RU2848334C2 - Real-time endonuclease activity assay associated with exonuclease - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a nucleic acid substrate for detecting endonuclease activity and use thereof. Disclosed is a nucleic acid substrate for detecting endonuclease activity comprising: (a) a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a pair for fluorescence resonance energy transfer (FRET); (b) at least one structure on at least one nucleic acid chain inhibiting cleavage of a substrate by an exonuclease used to detect endonuclease activity, where the structure that inhibits the cleavage of the substrate by exonuclease is selected from a hairpin, an overhanging chain and a nucleic acid modification, and where said structure is capable of inhibiting the activity of said exonuclease; and (c) an endonuclease recognition sequence. Invention also discloses a composition and a kit for detecting endonuclease activity, a method for detecting endonuclease activity, a method for detecting the presence of a target nucleic acid in a sample, a kit for carrying out a diagnostic procedure, a method for optimizing endonuclease cleavage reactions.

EFFECT: invention provides effective detection of endonuclease activity.

19 cl, 16 dwg, 14 ex

Description

Cross-references to related applications

[001] This application claims the benefit of U.S. Provisional Application Serial No. 63/272,091, filed October 26, 2021.

Field of invention

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

Federally Sponsored Research Statement

[003] Absent.

List of sequences

[004] Marked with a special icon.

Prerequisites for the creation of the invention

[005] Endonucleases are enzymes widely used in laboratory practice and in the field of molecular diagnostics. The process of designing and isolating new and improved endonucleases requires the development of rapid and convenient methods for assessing endonuclease activity. The most widely used endonuclease assays are very complex and have low efficiency. Furthermore, the commonly used methods are not quantitative. There are currently no generally accepted methods for assessing differences in activity between different sources, variants, or lots of endonucleases. Similarly, there are no simple and rapid methods for assessing different targets that allow for accurate determination of endonuclease specificity.

[006] Many current endonuclease assays are unsuitable for use with crude cell lysates or partially purified protein preparations because host nucleases rapidly degrade the target DNA. There is an unmet need for a rapid endonuclease assay that could be performed on crude lysates and semi-purified eluted fractions early in the purification process. Such an assay would improve enzyme purification and production, as well as accelerate the overall enzyme design process.

Description of the essence of the invention

[007] The present invention relates to methods, compositions, and kits for assessing endonuclease activity. The present invention utilizes a double-stranded nucleic acid substrate with a protected end labeled with a reporter fluorophore and a quencher fluorophore. An exonuclease hydrolyzes the substrate and provides fluorescence only after the endonuclease has cleaved the substrate.

[006] In one embodiment, the present invention relates to a nucleic acid substrate for detecting endonuclease activity, comprising: a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure on at least one strand of the nucleic acid that inhibits cleavage of the substrate by an exonuclease; and an endonuclease recognition sequence. The structure that inhibits cleavage of the substrate by an 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. The exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, T5 exonuclease, T7 exonuclease, lambda exonuclease, T exonuclease, 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 the acceptor fluorophore can be located at a distance of 1 to 12 nucleotides from each other on the same strand of the substrate or on different strands of the substrate. In some embodiments, the substrate is formed by a single strand. In some embodiments, one of the donor fluorophores and the 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 dyes, Pacific Orange dyes, Texas Red dyes, and BODIPY®. In some embodiments, the acceptor fluorophore is selected from the group consisting of tetramethyl-6-carboxyrrhodamine (TAMRA), tetrapropano-6-carboxyrrhodamine (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 (hexachlorofluorescein), TET (tetrachlorofluorescein), JOE (5'-dichlorodimethoxyfluorescein), BODIPY® dyes, Eclipse quencher (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-nitrophenyl)-azo)-yl-((2,5-dimethoxy-phenyl)azo)]aniline), and pyridinyl-isoquinoline-dione dyes. In some embodiments, the endonuclease is nickase. In some embodiments, the exonuclease is selected from exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease. In some embodiments, the endonuclease is a regulated endonuclease nucleic acid, such as a class I CRISPR endonuclease (CASCADE). 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-regulated endonuclease is a class II CRISPR endonuclease. In some embodiments, the endonuclease is CRISPR Cas9 endonuclease. In some embodiments, the endonuclease is 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-regulated endonuclease comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide molecule and a double-stranded guide molecule. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a region of a substrate. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nuclease (ZFN), Fok I-conjugated ZFN, transcription activator-like effector nuclease (TALEN), Endo TT 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 In some embodiments, the structure that inhibits cleavage of the substrate by the exonuclease is selected from a hairpin, a protruding strand, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages. In some embodiments, the endonuclease is a deoxyribonuclease and the substrate comprises DNA. In some embodiments, the endonuclease is a ribonuclease and the substrate comprises RNA. In some embodiments, the ribonuclease is selected from a ribozyme, a hammerhead ribozyme, a DNAzyme, a PNAzyme, or an engineered endoribonuclease.

[009] In one embodiment, the present invention relates to a composition for detecting endonuclease activity, comprising a nucleic acid substrate as described above and 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 single-strand break hydrolysis. In some embodiments, the exonuclease is selected from T5 exonuclease, T7 exonuclease, lambda exonuclease, exonuclease III, and Bal31 exonuclease. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In some embodiments, the nucleic acid-regulated endonuclease is a class I CRISPR endonuclease (CASCADE), 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 of the invention, the nucleic acid-regulated 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid targeting another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide molecule and a double-stranded guide molecule. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a substrate region. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), an Endo TT single-stranded endonuclease, an Argonaute endonuclease, an 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 cleavage of the substrate by the exonuclease is selected from a hairpin, an overhang, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages.

[0010] In one embodiment, the present invention relates to a method for detecting endonuclease activity, comprising: contacting an endonuclease and an exonuclease with a reaction mixture and with a nucleic acid substrate comprising a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure on at least one strand that inhibits cleavage of the substrate by the exonuclease; and an endonuclease recognition sequence; and evaluating fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates endonuclease activity. In some embodiments, the at least one structure that inhibits cleavage of the substrate by an 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 of these structures. 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 simultaneously contacted with the endonuclease and the exonuclease. In some embodiments, the reaction mixture is first contacted with an endonuclease and then with an 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-regulated endonuclease. In some embodiments, the nucleic acid-regulated endonuclease is a class I CRISPR endonuclease (CASCADE), 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 of the invention, the nucleic acid-regulated 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid targeting another nucleic acid (NATNA), such as a CRISPR guide RNA selected from single-stranded RNA and double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a substrate region. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, contacting comprises contacting a series of reaction mixtures containing the same NATNA with a series of different endonucleases. In some embodiments, contacting comprises contacting a series of reaction mixtures containing the same endonuclease with a series of different NATNAs. In some embodiments, contacting comprises contacting a series of reaction mixtures containing the same ingredients under different reaction conditions. In some embodiments, contacting comprises contacting a series of reaction mixtures containing the same ingredients with different endonuclease isolates. In some embodiments, 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 a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 cleavage of the substrate by the exonuclease is selected from a hairpin, an overhang and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages. In some embodiments, the nucleic acid substrate or endonuclease is present in an unpurified form.

[0011] In one embodiment, the present invention relates to a kit for detecting endonuclease activity, comprising: a nucleic acid substrate comprising a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure on at least one strand that inhibits cleavage of the substrate by an exonuclease; an endonuclease recognition sequence; and an exonuclease. In some embodiments, the at least one structure that inhibits cleavage of the substrate by an 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 of these structures. 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 being tested is a nucleic acid-regulated endonuclease. In some embodiments, the nucleic acid-regulated endonuclease is a class I CRISPR endonuclease (CASCADE), 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-regulated 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 kit further comprises a nucleic acid that targets another nucleic acid (NATNA) and is capable of forming a complex with the test endonuclease. In some embodiments, NATNA is a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a substrate region. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), an Endo TT single-stranded endonuclease, an Argonaute endonuclease, an 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 cleavage of the substrate by the exonuclease is selected from a hairpin, an overhang, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages.

[0012] In one embodiment, the present invention relates to a device for detecting endonuclease activity using a substrate according to claim 1, comprising: a reaction chamber for carrying out enzymatic reactions and a fluorescence detector.

[0013] In one embodiment, the present invention relates to a method for detecting the presence of a target nucleic acid in a sample, wherein said method comprises: 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, wherein said probe comprises: a donor fluorophore and an acceptor fluorophore, and wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage of the probe by the exonuclease; and an endonuclease recognition sequence, and evaluating 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 an 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 of these structures. 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 sequence characteristic of a disease or condition of a patient. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In some embodiments, the endonuclease is a class I CRISPR endonuclease (CASCADE), 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-regulated endonuclease comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises cr-RNA and tracr-RNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a probe region. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nuclease (ZFN), Fok I-conjugated ZFN, transcription activator-like effector nuclease (TALEN), Endo TT single-stranded endonuclease, Argonaute endonuclease, Arcus endonuclease, 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 inhibiting cleavage of the probe by an exonuclease is selected from a hairpin, an overhang, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude nucleic acid preparation.

[0014] In one embodiment, the present invention relates to 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 of which is capable of hybridizing to two or more target nucleic acids, wherein each probe comprises: a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage of the probes by the exonuclease; and an endonuclease recognition sequence, and evaluating 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 two or more nucleic acid probes comprise the same fluorophore. In some embodiments, the at least one structure that inhibits cleavage of the probes by an 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 of these structures. 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 a sequence characteristic of a bacterium, a sequence characteristic of a virus, a sequence characteristic of a parasite, and a sequence characteristic of a disease or condition of a patient. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In some embodiments, the endonuclease is a class I CRISPR endonuclease (CASCADE), 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 of the invention, the nucleic acid-regulated 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, the NATNA comprises crRNA and tracrRNA. In some embodiments, at least one NATNA is used for each of two or more probes, and each NATNA comprises a targeting region capable of hybridizing to a region of at least one 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 a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 cleavage of the probe by the exonuclease is selected from a hairpin, an overhang and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude nucleic acid preparation.

[0015] In one embodiment, the present invention relates to a method for detecting the presence of a target nucleic acid in a sample, wherein said method comprises: attaching to a target nucleic acid in a sample: a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one exonuclease cleavage inhibitory structure; and an endonuclease recognition sequence, to form a modified nucleic acid, and contacting the sample with the endonuclease and the exonuclease; and evaluating 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 exonuclease cleavage inhibiting structure is selected from a 3'-5' exonuclease cleavage inhibiting structure at each 3' end, a 5'-3' exonuclease cleavage inhibiting structure at each 5' end, 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 attachment is accomplished by ligating adaptors including a donor fluorophore, an acceptor fluorophore, and an exonuclease cleavage inhibitory structure. In some embodiments, prior to attachment, the target nucleic acid is amplified by PCR. In some embodiments, the attachment is accomplished by one or more rounds of extension using amplification primers comprising a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; and an exonuclease cleavage inhibitory structure. 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 sequence characteristic of a disease or condition of a patient. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In some embodiments, the endonuclease is a class I CRISPR endonuclease (CASCADE), 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 of the invention, 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid targeting another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing with a target nucleic acid or a modified target nucleic acid. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 exonuclease cleavage-inhibiting structure is selected from a hairpin, an overhang, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages. In some embodiments, the sample comprises a crude nucleic acid preparation.

[0016] In one embodiment, the present invention relates to a kit for performing a diagnostic procedure according to the method of claim 116, wherein said kit comprises: an endonuclease and a nucleic acid probe capable of hybridizing with a target nucleic acid, wherein the probe comprises: a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage of the probe by an exonuclease; and an endonuclease recognition sequence; and assessing fluorescence emitted by a reaction mixture, wherein a change in fluorescence indicates the presence of a target nucleic acid in a sample, 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 sequence characteristic of a disease or condition of a patient. In some embodiments, the at least one structure that inhibits cleavage of the probe by an 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 of these structures. 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, T exonuclease, 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-regulated endonuclease. In some embodiments, the nucleic acid-regulated endonuclease is a class I CRISPR endonuclease (CASCADE) 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 of the invention, the nucleic acid-regulated 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-regulated endonuclease is a CRISPR endonuclease in complex with a nucleic acid targeting another nucleic acid (NATNA), and NATNA is a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA is capable of interacting with the endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 exonuclease cleavage-inhibiting structure is selected from a hairpin, an overhang, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages.

[0017] In one embodiment, the present invention relates to a kit for detecting the presence of a target nucleic acid in a sample, wherein said method comprises one or more oligonucleotides capable of attaching to a target nucleic acid to form a modified nucleic acid, wherein the oligonucleotides comprise: a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure that inhibits cleavage by an exonuclease; wherein the modified nucleic acid comprises an endonuclease recognition sequence and an endonuclease, and 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 sequence characteristic of a disease or condition of a patient. In some embodiments, the at least one exonuclease cleavage inhibiting structure is selected from a 3'-5' exonuclease cleavage inhibiting structure at each 3' end, a 5'-3' exonuclease cleavage inhibiting structure at each 5' end, 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 attachment is accomplished by ligating oligonucleotides to a target nucleic acid, and the kit optionally includes a ligase. In some embodiments, the attachment is accomplished through one or more extension cycles, with the oligonucleotides acting as amplification primers, and the kit optionally includes reagents for carrying out the amplification. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In some embodiments, the endonuclease is a class I CRISPR endonuclease (CASCADE), 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 of the invention, 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid targeting another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a target nucleic acid or a modified target nucleic acid. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides. In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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, Ca3 and Cas7-11, and a restriction endonuclease. In some embodiments, the exonuclease cleavage inhibiting structure is selected from a hairpin, an overhanging strand, and a nucleic acid modification, such as one or more (5 or more) phosphorothioate linkages.

[0018] In one embodiment, the present invention relates to a method for optimizing endonuclease cleavage reactions, wherein the method comprises: preparing a series of reaction mixtures with an exonuclease and a nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure on at least one strand that inhibits cleavage of the substrate by the exonuclease; and an endonuclease recognition sequence; contacting each of the series of reaction mixtures with different amounts of endonuclease; assessing fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates endonuclease activity; selecting the amount of endonuclease that produces the highest fluorescence of the reaction mixture, or the highest rate of increase in fluorescence of the reaction mixture, as the optimal concentration of endonuclease. In some embodiments, the structure that inhibits cleavage of the substrate by an 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 of these structures. 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 simultaneously contacted with the endonuclease and the exonuclease. In some embodiments, the reaction mixture is first contacted with an endonuclease and then with an 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-regulated endonuclease such as a class I CRISPR endonuclease (CASCADE), 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', or a CRISPR Cas9 endonuclease or a CRISPR Cas12a endonuclease, and the substrate comprises a motif, adjacent to the protospacer (PAM) and 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, a nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), such as a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises crRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a substrate region. In some embodiments, NATNA is capable of interacting with an endonuclease. In some embodiments, NATNA comprises DNA and RNA nucleotides.

[0021] In some embodiments, the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), an Endo TT single-stranded endonuclease, an Argonaute endonuclease, an 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 cleavage of the substrate by the exonuclease is selected from a hairpin, an 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 5 or more phosphorothioate linkages.

[0022] In one embodiment, the present invention relates to a method for optimizing CRISPR endonuclease cleavage reactions, the method comprising: preparing a series of reaction mixtures with a CRISPR endonuclease, an exonuclease, and a nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore, wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair; at least one structure on at least one strand that inhibits cleavage of the substrate by the exonuclease; and an endonuclease recognition sequence; contacting each of the series with nucleic acids targeting another nucleic acid (NATNA); assessing fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates endonuclease activity; and selecting the NATNA that produces the highest fluorescence of the reaction mixture or the highest rate of increase in fluorescence of the reaction mixture as the optimal NATNA. In some embodiments, the at least one structure that inhibits cleavage of the substrate by an 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 of these structures. 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 simultaneously contacted with the endonuclease and the exonuclease. In some embodiments, the reaction mixture is first contacted with the endonuclease and then with the exonuclease.

[0023] In some embodiments of the invention, no purification steps are 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 endonuclease (CASCADE), 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, NATNA is a CRISPR guide RNA selected from a single-stranded guide RNA and a double-stranded guide RNA. In some embodiments, NATNA comprises scRNA and tracrRNA. In some embodiments, NATNA comprises a targeting region capable of hybridizing to a region of the substrate. In some embodiments of the invention, NATNA comprises DNA and RNA nucleotides.

[0025] In some embodiments, the structure that inhibits cleavage of the substrate by an exonuclease is selected from a hairpin, a protruding strand, 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 5 or more phosphorothioate linkages.

Brief description of the drawings

[0026] Figure 1 shows a diagram for a substrate and a method according to the invention.

[0027] Figure 2 illustrates experimental confirmation of the value of exonuclease-associated fluorescence detection (Example 2).

[0028] Figure 3 shows the cleavage of the substrate (Figure 1) by the endonuclease Cas12a (Example 3).

[0029] Figure 4 shows the experimental determination of the linear range of the assay as a function of Cas12a RNP concentration (Example 4).

[0030] Figure 5 illustrates experimental confirmation that exonuclease, but not Cas12a, hydrolyzes DNA (Example 5).

[0031] Figure 6 shows the experimental determination of the linear range of the assay as a function of DNA substrate concentration (Example 6).

[0032] Figure 7 shows a detailed schematic of the FAM-labeled substrate for CRISPR endonucleases.

[0033] Figure 8 shows the use of an exonuclease assay to cleave a FAM-labeled substrate (Figure 7) with the Cas12a endonuclease (Example 9).

[0034] Figure 9 shows the use of an exonuclease assay to cleave a TAMRA-labeled substrate with the Cas12a endonuclease (Example 10).

[0035] Figure 10 shows a schematic of FAM-labeled substrates with different arrangements of the FAM fluorophore.

[0036] Figure 11 shows the use of an exonuclease assay to cleave various FAM-labeled substrates (Figure 10) with the Cas12a endonuclease (Example 11).

[0037] Figure 12 shows the data shown in Figure 11 in a systematic manner.

[0038] Figure 13 shows the rate of change of fluorescence of reaction mixtures (Example 11).

[0039] Figure 14 shows the titration of exonuclease in an assay used to cleave the non-targeted substrate FAM with the endonuclease Cas12a (Example 12).

[004 0] Figure 15 shows the use of an exonuclease assay to cleave a FAM-labeled substrate with Cas9 endonuclease (Example 13).

[0041] Figure 16 shows the use of an exonuclease assay to cleave a FAM-labeled substrate with restriction endonucleases (Example 14).

Detailed description of the invention

[0042] Definitions

[0043] The following definitions are provided for a better understanding of the essence of the invention.

[0044] The term "endonuclease" refers to an enzyme that catalyzes the hydrolysis of a phosphodiester bond between two nucleoside residues within a polynucleotide (DNA or RNA) in which neither nucleoside residue is terminal.

[0045] The term "exonuclease" refers to an enzyme that catalyzes the hydrolysis of the phosphodiester bond between the terminal nucleoside residue and the penultimate nucleoside residue within a polynucleotide (DNA or RNA). Exonucleases can be processive or capable of stepwise removing multiple nucleoside residues from the end of a nucleic acid chain.

[0046] The term "CRISPR repeat" or "CRISPR repeat sequence" refers to the minimal CRISPR repeat sequence.

[0047] The term "endoribonuclease" refers to an enzyme that catalyzes the hydrolysis of a phosphodiester bond in RNA. In some embodiments, the endoribonuclease may be a site-directed polypeptide. The endoribonuclease may be a member of the CRISPR system (e.g., Type I, Type II, Type III). The endoribonuclease may belong to the repeat-associated prognostic protein (RAMP) superfamily of proteins (e.g., the Cas6, Cas6, Cas5 families). Endoribonucleases may also include RNase A, RNase H, RNase I, members of the RNase III family (e.g., Drosha, Dicer, RNase N), RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase VI, RNase V.

[0048] The term "inhibition" refers to the ability of a chemical structure to partially or completely inhibit a chemical reaction. One skilled in the art recognizes that partial or complete inhibition depends on the sensitivity of the detection methods. The term "inhibition of cleavage" in relation to a nuclease refers to the ability to significantly reduce the amount of cleavage product. The term "prevention of cleavage" in relation to a nuclease refers to the ability to reduce the amount of cleavage product below the detection level.

[0049] The term "NATNA" refers to a nucleic acid that targets another nucleic acid. NATNA may be part of a programmable endonuclease system, such as the CRISPR system. NATNA may consist of two polynucleotides that target a nucleic acid ("double-stranded guide molecule"), including CRISPR RNA (crRNA) and CRISPR trans-activating RNA (tracrRNA). NATNA may consist of an engineered polynucleotide that targets a single nucleic acid ("single-stranded guide molecule") and contains crRNA and tracrRNA connected by a fusion region (linker). NATNA may also consist of a naturally occurring single-stranded guide RNA (e.g., Cas12a guide RNA). CrRNA may contain a targeting region and an activating region. TracrRNA may contain a region capable of hybridizing with the activating region of crRNA. The term "non-targeting region" refers to the region capable of hybridizing with the target nucleic acid sequence. The term "activating region" refers to the region that interacts with a polypeptide, such as the CRISPR nuclease.

[0050] 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 enzymatic substrate. Fluorophore-labeled nucleic acids are a popular type of probe or enzymatic substrate. Probes and substrates labeled with two fluorophores, forming a FRET nappy, are particularly popular. Popular types of dual-labeled probes include Taqman and Molecular Beacon probes.

[0051] In the Taqman assay (US Patent No. 5,210,015), a dual-labeled oligonucleotide probe hybridizes to the resulting amplification product during PCR. Fluorescence is detected when the 5'-3' exonuclease activity of DNA polymerase hydrolyzes the probe between the two fluorophores.

[0052] Molecular beacons are dual-labeled hairpin probes in which binding of the probe to the target results in unwinding of the hairpin. This unwinding separates the FRET pair, allowing detection of the fluorescence of the donor fluorophore. Tyagi S, et al., (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 14(3):303.

[0 053] Nucleic acid substrates labeled with two fluorophores that form a FRET pair and are capable of transmitting conditional fluorescence can be used to detect enzymatic activity in vitro. Enzymatic activity can indicate, for example, the presence of an infectious agent in a sample. The use of probes 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 a microorganism in a sample results in cleavage of the probe by one or more nucleases (e.g., endonucleases or exonucleases). All possible cleavages result in physical separation of the fluorophore and quencher and the emission of detectable fluorescence, which indicates the presence of the microorganism in the sample.

[0054] A similar principle was used in U.S. Patent No. 10,653,800, where the RNA substrate includes 2'-O-methyl-modified pyrimidines and has unique sensitivity to mycoplasma RNase. The substrate is labeled with a fluorophore and a quencher. The presence of mycoplasma in the sample leads to cleavage of the probe by mycoplasma RNase and the emission of detectable fluorescence, indicating the presence of mycoplasma in the sample.

[0055] Fluorophore- and quencher-labeled double-stranded nucleic acid substrates 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, described the cleavage of such a substrate by the trans-cleavage (“trans-mincing”) activity of the CRISPR nuclease Cas12a. The trans-mincing activity is triggered by binding of the Cas12a-crRNA complex to a double-stranded DNA target. The trans-cleavage nuclease activity is directed at any double-stranded DNA in the vicinity of the Cas12a-crRNA-target complex. Milling of the fluorophore- and quencher-labeled substrate results in the emission of detectable fluorescence, indicating the formation of the Cas12a-crRNA-target complex in the sample.

[0056] CRISPR-Cas nuclease activity is typically assessed by incubating the ribonucleoprotein (RNP) complex with a simulated substrate, followed by agarose gel electrophoresis to separate cleaved fragments from the intact substrate. This approach is low throughput and is typically limited to endpoint analysis, thus providing little or no information on reaction kinetics.

[0057] A fluorescence assay has also been described for Cas12a; however, this assay only measures the nonspecific trans activity of the Cas12a enzyme (see Smith CW, Biochemistry. 2020, supra). When the Cas12a RNP interacts with a target sequence and cleaves that sequence (cis activity), Cas12a is activated and nonspecifically cleaves short DNA fragments (trans activity or "trans-mincing" activity). There are several limitations to assessing facet activity as an indicator of cis activity. First, the precise correlation between cis and trans activity is unknown. Second, the surrogate assay provides limited kinetic information about cis activity because the two reactions are performed by the same enzyme and cannot be separated. Finally, this assay does not have general applicability because many endonucleases do not have trans activity.

[0058] A fluorescence assay for assessing 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, 6913-6921). However, this assay only provides endpoint detection and does not provide real-time data, as it relies on denaturation of the DNA substrate after endonuclease digestion.

[0059] The present invention overcomes these drawbacks, providing a convenient, real-time assay for endonuclease activity. The present invention includes a simple, high-throughput fluorescence assay as a quality assessment and control tool.

[0060] The present invention further includes a diagnostic assay for specific endonuclease activity that indicates the presence of a diagnostic target in a sample.

[0061] 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 comprising a double-stranded region, such as a hairpin, through hybridization.

It will be apparent to those skilled in the art that hybridization and double-stranded region formation do not require 100% complementarity along the length of the nucleic acid strands. Under suitable conditions, determined by the ionic strength of the buffer and temperature, a stable hybrid (double-stranded region) can be formed with complementarity between the two nucleic acid strands that is less than 100%, such as 90%, 80%, 75%, or less.

[0062] As further shown in Figure 1, the nucleic acid substrate comprises a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair.

[0063] The fluorophore and quencher may be located at different locations on the double-stranded nucleic acid substrate. In addition, more than one fluorophore may be used. As shown in Figure 10, the fluorophore and quencher may be located on the target strand, on the non-target strand, or on both the target strand and the non-target strand.

[0064] Fluorescence resonance energy transfer (FRET), also known as Foerster resonance energy transfer, is the transfer of excitation energy from one molecule to another without fluorescence or reabsorption. A donor chromophore is converted to an electronically excited state after absorbing light at a specific wavelength. The donor transfers energy to an acceptor, and the acceptor becomes electronically excited. Subsequently, the acceptor's electronically excited state decays, resulting in the emission of detectable light. Because the acceptor reduces or quenches the fluorescence of the donor, it is sometimes called a quencher. The donor is sometimes called a reporter. In traditional FRET technology, 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 in close proximity to each other (e.g., 1-10 nm apart). In some embodiments, the donor fluorophore and acceptor fluorophore are located 0 to 12 nucleotides apart. The donor fluorophore and acceptor fluorophore are located on the same strand of the substrate or on different (opposite) strands of the substrate. Either the donor, the acceptor, or both can be located near the end of a nucleic acid strand, such as the 5' end or 3' end. The donor and acceptor fluorophores can be located on the same strand or on opposite strands. One skilled in the art can easily determine various arrangements of the donor fluorophore and acceptor fluorophore (or reporter fluorophore and quencher) within the double-stranded substrate to achieve the desired proximity of the fluorophores (e.g., 1-10 nm).

[0065] There is ample guidance in the existing literature for selecting appropriate FRET-capable reporter-quencher pairs, see, for example, U.S. Patent Nos. 5,538,848; 8,350,038; and 8,137,616. Typically, as recommended in U.S. Patent Application Publication US20060088855, donor fluorophores absorb energy in the range of 350-800 nm, preferably 350-600 nm or 500-750 nm, and the distance between the donor and acceptor is from 10 to 100 angstroms. 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 Edition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, 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); etc.

[0066] Many donors, acceptors, and donor/acceptor pairs that exhibit the FRET phenomenon are commercially available. Well-known 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, the Alexa Fluor family of dyes, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, and Texas Red. Well-known acceptors include rhodamine dyes such as tetramethyl-6-carboxyrrhodamine (TAMRA) and tetrapropano-6-carboxyrrhodamine (ROX), DABSYL, DABCYL, cyanine dyes including Cy5 and Cy5.5, anthraquinone, nitrothiazole, and nitroimidazole compounds. Additional acceptors include LC-Red 610, LC-Red 64 0, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ, and Iowa Black RQ, and the sulfonated cyanine dyes described in U.S. Patent No. 6,027,709. Well-known donor-acceptor combinations include fluorescein/rhodamine, and in particular carboxyfluorescein/tetramethylrhodamine (FAM/TAMRA). TAMRA as a quencher can also be combined 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. 97,967 6 and consists of an oxidized form of a first carba-NADH-based fluorophore and a second fluorophore that is excited by light at a wavelength of 445-540 nm and has a peak emission energy greater than 560 nm.

[0067] Another group of fluorescent compounds are the naphthylamines, which have an amino group in 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 3-phenyl-7-isocyanatocoumarin, acridines such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, and pyrenes.

[0068] Another category of fluorophores includes the BODIPY® dyes described in U.S. Patent No. 5,994,063. "BODIPY®" refers to a class of modified spectrally distinct fluorophores in which the parent heterocyclic molecule is a dipyrrometheneboron difluoride compound. Most BODIPY® fluorophores have a maximum absorption between approximately 450 and 700 and a maximum emission between approximately 450 and 700. Examples are BODIPY® 503/512-SE (4, 4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-3-propionic acid), BODIPY® 523/547 (4,4-difluoro-5-phenyl-4-bora-3,4a-diaza-3-indacene-3-propionic acid), BODIPY® 530/550 (4, 4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-3-indacene-3-propionic acid), BODIPY® 558/568 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), 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-3,4a-diaza-s-indacene-3-propionic acid) and BODIPY® 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3,4a-diaza-s-indacene-3-propionic acid).

[0069] One type of quencher is a "dark quencher." These non-fluorescent acceptors provide low background fluorescence and thus increase the sensitivity of the assay. When using a dark quencher, the donor fluorophore does not emit light until the quencher is removed from the donor. For example, if the donor and quencher are conjugated to an oligonucleotide, the donor fluorescence can only occur when the quencher is removed by hydrolysis of the oligonucleotide with a nuclease. One example of a dark quencher is DABCYL (4-[[4-(dimethylamino)phenyl]azo]benzoic acid), which quenches donor dyes in the range of 380 to 530 nm. Another dark quencher is Eclipse quencher (4-[[2-chloro-4-nitrophenyl]azo]aniline (available from Epoch Biosciences, Inc.), which has an absorption maximum at 530 nm and provides efficient quenching in the spectrum from 520 to 670 nm. 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-nitrophenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline) (all available from Biosearch Technologies, Inc.).

[0070] Another type of quencher includes the pyridinylisoquinolinedione derivatives disclosed in U.S. Patent No. 8,350,038. These compounds are characterized by low background signal and high quenching efficiency. Another category of quenchers are non-fluorescent cyanine quencher compounds attached to a nucleotide base via a linker compound, as described in U.S. Patent No. 6,348,596. Another category of quenchers are weakly luminescent cyanines that are substituted with one or more heteroaromatic quencher moieties, disclosed in U.S. Patent No. 8,093,411. These quenchers exhibit weak luminescence, or virtually no detectable luminescence, and effectively quench a broad spectrum of luminescent compounds.

[0071] Methods for synthesizing oligonucleotides and covalently attaching fluorophores to nucleic acids are known to those skilled in the art. See, e.g., U.S. Patents 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; and Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3'-thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research, 19:3019 (1991) (3'-sulfhydryl); Giusti et al., PCR Methods and Applications, 2:223-227 (1993); Agrawal et al., Tetrahedron Letters, 31:1543-1546 (1990) (attachment via phosphoramidate linkages); 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 an oligonucleotide using standard phosphoramidite chemistry. By incorporating such nucleotides into the desired position of the oligonucleotide, donor and acceptor fluorophores can be incorporated into any internal or terminal position of the oligonucleotide. In presynthesized fluorophore-labeled nucleotides, the label can be linked to a functional group attached, for example, to the amino group of the nucleotide base. In other embodiments, the label is attached to a portion of the nucleotide via a linker moiety. For example, in some embodiments, the nucleotide base is modified to allow conjugation with a label. For example, U.S. Patent No. 7,759,469 discloses substituted nitroindole nucleotides that can be conjugated with a fluorophore.

[0072] In some embodiments of the invention, the double-stranded nucleic acid substrate has a length of about 10 to about 90 base pairs. For example, for the Cas12a endonuclease, the substrate has a length of 35 to 90 base pairs and may contain a PAM (5 nucleotides), a spacer (20 nucleotides), an end protector (5 nucleotides at each end), with the addition of up to 35 base pairs. In general, endonucleases have different sizes and structures of recognition sequences.

Therefore, for each endonuclease tested, the optimal substrate length can be determined using the calculation shown above. The optimal length and sequence can be determined in silico or calculated empirically. This optimal length ensures the most efficient cleavage by the endonuclease without steric hindrance and is not excessive. Excessive length is associated with high production costs and requires the introduction of additional exonuclease units and additional time to complete the method described here. When selecting the optimal double-stranded nucleic acid substrate length for each endonuclease tested, one or more of these considerations can be taken into account.

[0073] In some embodiments, a nucleic acid substrate of the invention includes chemical modifications. In some embodiments, the modification increases the stability of the nucleic acid duplex. In some embodiments, the modification confers resistance to nuclease digestion or nuclease inhibition. In some embodiments, the modification confers resistance to exonuclease digestion or exonuclease inhibition.

[0074] In some embodiments, the modification is a backbone modification. One type of backbone modification is a modified internucleoside linkage. For example, the modification includes phosphorothioate linkages and internucleoside linkages of heteroatoms.

[0075] Another type of backbone modification is modification of the sugar moiety. In some embodiments, the modification includes the inclusion of a 6-membered morpholine ring in place of the ribose or deoxyribose ring. Another backbone modification includes the introduction of a cyclohexenyl ring in place of ribose or deoxyribose (ceNA). Another backbone modification includes the introduction of locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4'-carbon atom of ribose to form a bicyclic structure having a 2'-C,4'-C-oxymethylene linkage. LNA are characterized by duplex stability and resistance to 3'-5' exonuclease cleavage.

[0076] In some embodiments, the modification is a modification of a nitrogenous base. For example, the double-stranded nucleic acid substrate may include one or more of 5-methylcytosine (5-te-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-haluracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 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, 3-deazaadenine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b,4)benzoxazin-2(3)-one), phenothiazine cytidine (1H-pyrimido(5,4-b(1,4)benzothiazin-2(3)-one), G-complexes such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazine)-2(3)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindol cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-)g)rimidin-2-one), 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nitrogenous bases may be useful for enhancing the binding affinity of a polynucleotide compound. These may include 5-substituted pyrimidines, 6-azapyrimidines, and N-2-, N-6-, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. Substitutions of 5-methylcytosine can increase nucleic acid duplex stability by 0.6–1.2° and may be suitable base substitutions (e.g., in combination with modifications of the 2'-0-methoxyethyl sugar).

[0077] It will be apparent to one skilled in the art that nucleic acid modifications should not be included in the recognition site of the endonuclease being tested if such modification may interfere with the activity of the endonuclease, unless it has been established that the endonuclease is not inhibited by such modification. Similarly, it will be apparent to one skilled in the art that nucleic acid modifications that are known to inhibit exonuclease cleavage should not be included in the portion of the substrate located between the endonuclease recognition site and the fluorophore, so as not to block or inhibit the effectiveness of the method described herein.

[0078] The methods and compositions described herein utilize an exonuclease. Exonucleases are known to those skilled in the art, and many are commercially available, see, for example, Lovett S. T. (2011) The DNA Exonucleases of Escherichia coli. EcoSal Plus, 4(2) and Shevelev, I., Hiibscher, 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 on single-stranded DNA. RecJf is a 5'-3' exonuclease active on single-stranded DNA. Exonuclease III is a 3'-5' exonuclease active on single-stranded DNA and double-stranded DNA. T7 exonuclease, V exonuclease, VIII exonuclease, lambda exonuclease, and T5 exonuclease are 5'-3' exonucleases active on single-stranded DNA and double-stranded DNA. Exonuclease V (RecBCD) and BAL-31 are both 5'-3' and 3'-5' exonucleases active on single-stranded DNA and double-stranded DNA. Depending on the type of nucleic acid end formed by the endonuclease being tested, a skilled artisan can select a suitable exonuclease. The appropriate exonuclease will utilize the end or ends generated by the endonuclease and hydrolyze the nucleic acid substrate described herein. Exonuclease hydrolysis will separate a pair of FRET fluorophores (e.g., donor and acceptor or reporter and quencher) so that fluorescence or a change in fluorescence can be detected.

[0079] As shown in Figure 1, the substrate comprises a structure at at least one of its ends that inhibits or prevents cleavage by an exonuclease. The selection of an exonuclease for inclusion in the methods, compositions, and kits described herein determines the selection of an exonuclease-inhibiting and end-protecting structure in the nucleic acid substrate. For example, if the preferred exonuclease substrate is the 3' end, then the nucleic acid substrate has an exonuclease-inhibiting structure at each of its 3' ends. If the preferred exonuclease substrate is the 5' end, then the nucleic acid substrate has an exonuclease-inhibiting structure at each of its 5' ends. If the exonuclease is active at both the 3' and 5' ends, then the nucleic acid substrate has an exonuclease-inhibiting structure at each of the 3' and 5' ends.

[0080] The choice of exonuclease should also be consistent with the types of breaks and accessible ends generated by the endonuclease being tested. The exonuclease (or mixture of exonucleases) should be active against the types of ends (protruding, blunt or recessed, hydroxyl or phosphoryl) generated by the endonuclease being tested, and at the same time, it should be inhibited by inhibitory structures present at the ends of the nucleic acid substrate described in this application.

[0081] In some embodiments of the invention, the endonuclease is a nickase, i.e., an endonuclease that cleaves only one strand of a duplex and forms a nick. In this embodiment, the exonuclease can be, for example, exonuclease III, T5 exonuclease, T7 exonuclease, lambda exonuclease, and BAL31 exonuclease.

[0082] In some embodiments, an endonuclease cleaves both strands of a duplex and forms a double-strand break. In some embodiments, the double-strand break has blunt ends. In some embodiments, the double-strand break has staggered ends. In some embodiments, the staggered ends may have a 3' overhang. In some embodiments, the staggered ends may have a 5' overhang. One skilled in the art can select an exonuclease (or a mixture of two or more exonucleases) that is capable of initiating hydrolysis in the desired direction from the particular type of ends generated by the endonuclease being tested. For example, New England Biolabs (Ipswich, Mass.) provides a list of available exonucleases grouped by biochemical properties (e.g., type of ends desired and direction of hydrolysis).

[0083] As further shown in Figure 1, panel B, the substrate comprises a recognition sequence (or recognition site) for an endonuclease. Depending on the endonuclease being tested, the recognition site may comprise a cleavage site and have one or more additional elements. In some embodiments, the endonuclease being tested is a nucleic acid-regulated endonuclease. In some embodiments, the endonuclease being tested is a class I CRISPR endonuclease (CASCADE) or a class II CRISPR endonuclease. In some embodiments, the endonuclease being tested is a CRISPR Cas9 or CRISPR Cas12a (Cpf1) endonuclease.

[0084] 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, a hammerhead ribozyme, a DNAzyme, a PIAzyme, or an engineered endoribonuclease, such as those described in Choudhury, R. et al., (2012) Engineering RNA endonucleases with customized sequence specificities. Nature Comm., 3:1147.

[0085] In some embodiments, the endonuclease being tested is an endonuclease encoded by a CRISPR locus or an endonuclease related thereto. The CRISPR (clustered regularly repeated short palindromic repeats) genomic locus is found in many prokaryotic genomes and confers resistance to the incorporation of foreign nucleic acids. The structure, nomenclature, and classification of CRISPR loci are discussed in Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477.

[0086] Briefly, a typical CRISPR locus comprises a series of short repeats regularly interspersed with spacers. The CRISPR locus also includes sequences encoding CRISPR-associated (Cas) genes. The spacer-repeat sequence unit encodes crisprRNA (crRNA). In vivo, mature crRNAs are processed from a polycistronic transcript called pre-crRNA or the pre-crRNA array. Repeats in the pre-crRNA array are recognized by Cas-encoded proteins, which bind to the repeats and cleave them, releasing mature crRNAs. CRISPR systems cleave the target nucleic acid, with Cas proteins and crRNA forming CRISPR ribonucleoproteins (crRNPs). The crRNA molecule directs crRNP to a target nucleic acid (e.g., a foreign nucleic acid entering a bacterial cell), and the Ca3 nuclease proteins cleave the target nucleic acid.

[0087] Class 1, type I CRISPR systems include means for processing a pre-crRNA array, which includes a multiprotein complex called Cascade (CRISPR-associated complex for antiviral defense) and consists of CasA, B, C, D, and E subunits. The Cascade-crRNA complex recognizes a target nucleic acid by hybridizing the target nucleic acid to crRNA. The associated nucleoprotein complex recruits the Cas3 helicase/nuclease to facilitate cleavage of the target nucleic acid.

[0088] Class 2, type II CRISPR systems include transactivating CRISPR RNA (tracrRNA). TracrRNA hybridizes to the crRNA repeat in 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 by hybridizing the target nucleic acid to crRNA. Hybridization of crRNA to the target nucleic acid activates the Cas 9 nuclease to cleave the target nucleic acid.

[0089] Class 1, type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6), which cleave the pre-crRNA array via one or more CRISPR polymerase-like proteins.

[0090] Class 2, type V CRISPR systems contain another 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.

[0091] As shown in Figure 1, panel B, the substrate comprises a recognition sequence (or recognition site) of 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 CRSIPR Cas9 endonuclease, and the recognition sequence is a protospacer adjacent motif (PAM), while the cleavage site is adjacent to the PAM. In some embodiments, the endonuclease is CRISPR Cas12a endonuclease and the recognition sequence is a protospacer adjacent motif (PAM), while the cleavage site is 16-18 bases from the PAM on the non-target strand and 23-25 bases 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.

[0092] In some embodiments, the endonuclease is a nickase, i.e., an endonuclease that cleaves only one strand of a duplex and forms a nick. In some embodiments, the endonuclease cleaves both strands of the duplex and forms a double-strand break. The double-strand break may have blunt ends or staggered ends. The staggered ends may have a 3' overhang or a 5' overhang. The 5' end may have a 5'-phosphoryl or 5'-hydroxyl group, while the 3' end may contain a 3'-phosphoryl or 3'-hydroxyl group.

[0093] CRISPR nucleases do not cleave a fixed sequence, but are instead guided by a guide nucleic acid, as described above. In addition to the guide RNA, CRISPR nucleases recognize an additional sequence called a protospacer adjacent motif (PAM). In some embodiments, a substrate of the invention comprises a protospacer adjacent motif (PAM). In embodiments of the invention, if the endonuclease being tested is a class I CRISPR endonuclease (CASCADE), 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 those embodiments of the invention where the test endonuclease is a class II CRISPR 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'.

[0094] CRISPR nucleases do not cleave a fixed recognition sequence, but are instead guided by a guide nucleic acid, called a "guide RNA," also referred to herein as a "nucleic acid targeting another nucleic acid (NATNA)." The guide RNA (NATNA) contains a "spacer" sequence complementary to the cleavage site of the endonuclease. In embodiments where the test endonuclease is a CRISPR endonuclease, the substrate includes a target sequence capable of hybridizing to a portion (the "spacer") of the NATNA.

[0095] In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. The reaction mixture with such an endonuclease further requires a nucleic acid that targets another nucleic acid (NATNA). In some embodiments, the endonuclease is a CRISPR endonuclease and 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 NATNA comprises tracr RNA and cr RNA. In some embodiments, the endonuclease is a class 2, type V CRISPR endonuclease and NATNA comprises cr RNA.

[0096] In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, the NATNA may comprise, in the 5' to 3' direction, a spacer extension, a spacer, a minimal CRISPR repeat, a single guide connector, minimal tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some cases, a nucleic acid targeting another nucleic acid may comprise a tracrRNA extension, a 3' tracrRNA sequence, minimal tracrRNA, a single-stranded guide connector, a minimal CRISPR repeat, a spacer, and a spacer extension in any order.

[0097] In some embodiments of the invention, a nucleic acid targeting a guide nucleic acid may comprise a single guide NATNA. NATNA comprises a spacer sequence that can be designed to hybridize to the target nucleic acid sequence. NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, NATNA may have a spacer extension and a tracrRNA extension. These elements may include elements that can enhance the stability of NATNA. The CRISPR repeat and the tracrRNA sequence may interact with each other to form a double-stranded base-paired structure. Such a structure may facilitate the binding of an endonuclease to NATNA.

[0098] In some embodiments, the single-stranded guide NATNA comprises a spacer sequence located 5' of the first duplex that comprises a region of hybridization between the minimal CRISPR repeat and the minimal tracrRNA sequence. The first duplex may be interrupted by a "node." Such a "node" facilitates recruitment of the endonuclease to NATNA. The node may be followed by a first stem containing a linker connecting the minimal CRISPR repeat and the minimal tracrRNA sequence. The last base pair at the 3' end of the first duplex may be linked to a second linker connecting the first duplex to the middle tracrRNA. The middle tracrRNA may contain one or more additional hairpins.

[0099] In some embodiments of the invention, a NATNA may comprise a double-stranded guide nucleic acid structure. The double-stranded guide NATNA includes a spacer extension, a spacer, a minimal CRISPR repeat, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The double-stranded guide NATNA does not include a single-stranded guide connector. Instead, the minimal CRISPR repeat sequence comprises a 3' CRISPR repeat sequence, the minimal tracrRNA sequence comprises a 5' tracrRNA sequence, and the double-stranded guide NATNAs can hybridize via the minimal CRISPR repeat and the minimal tracrRNA sequence.

[00100] In some embodiments, NATNA is an engineered guide RNA comprising one or more DNA units (hybrid RDNA CRISPR or chRDNA). In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNA for use with a Class 2 CRISPR system may consist of two strands forming a secondary structure that includes an activating region consisting of an upper duplex region, a lower duplex region, a node, a targeting region, a strand, and one or more hairpins. The nucleotide sequence located immediately downstream of the targeting region may contain DNA and RNA in varying ratios. Another chRDNA can be a single-stranded guide D(R)NA for use with the Type II CRISPR system, comprising a targeting region and an activating region consisting of a lower duplex region, an upper duplex region, a fusion region, a node, a strand, and one or more hairpins. The nucleotide sequence located immediately downstream of the targeting region can contain DNA and RNA in varying ratios. For example, the targeting region can contain DNA or a mixture of DNA and RNA, and the activating region can contain RNA or a mixture of DNA and RNA.

[00101] In some embodiments of the invention, the guide RNA includes nucleic acid modifications, such as modifications that confer ribonuclease resistance. This feature is particularly useful in the crude lysate assays described below.

[00102] In some embodiments of the invention, the test endonuclease is a restriction endonuclease. In some embodiments, the test endonuclease is a type II, III, or IV restriction endonuclease. For each endonuclease, the substrate of the invention comprises a corresponding recognition sequence. For a type IV restriction endonuclease, the substrate of the invention also comprises one or more methylated residues required for cleavage by the endonuclease.

[00103] In embodiments where the test endonuclease 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, separated by a spacer of 4-18 nucleotides in length (see, e.g., Kim Y. G., et al., (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA. 93(3): 1156-1160.

[00104] In those embodiments of the invention in which the test endonuclease 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 consisting of 18-30 bases separated by a spacer of 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).

[00105] In some embodiments, the test endonuclease is Argonaute (Ago) endonuclease. Ago endonucleases do not have a recognition sequence but are guided by small interfering guide DNA (siDNA) to cleave complementary DNA. Hegge, et al., (2019) DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute, N.A. R. 47 (11):5809.

[00106] In some embodiments, the test endonuclease is Arcus endonuclease. Arcus is an I-Crel endonuclease with a 22-base target sequence (see, e.g., Durrenberger et al., (1991) Double-strand break induced recombination in Chlamydomonas reinhardtii chloroplasts, N.A.R. 24 (17):3323.

[00107] In some embodiments, the endonuclease being 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 an assay is an exodeoxyribonuclease or an exoribonuclease for cleaving the selected substrate. Examples of endoribonucleases that cleave one or more such substrates include RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z, and RNase P. Other examples of endoribonucleases include CRISPR endoribonucleases selected from Casl3 and Cas7-11.

[00108] Examples of exoribonucleases include exoribonucleases that cleave in the 3'-5' direction, such as RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, and that cleave in the 5'-3' direction, such as exoribonuclease I and exoribonuclease II.

[00109] In some embodiments, the present invention relates to a method for detecting endonuclease activity using a substrate described herein. As shown in Figure 1, panel B, the method comprises contacting a test endonuclease with a reaction mixture comprising a double-stranded nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein the fluorophores form a fluorescence resonance energy transfer (FRET) pair, and a recognition sequence of the test endonuclease. The reaction mixture is incubated under conditions suitable for endonuclease cleavage of its recognition sequence.

[00110] This method further comprises contacting the reaction mixture with an exonuclease. The exonuclease, substrate, and endonuclease may be added simultaneously or sequentially in any order.

[00111] As shown in Figure 1, panels B-C, the structure of the double-stranded nucleic acid substrate is such that the exonuclease does not hydrolyze the double-stranded nucleic acid substrate until cleavage by the endonuclease occurs. The respective ends of the double-stranded nucleic acid substrate contain structures that inhibit cleavage of the substrate by the exonuclease. As described herein, depending on the ends generated by the endonuclease, the exonuclease is a 3'-5' exonuclease, and the 3' end of the substrate is protected by an exonuclease inhibitory structure. In other embodiments, the exonuclease is a 5'-3' exonuclease, and the 5' end of the substrate is protected by an exonuclease inhibitory structure. In some embodiments, a mixture of a 3'-5' exonuclease and a 5'-3' exonuclease is used. In such embodiments, the 3' end and 5' end of the substrate are protected by an exonuclease-inhibiting structure.

[00112] As shown in Figure 1, panel C, the reaction mixture is then incubated under conditions suitable for exonuclease cleavage of the double-stranded nucleic acid substrate. In some embodiments, the conditions for endonuclease cleavage and subsequent exonuclease cleavage are the same. In such embodiments, no changes in buffers 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.

[00113] One advantage of this method is that it minimizes background signal, since the fluorophore and quencher can be placed very close to each other. This also avoids any steric effects that might arise if a fluorophore-quencher pair were embedded near the cleavage site, for example, when the fluorophore was on one side of the cleavage site and the quencher on the other.

[00114] In some embodiments of the invention, the endonuclease tested by the method is a nucleic acid-regulated endonuclease. In such embodiments, the method also uses NATNA. In some embodiments, the endonuclease is a CRISPR endonuclease and NATNA is a guide RNA.

[00115] In some embodiments, the endonuclease tested by the method is a class I CRISPR endonuclease (CASCADE), wherein 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 those embodiments of the invention where the endonuclease tested by the method is a class II CRISPR endonuclease, the double-stranded nucleic acid substrate used in the method 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' and 5'-NNNACA-3', 5'-TTN-3', 5'-TTTN-3' and 5'-TTTV-3'.

[00116] In some embodiments, the endonuclease tested by the method is one of a zinc finger nuclease (ZFN), a Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), a single-stranded endonuclease Endo TT, an Argonaute endonuclease, an 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 these embodiments, the double-stranded nucleic acid substrate used in the method comprises a suitable recognition site.

[00117] In some embodiments, the method comprises screening, testing, or comparing multiple endonucleases. In these embodiments, the method comprises contacting a series of reaction mixtures containing the same ingredients with a series of different endonucleases. In some embodiments, the series of endonucleases is a series of nucleic acid-regulated endonucleases, and the reaction mixtures in the series of reaction mixtures containing the same ingredients also contain 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 containing the same ingredients also contain the same guide RNA.

[00118] In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease, and the method comprises screening, testing, or comparing multiple NATNAs. In these embodiments, the method comprises contacting a series of reaction mixtures containing the same ingredients, including the same endonuclease, with a series of different NATNAs. In some embodiments, the endonuclease is a CRISPR endonuclease, and the NATNA is a guide RNA. In these embodiments, the method comprises contacting a series of reaction mixtures containing the same ingredients, including the same CRISPR endonuclease, with a series of different guide RNAs.

[00119] In some embodiments, the method comprises screening, testing, or comparing multiple preparations of the same endonuclease. In these embodiments, the method comprises contacting a series of reaction mixtures containing the same ingredients with a series of different preparations of the same endonuclease. The different preparations may be different isolates of the same endonuclease. The different preparations may be eluted aliquots obtained from a chromatography procedure aimed at isolating the endonuclease. The present invention includes a method of monitoring the elution of an endonuclease by performing the endonuclease activity assay described herein on the elution fractions obtained from the chromatography procedure and retaining the elution fractions with the highest endonuclease activity.

[00120] In some embodiments of the invention, the method includes screening, testing, or comparing multiple nucleic acid sequences to identify a preferred or optimal target sequence for an endonuclease. In these embodiments, the method includes contacting a series of reaction mixtures containing the same ingredients, 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 containing the same ingredients also contain the same guide RNA.

[00121] In some embodiments of the invention, the method includes screening, testing, or comparing several reaction conditions to determine preferred or optimal reaction conditions for the endonuclease. In some embodiments, the method includes contacting a series of reaction mixtures containing different buffer configurations with the same endonuclease. In some embodiments, the series of reaction mixtures are also exposed to different temperature profiles during the endonuclease cleavage step.

[00122] In some embodiments of the invention, the method includes screening, testing, or comparing several reaction conditions to identify preferred or optimal concentrations of endonuclease in nucleic acid cleavage reactions. In some embodiments of the invention, the method includes contacting a series of reaction mixtures that are identical but have different concentrations of the same test endonuclease.

[00123] In some embodiments, the method includes screening, testing, or comparing multiple CRISPR guide polynucleotides (guide RNAs or rRNAs) to identify a preferred or optimal rRNA for the CRISPR endonuclease. In these embodiments, the method includes contacting a series of reaction mixtures that are identical but have different guide RNAs. In some embodiments, the CRISPR guide polynucleotide comprises one or more DNA residues (hybrid CRISPR RDNA or chRDNA). In these embodiments, the method includes contacting a series of reaction mixtures that are identical but have different chRDNAs.

[00124] As shown in Figure 1, panel D, this method also includes assessing the fluorescence emitted by the reaction mixture, where a change in fluorescence indicates endonuclease activity. A change in fluorescence includes a change in the color (wavelength) of the fluorescent signal, as well as the appearance of fluorescence where fluorescence was not previously detected. In some embodiments, the assessment is qualitative and indicates the presence or absence of endonuclease activity. In other embodiments, the assessment is quantitative and indicates the relative amount of endonuclease activity. In some embodiments, the change in fluorescence further includes a change in fluorescence intensity and differences in fluorescence intensity between test samples.

[00125] In some embodiments, the method described herein is performed using isolated nucleic acid substrates and isolated polypeptides (e.g., endonuclease and exonuclease). In other embodiments, the assay is performed with crude mixtures without purification steps. In some embodiments, isolated or purified nucleic acid substrate is added to crude isolates or to resulting fractions of endonuclease, for example, to rapidly assess the production or purification process of endonuclease. In some embodiments, isolated or purified polypeptides (e.g., endonuclease and exonuclease) are added to crude nucleic acid isolates, for example, to minimally processed samples taken from patients, to rapidly detect the presence of an infectious agent in a patient.

[00126] In some embodiments, the present invention relates to a double-stranded nucleic acid substrate and a method for producing the substrate. The substrate has an exonuclease end protection, such as, for example, phosphorothioate linkages. 1, 2, 3, 4, or about 5 phosphodiester linkages can be replaced with phosphorothioate linkages. For example, if exonuclease III is used, the phosphodiester linkages can be replaced with phosphorothioate linkages at the 3' ends of both strands. Optionally, the terminal phosphate moiety (if present) at the 3' end of each strand can be replaced with a phosphorothioate.

[00127] 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 fluorophores and quenchers can be attached to the end of one strand. For example, if exonuclease III is used, one member of the fluorophore-quencher pair can be attached to the 5' end of the target strand, while this member can be attached to a nucleotide 1, 2, 3, 4, or approximately 5 nucleotides long from this end. For example, one strand can contain thymine-linked fluorescein at the 3rd nucleotide position from the 5' end and an Iowa Black® quencher at the 5' end.

[00128] In some embodiments, the test endonuclease recognizes a sequence on both strands (e.g., a palindromic sequence recognized by type II restriction endonucleases). In some embodiments, the test endonuclease 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 exemplary substrate for CRISPR Cas12a is shown in Figure 7. The target strand of the substrate includes a sequence targeted by the test endonuclease. For example, the Cas12a cleavage site (Cpfl) is described in Zetsche, B., et al., (2015) Cpfl is a single-RNA guided endonuclease of a Class II CRISPR-Cas system, Cell, 163:P759. As shown in Figure 7, the substrate further includes a spacer sequence recognized by CRISPR guide nucleic acids, a fluorophore, a quencher, and an exonuclease end protector such as phosphorothioate nucleotides at or near the ends of each strand.

[00129] In some embodiments, a fluorophore is placed on the target strand. In some embodiments, a fluorophore is placed on the non-target strand. In some embodiments, a fluorophore is placed on both the target and non-target strands. Figure 10 illustrates the substrate shown in Figure 7 with an alternative fluorophore placement. In Figure 10, the light and dark blocks on the top strand represent the recognition sequence (target) for CRISPR endonucleases (light block is a PAM, dark block is a spacer). Fluorophores are represented as asterisks, and quenchers are represented as dark crescents. Dark octagons represent exonuclease-inhibiting modifications, such as phosphorothioate nucleotides.

[00130] In some embodiments of the invention, the placement of the fluorophore affects the performance of the assay (see Example 11 and Figures 11, 12 and 13).

[00131] A double-stranded nucleic acid substrate can be prepared by combining the two strands (the target strand and the complementary non-target strand) in a reaction mixture containing a suitable buffer (e.g., TE). For optimal annealing, the mixture can be heated to >90°C and allowed to cool to room temperature.

[00132] In some embodiments, the effectiveness of exonuclease protection is tested for each exonuclease intended for use in the endonuclease assay disclosed herein. For each double-stranded nucleic acid substrate described herein, a control double-stranded nucleic acid substrate lacking exonuclease protection is prepared. Both substrates are exposed to the exonuclease in a suitable buffer and under suitable reaction conditions for both exonuclease activity and fluorescence (e.g., in NE buffer 1, pH 7.0 for exonuclease III and fluorescein) and fluorescence is assessed, for example, using a fluorescence reader. If the exonuclease protection is suitable for the exonuclease, then a fluorescent signal will be generated for the unprotected substrate, but not for the protected substrate.

[00133] In some embodiments, a double-stranded nucleic acid substrate described herein is used to detect endonuclease activity. The substrate has an exonuclease protector, a fluorophore, and a quencher (e.g., 1, 2, 3, 4, or about 5 phosphorothioate linkages at the 3' ends of both strands and thymine-linked fluorescein at the 3 nucleotide position from the 5' end and an Iowa Black® quencher at the 5' end of the target strand). The substrate is contacted with the exonuclease and endonuclease in a suitable buffer and under reaction conditions suitable for exonuclease activity, endonuclease activity, and fluorescence (e.g., NE buffer 1, pH 7.0 for exonuclease III, AsCas12a, and fluorescein). Guidance on the selection of a suitable buffer can be found in the manual available from endonuclease distributors (e.g., New England Biolabs for restriction endonucleases) or in published studies, such as Gasiunas, G., et al., (2020) A catalogue of biochemically diverse CRISPR-Cas9 orthologSf Nature Comm. 11, Article number: 5512 doi:10.103 8/s41467-020-193 44-1.

[00134] If the endonuclease is a nucleic acid-regulated 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 the nucleoprotein complex. The nucleoprotein complex comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), e.g., a CRISPR guide RNA (crRNA) such as crRNA for Cas12a (Cpfl), the relevant sequences of which can be found, e.g., in Yamano T., et al., (2016) Crystal structure of Cpf1 In complex with guide RNA and target DNA., Cell 165:P94 9. To assemble the nucleoprotein complex, NATNTA is incubated with the endonuclease under suitable conditions, e.g., at 37°C for 10 minutes. NATNTA can be pretreated to ensure proper secondary structure formation by heating (e.g., to 95°C for 2 minutes) and then allowing it to cool slowly to room temperature.

[00135] The fluorescence of the reaction mixture is then assessed. For exonuclease-protected substrates, a fluorescent signal is generated only when both exonuclease and endonuclease are present.

[00136] In some embodiments of the invention, the linear range of the assay with respect to the endonuclease concentration and the nucleic acid substrate concentration is tested to determine the optimal range of substrate concentration and sensitivity with respect to the endonuclease concentration.

[00137] In some embodiments, the present invention relates to a composition for detecting endonuclease activity. The composition comprises a nucleic acid substrate as described herein and further comprising a 3'-5' exonuclease (or a 5'-3 exonuclease, or both of these exonucleases) inhibited by structures at the 3' ends (or 5' ends, or both ends) of the substrate. The composition comprises a double-stranded nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein the fluorophores 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.

[00138] The composition may also comprise an exonuclease. Depending on the ends generated by the endonuclease, the composition comprises a 3'-5' exonuclease or a 5'-3' exonuclease, or a mixture of both. In some embodiments, the signal is enhanced by the presence of more than one fluorophore per substrate. In some embodiments, the specific properties of the endonuclease being tested are unknown. In such embodiments, both ends of both strands are labeled to account for all possible orientations and nucleic acid cleavage chemistries. In some such embodiments, the ends are labeled with different fluorophores emitting at different wavelengths. The emission wavelength indicates the identity of the cleaved strand and the cleavage chemistry. For 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. For 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 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 an exonuclease-inhibiting structure.

[00139] In some embodiments of the invention, the substrate is designed to accommodate multiple types of endonucleases, including endonucleases whose biochemical properties are not fully known at the time of testing. Such a substrate will have a fluorophore and quencher pair located at or near both ends of both strands. Such a doubly labeled substrate can be used in a reaction mixture containing an exonuclease capable of hydrolyzing in both directions (e.g., exonuclease V or exonuclease VII).

[00140] In some embodiments of the invention, the endonuclease tested by the composition is a nucleic acid-regulated endonuclease. In such embodiments, NATNA is also present in the composition. In some embodiments, the endonuclease is a CRISPR endonuclease and NATNA is a guide RNA.

[00141] In some embodiments of the invention, the endonuclease tested by the composition is a class I CRISPR endonuclease (CASCADE), wherein the double-stranded nucleic acid substrate in the composition includes a protospacer adjacent motif (PAM) and 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 those embodiments of the invention where the endonuclease tested by the composition is a class II CRISPR endonuclease, the double-stranded nucleic acid substrate in the composition includes a protospacer adjacent motif (PAM) and 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', 5'-TTN-3', 5'-TTTN-3' and 5'-TTTV-3'.

[00142] In some embodiments, the endonuclease tested by the composition is one of a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 these embodiments, the double-stranded nucleic acid substrate in the composition includes a suitable recognition site.

[00143] In some embodiments of the invention, the composition comprises exonuclease III, a double-stranded nucleic acid substrate comprising (i) both 3' ends containing one or more dsDNAs protected with phosphorothioate; (ii) a reporter fluorophore, and (iii) a compatible fluorescence quencher positioned to quench donor fluorescence if the double-stranded nucleic acid substrate is intact, and the endonuclease of interest.

[00144] In some embodiments, the present invention provides a method suitable for use as a convenient tool for evaluating, screening, testing, or comparing multiple endonucleases. This tool further allows for the evaluation of a set of endonuclease cleavage conditions, which allows for the determination of which condition provides the highest level or rate of endonuclease activity. This tool also allows for the evaluation of endonuclease isolation and purification methods. In particular, this tool can be used to compare protein isolation fractions to identify the fraction containing the isolated protein. In such embodiments, a modification is introduced to ensure that all components, for example, the endonuclease, exonuclease, NATNA (if used) will have activity and will be at least partially protected from enzymatic cleavage in the crude preparation. This tool can be rapidly applied to newly formed fractions, for example, to monitor the protein purification process. Moreover, this tool can be applied to screen multiple endonuclease substrates with different sequences to quickly identify the endonuclease target sequence.

[00145] The methods and compositions disclosed herein can be used in a diagnostic assay. In some embodiments, the present invention relates to a method for detecting the presence of a particular nucleic acid in a sample, wherein the nucleic acid comprises an endonuclease recognition sequence. In some embodiments, the sample is a sample taken from a patient. The nucleic acid can be characteristic of a microorganism, including a virus or bacterium. The nucleic acid can also contain a polymorphism or sequence, the presence of which is associated with a disease or condition that can be detected in the patient.

[00146] This method includes manipulating nucleic acids isolated from a sample. In some embodiments, the sample is obtained from an individual or a patient. In some embodiments, the sample may comprise a fragment of solid tissue or a solid tumor obtained from the individual or patient, such as by biopsy. The sample may also include bodily fluids that may contain nucleic acids (e.g., urine, sputum, serum, blood or blood fractions, i.e., plasma, lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid, or stool samples). In other embodiments, the sample is a cultured sample, such as a tissue culture containing cells and fluids from which nucleic acids can be isolated. In some embodiments of the invention, the nucleic acids of interest present or suspected to be present in the sample are derived from infectious agents such as viruses, bacteria, protozoa, or fungi.

[00147] In some embodiments, the method includes a pre-amplification procedure in which a nucleic acid in a sample is amplified by a polymerase chain reaction (PCR), resulting in the generation of a specific amplicon from each target nucleic acid. In some embodiments, a fluorophore, a quencher, and an end protection are incorporated into the amplicon by ligation of adapters. Formation of blunt ends, A-tailing, and adapter ligation can be performed using established methods, for example, in combination with the formation of a sequence library for massively parallel sequencing. In some embodiments, the fluorophore, quencher, and end protection are included directly in the amplification primers. Excess primers or adapters containing a fluorophore, quencher, and end protection can be removed by a purification procedure prior to endonuclease analysis. An amplicon comprising a fluorophore, a quencher and an end protection is a double-stranded nucleic acid substrate used directly in the method disclosed in this application.

[00148] In some embodiments, an endonuclease substrate, which is a probe comprising a fluorophore, a quencher, and an end protection, hybridizes to a target nucleic acid or an amplicon of a 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 becomes single-stranded before hybridization with the probe. The duplex formed by the nucleic acid=target hybridized to the probe comprising a fluorophore, a quencher, and an end protection becomes a double-stranded nucleic acid substrate used directly in the method disclosed herein.

[00149] A nucleic acid substrate obtained by any of the alternative methods described above contains a diagnostically significant nucleic acid sequence to be analyzed. In some embodiments of the invention, 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 a sequence the presence of which is associated with a disease or condition that can be detected in a patient. The endonuclease cleaves only if the sequence of interest, containing an endonuclease cleavage site, is present in the nucleic acid substrate.

[00150] In the diagnostic assay described herein, CRISPR endonucleases are particularly advantageous. A guide RNA for a CRISPR endonuclease (e.g., crRNA) can be designed to hybridize to any diagnostic sequence of interest. A sample is contacted with a probe containing a fluorophore, a quencher, and an end protector and capable of hybridizing to a target nucleic acid of diagnostic interest. The sample is then contacted with a guide RNA capable of hybridizing to the target nucleic acid. The CRSIPR endonuclease cleaves, and fluorescence becomes detectable only if a sequence capable of hybridizing to the probe and the designed guide RNA are present in the sample.

[00151] In some embodiments of the invention, the diagnostic method is multiplexed, meaning that multiple target sequences are detected in the same reaction mixture. In such embodiments, multiple nucleic acid probes are added to the sample. In those embodiments where the endonuclease is a CRISPR endonuclease, multiple guide RNAs are also added to the sample, and the same CRISPR endonuclease performs the cleavage resulting in the production of a detectable signal. In some embodiments, each of the different probes is labeled with different fluorophores. In some embodiments of the invention, all or some of the different probes are labeled with the same label, such as one label for a set of probes that hybridize to bacterial sequences and another label for a set of probes that hybridize to viral sequences, or one label for a set of probes that hybridize to Gram-positive bacterial sequences and another label for a set of probes that hybridize to Gram-negative bacterial sequences.

[00152] The endonuclease and exonuclease may be added to the sample sequentially or simultaneously. The endonuclease and exonuclease may be added to the sample before adding the double-stranded nucleic acid substrate. The exonuclease cleaves the strand (hydrolyzes) only if the endonuclease has previously cleaved, thereby creating an end accessible to the exonuclease. Bihydrolysis of the double-stranded nucleic acid substrate by the exonuclease separates the fluorophore and quencher, resulting in detection of a fluorescent signal. The presence of a fluorescent signal indicates the presence of a sequence of interest in the sample. In some embodiments, the assay includes the step of reporting that a sequence of interest (e.g., a sequence characteristic of a microorganism or polymorphism, or a sequence the presence of which is associated with a disease or condition that can be detected in a patient) is present in the sample.

[00153] In some embodiments, the present invention relates to a kit for detecting endonuclease activity. The kit includes an aliquot of a nucleic acid substrate described herein. The composition comprises a double-stranded nucleic acid substrate comprising: a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein the fluorophores 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 being tested.

[00154] The kit may further comprise an aliquot of an exonuclease. Depending on the ends generated by the endonuclease, the kit comprises a 3'-5' exonuclease or a 5'-3' exonuclease. For 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. For 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 comprise both a 3'-5' exonuclease and a 5'-3' exonuclease. The kit may contain a double-stranded nucleic acid substrate that has structures at both the 3' and 5' ends that inhibit cleavage of the substrate by an exonuclease.

[00155] In some embodiments of the invention, the endonuclease tested by the kit is a nucleic acid-regulated endonuclease. In such embodiments, the kit may also include an aliquot of NATNA. In some embodiments, the endonuclease tested is a CRISPR endonuclease, and the NATNA present in the kit is a guide RNA.

[00156] In some embodiments, the endonuclease to be tested by the kit is a class I CRISPR endonuclease (CASCADE), and the double-stranded nucleic acid substrate included in the kit includes 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 those embodiments of the invention where the endonuclease to be tested by the kit is a class II CRISPR endonuclease, the double-stranded nucleic acid substrate included in the kit comprises a protospacer adjacent motif (PAM) and 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'.

[00157] In some embodiments, the endonuclease to be tested by the kit is one of a zinc finger nuclease (ZFN), Fok I-conjugated ZFN, a transcription activator-like effector nuclease (TALEN), Endo TT 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 these embodiments, the nucleic acid substrate included in the kit comprises a suitable recognition site.

[00158] In some embodiments of the invention, the kit further comprises instructions for performing a method for testing endonuclease activity in the manner described herein.

[00159] In some embodiments, the present invention relates to a kit for performing a diagnostic procedure. The kit includes an aliquot of a probe capable of hybridizing with a target nucleic acid of diagnostic interest and comprising a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein the fluorophores 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 being tested. The kit may further comprise an aliquot of an exonuclease and an endonuclease. The exonuclease is capable of being inhibited by inhibitory structures present on the probe. The endonuclease is capable of binding and cleaving a duplex formed by the probe and the target sequence. In some embodiments, the endonuclease is a nucleic acid-regulated endonuclease. In such embodiments, the kit may also include an aliquot of NATNA capable of hybridizing to the target nucleic acid. In some embodiments, the test endonuclease is a CRISPR endonuclease, and the NATNA present in the kit is a guide RNA.

[00160] In some embodiments of the invention, the kit further comprises instructions for performing the diagnostic assay described herein.

[00161] The method disclosed herein can be implemented using a specialized device. In some of its embodiments, the present invention relates to a device for detecting endonuclease activity, comprising one or more reaction chambers for carrying out enzymatic reactions, and a fluorescence detector. This device may further comprise means for delivering and dosing components of the reaction mixtures described herein. This device can be adapted for high-throughput screening, for example, in multi-well plates (in microwell plates).

[00162] The device may include a multi-well plate fluorescence reader or a tube fluorometer, such as devices available from Tesap, ThermoFisher Scientific (BioTek Instruments), and Molecular Devices.

Examples

[00163] Example 1. Obtaining a double-stranded endonuclease substrate

[00164] In this example, a 60-base pair double-stranded nucleic acid construct was prepared in which the 3' ends of both strands were protected by a series of phosphorothioate linkages, and one strand was labeled with a fluorophore and a quencher. The last four phosphodiester linkages at the 3' ends of both strands were replaced with a phosphorothioate linkage. The terminal phosphate moiety at the 3' end of each strand was also replaced with a phosphorothioate. In addition, the target strand contained a thymine-linked fluorescein moiety incorporated at the 3 nucleotide position from the 5' end, and an Iowa Black® quencher was attached to the 5' end of the same strand. The dsDNA target included a well-characterized model targeting sequence of AsCas12a, which includes the cleavage site of the Acidaminococcus sp. Cas12a (AsCas12a) enzyme. Cleavage occurs 19–28 bp from the first thymine of the motif adjacent to the TTTC protospacer (PAM), which is located at position 16 from the 5' end of the substrate, and the final nucleotide of the spacer sequence is located at position 39 from the 5' end of the substrate. A second dsDNA target was generated that was identical to the first except that it lacked any end protection. Oligonucleotides with modifications were ordered from Integrated DNA Technologies (Coralville, Iowa).

[00165] The double-stranded substrate was assembled from the single-stranded SEQ ID NO: 1 and SEQ ID NO: 2. The double-stranded target DNA was prepared by combining the target strand and the complementary oligonucleotides of the non-target strands at a final concentration of 50 μM each in IX TE buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA), and to ensure proper annealing of the dsDNA, the mixture was heated to 95°C for 2 minutes and then slowly cooled to room temperature.

Upper chain (SEQ ID N0: 1):

5 ' -

Lower chain (SEQ ID N0: 2): 5 ' -

gcatacttagaggtctcgagcaaggagagatggctccaggaaatgggtcgaca* c* c* a*t* g-3 '

p ^L stands for Iowa Black® damper

nt stands for reporter fluorophore fluorescein

* denotes phosphorothioate bond

Underlined - protospacer adjacent motif (PAM) for Cas12a.

Double underline is the spacer region that hybridizes with the sr-RNA region.

[00166] A control double-stranded substrate lacking phosphorothioate protection was assembled from single-stranded SEQ ID NO: 3 and SEQ ID NO: 4.

Top strand (not exonuclease protected) (SEQ ID NO: 3): 5' -

Lower strand (not exonuclease protected) (SEQ ID NO: 4): 5' -

gcatacttagaggtctcgagcaaggagagagatggctccaggaaatgggtcgacaccatg-3' n" means quencher PF means fluorophore

Underlined - protospacer adjacent motif (PAM) for Cas12a.

Double underline is the spacer region that hybridizes with the sr-RNA region.

[00167] Example 2. Use of exonuclease-linked fluorescence detection

[00168] In this example, the substrates described in Example 1 were incubated at 100 nM with 0.5 U/μl exonuclease III in IX NEBuffer™ 1, pH 7.0, at 37°C (both from New England Biolabs, Ipswich, Mass.). 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. The signal was read using a Spectramax i3x plate (Molecular Devices, San Jose, Cal.). The results are shown in Figure 2. An increase in fluorescence was observed only on unprotected substrates in the presence of exonuclease III.

[00169] Example 3. Cleavage of substrate by endonuclease Cas12a

[00170] In this example, the authors demonstrated that cleavage of a phosphorothioate-protected dsDNA substrate by AsCas12a renders the phosphorothioate-protected substrate susceptible to exonuclease III cleavage, resulting in the production of a fluorescent signal that can be monitored in real time.

[00171]

[00172] A 60 bp double-stranded DNA target (dsDNA) was used that was protected with phosphorothioate as described in Example 1. Cas12a ribonucleoprotein (RNP), consisting of Cas12a and guide RNA (crRNA), was generated by incubating purified recombinant Cas12a protein at 37°C for 10 minutes with synthetic crRNA. Prior to RNP formation, crRNA was heated to 95°C for 2 minutes and slowly cooled to room temperature to ensure proper secondary structure formation. The crRNA component of the RNP provided specificity for the modeled AsCas12a target sequence present in the upper strand of the double-stranded substrate. Protected dsDNA target at 100 nM was incubated with either 2.5 U/μL exonuclease III alone or 2.5 U/μL exonuclease III and 112.5 nM AsCas12a RNP. Incubations were performed in IX NEBuffer™ 1, pH 7.0. A control reaction mixture included unprotected dsDNA target and exonuclease III, but no RNP. Reaction mixtures were incubated at 37°C, and fluorescence readings (excitation: 485 nm, emission: 520 nm) were recorded every 30 seconds for 600 minutes using a Spectramax i3x plate reader (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 RNP, but not in the RNP-free control, indicating that the end-protected dsDNA target was degraded after Cas12a cleavage. The unprotected dsDNA target was cleaved only by exonuclease III. During continuous fluorescence reading, a power outage occurred approximately 200 minutes into the experiment. Readings quickly resumed, but the interruption resulted in a sharp spike in fluorescence signal for all samples, which stabilized after approximately 10 minutes, and readings were restored.

[00173] Example 4. Linear range of the assay as a function of Cas12a RNP concentration.

[00174] In this example, the reaction between the double-stranded substrate, Cas12a RNP, and exonuclease was performed essentially the same as in Example 3, but using Outsmart® Buffer IX, pH 7.9 (New England Biolabs, Ipswich, Mass.). RNP concentrations ranged from 0.11 nM to 112.5 nM. The negative control did not include crRNA or Cas12a RNP. The positive control included exonuclease III with only the unprotected substrate (see Example 1). The results are shown in Figure 4. There is a direct correlation between the RNP concentration and the reaction rate. Reactions with lower RNP concentrations showed a plateau at a lower fluorescence intensity. A significant increase in fluorescence can be detected even for a sample concentration of 0.11 nM compared to the observed fluorescence for the reaction without Cas12a. The results indicate a linear dose-response relationship with Cas12a concentrations from 0.11 to 7 nM. The sensitivity of the assay over a wide range of Cas12a concentrations confirms the robustness of this quality control (QC) assay.

[00175] Example 5. Confirmation that an exonuclease, not Cas12a, hydrolyzes DNA.

[00176] Cas12a is unique among endonucleases in that it possesses non-specific exonuclease activity ("trans-cleavage"). In this example, the authors demonstrated that exonuclease III is responsible for cleavage of the dsDNA target after the initial cleavage of Cas12a-RNP, rather than for the trans-activity of Cas12a itself.

[00177] In this Example, the reaction between the double-stranded substrate, Cas12a RNP, and exonuclease was performed essentially the same as in Example 3, but using NEBuffer™ 1, pH 7.0. The concentration of dsDNA was varied. In addition, a control reaction mixture did not contain exonuclease. This control was included to determine whether Cas12a degrades the substrate on its own (without exonuclease) through a secondary non-specific nuclease activity known as trans-activity. The results are shown in Figure 5. We observed some increase in fluorescence in the absence of exonuclease III, indicating a possible limited contribution of Cas12a granule activity to the overall signal. However, the maximum fluorescence rate was approximately 3.3-fold higher in the presence of exonuclease III, suggesting that exonuclease III is present at sufficient concentration to ensure that the reaction rate is not limited by Cas12a transactivity. The maximum rate was observed for the unprotected dsDNA substrate with exonuclease III alone, indicating that exonuclease III activity is not rate-limiting.

[00178] Example 6. Linear range of the assay depending on the concentration of the DNA substrate

[00179] In this Example, the reaction between the double-stranded substrate, Cas12a RNP, and exonuclease was performed as described in Example 5. With all other reagents held constant, the DNA substrate concentration was varied from 2.06 nM to 500 nM. The results are shown in Figure 6. The inventors observed that the assay is sensitive enough to detect a dsDNA target sequence at a concentration of at least 4.1 nM and yields a linear dose-response over a wide range of dsDNA target concentrations.

[00180] Example 7 (prognostic). Cleavage of substrate by Cas9 endonuclease

[00181] In this Example, the experiment described in Example 3 was performed with Cas9 endonuclease instead of Cas12a endonuclease. The phosphorothioate-protected double-stranded DNA target (dsDNA) was similar to SEQ ID NO: 1/SEQ ID NO: 2, except that it contained a PAM sequence recognized by Cas9. In addition, the dsDNA target contained a fluorophore-quencher pair at the 5' end of the non-target strand as well as the target strand. Incubation was performed in Outsmart® Buffer IX. Cas9 ribonucleoprotein (RNP), consisting of SpyCas9 (Streptococcus pyogenes Cas9) and guide RNA (crRNA), was prepared by incubating purified recombinant Cas9 protein at 3°C for 10 minutes with synthetic crRNA. The cr-RNA RNP component provides specificity for SpyCas9 and the double-stranded substrate. The protected dsDNA target was incubated with either exonuclease III or the Cas9 RNP. One or more control reaction mixtures were included, such as no RNP, no exonuclease, or no exonuclease protection. The reaction mixtures were incubated at 37°C, and fluorescence readings corresponding to the reporter fluorophore emission wavelength were recorded every 30 seconds for 600 minutes using a Spectramax i3x plate reader (Molecular Devices, San Jose, Calif.).

[00182] Example 8 (prognostic). Cleavage of substrate by restriction endonuclease

[00183] In this Example, the experiment described in Example 3 was performed with a restriction endonuclease, such as a type II restriction endonuclease, instead of the Cas12a endonuclease. The phosphorothioate-protected double-stranded DNA (dsDNA) target is similar to SEQ ID NO: 1/SEQ ID NO: 2, except that it contains the recognition sequence of the restriction endonuclease being tested.

The protected dsDNA target was incubated with exonuclease III and restriction endonuclease in an appropriate buffer suitable for both restriction endonuclease and exonuclease III activity. One or more control reaction mixtures were included, such as without restriction endonuclease, without exonuclease, or without exonuclease protection. The reaction mixtures were incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore were recorded every 30 seconds for 600 minutes using a Spectramax i3x plate reader (Molecular Devices, San Jose, Calif.).

[00184] Example 9. Cleavage of FAM substrate by CRISPR Cas12a.

[00185] In this Example, we designed a substrate (the "BAM substrate") shown in Figure 7. The 60 bp target contained a protospacer adjacent motif (PAM) for Cas12a and a well-characterized AAVS1 spacer sequence. At the 5' end upstream of the PAM, the substrate contained deoxythymidine (c!T)-conjugated fluorescein (BAM) paired with the dark quencher Iowa Black. Both 3' ends were protected with phosphorothioate (Pt) linkages. The control substrate had no Pt protection at the 3' ends (the "unprotected BAM substrate").

[00186] The substrate (Figure 7) was cleaved in a reaction mixture containing the BAM substrate, Exo III, and the AsCas12a ribonucleoprotein (RNP) complex at 0.42-18 nM RNP, 2.5 kU/ml Exo III, and 100 nM dsDNA target in NEBuffer 1. The reaction was continued at 37°C and fluorescence data were collected as shown in Figure 8. The rate of increase in fluorescence over a period of 50-150 minutes was plotted against the concentration of Cas12a.

[00187] Example 10. Cleavage of TAMRA substrate by CRTSPR Cas12a.

[00188] In this Example, we constructed a substrate (the "TAMRA substrate") identical to the FAM substrate shown in Figure 7, except that a TAMRA-NHS ester was used instead of deoxythymidine (dT)-conjugated fluorescein (FAM). As with the FAM substrate (Figure 7), both 3' ends were protected with phosphorothioate (Pt) linkages. The control substrate had no Pt protection at the 3' ends (the "unprotected IAMRA substrate").

[00189] The substrate was cleaved in a reaction mixture containing TAMRA substrate, Exo III, and the AsCas12a ribonucleoprotein complex (RNP). The reaction mixture contained 18 nM RNP (or 1x NCA buffer control), 2.5 kU/ml Exo III, and 100 nM dsDNA substrate in NEBuffer 1. Reactions were continued at 37°C, and fluorescence data were collected as shown in Figure 9.

[00190] Example 11. Alternative placement of fluorophore

[00191] In this example, we developed a series of CRISPR Cas12a substrates (similar to those shown in Figure 7), but with alternative fluorophore placements. Figure 10 shows an "off-target FAM" substrate (the same as shown in Figure 7, having a FAM on the same strand and upstream of the PAM), an "on-target FAM" substrate (a FAM on the strand opposite the PAM), and a "dual FAM" substrate (a FAM on both the same strand and the opposite strand of the PAM).

[00192] Three substrates were cleaved in reaction mixtures containing one of the three FAM substrates, Exo III, and the AsCas12a ribonucleoprotein complex (RNP). Reaction mixtures contained 18 nM RNP (or 1x NCA buffer control), 2.5 kU/ml Exo III, 100 nM dsDNA substrate in NEBuffer 1. Reactions were continued at 37°C, and fluorescence data were collected (shown in Figure 11). Data are also presented as a comparison of all protected substrates (Figure 12) and the rate of fluorescence change in the different reaction mixtures (Figure 13).

[00193] Example 12. Optimization of Exonuclease Concentration [00194] In this Example, we titrated the exonuclease and optimized the exonuclease concentration. The digestion reactions were performed in NEBuffer 1 buffer, and the reaction mixtures contained the target strand FAM substrate (Figure 10), AsCas12a RNP at a concentration of 20.25 nM RNP (2:1 cas12a:cr-RNA), various amounts of ExoIII (156, 25-2500 U/mL), and FAM-labeled substrate. Control reaction mixtures contained unprotected substrate, no RNP, or no ExoIII. The reactions were continued at 37°C. The results are shown in Figure 14.

[00195] Example 13. Cleavage with Cas9 endonuclease

[00196] In this Example, the authors demonstrated the applicability of the assay to Cas9. This example provides a confirmation of the predictive Example 7. As described in Example 7, double-stranded DNA (dsDNA) was constructed as shown in Figure 1 and Figure 7. The substrate was protected with phosphorothioate and contained a PAM sequence recognized by Cas9, a spacer sequence, a fluorophore, and a quencher on the same strand as the PAM (the non-target strand). In this experiment, the Cas9 guide targeted the strand opposite the Cas12a guide.

[00197] We developed a single-stranded guide RNA (ssRNA) for the Cas9 endonuclease that combined crRNA and tracrRNA. As described in Example 7, incubations were performed in Cutsmartr® buffer IX. Cas9 ribonucleoprotein (RNP), consisting of SpyCas9 [Streptococcus pyogenes Cas9) and guide RNA (single-stranded guide RNA (ssRNA)), was prepared by incubating purified recombinant Cas 9 with ssRNA at 37°C for 10 minutes. Reaction mixtures included 50 nM SpyCas9, 1 kU/mL ExoI (New England Biolabs, Ipswich, Mass.), and dsDNA substrate at 110 nM in a 200 µL reaction volume. As described in Example 7, only a control reaction mixture without RNP (a reaction mixture containing only ExoIII) was included. The reactions were incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore were recorded every 30 minutes for 1000 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Calif. All reactions were performed in triplicate, and the averaged results for each data set are shown in Figure 15.

[00198] Example 14. Dissection by restriction endonucleases

[00199] In this example, the authors demonstrated the applicability of the assay to type II restriction endonucleases. This example represents a confirmation of the predictive Example 8.

[00200] As described in Example 8, phosphorothioate-protected double-stranded DNA (DSDNA) substrates were constructed as shown in Figure 1. The substrates contained recognition sequences for one of the restriction endonucleases Bsal, BcoDI, and Sail. As described in Example 7, each substrate was incubated with exonuclease III and a restriction endonuclease in Cutsmart® IX buffer. Each reaction mixture contained 300 units of one of Bsal HF v2, BcoDI, and Sail (New England Biolabs) and 1.25 kU ExoIII. The reaction mixtures were incubated at 37°C, and fluorescence readings corresponding to the emission wavelength of the reporter fluorophore were recorded every minute for 300 minutes using a Spectramax i3x plate (Molecular Devices, San Jose, Cal.). The results are shown in Figure 16.

[00201] Although the present invention has been described in detail with reference to specific examples, it will be obvious to one skilled in the art that various modifications can be made to the present description without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited by the examples described herein, but should be limited only by the claims presented below.

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<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q15">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>protospacer adjacent motif (PAM) for

Cas12a</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..55</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q16">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>catggtgtcgacccatttcctggagccatctctctctccttgctcgagacc

tctaag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>60</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..60</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q17">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Bottom strand (no exonuclease

protection)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..60</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gcatacttagaggtctcgagcaaggagagagatggctccaggaaatggg

tcgacaccatg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (41)

1. A nucleic acid substrate for detection of endonuclease activity, containing: (a) a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; (b) at least one structure on at least one strand of a nucleic acid that inhibits cleavage of a substrate by an exonuclease used to detect the activity of the endonuclease, wherein the structure that inhibits cleavage of the substrate by the exonuclease is selected from a hairpin, a protruding strand, and a modification of a nucleic acid, and wherein said structure is capable of inhibiting the activity of said exonuclease; and (c) endonuclease recognition sequence. 2. The substrate according to claim 1, where the exonuclease is selected from exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, exonuclease T5, exonuclease T7, exonuclease lambda, exonuclease T, exonuclease BAL-31, exonuclease RecJf, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I and exoribonuclease II. 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®. 4. The substrate according to any one of claims. 1-3, wherein 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 (hexachlorofluorescein), TET (tetrachlorofluorescein), JOE (5'-dichlorodimethoxyfluorescein), BODIPY® dyes, Eclipse quencher (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-nitrophenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]aniline), and pyridinyl isoquinolinedione dyes. 5. Substrate according to any one of paragraphs. 1-4, where the exonuclease is selected from exonuclease III, exonuclease T5, exonuclease T7, exonuclease lambda, exonuclease BAL-31, RNase R, RNase II, RNase D, RNase T, RNase BN, RNase PH, exoribonuclease I and exoribonuclease II. 6. The substrate according to any one of claims 1 to 5, wherein the endonuclease is a nucleic acid-regulated endonuclease. 7. The substrate of claim 6, wherein the nucleic acid-regulated endonuclease is a class I CRISPR endonuclease (CASCADE), 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'. 8. The substrate of claim 6, wherein the nucleic acid-regulated 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'. 9. The substrate of claim 6, wherein the nucleic acid-regulated endonuclease comprises an endonuclease and a nucleic acid that targets another nucleic acid (NATNA), and NATNA comprises DNA and RNA nucleotides. 10. The substrate of any one of claims 1 to 5, wherein the endonuclease is selected from the group consisting of zinc finger nuclease (ZFN), Fok I conjugated ZFN, transcription activator-like effector nuclease (TALEN), Endo TT endonuclease, Argonaute endonuclease, Arcus endonuclease, an endoribonuclease selected from RNase III, RNase A, RNase T1, RNase A, RNase H, RNase Z and RNase P, a ribozyme, a hammerhead ribozyme, a DNAzyme, a PNAzyme or an engineered endoribonuclease; Cas3 and Cas7-11, and a restriction endonuclease. 11. A composition for detecting endonuclease activity, comprising a nucleic acid substrate according to any one of claims 1-10 and an exonuclease. 12. A method for detecting endonuclease activity, comprising: (a) contacting an endonuclease with a reaction mixture comprising the composition of claim 11; and (b) measuring the fluorescence emitted by the reaction mixture, where a change in fluorescence indicates endonuclease activity. 13. The method according to claim 12, wherein the nucleic acid substrate or endonuclease is in an unrefined form. 14. A kit for detection of endonuclease activity, containing: (a) a substrate according to any one of claims 1 to 10; and (b) exonuclease. 15. A method for detecting the presence of a target nucleic acid in a sample, comprising: (a) contacting the sample with a substrate according to any one of paragraphs 1 to 10; and (b) measuring the fluorescence emitted by the reaction mixture, where a change in fluorescence indicates the presence of the target nucleic acid in the sample. 16. The method of claim 15, wherein the sample comprises an unpurified nucleic acid preparation. 17. A kit for carrying out a diagnostic procedure consisting of detecting a target nucleic acid in accordance with the method of claim 15, comprising the composition of claim 11, 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; and instructions for conducting diagnostic analysis. 18. A method for optimizing endonuclease cleavage reactions, wherein said method comprises: (a) preparing a series of reaction mixtures with an exonuclease and a nucleic acid substrate according to any one of claims 1-10; (b) contacting each of the series of reaction mixtures with different amounts of endonuclease; (c) measuring the fluorescence emitted by the reaction mixture, where a change in fluorescence indicates endonuclease activity; (d) selecting the amount of endonuclease that gives the highest fluorescence of the reaction mixture or the highest rate of increase in fluorescence of the reaction mixture as the optimal concentration of endonuclease. 19. A method for optimizing CRISPR endonuclease cleavage reactions, comprising: (a) preparing a series of reaction mixtures with a CRISPR endonuclease, an exonuclease, and a nucleic acid substrate, comprising: i. a donor fluorophore and an acceptor fluorophore, wherein said fluorophores form a fluorescence resonance energy transfer (FRET) pair; ii. at least one structure on at least one strand that inhibits cleavage of the substrate by an exonuclease, wherein the structure that inhibits cleavage of the substrate by an exonuclease is selected from a hairpin, a protruding strand, and a nucleic acid modification; and iii. endonuclease recognition sequence; (b) adding each of a series of nucleic acids targeting another nucleic acid (NATNA); (c) assessing the fluorescence emitted by the reaction mixture, where a change in fluorescence indicates endonuclease activity; (d) selection of the NATNA giving the highest fluorescence of the reaction mixture as the optimal NATNA.