substrate for each endonuclease tested can be determined using the calculations above. Optimal length and sequence can be determined in silico or found empirically. Such an optimal length allows for the most efficient digestion by the endonuclease without steric hindrance, without excessive length. Excessive length is associated with excessive production costs and requires additional units of exonuclease and additional time to perform the methods described herein. The optimal length of the double-stranded nucleic acid substrate for each endonuclease tested can take into account one or more of these considerations.

[0057] In some embodiments, the nucleic acid substrates of the present invention comprise chemical modifications. In some embodiments, the modifications increase the stability of the nucleic acid duplex. In some embodiments, the modifications confer resistance to nuclease digestion or inhibition of nucleases. In some embodiments, the modifications confer resistance to exonuclease digestion or inhibition of exonucleases.

[0058] In some embodiments, the modifications are backbone modifications. One type of backbone modification is a modified internucleoside linkage. For example, modifications include phosphorothioate linkages and heteroatom internucleoside linkages.

[0059] Another type of backbone modification is a modification of the sugar moiety. In some embodiments, the modification involves the incorporation of a six-membered morpholino ring in place of a ribose or deoxyribose ring. Another backbone modification involves the incorporation of a cyclohexenyl ring in place of a ribose or deoxyribose (ceNA). Yet another backbone modification involves the incorporation of locked nucleic acids (LNAs), in which a 2'-hydroxyl group is attached to the 4' carbon atom of ribose to form a bicyclic structure with a 2'-C,4'-C-oxymethylene bond. LNAs are characterized by duplex stability and resistance to 3'-5' exonuclease digestion.

[0060] In some embodiments, the modification is a nitrogenous base modification. For example, the double-stranded nucleic acid substrate may contain one or more of 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil, cytosine and and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine may be incorporated. Modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), and phenylalanine cytidine (2H-pyrimido(4,5-b)indol-2-one). Nucleobases may include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions can increase nucleic acid duplex stability by 0.6-1.2°C and may be a preferred base substitution (e.g., when combined with a 2'-O-methoxyethyl sugar modification).

[0061] As will be understood by those skilled in the art, a nucleic acid modification should not be included in the recognition site for the endonuclease being tested if the modification could interfere with endonuclease activity, unless it has been established that the endonuclease is not inhibited by that modification. Similarly, as will be understood by those skilled in the art, nucleic acid modifications known to inhibit exonuclease digestion should not be included in the portion of the substrate located between the endonuclease recognition site and the fluorophore, so as not to prevent or inhibit the performance of the methods described herein.

[0062] The methods and compositions described herein utilize exonucleases. Exonucleases are known in the art, and many are commercially available. See, for example, Lovett ST (2011). The DNA Exonucleases of Escherichia coli. EcoSal Plus, 4(2) and Shevelev, I., Hubscher, U. (2002) The 3'-5' exonucleases. Nat Rev Mol Cell Biol 3, 364-376. Many exonucleases are available from New England Biolabs (Ipswich, Mass.). For example, exonuclease I, exonuclease T, and exonuclease VII are 3'-5' exonucleases active against single-stranded DNA. RecJf is a 5'-3' exonuclease active against single-stranded DNA. Exonuclease III is a 3'-5' exonuclease active on single-stranded and double-stranded DNA. T7 exonuclease, exonuclease V, exonuclease VIII, lambda exonuclease, and T5 exonuclease are 5'-3' exonucleases active on single-stranded and double-stranded DNA. Exonuclease V (RecBCD) and BAL-31 are simultaneously 5'-3' and 3'-5' exonucleases active on single-stranded and double-stranded DNA. Depending on the type of nucleic acid terminus generated by the action of the endonuclease being tested, one skilled in the art can select an appropriate exonuclease. A suitable exonuclease utilizes one or more termini generated by the endonuclease to hydrolyze the nucleic acid substrate described herein. Hydrolysis by the exonuclease separates the FRET pair of fluorophores (eg, donor and acceptor or reporter and quencher) so that fluorescence or a change in fluorescence can be detected.

[0063] As shown in Figure 1, the substrate includes a structure at at least one of its termini that inhibits or prevents digestion by an exonuclease. The selection of the exonuclease included in the methods, compositions, and kits described herein informs the selection of the exonuclease inhibitor structure that provides end-protection in the nucleic acid substrate. For example, if the preferred substrate for the exonuclease is the 3' terminus, the nucleic acid substrate will have an exonuclease inhibitor structure at each of its 3' termini. If the preferred substrate for the exonuclease is the 5' terminus, the nucleic acid substrate will have an exonuclease inhibitor structure at each of its 5' termini. If the exonuclease has activity on both the 3' and 5' termini, the nucleic acid substrate will have an exonuclease inhibitor structure at each of its 3' and 5' termini.

[0064] The choice of exonuclease must also match the type of cleavage and available ends generated by the endonuclease being tested: the exonuclease (or mixture of exonucleases) must be inhibited by the inhibitory structures present at the ends of nucleic acid substrates described herein, and must have activity against the types of ends (protruding, blunt or recessed, hydroxyl or phosphoryl) generated by the endonuclease being tested.

[0065] In some embodiments, the endonuclease is a nickase, i.e., an endonuclease that cleaves only one strand of a duplex and generates a nick. In this embodiment, the exonuclease can be, for example, exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease.

[0066] In some embodiments, the endonuclease cleaves both strands of a duplex, generating a double-stranded break. In some embodiments, the double-stranded break has a blunt end. In some embodiments, the double-stranded break has a staggered end. In some embodiments, the zigzag end can have a protruding 3' end. In some embodiments, the zigzag end can have a protruding 5' end. One of skill in the art would be able to select an exonuclease (or a mixture of two or more exonucleases) capable of initiating hydrolysis in the desired direction from the particular type of end generated by the endonuclease being tested. For example, New England Biolabs, Inc. (Ipswich, Mass.) publishes a list of available exonucleases grouped by biochemical properties (e.g., the type of end required and the direction of hydrolysis).

[0067] As further seen in Figure 1, panel B, the substrate includes a recognition sequence (or recognition site) for an endonuclease. Depending on the endonuclease being tested, the recognition site may include a cleavage site and may have one or more additional elements. In some embodiments, the endonuclease being tested is a nucleic acid-guided endonuclease. In some embodiments, the endonuclease being tested is a CRISPR class I (CASCADE) endonuclease or a CRISPR class II endonuclease. In some embodiments, the endonuclease being tested is a CRISPR Cas9 or CRISPR Cas12a (Cpf1) endonuclease.

[0068] In some embodiments, the endonuclease is a deoxyribonuclease and the substrate is single-stranded or double-stranded DNA. In some embodiments, the endonuclease is a ribonuclease and the substrate is single-stranded or double-stranded RNA. In some embodiments, the endonuclease is a ribonuclease, such as a ribozyme, hammerhead ribozyme, DNAzyme, PNAzyme, or engineered endoribonuclease, for example, of the type described in Choudhury, R. et al. (2012) Engineering RNA endonucleases with customized sequence specificities. Nature Comm., 3:1147.

[0069] In some embodiments, the endonuclease to be tested is the endonuclease encoded by or related to CRISPR locus.CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome locus is found in many prokaryotic genomes and provides resistance to the invasion of foreign nucleic acid.The structure, nomenclature and classification of CRISPR locus are outlined in Makarova et al., Evolution and classification of the CRISPR-Cas systems.Nature Reviews Microbiology.2011 June;9(6):467-477.

[0070] Briefly, a typical CRISPR locus contains several short repeats with regularly spaced intervening sequences. CRISPR loci also contain coding sequences for CRISPR-associated (Cas) genes. The spacer-repeat sequence unit encodes the crisprRNA (crRNA). In vivo, mature crRNA is processed from a polycistronic transcript called the pre-crRNA or pre-crRNA array. Repeats in the pre-crRNA array are recognized by Cas-encoded proteins, which bind to the repeats and cleave them to release the mature crRNA. The CRISPR system performs target nucleic acid cleavage, and the Cas proteins and crRNA form the CRISPR ribonucleoprotein (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid entering a bacterial cell), and the Cas nuclease protein cleaves the target nucleic acid.

[0071] Class 1, or type I, CRISPR systems contain a means for processing the pre-crRNA array, including a multiprotein complex called Cascade (CRISPR-associated complex for antiviral defense), composed of subunits CasA, B, C, D, and E. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with the crRNA. The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of the target nucleic acid.

[0072] Class 2, type II CRISPR systems contain a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to the crRNA repeats within the pre-crRNA array and recruits endogenous RNase III to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, such as Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization between the target nucleic acid and the crRNA. When the crRNA hybridizes to the target nucleic acid, the Cas9 nuclease is activated and cleaves the target nucleic acid.

[0073] Class 1, Type III CRISPR systems comprise the RAMP superfamily of endoribonucleases (e.g., Cas6) that use one or more CRISPR polymerase-like proteins to cleave the pre-crRNA array.

[0074] Class 2, type V CRISPR systems contain a different set of Cas-like genes, including Csf1, Csf2, Csf3, and Csf4, which are distant homologs of the Cas genes in type I–III CRISPR systems.

[0075] As shown in Figure 1, panel B, the substrate includes a recognition sequence (or recognition site) for an endonuclease. In some embodiments, the recognition sequence and the cleavage site are the same. In some embodiments, the recognition site is a palindromic sequence characteristic of Type II restriction endonucleases, and cleavage occurs within the palindromic sequence. In some embodiments, the recognition sequence is different from the cleavage site. In some embodiments, the endonuclease is a CRSIPR Cas9 endonuclease, the recognition sequence is a protospacer adjacent motif (PAM), and the cleavage site is adjacent to the PAM. In some embodiments, the endonuclease is a CRSIPR Cas12a endonuclease, the recognition sequence is a protospacer adjacent motif (PAM), and the cleavage site is 16-18 bases away from the PAM on the non-target strand and 23-25 ​​bases away from the PAM on the target strand. See Strohkendl, I. et al. (2018) Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a, Mol. Cell, 71(5):816-824.e3.

[0076] In some embodiments, the endonuclease is a nickase, i.e., an endonuclease that cleaves only one strand of a duplex and generates a nick. In some embodiments, the endonuclease cleaves both strands of a duplex and generates a double-stranded break. The double-stranded break can have a blunt or zigzag end. The zigzag end can have a 3'- or 5'-overhanging end. The 5'-end can have a 5'-phosphoryl or 5'-hydroxyl group, and the 3'-end can have a 3'-phosphoryl or 3'-hydroxyl group.

[0077] CRISPR nucleases do not cleave fixed sequences, but are guided by a nucleic acid guide as described above. In addition to the guide RNA, CRISPR nucleases recognize an additional sequence called a protospacer adjacent motif (PAM). In some embodiments, the substrate of the present invention comprises a protospacer adjacent motif (PAM). In embodiments where the endonuclease being tested is a CRISPR class I (CASCADE) endonuclease, the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In embodiments where the endonuclease being tested is a CRISPR Class II endonuclease, the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0078] CRISPR nucleases do not cleave fixed recognition sequences, but are guided by a nucleic acid guide called a "guide RNA" (herein referred to as a "nucleic acid targeting nucleic acid (NATNA)"). The guide RNA (NATNA) contains a "spacer" sequence that is complementary to the endonuclease cleavage site. In embodiments where the endonuclease being tested is a CRISPR endonuclease, the substrate contains a target sequence that can hybridize to a portion (the "spacer") of the NATNA.

[0079] In some embodiments, the endonuclease is a nucleic acid-guided endonuclease. A reaction mixture containing such an endonuclease further requires a nucleic acid targeting nucleic acid (NATNA). In some embodiments, the endonuclease is a CRISPR endonuclease, and the NATNA is a guide RNA. The endonuclease is capable of forming a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a class 2, type II CRISPR endonuclease, and the NATNA comprises a tracrRNA and a crRNA. In some embodiments, the endonuclease is a class 2, type V CRISPR endonuclease, and the NATNA comprises a crRNA.

[0080] In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, the NATNA can include, in 5' to 3' order, a spacer extension, a spacer, minimal CRISPR repeats, a single guide connector, a minimal tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some cases, the nucleic acid targeting nucleic acid can include, in any order, a tracrRNA extension, a 3' tracrRNA sequence, a minimal tracrRNA, a single guide connector, minimal CRISPR repeats, a spacer, and a spacer extension.

[0081] In some embodiments, the guide nucleic acid targeting nucleic acid may comprise a single-guide NATNA. The NATNA comprises a spacer sequence that can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to the tracrRNA sequence. In some cases, the NATNA may have a spacer extension and a tracrRNA extension. These elements may include elements that can contribute to the stability of the NATNA. The CRISPR repeat and the tracrRNA sequence can interact to form a base-paired double-stranded structure. This structure can facilitate the binding of an endonuclease to the NATNA.

[0082] In some embodiments, the single-guide NATNA comprises a spacer sequence located 5' of the first duplex, which comprises a region of hybridization between the minimal CRISPR repeats and the minimal tracrRNA sequence. The first duplex may have a bulge in the middle. The bulge facilitates recruitment of an endonuclease to the NATNA. The bulge may be followed by a first stem comprising a linker connecting the minimal CRISPR repeats and the minimal tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex may be connected to a second linker connecting the first duplex to the mid-tracrRNA. The mid-tracrRNA may comprise one or more additional hairpins.

[0083] In some embodiments, the NATNA may comprise a double-guide nucleic acid structure. The double-guide NATNA comprises a spacer extension, a spacer, minimal CRISPR repeats, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The double-guide NATNA does not comprise a single-guide connector. Instead, the minimal CRISPR repeat sequence comprises a 3' CRISPR repeat sequence, and the minimal tracrRNA sequence comprises a 5' tracrRNA sequence, and the double-guide NATNA can hybridize via the minimal CRISPR repeats and the minimal tracrRNA sequence.

[0084] In some embodiments, the NATNA is an engineered guide RNA (CRISPR hybrid RDNA or chRDNA) containing one or more DNA residues. In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNAs used in Class 2 CRISPR systems may be composed of two strands forming a secondary structure including an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and an activation region composed of one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain various proportions of DNA and RNA. Other chRDNAs may be single-guide D(R)NAs used in Type II CRISPR systems, which include a targeting region and an activation region composed of a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain various proportions of DNA and RNA. For example, the targeting region can comprise DNA or a mixture of DNA and RNA, and the activation region can comprise RNA or a mixture of DNA and RNA.

[0085] In some embodiments, the guide RNA comprises a nucleic acid modification, such as a modification that confers resistance to ribonucleases, a feature that is particularly advantageous in the crude lysate assays described below.

[0086] In some embodiments, the endonuclease to be tested is a restriction endonuclease. In some embodiments, the endonuclease to be tested is a type II, type II, or type IV restriction endonuclease. The substrate of the present invention contains the appropriate recognition sequence for each endonuclease. In the case of a type IV restriction endonuclease, the substrate of the present invention also contains one or more methylated residues required for cleavage by the endonuclease.

[0087] In embodiments in which the endonuclease being tested is a zinc finger nuclease (ZFN) or a ZFN conjugated to the non-specific cleavage domain of the restriction endonuclease Fok I, the target sequence is approximately 22-52 bases in length and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides in length and separated by a spacer, which is 4-18 nucleotides in length (see, e.g., Kim YG et al. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA. 93(3):1156-1160).

[0088] In embodiments in which the endonuclease being tested is a transcription activator-like effector nuclease (TALEN) or a TALEN-Fok I fusion, the target sequence is approximately 48-85 nucleotides in length and comprises a pair of TALEN recognition sequences, each 18-30 bases in length and separated by a spacer, which is 12-25 bases in length (see, e.g., Christian M. et al. (2010) Targeting DNA double-strand breaks with TAL effector nucleases, Genetics. 186(2):757-61).

[0089] In some embodiments, the endonuclease tested is Argonaute (Ago) endonuclease. Ago endonucleases have no recognition sequence but are guided by small interfering DNA guides (siDNA) to cleave complementary DNA. Hegge et al. (2019) DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute, NAR47(11):5809.

[0090] In some embodiments, the endonuclease tested is Arcus endonuclease. Arcus is an I-CreI endonuclease with a 22-base target sequence (see, e.g., Durrenberger et al. (1991) Double-strand break induced recombination in Chlamydomonas reinhardtii chloroplasts, NAR24(17):3323).

[0091] In some embodiments, the endonuclease tested is an endoribonuclease and the substrate comprises RNA. In some embodiments, the substrate is single-stranded RNA. In some embodiments, the substrate is double-stranded RNA. In some embodiments, the substrate is an RNA-DNA hybrid. The exonuclease used in such assays is an exodeoxyribonuclease or an exoribonuclease, depending on the selected substrate. Examples of endoribonucleases that cleave one or more of such substrates include RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P. Another example of an endoribonuclease is a CRISPR endoribonuclease selected from Cas13 and Cas7-11.

[0092] Examples of exoribonucleases include those that cleave in the 3'-5' direction, such as RNase R, RNase II, RNase D, RNase T, RNase BN, and RNase PH, and those that cleave in the 5'-3' direction, such as exoribonuclease I and exoribonuclease II.

[0093] In some embodiments, the invention is a method for detecting endonuclease activity using the substrates described herein. As shown in Figure 1, panel B, the method includes contacting the endonuclease to be tested with a reaction mixture containing: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore) that form a fluorescence resonance energy transfer (FRET) pair, and a double-stranded nucleic acid substrate containing a recognition sequence for the endonuclease to be tested. The reaction mixture is incubated under conditions suitable for endonucleolytic cleavage of the recognition sequence.

[0094] The method further comprises contacting the reaction mixture with an exonuclease. The exonuclease, substrate, and endonuclease may be added simultaneously or sequentially in any order.

[0095] As shown in Figure 1, panels B-C, the structure of the double-stranded nucleic acid substrate prevents exonucleases from hydrolyzing the double-stranded nucleic acid substrate until endonucleolytic cleavage occurs. Suitable ends of double-stranded nucleic acid substrates include structures that inhibit cleavage of the substrate by exonucleases. As described herein, depending on the end generated by the endonuclease, the exonuclease is a 3'-5' exonuclease, and the 3' end of the substrate is protected by an exonuclease inhibitor structure. In other embodiments, the exonuclease is a 5'-3' exonuclease, and the 5' end of the substrate is protected by an exonuclease inhibitor structure. In some embodiments, a mixture of a 3'-5' exonuclease and a 5'-3' exonuclease is used. In such embodiments, both the 3' and 5' ends of the substrate are protected by exonuclease inhibitor structures.

[0096] As shown in Figure 1, panel C, the reaction mixture is further incubated under conditions suitable for exonucleolytic cleavage of the double-stranded nucleic acid substrate. In some embodiments, the conditions for endonucleolytic cleavage and subsequent exonucleolytic cleavage are the same. In such embodiments, no changes in buffer or incubation conditions are required. In some embodiments, no purification step is performed between contacting the reaction mixture with the endonuclease and contacting the reaction mixture with the exonuclease.

[0097] One advantage of this method is that the fluorophore and quencher can be placed in close proximity, minimizing background signal and avoiding steric effects that are likely to occur when a fluorophore-quencher pair is incorporated near the cleavage site, e.g., with the fluorophore on one side and the quencher on the other.

[0098] In some embodiments, the endonuclease tested by this method is nucleic acid-guided endonuclease.In such embodiments, NATNA is also used in this method.In some embodiments, endonuclease is CRISPR endonuclease, and NATNA is guide RNA.

[0099] In some embodiments, the endonuclease tested by the method is a CRISPR class I (CASCADE) endonuclease, and the double-stranded nucleic acid substrate used in the method comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In embodiments in which the endonuclease tested by the method is a CRISPR class II endonuclease, the double-stranded nucleic acid substrate used in the method comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0100] In some embodiments, the endonuclease tested by the method is one of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and restriction endonucleases. In these embodiments, the double-stranded nucleic acid substrate used in the method comprises a suitable recognition site.

[0101] In some embodiments, the method comprises screening, testing or comparing several endonucleases.In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same components with a series of different endonucleases.In some embodiments, the series of endonucleases is a series of nucleic acid-guided endonucleases, and the reaction mixtures in the series of reaction mixtures comprising the same components also comprise the same NATNA.In some embodiments, the series of endonucleases is a series of CRISPR endonucleases, and the reaction mixtures in the series of reaction mixtures comprising the same components also comprise the same guide RNA.

[0102] In some embodiments, the endonuclease is a nucleic acid-guided endonuclease, and the method comprises screening, testing or comparing several NATNAs.In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same components and containing the same endonuclease with a series of different NATNAs.In some embodiments, the endonuclease is a CRISPR endonuclease, and the NATNA is guide RNA.In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same components and containing the same CRISPR endonuclease with a series of different guide RNAs.

[0103] In some embodiments, the method involves screening, testing, or comparing several preparations of the same endonuclease. In these embodiments, the method involves contacting a series of reaction mixtures containing the same components with a series of different preparations of the same endonuclease. The different preparations can be different isolates of the same endonuclease. The different preparations can also be elution aliquots from a chromatography procedure aimed at isolating the endonuclease. The invention includes methods of monitoring the elution of an endonuclease by performing an endonuclease activity assay described herein on elution fractions emerging from the chromatography procedure and retaining the elution fractions with the highest endonuclease activity.

[0104] In some embodiments, the method comprises screening, testing or comparing several nucleic acid sequences to identify the preferred or optimal target sequence for endonuclease.In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same components, including the same endonuclease, with a series of double-stranded nucleic acid substrates having different sequences.In some embodiments, the endonuclease is a CRISPR endonuclease, and the reaction mixtures in the series of reaction mixtures comprising the same components also comprise the same guide RNA.

[0105] In some embodiments, the method involves screening, testing, or comparing several reaction conditions to identify preferred or optimal reaction conditions for the endonuclease. In some embodiments, the method involves contacting a series of reaction mixtures containing different buffer compositions with the same endonuclease. In some embodiments, the series of reaction mixtures are also subjected to different temperature profiles during the endonuclease digestion step.

[0106] In some embodiments, the methods involve screening, testing, or comparing several reaction conditions to identify a preferred or optimal endonuclease concentration in a nucleic acid cleavage reaction, hi some embodiments, the methods involve contacting a series of reaction mixtures that are identical except for varying concentrations of the same endonuclease being tested.

[0107] In some embodiments, the method involves screening, testing, or comparing several CRISPR polynucleotide guides (guide RNAs or gRNAs) to identify a preferred or optimal gRNA for a CRISPR endonuclease. In these embodiments, the method involves contacting a series of reaction mixtures that are identical except for the guide RNAs. In some embodiments, the CRISPR polynucleotide guides include one or more DNA residues (CRISPR hybrid RDNA or chRDNA). In these embodiments, the method involves contacting a series of reaction mixtures that are identical except for the chRDNAs.

[0108] As shown in Figure 1, panel D, the method then includes measuring fluorescence emitted by the reaction mixture, where a change in fluorescence indicates endonuclease activity. The change in fluorescence includes not only a change in color (wavelength) of the fluorescent signal, but also the appearance of fluorescence where no fluorescence was previously detectable. In some embodiments, the measurement is qualitative, indicating the presence or absence of endonuclease activity. In other embodiments, the measurement is quantitative, indicating the relative amount of endonuclease activity. In some embodiments, the change in fluorescence further includes a change in the intensity of fluorescence and differences in fluorescence between samples tested.

[0109] In some embodiments, the methods described herein are performed using isolated nucleic acid substrates and isolated polypeptides (e.g., endonucleases and exonucleases). In other embodiments, assays are performed using crude mixtures without substantial purification steps. In some embodiments, isolated or purified nucleic acid substrates are added to crude isolates or elution fractions of endonucleases, e.g., to rapidly evaluate endonuclease production or purification processes. In some embodiments, isolated or purified polypeptides (e.g., endonucleases and exonucleases) are added to crude isolates of nucleic acids, e.g., minimally processed patient samples, to rapidly detect the presence of infectious agents in patients.

[0110] In some embodiments, the present invention provides a double-stranded nucleic acid substrate and a method for producing the substrate. The substrate has exonuclease end protection, such as phosphorothioate bond. One, two, three, four, or about five phosphodiester bonds can be replaced with phosphorothioate bond. For example, when exonuclease III is used, the phosphodiester bond at the 3' end of both strands can be replaced with phosphorothioate bond. Optionally, the terminal phosphate moiety (if present) at the 3' end of each strand can be replaced with phosphorothioate.

[0111] The substrate also has a fluorophore (reporter) and a quencher. The reporter and quencher can be located near one end of one strand of the double-stranded nucleic acid substrate. One of the fluorophore and quencher can be attached to the end of one strand. For example, when using exonuclease III, one member of the fluorophore-quencher pair can be attached to the 5'-end of the target strand, and the member can be attached to a nucleotide 1, 2, 3, 4, or about 5 nucleotides away from the end. For example, one strand can have a thymine-linked fluorescein at the third nucleotide from the 5'-end and an Iowa Black® quencher at the 5'-end.

[0112] In some embodiments, the endonuclease being tested recognizes a sequence on both strands (e.g., a palindromic sequence recognized by a Type II restriction endonuclease). In some embodiments, the endonuclease being tested recognizes a sequence on one strand (e.g., a target sequence for a CRISPR Cas endonuclease). In such embodiments, the target nucleic acid has a target strand and a non-target strand. An example of a substrate for CRISPR Cas12a is shown in Figure 7. The target strand of the substrate contains a targetable sequence for the endonuclease being tested. For example, a cleavable site for Cas12a (Cpf1) is described in Zetsche, B. et al. (2015) Cpf1 is a single-RNA guided endonuclease of a Class II CRISPR-Cas system, Cell, 163:759. As shown in Figure 7, the substrate further includes a spacer sequence recognized by the CRISPR guide nucleic acid, a fluorophore, a quencher, and exonuclease end protection such as phosphorothioate nucleotides at or near the end of each strand.

[0113] In some embodiments, the fluorophore is located on the target strand. In some embodiments, the fluorophore is located on the non-target strand. In some embodiments, the fluorophore is located on both the target and non-target strands. Figure 10 shows the substrate of Figure 7 with an alternative fluorophore arrangement. In Figure 10, the light and dark blocks on the top strand represent the recognition (target) sequence for the CRISPR endonuclease (light blocks are PAMs, dark blocks are spacers). Fluorophores are represented as stars and quenchers are represented as dark half-moons. Dark octagons represent exonuclease-inhibiting modifications such as phosphorothioate nucleotides.

[0114] In some embodiments, the placement of the fluorophores affects the performance of the assay (see Example 11 and Figures 11, 12 and 13).

[0115] Double-stranded nucleic acid substrates can be prepared by combining two strands (a target strand and a complementary non-target strand) in a reaction mixture containing a suitable buffer (e.g., TE). For optimal annealing, the mixture may be heated to above 90°C and allowed to cool to room temperature.

[0116] In some embodiments, the effectiveness of exonuclease protection is tested for each exonuclease intended for use in the endonuclease assays disclosed herein. For each double-stranded nucleic acid substrate described herein, a control double-stranded nucleic acid substrate lacking exonuclease protection is generated. Both substrates are exposed to the exonuclease in a suitable buffer (e.g., NEBuffer1, pH 7.0 for exonuclease III and fluorescein) under suitable reaction conditions for both exonuclease activity and fluorescence, and fluorescence is measured, for example, by a fluorescence reader. If exonuclease protection is suitable for the exonuclease, a fluorescent signal will be generated for the unprotected substrate but not for the protected substrate.

[0117] In some embodiments, the double-stranded nucleic acid substrate described herein is used to detect endonuclease activity. The substrate has exonuclease protection, a fluorophore, and a quencher (e.g., one, two, three, four, or about five phosphorothioate bonds at the 3'-end of both strands, and a thymine-linked fluorescein at the third nucleotide from the 5'-end of the target strand, and an Iowa Black® quencher at the 5'-end). The substrate is contacted with an exonuclease and an endonuclease in a suitable buffer (e.g., NEBuffer1, pH 7.0 for exonuclease III, AsCas12a, and fluorescein) under suitable reaction conditions for exonuclease activity, endonuclease activity, and fluorescence. Guidance on selecting a suitable buffer can be obtained from endonuclease vendors (e.g., New England Biolabs for restriction endonucleases) or from published studies, e.g., Gasiunas, G. et al., (2020) A catalogue of biochemically diverse CRISPR-Cas9 orthologs, Nature Comm. 11, 5512 doi:10.1038 / s41467-020-19344-1.

[0118] When the endonuclease is a nucleic acid-guided endonuclease, a nucleoprotein complex (e.g., a ribonucleoprotein complex, RNP) is assembled and the endonuclease is added to the reaction mixture in the form of a nucleoprotein complex. The nucleoprotein complex includes the endonuclease and a nucleic acid targeting nucleic acid (NATNA), such as a CRISPR guide RNA (crRNA) for Cas12a (Cpf1). A suitable sequence of this crRNA can be found, for example, in Yamano T. et al. (2016) Crystal structure of Cpf1 in complex with guide RNA and target DNA, Cell 165:949. To assemble the nucleoprotein complex, NATNA is incubated with the endonuclease under suitable conditions, such as at 37°C for 10 minutes. NATNA can be pretreated by heating (e.g., to 95°C for 2 minutes) to allow for proper secondary structure formation, and then allowed to slowly cool to room temperature.

[0119] The fluorescence of the reaction mixture is measured. For exonuclease-protected substrates, a fluorescent signal is produced only in the presence of both the exonuclease and the endonuclease.

[0120] In some embodiments, the linear range of the assay with respect to endonuclease concentration and nucleic acid substrate concentration is tested to determine the optimal range of substrate concentration and sensitivity with respect to endonuclease concentration.

[0121] In some embodiments, the present invention is a composition for detecting endonuclease activity. The composition includes a nucleic acid substrate as described herein, further comprising a 3'-5' exonuclease (or a 5'-3-exonuclease, or both) that is inhibited by a structure at the 3' end (or the 5' end, or both) of the substrate. The composition includes a double-stranded nucleic acid substrate, which includes: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore) that form a fluorescence resonance energy transfer (FRET) pair; a structure at each end that inhibits cleavage of the substrate by the exonuclease; and a recognition sequence for the endonuclease to be tested.

[0122] The composition may also include an exonuclease. Depending on the terminus generated by the endonuclease, the composition may include a 3'-5' exonuclease, a 5'-3' exonuclease, or a mixture of both. In some embodiments, signal enhancement is achieved by having multiple fluorophores per substrate. In some embodiments, the exact identity of the endonuclease being tested is unknown. In such embodiments, both ends of both strands are labeled to accommodate all possible orientations and chemistries of nucleic acid cleavage. In some such embodiments, the ends are labeled with different fluorophores that emit at different wavelengths. The emission wavelength indicates the identity of the cleaved strand and the cleavage chemistry. In the case of a 3'-5' exonuclease, the double-stranded nucleic acid substrate has a structure at each 3' end that inhibits cleavage of the substrate by the exonuclease. In the case of a 5'-3' exonuclease, the double-stranded nucleic acid substrate has a structure at each 5' end that inhibits cleavage of the substrate by the exonuclease. In some embodiments, a mixture of 3'-5' and 5'-3' exonucleases is used, in which both the 3' and 5' ends of the substrate are protected by exonuclease inhibitor structures.

[0123] In some embodiments, the substrate is designed to accommodate several endonucleases, including endonucleases whose biochemical properties are not fully understood at the time of testing. Such substrates will have a fluorophore and quencher pair located at or near both ends of both strands. Such dual-labeled substrates can be utilized in reaction mixtures containing exonucleases capable of bidirectional hydrolysis (e.g., exonuclease V or exonuclease VII).

[0124] In some embodiments, the endonuclease tested by the composition is nucleic acid-guided endonuclease.In such embodiments, NATNA also exists in the composition.In some embodiments, the endonuclease is CRISPR endonuclease, and NATNA is guide RNA.

[0125] In some embodiments, the endonuclease tested by the composition is a CRISPR class I (CASCADE) endonuclease, and the double-stranded nucleic acid substrate in the composition comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In embodiments in which the endonuclease tested by the composition is a CRISPR class II endonuclease, the double-stranded nucleic acid substrate in the composition comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0126] In some embodiments, the endonuclease tested by the composition is one of zinc finger nucleases (ZFNs), ZFNs conjugated to Fok I, transcription activator-like effector nucleases (TALENs), EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and restriction endonucleases. In these embodiments, the double-stranded nucleic acid substrate in the composition comprises a suitable recognition site.

[0127] In some embodiments, the composition comprises exonuclease III, a double-stranded nucleic acid substrate comprising (i) both 3' ends comprising one or more phosphorothioate-protected dsDNA; (ii) a reporter fluorophore, and (iii) a compatible fluorescent quencher positioned to quench donor fluorescence when the double-stranded nucleic acid substrate is intact, and an endonuclease of interest.

[0128] In some embodiments, the present invention provides a method suitable for use as a convenient tool for evaluating, screening, testing, or comparing several endonucleases. This tool further allows for the evaluation of a set of endonuclease cleavage conditions by determining which conditions allow for the highest level or rate of endonuclease activity. This tool also allows for the evaluation of endonuclease isolation and purification methods. Specifically, this tool can be applied to compare protein isolation fractions to identify fractions containing isolated protein. In such embodiments, modifications are made to ensure that all components, e.g., endonucleases, exonucleases, and NATNAs (if used), are capable of activity and are at least partially protected from enzymatic degradation in crude preparations. This tool can be rapidly applied to nascent fractions, e.g., to monitor a protein purification process. Furthermore, this tool can be used to screen multiple endonuclease substrates with different sequences to rapidly identify the target sequence of an endonuclease.

[0129] The methods and compositions disclosed herein can be used in diagnostic assays. In some embodiments, the present invention is a method for detecting the presence of a specific nucleic acid in a sample, where the nucleic acid contains a recognition sequence for an endonuclease. In some embodiments, the sample is a patient sample. The nucleic acid can be characteristic of a microorganism, including a virus or bacteria. The nucleic acid can also contain a polymorphism or sequence whose presence is associated with a disease or condition to be detected in a patient.

[0130] The method involves manipulating nucleic acids from a sample. In some embodiments, the sample is obtained from a subject or patient. In some embodiments, the sample may include a solid tissue or a fragment of a solid tumor obtained from a subject or patient, for example, by biopsy. The sample may also include a bodily fluid that may contain nucleic acids (e.g., urine, sputum, serum, blood, or a blood fraction, i.e., plasma, lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cyst fluid, bile, gastric juice, intestinal fluid, or fecal sample). In other embodiments, the sample is a culture sample, such as a tissue culture, containing cells and fluids from which nucleic acids can be isolated. In some embodiments, the nucleic acid of interest present or suspected to be present in the sample is derived from an infectious agent such as a virus, bacteria, protozoan, or fungus.

[0131] In some embodiments, the method includes a pre-amplification step in which nucleic acids in a sample are amplified via polymerase chain reaction (PCR) to generate specific amplicons from each target nucleic acid. In some embodiments, fluorophores, quenchers, and end protections are incorporated into the amplicons by adapter ligation. Blunt-end formation, A-tailing, and adapter ligation can be performed, for example, by methods developed in conjunction with the formation of sequence libraries for massively parallel sequencing. In some embodiments, fluorophores, quenchers, and end protections are incorporated directly into the amplification primers. Excess primers or adapters containing fluorophores, quenchers, and end protections can be removed via a purification procedure before performing an endonuclease assay. The amplicons containing fluorophores, quenchers, and end protections are double-stranded nucleic acid substrates that are directly used in the methods disclosed herein.

[0132] In some embodiments, an endonuclease substrate is a probe comprising a fluorophore, a quencher, and end protection, which is hybridized to a target nucleic acid or an amplicon of the target nucleic acid in a sample. In some embodiments, the probe, target nucleic acid, or amplicon is single-stranded. In some embodiments, the probe, target nucleic acid, or amplicon is double-stranded, but is made single-stranded before being hybridized to the probe. The duplex formed by the target nucleic acid hybridized to the probe comprising a fluorophore, a quencher, and end protection becomes a double-stranded nucleic acid substrate that can be directly used in the methods disclosed herein.

[0133] The nucleic acid substrate formed by any of the above alternative methods contains a nucleic acid sequence of diagnostic importance to be interrogated. In some embodiments, the nucleic acid substrate is contacted with an endonuclease that targets a sequence of interest, such as a sequence characteristic of a microorganism, or a sequence containing a polymorphism or sequence whose presence is associated with a disease or condition to be detected in a patient. The endonuclease will cleave only if the sequence of interest containing the endonuclease cleavage site is present in the nucleic acid substrate.

[0134] Particularly advantageous in the diagnostic assay disclosed herein is CRISPR endonuclease.Guide RNA (e.g., crRNA) for CRISPR endonuclease can be designed to hybridize with any diagnostic sequence of interest.A sample is contacted with a probe that includes a fluorophore, a quencher, and end-protection, and can hybridize with the target nucleic acid of diagnostic interest.This sample is further contacted with a guide RNA that can hybridize with the target nucleic acid.Only when the sequence that can hybridize with the probe and the designed guide RNA exists in the sample, the CRISPR endonuclease will perform cleavage, and fluorescence will be detectable.

[0135] In some embodiments, the diagnostic method is multiplexed. That is, multiple target sequences are detected in the same reaction mixture. In such embodiments, multiple nucleic acid probes are added to the sample. In some embodiments, when the endonuclease is a CRISPR endonuclease, multiple guide RNAs are also added to the sample, and the same CRISPR endonuclease performs cleavage, resulting in the generation of a detectable signal. In some embodiments, each of the different probes is labeled with a different fluorophore. In some embodiments, all or part of the different probes are labeled with the same label, for example, a set of probes hybridizing to bacterial sequences is labeled with one label, a set of probes hybridizing to viral sequences is labeled with another label, or a set of probes hybridizing to Gram-positive bacterial sequences is labeled with one label, and a set of probes hybridizing to Gram-negative bacterial sequences is labeled with another label.

[0136] The endonuclease and exonuclease may be added to the sample sequentially or simultaneously. The endonuclease and exonuclease may be added to the sample before the addition of the double-stranded nucleic acid substrate. The exonuclease performs strand cleavage (hydrolysis) only if the endonuclease has previously cleaved an end accessible to the exonuclease. Hydrolysis of the double-stranded nucleic acid substrate by the exonuclease separates the fluorophore and quencher, producing a detectable fluorescent signal. The presence of the fluorescent signal indicates the presence of the sequence of interest in the sample. In some embodiments, the assay includes a step of reporting the presence of the sequence of interest in the sample (e.g., a sequence characteristic of a microorganism, or a polymorphism or sequence whose presence is associated with a disease or condition to be detected in a patient).

[0137] In some embodiments, the present invention is a kit for detecting endonuclease activity. The kit includes an aliquot of the nucleic acid substrate described herein. The composition includes a double-stranded nucleic acid substrate, which includes: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore) that form a fluorescence resonance energy transfer (FRET) pair; a structure at each end that inhibits cleavage of the substrate by an exonuclease; and a recognition sequence for the endonuclease to be tested.

[0138] The kit may further include an aliquot of exonuclease. Depending on the terminus generated by the endonuclease, the kit may include a 3'-5' exonuclease or a 5'-3' exonuclease. In the case of a 3'-5' exonuclease, the double-stranded nucleic acid substrate has a structure at each 3' end that inhibits cleavage of the substrate by the exonuclease. In the case of a 5'-3' exonuclease, the double-stranded nucleic acid substrate has a structure at each 5' end that inhibits cleavage of the substrate by the exonuclease. The kit may include both a 3'-5' exonuclease and a 5'-3' exonuclease. The kit may include a double-stranded nucleic acid substrate having structures at both the 3' and 5' ends that inhibit cleavage of the substrate by the exonuclease.

[0139] In some embodiments, the endonuclease tested by this kit is nucleic acid-guided endonuclease.In such embodiments, this kit can also comprise NATNA aliquot.In some embodiments, the endonuclease tested is CRISPR endonuclease, and the NATNA present in the kit is guide RNA.

[0140] In some embodiments, the endonuclease tested by the kit is a CRISPR class I (CASCADE) endonuclease, and the double-stranded nucleic acid substrate included in the kit comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'. In embodiments in which the endonuclease tested by the kit is a CRISPR Class II endonuclease, the double-stranded nucleic acid substrate included in the kit comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0141] In some embodiments, the endonuclease tested by the kit is one of a zinc finger nuclease (ZFN), a ZFN conjugated to Fok I, a transcription activator-like effector nuclease (TALEN), an endoribonuclease selected from EndoTT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, Cas3 and Cas7-11, and a restriction endonuclease. In these embodiments, the nucleic acid substrate included in the kit comprises a suitable recognition site.

[0142] In some embodiments, the kit further comprises instructions for performing a method for testing the activity of an endonuclease by a method described herein.

[0143] In some embodiments, the present invention is a kit for performing a diagnostic procedure. The kit includes an aliquot of a probe that can hybridize to a target nucleic acid of diagnostic interest and form a fluorescence resonance energy transfer (FRET) pair: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore); a structure at each end that inhibits cleavage of the substrate by an exonuclease; and a recognition sequence for the endonuclease to be tested. The kit may further include an aliquot of an exonuclease and an endonuclease. The exonuclease can be inhibited by the inhibitory structure present on the probe. The endonuclease can bind to and cleave the duplex formed by the probe and the target sequence. In some embodiments, the endonuclease is a nucleic acid-guided endonuclease. In such embodiments, the kit may also include an aliquot of NATNA that can hybridize to the target nucleic acid. In some embodiments, the endonuclease being tested is a CRISPR endonuclease and the NATNA present in the kit is a guide RNA.

[0144] In some embodiments, the kit further comprises instructions for performing the diagnostic assays described herein.

[0145] The methods disclosed herein can be performed using a dedicated device. In some embodiments, the present invention is a device for detecting endonuclease activity, comprising one or more reaction chambers for performing an enzymatic reaction and a fluorescence detector. The device may further comprise a means for delivering and distributing components of the reaction mixture described herein. The device can be applied to high-throughput screening, for example, in multiwell plates (microwell plates).

[0146] The equipment may include multiwell plate fluorescence readers or tube fluorometers such as those available from Tecan, ThermoFisher Scientific (BioTek instruments), and Molecular Devices. [Example]

[0147] Example 1. Preparation of double-stranded endonuclease substrate In this example, a 60-base pair double-stranded nucleic acid construct was prepared in which the 3' ends of both strands were protected with a series of phosphorothioate bonds, and one strand was labeled with a fluorophore and quencher. The last four phosphodiester bonds at the 3' end of both strands were replaced with phosphorothioate bonds. The terminal phosphate moiety at the 3' end of each strand was also replaced with phosphorothioate. Additionally, a thymine-linked fluorescein moiety was incorporated into the third nucleotide from the 5' end of the target strand, and an Iowa Black® quencher was attached to the 5' end of the same strand. The dsDNA target included a well-characterized model AsCas12a targetable sequence containing an Acidaminococcus sp. Cas12a enzyme (AsCas12a) cleavage site. Cleavage occurs 19–28 bp from the first thymine of the TTTC protospacer adjacent motif (PAM), with the PAM located 16 nucleotides from the 5′ end of the substrate and the final nucleotide of the spacer sequence located 39 nucleotides from the 5′ end of the substrate. A second dsDNA target was prepared that was identical to the first except for lacking any end-protection. Oligonucleotides containing modifications were ordered from Integrated DNA Technologies (Coralville, Iowa).

[0148] The double-stranded substrate was assembled from the single strands of SEQ ID NO: 1 and SEQ ID NO: 2. To ensure proper annealing of the dsDNA, the target strand and complementary non-target strand oligonucleotides were combined in 1×TE buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA) at a final concentration of 50 μM each, and the mixture was heated to 95° C. for 2 minutes, then slowly cooled to room temperature to produce the double-stranded DNA target.

[0149] [ka]

[0150] A control double-stranded substrate lacking phosphorothioate protection was assembled from the single strands of SEQ ID NO:3 and SEQ ID NO:4.

[0151] [ka] [Example]

[0152] Example 2: Utility of exonuclease-linked fluorescence detection In this example, 100 nM of the substrate described in Example 1 was incubated with 0.5 U / uL of Exonuclease III (both from New England Biolabs, Ipswich, Mass.) in 1X NEBuffer™ 1, pH 7.0, at 37°C. Fluorescence was monitored over time at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A fluorescent signal was generated for the unprotected substrate, but not for the protected substrate. Signals were read using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). The results are shown in Figure 2. An increase in fluorescence is observed only for the unprotected substrate in the presence of Exonuclease III. [Example]

[0153] Example 3: Cleavage of a substrate by Cas12a endonuclease This example demonstrates that cleavage of a phosphorothioate-protected dsDNA substrate by AsCas12a renders the phosphorothioate-protected substrate susceptible to digestion by exonuclease III, generating a fluorescent signal that can be monitored in real time.

[0154] A 60-bp double-stranded DNA (dsDNA) phosphorothioate-protected target, as described in Example 1, was used. Cas12a ribonucleoproteins (RNPs) consisting of Cas12a and guide RNA (crRNA) were formed by incubating purified recombinant Cas12a protein with synthetic crRNA at 37°C for 10 minutes. Prior to RNP formation, the crRNA was heated to 95°C for 2 minutes and slowly cooled to room temperature to allow for proper secondary structure formation. The crRNA component of the RNP provided specificity for a model AsCa12a targetable sequence present in the top strand of the double-stranded substrate. 100 nM of the protected dsDNA target was incubated with 2.5 U / uL of exonuclease III alone or with 2.5 U / uL of exonuclease III and 112.5 nM of AsCa12a RNP. Incubations were performed in 1X NEBuffer™ 1 at pH 7.0. Control reactions contained unprotected dsDNA targets and exonuclease III but no RNPs. Reactions were incubated at 37°C, and fluorescence readings (excitation: 485 nm, emission: 520 nm) were taken every 30 seconds for 600 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). The results are shown in Figure 3. A rapid increase in fluorescence was observed only in the presence of Cas12a RNPs, but not in the no-RNP control, indicating that the end-protected dsDNA targets were degraded after Cas12a cleavage. Unprotected dsDNA targets were degraded by exonuclease III alone. During continuous fluorescence readings, a power outage occurred approximately 200 minutes after the start of the experiment. Readings resumed immediately, but the power outage caused a spike in the fluorescence signal in all samples, which stabilized approximately 10 minutes after readings resumed. [Example]

[0155] Example 4. Linear range of the assay for Cas12a RNP concentration. In this example, reactions between double-stranded substrates, Cas12a RNP, and exonuclease were performed generally as in Example 3, except using 1X Cutsmart®, pH 7.9 buffer (New England Biolabs, Ipswich, Mass.). RNP concentrations ranged from 0.11 nM to 112.5 nM. Negative controls included no crRNA or no Cas12a RNP. Positive controls included unprotected substrate with exonuclease III only (see Example 1). Results are shown in Figure 4. There is a direct correlation between RNP concentration and reaction rate. Reactions at lower RNP concentrations plateau at lower fluorescence intensities. Even in the 0.11 nM sample, a measurable increase in fluorescence can be detected compared to that observed for reactions lacking Cas12a. The results demonstrate a linear dose response for Cas12a concentrations from 0.11 nM to 7 nM. The sensitivity of the assay over a wide range of Cas12a concentrations demonstrates the utility of this quality control (QC) assay. [Example]

[0156] Example 5. Confirmation that exonucleases hydrolyze DNA, but Cas12a does not hydrolyze DNA Cas12a is unique among endonucleases in that it possesses nonspecific exonuclease activity ("trans-shredding"). In this example, we demonstrate that exonuclease III, rather than the trans-activity of Cas12a itself, is responsible for degradation of dsDNA targets after initial cleavage by Cas12a-RNP.

[0157] In this example, reactions between double-stranded substrates, Cas12a RNPs, and exonuclease were performed generally as in Example 3, except using NEBuffer™ 1, pH 7.0. The concentration of dsDNA was varied. Additionally, control reactions contained no exonuclease. This control was included to determine whether Cas12a degrades the substrate alone (without exonuclease) through a secondary, nonspecific nuclease activity known as transactivation. The results are shown in Figure 5. Some increase in fluorescence was observed in the absence of exonuclease III, suggesting a possible limited contribution to the overall signal from Cas12a transactivation. However, the maximum rate of fluorescence was approximately 3.3-fold higher in the presence of exonuclease III, suggesting that exonuclease III was present at a sufficient concentration to ensure that the reaction rate was not limited by Cas12a transactivation. The highest rate observed was for the unprotected dsDNA substrate with exonuclease III alone, indicating that exonuclease III activity was not rate-limiting. [Example]

[0158] Example 6. Linear range of the assay with respect to DNA substrate concentration. In this example, a reaction between a double-stranded substrate, Cas12a RNP, and an exonuclease was performed similarly to Example 5. All other reagents were kept constant, and the DNA substrate concentration was varied between 2.06 nM and 500 nM. The results are shown in Figure 6. The assay was observed to be sensitive enough to detect target dsDNA sequences down to at least 4.1 nM and to exhibit a linear dose response over a wide range of target dsDNA concentrations. [Example]

[0159] Example 7 (Prophetic): Cleavage of a substrate by Cas9 endonuclease. In this example, the experiment described in Example 3 is performed using Cas9 endonuclease instead of Cas12a endonuclease. The double-stranded DNA (dsDNA) phosphorothioate-protected target is similar to SEQ ID NO:1 / SEQ ID NO:2, except that it contains a PAM sequence recognized by Cas9. Additionally, the dsDNA target contains a fluorophore and quencher pair at the 5' end of the non-target and target strands. Incubation is performed in 1X Cutsmart® buffer. Purified recombinant Cas9 protein is incubated with synthetic crRNA at 37°C for 10 minutes to form a Cas9 ribonucleoprotein (RNP) consisting of SpyCas9 (Streptococcus pyogenes Cas9) and a guide RNA (crRNA). The crRNA component of the RNP provides specificity for SpyCas9 and double-stranded substrates. The protected dsDNA target is incubated with exonuclease III and either a Cas9 RNP. One or more control reactions are included, e.g., those omitting RNP, exonuclease, or exonuclease protection. Reactions are incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore are taken every 30 seconds for 600 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). [Example]

[0160] Example 8 (Prophetic): Cleavage of a Substrate by a Restriction Endonuclease In this example, the experiment described in Example 3 is performed using a restriction endonuclease, such as a type II restriction endonuclease, instead of Cas12a endonuclease. The double-stranded DNA (dsDNA) phosphorothioate-protected target is similar to SEQ ID NO:1 / SEQ ID NO:2, except that it contains a recognition sequence for the restriction endonuclease being tested. The protected dsDNA target is incubated with Exonuclease III and the restriction endonuclease in an appropriate buffer that allows for both restriction endonuclease and Exonuclease III activity. One or more control reactions are included, e.g., those in which the restriction endonuclease is omitted, those in which the exonuclease is omitted, or those in which exonuclease protection is omitted. The reactions are incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore are taken every 30 seconds for 600 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). [Example]

[0161] Example 9. Cleavage of FAM substrate by CRISPR Cas12a In this example, the substrate shown in Figure 7 ("FAM Substrate") was designed. The 60 bp target contained the Cas12a protospacer adjacent motif (PAM) and the well-characterized AAVS1 spacer sequence. This substrate has a deoxythymidine (dT)-conjugated fluorescein (FAM) paired with an Iowa Black dark quencher at the 5' end upstream of the PAM. Both 3' ends are protected with phosphorothioate linkages (Pt). A control substrate lacked Pt protection at the 3' end ("Unprotected FAM Substrate").

[0162] The substrate (Figure 7) was cleaved in a reaction mixture containing FAM substrate, Exo III, and AsCas12a ribonucleoprotein complex (RNP) in NEBuffer1 at 0.42-18 nM RNP, 2.5 kU / mL Exo III, and 100 nM dsDNA target. The reaction proceeded at 37 °C, and the fluorescence data shown in Figure 8 was collected. The rate of fluorescence increase over a period of 50-150 min was plotted as a function of Cas12a concentration. [Example]

[0163] Example 10. Cleavage of TAMRA substrate by CRISPR Cas12a In this example, a substrate ("TAMRA substrate") was designed that was identical to the FAM substrate shown in Figure 7 except that a TAMRA-NHS ester was used instead of deoxythymidine (dT)-conjugated fluorescein (FAM). Like the FAM substrate (Figure 7), both 3' ends were protected with phosphorothioate linkages (Pt). A control substrate did not have Pt protection at the 3' end ("unprotected TAMRA substrate").

[0164] The substrate was cleaved in a reaction mixture containing the TAMRA substrate, Exo III, and AsCas12a ribonucleoprotein complex (RNP). The reaction mixture contained 18 nM RNP (or control 1x NCA buffer), 2.5 kU / mL Exo III, and 100 nM dsDNA substrate in NEBuffer 1. The reaction proceeded at 37°C, and the fluorescence data shown in Figure 9 was collected. [Example]

[0165] Example 11. Alternative fluorophore placement In this example, a series of CRISPR Cas12a substrates (similar to those shown in Figure 7) were designed with alternative fluorophore arrangements. Figure 10 shows a "non-targeted FAM" substrate (same as shown in Figure 7, with FAM upstream of the PAM on the same strand), a "targeted FAM" substrate (FAM on the opposite strand from the PAM), and a "dual FAM" substrate (FAM on both the same and opposite strands as the PAM).

[0166] The three substrates were cleaved in a reaction mixture containing one of the three FAM substrates, Exo III, and the AsCas12a ribonucleoprotein complex (RNP). The reaction mixture contained 18 nM RNP (or control 1x NCA buffer), 2.5 kU / mL Exo III, and 100 nM dsDNA substrate in NEBuffer1. The reaction proceeded at 37°C, and fluorescence data was collected (shown in Figure 11). The data are also presented as a comparison of all protected substrates (Figure 12) and the percentage fluorescence change in the various reaction mixtures (Figure 13). [Example]

[0167] Example 12. Optimization of exonuclease concentration. In this example, the exonuclease was titrated to optimize the exonuclease concentration. Cleavage reactions were set up in NEBuffer1 buffer and included the "target strand" FAM substrate (Figure 10), 20.25 nM AsCas12a RNP (cas12a:crRNA 2:1), various amounts of ExoIII (156.25-2500 U / mL), and FAM-labeled substrate. Control reactions included unprotected substrate, no RNP, or no ExoIII. Reactions proceeded at 37°C. Results are shown in Figure 14. [Example]

[0168] Example 13. Cleavage with Cas9 endonuclease In this example, we demonstrated the applicability of the assay to Cas9. This example consists of a validation of Predictive Example 7. As proposed in Example 7, double-stranded DNA (dsDNA) was designed according to Figures 1 and 7. The substrate was protected with phosphorothioates and contained a PAM sequence recognized by Cas9, a spacer sequence on the same strand as the PAM (non-target strand), a fluorophore, and a quencher. In this experiment, the Cas9 guide targeted the opposite strand from the Cas12a guide.

[0169] A single guide RNA (sgRNA) for the Cas9 endonuclease was designed in combination with the crRNA and tracrRNA. As proposed in Example 7, incubation was performed in 1X Cutsmart® buffer. Purified recombinant Cas9 was incubated with the sgRNA at 37°C for 10 minutes to form a Cas9 ribonucleoprotein (RNP) consisting of SpyCas9 (Streptococcus pyogenes Cas9) and a guide RNA (single guide RNA (sgRNA)). The reaction contained 50 nM SpyCas9, 1 kU / mL ExoIII (New England Biolabs, Ipswich, Mass.), and 110 nM dsDNA substrate in a 200 uL reaction volume. As proposed in Example 7, a control reaction omitting the RNP (ExoIII-only reaction) was included. Reactions were incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore were taken every 30 minutes for 1000 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). All reactions were performed in triplicate, and the average results for each data point are shown in Figure 15. [Example]

[0170] Example 14. Cleavage with restriction endonucleases This example demonstrates the applicability of the assay to type II restriction endonucleases. This example consists of a validation of predictive example 8.

[0171] As proposed in Example 8, double-stranded DNA (dsDNA) phosphorothioate-protected substrates were designed according to Figure 1. These substrates contained recognition sequences for one of the restriction endonucleases BsaI, BcoDI, and SalI. As proposed in Example 7, each substrate was incubated with Exonuclease III and a restriction endonuclease in 1X Cutsmart® buffer. Each reaction mixture contained 300 U of one of BsaI HF v2, BcoDI, and SalI (New England Biolabs) and 1.25 kU of ExoIII. The reactions were incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore were taken every minute for 300 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif.). The results are shown in Figure 16.

[0172] Although the present invention has been described in detail with reference to specific examples, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Accordingly, the scope of the present invention should not be limited by the examples described herein, but rather by the claims set forth below.

Claims

1. A composition when used to detect the activity of an endonuclease, the composition comprising: (a) a donor fluorophore and an acceptor fluorophore that form a fluorescence resonance energy transfer (FRET) pair; (b) at least one structure in at least one nucleic acid strand that inhibits cleavage of the substrate by an exonuclease; and (c) a recognition sequence and cleavage site for an endonuclease, wherein the endonuclease is selected from the group consisting of a nucleic acid-guided endonuclease, a zinc finger nuclease (ZFN), a ZFN conjugated to Fok I, a transcription activator-like effector nuclease (TALEN), an Argonaute endonuclease, an Arcus endonuclease, an engineered endoribonuclease; CAS3 and Cas7-11, and a type II restriction endonuclease. a nucleic acid substrate for detecting the activity of an endonuclease comprising the donor and acceptor fluorophores are not separated by an endonuclease cleavage site, The composition further comprises an exonuclease.

2. 2. The composition of claim 1, wherein the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease T, BAL-31 exonuclease, RecJf exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II.

3. 3. The substrate of claim 1 or 2, wherein the donor fluorophore is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), coumarin dyes, Alexa Fluor dyes, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, Texas Red, and BODIPY® dyes.

4. Acceptor fluorophores include tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL (4-[[4-(dimethylamino)-phenyl]-azo]-benzoic acid), Cy5 and Cy5.5, anthraquinone dyes, nitrothiazole dyes, nitroimidazole dyes, LC-Red 610, LC-Red 640, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ, and Iowa Black FQ.

4. The composition of any one of claims 1 to 3, wherein the fluorescein is selected from the group consisting of RQ, HEX (hexachloro-fluorescein), TET (tetrachloro-fluorescein), JOE (5'-dichloro-dimethoxy-fluorescein), BODIPY® dyes, Eclipse quenchers (4-[[2-chloro-4-nitro-phenyl]-azo]-aniline, BHQ-1 ([(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline), BHQ-2 ([(4-(1-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline), and pyridinyl-isoquinoline-dione dyes.

5. 5. The composition of claim 1, wherein the exonuclease is selected from exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I, and exoribonuclease II.

6. 2. The composition of claim 1, wherein the nucleic acid-guided endonuclease is a CRISPR class I (CASCADE) endonuclease and the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'.

7. the nucleic acid-guided endonuclease is a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease; 2. The composition of claim 1, wherein the substrate comprises a protospacer adjacent motif (PAM) consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNNCNNA-3', 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

8. 2. The composition of claim 1, wherein the nucleic acid-guided endonuclease comprises an endonuclease and a nucleic acid targeting nucleic acid (NATNA), wherein the NATNA comprises DNA and RNA nucleotides.

9. The composition of any one of claims 1 to 8, wherein the structure that inhibits cleavage of the substrate by an exonuclease is selected from a hairpin, a strand overhang, and a nucleic acid modification.

10. 1. A method for detecting endonuclease activity in a sample, comprising: (a) contacting a sample with a reaction mixture comprising the composition of any one of claims 1 to 9 under conditions suitable for endonuclease and exonuclease cleavage of the nucleic acid substrate; and (b) measuring the fluorescence emitted by the reaction mixture as a result of endonuclease cleavage and subsequent exonuclease cleavage, wherein a change in fluorescence indicates endonuclease activity. A method comprising:

11. The method of claim 10, wherein the nucleic acid substrate or the endonuclease is in an unpurified form.

12. A kit for use in detecting endonuclease activity, comprising the composition according to any one of claims 1 to 9.

13. 12. A device when used to detect the activity of an endonuclease by the method of claim 10 or 11, comprising a reaction chamber for carrying out an enzymatic reaction and a fluorescence detector.

14. 1. A method for detecting the presence of a target nucleic acid in a sample, comprising: (a) contacting a sample containing a single-stranded or single-stranded target nucleic acid with the composition of any one of claims 1 to 9, wherein the nucleic acid substrate is a probe capable of hybridizing to the target nucleic acid, the probe being single-stranded or single-stranded under conditions suitable for forming a duplex between the probe and the target nucleic acid and for endonuclease and exonuclease cleavage of the substrate; and (b) measuring the fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample. A method comprising:

15. 15. The method of claim 14, wherein the sample comprises a crude preparation of nucleic acid.

16. A kit for use in carrying out a diagnostic method comprising detecting a target nucleic acid according to the method of claim 14, the kit comprising the composition of claim 1, wherein the target nucleic acid is selected from a sequence characteristic of a bacterium, a sequence characteristic of a virus, a sequence characteristic of a parasite, and a patient sequence characteristic of a disease or condition of the patient.

17. 1. A method for optimizing an endonuclease digestion reaction, comprising: (a) preparing a series of reaction mixtures using an exonuclease and a nucleic acid substrate according to the composition of any one of claims 1 to 9; (b) contacting each of a series of reaction mixtures with a different amount of endonuclease; (c) measuring the fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of the endonuclease; (d) selecting the amount of endonuclease that results in the highest fluorescence of the reaction mixture or the highest rate of increase in fluorescence of the reaction mixture as the optimal endonuclease concentration. A method comprising:

18. 1. A method for optimizing a CRISPR endonuclease digestion reaction, comprising: (a) preparing a series of reaction mixtures using a CRISPR endonuclease, an exonuclease, and a nucleic acid substrate, wherein the nucleic acid substrate comprises: i. a donor fluorophore that forms a fluorescence resonance energy transfer (FRET) pair and acceptor fluorophore; ii. at least one structure in at least one strand that inhibits cleavage of the substrate by an exonuclease; and iii. Recognition sequence for endonuclease containing; (b) contacting each of the series of reaction mixtures with a series of nucleic acid targeting nucleic acids (NATNAs); (c) measuring the fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of the endonuclease; (d) Selecting the NATNA that results in the highest fluorescence of the reaction mixture as the optimal NATNA. A method comprising:

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